Models and methods for assessing immune recruitment

Abstract

A system for modeling organ-on-a-chip and other biological models includes a 3D- printed platform including a bioprinted vascular and interstitial infill capable of being seeded with active cells such that the functioning of organs (for example, human organs) may be replicated accurately. In particular, conditions may be replicated in a synthetic environment such that immune recruitment may be simulated, studied and better understood.

Description

Attorney Docket No.: PCT.1316MODELS AND METHODS FOR ASSESSING IMMUNE RECRUITMENTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority pursuant to 35 U. S. C. § 119 to U. S. Provisional Patent Application No. 63 / 748,429, filed January 22, 2025, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Drug discovery and development are time-consuming and costly endeavors. In vivo testing of new drugs involves a phased, highly -regulated approach that helps to ensure the efficacy of the underlying drugs and / or therapies, and helps to ensure the safety of patients upon whom they are being tested, but also often entails multi-billion dollar (and multi-year) regulatory approval processes. In silico models have the potential to accelerate drug discovery and development. However, the data used to train these models is predominantly derived from traditional preclinical approaches, such as animal models and two-dimensional cell cultures, which often lack translational relevance to human biology. Improved methods and systems for drug discovery and biological assays are needed.SUMMARY

[0003] The present disclosure presents systems, methodologies, and apparatuses that enable quick prototyping and modeling of organs, tissues, cells, biological processes, and other mechanisms, via a flexible platform that allows for adaptive and / or iterative build and refinements of models. The present embodiments include an ecosystem that produces a platform for creating physical, synthetic models of organs, tissues, cells, via bioprinting of scaffolds that supports internal passages that approximate the geometries of human vasculatures. The vasculatures may be coated with biological materials such as endothelial cells and may be operatively coupled to live tissues and cells seeded in an interstitial space within the scaffold. Accordingly, the synthetic physical models are seeded with live cells and / or tissues. Scaffolding enables delivery of biologically active materials to the cells and / or tissue(s) via fluid(s) flowing through the vasculature, and transport through vasculature walls. The ecosystem includes control of environmental factors used for assaying synthetic tissue and / or organ models, real-time tracking of biomarkers via sensors, ability to rapidly image tissues and / or cells, processing of assay data,Attorney Docket No.: PCT.1316machine learning tools for diagnosing results, and updating of scaffold print files such that updated models can be reprinted, assays may be rerun, and the results may be reanalyzed. Therefore, the entire ecosystem enables adaptive learning and quick re-prototyping such that learnings from assays being performed can be immediately incorporated into future scaffold designs for further assaying and examination. As a result, the disclosed platform and ecosystem allow high resolution synthetic organs to be rapidly created and assayed, thereby enabling expedited drug discovery and early drug candidate screening.

[0004] In certain aspects, the present disclosure is directed to methods of assessing immune cell recruitment and / or tumor cell killing. In some such embodiments, a method comprises providing a bioprinted scaffold comprising at least one perfusable internal passageway (or network) and an interstitial space. The interstitial space can be operatively or fluidically coupled to the at least one internal passageway (or network), such as in the sense that fluids traveling or contained within the internal passageway or network can diffuse or otherwise be transported into the interstitial space (e.g., by diffusing through the walls of the internal passageway or network). In addition, in some instances, the scaffold of a method described herein is configured to permit migration of immune cells from the internal passageway (or network) to the interstitial space, such as by passing through pores disposed in the internal passageway (or network), as described further herein.

[0005] Moreover, in some cases, a method described herein further comprises seeding tumor cells within the interstitial space. A method described herein, in some instances, may also comprise seeding endothelial cells within the at least one internal passageway (or network) to form a functional endothelium. In addition, in some cases, a method described herein also comprises culturing the seeded scaffold under perfusion conditions to establish a vascularized tumor model. It is to be understood that such a vascularized tumor model can have a vasculature (as described further herein) and can serve as a mimic or model for a biological tumor, including for purposes of carrying out methods (such as assays) described herein.

[0006] Further, in some embodiments, a method described herein also includes perfusing immune cells through the at least one internal passageway (or network). Additionally, in some instances, a method described herein comprises an assessing step. For example, in some embodiments, a method described herein comprises assessing at least one of (i) immune cell adhesion to the functional endothelium, (ii) transendothelial migration of immune cells into the interstitial space, (iii) immune cell infiltration into the tumor microenvironment, or (iv) tumor cell killing by infiltrating immune cells.Attorney Docket No.: PCT.1316

[0007] As described further herein, various tumor cells can be used in a method according to the present disclosure. For example, in some cases, the tumor cells comprise patient-derived tumor cells, such as may be obtained from a tumor biopsy or surgical resection. In some non-limiting example implementations, patient-derived tumor cells comprise patient-derived xenograft (PDX) cells. It is also possible for tumor cells used in a method described herein to be cells from a tumor cell line or immortal cell line, such as HeLa cells.

[0008] In addition, in some embodiments, endothelial cells used in a method described herein are also patient-derived cells. Moreover, in some such instances, the patient-derived endothelial cells are derived from the same patient as the tumor cells (in the event the tumor cells are also patient-derived). Further, in some cases, the endothelial cells comprise iPSC-derived endothelial cells, and such iPSC-derived endothelial cells can be derived from the same patient as the tumor cells in some instances. In still other embodiments, the endothelial cells of a method described herein comprise human umbilical vein endothelial cells (HUVECs) or tumor-associated endothelial cells.

[0009] As described further herein, it is also possible, in some implementations, to embed cells in a matrix, such as an extracellular matrix, prior to or during seeding. For example, in some instances, a method described herein further comprises embedding the tumor cells in an extracellular matrix prior to or during seeding within the interstitial space. In some such cases, the extracellular matrix comprises Matrigel. In other instances, the extracellular matrix comprises a tumor-relevant extracellular matrix, which in some cases may comprise one or more of collagen I, collagen IV, fibronectin, laminin, and hyaluronic acid. In some embodiments, the extracellular matrix comprises decellularized extracellular matrix (dECM) derived from tumor tissue. Moreover, in some such instances, the dECM is derived from the same patient as the tumor cells, or from a tumor tissue type-matched to the tumor cells seeded within an interstitial space described herein.

[0010] Various scaffold materials and architectures may be used in a method described herein. In some embodiments, for example, the scaffold of a method described herein comprises a porous hydrogel material configured to permit migration of immune cells. Moreover, in some preferred embodiments, the internal passageway (or network) of the scaffold comprises micropores connecting the passageway (or network) to the interstitial space. As described further herein, such micropores may be printed micropores, meaning that they are intentionally printed or created, such as may occur when the micropores are defined as part of a digital file used to form the scaffoldAttorney Docket No.: PCT.1316using an additive manufacturing process. That is, the micropores may be intentionally created as “voids'’ in a 3D printed entity. Additionally, the micropores can be selected to have a specific size and / or shape. For example, in some embodiments, the micropores (or population of micropores) have a diameter (or average diameter) in a range from about 5 μm to about 100 μm. In some cases, the micropores have a diameter or average diameter of 10-90 μm, 10-80 μm, 10-70 μm, 10-60 μm, 20-90 μm, 20-80 μm, 20-70 μm, 20-60 μm, 30-90 μm, 30-80 μm, 30-70 μm, or 30-60 μm. It is further to be understood that the “diameter'’ (or average “diameter”) described above can be the actual geometric diameter, such as in the case when the “openings” of the micropores have a circular or substantially circular cross section (e g., an only slightly oblate cross section, such as may occur when a cross section is non-circular but has an aspect ratio between 0.95 and 1.05). Alternatively, if the micropore or population of micropores does not have a circular or substantially circular cross section (e.g., the cross section is irregularly shaped), then the foregoing numerical ranges can apply to the equivalent circular area diameter (or average equivalent circular area diameter), where the “equivalent circular area diameter” is the diameter of a circle that has the same area as the irregular (or non-circular) shape.

[0011] Additionally, in some cases, the size of the micropores is selected based on a desired cell transport rate of a given cell type. For example, in some cases, the micropores are sized to permit passage of activated immune cells while maintaining endothelial coverage. For example, in some instances, the endothelial cells form a confluent layer over the micropores, and activated immune cells transmigrate through the endothelial layer and the micropores to reach the interstitial space of the scaffold. Similarly, it is also possible, in some implementations, for the scaffold to comprise or be formed from a hydrogel with tunable porosity to modulate immune cell migration rate.

[0012] Various immune cells may be used in a method described herein. For example, in some cases, the immune cells comprise T cells, such as cytotoxic CD8+ T cells, tumor-infiltrating lymphocytes (TILs), or chimeric antigen receptor T cells (CAR-T cells). Further, in some embodiments, the immune cells of a method described herein comprise natural killer (NK) cells, such as CAR-NK cells. In still other instances, the immune cells comprise peripheral blood mononuclear cells (PBMCs). Moreover, in some such embodiments, the PBMCs are derived from the same patient as the tumor cells used in the method (e g., in the seeding step). Additionally, in some implementations, the immune cells of a method described herein comprise macrophages, such as CAR-macrophages.Attorney Docket No.: PCT.1316

[0013] Methods described herein, in some cases, can further comprise perfusing at least one therapeutic candidate through the at least one internal passageway. As described further herein, a therapeutic candidate can be any species (e.g., small molecule, biologic, or other species) that is desired to be evaluated for therapeutic efficacy or performance Any therapeutic candidate not inconsistent with the technical objectives of the present disclosure may be used. For example, in some embodiments, the therapeutic candidate is selected from the group consisting of immune checkpoint inhibitors, bispecific T-cell engagers, bispecific antibodies, monoclonal antibodies, antibody drug conjugates (ADCs), cytokines, chemokines, small molecule immunomodulators, oncolytic viruses, and tumor vaccines. In some embodiments, the therapeutic candidate comprises an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti -LAG-3 antibodies, and anti -TIM-3 antibodies. In other cases, the therapeutic candidate comprises a bispecific T-cell engager (BiTE). Moreover, in some implementations, the therapeutic candidate is (as part of a method described herein) assessed for its ability’ to enhance immune cell recruitment, transendothelial migration, and / or tumor cell killing, including in a manner described in the present disclosure.

[0014] As stated above, a method described herein can comprise an assessing step. In some embodiments, such assessing comprises carrying out flow cytometry analysis. Moreover, in some cases, flow cytometry analysis comprises characterizing immune cell phenotype before and after perfusion through the scaffold. Additionally, in some implementations, flow cytometry’ analysis comprises quantifying immune cell activation markers selected from the group consisting of CD69, CD25, CD107a, granzyme B, perforin, IFN-y. and TNF-a. In other instances, flow cytometry analysis comprises distinguishing immune cells recovered from a vascular lumen of the scaffold, versus immune cells that infiltrated the interstitial space. Flow cytometry analysis may also comprise quantifying the ratio of immune cells to viable tumor cells following co-culture.

[0015] An assessing step of a method described herein may also comprise live-cell imaging of immune cell trafficking. In some such embodiments, live-cell imaging comprises tracking immune cell adhesion, rolling, arrest, and / or transmigration at an endothelial surface described herein. Further, in some instances, live-cell imaging comprises monitoring immune cell infiltration into a tumor microenvironment (e.g., the tumor microenvironment of, or created by, the tumor model described herein) over time.Attorney Docket No.: PCT.1316

[0016] Additionally, an assessing step of a method described herein, in some embodiments, comprises carrying out or performing confocal or multiphoton microscopy to visualize immune cell localization within the scaffold.

[0017] In some implementations of a method described herein, the method comprises assessing tumor cell killing. In some such cases, assessing tumor cell killing comprises measuring tumor cell viability. Moreover, in some such instances, tumor cell viability is measured by live / dead staining, ATP quantification, and / or LDH release. Additionally, assessing tumor cell killing can also comprise quantifying tumor cell apoptosis by Annexin V staining or caspase activation.

[0018] Turning again to some optional steps of some methods described herein, in some cases, a method according to the present disclosure can further comprise measuring cytokines and / or chemokines in perfusate to assess immune activation. In some such embodiments, the cytokines comprise one or more of IFN-y, TNF-a, IL-2, IL-6. IL-10, granzyme B, and perforin. In other instances, the chemokines comprise one or more of CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL4, and CCL5.

[0019] In still other embodiments, a method described herein further comprises carrying out or performing gene expression analysis of tumor cells and / or immune cells recovered from the scaffold. In some such instances, gene expression analysis comprises single-cell RNA sequencing (scRNA-seq), or spatial transcriptomics to assess regional patterns of immune infiltration and tumor response.

[0020] As stated above, in some implementations, a method described herein uses various cells derived from a patient or the same patient. For example, in some preferred embodiments, the tumor cells, endothelial cells, and immune cells used in the method are all derived from the same patient. Moreover, in some such instances, the method may be used to predict patient-specific response to an immunotherapeutic agent. It is also possible for some not but all cells to be derived from the same patient. For instance, in some cases, the tumor cells and immune cells are derived from the same patient, and the endothelial cells are derived from a different source (e.g., a different source described further herein).

[0021] In another example, a method described herein comprises assessing tumor cell killing, and the method comprises providing a bioprinted scaffold comprising at least one perfusable internal passageway (or network) and an interstitial space (e.g., as described above). For example, the interstitial space can be coupled to the at least one internal passageway (or network). The method can also comprise seeding tumor cells within the interstitial space and culturing the seededAttorney Docket No.: PCT.1316scaffold under perfusion conditions to establish a tumor model. As stated above, and as understood by the skilled person, such a “model” can mimic a tumor or have a structure that mimics a tumor in one or more respects. Further, in some embodiments, a method described herein also comprises perfusing at least one therapeutic candidate through the at least one internal passageway (or network) of the scaffold (e.g., in a manner described further herein), thereby exposing the tumor cells to the therapeutic candidate via transport from the passageway (or network) to the interstitial space (e.g., in a manner described hereinabove). Additionally, in some instances, such a method further comprises assessing tumor cell killing by at least one of (i) determining tumor cell viability, (ii) measuring tumor cell apoptosis, (iii) quantifying secreted biomarkers indicative of tumor cell death, or (iv) imaging tumor cell morphology and / or proliferation. Moreover, in some implementations of such a method, the method further comprises seeding endothelial cells within at least one internal passageway (or network) to form a functional endothelium (which, e.g., may have a surface as described above). Moreover, as noted above, various therapeutic candidates may be used in a method described herein. For example, in some cases, the therapeutic candidate is selected from the group consisting of antibody drug conjugates (ADCs), small molecule chemotherapeutics, targeted kinase inhibitors, monoclonal antibodies, bispecific antibodies, immunotoxins, oncolytic viruses, nucleic acid therapeutics, and radioligand therapeutic agents. In some preferred embodiments, the therapeutic candidate comprises an antibody drug conjugate (ADC) and the method assesses ADC-mediated tumor killing in a vascularized tumor model.

[0022] In another aspect, systems are also described herein. In some embodiments, a system for assessing immune cell recruitment and tumor cell killing is provided. Such a system, in some cases, comprises a bioprinted scaffold comprising or formed from a hydrogel material (e.g., as described above), at least one perfusable internal passageway (or network) lined with endothelial cells forming a functional endothelium (e.g., as described above), and an interstitial space containing tumor cells (e.g., as described above). Additionally, in some cases, the scaffold is configured to permit migration of immune cells from the internal passageway (or network) to the interstitial space (e.g., in a manner or using a structure described above). A system described herein can also comprise a perfusion apparatus configured to deliver fluid flow containing immune cells and / or therapeutic candidates through the at least one internal passageway (or network) of the scaffold at a controlled or pre-selected flow rate. For example, such a perfusion apparatus may comprise a pump and one or more connectors associated with the pump. Further, in some cases, a system described herein further comprises one or more reservoirs for supplying culture media, immuneAttorney Docket No.: PCT.1316cells, and / or therapeutic candidates to the perfusion apparatus (e.g., as described further hereinbelow). Moreover, in some embodiments, a system according to the present disclosure is configured to enable assessment of immune cell recruitment, transendothelial migration, tumor infiltration, and / or tumor cell killing in a manner described herein, such as by at least one of imaging, flow cytometry, collection of perfusate samples, and recovery of cells from the scaffold. It is further to be understood that a system described herein can include or be used with any of the components or features described for methods hereinabove. For example, in some instances, the tumor cells of a system described herein comprise patient-derived tumor cells. Additionally, in some cases, the tumor cells are embedded in an extracellular matrix comprising decellularized tumor ECM. In some instances, the endothelial cells, tumor cells, and / or immune cells of the system are derived from the same patient. As still another example, in some preferred embodiments of a system described herein, the at least one internal passageway (or network) of the scaffold comprises micropores sized to permit transendothelial migration of activated immune cells (e.g., having a size described above). A system described herein, in some cases, may further comprise an imaging system configured for real-time monitoring of immune cell trafficking within the scaffold, such as an imaging system further described herein.

[0023] In certain other aspects, the present embodiments are directed to: a method of performing an immune recruitment assay comprising: providing a printed, coated, endothelialized scaffold; stimulating an interstitial space and / or a vasculature of the endothelialized scaffold with CXCL12; perfusing the endothelialized scaffold with peripheral blood mononuclear cells (PBMCs); and capturing the resulting immune recruitment. In some embodiments, providing a printed, coated, endothelialized scaffold comprises: printing the scaffold; performing one or more post-print washes on the scaffold; performing UV sterilization on the washed scaffold; perfusing the scaffold with PBS; perfusing the scaffold with cross-linker; perfusing the scaffold with adhesive; perfusing the scaffold with ECM coating; and seeding the scaffold with live cells.

[0024] In some embodiments, the live cells comprise green fluorescent protein tagged human umbilical vein endothelial cells (GFP-HUVECs). In some embodiments, the ECM coating comprises fibronectin, collagen 1, and collagen 4.

[0025] In some embodiments, capturing the resulting immune recruitment comprises: capturing images of the endothelialized scaffold, and quantifying the immune recruitment activity by assessing cell viability on the endothelialized scaffold.

[0026] In some embodiments printing the scaffold comprises bioprinting a hydrogel scaffold.Attorney Docket No.: PCT.1316

[0027] In some embodiments, UV sterilization comprises using a UV clave.

[0028] In certain embodiments, the present disclosure is directed to a method of producing a component, the method comprising: inspecting a bioink vat to be used during the method of producing the component for dust and / or scratches; cleaning the bioink vat; preparing one or more build files for print; confirming a build platform temperature has been reached; bioprinting the component; submerging the printed component in a container containing filtered water; and performing at least one post-print washing steps on the component.

[0029] In some embodiments, the component is composed of or formed from a hydrogel material.

[0030] In some embodiments, the method includes filling the bioink vat with bioink prior to bioprinting the component. As described further here, in a bioink can be a material that may be used as a build material in an additive manufacturing process, such an additive manufacturing process intended for forming biologically relevant materials or materials that will be in contact with biological systems or components such as cells. A bioink described herein, for instance, may comprise a composition that forms a hydrogel as further described herein. In some embodiments, the bioink comprises 5-25% PEGDA. 0.5-3% photoinitiator, and 0.5-3% photoabsorber, and the bioink comprises a pH in a range from about 7.20 to about 7.40, + / - 0.02.

[0031] In some embodiments, the method includes warming the bioink to room temperature prior to filling the bioink vat with bioink.

[0032] In some embodiments, preparing one or more build files for print comprises at least one of 1) loading the one or more build files onto a bioprinting system associated bioprinting the component, and 2) adjusting an orientation of the component within the one or more build files.

[0033] In some embodiments, the one or more build files dictate at least a geometry of the component.

[0034] In some embodiments, the filtered water comprises 5 parts per billion (ppb) or less of total organic carbons (TOC).

[0035] In some embodiments, cleaning the bioink vat comprises cleaning the bioink vat with isopropanol or 2-propanol (IP A).

[0036] In some embodiments, confirming the build platform temperature has been reached comprises confirming that a measured temperature of the build platform is within an acceptable target temperature range, and the acceptable target temperature range comprises a range from about 39°C to about 41 °C.Attorney Docket No.: PCT.1316

[0037] In some embodiments, performing at least one post-print washing step comprises submerging the printed component into a liquid comprising at least one of filtered water and phosphate buffered saline (PBS) multiple times until respective concentrations of photoinitiator (PI) and photoabsorber (PA) within the liquid are each below 0.05%.

[0038] In some embodiments, the method includes monitoring swelling of the printed component following performing at least one post-print washing steps on the printed component, and monitoring swelling of the printed component comprises determining that the printed component has reached a steady-state geometry once it has de-swelled from a maximum geometry by an amount in a range from about 0.5% to about 3.0%

[0039] In addition, in certain embodiments, the present disclosure is directed to: a method of performing single-plane coating of a bioprinted scaffold comprising at least one internal passageway, the method comprising: priming the at least one internal passageway with phosphate buffered saline (PBS); perfusing the at least one internal passageway with PBS at a flow rate of at least 100% of the flow volume of the at least one internal passageway is perfused every minute; perfusing the at least one internal passageway with a cross-linker; perfusing the at least one internal passageway with an adhesive comprising at least one of poly-l-lysine, poly-ethylenimine (PEI), laminin and collagen; preparing an extracellular matrix (ECM) coating; perfusing the at least one internal passageway with the ECM coating; incubating the bioprinted scaffold; perfusing the bioprinted scaffold a first time with endothelial growth media (EGM); and perfusing the bioprinted scaffold a second time with endothelial growth media (EGM), wherein the perfusing the bioprinted scaffold with EGM the second time is performed at a lower flow rate than a flow rate associated with perfusing the bioprinted scaffold w ith EGM the first time.

[0040] In some embodiments, such a method includes perfusing the at least one internal passageway with PBS a second time following incubating the bioprinted scaffold.

[0041] In some embodiments, preparing the ECM coating comprises preparing a coating that comprises 65-70% fibronectin, 4-8% collagen 1, and 25-30% collagen 4.

[0042] In some embodiments, incubating the bioprinted scaffold comprises storing the scaffold overnight at a temperature of about 2° C to about 6° C.

[0043] In another aspect, the present disclosure includes a method of seeding a coated, bioprinted scaffold, the method comprising: providing a bioprinted scaffold coated with an extracellular matrix (ECM) comprising fibronectin, collagen 1, and collagen 4: checking the coated, bioprinted scaffold for bubbles; fluidly coupling the coated, bioprinted scaffold to a vacuum source, therebyAttorney Docket No.: PCT.1316performing a de-gas process on the coated, bioprinted scaffold; preparing green fluorescent protein tagged human umbilical vein endothelial cells (GFP-HUVECs); resuspending the GFP-HUVECs in endothelial growth media (EGM); and seeding the coated, bioprinted scaffold with GFP-HUVECs.

[0044] In some embodiments, the method includes re-seeding the coated, bioprinted scaffold with GFP-HUVECs if 1) bubbles are present and / or 2) the coated, bioprinted scaffold is not seeded uniformly.

[0045] In some embodiments, seeding the coated, bioprinted scaffold with GFP-HUVECs comprises perfusing the coated, bioprinted scaffold with the suspension at a rate of about 200 pL / min.

[0046] In some embodiments, such a method includes re-seeding the coated, bioprinted scaffold with GFP-HUVECs if the less than about 90% of an internal surface area of the coated, bioprinted scaffold is seeded.

[0047] In one aspect, the present embodiments are directed to a system for modeling organ-on-a-chip and other biological models including a 3D bio-printed scaffold with bioprinted vascular and interstitial infill capable of being seeded with living cells such that the functioning of organs (for example, human organs) may be replicated accurately.

[0048] In some embodiments, a system described herein may be used for assessing drug-induced vascular injury. In some such embodiments, a system comprises (1) a bioprinted scaffold comprising a hydrogel material and at least one perfusable internal passageway, wherein the internal passageway is coated with an extracellular matrix and seeded with endothelial cells forming a functional endothelium; (2) a perfusion apparatus configured to deliver fluid flow through the at least one internal passageway at a controlled flow rate; and (3) one or more reservoirs for supplying culture media and / or therapeutic candidates to the perfusion apparatus, wherein the system is configured to enable assessment of vascular injury by imaging, collection of perfusate samples, and / or recovery of cells from the scaffold.

[0049] In another aspect, the present embodiments are directed to a system that enables the modeling of an organ, a tissue, a cell, a biological process, and / or other mechanism comprising: a bioprinted entity; a cell associated with the bioprinted entity and / or seeded therein; and optionally comprising vasculature.

[0050] In another aspect, the present embodiments are directed to an ecosystem for designing, building, and refining physical models or platforms that replicate the functioning of an organ orAttorney Docket No.: PCT.1316system of organs that may be used for drug discovery and for assessing the effectiveness of various therapies comprising: a bioprinted entity; a cell associated with the bioprinted entity and / or seeded therein; and optionally comprising vasculature.

[0051] In another aspect, the present embodiments are directed to a system comprising a scaffold, a manifold, a sensor (or sensors), and / or a microfluidic cartridge, wherein the scaffold comprises: vasculature; an interstitial space; and wherein the system allows continuous monitoring.

[0052] In another aspect, the present embodiments are directed to an organ-on-a-chip platform assembly comprising: a platform base; a glass base sized to be seated within the platform base; at least one chip supported by the glass base; a case comprising at least one recess sized such that the chip may be inserted into the recess, the case comprising side walls, at least one fluid inlet, at least one fluid outlet, and multiple internal passages fluidly connecting the fluid inlet and / or the fluid outlet to the chip; a glass cover configured to be seated on top of the case and to cover the chip; and a cover retainer for holding the glass cover to the case.

[0053] In some embodiments, the assembly includes: at least one O-ring for sealing the chip to the glass cover; a clip slidably and laterally engageable with the case to hold the assembly together; a first vasculature comprising a first inlet, a first outlet, a first network of passages fluidly coupling the first inlet to the first outlet; and / or a second vasculature comprising a second inlet, a second outlet, a second network of passages fluidly coupling the second inlet to the second outlet, wherein the first network of passages is interlinked with and / or intertwined with the second network of passages, and wherein the first vasculature is not fluidly coupled to the second vasculature.

[0054] In some embodiments, the chip comprises a 3D bio-printed internal vasculature comprising a vasculature inlet and a vasculature outlet.

[0055] In some embodiments, the vasculature comprises a network of passages fluidly coupling the vasculature inlet to the vasculature outlet.

[0056] In some embodiments, the vasculature is formed within a 3D bio-printed hydrogel scaffold.

[0057] In some embodiments, the chip further comprises an interstitial space disposed within and / or around the network of passages.

[0058] In some embodiments, the chip further comprises interstitial infill disposed within the interstitial space, the interstitial infill comprising a 3D bio-printed repeating structure for supporting one or more active cells.Attorney Docket No.: PCT.1316

[0059] In some embodiments, the assembly includes living cells seeded within (1) interior walls of the vasculature, and (2) the interstitial space.

[0060] In some embodiments, the assembly includes at least one fluid disposed within, and / or flowing through, the internal vasculature.

[0061] In some embodiments, the assembly includes a first biological fluid disposed within and / or flowing through the first vasculature; and a second biological fluid disposed within and / or flowing through the second vasculature, wherein the first biological fluid is different than the second biological fluid, and wherein each of the first vasculature and second vasculature are operatively coupled to the interstitial space.

[0062] In another aspect, the present disclosure is directed to a system that enables the modeling of an organ, a tissue, a cell, a biological process, and / or other mechanism comprising: a bioprinted entity; a cell associated with the bioprinted entity and / or seeded therein; and optionally comprising vasculature.

[0063] In another aspect, the present disclosure is directed to an ecosystem for designing, building, and refining physical models or platforms that replicate the functioning of an organ or system of organs that may be used for drug discovery and for assessing the effectiveness of various therapies comprising: a bioprinted entity; a cell associated with the bioprinted entity and / or seeded therein; and optionally comprising vasculature.

[0064] In another aspect, the present disclosure is directed to a system comprising a scaffold, a manifold, and a sensor, wherein the scaffold comprises: vasculature; an interstitial space; and wherein the system allows continuous monitoring.

[0065] In some embodiments, the scaffold comprises or is formed from a hydrogel.

[0066] In some embodiments, the scaffold comprises a hydrogel comprising an inert polymer.

[0067] In some embodiments, the vasculature is perfused with a cell; and wherein, following perfusion, the cell is associated with the vasculature forming a cell layer.

[0068] In some embodiments, the cell is a mammalian cell.

[0069] In some embodiments, the cell is an endothelial cell.

[0070] In some embodiments, the interstitial space has a pattern selected from an orthorhombic pattern, a cubic pattern, a hexagonal lattice, and a pattern with spherical voids.

[0071] In some embodiments, the interstitial space comprises an interstitial infill.

[0072] In some embodiments, the interstitial infill comprises a hydrogel.

[0073] In some embodiments, the interstitial infill comprises a hydrogel and a cell.Attorney Docket No.: PCT.1316

[0074] In some embodiments, the hydrogel is bonded to the cell.

[0075] In some embodiments, the bond is selected from a covalent and an ionic bond.

[0076] In some embodiments, the bond is a covalent bond.

[0077] In some embodiments, the bond is an ionic bond.

[0078] In some embodiments, the cell is a mammalian cell.

[0079] In another aspect, the present disclosure is directed to a method of monitoring a characteristic of a biologically active material comprising a system comprising an organ-on-a-chip, wherein the organ-on-a-chip comprises: a bioprinted entity; and a cell associated with the bioprinted entity and / or seeded therein.

[0080] In some embodiments, the monitoring is continuous.

[0081] In another aspect, the present disclosure is directed to a method of modeling physiological conditions of an organ comprising an organ-on-chip, wherein the organ-on-a-chip comprises: a bioprinted entity; and a cell associated with the bioprinted entity and / or seeded therein.

[0082] In another aspect, the present disclosure is directed to a method of generating a 3D printed microphysiological system comprising a multi-cellular environment.

[0083] In another aspect, the present disclosure is directed to an organ-on-a-chip comprising: a bioprinted entity comprising a polymer; a cell associated with the bioprinted entity and / or seeded therein; and optionally further comprising vasculature.

[0084] In another aspect, the present disclosure is directed to a liver-on-a-chip comprising: a bioprinted entity comprising a polymer; a liver cell associated with the bioprinted entity and / or seeded therein; and optionally further comprising vasculature.

[0085] In another aspect, the present disclosure is directed to a kit comprising an organ-on-a-chip used for monitoring a detectable moiety, wherein the organ-on-a-chip models physiological conditions.

[0086] In another aspect, the present disclosure is directed to a tumor-on-a-chip comprising: a bioprinted entity comprising a polymer; a tumor cell associated with the bioprinted entity and / or seeded therein; and optionally further comprising vasculature.

[0087] In another aspect, the present disclosure is directed to a method of functionalizing a polymer and / or hydrogel surface, the method comprising: providing at least one polymer and / or hydrogel surface; pre-coating the polymer and / or hydrogel surface with a precursor; irradiating the pre-coated surface; coating the irradiated surface with a bioactive coating; performing at least one post-coating step to enable bioconjugation of the bioactive coating with the coated surface.Attorney Docket No.: PCT.1316

[0088] In some embodiments, the method includes pre-washing the polymer and / or hydrogel surface prior to precoating.

[0089] In some embodiments, the post-coating step comprises at least one of incubating, sterilizing, irradiating, and washing the coated surface.

[0090] In some embodiments, the precursor comprises at least one of acrylated-PEGlk-NHS, Acrylated-PEG-NHS derivatives, Maleimide-PEG, Vinyl sulfone-PEG, Methacrylated-PEG (PEG-MA), Hydrazide-PEG, Poly(lactic-co-glycolic acid) (PLGA) functionalized with NHS or Maleimide, Poly(ethylene glycol)-diacrylate (PEGDA), Functionalized polyacrylamide (PAM), Gelatin-methacryloyl (GelMA), Methacrylated hyaluronic acid (MeHA), Hydrazide-modified hyaluronic acid, Chitosan derivatives, Carbodiimide chemistry (e.g., EDC / NHS).

[0091] In some embodiments, irradiating the pre-coated surface comprises irradiating the precoated surface with a light source activated at a wavelength of 405 nm.

[0092] In some embodiments, the bioactive coating comprises at least one of gelatin methacrylate (GelMA), collagen methacrylate (ColMA), and collagen type I.

[0093] In some embodiments, the precursor comprises a cytocompatible photoinitiator.

[0094] In some embodiments, the cytocompatible photoinitiator is water soluble.

[0095] In some embodiments, the bioactive coating comprises at least one acrylate.

[0096] In some embodiments, the method includes seeding live cells on the coated surface.

[0097] In some embodiments, the polymer and / or hydrogel surface is part of a three-dimensional structure comprising at least one internal passage, the internal passage coated with a bioactive coating, the method further comprising: perfusing living cells through the internal passage, thereby seeding the live cells on one or more interior walls of the internal passage.

[0098] In some embodiments, the live cells comprise at least one of an endothelial cell, a biliary endothelial cell, a cholangiocyte, a liver parenchymal cell, a hepatocyte (HC), a primary human hepatocyte (PHH). aheptic stellate cell (HSCs). a Kupffer cell (KC), a liver sinusoidal endothelial cell (LSEC), a mucous cell, a parietal cell, a chief cell, an endocrine cell (e.g., a G cell, a D cell, a enterochromaffin cell, a EC-like cell, a X / A cell), a columnar epithelial cell, a cardiac fibroblast (CF), a cardiomyocyte, a smooth muscle cell, an enterocyte, a goblet cell, a Paneth cell, a stem cell, a neuron, a glia, a keratinocyte. a melanocyte, a Merkel cell, a Langerhan cell, a germ cell, a stromal cell, a seminiferous tubule, a Leydig cell, a tubule epithelial cell, a macula densa cell, a glomerular endothelial cell, a podocyte, a mesangial cell, a parietal epithelial cell, an immortalized cell (e.g., a 3T3 cell, a A549 cell, a HeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, aAttorney Docket No.: PCT.1316Jurkat cell, a OK cell, a Ptk2 cell, a Vero cell), a patient-derived cell (e.g., a tumor cell), a T cell, a peripheral blood mononuclear cell (PBMC), and / or an induced pluripotent stem cell (iPSC).

[0099] In some embodiments, a method described herein further includes using a peristaltic pump to seed live cells within the internal passage; and using the peristaltic pump to perfuse media through the internal passage.

[0100] In some embodiments, a lower volumetric flow rate is used for seeding live cells within the internal passage than for perfusing media through the internal passage.

[0101] In another aspect, the present disclosure is directed to a bioactive coating comprising: collagen type I in a range from about 0.2% to about 8.0% by volume; collagen type IV in a range from about 4.0% to about 40.0% by volume; fibronectin in a range from about 2.0% to about 90.0% by volume: and DPBS (IX) in a range from about 0.5% to about 95.0% by volume.

[0102] In another aspect, the present disclosure is directed to a bioactive coating for use in coating two-dimensional surfaces comprising: collagen type I in a range from about 0.2% to about 0.6% by volume; collagen type IV in a range from about 4.0% to about 13.0% by volume; fibronectin in a range from about 2.0% to about 7.0% by volume; and DPBS (IX) in a range from about 50.0% to about 95.0% by volume.

[0103] In another aspect, the present disclosure is directed to a bioactive coating for use in coating three-dimensional surfaces comprising collagen type I in a range from about 2.3% to about 8.0% by volume; collagen type IV in a range from about 12.0% to about 40.0% by volume; fibronectin in a range from about 35.0% to about 90.0% by volume; and DPBS (IX) in a range from about 0.5% to about 3.0% by volume.

[0104] In some embodiments, the vasculature described herein comprises micropores. In some embodiments, the micropores comprise or have a diameter (or average diameter) of about 40pm to about 60pm, about 35pm to about 65pm, about 30pm to about 70pm, or about 5pm to about 70pm, where the diameter is determined by the thickness of the channel walls. It is further to be understood that the "diameter" (or average ‘‘diameter’’) described above can be the actual geometric diameter, such as in the case when the micropores have a circular or substantially circular cross section (e g., an only slightly oblate cross section, such as may occur when cross section is non-circular but has an aspect ratio between 0.95 and 1.05). Alternatively, if the micropore or population of micropores does not have a circular or substantially circular cross section (e.g., the cross section is irregularly shaped), then the foregoing numerical ranges can apply to the equivalent circular area diameter (or average equivalent circular area diameter), where theAttorney Docket No.: PCT.1316“equivalent circular area diameter” is the diameter of a circle that has the same area as the irregular (or non-circular) shape. In some embodiments, the micropores comprise or have a length (or average length) of about 100pm, 150pm, or 200pm. In some embodiments, the micropores have a length or average length of 100-300µm.

[0105] In some embodiments, the scaffold is functionalized using the method(s) described herein.

[0106] In another aspect, the present disclosure is directed to an organ-on-a-chip cartridge comprising: at least one fluid inlet, at least one fluid outlet, at least one chip slot, at least one chip slot inlet, at least one chip slot outlet, and multiple internal channels fluidly connecting the fluid inlet and / or the fluid outlet to the chip slot inlet and / or the chip slot outlet.

[0107] In some embodiments, the chip inlet splits into two chip inlet sub-channels, and wherein the chip outlet channel split into two chip outlet sub-channels.

[0108] In some embodiments, the cartridge includes a media reservoir.

[0109] In another aspect, the present disclosure is directed to an organ-on-a-chip platform assembly comprising: a well plate, a cartridge comprising at least one fluid inlet, at least one fluid outlet, at least one chip slot, at least one chip slot inlet, at least one chip slot outlet, and multiple internal channels fluidly connecting the fluid inlet and / or the fluid outlet to the chip slot inlet and / or the chip slot outlet, wherein the cartridge is configured to be inserted into at least one well of the well plate; at least one chip supported by the chip slot and the well of the well plate, and a cover configured to be seated on top of the cartridge and to cover the chip.

[0110] In some embodiments, the cartridge further comprises a media reservoir.

[0111] In some embodiments, the cartridge includes: a first chip slot, a first fluid inlet, a first fluid outlet, and first multiple internal channels fluidly connecting the first fluid inlet and / or the first fluid outlet to the first chip slot inlet and / or the first chip slot outlet, a second chip slot, a second fluid inlet, a second fluid outlet, and second multiple internal channels fluidly connecting the second fluid inlet and / or the second fluid outlet to the second chip slot inlet and / or the second chip slot outlet.

[0112] In some embodiments, the assembly includes a first biological fluid disposed within and / or flowing through a first chip inserted in a first chip slot; and a second biological fluid disposed within and / or flowing through a second chip inserted in the second chip slot, wherein the first biological fluid is different than the second biological fluid.Attorney Docket No.: PCT.1316

[0113] In some embodiments, the assembly includes imaging inserts, wherein the imaging inserts secure and / or set the orientation of the chip.

[0114] In another aspect, the present disclosure is directed to a system comprising: a multi-part assembly comprising: at least one inlet flow line configured to deliver a fluid to an interior volume of the multi-part assembly; at least one outlet flow line configured to deliver the fluid from the interior volume of the multi-part assembly; a chip housed within the interior volume of the multipart assembly, the chip comprising: at least one inlet flow passage disposed therein and fluidly coupled to the inlet flow line; and at least one outlet flow passage disposed therein and fluidly coupled to the outlet flow line, wherein the inlet flow passage transitions to the outlet flow passage within an interstitial space disposed within the chip.

[0115] In some embodiments, each of the multi-part assembly and the chip are formed via an additive manufacturing process.

[0116] In some embodiments, each part of the multi-part assembly is composed of a metallic or a polymer material, the chip is composed of a hydrogel material, and each part of the multi-part assembly is composed of a harder material than the hydrogel material.

[0117] In some embodiments, each of the inlet flow passage and the outlet flow passage comprises at least two inlet or outlet flow passages, thereby forming at least two unconnected fluid flow passages through the chip, and each of the two unconnected fluid flow passages contains a different fluid.

[0118] In some embodiments, each of the inlet flow passage and the outlet flow passage comprises a single respective inlet or outlet portion which branches into a network of connected flow passages, the network of inlet flow passages fluidly connected to the corresponding network of outlet flow passages.

[0119] In some embodiments, at least one of the inlet passage and the outlet passage comprises at least one micropore disposed therein in a portion of the passage disposed within the interstitial space.

[0120] In some embodiments, the micropore comprises an internal diameter in a range described hereinabove, such as in range from about 40 pm to about 60 pm.

[0121] In some embodiments, the system includes at least one precursor comprising acrylated-PEGlk-NHS disposed on the chip.

[0122] In some embodiments, the precursor further comprises a photoinitiator.Attorney Docket No.: PCT.1316

[0123] In some embodiments, the system includes at least one bioactive coating comprising at least one of gelatin methacrylate (GelMA), collagen methacrylate (ColMA), and collagen type I.

[0124] In some embodiments, the system includes at least one live cell disposed within the interstitial space and / or attached to an interior surface of the inlet flow passage and / or the outlet flow passage.

[0125] In some embodiments, the live cell comprises at least one of an endothelial cell, a biliary endothelial cell, a cholangiocyte, a liver parenchymal cell, a hepatocyte (HC), a primary human hepatocyte (PHH), a heptic stellate cell (HSCs), a Kupffer cell (KC), a liver sinusoidal endothelial cell (LSEC), a mucous cell, a parietal cell, a chief cell, an endocrine cell (e.g., a G cell, a D cell, a enterochromaffin cell, a EC-like cell, a X / A cell), a columnar epithelial cell, a cardiac fibroblast (CF), a cardiomyocyte, a smooth muscle cell, an enterocyte, a goblet cell, a Paneth cell, a stem cell, a neuron, a glia, a keratinocyte. a melanocyte, a Merkel cell, a Langerhan cell, a germ cell, a stromal cell, a seminiferous tubule, a Leydig cell, a tubule epithelial cell, a macula densa cell, a glomerular endothelial cell, a podocyte, a mesangial cell, a parietal epithelial cell, an immortalized cell (e.g. a 3T3 cell, a A549 cell, a HeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, a Jurkat cell, a OK cell, a Ptk2 cell, a Vero cell), a patient-derived cell (e.g., a tumor cell), a T cell, a peripheral blood mononuclear cell (PBMC), and / or an induced pluripotent stem cell (iPSC).

[0126] In some embodiments, the system includes a fluid reservoir fluidly coupled downstream of the inlet flow line and upstream of the inlet flow passage.

[0127] In some embodiments, the system includes a pump disposed downstream of the fluid reservoir and upstream of the inlet flow passage.

[0128] In some embodiments, the system includes a covalent linker attached to the hydrogel material; and collagen attached to the covalent linker.

[0129] In another aspect, the present disclosure is directed to a method of functionalizing a hydrogel surface, the method comprising: providing at least one hydrogel surface; pre-coating the hydrogel surface with a precursor; irradiating the pre-coated surface; coating the irradiated surface with a bioactive coating comprising at least one of gelatin methacrylate (GelMA), collagen methacrylate (ColMA), and collagen type I; performing at least one post-coating step to enable bioconjugation of the bioactive coating with the coated surface.

[0130] In some embodiments, the post-coating step comprises at least one of incubating, sterilizing, irradiating, and washing the coated surface.Attorney Docket No.: PCT.1316

[0131] In some embodiments, irradiating the pre-coated surface comprises irradiating the precoated surface with a light source activated at a wavelength of 405 nm.

[0132] In some embodiments, the precursor comprises a cytocompatible photoinitiator.

[0133] In some embodiments, the system includes at least one sampling port and / or fluid sampling line fluidly coupled to at least one of the reservoirs, the outlet flow line, the outlet flow passage, the interstitial space, and / or another system flow passage.

[0134] In some embodiments, the system includes a removable top cover or lid enabling access to the interior volume, wherein the interstitial space being accessible when the top cover or lid is removed.

[0135] These and other embodiments are further described in the detailed description which follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figure 1 schematically illustrates an ecosystem for creating organ-on-a-chip and other biological models, according to aspects of the present embodiments.

[0137] Figure 2 illustrates a perspective, schematic view of a platform for modeling organ-on-a-chip and other biological models, according to aspects of the present embodiments.

[0138] Figure 3 illustrates a side view of a platform for modeling organ-on-a-chip and other biological models, according to aspects of the present embodiments.

[0139] Figure 4 illustrates an exploded perspective view of a platform assembly, according to aspects of the present embodiments.

[0140] Figure 5 illustrates an exploded perspective view of a platform assembly, according to aspects of the present embodiments.

[0141] Figure 6 illustrates a bottom perspective view of a platform assembly, according to aspects of the present embodiments.

[0142] Figure 7 illustrates a perspective view of a platform assembly, according to aspects of the present embodiments.

[0143] Figure 8 illustrates a top view of a platform assembly, according to aspects of the present embodiments.

[0144] Figure 9 illustrates a side view of a platform assembly, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0145] Figure 10 illustrates a front view of a platform assembly, according to aspects of the present embodiments.

[0146] Figure 11 illustrates an exploded perspective view of a platform assembly, according to aspects of the present embodiments.

[0147] Figure 12A illustrates a top view of a platform assembly, according to aspects of the present embodiments.

[0148] Figure 12B illustrates a perspective view of a platform assembly, according to aspects of the present embodiments.

[0149] Figure 13 illustrates a side view of a platform assembly, according to aspects of the present embodiments.

[0150] Figure 14 illustrates an exploded perspective view of a platform assembly, according to aspects of the present embodiments.

[0151] Figure 15A illustrates a perspective view of a platform assembly, according to aspects of the present embodiments.

[0152] Figure 15B illustrates a top view of a platform assembly, according to aspects of the present embodiments.

[0153] Figure 15C illustrates a bottom view of a platform assembly, according to aspects of the present embodiments.

[0154] Figure 15D illustrates a side view of a platform assembly, according to aspects of the present embodiments.

[0155] Figure 16 illustrates a perspective view of an organ chip, according to aspects of the present embodiments.

[0156] Figure 17 illustrates a perspective view of an organ chip, according to aspects of the present embodiments.

[0157] Figure 18 illustrates a perspective view of an organ chip, according to aspects of the present embodiments.

[0158] Figure 19 illustrates a side view of organ chips within a platform assembly, according to aspects of the present embodiments.

[0159] Figure 20A illustrates a top view of a vasculature configuration, according to aspects of the present embodiments.

[0160] Figure 20B illustrates a top view of a vasculature configuration, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0161] Figure 20C illustrates a top view of a vasculature configuration, according to aspects of the present embodiments.

[0162] Figure 21A illustrates a top view of a vasculature configuration, according to aspects of the present embodiments.

[0163] Figure 21B illustrates a perspective side view of a vasculature configuration, according to aspects of the present embodiments.

[0164] Figure 22 illustrates a sectional perspective view of a vasculature configuration, according to aspects of the present embodiments.

[0165] Figure 23A illustrates a top view of a vasculature configuration, according to aspects of the present embodiments.

[0166] Figure 23B illustrates a perspective side view of a vasculature configuration, according to aspects of the present embodiments.

[0167] Figure 24 illustrates a sectional perspective view of a vasculature configuration, according to aspects of the present embodiments.

[0168] Figure 25 illustrates views of interstitial infill, according to aspects of the present embodiments.

[0169] Figure 26A illustrates a perspective view of interstitial infill, according to aspects of the present embodiments.

[0170] Figure 26B illustrates a close-up perspective view of interstitial infill, according to aspects of the present embodiments.

[0171] Figure 27 illustrates a perspective view of a vasculature and interstitial space seeded with active cells, according to aspects of the present embodiments.

[0172] Figure 28 illustrates a flow chart of a method of creating, seeding, and operating an organ on a chip platform, according to aspects of the present embodiments.

[0173] Figure 29 illustrates a flow chart of a method of creating, seeding, and operating an organ on a chip platform, according to aspects of the present embodiments.

[0174] Figure 30 illustrates a perspective view of a liver-on-a-chip model, according to aspects of the present embodiments.

[0175] Figure 31 illustrates a perspective view of a liver-on-a-chip model, according to aspects of the present embodiments.

[0176] Figure 32 illustrates a perspective view of a liver-on-a-chip model, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0177] Figure 33 illustrates a perspective view of a liver-on-a-chip model, according to aspects of the present embodiments.

[0178] Figure 34 illustrates a fluorescent imaging image of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0179] Figure 35 illustrates a fluorescent imaging image of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0180] Figure 36 illustrates a fluorescent imaging image of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0181] Figure 37A illustrates a fluorescent imaging image of liver-on-a-chip vasculature, according to aspects of the present embodiments.

[0182] Figure 37B illustrates a fluorescent imaging image of liver-on-a-chip vasculature, according to aspects of the present embodiments.

[0183] Figure 37C illustrates a fluorescent imaging image of liver-on-a-chip vasculature, according to aspects of the present embodiments.

[0184] Figure 37D illustrates a fluorescent imaging image of liver-on-a-chip vasculature, according to aspects of the present embodiments.

[0185] Figure 37E illustrates a fluorescent imaging image of liver-on-a-chip vasculature, according to aspects of the present embodiments.

[0186] Figure 38 illustrates a perspective view of a bioprinted scaffold, according to aspects of the present embodiments.

[0187] Figure 39 illustrates a perspective view of bioprinted scaffolds inserted into a well plate, according to aspects of the present embodiments.

[0188] Figure 40A illustrates a top view of a bioprinted scaffold, according to aspects of the present embodiments.

[0189] Figure 40B illustrates a front view of a bioprinted scaffold, according to aspects of the present embodiments.

[0190] Figure 40C illustrates a side view of a bioprinted scaffold, according to aspects of the present embodiments.

[0191] Figure 41A illustrates a perspective view of examples of cartridges, according to aspects of the present embodiments.

[0192] Figure 41B illustrates a perspective view of exemplary cross sections of cartridges, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0193] Figure 41C illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0194] Figure 42A illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0195] Figure 42B illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0196] Figure 42C illustrates a perspective view of an exemplary view of a cartridge including internal channels, according to aspects of the present embodiments.

[0197] Figure 42D illustrates a perspective view of an exemplary view of a cartridge including wells within reservoirs, according to aspects of the present embodiments.

[0198] Figure 42E illustrates an exemplary diagram of a cartridge, according to aspects of the present embodiments.

[0199] Figure 43 illustrates a perspective view of printed examples of a cartridge, according to aspects of the present embodiments.

[0200] Figure 44A illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0201] Figure 44B illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0202] Figure 45 illustrates a perspective view of an imaging setup for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0203] Figure 46A illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0204] Figure 46B illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0205] Figure 46C illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0206] Figure 46D illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0207] Figure 47A illustrates a top view of an MPS platform structure, according to aspects of the present embodiments.

[0208] Figure 47B illustrates a perspective view of a three-dimensional chip organ, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0209] Figure 48A schematically illustrates a construction method of endothelial / epithelial interfacing MPS model, according to aspects of the present embodiments.

[0210] Figure 48B illustrates an exemplary image of Matrigel in a MPS model, according to aspects of the present embodiments.

[0211] Figure 48C illustrates an exemplary image fibronectin in a MPS model, according to aspects of the present embodiments.

[0212] Figure 48D illustrates an exemplary image of collagen-IV in a MPS model, according to aspects of the present embodiments.

[0213] Figure 48E is an exemplary image of an ECM-coated transendothelialization MPS model, according to aspects of the present embodiments.

[0214] Figure 48F illustrates an image of a functionalized vasculature with micropores, according to aspects of the present embodiments.

[0215] Figure 48G illustrates an image of a functionalized vasculature with micropores, according to aspects of the present embodiments.

[0216] Figure 49A schematically illustrates a construction method of an endothelial / epithelial interfacing MPS model, according to aspects of the present embodiments.

[0217] Figure 49B illustrates an exemplary image of Matrigel in a MPS model, according to aspects of the present embodiments.

[0218] Figure 49C illustrates an exemplary image of fibronectin in a MPS model, according to aspects of the present embodiments.

[0219] Figure 49D illustrates an exemplary image of collagen-IV in a MPS model, according to aspects of the present embodiments.

[0220] Figure 49E illustrates an exemplary image of an ECM-coated transendothelialization MPS model, according to aspects of the present embodiments.

[0221] Figure 50A schematically illustrates an AN14 hydrogel, according to aspects of the present embodiments.

[0222] Figure 50B schematically illustrates side view of a glass surface having a thin coating, according to aspects of the present embodiments.

[0223] Figure 50C schematically illustrates a side view of a glass surface having a thin coating, according to aspects of the present embodiments.

[0224] Figure 51A schematically illustrates a covalent linker incorporating an amine-reactive functional group tethered to a base polymer, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0225] Figure 51B schematically illustrates an example of an extracellular matrix (ECM), according to aspects of the present embodiments.

[0226] Figure 52A schematically illustrates an AN14 hydrogel interacting with a bioactive coating, according to aspects of the present embodiments.

[0227] Figure 52B illustrates a fluorescence image of HepG2 attached to an A14 hydrogel, according to aspects of the present embodiments.

[0228] Figure 53A schematically illustrates an example of a glass surface functionalized with a linker, according to aspects of the present embodiments.

[0229] Figure 53B illustrates a fluorescence image of an example of glass thin coating having moderate fluorescence, according to aspects of the present embodiments.

[0230] Figure 53C illustrates a fluorescence image of an example of glass thin coating having moderate to high fluorescence, according to aspects of the present embodiments.

[0231] Figure 54A schematically illustrates an example of a base polymer functionalized with an acrylate, according to aspects of the present embodiments.

[0232] Figure 54B illustrates a fluorescence image of liver cancer cells, according to aspects of the present embodiments.

[0233] Figure 54C illustrates a fluorescence image of liver cancer, according to aspects of the present embodiments.

[0234] Figure 55A illustrates a dot plot depicting the results of an assay measuring ATP concentration of cells, according to aspects of the present embodiments.

[0235] Figure 55B illustrates an example fluorescence microscopy image of HUVEC cells attached to hydrogels, according to aspects of the present embodiments.

[0236] Figure 55C illustrates an example fluorescence microscopy image of HUVEC cells attached to hydrogels, according to aspects of the present embodiments.

[0237] Figure 55D illustrates an example fluorescence microscopy image of HUVEC cells attached to hydrogels, according to aspects of the present embodiments.

[0238] Figure 55E illustrates an example fluorescence microscopy image of HUVEC cells attached to hydrogels, according to aspects of the present embodiments.

[0239] Figure 55F illustrates an example fluorescence microscopy image of HUVEC cells attached to hydrogels, according to aspects of the present embodiments.

[0240] Figure 56A illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0241] Figure 56BA illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.

[0242] Figure 56C illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.

[0243] Figure 57A illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.

[0244] Figure 57B illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.

[0245] Figure 57C illustrates an example fluorescence microscopy image of HUVEC cells attached to a synthetic hydrogel, according to aspects of the present embodiments.

[0246] Figure 58 schematically illustrates an example of a glass surface after functionalization, according to aspects of the present embodiments.

[0247] Figure 59A illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen, according to aspects of the present embodiments.

[0248] Figure 59B illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen, according to aspects of the present embodiments.

[0249] Figure 60A illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen.

[0250] Figure 60B illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen.

[0251] Figure 60C illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen.

[0252] Figure 60D illustrates an example fluorescence microscopy image of HUVEC cells attached to a 3-D printed lumen.

[0253] Figure 61A illustrates a microscopy image of an example of endothelization of a structure, according to aspects of the present embodiments.

[0254] Figure 61B illustrates a microscopy image of an example of endothelization of a structure, according to aspects of the present embodiments.

[0255] Figure 62 illustrates a flowchart of a construction method of endothelial / epithelial interfacing MPS model, according to aspects of the present embodiments.

[0256] Figure 63 illustrates a flowchart of a construction method of an endothelial / epithelial interfacing MPS model using thiolated polymer, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0257] Figure 64 illustrates a top view of an example scaffold used in a cartridge, according to aspects of the present embodiments.

[0258] Figure 65 illustrates a sectional perspective view of an example of a setup to fluidly couple two cartridges, according to aspects of the present embodiments.

[0259] Figure 66 illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0260] Figure 67A illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0261] Figure 67B illustrates a side view of an example of a setup to fluidly couple reservoirs of a cartridge, according to aspects of the present embodiments.

[0262] Figure 68A illustrates a perspective view of an example of a setup for a recirculatory flow through a cartridge, according to aspects of the present embodiments.

[0263] Figure 68B illustrates a perspective view of an example of a setup for a bi-directional flow through a cartridge, according to aspects of the present embodiments.

[0264] Figure 69 illustrates a perspective view of an assembly for modeling organ-on-a-chip, according to aspects of the present embodiments.

[0265] Figure 70 illustrates an exemplary cross section view of a cartridge, according to aspects of the present embodiments.

[0266] Figure 71A illustrates a perspective view of an exemplary scaffold to be used in a cartridge, according to aspects of the present embodiments.

[0267] Figure 71B illustrates a perspective view of an exemplary scaffold to be used in a cartridge, according to aspects of the present embodiments.

[0268] Figure 72A illustrates an exploded perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0269] Figure 72B illustrates a perspective view of an example of a cartridge, according to aspects of the present embodiments.

[0270] Figure 73 illustrates a flow chart of a method of seeding and functionalizing a synthetic vascular network, according to aspects of the present embodiments.

[0271] Figure 74A illustrates a flowchart of a method of bioprinting a scaffold, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0272] Figure 74B illustrates a plot of the concentrations of photoinitiator and photoabsorber as function of the number of washes being performed, according to some embodiments described herein.

[0273] Figure 74C illustrates a plot of the Y and Z dimensions of a printed scaffold plotted as a function of days in solution, according to some embodiments described herein.

[0274] Figure 74D illustrates a perspective view of an example of an array of scaffolds according to aspects of the present embodiments.

[0275] Figure 75 illustrates a flowchart of a method of performing single-plane coating (e.g., of printed scaffolds), according to aspects of the present embodiments.

[0276] Figure 76 illustrates a flowchart of a method of endothelializing and seeding printed scaffolds, according to aspects of the present embodiments.

[0277] Figure 77 illustrates a flowchart of a method of expanding T-cells, according to aspects of the present embodiments

[0278] Figure 78 illustrates a flow chart of a method of performing an immune recruitment assay, according to aspects of the present embodiments

[0279] Figure 79A illustrates a flurorescence microscopy image of a vasculature endothelialized with GFP expressing cells, according to aspects of the present embodiments.

[0280] Figure 79B illustrates a flurorescence microscopy image of a vasculature endothelialized with GFP expressing cells, according to aspects of the present embodiments.

[0281] Figure 79C illustrates a flurorescence microscopy image of a vasculature endothelialized with GFP expressing cells, according to aspects of the present embodiments.

[0282] Figure 80 illustrates fluorescence microcscopy images of perfusion of T-cells, according to aspects of the present embodiments.

[0283] Figure 81 illustrates fluorescence microcscopy images of T cells exerting cytotoxic effects on the endothelium, according to aspects of the present embodiments.

[0284] Figure 82 illustrates fluorescence microcscopy images of T cell migratory response in the absence of CXCL12, according to aspects of the present embodiments.

[0285] Figure 83 illustrates fluorescence microcscopy images of T cell migratory response in the presence of CXCL12, according to aspects of the present embodiments.

[0286] Figure 84 illustrates fluorescence microcscopy images of T cell migratory response in the presence of high concentration of CXCL12, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0287] Figure 85A illustrates a perspective view of a scaffold according to one embodiment described herein.

[0288] Figure 85B illustrates a graph of quantification of T cell migration upon CXCL12 stimulation, according to aspects of the present embodiments.

[0289] Figure 86A illustrates a perspective view of a scaffold according to one embodiment described herein.

[0290] Figure 86B illustrates a graph of quantification of T cell migration upon CXCL12 stimulation, according to aspects of the present embodiments.

[0291] Figure 87 illustrates fluorescence microcscopy images of T cell migration activity in a 3D complex TEM model, according to aspects of the present embodiments.

[0292] Figure 88A illustrates a fluorescence microcscopy image of T cell migration quantification in a 3D complex TEM model, according to aspects of the present embodiments.

[0293] Figure 88B illustrates a fluorescence microcscopy image of T cell migration quantification in a 3D complex TEM model, according to aspects of the present embodiments.

[0294] Figure 88C illustrates a fluorescence microcscopy image of T cell migration quantification in a 3D complex TEM model, according to aspects of the present embodiments.

[0295] Figure 88D illustrates a perspective view of a scaffold according to one embodiment described herein, in connection with Figures 88A-C.

[0296] Figure 88E illustrates a graph of T cell migration quantification in a 3D complex TEM model, according to aspects of the present embodiments.DETAILED DESCRIPTION OF THE DISCLOSUREDefinitions

[0297] In order for the present disclosure to be more readily understood, certain terms are defined below. Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0298] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.Attorney Docket No.: PCT.1316

[0299] A, an, at least one, one or more: It is to be noted that the term “a” or “an” entity refers to one or more of that entity. For example, “a cell” is understood to represent one or more cells. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein, unless the context of a particular use requires otherwise.

[0300] Additive manufacturing: The terms “additive manufacturing,” “three-dimensional printing system,” “three-dimensional printer,” “printing,” and the like generally describe various solid freeform fabrication techniques for making three-dimensional articles or objects by selective laser sintering (SLS), stereolithography (SLA), dynamic light projection (DLP), selective deposition, jetting, fused deposition modeling (FDM), multijet modeling (MJM), and other additive manufacturing techniques now known in the art or that may be known in the future that use a build material or ink to fabricate three-dimensional objects.

[0301] Agent. As used herein, the term “agent”, may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0302] Aliphatic. As used herein, “aliphatic” means a straight-chain (i.e.. unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon, bicyclic hydrocarbon, or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.Attorney Docket No.: PCT.1316

[0303] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0304] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to. severity of, stage of. etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, electrostatic, magnetism, and combinations thereof.

[0305] Biologically active: As used herein, the term “biologically active” or “bioactive” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological or physiological effect on that organism, is considered to be biologically active. In some embodiments, a “bioactive” interacts with a biological system and / or organism. In some embodiments, a “bioactive” interacts with an organism and a surface. In some embodiments, a “bioactive” is sandwiched between a surface (e.g., glass, plastic) and an organism (e.g., cell). In some embodiments, a “bioactive” is permeable to components of a biological system (e.g., cell, macromolecule, steroid, lipophilic molecule, polar molecule). In some embodiments, a “bioactive” interacts with focal adhesions of a cell (e.g., macromolecule assemblies). In some embodiments, a “bioactive” facilitates interaction within a cell microenvironment that consists of other cells and an extracellular matrix. In some embodiments, a “bioactive” regulates cell polarization. In someAttorney Docket No.: PCT.1316embodiments, a “bioactive” regulates cell protrusion. In some embodiments, a “bioactive” regulates cell migration.

[0306] Biological sample. As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gy necological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0307] Biomarker. The term “biomarker” is used herein, consistent with its use in the art, to refer to a to an entity, event, or characteristic whose presence, level, degree, ty pe, and / or form, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state. To give but a few examples, in some embodiments, a biomarker may be or comprise a marker for a particular disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or re-occur. In some embodiments, a biomarker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus,Attorney Docket No.: PCT.1316in some embodiments, a biomarker is predictive, in some embodiments, a biomarker is prognostic, and in some embodiments, a biomarker is diagnostic of the relevant biological event or state of interest. A biomarker may be or comprise an entity of any chemical class, and may be or comprise a combination of entities. For example, in some embodiments, a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g., a metal or ion), or a combination thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is intracellular. In some embodiments, a biomarker is detected outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc. In some embodiments, a biomarker may be or comprise a genetic or epigenetic signature. In some embodiments, a biomarker may be or comprise a gene expression signature.

[0308] Bioprinting: The term "bioprinting." as used herein, refers to 3D printing or additive manufacturing with biocompatible materials such as hydrogels onto which live cells may be stably adhered and / or otherwise functionalized. It is to be understood that a scaffold described herein, in some cases, may be bioprinted. That is, in some embodiments, 3D printing or additive manufacturing (e.g., using a hydrogel as a "‘build material" or “ink”) may be used to form a scaffold described herein.

[0309] Comprising: A composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are required or essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any system, device, composition, or method described as “comprising” (or which “comprises”) one or more named elements or steps also describes the corresponding, more limited system, device, composition, or method “consisting essentially of’ (or which “consists essentially of’) the same named elements or steps, meaning that the system, device, composition, or method includes the named required or essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the system, device, composition, or method. It is also understood that any system, device, composition, or method described herein as “comprising” or “consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method “consisting of’ (or “consists of’) the named elements or steps to the exclusion of any other unnamed element or step. In any system, device, composition, orAttorney Docket No.: PCT.1316method disclosed herein, known or disclosed equivalents of any named required or essential element or step may be substituted for that element or step.

[0310] Conservative and non-conservative substitution: A ‘conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g.. aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine (G); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)); nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), menine (M), tryptophan (W), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)); and aromatic side chains (e.g., ty rosine (Y), phenylalanine (F), try ptophan (W), histidine (H)). For example, substitution of a phenylalanine for a ty rosine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand a target of interest. In some embodiments, conservative amino acid substitutions in the sequences of a ligand do not reduce or abrogate the binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of a ligand to a target of interest. Methods of identifying nucleotide and amino acid conservative substitutions and non-conservative substitutions which confer, alter or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997)). In some embodiments, non-conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand a target of interest. In some embodiments, non-conservative amino acid substitutions in the sequences of a ligand do not reduce or abrogate the binding of the ligand to a target of interest. In some embodiments, non-conservative amino acid substitutions do not significantly affect specific binding of a ligand to a target of interest.

[0311] Continuous: A ‘‘continuous"’ process (such as a continuous build process or manufacturing process) refers to a single process that proceeds in a single manner or mode (e.g., additive manufacturing carried out by DLP, in a layer-by-layer manner), as opposed to a “batch” process or “discontinuous” or “multi-stage” process in which the manner or mode changes. For instance, an exemplary non-continuous process would be injection molding to form one structure, followed by surface treatment of the structure. These two steps together would not constitute a “continuous” process as used herein. Similarly, another example of a non-continuous process is aAttorney Docket No.: PCT.1316process that first forms a structure using additive manufacturing, and then forms a different structure using a different additive manufacturing process or ‘‘print job,” and then joins the two structures together in a separate step (e.g., using an adhesive or mechanical fastener). The term “continuous with,” when used in reference to a structure, is synonymous with “integral with” as described herein.

[0312] Digital light processing (DLP): As used herein and as understood by a person of ordinary skill in the art, the term, “digital light processing” (DLP) refers to a 3D printing technology used to rapidly produce photopolymer parts using a projected light source to cure an entire layer at once. In some cases, DLP uses a range of wavelengths from 360 nm to 405 nm, typically as the light source in connection with a photocurable polymer resin.

[0313] Directions or orientations: As one example, it is to be understood that a “vertical” orientation or a “vertical” direction is an “up and down” orientation or direction (e.g.. based on the orientation of the ‘long axis’ of the relevant structure, as compared to the ‘short axis’ of the relevant structure). Moreover, such an “up and down” or “vertical” orientation or direction can be relative to the force of gravity exerted by the earth, such that "dow n" is tow ard the ground, and “up” is toward the sky, when the relevant platform, component, device, or system is used as intended and as described herein. However, it is further to be understood that terms such as “vertical” and “horizontal” as used herein are to be understood as terms relative to one another to indicate different directions or orientations, analogous to the standard use of terms such as “x-direction” and “y -direction” and “z-direction” in a Cartesian coordinate system, or “length” and “width” and “height” in geometry. Thus, for instance, unless the context clearly indicates otherwise, a “vertical” direction or orientation described herein is orthogonal or perpendicular to a “horizontal” direction or orientation described herein. These terms may also be replaced, in general, with Cartesian directions or orientations, such as “z” (e.g., for “height” or the “vertical” direction or orientation) and “x” or “y” (e.g., for “length” or “width” or the “horizontal” direction or orientation).

[0314] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety' with w hich it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotideAttorney Docket No.: PCT.1316comprises a regulatory' sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0315] Hydrogel: As used herein, the term, “hydrogel” refers to a three-dimensional network composed of or formed from polymers (in some cases, hydrophilic polymers), such as may be synthesized by crosslinking water-soluble polymers. Hydrogels can retain a large quantity of water within their network (e.g., such that water constitutes most or all of the fluid phase of the biphasic gel), including without destroying the original structure (e.g., of the non-fluid phase). Hydrogels can have flexibility and swelling or non-swelling properties.

[0316] Improve, increase, inhibit, and reduce: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in absence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.Attorney Docket No.: PCT.1316

[0317] Integral with: As used herein, the term “integral with" refers to two entities or components that are seamlessly j oined to one another, without use of an attachment mechanism or means such as an adhesive or mechanical fastener. Such “integral” entities or components, for example, may be formed by producing both entities in the same additive manufacturing “print job” or other continuous, single process. That is, in some cases, a component, structure, or portion that is “integral with” another component, structure, or portion (or that has an “integral structure”) may be formed from the same material as the other component, structure, or portion (including as may occur if both are formed from the same build material in the same additive manufacturing print job or process).

[0318] Interstitial space and infill: As used herein, the term “interstitial space” refers to a space, void, or volume in between other structures described herein, such as wherein one or more cells in a fluid(s) and / or a polymer (e.g., a hydrogel) are seeded to create a tissue. An "interstitial infill” refers to a structure or material (e.g., a hydrogel or hydrogel structure) disposed within an interstitial space. As one example, an “interstitial” space may be a volume or void wherein a cell in a fluid(s) and / or a polymer (e.g., a hydrogel) is seeded to create a tissue or tissue-like structure. In some cases, the interstitial infill can enable creation of a two-dimensional or three-dimensional tissue through stimulation of cell association. The tissue or tissue-like structure, in this example, may be described as an “infill.” Infills are further described below in the context of “scaffolds.”

[0319] In vitro’. The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0320] In vivo’. The term “in vivo” as used herein refers to events that occur within a multicellular organism, such as a human and anon-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0321] Lattice or Lattice Structure: As used herein, the term, “lattice” or “lattice structure” refers to a repeating two-dimensional or three-dimensional pattern of structural members formed of 3D-printed hy drogels. The pattern may occur on a nanoscale (10 nm or greater), microscale (e.g., ones of microns or tens of microns), millimeter scale, and / or macroscale (i.e., as distinguished from crystal lattice structures, which may be formed and / or repeating on atomic or molecular level, on the order of ones to tens of Angstroms).Attorney Docket No.: PCT.1316

[0322] Linker: As used herein, “linker” is used to refer to that portion of a multi-element agent that connects different elements to one another.

[0323] Network: See the description of “vasculature” below for further description of a “network,” as used herein.

[0324] Operably linked: The term “operably linked,” as used herein, indicates that two or more components are arranged such that the components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. Two molecules are “operably linked” whether they are attached directly or indirectly.

[0325] Optionally Substituted: As described herein, compounds may sometimes contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit orimplicit from the structure (e.g.,refers to“optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R°; -(CH2)O4ORO; -0(CH2)O-4R°, -0-(CH2)O4C(O)ORO; -(CH2)O4CH(0RO)2; -(CH2)O-4SRO; -(CH2)o4Ph, which may be substituted with R°; -(CH2)o-40(CH2)o iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o40(CH2)o i -pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)o-4N(R°)2; -(CH2)o-4N(R°)C(0)R°: -Attorney Docket No.: PCT.1316N(R°)C(S)R°; (CH2)o^N(R°)C(0)NR°2; -N(RO)C(S)NR°2; (CH2)O4N(RO)C(O)OR°; N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)o4C(O)R°; -(CH2)o4C(O)CH2R°; -C(S)R°; -(CH2)O4C(O)ORO; -(CH2)O4C(O)SRO; -(CH2)0^C(O)OSiR°3j -(CH2)o4OC(O)R°; -OC(0)(CH2)O4SR- SC(S)SR°; –(CH2)0–4SC(O)R°; –(CH2)0–4C(O)NR°2; -C(S)NR°2; -C(S)SR°; -SC(S)SR°. -(CH2)O40C(0)NRO2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)O4SSRO: -(CH2)O4S(O)2RO; -(CH2)O4S(O)2ORO; -(CH2)O40S(0)2RO; -S(O)2NRO2; -(CH2)O^S(0)R°; -N(R°)S(O)2NRO2; -N(R°)S(0)2RO; -N(OR°)R°; -C(NH)NR°2; -P(0)2RO; -P(0)RO2; -OP(O)RO2; -OP(O)(ORO)2: SIR°3; –(C1–4straight or branched alkylene)O–N(R°)2; or –(C1–4straight or branched alkylene)C(O)O–N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6aliphatic, -CH2Ph, -0(CH2)o iPh, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s). form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below. Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)o-2OH, -(CH2)O2OR*, -(CH2)O2CH(OR*)2; -O(haloR*), -CN, -N3, -(CH2)o-2C(0)R*, -(CH2)o2C(O)OH, (CH2)O2C(O)OR*, (CH2)O2SR*, (CH2)O2SH, (CH2)O2NH2, (CH2)O2NHR’, (CH2)O2NR’2, — NO2, -SiR*3, -OSiR*3, -C(O)SR* -(Ci-4straight or branched alkylene)C(O)OR*. or -SSR* wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from Ci-4aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S. Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0 (“oxo”), =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =N0R*, -O(C(R*2))2 30-, or -S(C(R*2))2-3S- wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Attorney Docket No.: PCT.1316Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR* 2)2 3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2. or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH2PI1, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -RT, -NRT2, -C(O)Rt, -C(O)CH2R^, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)Rt -S(O)2Rt. -S(O)2NRt2, -C(S)NRt2, -C(NH)NRt2or –N(Rt)S(O)2Rt; wherein each R1' is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R1', taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on the aliphatic group of R:are independently halogen, -R*. -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2. -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0326] For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5thEd., Ed.: Smith, M. B. and March, J., John Wiley & Sons. New York: 2001, the entire contents of which are hereby incorporated by reference.

[0327] Patient: As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic,Attorney Docket No.: PCT.1316and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0328] Paterned Hydrogel: As used herein, the term, “patterned hydrogel” refers to a hydrogel structure with at least one repeating pattern disposed therein, the repeating pattern at least partially defined by one or more voids and repeating multiple times and defined by a fixed spacing.

[0329] Perfusion conditions: As appreciated by the skilled person, perfusion conditions include volume and flow conditions (and optionally temperature conditions, such as biological temperature conditions) associated with perfusing a network, vasculature, or scaffold in a manner described herein. Carrying out a step or action “under perfusion conditions” can refer to carrying out the step or action while perfusing (e.g., a network, vasculature, or scaffold) or while an entity (e.g., a network, vasculature, or scaffold) is perfused.

[0330] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.Attorney Docket No.: PCT.1316

[0331] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier’" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository' waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0332] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,Attorney Docket No.: PCT.1316laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

[0333] Physiological conditions: As used herein, “physiological conditions” has its art-understood meaning referencing conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to conditions of the external or internal milieu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and / or within a surgical site. Physiological conditions ty pically include, e.g., a temperature range of 20 - 40°C, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity' as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and / or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[0334] Polypeptide: The term “polypeptide” as used herein refers to a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising tyvo amino acids linked together via a peptide bond. As is knoyvn to those skilled in the art, polypeptides may be processed and / or modified.

[0335] Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary- physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.Attorney Docket No.: PCT.1316

[0336] Prodrug: As used herein, the term “prodrug” refers to a compound that is a drug precursor which, following administration, releases (e.g., is converted into) the drug in vivo via a chemical or physiological process (e.g.. via cleavage as a result of exposure to a particular pH or through action of a particular enzyme or enzymes).

[0337] Ranges: All ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9. All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” should generally be considered to include the end points 5 and 10. Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0338] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art. a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0339] Scaffold: As used herein and as understood by the skilled person, the term, “scaffold” refers to a structural framework or matrix, sometimes used within physical models of living tissues (e.g., synthetic tissues), that may also be used for drug discover}7and for assessing the effectiveness of various therapies. In some cases, a scaffold supports internal passages that approximate the geometries of human vasculatures, tissues, and other structures, and may be operatively coupled to cells seeded in an interstitial space within the scaffold. A scaffold can thus enable delivery of biologically active materials to the cells and / or tissue(s) via fluid(s) flowing through the vasculature, and transport through vasculature walls. More particularly, a scaffold describedAttorney Docket No.: PCT.1316herein, in some cases, may be formed from a hydrogel, such as a bioprinted hydrogel. Moreover, in some preferred embodiments, a scaffold described herein has a continuous or integral structure, or is printed all at once (as one "print job" in an additive manufacturing process). Further, in some preferred embodiments, a scaffold described herein has two primary components, substructures, or portions, both of which can be essentially defined by printing an overall structure that has voids arranged in a desired manner: one primary component can be a vascular structure, and the other primary’ component can be an infill that surrounds the vascular structure and is “fed” by the vascular structure. An infill can be “fed” by a vascular structure in the sense that the vascular structure transports media (e.g., nutrients, pharmaceutically active agents, etc.) to and from the infill (e.g., due to diffusion of the media through walls of the vascular structure). Moreover, in some cases, as described above, the infill is organ-specific or has an organ-specific structure, meaning a structure that is intended to mimic or simulate a specific organ. For example, a liverspecific infill may comprise microspheres.

[0340] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting / inhibitory agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., compnses at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds describedAttorney Docket No.: PCT.1316herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;, " C.13C or14C for 12C;,13N or15N for 14N;17O or18O for 160;36C1 for XXC;18F for XXF; 131I for XXXI; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compoundAttorney Docket No.: PCT.1316may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0341] Substantially: As used herein, the term “‘substantially'’ refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially’’ is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0342] Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art-understood meaning referring to administration of a compound and / or composition such that it enters the ecosystem.

[0343] Tumor Microenvironment: As used herein, the term “tumor microenvironment” refers to a complex ecosystem surrounding a tumor, composed of cancer cells, stromal tissue (including blood vessels, immune cells, fibroblasts and signaling molecules), molecules, and / or the extracellular matrix. Mutual interaction between cancer cells and the different components of the tumor microenvironment support its growth and invasion in healthy tissues which correlates with tumor resistance to current treatments and poor prognosis. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads.

[0344] Units, prefixes, and symbols: Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, unless expressly stated otherwise.

[0345] Variant and Mutant: The term “variant” is usually defined in the scientific literature and used herein in reference to an organism that differs genetically in some way from an accepted standard, “Variant” can also be used to describe phenotypic differences that are not genetic (King and Stansfield, 2002, A dictionary of genetics, 6th ed., New York, New York, Oxford University Press.Attorney Docket No.: PCT.1316

[0346] The term “mutation” is defined by most dictionaries and used herein in reference to the process that introduces a heritable change into the structure of a gene (King & Stansfield, 2002) thereby producing a “mutant.” The term “variant” is increasingly being used in place of the term “mutation” in scientific and non-scientific literature. The terms are used interchangeably herein.

[0347] Vasculature, Vascularized, and Vascular: As used herein, the terms “vasculature”, “vascularized”, and “vascular” refer to a single vessel or a network (i.e., a two-dimensional network, a three-dimensional network) of vessels that enable distribution of drugs, biologies, biological fluids, and / or other substances to a cell. As understood by a person of ordinary skill in the art, the term “vasculature” refers to a perfusable 2D or 3D interconnected tubular transport system. More particularly, a “vasculature” or “vascular structure” can convey biological fluids, nutrients, drugs, biologies, and / or gases or other substances to a cell, cellular aggregate, or tissue, including to maintain viability of the cell / tissue. " Vasculature” or a “vascular structure” may also convey waste away from a cell, cellular aggregate, or tissue. In some cases, “vasculature” or a “vascular structure” described herein interpenetrates or is embedded within another network or within a tissue or a tissue mimic or substitute or extracellular matrix, such as a hydrogel structure described herein. For instance, in some instances, a vasculature or vascular structure described herein comprises one or more channels layered with, partially or completely overlapping, surrounding, embedded within, and / or interwoven with one or more channels of a tissue or other network, or embedded within a hydrogel structure. Further, in some embodiments, the “vasculature” or “vascular structure” of a device, system, or method described herein does not consist of or comprise regular cylinders or other regular geometric shapes (e.g., such regular geometric shapes joined together to form a network), but instead has an irregular geometric structure, shape, or cross-section. Additionally, in some cases, the “vasculature” or “vascular structure” of a device, system, or method described herein has a perfusable architecture or structure mimicking a mammalian (e.g., human) vascular system that includes an artery, arteriole, capillary bed, venule, vein, or a combination of two or more of the foregoing. In some cases, a vasculature or vascular structure described herein obeys or corresponds to Murray’s Law, as further described below. It is further to be understood that an entity that is “vascularized” has a vasculature or vascular structure as described above.Attorney Docket No.: PCT.1316“Ecosystem”

[0348] Among other things the present disclosure describes an ecosystem for designing, building, and refining physical models or platforms that replicate the functioning of organs (for example, human organs) that may be used for drug discovery and for assessing the effectiveness of various therapies. Fig. 1 illustrates an ecosystem 10 for creating organ-on-a-chip and other biological models, according to aspects of the present embodiments. The ecosystem 10 may include bioprinting equipment (for example, 3D printers that print using biological materials, bio-inks, etc.), conventional (non-bio) 3D printers (for example, that print using polymers, metals, ceramics, etc.), post-processing equipment such as heat treat ovens, autoclaves, rinse stations, cell seeding equipment (such as pipettes for seeding live cells in bioprinted scaffolds), cell culture equipment such as bioreactors, drug perfusion equipment (including pumps, filtration equipment, flow channels, valves), assaying equipment for performing assays on perfused cells, live imaging equipment, sensors and / or kits for quantification of analytes, image processing equipment (such as computers including memory and processors), machine learning capabilities (including cloud / network-based machine learning algorithms, local algorithms. CPU, GPU, and / or tensor processing unit (TPU)-based algorithms, etc.), and computer equipment and software for designing and refining computer models to be printed as physical models, etc. The ecosystem 10 may also include equipment for glass room, clean rooms for cell incubation, shaker plates used in postprocessing of printed components, refrigerators for storing bio-inks, PCR (polymerase chain reaction) equipment for amplifying cells, next generation sequencing equipment, flow cytometry equipment, analytical equipment, diagnostic tooling, microscopy equipment, incubators, and other equipment. In some embodiments, bioprinting methodologies, systems, and technologies described herein may be overlapping with, similar to, substantially similar to, and / or identical to those described in United States Patent Nos. 10.639,880 and / or 10,828.833.ScaffoldPolymer Composition

[0349] The present disclosure provides technologies that, in some embodiments, utilize a polymeric material (and / or materials, such as bioprinted entities, generated from them, and / orAttorney Docket No.: PCT.1316ecosystems that include them), for example to model an organ system (e.g., a diseased or healthy organ), including in diagnostic applications (e.g., to identify and validate a new disease target). In some aspects, the present disclosure provides methods to generate an ecosystem, including a polymer (e.g., a hydrogel), that includes control of environmental factors used for assaying synthetic tissue and / or organ models, real-time and / or continuous tracking of biomarkers via sensors, an ability to rapidly image tissues and / or cells, processing of assay data, machine learning tools for diagnosing results, and updating of scaffold print files such that updated models can be reprinted, assays may be rerun, and the results may be reanalyzed.

[0350] It is to be understood that a bioprinted entity or other entity described herein (e.g., a scaffold) may be formed from any hydrogel not inconsistent with the technical objectives of the present disclosure. For example, in some cases, the hydrogel is formed from the polymerization or gelation of a composition comprising an acrylate component (e.g., one or more hydroxyalkylacrylates present in the hydrogel or hydrogel -forming composition in an amount of 10-90 weight percent, based on the total weight of the hydrogel or hydrogel-forming composition), a photoinitiator component (e g., a monoacylphosphine oxide (MAPO) salt or bisacylphosphine oxide (BAPO) salt, which may in some instances be a sodium or lithium MAPO or BAPO salt, and which may be present in an amount of 0.1-5 weight percent, based on the total weight of the hydrogel or hydrogel-forming composition), an optional non-curable absorber component (e.g., sulfonated quinoline yellow, which may be present in an amount of 0.1 -5 weight percent, based on the total weight of the hydrogel or hydrogel-forming composition), and water (e.g., as the “balance’' of the hydrogel or hydrogel-forming composition, or in an amount of 20-80 weight percent). Nonlimiting examples of hydrogels which may be suitable for bioprinting a scaffold in some embodiments described herein include those disclosed in U. S. Patent 12,384,935 or U. S. Patent Application Publication 2024 / 0091412 A1, the entireties of which are hereby incorporated by reference.

[0351] The present disclosure teaches that, in some embodiments, a bioprinted entity provided and / or utilized in accordance with the present disclosure includes a polymer moiety, such as a hydrogel moiety (e.g., formed from a polyethylene glycol diacrylate, or PEGDA) and a coating moiety (e.g., a polypeptide), covalently linked to one another, optionally via a linker.

[0352] Indeed, in some aspects, the present disclosure provides improvements to useful polymers achieved by conjugating them to a coating moiety (e.g., a polypeptide) as described herein to facilitate association of a cell with the bioprinted entity. In some embodiments, the presentAttorney Docket No.: PCT.1316disclosure provides conjugates of a polymer moiety that show improved cellular distribution relative to a comparable preparation of the same polymer moiety when not conjugated. Those skilled in the art. reading the present disclosure, will appreciate that conjugates as provided and / or utilized herein are typically produced by linking a coating moiety (e.g., a polypeptide) as described herein with a preparation of a polymer moiety or polymeric moiety. Those skilled in the art will further appreciate that such polymer moiety preparations are typically not perfectly uniform compositions but rather include some structural diversity of the polymers within them. Those skilled in the art will appreciate that polymer preparations (including, e.g., as may be purchased commercially or prepared) are ty pically characterized by a molecular weight, or molecular weight range or distribution, which is typically representative of an average or median molecular weight of polymer in the preparation.

[0353] Still further, those skilled in the art will appreciate that polymer moieties are typically characterized by a poly dispersity’ index, reflecting the weight average molecular weight divided by the number average molecular weight (Mw / Mn), which provides an assessment of the molecular weight distribution in a polymer moiety.

[0354] In some embodiments, conjugates as described and / or utilized herein are prepared from polymer moiety with a PDI within a range of about 1 to about 5. In some embodiments, conjugates as described above and / or utilized herein are prepared from polymer moieties with a PDI of about 1 to about 3. In some embodiments, conjugates as described above and / or utilized herein are prepared from polymer moieties with a PDI of about 3 to about 5. In some embodiments, conjugates as described and / or utilized herein are prepared from a polymer moiety with a PDI of about 1 to about 2.

[0355] In some embodiments, a polymer moiety may be characterized by an optimal diffusion coefficient, reflecting the diffusivity of a solute with hydrodynamic radius in a liquid, wherein it provides an assessment of the rate of diffusion of one material through another. In some embodiments, a polymer moiety as described and / or utilized herein may have a diffusion coefficient of about 5 μm2 / sec to about 120 μm2 / sec.

[0356] The present disclosure particularly teaches that provided technologies are particularly applicable to (e.g., beneficial when applied to) polymer moieties that are or formed from acrylated hydrogel moieties or components or starting materials, such as PEGDA.Attorney Docket No.: PCT.1316

[0357] In some embodiments, the present disclosure provides an insight that certain polymer moieties surprisingly can impart to a conjugate as described herein an abi li ty to exhibit a desired cell distribution.

[0358] In some embodiments, the present disclosure provides an insight that certain bioprinted entities exhibit a desired cell distribution.

[0359] In those embodiments that may comprise a plurality of polymer moieties, such polymer moieties may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality of distinct polymer moieties.

[0360] For example, in some embodiments, a polymer moiety useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular coating moiety (e.g., a polypeptide).

[0361] In some embodiments, a bioprinted entity may be or comprise an optionally substituted polymer moiety. In some embodiments, a polymer moiety is optionally substituted with –(CH₂)₀₋₄C(O)CH₂R°. In some embodiments, a polymer moiety is optionally substituted with - C(O)CH2R°. In some embodiments, a polymer moiety is optionally substituted with -C(O)CH2R°, wherein R° is C1-6aliphatic. In some such embodiments, R° is an unsaturated C1-6alkyl. In further embodiments, R° is CH2.

[0362] In some embodiments, a polymer moiety is optionally substituted with -C(O)CH2R:. In some embodiments, a polymer moiety is optionally substituted with -C(O)CH2R:. wherein Rfis C1-6 aliphatic. In some such embodiments, R1’ is an unsaturated C1-6 alkyl. In further embodiments, R:is CH2.

[0363] In some embodiments, a polymer moiety is optionally substituted with at least one O, wherein ” represents a point of attachment to the polymer moiety.

[0364] In some embodiments, a polymer moiety is optionally substituted with at least one 0, wherein represents a point of attachment to the polymer moiety.

[0365] In some embodiments, a polymer moiety is optionally substituted with at least one O OHO, wherein” represents a point of attachment to the polymer moiety.Attorney Docket No.: PCT.1316

[0366] In some embodiments, a polymer moiety may be an acrylated, methacrylated, or diacrylated hydrogel.

[0367] In some embodiments, a bioprinted entity may be or comprise a polymer moiety selected from the group comprising PEGDA, alginate, gelatin, GelMA, ColMA, hyaluronic acid, and / or polyethylene glycol (PEG).Coating Moieties

[0368] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties (e.g., a polypeptide), optionally associated with one another via a linker(s).

[0369] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s), wherein the coating moiety is selected from a group consisting of a polypeptide, a small molecule, a peptidomimetic, a lipid, a lipid nanoparticle, a nucleic acid, a (poly)saccharide, or a combination thereof.

[0370] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s). wherein the coating moiety is a polypeptide.

[0371] In some embodiments, the present disclosure provides an insight that certain polypeptides surprisingly can impart to a conjugate as described herein an ability to exhibit a desired cell distribution.

[0372] In those embodiments that may comprise a plurality’ of polypeptides, such polypeptides may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality of distinct polypeptides.

[0373] For example, in some embodiments, a polypeptide useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular polymer moiety.

[0374] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moietyAttorney Docket No.: PCT.1316may be adjusted, for example through linkage of a plurality of polypeptides, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.

[0375] In some embodiments, a polypeptide as described herein may, for example, be selected from the group consisting of a collagen, a fibrin, an integrin, a selectin, a cadherin, a member of the immunoglobulin superfamily (IgSF) (e g., a nectin, a mucin), a laminin, Matrigel, an extracellular matrix (ECM) protein, an antibody, an antibody fragment, etc. In some embodiments, a polypeptide is a collagen. In some embodiments, a polypeptide is a fibrin. In some embodiments, a polypeptide is an integrin. In some embodiments, a polypeptide is a selectin. In some embodiments, a polypeptide is a cadherin. In some embodiments, a polypeptide is an IgSF. In some embodiments, a polypeptide is a nectin. In some embodiments, a polypeptide is a fibronectin. In some embodiments, a polypeptide is a mucin. In some embodiments, a polypeptide is a laminin. In some embodiments, a polypeptide is a Matrigel. In some embodiments, a polypeptide is an ECM protein. In some embodiments, a polypeptide is an antibody. In some embodiments, a polypeptide is an antibody fragment.

[0376] In some embodiments, a polypeptide as described above and herein may be naturally occurring. In some embodiments, a polypeptide as described above and herein may be engineered. In some such embodiments, an engineered polypeptide may have one or more conservative amino acid substitutions. In some such embodiments, an engineered polypeptide may have one or more non-conservative amino acid substitutions. In further embodiments, an engineered polypeptide may have a combination of one or more conservative and / or non-conservative amino acid substitutions.

[0377] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s), wherein the coating moiety is a small molecule.

[0378] In some embodiments, the present disclosure provides an insight that certain small molecules surprisingly can impart to a conjugate as described herein an ability7to exhibit a desired cell distribution.

[0379] In those embodiments that may comprise a plurality of small molecules, such small molecules may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality7of distinct small molecules.Attorney Docket No.: PCT.1316

[0380] For example, in some embodiments, a small molecule useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular polymer moiety.

[0381] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of small molecules, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.

[0382] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s). wherein the coating moiety is a peptidomimetic.

[0383] In some embodiments, the present disclosure provides an insight that certain peptidomimetics surprisingly can impart to a conjugate as described herein an ability to exhibit a desired cell distribution.

[0384] In those embodiments that may comprise a plurality of peptidomimetics, such peptidomimetics may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality of distinct peptidomimetics.

[0385] For example, in some embodiments, a peptidomimetic useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular peptidomimetic moiety.

[0386] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular peptidomimetic moiety may be adjusted, for example through linkage of a plurality of peptidomimetics, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.

[0387] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s), wherein the coating moiety is a lipid.Attorney Docket No.: PCT.1316

[0388] In some embodiments, the present disclosure provides an insight that certain lipids surprisingly can impart to a conjugate as described herein an ability to exhibit a desired cell distribution.

[0389] In those embodiments that may comprise a plurality of lipids, such lipids may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality of distinct lipids.

[0390] For example, in some embodiments, a lipid useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e g., linked) with a particular polymer moiety.

[0391] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of lipids, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.

[0392] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s), wherein the coating moiety is a lipid nanoparticle.

[0393] In some embodiments, the present disclosure provides an insight that certain lipid nanoparticles surprisingly can impart to a conjugate as described herein an ability to exhibit a desired cell distribution.

[0394] In those embodiments that may comprise a plurality of lipid nanoparticles, such lipid nanoparticles may, in some embodiments, all be the same; in other embodiments, a provided system may comprise a plurality of distinct lipid nanoparticles.

[0395] For example, in some embodiments, a lipid nanoparticle useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular polymer moiety.

[0396] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of lipid nanoparticles, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.Attorney Docket No.: PCT.1316

[0397] In some embodiments, a lipid nanoparticle as described above and herein may comprise a payload. In some embodiments, the payload comprises a naturally occurring and / or engineered nucleic acid. In some such embodiments, an engineered nucleic acid may have one or more conservative substitutions. In some such embodiments, an engineered nucleic acid may have one or more non-conservative substitutions. In further embodiments, an engineered nucleic acid may have a combination of one or more conservative and / or non-conservative substitutions. In some embodiments, the nucleic acid may be selected from a group consisting of DNA, siRNA, mRNA, tRNA, rRNA, etc. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is tRNA. In some embodiments, the nucleic acid is rRNA.

[0398] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via a linker(s), wherein the coating moiety is a nucleic acid (e.g., DNA, siRNA, mRNA, tRNA, rRNA).

[0399] In some embodiments, the present disclosure provides an insight that certain nucleic acids surprisingly can impart to a conjugate as described herein an ability' to exhibit a desired cell distribution.

[0400] In those embodiments that may comprise a plurality of nucleic acids, such nucleic acids may, in some embodiments all be the same; in other embodiments, a provided system may comprise a plurality of distinct nucleic acids.

[0401] For example, in some embodiments, a nucleic acid useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular polymer moiety.

[0402] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of nucleic acids, which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.

[0403] In some embodiments, a coating moiety comprises a naturally occurring and / or engineered nucleic acid. In some such embodiments, an engineered nucleic acid may have one or more conservative substitutions. In some such embodiments, an engineered nucleic acid may have one or more non-conservative substitutions. In further embodiments, an engineered nucleic acidAttorney Docket No.: PCT.1316may have a combination of one or more conservative and / or non-conservative substitutions. In some embodiments, the nucleic acid may be selected from a group consisting of DNA, siRNA, mRNA, tRNA. rRNA, etc. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is tRNA. In some embodiments, the nucleic acid is rRNA.

[0404] In some embodiments, the present disclosure provides and / or utilizes conjugates (and / or materials, such as hydrogels, generated from them, and / or systems that include them) which comprise one or more polymer and coating moieties, optionally associated with one another via one or more linker(s), wherein the coating moiety is a (poly)saccharide.

[0405] In some embodiments, the present disclosure provides an insight that certain (poly)saccharides surprisingly can impart to a conjugate as described herein an ability to exhibit a desired cell distribution.

[0406] In those embodiments that may comprise a plurality of (poly)saccharides, such (poly)saccharides may, in some embodiments all be the same; in other embodiments, a provided system may comprise a plurality of distinct (poly)saccharides.

[0407] For example, in some embodiments, a (poly)saccharide useful in accordance with the present disclosure is characterized by a particular degree of interaction with a cell, for example, when associated (e.g., linked) with a particular polymer moiety.

[0408] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of (poly)saccharides. which may be the same or different and which, individually or together, may be considered or constitute a bioprinted entity as described herein.Linkers

[0409] In some embodiments, a conjugate, as described in the present disclosure, comprises one or more linkers.

[0410] For example, in some embodiments, a conjugate includes a linker which conjugates a polymer moiety and a coating moiety (e g., a polypeptide).

[0411] In some embodiments, a linker moiety is referred to as “L”. In some embodiments, a linker may be cleavable or degradable under biological conditions. In some embodiments, a linker may be non-cleavable and / or non-degradable under biological conditions. In some embodiments,Attorney Docket No.: PCT.1316a linker may degrade via hydrolysis or enzymatic reaction. In some embodiments, a linker may be cleavable through application of a cleavage promoter (e.g., an electrical, chemical, and / or enzymatic stimulus). In some embodiments, a linker degrades (e.g., over and / or within a specified period of time, such as within hours, days, weeks, or months) after administration of the system.

[0412] In some embodiments, a linker may be associated with a moiety as described herein (e.g., with a polymer moiety, a polypeptide, and / or an “other” moiety ) via chemical conjugation; in some embodiments chemical conjugation may be or comprise click chemistry’. Thus, in some embodiments, a conjugate as described and / or utilized herein may be formed by and / or may participate in a chemical linkage reaction, e.g., a click chemistry reaction.

[0413] In some embodiments, conjugation of two or more moieties with one another can be mediated by a chemical reaction that involves amine-reactive click chemistry. In some embodiments, conjugation of two or more moieties with one another can be mediated by a chemical reaction that involves an N-hydroxysuccinimide (NHS). In some embodiments, conjugation of two or more moieties with one another can be mediated by an NHS-ester click chemistry reaction. In some embodiments, conjugation of two or more moieties with one another can be mediated by an NHS-ester click chemistry reaction at physiological conditions.

[0414] In some embodiments, a linker isO,wherein n is 0-100; wherein the NHS-ester portion of the linker forms a covalent bond w ith a free amine on a coating moiety; and wherein the acrylate portion of the linker forms a covalent bond with a polymer moiety.

[0415] In some embodiments, a linker is acrylated polyethylene glycol succinimidyl valerate (acrylated PEG SV A).

[0416] In some embodiments, a linker is PEG-NHS.

[0417] In some embodiments, a linker may be a bond.

[0418] In some embodiments, L is polyethylene glycol (PEG). In some embodiments, L may be an ethylene diamine, e.g., a polyethylene glycol diamine, etc.

[0419] In some embodiments, L comprises a moiety which results from a “click" reaction. In some embodiments, L comprises a triazole. In some embodiments, L comprises an imine. In some embodiments, L comprises an oxime. In some embodiments, L comprises a hydrazine. In someAttorney Docket No.: PCT.1316embodiments, L comprises a moiety which results from a nucleophilic addition. In some embodiments, L comprises a moiety which results from a Michael addition. In some embodiments, L comprises a thiol-ene.

[0420] In some embodiments, L is an optionally substituted C1-6alkylene chain wherein one, two, or three methylene units of L are optionally and independently replaced by -NH-, -O-, -S-, — S(O)~, -S(O)2-, or — C(O)~. A variety of techniques may be used for conjugating or associating the polymer moiety to a coating moiety (e.g., a polypeptide).Cells

[0421] The present disclosure teaches that, in some embodiments, a bioprinted entity provided and / or utilized in accordance with the present disclosure includes a polymer moiety, such as a hydrogel moiety (e.g.. PEGDA) a coating moiety (e.g., a polypeptide), covalently linked to one another, optionally via a linker (e.g., acrylated PEG SV A), wherein the coating moiety facilitates the association of a cell.

[0422] In some embodiments, a cell associated with the bioprinted entity may be selected from a naturally occurring cell and / or an engineered cell. In some embodiments, a cell associated with the bioprinted entity may be a naturally occurring cell. In some embodiments a cell associated with the bioprinted entity may be an engineered cell. In some embodiments, one or more cells associated with the bioprinted entity may be a combination of a naturally occurring and an engineered cell.

[0423] In some embodiments, a cell associated with the bioprinted entity and / or seeded therein is selected from a group consisting of an endothelial cell, a biliary endothelial cell, a cholangiocyte, a liver parenchymal cell, a hepatocyte (HC), a primary human hepatocyte (PHH), a hepatic stellate cell (HSCs), a Kupffer cell (KC), a liver sinusoidal endothelial cell (LSEC), a mucous cell, a parietal cell, a chief cell, an endocrine cell (e.g., a G cell, a D cell, a enterochromaffin cell, a EC-like cell, a X / A cell), a columnar epithelial cell, a cardiac fibroblast (CF), a cardiomyocyte, a smooth muscle cell, an enterocyte, a goblet cell, a Paneth cell, a stem cell, a neuron, a glia, a keratinocyte, a melanocyte, a Merkel cell, a Langerhans cell, a germ cell, a stromal cell, a seminiferous tubule, a Leydig cell, a tubule epithelial cell, a macula densa cell, a glomerular endothelial cell, a podocyte, a mesangial cell, a parietal epithelial cell, an immortalized cell (e.g. a 3T3 cell, a A549 cell, a HeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, a Jurkat cell, aAttorney Docket No.: PCT.1316OK cell, a Ptk2 cell, a Vero cell), a patient-derived cell (e.g., a tumor cell), a T cell, a peripheral blood mononuclear cell (PBMC), and / or an induced pluripotent stem cell (iPSC).

[0424] In some embodiments, a cell associated with the bioprinted entity is an endothelial cell.

[0425] In some embodiments, a cell associated with the bioprinted entity is a biliary endothelial cell.

[0426] In some embodiments, a cell associated with the bioprinted entity is a cholangiocyte.

[0427] In some embodiments, a cell associated with the bioprinted entity is a liver parenchymal cell.

[0428] In some embodiments, a cell associated with the bioprinted entity is a hepatocyte (HC).

[0429] In some embodiments, a cell associated with the bioprinted entity is a primary human hepatocyte (PHH).

[0430] In some embodiments, a cell associated with the bioprinted entity is a hepatic stellate cell (HSCs).

[0431] In some embodiments, a cell associated with the bioprinted entity is a Kupffer cell (KC).

[0432] In some embodiments, a cell associated with the bioprinted entity is a liver sinusoidal endothelial cell (LSEC).

[0433] In some embodiments, a cell associated with the bioprinted entity is a mucous cell.

[0434] In some embodiments, a cell associated with the bioprinted entity is a parietal cell.

[0435] In some embodiments, a cell associated with the bioprinted entity is a chief cell.

[0436] In some embodiments, a cell associated with the bioprinted entity is an endocrine cell (e.g.. a G cell, a D cell, an enterochromaffin cell, a EC-like cell, a X / A cell).

[0437] In some embodiments, a cell associated with the bioprinted entity is a columnar epithelial cell.

[0438] In some embodiments, a cell associated with the bioprinted entity is a cardiac fibroblast (CF).

[0439] In some embodiments, a cell associated with the bioprinted entity is a cardiomyocyte.

[0440] In some embodiments, a cell associated with the bioprinted entity is a smooth muscle cell.

[0441] In some embodiments, a cell associated with the bioprinted entity is an enterocyte.

[0442] In some embodiments, a cell associated with the bioprinted entity is a goblet cell.

[0443] In some embodiments, a cell associated with the bioprinted entity is a Paneth cell.

[0444] In some embodiments, a cell associated with the bioprinted entity is a stem cell.

[0445] In some embodiments, a cell associated with the bioprinted entity is a neuron.Attorney Docket No.: PCT.1316

[0446] In some embodiments, a cell associated with the bioprinted entity is a glia.

[0447] In some embodiments, a cell associated with the bioprinted entity is a keratinocyte.

[0448] In some embodiments, a cell associated with the bioprinted entity is a melanocyte.

[0449] In some embodiments, a cell associated with the bioprinted entity is a Merkel cell.

[0450] In some embodiments, a cell associated with the bioprinted entity is a Langerhans cell.

[0451] In some embodiments, a cell associated with the bioprinted entity is a germ cell.

[0452] In some embodiments, a cell associated with the bioprinted entity is a stromal cell.

[0453] In some embodiments, a cell associated with the bioprinted entity is a seminiferous tubule.

[0454] In some embodiments, a cell associated with the bioprinted entity' is a Leydig cell.

[0455] In some embodiments, a cell associated with the bioprinted entity is a tubule epithelial cell.

[0456] In some embodiments, a cell associated with the bioprinted entity is a macula densa cell.

[0457] In some embodiments, a cell associated with the bioprinted entity is a glomerular endothelial cell.

[0458] In some embodiments, a cell associated with the bioprinted entity is a podocyte.

[0459] In some embodiments, a cell associated with the bioprinted entity is a mesangial cell.

[0460] In some embodiments, a cell associated with the bioprinted entity' is a parietal epithelial cell.

[0461] In some embodiments, a cell associated with the bioprinted entity is an immortalized cell (e.g.. a 3T3 cell, a A549 cell, a HeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, a Jurkat cell, an OK cell, a Ptk2 cell, a Vero cell).

[0462] In some embodiments, a cell associated with a bioprinted entity' is a patient-derived cell (e.g., a tumor cell).

[0463] In some embodiments, a cell associated with a bioprinted entity is a T cell.

[0464] In some embodiments, a cell associated with a bioprinted entity is a peripheral blood mononuclear cell (PBMC).

[0465] In some embodiments, a cell associated with a bioprinted entity is an induced pluripotent stem cell (iPSC).

[0466] In some embodiments, a cell associated with the bioprinted entity is a combination of one or more cells as described above and herein. In some embodiments, a combination of cells mayAttorney Docket No.: PCT.1316comprise an endothelial cell and / or a cell other than an endothelial cell. In some embodiments, a combination of cells may comprise a liver parenchymal cell, a PHH, and / or an endothelial cell.

[0467] Those skilled in the art. reading the present disclosure, will appreciate that, in some embodiments, interaction of a cell with a bioprinted entity comprising a particular polymer moiety may be adjusted, for example through linkage of a plurality of cells, which may be the same or different.

[0468] In some embodiments, a cell associated with a bioprinted entity as described above and herein forms a tissue.Scaffold Formation

[0469] In some embodiments, a bioprinted entity comprises a biocompatible resin and a bioprinted vascularized scaffold. In some such embodiments, a bioprinted entity comprises at least one chamber into which a cell in media may be pipetted.

[0470] In some embodiments, the vascularized scaffold may go through the chambers. In some embodiments, aspects of the vascularized scaffold may be controlled (e.g., wall thickness, lumen diameter, porosity, architecture) to provide alternate properties. It is also understood that the vascularized scaffold may comprise more than one independent network (e.g., vasculature, a bile duct). In some embodiments, bioprinting methodologies, systems, and technologies described herein may be overlapping with, similar to, substantially similar to, and / or identical to those described in United States Patent Nos. 10,639,880 and / or 10,828,833.

[0471] Fig. 28 illustrates a method 800 of creating, seeding, and operating an organ on a chip platform, according to aspects of the present embodiments. At step 802, the method 800 may include loading an electronic build or print file onto a 3D printer (for example, a printer that uses digital light processing (DLP) to print polymer parts). At step 804, the method 800 may include 3D printing (i.e., additively manufacturing) resin manifold parts. At step 806. the method 800 may include post processing steps (such as deburring to remove any abnormalities, smoothing, finishing steps, etc.). At step 808, the method 800 may include autoclaving the printed parts to help encourage and expedite curing. At step 810, the method 800 may include loading an electronic build or print file onto a 3D printer (for example, a 3D-bio printer that uses DLP to print bioactive parts). At step 812, the method 800 may include 3D printing (i.e., additively manufacturing) bioprinted scaffolding. At steps 814 and 816, the method 800 may include washing and equilibrating the bioprinted scaffolding. At step 816, the method 800 may include assembling theAttorney Docket No.: PCT.1316bioprinted scaffolding within a hydrogel chip (for example, via an access port in the chip). At step 820, the method 800 may include connecting the vasculature to a system (i.e., perfusion system, flow rig, bioreactor, etc.) that fluidly couples flow lines such that bio-fluids may flow through the vasculature. At step 822, the method 800 may include coating the internal walls of the vasculature with a chemical linker. At step 824, the method 800 may include coating the interstitial space with a chemical linker.

[0472] Referring still to Fig. 28, the method may include washing the internal walls of the vasculature and the interstitial space, after the chemical linker is added. At step 828, the method 800 may include adding a bioactive molecular coating to the interior walls of the vasculature and the interstitial space, which again then may be washed leaving behind a homogenous layer of coating. At step 832, the method 800 may include leaving the cells in place for a period of time (for example, 2 hours, 4 hours. 6 hours, 8 hours. 10 hours, overnight, 12 hours, 16 hours, 24 hours, 48 hours, etc.) allowing the cells to attach to the respective surfaces. At step 834, the method 800 may include initiating perfusion. At step 836, the method 800 may include monitoring sensor data. At step 838, the method 800 may include collecting effluent flow from the cell chamber and vasculature. At step 840, the method 800 may include assessing key parameters. At step 842, the method 800 may include repeating any of the previous steps.

[0473] Fig. 29 illustrates a method 900 of creating, seeding, and operating an organ on a chip platform, according to aspects of the present embodiments. Several of the steps of the method 900 shown in Fig. 29 are similar to or the same as steps of method 800 illustrated in Fig. 28. At step 916, the method 900 may include rotating the printed platform to encourage cell attachment. At step 922, the method 900 may include monitoring the process concurrent with imaging and effluent collection. At step 924, the method 900 may include introducing a drug through the vasculature (such that, in some embodiments, it may transport across the vasculature walls into the cells in the interstitial space). At step 926, the method 900 may include monitoring the process concurrent with imaging and effluent collection following introduction of the drug(s). At step 928, the method 900 may include performing one or more terminal assays (for example, histology, IHC, omics, etc.). At step 930, the method 900 may include feeding data into a machine learning (ML) algorithm. At step 932, the method 900 may include creating an in silico model. At step 932, the method 900 may include refining the geometry of the scaffold model (i.e., to be reprinted) based on the in silico model. At step 936, the method 900 may include repeating any of the previous steps.Attorney Docket No.: PCT.1316

[0474] Fig.2 and Fig. 3 illustrate a system 20 and platform 30 for modeling organ-on-a-chip and other biological models, according to aspects of the present embodiments. The system 20 may include a platform 30 (for example, a 3D printed assembly as described herein) as well as other components such as an inlet line 12 coupled to an inlet port 14 (or inlet flow line) of the platform 30, and outlet line 18 coupled to an outlet port 16 (or outlet flow line) of the platform, one or more sensors 22, 24 disposed in the inlet and / or outlet lines 12, 18, a reservoir, a pump, and other potential components such as filters, fluid replenishment systems, blood / fluid warmers, etc. The platform 30 may include one or more internal chips that include internal vasculatures and are fluidly coupled to the inlet and outlet ports 14, 16. The system 20 may be used, for example, to perfuse organs and / or to study the functioning of organs in a simulated and active (i.e., including live cells) environment.

[0475] Fig. 3 illustrates a side view of platform 30 for modeling organ-on-a-chip and other biological models, according to aspects of the present embodiments. Fig. 4 illustrates a platform assembly 30, according to aspects of the present embodiments. The platform / platform assembly may include a base 42, a glass based 38 configured to be seated in the base 42 and to support one or more chips 40A, 40B, O-rings 36 for sealing the chips 40A, 40B to a glass cover 26, a clip 34 laterally slidable across a case 32, and a cover retainer 28 for holding the glass cover 26 in place. The fluid inlet 14 and fluid outlet 16 are visible in Fig. 4. With the exception of the chips 40A, 40B, the glass base 42, the glass cover 26, and the O-rings 36, each of the other components of the platform assembly 30 (i.e., the base 42, the clip 34, the case 32, and the cover 28) may be formed via 3D printing and may be composed of a polymer material.

[0476] Referring again to Fig. 3, the platform 30 (specifically the base 42) may include a base clip 46 that is shaped to be engaged with a corresponding case clip 44 (i.e., integrally formed with the case 32) for holding the base 42 to the case 32. In some embodiments, the platform may include a base and case clips 46. 44 on each end. Each of the chips 40A, 40B may be disposed within the case 32 via corresponding recesses 54 disposed within the case 32. Each chip 40A, 40B may include an internal 3D printed scaffold 70 with an internal vasculature 60 disposed therein. The scaffold 70 may be formed of hydrogel material. The vasculature 60 may include a vasculature inlet 48 and a vasculature outlet 52, both being fluidly coupled (via microchannels disposed within the case 32 (not shown)) to the respective fluid inlet 14 and fluid outlet 16 disposed in the case 32. The vasculature 60 may include a network of flow passages fluidly connecting the vasculature inlet 48 (or inlet flow passage 48) and vasculature outlet 52 (or outlet flow passage 52), andAttorney Docket No.: PCT.1316geometrically defining the boundaries of an interstitial space defined therewithin. The platform 30 and chips 40A, 40B, may further comprise an interstitial infill 58 that includes an internal structure (for example, a 3D-printed repeating structure) enabling active cells to be seeded thereto. Cells may be added into the interstitial space via an access port 56 disposed within the scaffold 70.

[0477] Fig. 5 illustrates a platform assembly 30, according to aspects of the present embodiments. In the embodiment of Fig. 5, the platform 30 includes two fluid inlets 14 and two fluid outlets 16. This configuration enables different fluids to be routed to the two cells. This configuration also enables two different fluids to be routed to each of the two cells, as further described herein in connection with Figures 30-33.

[0478] Fig. 6 illustrates a bottom view of a platform assembly 30, according to aspects of the present embodiments. The platform may be configured to accommodate different numbers of cells including but not limited to 1, 2, 3, 4, 5, 8. 10, 15, 16, 20, 50, 100. 1000, and more than 1000 cells.

[0479] Fig. 7 illustrates a perspective view of a platform assembly 30, according to aspects of the present embodiments. In the illustration of Fig. 7, a grip surface 62 is disposed in the clip 34 to help facilitate sliding the clip 34 on and off.

[0480] Fig. 8 illustrates a top view of a platform assembly, according to aspects of the present embodiments.

[0481] Fig. 9 illustrates a side view of a platform assembly, according to aspects of the present embodiments.

[0482] Fig. 10 illustrates a front view of a platform assembly, according to aspects of the present embodiments.

[0483] Fig. 11 illustrates a view of a platform assembly 50, according to aspects of the present embodiments. The platform assembly 50 includes a 4x4 grid of recesses enabling 16 cells to be disposed therein. In some embodiments, the platform 30 includes an internal micro-fluid framework that acts as a manifold such that fluids can be route to and from each of the (in this case) 16 cells 40, via only a single fluid inlet 14 and a single fluid outlet 16.

[0484] Figs. 12A and 12B illustrate views of a platform assembly 50, according to aspects of the present embodiments.

[0485] Fig. 13 illustrates a view of a platform assembly 50, according to aspects of the present embodiments.

[0486] Fig. 14 illustrates a view of a platform assembly 50, according to aspects of the present embodiments.Attorney Docket No.: PCT.1316

[0487] Figs. 15A, 15B, 15C, and 15D illustrate views of a platform assembly 50, according to aspects of the present embodiments.

[0488] Fig. 16 illustrates a view of an organ chip 40. according to aspects of the present embodiments. As shown in Fig. 16, the vasculature 60 disposed within the hydrogel scaffold 70 of the chip 40 may have a larger diameter at each of the inlet and outlets, and may include a plurality or network of smaller vasculature passages 60A connecting therebetween. In some embodiments, the internal diameter of the vasculature decreases from the vasculature inlet to a center of the chip 40, and then increases from the center of the chip 40 to the vasculature outlet.

[0489] Fig. 17 illustrates a view of an organ chip 40, according to aspects of the present embodiments.

[0490] Fig. 18 illustrates a view of an organ chip 40, according to aspects of the present embodiments.

[0491] Fig. 19 illustrates a view of organ chips 40A, 40B within a platform 30 assembly, according to aspects of the present embodiments. The platform 30 may include a first chip 40A that includes a scaffold including a vasculature that is representative of a tumor, and a second chip 40B that includes a scaffold including a vasculature that is representative of a liver. The platform 30 may include one or more sensors 64 disposed within the vasculature, within the interstitial space, as well as in other potential locations such as in the inlet and outlet lines 12, 18 (shown in Fig.2). The one or more sensors 64 may include sensors for measuring luminescence, colorimetry, electrochemical activity, fluorescence, and / or metabolic activity'.

[0492] Figs. 20A, 20B, and 20C illustrate views of vasculature configurations, according to aspects of the present embodiments. In the configuration shown in Fig. 20A, the vasculature 60 of the scaffold 70 includes a single passageway or channel through the interstitial infill 58. The vasculature 60 may take a serpentine path making several turns (for example 90, 180, 270, and / or other number of degree turns) through the interstitial infill 58. In the configuration shown in Fig.20B, the scaffold 70 may include multiple vasculatures 60 (or channels, or passageways), each vasculature 60 including an internal network of connections or passageways disposed within the interstitial infill 58 between a vasculature inlet and a vasculature outlet. In the configuration shown in Fig. 20B, the multiple vasculatures 60 may not connect to each other. In the configuration shown in Fig.20C, the scaffold 70 may include an open vasculature 60 where passageways connect to adjacent passageways within the interstitial infill 58 forming a two dimensional and / or three dimensional interconnected vascular 60.Attorney Docket No.: PCT.1316

[0493] Figs.21A-B and 22 illustrate views of vasculature configurations 60, according to aspects of the present embodiments. In the embodiments illustrated in Figs. 21A, 21B, and 22, the interstitial infill 58 includes a repeating lattice structure or framework.

[0494] Figs. 23A, 23B, and 24 illustrate views of vasculature configurations 60, according to aspects of the present embodiments. In the embodiments illustrated in Figs.23A, 23B, and 24, the interstitial infill 58 includes a plurality of packed and interconnected microspheres.

[0495] Fig. 25 illustrates views of interstitial infill 58. according to aspects of the present embodiments. In some configurations, the interstitial infill 58 may include one or more integral handles 66 and / or other features to facilitate handling of the interstitial infill 58.

[0496] Figs. 26A and 26B illustrate a view of interstitial infill 58, according to aspects of the present embodiments. In the embodiments illustrated in Figs. 26A and 26B, the interstitial infill may include a lattice of interconnected spherical pores 68. connected via a plurality of interconnects 72. In some embodiments, the spherical pores 68 are connected to every adjacent spherical pore 68 via the interconnects 72. In some embodiments, the spherical pores 68 are not connected to every adjacent spherical pore 68 but are connected to 1) at least two adjacent spherical pores 68 in the same horizontal plane, 2) at least one adjacent spherical pore 68 in the next vertical layer higher of spherical pores, and 3) at least one adjacent spherical pore 68 in the next vertical layer lower of spherical pores. Accordingly, in some embodiments, each spherical pore 68 is connected to at least 4 adjacent spherical pores 68 via at least 4 corresponding interconnects 72.

[0497] Referring still to Figs. 26A and 26B, in some embodiments, the framework 58 (or interstitial infill 58) illustrated in Figs. 26A and 26B may be well suited for creating models of (e g., synthetic) tissue such as liver tissue. In some embodiments, the each of the spherical pores 68 may have a diameter of about 300 pm (i.e., 300 microns) or from about 250 pm to about 350 pm, or from about 200 pm to about 400 pm, or from about 150 pm to about 450 pm. In some embodiments, each of the interconnects 72 may have a maximum dimension (i.e., diameter, length, major axis, etc.) of about 60 pm, or from about 55 pm to about 65 pm, or from about 50 pm to about 70 pm, or from about 45 pm to about 75 pm, or from about 40 pm to about 80 pm. The framework 58 (or interstitial infill 58) may be seeded with active cells via pipette. In some embodiments, the framework 58 (or interstitial infill 58) may be seeded with primary human hepatocyte and / or may be co-cultured with stellate cells and / or Kupffer cells. In some embodiments, the vasculature 60 that in which the framework 58 (or interstitial infill 58) is disposed may be seeded with sinusoidal endothelial cells. In some embodiments, the frameworkAttorney Docket No.: PCT.131658 (or interstitial infill 58) may also be disposed within a second vasculature (i.e., a bile duct) that is seeded with cholangiocytes lining the ducts (i.e., lining the interior vasculature 60 walls). In some embodiments, the framework 58 (or interstitial infill 58) is not vascularized (i.e.. not disposed within a vasculature 60). In some embodiments, the framework 58 (or interstitial infill 58) may be seeded at a density of from about 200,000 to about 1 million cells per chip.

[0498] Fig.27 illustrates a view of a vasculature 60 and interstitial space seeded with active cells 76, 74, according to aspects of the present embodiments. The interstitial infill 58 is also shown in Fig.27. In some embodiments, a first cell type 74 is seeded within the interior walls of the vascular 60 while a second cell type 76 is seeded in the interstitial space (and is supported by the interstitial infill 58).Endothelial coating, seeding and functionalization

[0499] Among other things, the present disclosure is directed to systems and methods for functionalizing and characterizing the inner walls of a complex, high-resolution, multi-planar vascular network of 3D-printed synthetic tissues using cell-specific ECM components aimed at instructing human umbilical vein endothelial cells (HUVECs) and / or for creating and maintaining an endothelialized vascular network for up to 21 days. The disclosed systems and methods can be used to functionalize surfaces of high-resolution 3-D printed constructs of synthetic origins with cell-specific ECM materials, by adopting a photo-reactive chemistry, including the ability to functionalize a complex multi-planar architecture of micron-scale channels, with extracellular matrix proteins of interest. The disclosed systems and methods enable the use of high resolution, high-fidelity printing methodologies (for example, via 3D Systems DLP printers FS10, FS20, and equivalents) in connection with methodologies that also enable cell-adhesion, thereby allowing for the generation of human-relevant data. When bioconjugated with the appropriate native ECM proteins, these vascularized tissues have applications that can include drug delivery, immunooncology, and in vitro modeling of drug-induced tissue injury, among other uses.

[0500] At a high level, the disclosed methods and systems may include providing a hydrogel or polymer chip, scaffold or vasculature network, coating the scaffold with ECM formulations via procedures to coat internal and external surfaces thereof, irradiating the coated surfaces to covalently bond the coating to the surfaces, seeding the coated surfaces with one or more bioactive coatings (e g., acrylates), and irradiating the coated surfaces to enable functionalization of live cells thereon. Various assays can then be performed on the platform to mimic human in vivo conditions.Attorney Docket No.: PCT.1316

[0501] The present disclosed methods can be used for functionalizing and characterizing the inner walls of a complex, high-resolution, multi-planar vascular network. Fig. 73 shows a method 750 of coating surfaces of a complex, high-resolution, multi-planar vascular network, such that live cells may be functionalized thereon. At step 734, the method 750 may include printing the organ chip / scaffold / vasculature network as described herein. At step 736, the method 750 may include performing a pre-wash on the organ chip I scaffold / vasculature network. In some embodiments, step 736 may include briefly immersing (that is, for about 5 to about 10 seconds) on the organ chip / scaffold / vasculature network in a tub containing autoclaved Milli-Q at 37°C to remove any excess bioink present on the print platform and on the surface of any printed samples. In some embodiments, step 736 may be repeated one or more times. In some embodiments, step 736 may also include immersing any print samples, chips, etc. in autoclaved Milli-Q filled containers covered with foil to prevent undesired polymerization of unreacted acrylate groups due to stray light. In some embodiments, step 736 may include washing the chip / samples in autoclaved Milli-Q for 1 day and sterile DPBS for 1 day with media changes at least 3 times per day. At step 738, the method 750 may include adding precursor(s) to the organ chip / scaffold / vasculature network. In some embodiments, the precursor(s) may include acrylated-PEGlk-NHS (i.e., Acr-PEG-NHS). In some embodiments, the precursor(s) may include from about 1.5 mg / mL to about 4.0 mg / mL (for example, from about 2.0 mg / mL to about 3.5 mg / mL, from about 2.0 mg / mL to about 3.0 mg / mL, from about 2.25 mg / mL to about 2.75 mg / mL, and / or about 2.5 mg / mL) of Acr-PEG-NHS to 50 mM NaHCO3. In some embodiments, precursor(s) may include from about 0.5% to about 3% of a water soluble, cytocompatible. photoinitiator (e.g., LAP).

[0502] Referring still to Fig. 73, at step 740, the method 750 may include irradiating the coated organ chip / scaffold / vasculature network / sample. In some embodiments, irradiating may include using an LED activated at a wavelength of 405 nm for about 5 to 15 minutes (i.e., about 8-12 minutes, e.g., about 10 minutes). In some embodiments, irradiating may include doing so for a sufficient period of time to covalently tether the precursor (e.g., Acr-PEG-NHS) to the surface of the organ chip / scaffold / vasculature network / sample. At step 742, the method 750 may include removing excess cells. In some embodiments, step 742 may include removing any excess Acr-PEGlk-NHS solution. At step 744, the method 750 may include preparing stock of one or more bioactive coatings (e.g., acrylates). In some embodiments, bioactive coatings (acrylates) may include GelMa, ColMA, collagen type 1 and / or acrylate-PEG. In some embodiments, preparation of ColMA stock may include: providing collagen methacrylate; making a 3 mg / mL stock usingAttorney Docket No.: PCT.1316100 mg of lyophilized ColMA and 33.3 mL of 20 mM acetic acid; manually shaking the mixture for about 2 hours (or until all of the collagen has dissolved and / or is no longer visible to the human eye); placing the mixture on ice intermittently to ensure the mixture does not exceed 4°C; and placing the mixture in a refrigerator overnight to allow any bubbles to dissipate. In some embodiments, ColMA stock may include a concentration in a range from 50 pg / mL - 200 pg / mL.

[0503] Referring still to Fig. 73, at step 746, the method 750 may include coating organ chip / scaffold / vasculature network with one or more bioactive coatings (e.g., acrylates). In some embodiments used in connection with bioconjugation of the surfaces with collagen type I, coating 746 may include adding about 10, 20, 100 or 200 pg / mL collagen-I to the surfaces and incubating for 2 hours at 37°C followed by hydrating with water and sealing to prevent dehydration. In some embodiments used in connection with bioconjugation of the surfaces with ColMA, coating 746 may include adding a precursor solution containing ColMA 50, 100, or 250 pg / mL and from about 0.5% to about 3% of a water soluble, cytocompatible, photoinitiator (e.g., LAP) to the surfaces and incubating for 2 hours over ice, following by washing twice with PBS and irradiating at 405 nm for about 3-7 minutes (e.g., about 5 minutes). In some embodiments used in connection with bioconjugation of the surfaces with GelMA, coating 746 may include adding about 150 pL of precursor containing GelMA (in a wight percent range from about 2.5% to about 5%) and from about 0.5% to about 3% of a water soluble, cytocompatible, photoinitiator (e.g., LAP) to the surfaces and incubating for about 20-35 minutes (e.g., from about 25 to about 30 minutes) at 37°C, followed by hydrating and irradiating at 405 nm for 10 minutes (e.g., thereby covalently tethering GelMA to the hydrogel’s surface via unreacted -NH2in the GelMA backbone and acrylate functionalization) as described herein. At step 748, the method 750 may include allowing bioconjugating of the bioactive coating to occur on the organ chip / scaffold / vasculature network surfaces. At step 752, the method 750 may include washing, sterilizing and / or incubating the bioconjugated organ chip / scaffold / vasculature network. In some embodiments, sterilization may include use of an ultraviolet light source activated at a wavelength of 254 nm for about 2 hours. In some embodiments, washing may include washing one or more times with PBS at a temperature range from about 37°C to about 40°C. In some embodiments, incubation may include incubation for 2 hours at about 37°C. At step 754, the method 750 may include seeding and / or perfusing the surfaces and / or channels / flow passages / vasculatures / lumens with live cells, as described herein. At step 756, the method 750 may include selectively adjusting the in vitro conditions of the organ chip / scaffold / vasculature network according to the requirements of the assay being performed.Attorney Docket No.: PCT.1316

[0504] In some embodiments, cells (e.g., HepG2) attach to a functionalized surface (e.g., a surface having a thin coating of an acrylate such as GelMA, ColMA, acrylate-PEG) (as shown in Figs. 52B, 53B, 53C, 54B, and 54C)

[0505] In some embodiments cells attach and migrate on a functionalized surface. In some embodiments, cells attach and divide (e.g., multiply) on a functionalized surface. In some embodiments, cells remain viable on a functionalized surface for at least 1 day, at least 3 days, at least 6 days, at least 7 days, at least 9 days, at least 12 days, at least 15 days, or at least 18 days.

[0506] In some embodiments, an interstitial space may for example, contain cells attached to a functionalized surface according to aspects of the present embodiments. For example, interstitial spaces, vasculatures, and other scaffolds or surfaces described herein may contain a functionalized surface with cells attached thereto.

[0507] In some embodiments cells attach to a functionalized surface of a vascular channel (e.g., lumen). In some embodiments, one cell (or a population of cells) attach to a functionalized surface of the interstitial space and a second cell (or second population of cells) attach to a functionalized surface of the vascular channel. In some embodiments, a cell can migrate across a functionalized surface. For example, a cell can migrate across a functionalized surface in response to a stimulus. Stationary cells or cells in migration can be visualized using microscopy and / or detection methods familiar to a skilled artisan.

[0508] In some embodiments, a medium (e.g., solution, buffer) can flow (e.g., be perfused) across a functionalized surface. For example, perfusing media across a layer of cells maintains viability of said cells. In some embodiments, a cell attached to a functionalized surface is selected from a group consisting of an endothelial cell, a biliary endothelial cell, a cholangiocyte, a liver parenchymal cell, a hepatocyte (HC), a primary human hepatocyte (PHH), a heptic stellate cell (HSCs), a Kupffer cell (KC), a liver sinusoidal endothelial cell (LSEC), a mucous cell, a parietal cell, a chief cell, an endocrine cell (e.g.. a G cell, a D cell, a enterochromaffin cell, a EC-like cell, aX / A cell), a columnar epithelial cell, a cardiac fibroblast (CF), a cardiomyocyte, a smooth muscle cell, an enterocyte, a goblet cell, a Paneth cell, a stem cell, a neuron, a glia, a keratinocyte, a melanocyte, a Merkel cell, a Langerhan cell, a germ cell, a stromal cell, a seminiferous tubule, a Leydig cell, a tubule epithelial cell, a macula densa cell, a glomerular endothelial cell, a podocyte, a mesangial cell, a parietal epithelial cell, an immortalized cell (e.g. a3T3 cell, a A549 cell, aHeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, a Jurkat cell, a OK cell, a Ptk2 cell, a Vero cell), a patient-derived cell (e.g., a tumor cell), a T cell, a peripheral blood mononuclear cellAttorney Docket No.: PCT.1316(PBMC), and / or an induced pluripotent stem cell (iPSC). In some embodiments, one cell type is attached to a functionalized surface. In some embodiments, multiple cell types are attached to a functionalized surface.

[0509] In some embodiments, an AN14 hydrogel containing polyethylene glycol diacrylate and gelatin methacrylate includes amino groups on a surface of the AN14 hydrogel that are capable of binding to a bioactive coating. Fig. 50A is a diagram of an AN14 hydrogel having at least one amino group on a surface of the hydrogel. In some embodiments, the AN 14 hydrogel comprises less than 50% gelatin methacrylate, less than 40% gelatin methacrylate, less than 30% gelatin methacrylate, less than 25% gelatin methacrylate, less than 20% gelatin methacrylate, less than 15% gelatin methacrylate, less than 10% gelatin methacrylate, less than 5% gelatin methacrylate, or less than 3% gelatin methacrylate. In some embodiments, a functionalized surface is a glass surface that includes a coating of gelatin methacrylate (" Gel A") having amino groups capable of binding to a bioactive coating. Fig. 50B is a diagram of a glass surface having a thin coating composed of 2.5% volume percent GelMA. The GelMA glass thin coating includes abundant amino groups on its surface. The GelMA glass thin coating is capable of intermolecular crosslinking with denatured collagen. In some embodiments, a GelMA glass thin coating has a degree of functionalization (DOF) of about 50%. In some embodiments, a functionalized surface is a glass surface that includes a coating of collagen methacrylate (“ColMA”) having amino groups capable of binding to a bioactive coating. Fig. 50C is a diagram of a glass surface having a thin coating of 250 pg / ml ColMA. The ColMA glass thin coating includes amino groups on its surface and is capable of intermolecular crosslinking with fibrils. In some embodiments, the ColMA glass thin coating has a DOF of about 20%.

[0510] In some embodiments, an acrylate-based hydrogel surface (e.g., a functionalized surface having a thin coating of an acrylate such as GelMA, ColMA, and / or polyethylene glycol diacrylate (PEGDA)) is capable of binding to a bioactive coating within an extracellular matrix. In Fig.51A, a covalent linker (e.g., GelMA, ColMA acrylate-PEG) incorporating an amine-reactive functional group (e.g., NHS) is tethered to a base polymer. The covalent linker is capable of reacting with an extracellular matrix (“ECM”) protein (e.g., collagen). In some embodiments, the covalent linker reacts with an ECM protein at a pH of about 7-9. In this reaction, the protein forms a covalent bond with the covalent linker by replacing the amine-reactive functional group on the covalent linker. Fig. 51B shows the ECM covalently bound to a base polymer via the covalent linker.Attorney Docket No.: PCT.1316

[0511] Embodiments of the present disclosure may bind cells to various surfaces through photoreactive chemistry. Fig. 52A shows a diagram of an AN14 hydrogel interacting with a bioactive coating. The bioactive coating is capable of binding to the amino groups on the hydrogel.Figs. 52B, 53B, 53C, 54B, and 54C depict fluorescence images of liver cancer cells (HepG2) attached to various surfaces. HepG2 are imaged using fluorescence microscopy. HepG2 nuclei are stained in blue using DAPI and F-actin fdaments are stained in green using Phalloidin. HepG2 cells are imaged on day 1 after seeding on the surface. Fig. 52B depicts a fluorescence image of liver cancer cells attached to an AN 14 hydrogel of the present disclosure through a bioactive coating. The fluorescence visible in Fig. 52B shows the AN14 hydrogel is capable of some cell attachment. In Fig.53A, a glass surface is functionalized with a linker comprising an acrylate (e.g., GelMA, ColMA, polyethylene glycol diacrylate (PEGDA), etc.) having a silyl group. The silyl group of the acrylate is bound to the glass surface. The acrylate is capable of binding to a bioactive coating, thereby tethering the liver cancer cells to the functionalized glass surface via photoreactive chemistry. FIG. 58 illustrates the glass surface of 53A after functionalization. Fig. 53B shows a 2.5% GelMA glass thin coating having moderate fluorescence, indicating it is capable of moderate cell attachment. Fig. 53C shows a 250 pg / mL ColMA glass thin coating having moderate to high fluorescence, indicating a capability for moderate to high cell attachment. In Fig. 54A, a base polymer is functionalized with an acrylate (e.g., acrylate-PEGlk-NHS) that is covalently bound to a bioactive coating via photocrosslinking chemistry. In some embodiments, the base polymer is an AN14 hydrogel. In some embodiments, the bioactive coating is or comprises collagen ECM proteins. Fig. 54B shows the fluorescence of liver cancer cells attached to the functionalized base polymer at a concentration of 20 pg / ml of collagen, the attached liver cells having high fluorescence. Fig.54C shows the fluorescence of liver cancer cells attached to the functionalized base polymer at a concentration of 200 pg / ml of collagen, the atached liver cells having high fluorescence. Figs. 54B and 54C indicate that a functionalized base polymer of the present disclosure is capable of high cell atachment.Coating Solutions & Formulations

[0512] According to aspects of the present disclosure, bioactive coatings may be used to encourage cell growth and binding to scaffold / vasculature surfaces. In some embodiments, coating solutions and formulations may include collagen type I, collagen type IV, fibronectin, and / or DPBS (IX) (that is, Dulbecco’s Phosphate Buffered Saline (DPBS) IX, or equivalent PBS).Attorney Docket No.: PCT.1316In some embodiments, coating solutions and formulations may include different relative amounts of these constituents depending on if the surface being coated is two-dimensional (2D) or includes a three-dimensional geometry (for example, 3D vasculatures and / or other three-dimensional scaffolds). Without wishing to be bound by theory, it is contemplated that a higher relative volume content of fibronectin may be beneficial when coating three-dimensional geometries (i.e., as compared to two-dimensional geometries) in order to promote binding and / or adhesion of bioactive cells with the surface, scaffold, and / or geometry being coated.

[0513] Table 1 below shows nominal volume per 1000 pL and corresponding volume percents for each of the four coating constituents (collagen type I, collagen type IV, fibronectin, and / or DPBS (IX)) for 2D surfaces and 3D surfaces / geometries. In some embodiments, the coatings illustrated below may be particularly well-suited for helping human umbilical vein endothelial cells (HUVECs) to bond to hydrogel scaffolds / surfaces. As can be seen in Table 1, fibronectin is present in a much higher volume percent in coatings used for 3D surfaces I geometries than in coatings used for 2D surfaces.Table 1 - Coating constituents for 2D and 3D surfaces2D surfaces 3D geometriesVol (pL) Volume Vol (pL) Volume / 1000 pL Percent / 1000 pL PercentCol l 4.2 0.42% 47.3 4.73% Col IV 88.0 8.80% 268.4 26.84% Fibronectin 44.0 4.40% 671.1 67.11%DPBS (IX) 863.8 86.38% 13.2 1.32%

[0514] Tables 2 and 3 below show nominal, approximate minimum and approximate maximum volume percents for each of the four coating constituents, for 2D surface coatings and 3D surface coatings respectively. Accordingly, bioactive coatings of the present disclosure may include collagen ty pe I in a range from about 0.2% to about 8.0%, collagen ty pe IV in a range from about 4% to about 40%, fibronectin in a range from about 2% to about 90%, and DPBS (IX) in a range from about 0.5% to about 95%. Each of the constituents shown in Tables 1-3 may be present at concentrations equal to, or approximately equal to, their respective stock concentrations, prior to mixing with the other constituents.Attorney Docket No.: PCT.1316Table 2 - Nominal, minimum and maximum coating constituents for 2D surfaces 2D surfacesVol (pL) Nom. Min Max / 1000 pL Vol. % Vol. % Vol. %Col l 4.2 0.42% 0.2% 0.6%Col IV 88.0 8.80% 4.0% 13.0% Fibronectin 44.0 4.40% 2.0% 7.0%DPBS (IX) 863.8 86.38% 50.0% 95.0%Table 3 - Nominal, minimum and maximum coating constituents for 3D surfaces3D geometriesVol (pL) Nom. Min Max / 1000 pL Vol. % Vol. % Vol. %Col l 47.3 4.73% 2.3% 8.0%Col IV 268.4 26.84% 12.0% 40.0% Fibronectin 671.1 67.11% 35.0% 90.0%DPBS (IX) 13.2 1.32% 0.5% 3.0%Preparation of Materials

[0515] Bis-NHS-PEG: The preparation may begin with dissolving stock material in an alkaline buffer, followed by applying a sterile filter. Afterwards, the solution is added to the hydrogel well and allowed a period of five minutes for adsorption to occur. Subsequently, the excess is removed, and the solution is washed with DPBS. Next steps include a collagen-I coating, followed by an incubation in 37°C for two hours. Lastly, the solution is washed with DPBS and Cell seeding is performed.

[0516] Acr-PEGlk-NHS: The preparation may begin with dissolving stock material in an alkaline buffer and adding PI, followed by applying a sterile filter. Afterwards, the solution is added to the hydrogel well and allowed a period of five minutes for adsorption to occur. Subsequently, the excess is removed, and the solution is washed with DPBS. Next steps include aAttorney Docket No.: PCT.1316collagen-I coating, followed by an incubation at 37°C for two hours. Lastly, the solution is washed with DPBS and cell seeding is performed.

[0517] ColMA: The preparation may begin with a 3mg / mL stock in sterile acetic acid. Next, a dilution is made using sterile DPBS on ice. Afterwards, PI is added and gently shaken until dissolved, followed by a sterile filter. Subsequently, samples are added (on ice) and covered with foil. Next steps include an incubation in 37°C for two hours, followed by irradiating using a 405 nm LED box for a period of 10 minutes. Lastly, the solution washed with DPBS and cell seeding is performed.

[0518] GelMA: The preparation may begin with a 20% sterile stock, filtered in PBS. Next, a dilution is made using sterile DPBS, and PI is added. Afterwards, a sterile filter is applied, followed by placing back in warming bath. Subsequently, samples are added and covered with foil. Next step includes irradiating using a 405 nm LED box for a period of 10 minutes. Lastly, the solution is washed with DPBS and cell seeding is performed.

[0519] PDA: The preparation may begin with dissolving DH at basic pH, followed by a sterile filter. Subsequently, inner chamber is coated. Lastly, PDA is washed with PBS and cell seeding is performed.

[0520] TMSPMA: The preparation may begin with glass bottom plates washed in a detergent and sonicated in Milli-Q. Next steps include plasma activation, followed by addition of HCL / H2O2. Afterwards, toluene is added to the solution and treated for a period of two hours. Subsequently, the solution is w ashed in EtOH, dried in a fume hood, and stored at Store at 4°C until use, when desired amounts of ColMA / GelMA and PI are added. Next step includes irradiating using a 405 nm LED box for a period of 10 minutes, followed by irradiation at 254nm to sterilize.Transendothelial migration (TEM)

[0521] Organ-on-a-chip platform is considered the most promising substitute for conventional animal model-based drug discovery approach in pharmaceutical industry. The endothelialization of tissue modeling to achieve a more in vivo-like organ and / or tissue structure has become more significant in the tissue engineering field, and therefore introduction of highly engineered endothelial layer to in vitro tissue modeling may render organ on-a-chip-based drug discovery7more desirable by the pharmaceutical industry. In addition, due to increased interest in immune therapy for cancer treatment, transendothelial migration (TEM) of immune cells to tumor tissue is extensively researched in academia and industry. Consequently, development of in vitro models that can realistically recapitulate TEM is highly desirable. Conventional microphysiologicalAttorney Docket No.: PCT.1316systems (MPS) for TEM studies exhibit limitations such as difficulty to apply to 3D tissue modeling, randomly distributed vasculatures resulting in poor connectivity and short culturing periods, as well as complicated fabrication methods. To overcome these challenges, a scalable fabrication method is disclosed for construction of MPS including in vitro 3D vascularized tissue structures interfaced with endothelial conduits for TEM studies.

[0522] In some embodiments, a full-scale vasculature microfluidic channel is constructed to recapitulate an in vivo vascularized tissue environment in a more reproducible manner. The vasculature microfluidic channel, for example, is superior to conventional angiogenesis -based methods which result in random vascularization. In some embodiments, the vasculature microfluidic channel includes micropores to maximize the interfacing area between an endothelial layer and epithelial and / or tumor tissue. In some embodiments, a covalent bonding-based biochemical surface modification approach is used to incorporate bio-compatible and bioactive molecules on a surface of vasculature channel and surface surrounding interstitial space, to achieve an in vzvo-like tissue environment in a physio-chemically stable manner.MPS model

[0523] In some embodiments, a MPS platform may be constructed using a highly biocompatible. O2-permeable and nutrient-permeable hydrogel via a simple and scalable 3D printing approach. In some embodiments, a MPS platform may include a micro-scaled interstitial space and a branched microfluidic channel. An interstitial space may be loaded with an ECM hydrogel to achieve 3D epithelial tissue and / or tumor tissue formation. A branched microfluidic channel may include an endothelial cell layer to form a vasculature lumen structure, through which cell culture media and / or immune cells may be introduced to mimic the in vivo circulation condition.

[0524] In some embodiments, surface of interstitial space and vasculature channel may be functionalized with protein-reactive chemical moiety to immobilize ECM hydrogel and ECM protein with robust physical stability. An interstitial space surface, for example, may incorporate an amine-reactive functional group (e.g., N-hydroxysuccinimidyl ester; NHS) via covalent bonding to allow mechanically stable immobilization of ECM hydrogels such as Matrigel and GelTrex. A vasculature channel, for example, may include an ECM protein (e.g., Collagen, Fibronectin and / or Gelatin)-reactive surface to promote the endothelial cell layering. An ECM protein-reactive surface may include NHS, poly-L-Lysine, thiolated gelatin, thiolated heparin, Thiolated hyaluronic acid and / or thiolated collagen.Attorney Docket No.: PCT.1316

[0525] In some embodiments, microscale pores may be introduced along a 3D microfluidic channel to facilitate a three-dimensional interface between the interstitial space and an endothelial layer. The micropores may serve as a conduit for the therapeutics or immune cells (for example, T-cells) to migrate through the endothelial cell layer and a soft hydrogel extracellular matrix (Matrigel) into the interstitial space containing tumor cells or tumoroids. The cells, however, cannot migrate through vasculature walls. The hydrogel material used for the vasculature walls selectively permeable to molecules lower than ~10kDa.

[0526] In some embodiments, the micropores may be monolithic with the rest of the vasculature, i.e., the same printing process and material properties is used, however, slightly different dosages may be implemented to print finer details. In some embodiments, the micropores may be printed using light-based DLP 3D printing technology, such as FS-series of bioprinters offered by 3D Systems, or BioNanoOne, a 2-photon 3D DLP technology offered by UpNano. In some embodiments, design with micropores may be printed using FS-10. The FS-10 may include a pixel size of 10 pm, and therefore provides a practical printing resolution of 30 pm. Conventional methods of introducing pores in a Transwell may include creation of a master mold.

[0527] In some embodiments, a 3D vasculature channel that is perfused with media via a syringe pump system, may allow epithelial tissue and endothelial cell layer to be cultured for longer time (e.g., at least 3 weeks) while maintaining high tissue functionality. An endothelialized vasculature may create a native barrier possessed by a blood vessel. Perfusion of media through this vessel provides nutrients and oxygen to the cells forming the barrier (for example, endothelial cells). Additionally, flowing immune cells through vasculature introduces an additional level of complexity to the MPS model wherein these cells need to permeate across the barrier, travel through the microporous channel to invade the tumor in the interstitial space.

[0528] In some embodiments, a MPS model may include one inlet and one outlet, allowing one therapeutic to be perfused through the entire system. In some embodiments, a MPS model may be modified for targeted delivery of therapeutics to localized regions.

[0529] In some embodiment, a MPS model includes a large-scale vasculature network with micropores connecting endothelial layer in a channel and epithelial (or tumor) tissues in interstitial space. Fig.47A is 2D image of a MPS platform structure 250 including an inlet 254, an outlet 256, a vasculature microfluidic channel 252, interstitial spaces 258 and micropores 260 connecting vasculature channel 252 and interstitial spaces 258. The MPS platform structure 250 may be expanded three-dimensionally into a chip organ 262 to recapitulate a 3D in vivo tissue, as shownAttorney Docket No.: PCT.1316in Fig. 47B. The chip organ 262 may include an inlet 254, an outlet 256, and vasculature 252. A closeup area 264 shows the micropores 260 connecting vasculature channel 252 and interstitial spaces 258. In some embodiments, the micropores 260 may include a diameter of about 40 pm to about 60 pm. In some embodiments, the number of micropores 260 is directly proportional to the surface area of the vasculature channel 252. For example, a channel with a surface area of X cm2may include X number of micropores 260 (for example, one micropore per unit area). The length of the micropores 260 is determined by the thickness of a vasculature channel wall. In some embodiments, the micropores 260 may include a length of 100 pm. In some embodiments, the micropores 260 may include a length of 150 pm. In some embodiments, the micropores 260 may include a length of 200 pm.

[0530] In some embodiments, a MPS platform enables evaluation of key functions of immune cells including activation of immune cells by an external agent, migration of immune cells from vasculature into a tissue compartment, and interaction of immune cells with a tissue. When immune cells (e.g., T-cells) are activated by an immunotherapeutic agent (e.g., a peptide, an antibody -based agent), the immune cells move into a tissue compartment (for example a tumor) and activities within interstitial space may be monitored. In some embodiments, a MPS platform may be used in development of cancer therapy. Cancer treatments, for example, focus on killing tumor cells (e.g., cancerous cells) without damaging the surrounding healthy cells. Immune cells, however, may exhibit exhaustion due to lack of nutrients and / or oxygen, and therefore become unable to kill tumor cell. A MPS platform may be used to evaluate the ability of activated immune cells in killing tumor cells. In some embodiments, a MPS platform may be used in development of gene therapies (for example, mRNA in lipid nanoparticles) that are drained by liver. A MPS platform may assess whether therapeutic agents reach a tissue of interest, and / or there is drainage from vasculature into other organs, and thus, enabling evaluation of delivery and efficacy of gene therapies. In some embodiments, a MPS platform may be used to assess safety of vascular delivery of therapeutic agents. Some therapeutic agents, not only kill surrounding healthy tissue of a tumor cell, but they may also kill vascular tissue cells during delivery. And therefore, requiring further assessments to ensure safe vascular delivery'.

[0531] In some embodiments, immune cells may be perfused through a vasculature and migrate into the tumor compartment in the interstitial space. Unattached or free immune cells may either be collected by washing the vasculature or by collecting the medium in the interstitial well. In some embodiments, immune cells are recruited by the tumor compartment, and therefore can be isolatedAttorney Docket No.: PCT.1316by digesting the Matrigel using digestion reagents, such as organoid harvesting solution and cell recovery solutions. In some embodiments, the immune cells are recruited to the endothelial cells lining the vasculature, and therefore may be dissociated from the hydrogel scaffold by perfusing dissociation reagents, such as trypsin and Accutase. The trypsinized cells may further be processed and analyzed for protein or RNA quantification.

[0532] In transendothelial migration experiments, the destination of immune cells is interstitial space where tumoroids and Matrigel are located. In some embodiments, a portion of migrated immune cells may escape the Matrigel layer, reaching the media. The immune cell fractions may be collected from the media, and the phenotype can be efficiently analyzed and classified by FACS. In some embodiments, migrated immune cell fractions may be collected by scraping Matrigel in the interstitial space.MPS construction

[0533] In some embodiments, MPS construction includes an ECM coating approach using a positively charged polymer. A covalent bonding (e.g., amide bonding)-based biochemical conjugation technique may allow stable bioconjugation of biomaterial (for example, ECMs and ECM-derived hydrogels) to the surface of MPS substrate. Fig. 62 is a flowchart of a construction method 700 of Endothelial / epithelial interfacing MPS model. At step 702, the method 700 may include a MPS substrate including vasculature microfluidic channel, micropores and an interstitial space. At step 704, the method 700 may include applying a first layer of a photo crosslinker (for example, by a SVA treatment) to functionalize the surface of MPS substrate through aminereactive chemical moieties. At step 706, the method 700 may include loading Matrigel and fluorescent beads into the interstitial space. The protein fraction of Matrigel may covalently adhere to the surface of MPS substrate which is activated by amine-reactive groups. The Matrigel may fill the micropores which renders the vasculature channel operational (i.e., the microfluidic channel is completed when micropores are closed). At step 708, the method 700 may include applying a second layer of a photo crosslinker (for example, through a SVA treatment) to the vasculature microfluidic channel to achieve a second surface modification (amine-reactive activation). At step 710, the method 700 may include immobilizing a positively charged and amine-rich polymer, for example, poly-L-Lysine (PLL) via covalent bonding (such as amide bond formation). At step 712, the method 700 may include promoting, by the immobilized PLL, the binding of ECM proteinsAttorney Docket No.: PCT.1316such as fibronectin via electrostatic interaction between positively charged PLL-surface and negatively charged fibronectin under physiological pH condition.

[0534] Fig.48A is a schematic of a construction method 270 of Endothelial / epithelial interfacing MPS model. At step 271, the method 270 may include a MPS substrate 280, vasculature microfluidic channel 252, micropores 260 and an interstitial space 258. At step 272, the method 270 may include applying a first layer of a photo crosslinker 282 (for example, by a SVA treatment) to functionalize the surface of MPS substrate 280 through amine-reactive chemical moieties. At step 273, the method 270 may include loading Matrigel 284 and fluorescent beads 286 into the interstitial space 258. The protein fraction of Matrigel 284 may covalently adhere to the surface of MPS substrate 280 which is activated by amine-reactive groups. The Matrigel 284 may fill the micropores 260 which renders the vasculature channel 252 operational (i.e., the microfluidic channel is completed when micropores are closed). At step 274. the method 270 may include applying a second layer of a photo crosslinker 288 (for example, through a SVA treatment) to the vasculature microfluidic channel 252 to achieve a second surface modification (amine-reactive activation). At step 275, the method 270 may include immobilizing a positively charged and amine-rich polymer, for example, Poly-L-Lysine (PLL) 290 via covalent bonding (such as amide bond formation). At step 276, the method 270 may include promoting, by the immobilized PLL 290 the binding of ECM proteins 292 such as fibronectin via electrostatic interaction between positively charged PLL-surface and negatively charged fibronectin under physiological pH condition.

[0535] Figs.48B-E are exemplary images of Matrigel, fibronectin, collagen-IV and ECM-coated transendothelialization MPS model, respectively. Images of Figs. 48B-E may include immunofluorescent staining results, confirming that Matrigel and ECMs proteins (fibronectin, collagen-IV) are immobilized at designated locations.

[0536] Figs. 48 F-G are exemplary images of vasculature channel-micropore-interstitial space connection. Figs. 48F-G may include vasculature channels 252, channel walls 294, micropores 260, and interstitial spaces 258. The Matrigel may be removed from the interstitial spaces 258 by pipetting. The micropores 260 may remain filled with Matrigel which allows a leakage-free ECM coating in vasculature channels 252.

[0537] In some embodiments, MPS construction includes an ECM coating approach using thiolated polymer. Fig.63 is a flowchart of a construction method 720 of an Endothelial / epithelial interfacing MPS model using thiolated polymer. At step 724, the method 720 may include a MPS substrate including vasculature microfluidic channel, micropores and an interstitial space. At stepAttorney Docket No.: PCT.1316724, the method 720 may include applying a first layer of a photo crosslinker (for example, by a SVA treatment) to functionalize the surface of MPS substrate through amine-reactive chemical moieties. At step 726, the method 720 may include loading Matrigel and fluorescent beads into the interstitial space. The protein fraction of Matrigel may covalently adhere to the surface of MPS substrate which is activated by amine-reactive groups. The Matrigel may fill the micropores which renders the vasculature channel operational (i.e., the microfluidic channel is completed when micropores are closed). At step 728, the method 720 may include addition of thiolated biopolymer (for example, thiolated gelatin and / or thiolated heparin) to the vasculature channel, to achieve a second surface modification. The added thiolated biopolymer may be covalently conjugated to the substrate, resulting in a Michael-ty pe addition between thiolated polymer and acry late of the substrate. At step 730, the method 720 may include promoting, by surface-conjugated biopolymer the binding of ECM proteins such as fibronectin via its biophysical affinity to ECMs.

[0538] Fig. 49A is a schematic of a construction method 300 of an Endothelial / epithelial interfacing MPS model using thiolated polymer. At step 301, the method 300 may include a MPS substrate 280, vasculature microfluidic channel 252, micropores 260 and an interstitial space 258. At step 302, the method 300 may include applying a first layer of a photo crosslinker 282 (for example, by a SVA treatment) to functionalize the surface of MPS substrate 280 through aminereactive chemical moieties. At step 303, the method 300 may include loading Matrigel 284 and fluorescent beads 286 into the interstitial space 258. The protein fraction of Matrigel 284 may covalently adhere to the surface of MPS substrate 280 which is activated by amine-reactive groups. The Matrigel 284 may fill the micropores 260 which renders the vasculature channel 252 operational (i.e., the microfluidic channel is completed when micropores are closed). At step 304, the method 300 may include addition of thiolated biopolymer 306 (for example, thiolated gelatin and / or thiolated heparin) to the vasculature channel 252, to achieve a second surface modification. The added thiolated biopolymer 306 may be covalently conjugated to the substrate, resulting in a Michael-type addition between thiolated polymer and acrylate of the substrate 280. At step 305, the method 300 may include promoting, by surface-conjugated biopolymer 306 the binding of ECM proteins 292 such as fibronectin via its biophysical affinity to ECMs.

[0539] Figs.49B-E are exemplary images of Matrigel, fibronectin, collagen-IV and ECM-coated transendothelialization MPS model, respectively. Images of Figs. 49B-E may include immunofluorescent staining results, confirming that Matrigel and ECMs proteins (fibronectin and collagen-IV) are immobilized at designated locations.Attorney Docket No.: PCT.1316

[0540] Various biocompatible hydrogel materials may be used to 3D print a MPS platform. In some embodiments, a hydrogel material may include PEGDA (Polyethylene Glycol Diacrylate), PEGMA (Polyethylene glycol methyl ether methacrylate), Acrylamide, Bis-acrylamide, Gelatin, Methacrylated gelatin, Thiolated gelatin, Thiolated collagen, Methacrylated collagen, Methacrylated chitosan, Thiolated chitosan, Methacrylated heparin, Thiolated Heparin, Methacry lated hyaluronic acid, Thiolated hyaluronic acid, Matrigel, Methacrylated alginate, Multiarm PEG-Maleimide, Multi-arm PEG-ThioL Multi-arm PEG-DBCO, Multi-arm PEG-N3, and Multi-arm PEG-Biotin with streptavidin.

[0541] Several 3D printing methods may be utilized to generate interconnected vasculature channel and interstitial space including widely distributed micropores. In some embodiments, 3D printing may include photopolymerization method based on Digital Light Processing (DLP) approach using basal material PEGDA, photoinitiator (e.g., Irgacure 2959 and Lithium phenyl-2,4,6-trimethylbenzoylphosphinate; LAP), and photoabsorber (e.g., tartrazine). In some embodiments, 3D printing may include Stereolithography (SLA)-based bioprinting, Extrusion bioprinting or Ink-jet bioprinting and both one photon and two photon DLP.

[0542] In some embodiments, a MPS substrate may include an acrylate-rich hydrogel, for example, a PEGDA-based acylate-rich hydrogel. In some embodiments, a bioconjugation approach to immobilize ECM proteins and / or gel-embedded tissues for a PEGDA-based acylate-rich hydrogel may include a combination of photo crosslinking and amine-targeting using Acrylate-PEG-NHS (SV A). In the presence of a photoinitiator, acrylate group of SVA may covalently adhere to a free acrylate group of a MPS substrate via photo crosslinking. Successively, NHS group which is a chemical moiety that interacts with primary amine group of a protein via amide bond formation, may be functionalized on a surface of MPS. Afterwards, ECM proteins and / or ECM-based hydrogel may be applied, and consequently immobilized on the surface of MPS. In some embodiments, photo crosslinking reagent may include Sulfo-SANPAH. Sulfo-NHS-Diazirine, and NHS-Diazirine.

[0543] In some embodiments, a MPS substrate may include a non -aery I ate hydrogel. In some embodiments, a non-acrylate hydrogel may contain primary amine groups, for example, a gelatinbased gel, a collagen-based gel, and a chitosan-based gel. In some embodiments, a homobifunctional amine-reactive crosslinker may be used for a hydrogel that contains primary amine groups. Homo-bifunctional amine-reactive crosslinkers contain two amine-reactive moieties including n-hydroxysuccinimidyl ester (NHS) and N-hydroxysulfosuccinimidyl ester (SulfoAttorney Docket No.: PCT.1316NHS), and an aldehyde group, enabling covalently crosslinking of amine-rich surface of MPS and ECM proteins via forming an amide bond (amine and NHS / Sulfo-NHS) or an imine bond (amine and aldehyde). Examples of homo-bifunctional amine-reactive crosslinkers include bis(sulfosuccinimidyl)suberate (BS3), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate) (DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and Glutaraldehyde. In some embodiments, a non-acrylate hydrogel may contain thiol groups. In some embodiments, a hetero-bifunctional maleimide reactive and amine-reactive crosslinker may be used for a hydrogel that contains thiol groups. Maleimide reactive groups may be conjugated to thiol groups on a surface of MPS and amine-reactive groups may interact with primary amine groups in ECM proteins. Examples of ahetero-bifunctional maleimide reactive and amine-reactive crosslinker may include Succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (Sulfo-SMCC), N-y-maleimidobutyryl-oxysuccinimide ester (GMBS), and N-y-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS).Interstitial Infill and Interstitial Space

[0544] The present disclosure teaches that, in some embodiments, an ecosystem that produces a platform for creating physical, synthetic models of an organ, a tissue, and / or a cell, via bioprinting of a scaffold comprises a bioprinted entity, vasculature, an interstitial infill, and / or an interstitial space.

[0545] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, the interstitial space may be a void wherein a cell in a fluid(s) and / or a polymer (e.g., a hydrogel) are seeded to create a tissue. In some such embodiments, the cell in a fluid(s) may be an endothelial cell optionally coupled to live tissues and / or a cell other than an endothelial cell.

[0546] In some embodiments, the interstitial infill may be a polymer anchor and / or structure (e.g., a hydrogel) that enables creation of a two-dimensional or three-dimensional tissue through stimulation of cell association. In some such embodiments, the cell in a fluid(s) may be an endothelial cell optionally coupled to live tissues and / or a cell other than an endothelial cell.

[0547] In some embodiments, the interstitial infill comprises an interstitial pattern that approximates the geometries of the cell and / or tissue(s) native format. In some embodiments, the interstitial pattern is selected from a fibrous and / or a spherical pattern.Attorney Docket No.: PCT.1316

[0548] In some embodiments, the interstitial pattern is a fibrous pattern. In some embodiments, the fibrous pattern may be modified according to different parameters (e.g., density, thickness, orientation).

[0549] The present disclosure teaches, that at least in some embodiments, the interstitial space is perfused with a coating moiety through a chamber. In some such embodiments, the coating moiety is a cell and / or plurality of cells, which may be the same or different. In further embodiments, the cell is an endothelial cell and a cell other than an endothelial cell. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is other than an endothelial cell.

[0550] In some embodiments, the interstitial pattern is a spherical pattern, which may comprise pores and / or windows. In some embodiments, the spherical pattern may be modified according to different parameters (e.g., size, wall thickness, and number of microspheres).Cartridge

[0551] In some embodiments, an assembly for modeling organ-on-a-chip may include a cartridge that enables high throughput along with imaging capabilities. A cartridge may include a 3D printed part (resin) that houses a scaffold and creates the microfluidic interface between the scaffold and a pump. Such cartridge may be used to model organ-on-a-chip using well plates, and handled by currently available robot systems. Fig. 41A illustrates examples of cartridges 90A and 90B, according to aspects of the present embodiments. Cartridge 90A may include a reservoir 41 A, an inlet 14A, an outlet 16A, and an access port 56A. Cartridge 90B may include a reservoir 41B, an inlet 14B, an outlet 16B, and an access port 56B. The inlets 14A, 14B and the outlets 16A, 16B may each include a hosebarb 15 which connects to a flexible hose and / or tube. Fig.41B illustrates exemplary cross sections of cartridges 90A and 90B, also show n in Fig. 41 A. Cartridge 90A may include well 92A, and internal channels 91 A fluidly connecting inlet 14A and outlet 16A to the well 92A. Cartridge 90B may include well 92B, chip slot 43, and internal channels 91B fluidly connecting inlet 14B and outlet 16B to the chip slot 43. Fig. 41C illustrates an assembly 95 for modeling organ-on-a-chip, according to aspects of the present embodiments. The platform 95 may include a well plate 96 and cartridges 90A and 90B positioned within wells 97 of the well plate 96.

[0552] Fig. 64 shows an example of a scaffold 70 used in cartridge 90B, according to aspects of the present embodiments.

[0553] Fig.65 illustrates an example of a setup 350 to fluidly couple cartridge 90A and cartridge 90B, according to aspects of the present embodiments. The output 16B of cartridge 90B may beAttorney Docket No.: PCT.1316fluidly connected to the input 14A of the cartridge 90A by a tube, while the output 16A of cartridge 90A may be fluidly connected to the input 14B of the cartridge 90B by a pump 93. The well 92 may be used to store media 352 for perfusion of a scaffold 70 while the cartridges 90A, 90B are seated within wells of a well plate. Fig. 66 illustrates an assembly 95 for modeling organ-on-a-chip, according to aspects of the present embodiments. The assembly 95 may include a well plate 96 and cartridges 90A and 90B positioned within wells 97 of the well plate 96. The output 16B of cartridge 90B may be fluidly connected to the input 14A of the cartridge 90A by a tube 94, while the output 16A of cartridge 90A may be fluidly connected to the input 14A of the cartridge 90A through a pump 93.

[0554] In some embodiments, a single cartridge may include two reservoirs to house and interface the microfluidic flow to one or two scaffolds, depending on the configuration. Multiple of such cartridges can be assembled in a well plate, ensuring the scaffolds and tubing are equidistant / symmetrically distributed, which is highly desirable in a high-throughput system given the complexity and number of tubes. Fig. 42A illustrates an example of a cartridge 100, according to aspects of the present embodiments. Cartridge 100 may include reservoirs 41A, 41B, an inlet 14, and an outlet 16. Each inlet 14 and outlet 16 may include a hosebarb 15 which connects to a flexible hose and / or tube. Fig.42D illustrates an exemplary view of a cartridge 100 including wells 110 and 112 within reservoirs 41 A and 41B, respectively. The well 110 may include a volume of 2358.16 mm3and a surface area of 7.21 cm2. The well 112 may include a volume of 4965.6 mm3and a surface area of 13.48 cm2.

[0555] Fig 42C illustrates an exemplary view of a cartridge 100 including internal channels 104, 106 and 108. The internal channel 104 may fluidly connect the inlet 14 to reservoir 41A, the internal channel 106 may fluidly connect reservoir 41 A to reservoir 41B, and the internal channel 108 may fluidly connect reservoir 41B to the outlet 16. The internal channel 104 may include a volume of 19.94 mm3and a surface area of 1.05 cm2. The internal channel 106 may include a volume of 23.41 mm3and a surface area of 1.22 cm2. The internal channel 108 may include a volume of 45.39 mm3and a surface area of 2.34 cm2. Each of the internal channels 104 and 106 may split into two sub-channels 105 inside reservoir 41 A, forming a bifurcation (e.g., fork shape). As a result, the microfluidic interface between the cartridge 100 and a scaffold is established through four sub-channels 105. Each sub-channel 105 may be connected to a separate fluidic channel, allowing for individual control. In some embodiments, the division of internal channels 104, 106 into four sub-channels 105 may enable the use of scaffolds with high vasculature density.Attorney Docket No.: PCT.1316This is achieved by partitioning the flow at the cartridge level, prior to interfacing with the scaffold, rather than within the scaffold itself. In such cases, creating flow bifurcation within the scaffold may not be optimal due to limited bulk gel volume. In some embodiments, the division of internal channels 104, 106 into four sub-channels 105 may be used in scaffolds that are perfused with two different types of media. In some embodiments, the division of internal channels 104, 106 into four sub-channels 105 may be used to collect by-products of cell function, such as bile. Since the reservoir 41B may be used to store media, the internal channels 106, 108 may not split as they act solely as media inlet or outlet.

[0556] Fig. 67A illustrates an example of a cartridge 100, according to aspects of the present embodiments. Cartridge 100 may include reservoirs 41 A and 41B. The reservoir 41A may include a chip slot 43, and four hosebarbs 107. Fig. 67B illustrates an example of a setup 360 to fluidly couple reservoirs 41 A and 41B of a cartridge 100, according to aspects of the present embodiments. A scaffold 70 may be inserted into the bottom of reservoir 41 A, and subsequently cartridge 100 is placed into two wells of a well plate 96. The glass bottom 363 of the well plate 96 compresses the scaffold 70 against the chip slot 40, ensuring a microfluidic seal at the scaffold-to-cartridge interface. A set of O-rings 144 creates a seal between the cartridge 100 and the well plate 96, preventing contaminants from entering and securing the cartridge 100 in place. A sealed lid 362 with O-rings 144 and air filters is used to seal the cartridge 100, preventing the ingress of contaminants while allowing gas exchange. The well 112 of reservoir 41 B may be used to store media 352 for perfusion of the scaffold 70 within the reservoir 41 A. Closeup 364 shows the interface between the scaffold 70 and the hosebarb 117, and the direction of flow 366 of media 352 from the cartridge 100 into the scaffold 70.

[0557] Fig.68A illustrates an example of a setup 370 for a recirculatory flow through a cartridge 100, according to aspects of the present embodiments. The setup 370 may include a cartridge 100 and a peristaltic pump 372. Fig. 68B illustrates an example of a setup 374 for a bi-directional flow through a cartridge 100, according to aspects of the present embodiments. The setup 374 may include the cartridge 100 with a capped outlet 378 and a syringe pump 376.

[0558] Fig. 42B illustrates an assembly 102 for modeling organ-on-a-chip, according to aspects of the present embodiments. The assembly 102 may include a well plate 96 and a cartridge 100 positioned within wells 97 of the well plate 96. Fig. 69 illustrates an assembly 102 for modeling organ-on-a-chip, according to aspects of the present embodiments. The assembly 102 may include a well plate 96 and a plurality of cartridges 100 positioned within wells 97 of the well plate 96.Attorney Docket No.: PCT.1316

[0559] Fig. 42E illustrates an exemplary diagram 120 of a cartridge 100, according to aspects of the present embodiments. The diagram 120 may include a top view 140, a bottom view 122, a back view 134, a side view 136. and a front view 138. The top view 140 may include access ports 56A, 56B. Section A-A 142 corresponds to a cross section at line A-A 141 of the top view 140. The Section A-A 142 may include reservoirs 41 A, 41B, the wells 110 and 112, the internal channels 104, 106 and 108, and O-ring slots 144. The chip slot 43 may include a thickness of 4.5 mm. The bottom view 122 may include the well 110 and the chip slot 43. Section B-B 124 corresponds to a cross section at line B-B 123 of the bottom view 122. The Section B-B 124 may include two hosebarbs 107. A close-up of Section B-B may include a channel 104 which splits into two channels 105. The channel 104 may include a diameter of 0.8 mm and the channels 105 may include a diameter of 0.5 mm. The back view 134 may include reservoir 41B. The reservoir 41B may include a height 135 of 24.6 mm and a diameter 137 of 25.2 mm. The access port 56B of reservoir 41B may include a diameter 139 of 21.9 mm. The side view 136 may include a total cartridge length 143 of 68.0 mm and a cover length 145 of 44.7 mm. The front view 138 may include reservoir 41A, inlet 14, outlet 16 and hosebarbs 15. The hosebarb 15 may include a diameter of 1 / 16 inches.

[0560] Fig. 43 shows printed examples of a cartridge 100, according to aspects of the present embodiments. The cartridge 100 may be 3D printed using a metal. A metallic cartridge 100 may exhibit durability', heat resistance, low drug sorption and reusability. For example, a metallic cartridge 100 may be autoclaved for maintaining cleanliness and / or sterility. In some embodiments, the cartridge 100 may be 3D printed using Stainless Steel 316L. Stainless Steel 316L may provide biocompatibility, corrosion resistance, and mechanical properties which enable the use of the cartridge 100 in Cytotoxicity and absorption studies. A close-up of a selected area 150 of the reservoir 41 A may include chip slot 43 and hosebarbs 107. In some embodiments, the cartridge may also and / or instead be coated withL‘parylene’?to enable low absorption.

[0561] Fig. 44A illustrates an example of a cartridge 160, according to aspects of the present embodiments. The cartridge 160 may include two reservoirs 41A, 41B, two inlets 14A,14B, and two outlets 16A, 16B. The internal channel 161A may fluidly connect the inlet 14A to the reservoir 41A, and the internal channel 163A may fluidly connect reservoir 41A to the outlet 16A. The internal channel 161B may fluidly connect the inlet 14B to the reservoir 41B, and the internal channel 163B may fluidly connect reservoir 41B to the outlet 16B. The internal channels 162A and 162B provide internal interfaces inside reservoirs 41A and 41B, respectively. Fig. 70 illustrates anAttorney Docket No.: PCT.1316exemplary cross section of a cartridges 160, according to aspects of the present embodiments. The cartridge 160 may include two reservoirs 41 A, 41B, two chip slots 43A, 43B, and two wells 164A, 164B. The wells 164A, 164B, located above the chip slots 43 A, 43B. may be used to store media. The internal channel 161 A may receive the media from a pump outlet and direct it to the chip slot 43 A where a scaffold can be inserted. The media may travel through the scaffold’s vasculature and subsequently directed the well 164 A by the internal channel 162 A. The internal channel 163 A may direct the media from well 164A to the pump inlet which feeds it back into internal channel 161 A, thereby completing a perfusion cycle. The internal channel 161B may receive the media from a pump outlet and direct it to the chip slot 43B where a scaffold can be inserted. The media may travel through the scaffold’s vasculature and subsequently directed the well 164B by the internal channel 162B. The internal channel 163B may direct the media from well 164B to the pump inlet which feeds it back into internal channel 161B, thereby completing a perfusion cycle.

[0562] Fig. 44B illustrates an assembly 165 for modeling organ-on-a-chip, according to aspects of the present embodiments. The platform 165 may include a well plate 98 and one or more cartridges 160 positioned within wells 99 of the well plate 98.

[0563] Fig. 45 illustrates an imaging setup 166 for modeling organ-on-a-chip, according to aspects of the present embodiments. The imaging setup 166 may include one or more imaging inserts 168, and a well plate 98. The imaging inserts 198 may secure and / or set the orientation of the scaffolds. The imaging setup 166 setup may enhance the efficiency of imaging process, allowing for a higher throughput. According to aspects of the present embodiments, imaging setup 166 may also be used to extract samples (e.g., from the reservoir and / or interstitial space) to enable downstream testing.

[0564] Fig. 71 illustrates exemplary scaffolds 70A, 70B to be used in a cartridge, according to aspects of the present embodiments. The scaffold 70A may include a vasculature 60, a vasculature inlet 48 and a vasculature outlet 52. The scaffold 70A, for example, includes a simpler design where one inlet and one outlet is sufficient. The scaffold 70A may be used inside a cartridge 90B. The scaffold 70B may include two vasculatures 60 A, 60B, two vasculature inlets 48A, 48B and two vasculature outlets 52A, 52B. The scaffold 70B, for example, may be used in complex designs, e g., high vasculature density, where two inlets and two outlets are desirable. Additionally, the scaffold 70B may be perfused with two different types of media or used to collect by-products of cell function, such as bile. The scaffold 70B may be used inside a cartridge 100 or a cartridge 160.Attorney Docket No.: PCT.1316

[0565] In some embodiments, a cartridge may not include any reservoirs, resulting in a shorter perfusion path to a scaffold. Such cartridge, for example, may be used for endothelial experiments.Fig. 46A illustrates an example of a cartridge 170. according to aspects of the present embodiments. The cartridge 170 may be used to perfuse a single scaffold. Fig. 46B illustrates an example of a cartridge 180, according to aspects of the present embodiments. The cartridge 180 may be used to perfuse three scaffolds. Fig. 46C illustrates an example of a cartridge 190, according to aspects of the present embodiments. The cartridge 190 may be used to perfuse two scaffolds. Fig.46D illustrates an assembly 200 for modeling organ-on-a-chip, according to aspects of the present embodiments. The assembly 200 may include a cartridge 190, a case 202, and a holder 204. The cartridge 190 may be positioned on top of the case 202, such that three scaffolds are disposed within the case 202. The cartridge may be sealed by a lid to maintain scaffold hydration and sterility. The holder 204 may include a holder clip 206 to secure the cartridge 190 and case 202. The holder 204 may be used in a robot system. Figs.72A and 72B illustrate examples of a cartridge 190, according to aspects of the present embodiments. The cartridge 190 may be used to perfuse three scaffolds.Organ Systems

[0566] The present disclosure provides technologies to generate a model organ and / or tissue. In some aspects, the present disclosure provides methods to generate a model organ and / or tissue that includes a bioprinted entity, comprising a polymer (e.g.. a hydrogel), and a cell associated with the bioprinted entity and / or seeded therein. In some embodiments, the model organ and / or tissue is one aspect of an ecosystem (e.g., organ-on-a-chip) which may be used for drug discovery and for assessing the effectiveness of various therapies. In some aspects, the model organ and / or tissue is optionally vascularized.

[0567] In some embodiments, the model organ and / or tissue is selected from an organ or tissue of the integumentary system, skeletal system, muscular system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory' system, digestive system, urinary' system, and / or reproductive system.

[0568] In some embodiments, the model organ and / or tissue is an organ or tissue of the integumentary system. In some embodiments, the model organ is selected from the group consisting of an epidermis, a dermis, a hypodermis, a gland, a hair, and a nail.Attorney Docket No.: PCT.1316

[0569] In some embodiments, the model organ and / or tissue is an organ or tissue of the skeletal system. In some embodiments, the model organ is selected from the group consisting of a bone, a cartilage, a ligament, and a tendon.

[0570] In some embodiments, the model organ and / or tissue is an organ or tissue of the muscular system. In some embodiments, the model organ is selected from the group consisting of a skeletal muscle, a smooth muscle, and a cardiac muscle.

[0571] In some embodiments, the model organ and / or tissue is an organ or tissue of the nervous system. In some embodiments, the model organ is selected from the group consisting of a brain, a spinal cord, and a nerve.

[0572] In some embodiments, the model organ and / or tissue is an organ or tissue of the endocrine system. In some embodiments, the model organ is selected from the group consisting of a hypothalamus, a pineal gland, a pituitary gland, a thyroid gland, a parathyroid gland, a thymus, an adrenal gland, and a pancreas.

[0573] In some embodiments, the model organ and / or tissue is an organ or tissue of the cardiovascular system. In some embodiments, the model organ is selected from the group consisting of an artery, a vein, a blood vessel, a heart, and a lung.

[0574] In some embodiments, the model organ and / or tissue is an organ or tissue of the lymphatic system. In some embodiments, the model organ is selected from the group consisting of bone marrow, a spleen, a thymus, a lymph node, and a lymphatic vessel.

[0575] In some embodiments, the model organ and / or tissue is an organ or tissue of the respiratory system. In some embodiments, the model organ is selected from a lung, a nose, and a trachea.

[0576] In some embodiments, the model organ and / or tissue is an organ or tissue of the digestive system. In some such embodiments, the model organ is selected from a stomach, a gallbladder, a liver, a small intestine, a large intestine, a rectum, and an esophagus.

[0577] In some embodiments, the model organ and / or tissue is an organ or tissue of the urinary system. In some embodiments, the model organ is selected form a kidney and a bladder.

[0578] In some embodiments, the model organ and / or tissue is an organ or tissue of the reproductive system. In some embodiments, the model organ is selected from an ovary, a fallopian tube, a uterus, a cervix, a vagina, a prostate, and a teste.

[0579] In some embodiments, the model organ and / or tissue is a tumor.Attorney Docket No.: PCT.1316

[0580] In some embodiments, the model organ and / or tissue as described above and herein is patient-derived.

[0581] In some embodiments, the model organ and / or tissue as described above and herein is diseased or healthy. In some embodiments, the model organ and / or tissue as described above and herein is diseased. In some embodiments, the model organ and / or tissue as described above and herein is healthy.

[0582] In some embodiments, the model organ and / or tissue as described above and herein is an organ-on-a-chip.

[0583] In some aspects, the present disclosure provides methods to generate a model organ and / or tissue.Liver System

[0584] In some embodiments, the model organ is a liver. In some aspects, a model liver comprises a cell chamber (to metabolize a biologically active material, e.g., a drug), an access point (to enable delivery or sampling of a fluid(s) comprising a cell and / or a biologically active material), an inlet, and an outlet. In some embodiments, the model liver is vascularized. In some embodiments, the model liver is optionally vascularized. In some embodiments, the model liver is not vascularized.

[0585] Figs. 30-33 illustrate a model liver, according to aspects of the present embodiments. The model liver may include a scaffold comprising a bioprinted entity, a bile duct, a chamber, an inlet, an outlet, and optionally vasculature. In some such embodiments, the model liver may comprise a cell associated with the scaffold and / or seeded therein, wherein the cell is optionally an endothelial cell. In some embodiments, the model liver optionally comprises a cell associated with the vasculature and / or seeded therein, wherein the cell is optionally an LSEC. In some embodiments, the model liver optionally comprises a cell associated with the bile duct and / or seeded therein, wherein the cell is optionally a cholangiocyte. It will be appreciated by one of skill in the art that the association of a cell may occur with a combination of one or more aspects of the scaffold (e.g., bioprinted entity, bile duct, chamber, inlet, outlet, vasculature), and furthermore that a plurality of cell types may be associated with said scaffold.

[0586] Referring still to Figs. 30-33, the chip 40 may include a first fluid network (e.g., vasculature 60) and a second fluid network (e.g., bile duct 80). The first and second fluid networksAttorney Docket No.: PCT.131660, 80 may be intertwined and or interconnected (e.g., with interlinking fluid passageways) without actually being fluidly coupled. Both the first and second fluid networks 60, 80 may be operatively coupled (for example functionally coupled) to the interstitial space and interstitial infill 58 disposed therein such that drugs and / or other biological substances that flow through either the first and / or second fluid networks 60, 80 may pass through the vasculature walls or bile duct walls and into the interstitial infill (e.g., to the active cells seeded therein).

[0587] Figs.34-37 E illustrate fluorescence imaging of a model liver, according to aspects of the present embodiments. The model liver may include a vasculature associated with a cell that comprises an interstitial pattern which may be characterized by fluorescence imaging, optionally wherein the interstitial pattern approximates the geometries of the cell and / or tissue(s) native format. In some embodiments, the interstitial pattern is spherical. In some embodiments, the interstitial pattern is fibrous. In some embodiments, the present disclosure provides a liver model (e.g., liver-on-a-chip) that shows improved cellular distribution relative to a comparable liver model (e.g., when comparing interstitial patterns via fluorescence imaging).

[0588] Fig.38 illustrates a bioprinted scaffold, according to aspects of the present embodiments. The bioprinted scaffold may be inserted into well plates for 3D culture applications.

[0589] Fig. 39 illustrates bioprinted scaffolds inserted into a well plate, according to aspects of the present embodiments. In some embodiments, the well plate may include various numbers of wells (for example, from about 6 wells to about 1536 wells), and may include bioprinted hydrogel scaffolds inserted in the wells, the scaffolds being coated with two reagents.

[0590] Figs. 40A-40C illustrate top. front, and side views of a bioprinted scaffold, according to aspects of the present embodiments.Delivery of Biologically Active Materials

[0591] In some embodiments, a scaffold, as described in the present disclosure, enables delivery of a biologically active material to a cell and / or tissue(s) via fluid(s) flowing through the scaffold. In some embodiments, a scaffold, as described in the present disclosure, enables delivery of a biologically active material to a cell and / or tissue(s) via fluid(s) flowing through the vasculature. In some such embodiments, delivery of the biologically active material occurs through vasculature walls.Attorney Docket No.: PCT.1316

[0592] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is selected from a group comprising a drug, a small molecule, a polypeptide, a peptidomimetic, a nucleic acid, a lipid, a lipid nanoparticle, an immunotherapeutic, a viral vector, and / or a cell. In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a drug. In some such embodiments, the drug is anti-inflammatory (e.g., Met-CCL5, cenicriviroc, belapectin, aspirin, GS-0976-WZ66, liraglutide, resmetirom). In some embodiments, the drug inhibits oxidative stress (e.g., oroxylin, methyl ferulic acid, GKT137831, losartan). In some embodiments, the drug inhibits hepatocyte apoptosis (e.g., VX-166, emricasan, pentoxifylline, |3-elemene, selonsertib). In some embodiments, the drug (e.g., halofuginone, FR(EtOH), BMS986263, simtuzumab) inhibits extracellular matrix (ECM) growth. In some embodiments, the drug inhibits HSCs activation and proliferation (e.g., pirfenidone, fluorofenidone, praziquantel, ferulic acid, sorafenib, AZD6244, nilotinib, rilpirivine, saracatinib, pioglitazone, curcumin, pegbelfermin, NGM282, hydronidone, ICG001, PrI-724, octreotide, obeticholic acid, cilofexor, PX20606, rimonaban, SR141716A, JD5037, elafibranor, rosiglitazone, statins, sitagliptin, alogliptin).

[0593] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a small molecule.

[0594] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a polypeptide and / or variant thereof. In some embodiments, the polypeptide is selected from the group consisting of an antibody, an antibody fragment, a peptide, a protein, and / or variant thereof.

[0595] In some embodiments, the polypeptide is an antibody fragment and / or variant thereof.

[0596] In some embodiments, the polypeptide is a peptide and / or variant thereof.

[0597] In some embodiments, the polypeptide is a protein and / or variant thereof.

[0598] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a peptidomimetic or variant thereof.

[0599] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a nucleic acid or variant thereof. In some embodiments, the nucleic acid is selected from the group consisting of siRNA, tRNA, rRNA, mRNA. miRNA, gRNA, DNA, and / or variant thereof. In some embodiments, the nucleic acid is a siRNA and / or variant thereof.

[0600] In some embodiments, the nucleic acid is a tRNA and / or variant thereof.

[0601] In some embodiments, the nucleic acid is a rRNA and / or variant thereof.Attorney Docket No.: PCT.1316

[0602] In some embodiments, the nucleic acid is a mRNA and / or variant thereof.

[0603] In some embodiments, the nucleic acid is a miRNA and / or variant thereof.

[0604] In some embodiments, the nucleic acid is a gRNA and / or variant thereof.

[0605] In some embodiments, the nucleic acid is a DNA and / or variant thereof.

[0606] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a lipid and / or variant thereof.

[0607] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a lipid nanoparticle and / or variant thereof. In some embodiments, the lipid nanoparticle may or may not comprise a payload, wherein the payload is selected from a group consisting of a small molecule, a nucleic acid (e.g., mRNA), and / or a polypeptide.

[0608] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is an immunotherapeutic or variant thereof.

[0609] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a viral vector. In some embodiments, the viral vector may or may not comprise a payload, wherein the payload is selected from a group consisting of a small molecule, a nucleic acid (e.g., mRNA), and / or a polypeptide. In some embodiments, the viral vector is an adeno-associated virus (AAV) and / or variant thereof. In some such embodiments, the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVDJ, AAVDJ / 8, AAVPHP.eB, AAVPHP. S, AAV2-QuadYF, and / or AAV2.7m8. In some embodiments, the AAV is AAV1. In some embodiments, the AAV is AAV2. In some embodiments, the AAV is AAV3. In some embodiments, the AAV is AAV4. In some embodiments, the AAV is AAV5. In some embodiments, the AAV is AAV6. In some embodiments, the AAV is AAV6.2. In some embodiments, the AAV is AAV7. In some embodiments, the AAV is AAV8. In some embodiments, the AAV is AAV9. In some embodiments, the AAV is AAVrh10. In some embodiments, the AAV is AAVDJ. In some embodiments, the AAV is AAVDJ / 8. In some embodiments, the AAV is AAVPHP.eB. In some embodiments, the AAV is AAVPHP. S. In some embodiments, the AAV is AAV2-QuadYF. In some embodiments, the AAV is AAV2.7m8.

[0610] In some embodiments, the biologically active material delivered via fluid(s) through the vasculature is a cell and / or variant thereof. In some such embodiments, the cell is selected from the group consisting of a T cell, a PBMC, a pluripotent stem cell (PSC), an adult stem cell (ASC), an embry onic stem cell, a cancer stem cell (CSC), a hematopoietic progenitor cell (HPC), a myeloidAttorney Docket No.: PCT.1316stem cell, a monocyte, a lymphocyte, a granulocyte, a patient-derived cell (e.g., a tumor cell), a chimeric antigen receptor (CAR)-positive T cell, and / or a CD54+cell.

[0611] Those skilled in the art. reading the present disclosure, will appreciate that, in some embodiments, the biologically active material delivered via fluid(s) through the vasculature may be adjusted, for example to comprise a plurality of materials (e.g., a combination of a polypeptide and / or a small molecule), which may be the same or different.

[0612] As described herein, use of a variety of biologically active materials is contemplated as compatible with various embodiments. In some embodiments, a biologically active material is or comprises a detectable moiety.Detection of a Detectable Moiety

[0613] As is described herein, detecting (e.g., detecting a signal, such as a biomarker or detectable moiety), as applicable to methods and compositions described herein, may be achieved in any application-appropriate manner. For example, in some embodiments, a step of detecting is or comprises an immunological assay, a nucleic acid amplification assay, a fluorescence assay (e.g., a calf intestinal alkaline phosphatase (CIP) assay), a luminescence assay, a colorimetric assay (e.g., a urea-associated assay), an albumin binding assay, genomics, proteomics, transcriptomics (e.g., spatial transcriptomics), an RNA expression assay, metabolomics (e.g., an ATP-associated assay), and / or immunohistochemistry.

[0614] As is described herein, many embodiments include the use of one or more biological samples (e.g., a sample of fluid or tissue taken from an organ system), and the manner of detecting a biomarker and / or detectable moiety may vary depending upon the biological sample used in a particular embodiment. In accordance with the present disclosure, any of a variety of biological samples are contemplated as compatible with various embodiments. For example, in some embodiments, a biological sample is or comprises a fluid(s) from the chamber of the organ system.

[0615] In accordance with various embodiments, and depending upon the specific biomarker(s) used, one of skill in the art will envision one or more appropriate methods of detecting or determining the presence and / or quantity / level of a biomarker and / or detectable moiety in a biological sample. In some embodiments, a biomarker and / or detectable moiety may be detected using any of a variety’ of modalities including fluorescence, radioactivity, chemiluminescence, electrochemiluminescence, colorimetry, FRET, HTRF, isotopic methods, partner binding (e.g.,Attorney Docket No.: PCT.1316biotin / avidin, antibodies, hybridization), or any other known manner of detecting a biomarker and / or detectable moiety. In some embodiments, a biomarker and / or detectable moiety' may be detected through binding of a detectable moiety (e.g., an exogenously added detectable moiety) such as an antibody that includes, for example, a tag in accordance with one or more of the above modalities, or an enzyme (e.g., luciferase, (3-gal).

[0616] Those skilled in the art will readily appreciate that the methods described herein can be useful in a variety of applications involving monitoring a detectable moiety within a model organ-on-a-chip (e.g., a healthy or disease model) after the delivery of a biologically active material. In some embodiments, methods described herein may be useful in assessing whether or not a detectable moieties levels are abnormal, relative to a desired or ‘‘normal” level of expression. In some embodiments, methods described herein may be used to predict or characterize a potential reaction to a biologically active material (e.g., a disease model responds to the biologically active material favorably by exhibiting a decrease in the metric used to determine said diseased state), thus potentially allowing for the enablement, prevention and / or mitigation of the reaction, based on the desired outcome.Characterization of a Scaffold

[0617] Those skilled in the art, reading the present specification, will appreciate that it may be desirable to characterize one or more features of bioprinted entities independent of a cell and / or associated with a cell, and / or of components or combinations thereof, for example when designing (e.g., selecting appropriate components of) or producing a provided system and / or when monitoring or assessing a preparation thereof. Alternatively or additionally, in some embodiments, it may be desirable to assess one or more features of a provided system as administered, for example in order to monitor a subject and / or treatment thereof.

[0618] In some embodiments, cellular distribution is a characteristic property of a system and / or bioprinted entity associated with a cell and / or seeded therein. In some embodiments, the present disclosure provides systems and / or bioprinted entities that show improved cellular distribution relative to a comparable system and / or bioprinted entity.

[0619] Those skilled in the art, reading the present specification, will appreciate that it may be desirable to characterize one or more features of bioprinted entities, and / or of components or combinations thereof, for example when designing (e.g., selecting appropriate components of) orAttorney Docket No.: PCT.1316producing a provided system and / or when monitoring or assessing a preparation thereof. Alternatively or additionally, in some embodiments, it may be desirable to assess one or more features of a provided system as administered, for example in order to monitor a subject or treatment thereof.Bioprinting Methods

[0620] Fig. 74A illustrates a method 734 of bioprinting a scaffold, according to aspects of the present embodiments. At step 758, the method 734 may include warming the bioink to room temperature. In some embodiments, the bioink may be stored in a refrigerator prior to use. In some embodiments, the bioink may include alginate, collagen, gelatin, fibrin, hyaluronic acid, chitosan, silk, agarose. Matrigel. photocurable resins, and / or mixes thereof. In some embodiments, warming the bioink to room temperature includes warming the bioink vat / container in a warm water bath. In some embodiments, the method 734 may include warming up the build platform / bioprinting system to a temperature of about 40 °C (+ / -1 °C). In some embodiments, the bioprinting system may include a light projector (for example, to enable digital light processing (DLP) in connection with the bioprinting modality / build process). In some embodiments, the bioprinting system uses a bioink vat that includes a hydrophobic bottom membrane. At step 760, the method 734 may include inspecting the bioink vat / container for dust, scratches, and / or other irregularities. In some embodiments, inspecting the bioink vat may include placing the vat over a light projector such that scratches, dust, and / or other deposits may be more easily identified. At step 762, the method 734 may include cleaning the bioink vat. In some embodiments, cleaning the bioink vat includes the use of filtered water. In some embodiments, filtered water includes filtering with a 0.22 um filter such that no particles greater than 0.22um are present. In some embodiments, filtered water includes 5 parts per billion (ppb) or less of total organic carbons (TOC). In some embodiments, filtered water includes Milli-Q water. In some embodiments, cleaning the bioink vat includes spraying the bioink vat with filtered water from a distance of 3-10 inches (for example, 3-8 inches, 3-7 inches, 3-6 inches, 4-6, inches, and / or 4-5 inches), followed by wiping the bioink vat gently using a membrane wipe, so as not to damage the bioink vat. In some embodiments, cleaning the bioink vat (step 762) includes saturating a membrane wipe with Milli-Q water following by wiping the bioink vat. In some embodiments, cleaning the bioink vat includes spraying interior or exterior surfaces of the bioink vat with Isopropanol, 2-propanol (IP A) followedAttorney Docket No.: PCT.1316by one or more drying steps. In some embodiments, drying steps include using a hair dryer at medium heat (or a blower) at a distance of about 3 to about 10 inches. In some embodiments, drying the bioink vat includes using a hair dryer / blower to dry the interior portions of the bioink vat and using a glass wipe to dry the exterior portions of the bioink vat. In some embodiments, drying and / or cleaning the bioink vat includes inverting the bioink vat such that IPA does not accumulate near, or come in contact with, the hydrophobic bottom surface.

[0621] Referring still to Fig. 74A, at step 764, the method 734 may include preparing the build file(s) (i.e.. digital build files detailing the scaffold geometry, support structures, build sequence (e.g., layer by layer print patterns), scaffold orientation during build, and other build details) for print. In some embodiments, preparing the build file for print includes rotating the digital file 90-degrees from the "normal" orientation when the scaffold is in use such that a larger number of scaffolds can fit within the build area during each print. According to aspects of the present embodiments, scaffolds may be printed individually or in batches. In some embodiments, the method 734 may include filling the bioink vat with the warmed bioink via pipette after the bioink vat has been loaded into the bioprinting system / printer. At step 766, the method 734 may include confirming the build temperature (i.e., build platform target temperature) has been reached (e.g., 40 °C + / -1 °C, in some embodiments). For example, in some embodiments the bioprinting system / build platform should be at a temperature in a range from about 39°C to about 41°C prior to initiating build sequence / printing process. At step 767, the method 734 includes printing the scaffold. At step 768, the method 734 may include filling one or more specimen vessels (or jars) with filtered water. In some embodiments, the filtered water includes Milli-Q water. When the scaffold print job is complete, the scaffolds may be removed from a platform of the bioprinting system using a plastic razor (e.g., rather than a metal razor), so as to not risk damaging the bioprinting system. At step 770, the method 734 may include placing the printed scaffold(s) in the vessel or container (or jar) such that they are submerged in filtered water (e.g., Milli-Q water). At step 772, the method 734 may include cleaning the build platform / system (e.g., using IPA in some embodiments, followed by drying via blow dryer, in some embodiments). The bioink vat, in some embodiments, may be cleaned post-print via the same cleaning processes as those used for the preprint vat cleaning. At step 773, the method 734 may include performing post-print washing of the printed scaffold. At step 774, the method 734 may include monitoring hydrogel swelling.

[0622] Still referring to Fig. 74A, post-print washing 773 of the printed scaffold is beneficial to ensure any excess photoinitiator and / or photoabsorber are removed from the scaffold. In someAttorney Docket No.: PCT.1316embodiments according to the present disclosure, the photoinitiator is activated at a wavelength of about 375 nm, and the photoabsorber is activated at a wavelength of about 425 nm. In some embodiments, the photoinitiator may include one or more of benzophenone. Phenyl bis (2,4,6-trimethyl benzoyl) phosphine oxide (BAPO, Irgacure 819), 2-hydroxy-2-methyl-1- phenyl-propan-1-one (Irgacure 1173), 2-Hydroxy-4'-(2-hydrox yethoxy )-2 -methylpropiophenone (Irgacure 2959), 2,2'-azobis[2-methyl-n-(2-hydroxyethyl) propionamide] (VA-086), 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651 or DMPA). Diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (Darocure TPO; Lucirin IPO), lithium phenyl(2,4,6-trimethylbenzoyl) phosphinnate (LAP), and Ethyl (2,4,6-trimethylbenzoyl) phenylphosphinate (Lucirin TPO-L) In some embodiments, the photoinitiator is activated at a wavelength in a range from about 245 nm to about 393 nm. In some embodiments, the photoabsorber comprises at least one of tartrazine, curcumin, anthocyanin, 2-hydroxy-4-methoxy-benzophenone-5-sulfonic acid (HMBS), reactive orange 16 (RO16) (disodium(3Z)-6-acetamido-4-oxo-3-[[4-(2-sulfonatooxy ethyl sulfonyl) phenyl]hydrazinylidene] naphthalene-2-sulfonate), anionic azo dye, and / or food dye. In some embodiments, post-print washing 773 may include creating a cleaning solution (for example, including filtered water (such as Milli-Q water), or phosphate buffered saline (PBS), or a mixture thereof). In some embodiments, post-print washing 773 may include submerging the printed scaffolds into the cleaning solution for a period of time, and then repeating the process one or more times, as needed, ■with fresh cleaning solution. In some embodiments, post-print washing 773 may include measuring the concentration of photoinitiator and / or photoabsorber in the cleaning solution following a wash, and then repeating the cleaning process as many times as are needed until the respective concentration of photoinitiator and / or photoabsorber is below a pre-selected threshold. In some embodiments, the pre-selected threshold includes a concentration of about 0.01% or lower. In some embodiments, post-print washing 773 may include washing the scaffolds (e.g., with fresh cleaning solution each time) a total of at least 3 times (for example, 4 times) over a period of 24 hours or greater (for example, 48 hours).

[0623] In some embodiments, various steps of the method 734 can be executed in a different order than what is shown in Fig. 74A, or in some embodiments, simultaneously. (This applies to other methods disclosed herein as well). For example, in some embodiments, warming the bioink to room temperature (step 758), inspecting and cleaning the bioink vat (steps 760 and 762), preparing the build file(s) for print (step 764), and confirming the platform target build temperature has been reached (step 766) can be performed in any order and / or can be performed at the sameAttorney Docket No.: PCT.1316time. Step 772 (cleaning the build platform) can be performed any time after the build process is complete and the printed scaffold(s) have been removed from the build platform. Similarly, step 768 (filling the specimen vessel with filtered water) can be performed any time prior to step 770 (placing the scaffolds(s) in the specimen vessel).

[0624] Fig. 74B illustrates the concentrations of photoinitiator (PI) 769 and photoabsorber (PA) 771 as function of the number of washes being performed. It is noted that the Y-axis scale is logarithmic and thus every’ successive wash removes the majority of whatever photoinitiator (PI) 769 and photoabsorber (PA) 771 are present (e.g., each wash removes 50%-99% (e.g., 50%-95%, e.g., 50%-90%, e g., 50%-80%) of the remaining photoinitiator (PI) 769 and photoabsorber (PA) 771). As illustrated, after 4 washes, there is less than 0.01% of each of the photoinitiator (PI) 769 and photoabsorber (PA) 771 residing on the scaffold (e.g., as measured as a concentration of the cleaning solution). In some embodiments, determining the concentration of photoinitiator (PI) 769 and / or photoabsorber (PA) 771 in the cleaning solution includes using ultraviolet-visible (UV-Vis) spectroscopy. In some embodiments, during each cleaning step, the printed scaffold are submerged in a container or vessel containing form about 100 mL to about 150 mL of cleaning solution.

[0625] Referring again to Fig. 74A and also to Fig. 74C, the printed (hydrogel) scaffold may initially swell for a first period of time, followed by a period of de-swell for a second period of time, after which the printed (hydrogel) scaffold remains at a substantially unchanged, static geometry’. For example, referring to Fig. 74C (which shows the Y and Z dimensions of a printed scaffold plotted as a function of days in solution) the printed (hydrogel) scaffold may initially swell from day 0 to day 3 (i.e., the first period of time 777), and then may de-swell from day 3 to day 4 (the second period of time 779) after which the scaffold has reached a static geometry. According to one illustrative embodiment, the scaffold may swell by about 2.5 mm during the first period of time 777, and then may de-swell by about 150 pm during the second period of time (for example, for a scaffold that initially has a dimension in a range from about 7.5 mm to about 12.5 mm). As illustrated in Fig. 74C, the swelling amounts (on both a relative and absolute basis) may be different in one direction (i.e., a build direction, “Z” in this case) than in another direction (i.e., a non-build direction, “Y"’ in this case). In some embodiments, once a printed scaffold has deswelled a certain percentage from a maximum, swelled dimension (or geometry), it may be considered to have reached a steady state geometry. For example, according to the present disclosure, a printed scaffold may be deemed to have reached a steady state geometry once it was de-swelled from a maximum geometry’ by an amount in a range from about 0.5% to about 3.0%,Attorney Docket No.: PCT.1316or in a range from about 0.5% to about 2.5%, or in a range from about 0.5% to about 2.0%, or in a range from about 1.0% to about 3.0%, or in a range from about 1.0% to about 2.0%, or in a range from about 0.75% to about 2.5%, or in a range from about 0.75% to about 2.0%, or in a range from about 0.75% to about 1.75%, and / or in a range from about 1.0% to about 1.5%. The time required for a printed work product to reach equilibrium will vary depending on the geometry of the work product, with larger work products generally taking longer to reach equilibrium. Accordingly, using a constant or pre-selective de-swell percentage or range of percentages provides a good way to assess when a work product had reached equilibrium that is not dependent on work product geometry.

[0626] Fig. 74D illustrates an example of an array of scaffolds 70A, each scaffold 70A being oriented vertically (e g., rotated 90 degrees from the orientation each is in when in use) during print to maximize the number of scaffolds 70A that can be built (i.e., that can fit within the print area) during each print (i.e., in each batch). As shown in Fig.74D, and as described herein, each scaffold 70A includes a fluid inlet 48, a fluid outlet 52, and interstitial infill 58. Fig. 74D also illustrates printed support structures 59 that are printed as part of the build process, and that help to support the scaffolds 74A while they are being printed.

[0627] According to the present disclosure, the printing (e.g., bio-printing), post-print washing, and swell-monitoring steps, as well as the UV clave, cell seeding, sterilizing, coating, endothelializing and / or functionalizing steps are each disclosed in detail due to the importance of ensuring that proper conditions are maintained for cell culturing, and to avoid the risk of contamination. In order to achieve the assay results disclosed herein, diligence must be taken to follow the process-steps as disclosed. In some embodiments, certain processes should be repeated multiple times. In some embodiments, process steps may occur in a different order than the order in which they are described herein. In some embodiments, one or more steps may be able to be performed concurrently. In some embodiments, processes may need to be adjusted according to the specific tests and assays being performed. However, skipping steps and / or deviating greatly from the processes described herein increases the likelihood that the disclosed performance results may not be able to be achieved.

[0628] Referring again to Fig. 74A, in some embodiments, the bioink includes 5-25% PEGDA, 0.5-3% photoinitiator, and 0.5-3% photoabsorber. In some embodiments, the bioink includes a pH in a range from 7.2 to 7.4. In some embodiments, the bioink includes a viscosity in a range from about 0.005 Pas to about 0.02 Pas. In some embodiments, the bioink is transparent. In someAttorney Docket No.: PCT.1316embodiments, exhibits no auto-fluorescence. In some embodiments, the bioink includes a photocrosslinkable monomer, photoabsorber, and a photoinitiator. In some embodiments, photocrosslinkable monomers include polyethylene glycol) compounds functionalized with acrylate groups such as poly(ethylene glycol) diacrylate (PEGDA; two chains) and four-arm PEG acrylate (four chains) with molecular weights in a range from 700 g / mol to 35,000 g / mol. In some embodiments, photocrosslinkable monomers include hydroxyalkyl compounds which are functionalized with acrylate groups, methacrylate groups, and / or acrylamide group on their other end such as hydroxy ethyl methacrylate (HEMA), hydroxypropyl acrylate (HP A), and / or hydroxyethyl acrylamide (HEAA). In some embodiments, photocrosslinkable monomers include methacrylate or acrylate-terminated polydimethyl siloxanes (PDMS). In some embodiments, bioprinting systems may include digital light projection (DLP), stereolithography (SLA), or two photon polymerization (2PP)-based bioprinting systems and / or Multi Jet printing (MJP) systems capable of printing with an X-Y resolution of 10 pm. In some embodiments, while the scaffold is being printed (for example, at step 767) or thereafter the system may record and / or monitor certain parameters such as LED intensity, temperature and position of the printer arm, and forces experienced by the platform (load cell) throughout the printing process.EXAMPLESExample 1Hydrogel Scaffolds

[0629] The present example demonstrates that hydrogel scaffolds described herein achieve cell growth (e g., cell culture) for a sustained duration of time and under conditions that reproduce a physiological (e.g., natural, endogenous) environment.Preparing scaffolds for coating

[0630] Hydrogel scaffolds described herein are placed in a 12 well plate in phosphate buffered saline (PBS) and exposed to UV for 2 hours. The hydrogel scaffolds are carefully removed from the wells and placed upside down in a sterile petri-dish. H-VIOS2-DF chips are primed with IX PBS to ensure there are droplets at both the inlet and outlet. The hydrogel is assembled in the chip and 150-600 uL of collagen type I (Col 1) is added to the well of the hydrogel. Collagen calculations w ere performed as previously described. Female luers are assembled at the inlets and outlets of the h-Vios2chips and capped with male caps. Chips are placed in the incubator at 37°C for 1 hour.Attorney Docket No.: PCT.1316Vasculature washing and ECM coating

[0631] Using a syringe pump, 250-750 uL IX PBS was perfused through each hydrogel to remove air trapped in the vasculature followed by perfusion of 100-300 uL 10X penicillin / streptomycin (pen / strep) and 10X amphotericin B (Amp B). The vasculature was washed with 250-750 uL IX PBS and 20-60mg of Acrylated-PEG-OCH2CH2CH2CH2CO2-NHS (SV A) was dissolved in l-4mL of 1% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) solution (Dulbecco’s PBS). The SVA solution was added to the vasculature using a syringe pump at 50-200 ul / min. The hydrogel scaffolds were then placed under UV for 20 minutes. The channels (e.g., vasculature) were washed with 200-700 uL IX Dulbecco’s PBS (DPBS). A collagen coating (e.g., ECM) solution was prepared as previously described. Each hydrogel was perfused with 50-300 uL ECM solution at 20-75 uL / min. Excess liquid from the (+) side of the chip aspirated. The inlets and outlets were capped with male caps and the hydrogel scaffolds incubated for 1-3 hours at 37°C. The vasculature was washed with 200-500 uL DPBS at 50 - 200 uL / min. Hydrogel scaffolds were filled with 300uL-1100uL EGM (cAP-02, Angio-Proteomie) medium containing P / S and AmpB for equilibration before being placed in an incubator at 37°C overnight. The EGM solution was emptied form the hydrogel wells while cells were prepared.Preparing GFP tagged human umbilical vein endothelial cells (HUVEC)

[0632] HUVEC cells were washed 2X with PBS and detached using 0.05% Trypsin. EGM was then added to the well at an equal volume to trypsin. The cells were centrifuged at 200 g for 5 minutes and then resuspended in medium. The cells were subsequently counted.Preparing EGM medium for cell

[0633] AmpB was added to EGM to prevent fungal contamination of hydrogels. HUVECs were resuspended in EGM at 200,000 cells / cm2.Seeding HUVECs

[0634] The chip was attached to the syringe pump and 200 uL of cell suspension was slowly perfused at the (-) side of the chip at 50 uL / min and through the vasculature. This was carried out for all hydrogel scaffolds. The inlets and outlets are then capped and the lids of the chips are closed.Attorney Docket No.: PCT.1316The chips were then loaded on a programmable rotator for rotation at 1 rotation per minutes for 4 hours.Imaging D2 GFP-HUVEC cells

[0635] Stable attachment of GFP-tagged HUVECs were assessed using a 4x objective (EVOS microscope) using a Z-stack and a custom imaging plate. Fresh EGM was added to the wells of the hydrogel after imaging. On day 3, cells were imaged as previously described while the chip was connected to a syringe pump. While imaging, the syringe pump was set at a flow rate of 5 uL / min to 70 uL / min to determine the flow rate which the cells could tolerate (e.g., did not detach). Once the optimal flow rate was determined, tubing of a peristaltic pump was primed with EGM and connected to the chip inlets and outlets. Perfusion was started, with 150 uL to 450 uL fresh EGM medium added to the wells every 24 hours. The reservoir was replenished with fresh medium every 48 hours. Hydrogels containing cells were imaged on day 1, day 3, day 7, day 10, and day 14. On day 14, cells were fixed by perfusing 4% paraformaldehyde through the hydrogels. Fixation occurred overnight at 4°C. Assessment of cell morphology and coverage (e.g., confluency) was assessed using fluorescence confocal microscopy to visualize GFP. actin, ZO-1, CD-31 and Hoechst staining.Example 2Single-Plane Coating Methods

[0636] Fig. 75 illustrates a method 775 of performing single-plane coating (i.e.. of printed scaffolds), according to aspects of the present embodiments. Once the scaffolds have been printed, cleaned, and swelled / de-swelled as described herein, they are ready to be coated. At step 776, the method 775 may include bringing the scaffold to a temperature of 37°C, and sterilizing the printed scaffold(s) via UV clave (for example, for about 30-60 minutes, e.g.. about 35 minutes, about 40 minutes, about 45 minutes, about 35-55 minutes, about 50 minutes, about 55 minutes, etc., for example, at a UV intensity of at least 35 mJ / cm2to 55 mJ / cm2, or from about 40 mJ / cm2to 45 mJ / cm2). At step 778, the method 775 may include preparing the scaffold for coating. In some embodiments, preparing the scaffold(s) for coating includes priming each of the scaffold inlets 60 (shown in Figs. 16-18) with Dulbecco’s Phosphate Buffered Saline (DPBS) e.g., using a pipette or syringe (e.g., a 4 mL syringe with a 21 -gauge needle) until DPBS is observed flowing out of the corresponding outlets. In some embodiments, preparing the scaffold(s) for coatingAttorney Docket No.: PCT.1316includes assembling the scaffolds into the corresponding chip after each scaffold is primed, and filling the interstitial space of each assembly with 200 pL of DPBS. At step 780, the method 775 may include perfusing the scaffold with PBS (for example, DPBS). In some embodiments, perfusing the scaffold with PBS includes using a properly primed syringe pump (e.g., and manually removing any air bubbles if needed). In some embodiments, perfusing the scaffold with PBS includes perfusing at a rate of about 200 pL / min. In some embodiments, perfusing the scaffold with PBS includes perfusing at a rate that ensures approximately 100% of the flow volume is perfused every minute. The scaffolds can be visibly observed, in some embodiments, during perfusing to assess if leaks are present (such that any leaky scaffolds can be discarded). At step 782, the method 775 may include performing quality control (QC) steps on the printed scaffold. In some embodiments, performing quality control (QC) steps on the printed scaffold includes using a microscope (for example, an inverted microscope, e.g., an Evo cell imaging system (EVOS) microscope) to observe if any air bubbles are present / trapped in the scaffold passageways / vasculature. If air bubbles are observed, they can be manually removed (e.g., via syringe) and / or removed via further PBS perfusion.

[0637] Referring still to Fig. 75. at step 784, the method 775 may include perfusing the scaffold with cross-linker. In some embodiments, the cross-linker includes an Acrylated-PEG-OCH2CH2CH2CH2CO2-NHS (i.e., succinimidyl valerate (SV A)) cross linker (for example, a 4% SVA cross-linker). In some embodiments, the cross-linker is prepared by dissolving the crosslinker in working 1% LAP (i.e., Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) solution and subsequently filtering (e.g., 22 pm filtering) the solution. In some embodiments, the 4% SVA cross-linker includes a molecular weight of about 1900 g / mol to about 2100 g / mol (for example, from about 1950 g / mol to about 2050 g / mol). At step 786, the method 775 may include performing another UV clave process on the printed scaffold. In some embodiments, performing another UV clave process on the printed scaffold (e.g., following perfusion with the cross-linker) includes claving at 365 nm for about 10 minutes, flipping the printed scaffold over to expose the opposite side, claving for another 10 minutes at 365 nm with the second side exposed, and then subsequently re-filling the interstitial space with 200 pL of DPBS. At step 788, the method 775 may include perfusing the scaffold with an adhesive. In some embodiments, the adhesive may include a Poly-1-lysine solution. In some embodiments, other types of adhesive solutions such as those that include poly-ethylenimine (PEI), laminin and / or collagen may be used. In some embodiments, steps 784 through 788 should be performed within a total time period of about 2 hours to avoidAttorney Docket No.: PCT.1316noticeable degradation of the cross-linker. In some embodiments, the printed scaffolds may be incubated for 2 hours at room temperature following step 788 (adhesive perfusion).

[0638] Still referring to Fig. 75, at step 790, the method 775 may include preparing an extracellular matrix (ECM) coating. In some embodiments, preparing an ECM coating includes preparing a coating that includes roughly 65-70% fibronectin, roughly 4-8% collagen 1, and roughly 25-30% collagen 4. In some embodiments, the ECM coating may be mixed using an autoclaved microcentrifuge tube, which itself may be pre-chilled prior to addition of each of the constituents. The mixture may be centrifuged to ensure homogeneity. At step 792, the method 775 may include perfusing the printed scaffold with the ECM coating. In some embodiments, perfusing the printed scaffold includes perfusing multiple scaffolds with the ECM, one at a time. At step 794, the method 775 may include incubating the printed scaffold (e.g., overnight at about 4° C (e.g., at about 2° C to about 6° C)). At step 796 the method 775 may include perfusing the scaffold (e.g., at about 50-120 pl / min) with PBS (e.g., DPBS) to wash the scaffolds. In some embodiments, if the PBS has been in storage and / or refrigeration for more than 2 days, 0.22 pm filtering may be performed. At step 798, the method 775 may include performing a fast (or relatively higher flow rate) perfusion of the printed scaffold with endothelial growth media (EGM) (for example, at a flow rate of about 40-60 pl / min). At step 799, the method 775 may include performing a slow (or relatively lower How rate) perfusion of the printed scaffold with EGM (for example, at a flow rate of about 20-50 µl / hr.). In some embodiments, EGM includes Lonza EGM-2 media. In some embodiments, the step 799 (slow perfusion) is performed for 1-3 days (for example. 30-64 hours, or 36-60 hours, or about 42 to about 54 hours, or about 48 hours) in order to incubate the coated scaffolds.Example 3Endothelializing and Seeding Protocols

[0639] Fig. 76 illustrates a method 1000 of endothelializing and seeding printed scaffolds, according to aspects of the present embodiments. The method 1000 is to be performed following the single-plane coating method described in Fig. 75. At step 1002, the method 1000 may include checking the coated scaffolds for bubbles, following the 48-hour incubation of the printed scaffold in EGM. In some embodiments, fetal bovine serum or human serum may be used instead of EGM. At step 1004, the method 1000 may include performing a de-gas process by fluidly coupling the coated scaffold to a vacuum source, initially for less than 60 seconds (for example, 30 second, 20Attorney Docket No.: PCT.1316seconds, 15 seconds, or in some embodiments, about 10 seconds). Following the initial de-gas, the vacuum source may remain coupled to the coated scaffold while filtration (for example, 0.45pm filtration) is also performed, for a period of about 5 minutes. At step 1006, the method 1000 may include preparing human umbilical vein endothelial cells (HUVECs) e.g., green fluorescent protein (GFP)-tagged HUVECs. In some embodiments, preparing GFP-HUVECs includes: 1) detaching the HUVECs using 0.05% try psin (e.g., and allowing the try psin to persist / be activated for about 3 minutes), 2) adding EGM to the HUVECs, 3) centrifuging the mixture for about 5 minutes, 4) resuspending the HUVECs in EGM, 5) trypsinizing the suspension, and 6) resuspending in about 500 pL of EGM. In some embodiments, cell counting may then be performed such that if a higher cell density than desired is observed, the cells can be centrifuged again. At step 1008, the method 1000 may include resuspending the GFP-HUVECs in EGM (e.g., to achieve a cell density of about 200,000 cells / cm2(for example, to achieve a density in a range from about 150,000 cells / cm2to about 250,000 cells / cm2). In some embodiments, an endothelial call coverage of at least 90% (i.e., of the surface area) may be used in place of a specific cell seeding density. At step 1010, the method 1000 may include seeding the scaffold with GFP-HUVECs. In some embodiments, seeding the scaffold with GFP-HUVECs includes first using microscopy to identify any air bubbles in the coated scaffold (and then removing any air bubbles that are present). In some embodiments, seeding the scaffold with GFP-HUVECs includes resuspending the cell suspension followed by loading the suspension (e.g., via pipette) into a syringe, followed by priming the syringe. In some embodiments, seeding the scaffold with GFP-HUVECs includes doing so in 100 pL increments to reduce the likelihood of cloggage. In some embodiments, seeding the scaffold with GFP-HUVECs includes perfusing the suspension at a rate of 200 pL / min. At step 1012, the method 1000 may include re-seeding the scaffold with GFP-HUVECs, as needed, if the seeding does not appear uniform (upon inspection via microscope) and / or if air bubbles are present.

[0640] Still referring to Fig. 76. the seeding process 1000 described herein can be repeated for each of the printed scaffolds until all of the scaffolds are seeded. In some embodiments, seeding and re-seeding can be performed as many times as is needed to ensure uniform distribution of the GFP-HUVECs on or within the scaffolds. In some embodiments, once the printed, coated scaffolds are seeded, they can be continuously rotated and / or perfused with EGM at a low flow rate (e.g., 20-40 pL / hr.). In some embodiments, the seeded scaffolds are washed periodically (for example, every 1-3 days, (e.g., every 48 hours)) at a flow rate in a range of about 30-70 pL / min for 5-10 minutes to remove any cell debris. In some embodiments, the seeded scaffolds may be imagedAttorney Docket No.: PCT.1316daily and / or weekly (for example, after days 1, 3, 7, 14, 21, etc.). In some embodiments, the seeded scaffolds may be re-seeded periodically (for example, on days 8 and 15) following the initial seeding.Example 4T-Cell Expansion

[0641] Fig.77 illustrates a method 1020 of expanding T-cells, according to aspects of the present embodiments. At step 1014, the method 1020 may include preparing a media composition. In some embodiments, preparing the media composition comprises adding T Cell activator (e.g., CD3 / CD28 T Cell activator) to medium (e.g., ImmunoCult medium), along with Recombinant Human IL-2 (cytokines), and gentamicin for 2 days, followed by adding additional medium, IL-2 cytokines, and gentamicin on Day 3, and incubating until Day 10. At step 1016, the method 1020 may include thawing the media vial, as well as providing fresh expansion medium. In some embodiments, a water bath may be used to thaw the media vial and warm it to, for example 37°C. At step 1018, the method 1020 may include decontaminating the media vial. At step 1022, the method 1020 may include centrifuging the media (for example, at 1800 rpm for 5 minutes). Following step 1022, the method 1020 may include performing immunophenotype characterization, as shown in steps 1050-1070 of Fig. 77. In some embodiments, immunophenotype characterization may be performed in connection with PBMC-derived T cells. At steps 1050, 1052, and 1054 the method 1020 may include preparing samples (for example, 8M total with 0.5 million cells per sample), centrifuging the samples (for example, at 1800 rpm for 5 minutes), and resuspending the cells, respectively. At step 1056, the method 1020 may include setting aside unstained cells. At step 1058, the method 1020 may include preparing an Fc (i.e., fraction crystallizable) block (e.g., of the stain buffer equipment) followed by incubation (e.g., for 10 minutes at room temperature). In some embodiments, immunophenotype characterization as described herein includes the use of antibodies that bind to CD3, CD4, CD8, CD28, and / or PD1 cell surface markers. At step 1060, the method 1020 may include antibody staining (e.g., followed by 20 minutes of room temperature incubation at a location that is not exposed to light). The cells may then be washed using stain buffer and centrifuged again for 10 minutes at 1800 rpm. At step 1062, the method 1020 may include discarding supernatant. At step 1064 the method 1020 may include resuspending the cells, followed by a 15-minute incubation at room temperature. At step 1066, the method 1020 may include washing the cells (again with stain buffer), centrifuging for 10Attorney Docket No.: PCT.1316minutes at 4500 rpm), and discarding the supernatant. At step 1068, the method 1020 may include adding buffer (e.g., stain buffer) and storing at 4 °C in a location that is not exposed to light. At step 1068. the method 1020 may include performing flow cytometry (for example, using fluorescent antibodies that may include, for example, BV421 (VI), APC-Cy7 (R3), PerCP-Cy5.5 (B5), PE (B2), and / or BV650 (V5)).

[0642] Referring still to Fig. 77, once immunophenotype characterization is complete, at step 1024, the method 1020 may include incubating the cells again (e.g., at a temperature of 37 °C in a 5% CO2 environment, for three days). At steps 1026 and 1028, the method 1020 may include centrifuging (at 1800 rpm for 5 minutes), doing an 8-fold increase in the culture medium, and incubating the cells (at a temperature of 37 °C in a 5% CO2 environment), starting on Day 3, thereby performing an 8:1 expansion of the cell population. At steps 1030 and 1032, the method 1020 may include collecting the cell suspension, doing a 4-fold culture medium volume incre...

Claims

Attorney Docket No.: PCT.1316CLAIMS1. A method of assessing immune cell recruitment and / or tumor cell killing, the method comprising:providing a bioprinted scaffold comprising at least one perfusable internal passageway and an interstitial space, wherein the interstitial space is operatively coupled to the at least one internal passageway, and wherein the scaffold is configured to permit migration of immune cells from the internal passageway to the interstitial space;seeding tumor cells within the interstitial space;seeding endothelial cells within the at least one internal passageway to form a functional endothelium;culturing the seeded scaffold under perfusion conditions to establish a vascularized tumor model;perfusing immune cells through the at least one internal passageway; and assessing at least one of (i) immune cell adhesion to the functional endothelium, (ii) transendothelial migration of immune cells into the interstitial space, (iii) immune cell infiltration into the tumor microenvironment, or (iv) tumor cell killing by infiltrating immune cells.

2. The method of claim 1, wherein the tumor cells comprise patient-derived tumor cells.

3. The method of claim 2, wherein the patient-derived tumor cells are obtained from a tumor biopsy or surgical resection.

4. The method of claim 2, wherein the patient-derived tumor cells comprise patient-derived xenograft (PDX) cells.

5. The method of claim 1, wherein the tumor cells comprise tumor cell lines.

6. The method of claim 1, wherein the endothelial cells comprise patient-derived endothelial cells.Attorney Docket No.: PCT.13167. The method of claim 6, wherein the patient-derived endothelial cells are derived from the same patient as the tumor cells.

8. The method of claim 1, wherein the endothelial cells comprise iPSC-derived endothelial cells.

9. The method of claim 8, wherein the iPSC-derived endothelial cells are derived from the same patient as the tumor cells.

10. The method of claim 1, wherein the endothelial cells comprise human umbilical vein endothelial cells (HUVECs).

11. The method of claim 1, wherein the endothelial cells comprise tumor-associated endothelial cells.

12. The method of claim 1. further comprising embedding the tumor cells in an extracellular matrix prior to or during seeding within the interstitial space.

13. The method of claim 12, wherein the extracellular matrix comprises Matrigel.

14. The method of claim 12, wherein the extracellular matrix comprises a tumor-relevant extracellular matrix.

15. The method of claim 14, wherein the tumor-relevant extracellular matrix comprises one or more of collagen I, collagen IV, fibronectin, laminin, and hyaluronic acid.

16. The method of claim 12, wherein the extracellular matrix comprises decellularized extracellular matrix (dECM) derived from tumor tissue.

17. The method of claim 16, wherein the dECM is derived from the same patient as the tumor cells.Attorney Docket No.: PCT.131618. The method of claim 16, wherein the dECM is derived from a tumor tissue type-matched to the tumor cells.

19. The method of claim 1, wherein the scaffold comprises a porous hydrogel material configured to permit migration of immune cells.

20. The method of claim 1, wherein the at least one internal passageway comprises printed micropores connecting the passageway to the interstitial space.

21. The method of claim 20, wherein the micropores have a diameter in a range from 5 pm to 100 pm.

22. The method of claim 20, wherein the micropores are sized to permit passage of activated immune cells while maintaining endothelial coverage.

23. The method of claim 20, wherein the endothelial cells form a confluent layer over the micropores, and wherein activated immune cells transmigrate through the endothelial layer and the micropores to reach the interstitial space.

24. The method of claim 1. wherein the scaffold comprises a hydrogel with tunable porosity to modulate immune cell migration rate.

25. The method of claim 1, wherein the immune cells comprise T cells.

26. The method of claim 25, wherein the T cells comprise cytotoxic CD8+ T cells.

27. The method of claim 25, wherein the T cells comprise tumor-infiltrating lymphocytes (TILs).

28. The method of claim 25, wherein the T cells comprise chimeric antigen receptor T cells (CAR-T cells).

29. The method of claim 1, wherein the immune cells comprise natural killer (NK) cells.Attorney Docket No.: PCT.131630. The method of claim 29, wherein the NK cells comprise CAR-NK cells.

31. The method of claim 1, wherein the immune cells comprise peripheral blood mononuclear cells (PBMCs).

32. The method of claim 31, wherein the PBMCs are derived from the same patient as the tumor cells.

33. The method of claim 1, wherein the immune cells comprise macrophages.

34. The method of claim 33, wherein the macrophages comprise CAR-macrophages.

35. The method of claim 1, further comprising perfusing at least one therapeutic candidate through the at least one internal passageway.

36. The method of claim 35, wherein the therapeutic candidate is selected from the group consisting of immune checkpoint inhibitors, bispecific T-cell engagers, bispecific antibodies, monoclonal antibodies, antibody drug conjugates (ADCs), cytokines, chemokines, small molecule immunomodulators, oncolytic viruses, and tumor vaccines.

37. The method of claim 35, wherein the therapeutic candidate comprises an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, and anti-TIM-3 antibodies.

38. The method of claim 35, wherein the therapeutic candidate comprises a bispecific T-cell engager (BiTE).

39. The method of claim 35, wherein the therapeutic candidate is assessed for its ability to enhance immune cell recruitment, transendothelial migration, and / or tumor cell killing.

40. The method of claim 1, wherein assessing comprises flow cytometry analysis.Attorney Docket No.: PCT.131641. The method of claim 40, wherein flow cytometry analysis comprises characterizing immune cell phenotype before and after perfusion through the scaffold.

42. The method of claim 40, wherein flow cytometry analysis comprises quantifying immune cell activation markers selected from the group consisting of CD69, CD25, CD107a, granzyme B, perforin, IFN-y, and TNF-a.

43. The method of claim 40, wherein flow cytometry analysis comprises distinguishing immune cells recovered from a vascular lumen of the scaffold, versus immune cells that infiltrated the interstitial space.

44. The method of claim 40, wherein flow cytometry analysis comprises quantifying the ratio of immune cells to viable tumor cells following co-culture.

45. The method of claim 1, wherein assessing comprises live-cell imaging of immune cell trafficking.

46. The method of claim 45, wherein live-cell imaging comprises tracking immune cell adhesion, rolling, arrest, and / or transmigration at the endothelial surface.

47. The method of claim 45, wherein live-cell imaging comprises monitoring immune cell infiltration into the tumor microenvironment over time.

48. The method of claim 1. wherein assessing comprises confocal or multiphoton microscopy to visualize immune cell localization within the scaffold.

49. The method of claim 1, wherein assessing tumor cell killing comprises measuring tumor cell viability.

50. The method of claim 49, wherein tumor cell viability is measured by live / dead staining, ATP quantification, or LDH release.Attorney Docket No.: PCT.131651. The method of claim 1, wherein assessing tumor cell killing comprises quantifying tumor cell apoptosis by Annexin V staining or caspase activation.

52. The method of claim 1, further comprising measuring cytokines and / or chemokines in perfusate to assess immune activation.

53. The method of claim 52, wherein the cytokines comprise one or more of IFN-y, TNF-a, IL-2, IL-6, IL- 10, granzyme B, and perforin.

54. The method of claim 52, wherein the chemokines comprise one or more of CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL4. and CCL5.

55. The method of claim 1, further comprising gene expression analysis of tumor cells and / or immune cells recovered from the scaffold.

56. The method of claim 55, wherein gene expression analysis comprises single-cell RNA sequencing (scRNA-seq).

57. The method of claim 55, wherein gene expression analysis comprises spatial transcriptomics to assess regional patterns of immune infiltration and tumor response.

58. The method of claim 1, wherein the tumor cells, endothelial cells, and immune cells are all derived from the same patient.

59. The method of claim 58, wherein the method is used to predict patient-specific response to an immunotherapeutic agent.

60. The method of claim 1, wherein the tumor cells and immune cells are derived from the same patient, and the endothelial cells are derived from a different source.

61. A system for assessing immune cell recruitment and tumor cell killing, the system comprising:Attorney Docket No.: PCT.1316a bioprinted scaffold comprising a hydrogel material, at least one perfusable internal passageway lined with endothelial cells forming a functional endothelium, and an interstitial space containing tumor cells, wherein the scaffold is configured to permit migration of immune cells from the internal passageway to the interstitial space;a perfusion apparatus configured to deliver fluid flow containing immune cells and / or therapeutic candidates through the at least one internal passageway at a controlled flow rate; andone or more reservoirs for supplying culture media, immune cells, and / or therapeutic candidates to the perfusion apparatus;wherein the system is configured to enable assessment of immune cell recruitment, transendothelial migration, tumor infiltration, and / or tumor cell killing by at least one of: imaging, flow cytometry, collection of perfusate samples, and recovery of cells from the scaffold.

62. The system of claim 61, wherein the tumor cells comprise patient-derived tumor cells.

63. The system of claim 61, wherein the tumor cells are embedded in an extracellular matrix comprising decellularized tumor ECM.

64. The system of claim 61, wherein the at least one internal passageway comprises micropores sized to permit transendothelial migration of activated immune cells.

65. The system of claim 61, wherein the endothelial cells, tumor cells, and / or immune cells are derived from the same patient.

66. The system of claim 61, further comprising an imaging system configured for real-time monitoring of immune cell trafficking within the scaffold.

67. A method of assessing tumor cell killing, the method comprising:providing a bioprinted scaffold comprising at least one perfusable internal passageway and an interstitial space, wherein the interstitial space is coupled to the at least one internal passageway;seeding tumor cells within the interstitial space;Attorney Docket No.: PCT.1316culturing the seeded scaffold under perfusion conditions to establish a tumor model; perfusing at least one therapeutic candidate through the at least one internal passageway, thereby exposing the tumor cells to the therapeutic candidate via transport from the passageway to the interstitial space; andassessing tumor cell killing by at least one of (i) determining tumor cell viability, (ii) measuring tumor cell apoptosis, (iii) quantifying secreted biomarkers indicative of tumor cell death, or (iv) imaging tumor cell morphology and / or proliferation.

68. The method of claim 67, further comprising seeding endothelial cells within at least one internal passageway to form a functional endothelium.

69. The method of claim 67, wherein the therapeutic candidate is selected from the group consisting of antibody drug conjugates (ADCs), small molecule chemotherapeutics, targeted kinase inhibitors, monoclonal antibodies, bispecific antibodies, immunotoxins, oncolytic viruses, nucleic acid therapeutics, and radioligand therapeutic agents.

70. The method of claim 67, wherein the therapeutic candidate comprises an antibody drug conjugate (ADC) and the method assesses ADC-mediated tumor killing in a vascularized tumor model.

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