Mature endothelial tissue models, microchannel formation and perfusion, and vascularization of tissue beds

Abstract

Microfluidic in vitro endothelial tissue model devices and methods are described. A microfluidic device body comprising a microchannel cavity formed in the body, the microchannel cavity including a main microchannel channel connected to inlet and outlet ports. A plurality of fully mature peripheral blood-derived endothelial cells are deposited or otherwise situated within a matrix material on an inner surface of the main microchannel, the matrix material suitable for promoting or maintaining viability of the peripheral blood-derived endothelial cells and allowing the peripheral blood-derived endothelial cells to self-assemble into one or more tube. Method include generating tissue-penetrating vascular networks, and methods of improving yield and ensuring robust and reproducible peripheral blood-derived endothelial cells isolation while generating mature endothelial cells from patient peripheral blood-derived endothelial progenitor cells. Some methods include isolating proliferative peripheral blood-derived endothelial cells from limited blood sample volumes and exposing progenitor cells to arterial hemodynamic conditions.

Description

MATURE ENDOTHELIAL TISSUE MODELS, MICROCHANNEL FORMATION AND PERFUSION, AND VASCULARIZATION OF TISSUE BEDSCross-Reference to Related ApplicationsThis application is entitled to and claims the benefit and priority of earlier filed United States provisional patent application No. 63756213, filed February 9, 2025, under 35 U.S.C. § 119(e). which earlier filed application is incorporated by reference herein in its entirety.

[0001] BACKGROUND INFORMATION

[0002] Technical Field

[0003] The present disclosure relates generally to biotechnological devices and methods, and more particularly to endothelial tissue models, passive cylindrical microchannel formation, unidirectional perfusion of microchannel arrays, and vascularization of tissue beds.

[0004] Background Art

[0005] There is a substantial need to model human physiology in nonclinical settings to study human medicine, identify contributors to and mechanisms of diseases, discover disease-correlated biomarkers, test new and existing therapeutics and medical devices, and inform clinical decision making and treatment strategies. Moreover, due to genetic and environmental differences and inherent biological stochasticity, there are substantial heterogeneities in physiological characteristics, manifestations of diseases, and performances of and responses to pharmaceutical agents across the human population. In nonclinical biomedical research and drug development, there has historically been and remains a strong reliance on animal models to represent human physiology, pathophysiology, and response to stimuli. Results from compound testingin these models are used in applications to regulatory organizations seeking approval for in-human clinical trials. However, due to the inherent differences between animals and humans at physiological, tissue, cellular, and genetic levels, conclusions drawn from testing in animals often do not agree with results from testing in humans. Moreover, animal models cannot capture the patient-to-patient variations in manifestations or governing mechanisms of diseases, clinically observed disease severity, or individual patients’ responses to drugs and other therapeutic interventions.

[0006] In clinical trials of new drugs or medical devices, a limited number of patients are recruited wherein therapeutic candidates are tested in sample groups intended to represent the human population. However, patient recruitment and confirmation of mechanisms of disease alteration in patients with varying physiologies and coexisting conditions by prospective therapeutic agents remain significant challenges. As a result of the limitations of current preclinical and clinical testing methods, there is a high failure rate of new prospective therapeutics (some estimates indicate that over 90% of drugs entering clinical trials ultimately fail).

[0007] Human-based advanced in vitro technologies like spheroid, organoid, and organ-on-chip (i.e., microphysiological) models have been developed in efforts to bridge gaps between preclinical testing methods with limited human relevance and clinical in-human testing. However, existing models typically rely on non-diseasespecific, healthy cell sources or cells altered by methods that differ substantially from natural occurrences. That is, these models often use pooled cell batches or stem cell-derived cells that lack patient- specificity and / or require expensive, time-consuming processing that dilute contributors to tissue-, disease-, and patient-specific characteristics.

[0008] In the context of vascular biology and relevant applications in drug testing, models of blood vessels typically use primary endothelial cells sourced directly from tissue / organ donors. These sources are highly limited and only represent small subsets of the human population. Additionally, primary cell isolation methods are inherently invasive and therefore are limited to donors at end of life or those undergoing invasive medical procedures for other reasons. Common examples include: 1) human umbilical vein endothelial cells (HUVECs), widely used due to their accessibility, often involve pooling from multiples donors and are commonly employed to represent endothelia throughout the body; 2) human microvascular endothelial cells (HMVECs), derived from a range of tissues such as the skin, lungs, or heart; and human pulmonary artery endothelial cells (HPAECs). human aortic endothelial cells (HAECs), and human saphenous vein endothelial cells (HSaVEC), which have highly limited availability.

[0009] While these products address certain research needs, their inherent limitations (limited accessibility, donor cell pooling) underscore a demand for more accessible sources of endothelial cells that can be used to create accurate models of patient vasculature for biomedical research and product screening.

[0010] Advances in induced pluripotent stem cell (iPSCs) technology have led to the creation of stem cell-derived endothelial cells (iPSC-ECs or iECs). However, there are many drawbacks of using these cells in preclinical research that limit their adoption and the accuracies of resulting predictions of physiology and drug response: 1) methods of generating precursor / stem cells and differentiating cells effectively are timeconsuming and require highly sophisticated skills to obtain a phenotypically pure cell population; iPSC differentiation protocols are highly sensitive to applied differentiation factors, and their administration must be done sequentially in carefully timed procedures; iPSC-derived cells are extremely sensitive, and differentiation proceduresgenerally have low success rates; iPSC differentiation processes are difficult to scale due to process duration, procedural precision, and the resource requirements; protocols for differentiating stem cells yield “immature” cell phenotypes and are highly limited in their abilities to faithfully emulate physiological hallmarks of mature adult cells for extended passages; de-differentiation of starting cell sources and re-differentiation of stem cells cause cell populations to lose important transcriptomic and epigenetic influences in the body.

[0011] Despite having advantages over healthy and pooled cell sources, these challenges and limitations make iPSC-derived ECs less suitable for use in high throughput research and testing and highlight the need for an alternative cell source that is easy to access and can therefore achieve sufficient scale to study diseases and drug responses across large patient populations.

[0012] Alternatively, researchers have identified endothelial-like cell types in umbilical cord blood and peripheral blood. These biofluids contain endothelial progenitor cells (EPCs), which are immature cells that, in the body, are involved, either through directly replenishing or by stimulating replenishment through paracrine signaling, in wound healing processes and growth of new blood vessels. Most EPCs found in circulating blood are of hematopoietic lineage, but some subpopulations are considered to be non-hematopoietic (there is some disagreement in scientific literature on this). When cultured in vitro, these progenitor cells are often referred to as endothelial colony forming cells (ECFCs) or outgrowth endothelial cells (OECs), which are, broadly, immature unipotent stem cells that have committed to endothelial differentiation paths. Importantly, EPCs and their in vitro analogs express key markers that reveal their commitment to endothelial cell types and their immature state. Specifically, these cells typically express proteins including hematopoietic marker CD34 and some standardendothelial markers (often at lower levels than mature cells, particularly at early stages of differentiation). However, they also express markers of immaturity including surface and / or intracellular expression of CD 133 and c-kit (CD117) and lack proteins found in fully mature endothelial cells like CD31 and high expression of Von Willebrand Factor (VWF). There is scientific literature covering the use of EPCs and ECFCs on various kinds of medical devices and in vitro culture systems, predominantly with the intention of exploring uses of these cell types as therapeutic and regenerative medicine modalities. Notably. Mathur et al. demonstrated the use of OECs. defined specifically as a form of EPCs, on a microfluidic device to model endothelial function and dysfunction. However, this device, and other applications of ECFCs for in vitro research and compound testing specifically use cells that are still in immature progenitor states. Conversely, the generation and use mature cells from peripheral blood-derived EPCs has not been explored. As a result, the accuracy of their behaviors and responses to applied molecules is limited.

[0013] Hemodynamic forces, including shear stress, are well known to significantly influence stem cell differentiation and phenotypes of immature and mature cell and tissue cultures across the . Studies show that applying organotypic flow patterns to ECs contributes to induction of tissue-specific characteristics. For example, high shear stress can condition ECs towards an arterial fate whereas lower shear conditions direct ECs towards a venous fate. Co-culturing with tissue-specific cell types has also been shown to enhance organotypic characteristics of the endothelium. For example, coculturing ECs with pericytes and astrocytes strengthens tight junction formation and selective permeability, characteristics associated with neurovascular units. These principles provide valuable insights for advancing tissue-specific endothelial biology, but their applications have largely been demonstrated in isolation and have never beenapplied to cell sources that can be acquired with minimal invasiveness and without requiring use of stem cells.

[0014] In addition to cell sources, cell culture systems and engineered tissue culture device architectures are highly important in dictating value of advanced in vitro model technologies. Microfluidic platforms are often employed to model many tissues throughout the body, owing to their ability to introduce biophysical stimuli such as intraluminal shear stress and biochemical factors like exogenous growth factors or cytokines. Various strategies are employed but can be broadly summarized as follows:

[0015] Monolayers seeded in microchannels, where cells are cultured directly onto the surfaces of microchannels to form monolayers, is a simple and very common approach wherein fluid flow can be used to improve traditional static cell culture methods. As an example, Ibidi GmbH offers devices and rudimentary perfusion systems that push fluid through small fluidic chambers on which users can place cell monolayers to culture under fluid flow conditions. However, two-dimensional monolayers do not replicate the 3D tissue structures, which limits these approaches’ predictive power for modeling health and disease processes. Further, these monolayers are often situated on plastic substrates that are stiffer than the typical extracellular matrix supporting in vivo tissues, which is known to decrease the accuracy of cell and tissue culture behaviors.

[0016] Monolayers patterned in channels within hydrogels: to address the previous limitation related to substrate stiffness, some groups pattern hydrogels within microfluidic devices to feature hollow channels. For example, some researchers and companies have devices upon which acupuncture or similar needles are fixed, hydrogel solutions are poured around the needles and cured, and the needles are removed leaving small hollow cavities within larger hydrogel bodies. Cells can be seeded on the innersurface of these channels, replicating the cylindrical shape many cell layers in the body have. These structures and hydrogel biomaterials better replicate tissue microenvironments but at the cost of scalability and limited complexity; current patterning processes are typically labor-intensive, incompatible with standard cell culture plastics, and / or constrained to diameters that limit their applicability across different tissue types. Further, these approaches can only create single cell type monolayers thereby restricting model accuracy due to absence of supporting and communicating cell / tissue types.

[0017] Monolayers situated across barriers: Many device designs implement synthetic barriers or tissue separation regions to provide mechanisms by which multiple cell types can be cultured in some form of communication with one another. Typically, cells are cultured on either side of a porous membrane or other separating structure opposite other cell types. For example, some use porous plastic materials where cells can be seeded on either side of thin sheets. In other cases, plastic micropillar structures or microchannels filled with viscous hydrogel materials are used to separate independent microchannels containing different cell types found in close proximity or indirect communication in the body. These arrangements create conditions for indirect and often inaccurate cell-cell communication processes to occur. While these methods are compatible with traditional cell culture plasticware (e.g., Transwell inserts in wellplates), they do not typically create tissue structures that match those in the body and involve separating different cell types by non-physiologic distances or artificial materials and device geometries.

[0018] In 2012, Bischel et al. published a study in which viscous finger patterning - a natural physical process by which less viscous fluids, driven by capillary and / or other pressures, “tunnel” through more viscous fluids creating fluid-filled cavities therein.This process was demonstrated on a simple microfluidic device wherein a horizontal microchannel was connected by two vertical cylindrical ports and later demonstrated to be capable of achieving multi-step viscous finger patterning to create multiple cell layers surrounding a single microchannel. Despite the potential of this approach to create cylindrical or other rounded microchannel shapes, adoption has been very limited due to the inherent instability of the viscous fingering process (dictated by a principle called Saffman-Taylor instability) leading to inconsistent channel formations. Since then, multiple studies have been published the use of this approach wherein pipette tips are used to extend the distance that the Saffman-Taylor instability fluid front travels prior to entering the horizontal microchannel region to achieve an increase in viscous finger stability. However, this approach comes with a substantial drawback as there is a substantial requirement for careful manual manipulation of inserted pipette tips upon injection and tip removal to avoid microchannel collapse resulting from hydrogel disruption.

[0019] Another property governing a microfluidic cell / tissue culture system’s accuracy, longevity, etc. is the presence and nature of transport phenomena like fluid movement and associated biophysical cues that mimic native tissue microenvironments. These phenomena and biophysical cues can include:

[0020] Intraluminal flow: the application of intraluminal flow is essential for replicating the nutrient, oxygen, and signaling molecule transport and the shear stress experienced by vasculature in vivo. Normal shear stress has been shown to regulate EC alignment, tight junction integrity, and homeostatic processes while abnormal shear stress can induce severe tissue dysfunction and disease. Traditional methods for generating flow in vitro include syringe pumps and hydrostatic pressure heads.However, these often fail to achieve the sustained, controlled fluid flow profiles required for effective, long-term tissue culture.

[0021] Interstitial flow: mimicking fluid movement through an extracellular matrix can play a vital role in nutrient transport, waste removal, and the distribution of biochemical signals within a tissue culture system. For example, interstitial flow in tumor microenvironment models has been shown to influence cancer cell migration, drug delivery, and immune cell trafficking. These same phenomena significantly affect organoid development, tissue polarization, and culture longevity. However, bioreactor platforms that reliably produce controlled interstitial flow rates and patterns are not currently available.

[0022] Cyclic strain and mechanical loading: many tissues in vivo experience mechanical forces such as cyclic strain and compression. These forces induce tissue remodeling and affect cellular gene expression in health and disease states. For example, cyclic strain has been demonstrated to upregulate genes involved with extracellular matrix remodeling that is critical for vascular integrity and wound healing. However, scalability and reproducibility of culture platforms with cyclic strain capabilities remain challenges, particularly in high-throughput applications.

[0023] Importantly, many of these biophysical elements are supplied by external and ancillary equipment, which severely limits scalability of a set of models. Common methods of increasing scalability often rely on eliminating external hardware and instead controlling flow and pressure distribution within the microfluidic systems themselves.

[0024] Tissue engineering and regenerative medicine have experienced significant advancements in developing three-dimensional (3D) tissue constructs that mimic some structural and functional properties of native tissues. These constructs are valuable for applications in developmental and systems biology research, drug screening, disease modeling, and therapeutic implantation. A critical challenge in the development and maintenance of 3D tissue constructs is achieving necessary nutrient and oxygen transport, particularly in larger or densely populated tissue models. To address this challenge, vascularization - the formation of networks of interconnected micro vessels - has emerged as a critical focus area in research and development. Self-assembly of tissues is one method by which perfusable vasculature may be assembled on in vitro platforms.

[0025] Tissue self-assembly is often used to act as mimics of naturally occurring biological processes of angiogenesis and vasculogenesis. In angiogenesis methods, a parent blood vessel is typically patterned in a device, and new daughter blood vessels are coaxed and directed towards a growth factor source, either exogenously supplied factors or paracrine signaling by other cell types. After some time, these sprouts can form interconnected networks, anastomosing to build a perfusable capillary network. Vasculogenesis, on the other hand, is a bottom-up tissue engineering strategy, where individual ECs are embedded in a hydrogel, sometimes with supporting stromal cell types such as fibroblasts or multipotent stromal cells and are coaxed to form a perfusable capillary network via paracrine signaling from supporting cells or exogenous growth factor supplementation in the cell culture medium. This strategy is more commonly employed due to the low user input required as model assembly typically only requires injecting a cell-laden hydrogel into a microchannel and then maintaining the device with media changes. While these requires more tissueengineering than microfluidics engineering, microfluidic devices are commonly employed because they enable easy cell compartmentalization and a route for perfusing the self-assembled vasculature.

[0026] Another method to increase model complexity is by directly inducing angiogenesis or vasculogenesis to fenestrate a secondary cell construct with capillary blood vessels, mimicking a tissue and the relevant microenvironment. The secondary construct, often a spheroid composed of a single cell type or a more complex multicellular organoid model, is embedded within a hydrogel. In these systems, parent blood vessels sprout toward the construct (angiogenesis), or hydrogel-embedded ECs self-assemble in place to form a capillary network surrounding the construct (vasculogenesis). These vessels are typically demonstrated to be perfusable by perfusing fluorescent Dextran or microbeads, but the ability to induce vascularization through the secondary construct remains highly limited. For instance, vascularization frequently forms around the outer surface of the construct, but this configuration fails to accurately facilitate material transport into the construct's interior, leaving it vulnerable to hypoxia and necrotic core formation. In other cases, vascular integration into the construct is achieved during embryoid body formation by co-seeding vascular precursors or fragments. These constructs are subsequently transferred to microfluidic devices. However, connections between the microfluidic system and the vascular fragments are often incomplete, leaving the fragments inaccessible by perfusion and reducing their functional relevance. Some researchers have reported the use of “Blood Outgrowth Endothelial Cells” (BOECs) that are “isolated from patients” and seeded on inner surfaces of microfluidic channels. These BOECs are defined specifically as endothelial progenitor cells (EPCs). If the definition in the patent application referencing this work is taken, BOECs simply refer to immature EPCs taken from blood. OrbusNeich developed the COMBO Stent that, after implantation, captures apatient’s circulating PB-EPCs and promotes their differentiation to mature ECs for the goal of re-endothelializing the implanted stent. This does not involve any microfluidic device applications or tissue models and occurs in vivo.

[0027] In the art of microfluidic devices, conventional microfluidic devices feature cylindrical vertical inlet ports with uniform diameters. Multiple organ-chip researchers and companies cast gels around acupuncture or similar needles which act as sacrificial molds for creating channels when the needles are removed. Tissue engineered blood vessels (TEBVs) are created using layer-by-layer gelation of cell laden collagen gels around a steel mandrel, similar to the acupuncture needle method described above. Some have demonstrated formation of cylindrical microchannels by viscous finger patterning wherein low viscosity fluids were placed on top of cylindrical ports connected to microchannels containing high viscosity fluids. Some other researchers modified the viscous finger patterning method above whereby pipette tips containing viscous fluids were placed in microfluidic device inlet ports prior to application of lower viscosity fluids (inside the pipette tips) to induce viscous finger patterning. After the viscous fingering process occurs, pipette tips are removed from ports (which often causes gel collapse). Multiple bioprinting groups use bioinks containing cells to print cylindrical structures.

[0028] In the art of microfluidic platforms, companies like Draper and CN Bio market microfluidic platforms that have pumps built into their devices. Draper uses electrically actuated micropumps with two pumps per microchannel, and CN Bio uses sequential membrane deflections to move fluid for one tissue unit per pumping unit. Other similar technologies, such as Mimetas’ OrganoPlates and Hesperos’s body-on-a-chip, create perfusion through a rocking mechanism that alternates the hydrostatic pressure at the microchannel inlets and outlets. ElveSys’s ElveFlow system uses pneumatic pressureapplied to large fluid reservoirs to drive fluid. However, among other limitations, this approach does not enable on-device perfusion or recirculation of individually addressable channels.

[0029] Competitor technologies include Mimetas’ OrganoPlate, Qureator’s CurioChips and U-IMPACT platform, AIM Biotech’s idenTx, and Acari Biosciences’ Vascularized Micro-Organ platforms. In each of these, a microchannel housing a tissue bed - with organoids or similar cell constructs - is patterned adjacent to one or more microchannels containing “parent vessels” that serves as the source of fluid perfusion to microvascular networks. Importantly, the flow direction through the microvascular network is perpendicular to the perfusion source, which limits reproducibility and control of important conditions. Qureator, VivoDyne, AIM Biotech, and Aracari Biosciences also use a vasculogenesis method of vascularizing hydrogels that involves embedding endothelial cells and supporting cells in a hydrogel solution, injecting the solution into the microfluidic device, allowing hydrogel solidification, and then hydrating with static cell culture medium. In some cases, other tissues are mixed into the applied hydrogel solution. In each case, the endothelial cells self-assemble into vascular networks, and in the case of co-culture with organoids, form in the region adjacent to the tissue culture without penetrating into the tissue construct.

[0030] As may be seen, current practices may not be adequate for all circumstances, and do not address the noted problems with respect to tissue models, passive cylindrical microchannel formation, unidirectional perfusion of microchannel arrays, and vascularization of tissue beds. There remains a need for more robust devices and methods. The devices and methods of the present disclosure are directed to these needs.

[0031] SUMMARY

[0032] In accordance with the present disclosure, devices and methods are described which reduce or overcome many of the faults of previously known devices and methods.

[0033] A first aspect of the disclosure are microfluidic in vitro endothelial tissue models (devices) comprising (or consisting essential of. or consisting of) mature peripheral blood-derived endothelial cells (PBECs), and methods of using same. The devices and methods of the present disclosure mark the first application of fully mature cells acquired by differentiation of progenitor cells from peripheral blood on microfluidic devices to create in vitro endothelial tissue models. Here, we cover the use of any fully mature ECs (defined based on surface markers) generated from PB-EPCs on microfluidic devices to create tissue models. To our knowledge, although these concepts have been explored separately for different reasons, there has been no suggestion of complete in vitro differentiation / maturation of cells from blood-derived progenitors and subsequent application on microfluidic devices.

[0034] A second aspect of the disclosure are devices (referred to herein as microdevices) comprising (or consisting essential of, or consisting of) a conical inlet port for passive formation of cylindrical microchannels in viscous fluids, and methods of using the same to create cylindrical microchannels in viscous fluids. The microdevices of this aspect of the present disclosure allow passive methods for creating cylindrical microchannels using a conical inlet port structure that directs and focuses a passive viscous finger patterning (VFP) technique. Conventional microfluidic devices use cylindrical inlets, and variations from this shape is highly uncommon for any purpose. While it has been proposed that adding pipette tips at the inlet ports providesextra traveling length for the patterning flow to stabilize, patterning conical shapes directly into the body of the devices to avoid the need for often disruptive device handling and manipulation has never been suggested. The conical structure achieves unique phenomena not previously explored including harnessing differences in hydrophilicity between materials, implementing passive patterning not requiring user interfacing with the device, and a reduction in Saffman-Taylor instability by fluid focusing and entropy reduction.

[0035] Another aspect of the present disclosure are microfluidic devices enabling onboard pneumatic unidirectional fluid recirculation. This device utilizes an innovative combination of pneumatic pressurization of fluids, well plates as reservoirs, and pneumatic valve-operated recirculation lines to achieve controlled recirculation. Using only two controlled actuation elements, the proposed platform can create unidirectional flows and fluid recirculation of arrays of perfusable channels (with or without gels in the channels). The process used in this device involves a unique design and operating principle whereby ports for two microchannels share a common overhead fluid reservoir, rely on precise design of microchannel diameters to create a large difference in flow resistances, utilize open-top wells to actuate membrane-based valves that dictate fluid paths, and includes a device lid with manifolds for air distributions to fluid reservoirs, thereby combining multiple uncommon or unprecedented features in microfluidics engineering.

[0036] Still another aspect of the present disclosure are methods of generating tissuepenetrating vascular networks using hydrogel pre-vascularization and a removable mold for tissue injection. The methods of this aspect of the present disclosure represent a new approach for integrating microvascular networks and 3D tissue culture wherein a sequential process of vascular network formation around a biocompatible moldingrod is followed by tissue culture injection and vascular network permeation into the tissue. These methods are non-obvious because they apply existing techniques in an unusual sequence never previously suggested and by using a new device design and method for creating a removable mold in a pre-vascularized hydrogel.

[0037] In certain embodiments a logic device may be provided to control all or portions of the devices and methods of the present disclosure, and the logic device may be configured to be operated and / or viewed from a Human / Machine Interface (HMI) wired or wirelessly connected to the logic device. Certain embodiments may include one or more audio and / or visual warning devices configured to receive communications from the logic device upon the occurrence of a pressure rise (or fall) in a sensed pressure above (or below) a set point pressure, or a change in concentration of one or more sensed concentrations or temperatures, or both, above one or more set points. The occurrence of a change in other measured parameters outside the intended ranges may also be alarmed in certain embodiments. Other measured parameters may include, but are not limited to, liquid or gas flow rate, and liquid density.

[0038] Certain device and method embodiments of this disclosure may operate in modes selected from the group consisting of automatic continuous mode, automatic periodic mode, and manual mode.

[0039] In certain embodiments, pressure (P), temperature (T), density, and / or mass flow may be sensed inside or at any location on the devices, or prior to entering a device, or after exiting a device. Mass flow sensors may be employed. All combinations of sensing T, P, density, and / or mass flow are disclosed herein and considered within the present disclosure.

[0040] These and other features of the devices and methods of the present disclosure will become more apparent upon review of the brief description of the drawings, the detailed description, and the claims that follow. It should be understood that wherever the term “comprising” is used herein, other embodiments where the term “comprising” is substituted with “consisting essentially of’ are explicitly disclosed herein. It should be further understood that wherever the term “comprising” is used herein, other embodiments where the term “comprising” is substituted with “consisting of’ are explicitly disclosed herein. Moreover, the use of negative limitations is specifically contemplated; for example, certain devices may be devoid of carcinogenic compounds.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The manner in which the objectives of this disclosure and other desirable characteristics can be obtained is explained in the following description and attached drawings in which:

[0043] FIG. 1 is a schematic illustration depicting one embodiment wherein PBECs (labeled here as ECs) can be applied to the inner surface of a square or rectangular microfluidic device;

[0044] FIGS. 2A and 2B schematically illustrate that in some embodiments, PBECs can self-assemble within hydrogel matrices to create microvascular networks under influences of growth factor gradients, mural cell components or tissue organoids, and multichannel microfluidic devices can be leveraged for de novo creation of new microvascular networks in vitro;

[0045] FIG. 3 is a schematic illustration overview of one VFP process in accordance with the present disclosure;

[0046] FIG. 4 is a schematic sectional view of a microchannel with truncated cone shaped ports, the specific geometry and dimensions shown here represent one possible design;

[0047] FIGS. 5 A, 5B, 5C, and 5D are schematic cross-sectional views illustrating the effects of the conical shape ports in directing the path of the viscous finger patterning process;

[0048] FIG. 6 schematically illustrates steps associated with applying a viscous finger patterning process of the present disclosure using the truncated conical ports in accordance with the present disclosure;

[0049] FIGS. 7A and 7B are schematic illustrations of a 96 well plate lid with overhead control lines A and B, with FIG. 7A being a schematic bottom plan view of the lid and patterned control lines within the body, and FIG. 7B being a sectioned view along line A-A’ showing 8 outlets that align over the centers of the wells;

[0050] FIG. 8 is a schematic illustration of a recirculation device microchannel format, with main channels, recirculation lines with valve pads, and pneumatic lines that actuate valves;

[0051] FIGS. 9A and 9B schematically illustrate one assembly and configuration of the device for dual vascularization of tissues and tissue beds;

[0052] FIGS. 10A, 10B, and IOC are schematic illustrations and description of one process for creating a cavity (using a mandrel), pre-vascularizing a gel, and introducing a tissue for vascularization;

[0053] FIGS. 11A, 1 IB, and 11C are schematic illustrations of a variation of the device wherein a barrier layer is fabricated in the lower chamber;

[0054] FIGS. 12A, 12B, and 12C are schematic illustrations of a variation of the device wherein multiple tissues are connected and vascularized; and

[0055] FIGS. 13A, 13B, and 13C are schematic illustrations of a variation of the device wherein a skin graft is placed on top of the device and introduced to UV to study its effects on skin diseases.

[0056] It is to be noted, however, that the appended drawings are not to scale, and illustrate only typical device and method embodiments of the present disclosure. Therefore, the drawing figures are not to be considered limiting in scope, for the disclosure may admit to other equally effective embodiments. Identical reference numerals are used throughout the several views for like or similar elements.

[0057] DETAILED DESCRIPTION

[0058] In the following description, numerous details are set forth to provide an understanding of the disclosed devices, combinations, and methods. However, it will be understood by those skilled in the art that the devices and methods disclosed herein may be practiced without these details and that numerous variations or modifications from the described embodiments may be possible. All technical articles, published andnon-published patent applications, standards, patents, statutes and regulations referenced herein are hereby explicitly incorporated herein by reference, irrespective of the page, paragraph, or section in which they are referenced. Where a range of values describes a parameter, all sub-ranges, point values and endpoints within that range or defining a range are explicitly disclosed herein. All percentages herein are by weight unless otherwise noted. In the event definitions of terms in the referenced patents and applications conflict with how those terms are defined in the present application, the definitions for those terms that are provided in the present application shall be deemed controlling. Where a range of values describes a parameter, all sub-ranges, point values and endpoints within that range are explicitly disclosed herein. This document follows the well-established principle that the words “a” and “an” mean “one or more” unless we evince a clear intent to limit “a” or “an” to “one.” For example, when we state “a microchannel”, we mean that the specification supports a legal construction of “a microchannel” that encompasses structure distributed among multiple physical structures.

[0059] Microfluidic in vitro endothelial tissue models using mature peripheral blood-derived endothelial cells (PBECs)

[0060] The present disclosure describes the use of mature endothelial cells generated from endothelial progenitor cells (EPCs) acquired from peripheral blood on microfluidic devices for the purpose of modeling the biology and physiology of endothelial cells, endothelial tissues, and interactions between endothelial cells and other cell and tissue types. Creating microfluidic devices with mature peripheral blood-derived endothelial cells on them begins with the collection and isolation of EPCs from peripheral blood containing many cell types and other particles. There are multiple methods that have been previously demonstrated for the isolating EPCs from peripheralblood. These methods, and others not described here, can be used to isolate this progenitor cell type for downstream processing into mature endothelial cells as used for the embodiments of the present disclosure described herein.

[0061] Density gradient centrifugation (DGC): The most common approach for collecting EPCs from whole blood uses DGC. This methodology involves addition of particular solutions with known densities to samples of interest to generate layers of materials of differing densities followed by centrifugation steps that cause cells and molecules within the starting solution to separate according to their density. There are multiple protocols whereby density gradient centrifugation can be applied to isolate populations of cells containing EPCs. The information below represents only one such method and does not represent the invention described in this section. Briefly, some volume (typically, 30-50 mL) of whole blood collected (usually in the presence of an anticoagulant (for example, blood can be collected in heparin-functionalized blood collection tubes that can be purchased for this purpose) is mixed with an equal volume of IX PBS or Hanks Buffered Saline Solution (HBSS) using mechanical pipettes, vortex instruments, or manual shaking of the solution. Then, 30 mL of the diluted blood is carefully layered over 15 mL of DGC medium in separate 50 mL centrifugation tubes. Once all the blood has been layered across multiple tubes, the tubes are centrifuged at 540 x g for 40 minutes at room temperature without special break or acceleration settings. After centrifugation, a distinct white “buffy coat” layer of peripheral blood mononuclear cells (PBMCs) can be observed above the DGC medium layer. The buffy coat is carefully (avoiding collecting any of the layer below) extracted using pipetting and added to fresh centrifuge tubes. An equal volume of IX PBS or HBSS is then mixed in the tubes, and tubes are centrifuged at 300 x g for 20 minutes to separate the PBMCs and remove and traces of cross-contaminating red blood cells (RBCs) or platelets. The supernatant is discarded, and collected cells are resuspendedin growth media. At this point, the cell populations are ready for plating. This method results in collection of all PBMCs (of which only a fraction are EPCs).

[0062] Using magnetic assisted cell sorting (MACS) kits: Another common approach of collecting EPCs is to use MACS kits (many of which can be purchased). Briefly, whole blood is collected and diluted with an equal volume of IX PBS or HBSS. Magnetic beads functionalized with CD34 antibodies are then introduced in the diluted blood samples and incubated according to protocols established by magnetic bead manufacturers. After incubation, the tubes containing diluted blood and beads are placed over or adjacent to magnetic arrays that separate the magnetic beads and cells that are attached to them (CD34+ cells) from the rest of the solution. The rest of the solution is carefully aspirated, and bead-cell suspensions are washed thoroughly with IX PBS or HBSS, after which the magnetic separation can be repeated to remove any traces of unconjugated cell types. The collected cells can then be detached from the magnetic beads using buffers provided or recommended by manufacturers corresponding to the types of beads used. The cells are then ready to be plated for downstream culture. In some cases, this process can be merged with DGC to first collect PBMCs and then purify the PBMC population to selectively collect EPCs cells using MACS.

[0063] Using cell capture micro- or milli-fluidic bioreactors: Cell capture on micro- or millifluidic devices is a technique that can be used to isolate specific cell populations. Briefly, a microfluidic or millifluidic bioreactor can be fabricated using standard microfabrication approaches (e.g., photo- and soft lithography, 3D printing, milling). Using common surface functionalization techniques (e.g., EDC-NHS coupling), the inner surfaces of microfluidic bioreactors can be functionalized with antibodies matching cell surface markers (e.g., CD34 in the case of hematopoietic stem cells andendothelial progenitor cells). Once the functionalization is complete, blood, huffy coats, or PBMC populations (for instance, collected after DGC) can be introduced in the channels / chambers and allowed to conjugate with the functionalized surfaces. Incubating the device upright will allow cells to attach preferentially to the bottom surface due to gravity. After incubation in an incubator, the unattached cell can be washed away by perfusing fluid using pipettes, gravity, or other means.

[0064] Conventionally, once PBMCs or EPCs have been collected using any of the methods described above, these cell populations are cultured in standard plastic containers (e.g., culture flasks or well plates) under static fluid conditions with traditional endothelial cell culture media. One common existing method to culture collected EPCs into robust endothelial cells is to plate EPCs on collagen-coated flasks and maintain cells with isolation medium (EGM2+20% fetal bovine serum) using standard cell culture practices. In cases where PBMCs containing EPC subpopulations are collected, prolonged culture of these populations with endothelial cell culture media, followed by lifting cells (using cell detachment enzyme solutions) and replating them with endothelial culture media, typically induces death of non-endothelial cell types and emergence of EPCs as the dominant cell type in the culture environment. Once these EPCs are isolated, they are traditionally maintained using standard culture methods and studied / applied in their immature progenitor form.

[0065] Conversely, this invention involves, specifically, the use of mature endothelial cells derived from PB-EPCs. There are methods for generating mature ECs from these progenitors that have been studied and published in scientific literature. Supplements like osteoprogerin (OPG), soluble CD 146, VEGF, and / or SDF-1 can be supplemented to culture media. Studies have demonstrated that these factors can reduce the time for emergence of early EPC colonies and / or increase colony yields. Studies have alsodemonstrated that using xenofree conditions and the added potency of platelet lysate-derived growth factors can yield twice the number of colonies per mL of blood. Replacing animal derived fetal bovine serum with human platelet lysate (HPL) as a suitable serum substitute can improve culture quality and maturation. Finally, fluid shear stress has been shown to improve maturity by better replicating forces that endothelial cells experience in the body, particularly in later stages of attachment during tissue repair. The “maturity” of cell populations after these treatments, and others not described here, are applied can be explored based on some cell surface markers and functional characteristics of endothelial cells. However, these analyses and the studies upon which they are based have been addressed specifically to understanding maturation processes and the factors influencing them in the context of using EPCs in vivo as agents for regenerative medicine to repair and replace damaged blood vessels. The use of mature cells derived from EPCs as sources for in vitro models of human vascular tissue has not yet, to our knowledge, been suggested even conceptually.

[0066] Distinct from the few existing applications of peripheral blood-derived endothelial progenitor cells on microfluidic devices, we have identified and utilize mature endothelial cells derived from peripheral blood, which we refer to herein as peripheral blood endothelial cells (PBECs), acquired by differentiation / maturation of peripheral blood endothelial progenitor cells (PB-EPCs) on microfluidic devices. These mature PBECs are characterized by the absence, or minimal expression of. stem cell markers that can be measured using standard molecular biology techniques that analyze ribonucleic acid (RNA) and / or protein content in cells (such as quantitative polymerase chain reaction techniques, western blotting, or other similar methods). Specifically, immature endothelial progenitors maintain expression of one or both of stem cell markers CD133 and CD117 (c-kit). Conversely, mature endothelial cells express none,or very low, levels of both of these markers; that is, they are CD133 CD117’, CD133" CD1171OW, CD133lowCD117’, or CD133lowCD117low. We use these characterizations to define mature PBECs that are applied on microfluidic devices as described below. One skilled in the art can also appreciate that absence of or increased expression of other markers may be found on mature endothelial cells derived from EPCs including, but not limited to, markedly increased expression (relative to levels observed in EPCs after isolation from blood) of CD 146, Von Willebrand Factor (VWF), and / or vascular endothelial cadherin (VE-Cad).

[0067] In certain embodiments, mature PBECs may be used on microfluidic devices to create in vitro models of human vasculature. These embodiments can be divided into three approaches:

[0068] Replicating individual vessels: In these embodiments, a layer of PBECs is formed on the inner surface of a channel or cavity within a microfluidic device body. These channels, and the PBEC layer, can be formed by multiple possible methods. Some, but not all, possible methods / forms include:

[0069] Using one or more prefabricated microchannel created by standard microfluidic device fabrication methods such as photo- and soft lithography, 3D printing, and milling. In some cases, pre-formed microchannels are filled and coated with dilute solution of gelatin, collagen, fibronectin, and / or other materials to increase potential for cell attachment and mimic physiological basement structure of human blood vessels. PBEC suspensions can be injected into access ports or other cavities that are used to access microfluidic channels in device bodies using pipettes, syringes, or other injection devices. These injection devices can involve application of positive pressure to drive the fluids into microchannels, or, in embodiments where device dimensionsallow, PBEC-containing solutions can be allowed to move into the microchannel by gravitational or capillary forces.

[0070] 3D bioprinting can be used to pattern tubular structures with PBEC layers using programmable printers and circular nozzles that produce cylindrical tubular structures.

[0071] A cylindrical vascular lumen can be patterned through a thicker volume of matrix material and / or gels using principles of viscous patterning or by applying viscous fluids or gels around previously injected small rods or needles.

[0072] Fundamentally, the components of these embodiments include, but are not limited to the following.

[0073] A microfluidic device comprising a cavity formed in a body including a main channel connected to inlet and outlet ports; and a plurality of PBECs on the inner surface of the cavity.

[0074] Replicating vascular networks: Referring to FIG. 1, embodiment 100 is illustrated schematically in flowchart format, illustrating an expanded cross-sectional view of a rectangular microchannel cavity 102 lined on its inner surface with PBECs (referred to as ECs in FIG. 1). In embodiment 100 and similar embodiments, PBECs are deposited or otherwise situated within a matrix material suitable for promoting or maintaining cell viability and allowed to self-assemble into a set of more than one tube. These PBECs may or may not be cultured with other cell types to either support the vasculature formation, viability, and function or with non-vascular cells or tissues (e.g., spheroids or organoids). In some cases, microfluidic devices containing independent regions for loading hydrogels laden with various vascular cells and media channels areemployed. First, a mixture of hydrogel and endothelial and / or mural cells is prepared by mixing cells at known concentrations with cell-compatible hydrogels that can be polymerized within the hydrogel chamber after loading on to a microfluidic device 101. In FIG. 1, “ECM” refers to extracellular material or matrix, in certain embodiments self-assembling matrix materials, and “PBS” refers to phosphate-buffered saline. PBS mimics body fluids to maintain stable pH and salt balance. Usually, a fibrin matrix created by polymerization of fibrinogen via thrombin is used, but one skilled in the art will appreciate that there are many matrix materials that can be used and more that will be developed in the future. The cell and hydrogel mixtures are immediately mixed and loaded in the hydrogel (or designated) channels and allowed or stimulated to polymerize and crosslink according to the appropriate method(s) corresponding to the matrix material used. Fundamentally, the components of this application include, but are not limited to (see FIG. 1): a microfluidic device 101 comprising a cavity 102 formed in the body of the device 101 in which a matrix 103 is deposited or otherwise formed; and seed PBECs 104 are mixed into or applied to matrix 103 and a plurality of PBECs 106 are embedded within matrix 103 within the body of device 101. One example of a device and method for using PBECs for multichannel self-assembly of vascular networks is illustrated schematically in FIGS. 2A and 2B as embodiment 200. One skilled in the art will appreciate that this is just one of many possible multichannel designs. Device embodiment 200 includes a microfluidic device body 202 having fluid inlets 204, 206, and fluid outlets 208, 210. A hydrogel compartment 212 is formed around fluid compartments 214, 216. where fluid compartment 214 connects fluid inlet 204 to fluid outlet 208, and fluid compartment 216 connects fluid inlet 206 to fluid outlet 210. The hydrogel may be admitted through an inlet 218, while growth factors or other ingredients fed through one or more inlets 220.

[0075] Replicating vascular network formation from parent vessel(s): In this form of application, the previous two methods are combined in such a way that PBECs are formed on the inner layer of a channel or cavity within a microfluidic device which is positioned adjacent to a matrix suitable for promoting or maintaining cell viability. In this case, vascular networks form by self-assembly of PBECs in the cell-supporting matrix from the microfluidic channel / cavity by perfusion of cells from the parent vessel(s). One example of a form of applying this approach is illustrated below. One skilled in the art will appreciate that there are many possible methods for implementing this vascular formation form.

[0076] Microdevices with a conical inlet port for passive formation of cylindrical microchannels in viscous fluids are another aspect of the present disclosure.

[0077] Viscous finger patterning (VFP) has been demonstrated previously as a way of harnessing a natural physical phenomenon to create cylindrical or ellipsoidal microchannels in viscous fluids and gels on microfluidic devices. Referring to FIG. 3 and embodiment 300, this process involves the application of, first, a viscous fluid 306 in a prefabricated microfluidic channel 303 within a microfluidic device body 302. In general, microfluidic device body 302 contains horizontal microchannel(s) with vertical ports 304A, 304B on each end extending up to a top surface of device body 302. The VFP process begins with the deposition of a low viscosity fluid 308 into vertical port 304B on device body 302 such that a droplet is formed. The capillary pressure exerted by the surface tension of the low or less viscous fluid 308 pushes the less viscous fluid 308 through the more viscous fluid 306 (as long as the fluidic resistance of the microchannel and the relative difference between the viscosities of the fluids is adequate), which creates a channel 310 with a round cross section. The process of the less viscous fluid 308 tunneling through the more viscous fluid 306 is governedby a principle known as Saffman-Taylor Instability. That is, the process of a less viscous fluid tunneling through a more viscous fluid is inherently unstable due to interactions between the two fluids.

[0078] In all previous devices and methods, standard microfluidic devices with cylindrical inlet and outlet ports have been used to apply this process. In some cases, droplets have been applied on top of these cylindrical ports, and in others, pipette tips have been inserted and left inside inlet ports to increase fluid travel distance. However, each of these approaches, and the use of these cylindrical ports, has significant disadvantages. The inconsistency of fluid tunneling resulting from Saffman-Taylor Instability results in a process whereby formation of microchannels is unreliable and requires precise fluid layering. The use of pipette tips to extend the fluid travel distance does appear to offer some increase in consistency of tunneling, but it adds a source of failure wherein the removal of pipette tips often causes microchannels to collapse due to disturbance of the viscous fluid in the microchannel.

[0079] The microfluidic devices in accordance with the present disclosure are characterized by an uncommon (or, perhaps, unprecedented) port geometry where one or more ports (304A and 304B in FIG. 3) for accessing microchannels are conical in shape. The conical ports are vertical geometries with one end open to atmosphere and the other in contact with, or progressing to, one or more horizontal microchannels. The cones are designed such that the larger diameter is at the top (upper) end of the shape, and they may be truncated to some nonzero minimum diameter.

[0080] Referring to FIG. 4, in certain embodiments (but not all), the geometries of ports 304A, 304B are composed of two sections: an inverted cone with top section 320 diameter ranging from about 2 to about 4 mm (or from 2-4 mm) and a bottom 322having diameter ranging from about 0.5 to about 1 mm (or from 0.5-1 mm); and a straight cylindrical port 324 having a diameter ranging from about 0.5 to about 1 mm (or 0.5-1 mm). The height of the individual conical sections 320, 322 and cylindrical sections 324 can be any height. In some cases, the combined height of the port (including conical and a cylindrical section, if any) ranges from about 4 to about 6 mm (or 4-6 mm). In some cases, the distribution of heights of the two sections can be 0.5 mm for the inverted conical part and 4-5 mm for the cylindrical part.

[0081] Devices with these features can be produced by multiple manufacturing processes. For example, microfluidic device bodies can be produced by standard photolithography and soft lithography whereby multiple layers - a flat bottom layer, the horizontal microchannel layer with monolithic portions of the vertical port structures fabricated into molds or punched out of the cast material, with or without a cylindrical region depending on the particular truncated conical shape used, and with a top layer consisting of the conical shape. The same layers can also be created by other manufacturing methods including, but not limited to, milling, three-dimensional (3D) printing, and injection molding. Separate layers can be combined by multiple means including plasma bonding, adhesives, and physical clamping.

[0082] Ports with conical sections that are pre-patterned into the body of microchannels allow improved access for fluid deposition, remove the need for inserting and subsequently removing other parts like pipette tips, and can more readily be scaled up compared to previous methods for patterning microchannels in viscous fluids (a more detailed description of advantages is outlined in the “Benefits” section below). Referring to FIGS. 5A, 5B, 5C, and 5D, and FIGS. 6A and 6B, one operating procedure and device (embodiment 400) of the present disclosure and associated methodology is as follows.

[0083] Referring to FIG. 6A, a precise volume of matrix material 306 (such as a collagen hydrogel) is calculated and injected into a microfluidic device body 302 having wells 360, 362, a first conical port 320, a second conical port 321, and a connecting microchannel 323. The precise volume is calculated according to first and second ports and microchannel void volumes. Matrix material 306, sometimes referred to herein as the viscous fluid, is injected into first conical port 320 and microchannel 323 such that matrix material 306 level reaches a top of the second cone, as illustrated schematically in the middle portion of FIG. 6A. Unpolymerized matrix material 306 can be injected into microchannels via a pipette 280 or robotic fluid handlers. In some embodiments, this injection is performed from conical outlet port 320 to conical inlet port 321, but inlet to outlet injection can also be used. In some embodiments, matrix material 306 injected in the first conical port 320 (outlet) to second conical port 321 (inlet) format as illustrated in FIG. 6A (and allowed to reach the top surface of the inlet port 321) constitutes a fluid / gel with a much higher hydrophilicity than the material making up the microfluidic device body. Once the pipette 280 tip is removed from first conical port 320, surface tension and gravity forces equilibrate the levels of collagen between first and second conical ports 320 and 321. Matric material 306 should completely fill first and second conical ports 320 and 321 and microchannel 323 without any need for resupplementation.

[0084] Referring now to FIG. 6B, exploiting the hydrophilicity of injected matrix material 306 and an opposing hydrophobicity of the surrounding material used for manufacturing microfluidic device body 302, a desired volume of PBS or some other low-viscosity fluid 308 can be deposited into second conical port 321 using a pipette 350, syringe, or similar instrument without bringing the tip in contact with second conical port 321 and matrix material 306 therein. The interplay between hydrophilicand hydrophobic forces “pulls” the drop of less viscous fluid 308 precisely over second conical port 321. The capillary force exerted by the fluid surface tension and curvature of the droplet will exert a pressure that will push the low viscosity fluid 308 and, in combination with gravitational force, will induce the viscous finger patterning process. The conical shape of port 321 will direct less viscous fluid 308 on a path into a central region of the conical port (as illustrated schematically in FIGS. 5A-D), and subsequently into microchannel 323, whereby Saffman-Taylor instability is reduced. That is, the restriction of available volume that low viscosity fluid 308 path can take by the normal and buoyant forces created by the tapered cone walls of second conical port 321 and high viscosity matrix material fluid 306 therein restricts the viscous finger pattern to a defined region and substantially reduces the instability of the phenomenon.

[0085] Once microchannel(s) 323 have been layered with PBS or other low viscous fluid and VFP has occurred, matrix material 306 polymerization can be induced according to the appropriate method(s) corresponding to the matrix material used. In some cases, this involves placing the entire device in a standard cell culture incubator (not illustrated).

[0086] The process (steps 1-3) can be repeated multiple times by injecting fresh matrix solutions from conical outlet(s) 320 towards conical inlet(s) 321 and layering fresh PBS, resulting in multilayered tubular structures. Adding one or more cell seeding solutions 380 can be performed in the same way media is added in wells 360, 362, for example, using a pipette 370. Hydrostatic pressure will push the cell suspension 380 through.

[0087] In certain embodiments, referring again to FIG. 4, the tops of conical ports 320, 321 may be patterned into microfluidic device bodies 302 to have a 2-4 mm diameter,allowing a higher range of PBS volumes that can be layered, in contrast to existing cylindrical opening ports. The wide range of PBS layering volumes offers more control, reproducibility and tunability over the dimensions of patterned microchannels.

[0088] In certain embodiments, the matrix material (high viscosity fluid) injected into the microchannel(s) can contain embedded cells. In some cases, after the microchannels are formed, cell suspensions can be injected to induce cells to adhere to the inner surface of the microchannel formed within the higher viscosity fluid. In some cases, when microchannel formation processes and cell suspension injections are repeated, multilayered tissue structures can be created wherein cells are patterned in concentric layers.

[0089] Microfluidic devices enabling on-board pneumatic unidirectional fluid recirculation are another aspect of the present disclosure.

[0090] The devices in accordance with this aspect of the present disclosure create unidirectional fluid flow and recirculation for continuous perfusion of fluid through microfluidic channels using a new combination of a unique microfluidic device design wherein inlet and outlet ports for pairs of corresponding channels are connected by common wells, a device lid with manifolds and channels engineered for air distribution and venting, and on-device pneumatic-actuated valves. The core components of these embodiments, and some specific examples of applications and variations thereof, are now explained with reference to FIGS. 7A, 7B, and 8.

[0091] Major Component 1: A lid 475 having a lid body 401 and 96 individual well lids 363, illustrated in FIG. 7A in bottom plan view for a 96 well device, positionable on top of the microfluidic device (not illustrated in FIGS. 7A and 7B). Lid 475 containstwo independent sets of overhead air control lines 402 and 404 - where the term “air lines” describes open channels / cavities or conduits through which air can travel or wherein air can be pressurized - embedded / patterned within lid body 401 of lid 475 (Fig. 7A). Overhead air control lines 402 and 404 each have a single port (406, 408 respectively) to which external pumps or compressors (not illustrated) may be connected, and common manifold lines (412, 414, respectively) that can be connected to external pressure sources such as pumps or air compressors or other actuation elements, allowing the lines to distribute pneumatic pressure to wells 360 and / or to vent air to the device’s surroundings.

[0092] Overhead Control Line 402 - A set of lines connecting an external pump (or other actuation element) to the wells on the platform (described below) is patterned into the plastic lid. The overhead control line 402 has a manifold line with a single access point 406 (from outside of the lid) where an external pressure source can be connected. In some cases, manifold line 402 has nodes that split into a set of parallel lines 412 (spanning across the length of a column or row of wells).

[0093] For example, for a 96 well plate format containing 48 independent microchannels 450 (FIG. 8), there are a total of 6 parallel subchannels 412 that span across columns 1, 3, 5, 7, 9 and 11 (FIG. 7A); for a 384 well plate format (not illustrated) with 192 individual microchannels, there are a total of 12 parallel subchannels spanning across columns 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

[0094] Each subchannel 412 from the primary manifold in overhead control line 402 branches into individual outlet conduits 413 (FIG. 7B) that deliver or pressurize air above each well in each row down each column. These individual lines 412 have outlets 413 for delivering air to each well along the column.

[0095] For example, for a 96 well plate format (48 microchannels), each subchannel within a column has a total of 8 air outlets placed 9 mm apart (aligning with the center of each well situated below, Fig. 7B); for a 384 well plate format (192 microchannels), each subchannel has a total of 16 air outlets placed 4.5 mm apart (aligning with the center of each well situated below). Hence, the overhead control line 402 has a single inlet 406 where an external pressure source feeds pneumatic pressure that is delivered to 6 / 12 columns of 8 / 16 wells simultaneously (a total of 48 / 192 wells).

[0096] Overhead Control Line 404 - Like overhead control line 402, another set of air lines, overhead control line 404, runs parallel to overhead control line 402 (FIGS. 7A and 7B). A separate manifold line access point 408 facilitates connection to another external pressure source or allows one end of the apparatus to be left open to atmosphere. FIG. 7A is a schematic bottom plan view of a 96 well plate lid with overhead control lines 402 and 404 (patterned control lines within the body). FIG. 7B is a sectioned view along line A-A’ of FIG. 7 A illustrating 8 outlets 413 that align over the centers of the wells.

[0097] Major Component 2: The second layer in the assembly is a bottomless well / reservoir layer wherein geometries are shaped to encompass a pair of microfluidic device access ports within each well / reservoir. These shapes effectively include a wall or walls (which can be square, rectangular, circular, or some other geometry) that are patterned in an array that matches the geometry of the microfluidic device to be situated below Major Component 2.

[0098] For example, these reservoirs can be patterned in standard 96- or 384-well plate format following specifications set by Standards ANSVSLAS 1-2004 throughANSI / SLAS 4-2004. These wells are open at the top and bottom facilitating integration with other invention components. The open-top allows direct access for fluid handling, tissue seeding, and sampling, while the open-bottom interface enables fluid or material exchange with the other Major Components assembled below this layer. Importantly, the dimensions of these reservoirs are constrained such that the walls of each reservoir must be outside of, and therefore encompass, one access port of each pair of channels (therefore, two access ports per well / reservoir) included in Major Component 3.

[0099] Major Component 3: Referring to FIG. 8, the third component, 500, within the device assembly is where all the fluidic microchannels 450 are situated. Component 500 includes a layer 502 of plastic or other biocompatible materials. Within layer 502, there are three types of microchannels with an additional membrane layer.

[0100] Main microchannels 450 (Layer 1) - Main microchannels 450 are designated as the microfluidic channels that will contain the cells / tissue(s) under study. For example, in the 96 and 384 well plate formats, there are a total of 48 / 192 main microchannels 450 each of which, when combined with its corresponding recirculation channel (explained below), encompasses a single tissue unit. The inlet ports 460 and outlet ports 462 of channels 450 are situated inside wells (Major Component 2).

[0101] Recirculation channels (Layer 1) - Each main channel 450 is also accompanied by a corresponding recirculation channel 470 that is situated within each tissue unit footprint. Recirculation channels 470 are wider and / or taller than the main channels, which makes their fluidic resistance lower than the main channels. Each recirculation channel 470 is connected to a valve 472 (Major Component 4) that enables opening and closing of recirculation channel 470 for fluid flow. The access ports for corresponding main and recirculation channel pairs are arranged such that each mainchannel inlet and recirculation channel outlet share a common overhead well / reservoir (Major Component 2) and each main channel outlet and recirculation channel inlet share a common overhead well / reservoir.

[0102] Deflectable membrane (Layer 2) - A deflectable membrane made up of a thin polymer material is assembled between Layers 1 and 3. This can be made of a range of possible materials including, but not limited to, polydimethylsiloxane (PDMS) or cyclic olefin copolymers (COC). The thickness and material properties of the membrane must be such that application of pneumatic pressure in the layer below can deflect the membrane up into Layer 1 to restrict the flow path in the recirculation line sufficiently that the fluidic resistance through the main channels is significantly lower than that of the recirculation channels. These membranes may be applied such that they encompass the entire device footprint, or they may be selectively patterned such that they only cover the valve control element regions.

[0103] Valve control elements (Layer 3) - Microchannels and ports facilitating connection of another external pump or other actuating element are patterned in this layer. There are multiple achievable arrangements to create this capability. One is highlighted below.

[0104] In one application, valve control lines are patterned into the microfluidic device such that a pneumatic line is patterned below the membrane (Layer 2) for application of pressure to deflect the membrane up. The components of the valve control elements in this form include:

[0105] Valve control line component 480 - A channel 480 having a single inlet 482 is patterned into Layer 3 (and possible other layers of the device). The inlet482 of line 480 connects to an actuator of some kind that supplies pressure for operating the microvalves.

[0106] Valve control line component 484 - Valve control lines 484 proceed from and are orthogonal to valve control line 480. This results in horizontally spanning valve control lines that supply the pressure provided by the external actuator through valve control line component 480. Valve control line component 484 is patterned such that it underlaps recirculation lines 470 and has geometries such that the contact area underneath each recirculation line 470 is larger than the rest of the line to increase the deflection of the membrane in that area.

[0107] The design of these components comprising the entire platform is engineered to create a unidirectional recirculating flow within each tissue unit system of main and recirculation channels connected in a parallel fluidic connection. The device operates in two different cycles, perfusion and recirculation, which are now described.

[0108] When fluid is introduced into the inlet wells of each device and the lid 475 (Major Component 1) is installed on top, the air supplied via the overhead control lines 402 and / or 404 create a pressured environment across each inlet well (where the term “inlet well” refers to each well including the inlet port of the main channels). This causes fluid movement from the wells corresponding to the inlets of the main channels towards the main channel outlets, where it is collected in the opposite wells / reservoirs. During the perfusion stage, the microvalves 472 (Major Component 4) are closed, making the recirculation channel unavailable for fluid perfusion. The perfusion cycle time can be calculated using the volume of fluid that was introduced in each inlet well and the rate of perfusion generated by the pressure applied.

[0109] Once fluid has been perfused through the main channel(s) 450, the recirculation cycle is initiated by opening microvalves 472 (Major Component 4) through actuation and / or release of the pneumatic pressure operating that valve. Using overhead pneumatic pumps, the pressure difference is reversed, either by using vacuum or reversing the relative magnitude of positive pressures between two overhead pumps. Owing to the lower fluidic resistance in recirculation channels 470 compared to main channels 450, recirculation channels 470 with open valves 472 become the preferred flow path for recirculation. Like the perfusion cycle, the recirculation cycle time can be calculated using the pulling rate and volume information. Referring to FIG. 8, once fluid has been recalled from the wells with the outlet ports of main channels 450, the recirculation cycle ends via closing microvalves 472 (Major Component 4). The operation can then be repeated continually for extended durations without the need for manual exchange of fluid.

[0110] Certain device embodiments in accordance with the present disclosure are designed to deliver pneumatic pressure to the wells situated below the lid. Two independent control lines 402, 404, allow application of different pressures at the inlet and outlet wells of each tissue unit (comprising a patterned channel containing human tissue). This can be exploited to create different fluid flow regimes through patterned tissues, while independently allowing users to vary flow, pressure or both, as now described.

[0111] Constant flow, varying absolute pressure: Hagen-Poiseuille’s law states that the flow rate of a fluid across a channel is equal to the ratio of pressure difference across the inlet and outlet and the fluidic resistance:P1p inlet —1p outletQ Flow rate) = —R RIf the pressure difference across a channel is conserved, the flow rate will be constant. Since the pressures in the inlet and outlet wells are equal to the respective control line pressures, the pressure drop across a 1x2 well subsystem isP inlet ~ P outlet — PA “ PBThe device allows varying the absolute pressures in control line 402 (PA) and control line 404 (PB) such that both pressures increase or decrease, while keeping their difference a constant value. Hence, this device helps achieve a constant flow while varying the absolute pressures across the inlet and outlets of channels.

[0112] Varying flow rate: Conversely, the devices can help achieve a range of different flow rates or flow profiles by varying the pressure drop across the inlet and outlet wells. By varying one or both the pressures across the microchannels, the device enables creation of unique pressure drops and hence, different flow rates.

[0113] Methods of generating tissue-penetrating vascular networks using hydrogel prevascularization and a removable mold for tissue injection are other aspects of the present disclosure.

[0114] We now describe, with reference to FIGS. 9A and 9B and embodiment 600, microfluidic devices and operating procedures that enable injection of three-dimensional (3D) tissue cultures into pre-vascularized hydrogels and subsequent vascularization of the newly injected tissue bed. The microfluidic devices and operating procedures of these aspects of the disclosure accomplish this via unique designs ofmicrochannels and new molding processes that enable tissue injection and rapid vascularization.

[0115] Major Component 1 - The first layer 602 in assembly embodiment 600 is a bottomless well / reservoir layer wherein geometries are shaped to encompass a pair of microfluidic device access ports within each well / reservoir. These shapes effectively include a wall or walls (which can be square, rectangular, circular, or some other geometry) are patterned in an array that matches the geometry of a microfluidic device to be situated below Major Component 1.

[0116] For example, these reservoirs can be patterned in standard 96- or 384-well plate format following specifications set by Standards ANSI / SLAS 1-2004 through ANSI / SLAS 4-2004. These wells are open at the top and bottom facilitating integration with other components of the present disclosure. The open-top allows direct access for fluid handling, tissue seeding, and sampling, while the open-bottom interface enables fluid or material exchange with the other Major Components assembled below this layer.

[0117] Major Component 2 - the middle layer component, 604, serves as the first instance of microchannels (FIG. 9A). Here, a transparent plastic is machined, molded, 3D-printed, or otherwise fabricated, to have cut-outs of angled microchannels 606, wherein each tissue unit footprint (constituting a single sub-system of the entire array of tissue units) contains a microchannel 606 that runs from the top-most-well in each tissue unit (FIG. 9B, Well A-l) across to the top-middle well (Well A-2) and then down to the bottom-right-most well (Well B-3). The portions of microchannel 606 directly underneath Major Component 1’s open-bottom wells share the same diameter, whereas portions of microchannel 606 spanning between the open-bottom wells can be differentsizes (in some cases, 1-mm wide channels are used). The angle between the microchannel portion between Wells A-2 and B-3 can be 45°, but other angles and shapes are also possible.

[0118] In some embodiments (but not all), a second set of microchannels is patterned within this layer to facilitate unidirectional system perfusion. This is accomplished onboard the microfluidic device by returning fluid from the main microchannel’s outlet (Well B-3) back to the inlet (Well A-l) while preventing backflow into the main microchannel (running Well A-l to B-3). To accomplish this, the secondary microchannel design, again situating microchannels withing the same sub-system footprints, consists of a microchannel running from Well B-3 to Well B-2, then turning 45° towards well A-l with the same width.

[0119] In some embodiments, there is a valve element consistent with the structure described herein above wherein pneumatic pressure from a lower layer deflects a membrane up into the recirculation line corresponding to the main channel.

[0120] In other embodiments, the recirculation microchannel has a discontinuity at well B-2, making this channel imperfusable at the baseline setting. The discontinuity is altered by integration of valves, either surface-mounted or within a fluidic layer (as described herein above), opening at predefined intervals to facilitate fluid flow from well B-3 to well A-l, thus replenishing the input reservoir to re-start the unidirectional perfusion.

[0121] On a multi-system well plate (z.e., a plate with 16 tissue in the 96-well format or a plate with 64 sub-systems in the 384-well format), the valve pneumatic controllines may be patterned as a chain and / or in a parallelized format such that each of the valves are operated by a single increase in pressure from the pneumatic line at once.

[0122] Major Component 3 (optional) - A second cutout 608 similar to Major Component 2 (layer 604) can be included which situates a second set of microchannels 610 under the first set. Importantly, the microchannel 610 footprint is a mirror of the microchannels 606 described in Major Component 2 taken on a vertical axis. That is, the Major Component 3 microchannel 610 designs can be described using a single tissue unit, where a microchannel begins at well B-l (FIG. 9B), travels upwards and to the right towards Well A- 2 at some angle, and then terminates at Well A-3. Importantly, microchannels 610 overlap with microchannels 606 only under a single central well per each sub-system (Fig. 9B, Well A-2). Like Major Component 2, this component can sometimes feature a secondary microchannel for fluid recirculation, also having the Major Component 2 design but mirrored about the vertical axis.

[0123] These embodiments may be assembled as follows: a baseplate of transparent plastic material with some thickness serves as the bottom of the assembly atop of which subsequent platform layers are assembled. Another layer of plastic having prepatterned microchannels of the Major Component 3 is bonded to the baseplate and topped by a thin plastic membrane. When assembled, fully enclosed microchannels are formed with the microchannel floor provided by the baseplate and the microchannel ceiling provided by the membrane. Major Component 2, if used and having the same major dimensions but an alternate microchannel footprint cutout, is bonded or otherwise joined to the previous assembly. This is topped by a second membrane, thus sealing the second layer of microchannels with the previous sandwich serving as the new microchannel’s floor and the newly added membrane serving as this microchannel’s ceiling. Major Component 1, having the same major dimensions but analternate cutout of features, is bonded to the previous assembly. This component is left open to the air to facilitate microchannel access by users (or automated equipment). In configurations requiring unidirectional perfusion, valves are positioned either within the layers associated with Major Component 2 or within Major Component l’s reservoirs, interfacing with the microchannels in Major Components 2 and 3.

[0124] The design of these components comprising the platforms of the present disclosure are engineered to deliver uniform fluid flow throughout the microchannels. In embodiments where microchannels are filled with hydrogels supporting vascularized tissue beds, the flow can be supplied intraluminally (z.e., through the vasculature) by depositing (or pumping) fluid into wells A-l and B-l (inlets) which then perfuse within the capillary networks towards wells B-3 and A-l (outlets), respectively. Interstitial flow can be applied by depositing fluid or pumping air or fluid at the overlap wells (e.g.. Well A-2), driving flow in both directions (z.e., towards wells A-l, B-l, B-3, and A-3). In embodiments where microchannels are filled with fluid or air instead of capillary-embedded hydrogels, these same strategies might be employed. In any case, the uniformity is achieved via these characteristics:

[0125] Channel geometry - each of the microchannels ensure a single, defined pathway for fluid movement that prevents unwanted lateral flow or redistribution to adjacent compartments. Uniform flow is further supported by the absence of bifurcations within individual sub-systems, eliminating variations in flow and / or pressure velocity and distribution that is often observed in single-planed and multi-compartmented microfluidic platforms.

[0126] Optional unidirectional recirculation - for experiments needing sustained and automated perfusion, the platform can be configured with secondary recirculation microchannels. These are not continuous, as a surface-mounted quake valve controls when the secondary channel is open or active (and thus facilitating media refilling from major microchannel outlets to inlets).

[0127] In certain embodiments, device structures and operational variants described above can be combined with new tissue engineering methods to create pre- vascularized regions into which tissue cultures can be added and induce vascularization of those tissue constructs and the surrounding regions. These tissues can house spheroids, organoids, or similar structures. The methods of the present disclosure enable formation of perfusable capillary networks throughout these tissue constructs, addressing challenges of achieving full construct vascularization, which in turn should provide nutrient transport and waste removal that is critical in maintaining complex tissue structures.

[0128] To enable this approach, a structure is used to pattern open spaces within the overlap wells (e.g. Fig. 9B, Well A-2) to facilitate tissue injection into the system. This structure can be fabricated as a lid that can be placed on top of the microfluidic device body or in some other form wherein a mandrel (see FIG. 10A) can be precisely positioned over each central tissue well in an array of tissue units. These mandrels consist of plastic or some other material fashioned into some shape (cylindrical, cuboidal. or otherwise) that is positioned in the devices prior to hydrogel injection to create an open space for later tissue injection.

[0129] First, a hydrogel precursor solution should be generated.

[0130] In some embodiments, a fibrinogen precursor solution is prepared by layering 30 mg lyophilized fibrinogen over 2 mL warmed IX PBS (yielding 15 mg / mL fibrinogen solution). Depending upon lot characteristics (z.e., proportion of fibrinogenprotein in lyophilized protein, proportion of clottable fibrinogen in lyophilize protein), the solution is adjusted to 5 mg / mL via dilution with a volume of IX PBS or other factors, as experimental needs dictate (e.g., collagen solution can be added to increase extracellular matrix content). Separately, thrombin solutions, which cleave fibrinogen to form the fibrin hydrogel, are prepared by diluting bovine thrombin to 2 U / mL in cell culture medium (type dictated by EC type as specified by experimental needs). Thrombin solutions are set aside while ECs but also sometimes supporting cells (e.g., fibroblasts or multipotent stromal cells) are lifted from traditional cell culture plasticware after reaching 80% confluency. After lifting, each cell type is counted and subsequently resuspended in thrombin-containing solution at a cell concentration that is twice the final concentration destined for the hydrogel (this number changes based on experimental needs). These solutions are mixed in a 1:1 ratio with the fibrinogen solutions and then immediately injected into the microchannels. Over 3-4 minutes, the hydrogel crosslinks and becomes solid, indicated by a color transition from clear to opaque. Once solidified, the system can be hydrated with cell culture medium as experimental needs dictate, but a common recipe employs PromoCell EGM-MV2 medium (cat. no. C-22022) supplemented with growth factors including VEGF-A, VEGF-C, bFGF, SIP, and ESM-1 (where “VEGF” refers to vascular endothelial growth factor; “bfGF” refers to basic fibroblast growth factor. One skilled in the art will appreciate that other hydrogels are possible for this purpose and that the approach described here represents only one of many applicable methods for generating hydrogel precursor solutions.

[0131] Once created, the hydration medium is dispensed across wells of the hardware and allowed to move into the channels to hydrate the channels and any materials therein. For example, in a 2 x 3 well footprint system, as illustrated in FIG. 9B, 200 pL medium is dispensed in wells A-l and B-l, whereas 100 pL is dispensed in wells A- 3and B-3. The differences in fluid reservoir height formed via differential volumes induces an interstitial flow that travels along each of the two microchannels until the fluid levels across the inlet / outlet reservoirs equilibrate. This approach has been shown to be beneficial to self-assembling capillary networks as the flow increases matrix metalloprotease secretion that aids in extracellular matrix remodeling. Fluid reservoirs can be re-established at certain time intervals (such as every 24 hours), or as a user’s experimental needs dictate, by removing all media from the system via simple aspiration from the reservoirs followed by fresh medium injection.

[0132] Referring to FIGS. 10A, 10B, and 10C, for the purpose of vascularizing spheroids, organoids, or similar cell constructs, use of the main platform as well as the mandrel structures are employed. Here, the hydrogel injection protocol is performed after mandrels are positioned in the system. After hydrogel injection, the mandrels are left in place while the capillaries self-assemble (in some cases, self-assembly is allowed to proceed for 4 days). Then, the lid or other mandrel holding structure is carefully removed from the system, effectively removing the mandrels and revealing molded pockets within the hydrogel in the overlap well region (e.g., Fig. 9B, Well A-2). Separately, spheroid, organoid, or similar tissue solutions are prepared according to procedures corresponding to each tissue model.

[0133] For example, referring again to FIGS. 10A, 10B, and 10C, the tissue constructs can be embedded in a fibrin hydrogel as previously described for the ECs and their supporting (stromal) cells. Once embedded, the tissues can be injected into the pocket formed by the mandrels by pipette or other methods, and the gels can be allowed or stimulated to crosslink. Depending upon user needs, sometimes the organoids may be co- seeded with ECs of vascular fragments to initiate intra-organoid vascularization as well as anastomosis with the existing, pre-vascularized tissue bed. In FIG. 10A,“BECs” and “LECs” refer to blood-derived endothelial cells and lymph-derived endothelial cells, respectively.

[0134] The microfluidic cell culture systems described herein can be adapted for a range of other experimental applications. For example, one variation involves configuring the systems as traditional transwell-like systems, where polyurethane membranes serve as surfaces for seeding multiple monolayers on opposite sides. In this configuration, an epithelium can be seeded on the bottom side of a membrane layer while an endothelium can be seeded on the top of the same membrane layer (or vice versa depending on user needs), which would then be useful for studies of tissue-tissue interfaces such as alveolar-capillary barriers or the blood-brain barrier. As the variation is related mostly to different configurations of cell compartmentalization, the system preserves its ability to support controlled and uniform flow through each of the microchannels, allowing researchers the opportunity to study the effects of shear stress, permeability, or cell-cell interactions in barrier models.

[0135] A second variation involves modifying the microchannel dimensions and layout to accommodate multiple overlap wells within a single sub-system. While this effectively reduces the number of sub-systems per well-plate, this variation allows for multi- organoid integration where organoids of different tissues can be placed in individual overlap wells within the same system (refer to FIGS. 11A, 1 IB, 11C, 12A, 12B, and 12C). With this variation, researchers can study systems biology phenomenon, such as drug metabolism by one organoid (e.g., liver) and subsequent delivery and action in another (e.g., brain). Such an experimental setup enables investigation into metabolic coupling, inter-organ signaling, and tissue-specific responses under controlled conditions. Additionally, each overlap well can be configured with distinct environmental factors such as nutrient gradients, oxygenconcentrations, or cytokine / growth factor supplementation, enabling experiments that simulate localized microenvironments or dynamic inter-organ interactions.

[0136] Methods of engineering tissues described herein can be adapted to accommodate alternative tissue integration strategies. One notable variation involves the use of ex vivo tissue biopsies, such as skin (FIGS. 13 A, 13B, and 13C), in place of organoids. Instead of embedding the tissue directly into the pre- vascularized region via the mandrel, these biopsies can be laid atop the hydrogel scaffold. The vascularized hydrogel provides support and nutrient exchange for the ex vivo tissue, extending its lifetime for an increased experimentation window. This approach is particularly valuable for modeling diseases or testing therapeutics in personalized medicine applications, where the patient- specific biopsy can retain its native architecture while benefiting from vascular support by the engineered system.

[0137] Methods of generating mature endothelial cells from patient peripheral blood-derived endothelial progenitor cells are other aspects of the present disclosure.

[0138] Isolation of proliferative PBECs from limited blood sample volumes can be challenging. To improve yield and ensure robust and reproducible PBEC isolation, we contemplate supplementing with the following:

[0139] Flow-based programming and differentiation of EPCs into arterial or venous mature endothelial subtypes: We hypothesize and contemplate that, during the culture of early progenitors (on their way to maturation into PBECs), exposure of physiological flow patterns and specific molecular factors to these cells can alter the differentiation pathways and polarize cells towards a more arterial or venous phenotype. We hypothesize and contemplate that exposure of progenitors to arterial hemodynamic conditions (shear stress -10-20dynes / cm2) and increased VEGF concentration can yield cells with increased expression of arterial specific markers (EphrinB2, SOX17 etc.). Conversely, exposure of progenitors to low shear flows (shear stress ~l-5 dynes / cm2) with low / no VEGF can drive the differentiation of PBECs into a more venous subtype with increased expression of venous markers (COUP-TFII, EphB4 etc.).

[0140] Tissue- specific maturation of EPC derived endothelial cells: We hypothesize and contemplate that during the maturation of EPC derived cells, co-culturing EPCs with tissue-specific cells will create mature tissue- specific endothelial cells. For example, co-culturing EPCs during the maturation process with brain astrocytes and neural progenitors will create endothelial cells that are phenotypically close to brain microvascular endothelial cells (BMVECs). Similarly, culturing patient-derived EPCs with liver hepatocytes and stellate cells will create endothelial cells with similar functional and phenotypic profiles as liver sinusoid endothelial cells (LSECs). Creation of these tissue-specific endothelial cells that are also patient-derived will allow us to create autologous tissue endothelial cell banks without the need of invasive biopsies. This approach will also enable creation of more physiologically relevant tissue models for drug screening and biomarker discovery applications, predicting patient-specific tissue responses.

[0141] From the foregoing detailed description of specific embodiments, it should be apparent that patentable devices, systems, and methods have been described. Although specific embodiments of the disclosure have been described herein in some detail, this has been done solely for the purposes of describing various features and aspects of the devices, systems, and methods, and is not intended to be limiting with respect to their scope. It is contemplated that various substitutions, alterations, and / or modifications,including but not limited to those implementation variations which may have been suggested herein, may be made to the described embodiments without departing from the scope of the appended claims. For example, some devices of this disclosure may be devoid of certain components and / or features: for example, devices devoid of filters; systems devoid of hydraulics; systems devoid of threaded fittings; systems devoid of welded fittings; systems devoid of carcinogenic materials. Some methods may employ pulsed flows, including sinusoidal pulses and pulses other than sinusoidal pulses, for example, but not limited to. step pulses. Some methods are devoid of biopsies.

Claims

What is claimed is:

1. A microfluidic in vitro endothelial tissue model device comprising:a) a microfluidic device body comprising a microchannel cavity formed in the body, the microchannel cavity including a main microchannel channel connected to inlet and outlet ports; andb) a plurality of fully mature peripheral blood-derived endothelial cells deposited or otherwise situated within a matrix material on an inner surface of the main microchannel, the matrix material suitable for promoting or maintaining viability of the peripheral blood-derived endothelial cells and allowing the peripheral blood-derived endothelial cells to self-assemble into one or more tube.

2. The microfluidic in vitro endothelial tissue model device of claim 1, wherein the fully mature peripheral blood-derived endothelial cells are acquired by differentiation of progenitor cells from peripheral blood.

3. The microfluidic in vitro endothelial tissue model device of claim 1, wherein the fully mature peripheral blood-derived endothelial cells are defined based on surface markers.

4. The microfluidic in vitro endothelial tissue model device of claim 1, wherein the fully mature peripheral blood-derived endothelial cells are generated from peripheral blood endothelial progenitor cells.

5. A multichannel microfluidic in vitro endothelial tissue model device comprising: a) a microfluidic device body having a plurality fluid inlets, and a plurality of fluid outlets;b) a hydrogel compartment formed around a plurality of main microchannels, where each of the main microchannels connects one of the fluid inlets to one of the fluid outlets;c) the hydrogel admitted through a hydrogel inlet port in the microfluidic device body; andd) the microfluidic device body having one or more additional inlet ports for injecting growth factors or other ingredients into the hydrogel and intersecting with fluid flowing through the plurality of main microchannels.

6. The multichannel in vitro endothelial tissue model device of claim 5, wherein the fluid flowing through the plurality of main microchannels comprises fully mature peripheral blood-derived endothelial cells that can self-assemble within the plurality of main microchannels formed in the hydrogel to create microvascular networks under influences of growth factor gradients, mural cell components or tissue organoids.

7. A microfluidic in vitro endothelial tissue model device comprising:a) a microfluidic device body comprising a microchannel cavity formed in the body, the microchannel cavity including a main microchannel channel fluidly connected to a conical inlet port and conical outlet port;b) the main microchannel and the conical inlet and outlet ports having internal surfaces coated with a layer of viscous fluid, the layer of viscous fluid formed by passive formation of cylindrical microchannels in the viscous fluid by viscous finger patterning technique employing a less viscous fluid.

8. A multichannel microfluidic in vitro endothelial tissue model device comprising: a) a microfluidic device body comprising a plurality of microchannel cavities formed in the body, each microchannel cavity including a main microchannel channel fluidly connected to one of a plurality of conical inlet ports and one of a plurality of conical outlet ports;b) the main microchannels and the conical inlet and outlet ports having internal surfaces coated with a layer of viscous fluid, the layer of viscous fluid formed by passive formation of cylindrical microchannels in the viscous fluid by viscous finger patterning technique employing a less viscous fluid.

9. A multichannel microfluidic in vitro endothelial tissue model control device enabling on-board pneumatic unidirectional perfusion and fluid recirculation, comprising:a) a lid having a lid body and a plurality of individual well lids, the lid positionable on top of a microfluidic device, the lid comprising first and second sets of overhead air control conduits through which air can travel or wherein air can be pressurized, the overhead air control conduits embedded / patterned within the lid body of the lid;b) the overhead air control conduits each having a single port to which an external pump or compressor may be connected, and common manifold conduits allowing the overhead air control conduits to distribute pneumatic pressure to wells and / or to vent air to the device’s surroundings.

10. The multichannel microfluidic in vitro endothelial tissue model control device of claim 9, wherein the first set of overhead control conduits comprises a set of conduits configured to connect an external pump (or other actuation element) to wells on a platform is patterned into the lid, the first set of overhead control conduits comprising a first manifold conduit with a single access point from outside of the lid where anexternal pressure source can be connected, and first nodes that split into a first set of parallel subchannel conduits spanning across a length of a column or row of wells.

11. The multichannel microfluidic in vitro endothelial tissue model control device of claim 9 configured to control a 96- well plate, 8 row by 12 column format, containing 48 independent main microchannels, wherein the first set of overhead control conduits comprises 6 parallel subchannel conduits that span across columns 1, 3, 5, 7, 9 and 11.

12. The multichannel microfluidic in vitro endothelial tissue model control device of claim 9 configured to control a 384-well plate, 16 row by 24 column format containing 192 independent main microchannels, wherein the first set of overhead control conduits comprises a total of 12 parallel subchannels spanning across columns 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

13. The multichannel microfluidic in vitro endothelial tissue model control device of claim 10, wherein each subchannel conduit branches into individual outlet conduits that deliver or pressurize air above each well in each row down each column.

14. The multichannel microfluidic in vitro endothelial tissue model control device of claim 9, wherein the second set of overhead air control conduits comprises a set of conduits configured to connect an external pump (or other actuation element) to wells on a platform is patterned into the lid, the second set of overhead control conduits comprising a second manifold conduit with a single access point from outside of the lid where an external pressure source can be connected, and second nodes that split into a second set of parallel subchannel conduits spanning across a length of a column or row of wells.

15. The multichannel microfluidic in vitro endothelial tissue model control device of claim 10. wherein the first set of parallel subchannel conduits include first outlets that align over centers of a first portion of the wells.

16. The multichannel microfluidic in vitro endothelial tissue model control device of claim 14, wherein the second set of parallel subchannel conduits include second outlets that align over centers of a second portion of the wells.

17. A multichannel microfluidic in vitro endothelial tissue model assembly, comprising:a) a lid having a lid body and a plurality of individual well lids, the lid positionable on top of a microfluidic device, the lid comprising first and second sets of overhead air control conduits through which air can travel or wherein air can be pressurized, the overhead air control conduits embedded / patterned within the lid body of the lid;b) a second component comprising a bottomless well / reservoir layer having a plurality of wells, wherein well geometries are shaped to encompass a pair of microfluidic device access ports within each well / reservoir, the wells open at the top and bottom facilitating direct access for fluid handling, tissue seeding, and sampling, while the open-bottom interface enables fluid or material exchange with a third component assembled below this layer; and c) a third component comprising a layer of plastic or other biocompatible material having a plurality of fluidic microchannels formed therein, the layer of plastic comprising three types of microchannels with a deflectable membrane layer:i) a first layer comprising;A) a plurality of main microchannels designated as the microfluidic channels configured to contain thecells / tissue(s) under study, comprising inlet ports 460 and outlet ports 462 of channels 450 are situated inside wells, andB) a corresponding plurality of recirculation channels that are situated within each tissue unit footprint, wherein the recirculation channels are wider and / or taller than the main microchannels, having fluidic resistance lower than the main microchannels, each recirculation channel connected to a valve that enables opening and closing of respective recirculation channels for fluid flow, wherein the ports for corresponding main microchannel and recirculation channel pairs are arranged such that each main microchannel inlet and recirculation channel outlet share a common overhead well / reservoir, and each main microchannel outlet and recirculation channel inlet share a common overhead well / reservoir;ii) a second layer comprising a deflectable membrane comprising a thin polymer material assembled between the first layer and a third layer, the thin polymer material having thickness and material properties such that application of pneumatic pressure below can deflect the membrane up to restrict flow path in the recirculation channels sufficiently that the fluidic resistance through the main microchannels is significantly lower than that of the recirculation channels; and iii) the third layer comprising valve control elements including air microchannels and air ports facilitatingconnection of an external pump or other actuating element, the valve control element patterned in the third layer,18. The multichannel microfluidic in vitro endothelial tissue model assembly of claim 17, wherein the valve control element patterned in the third layer includes a plurality of secondary valve control conduits fluidly connected orthogonal to a primary valve control conduit, the secondary valve control conduits patterned such that they underlap the recirculation conduits and have geometries such that contact area underneath each recirculation conduit is larger than a rest of the conduit to increase deflection of the membrane in that area.

19. A method of generating tissue-penetrating vascular networks comprising:using hydrogel pre-vascularization and a removable mold for tissue injection, integrating microvascular networks and 3D tissue culture wherein a sequential process of vascular network formation around a biocompatible molding rod is followed by tissue culture injection and vascular network permeation into the tissue.

20. A method of improving yield and ensuring robust and reproducible peripheral blood-derived endothelial cells isolation while generating mature endothelial cells from patient peripheral blood-derived endothelial progenitor cells, comprising: a) isolating proliferative peripheral blood-derived endothelial cells from limited blood sample volumes;b) exposing progenitor cells to arterial hemodynamic conditions (shear stress -10-20 dynes / cm2) and increased vascular endothelial growth factor (VEGF) concentration, yielding cells with increased expression of arterial specific markers (EphrinB2, SOX17 etc.), or alternatively exposing progenitor cells to low shear flows (shear stress -1-5 dynes / cm2) with low / no VEGF, driving the differentiation of peripheral blood-derived endothelial cells into a more venous subtype with increasedexpression of one or more venous markers selected from COUP-TFII. EphB4, and the like.

21. A method of tissue-specific maturation of EPC derived endothelial cells, comprising:maturing of EPC derived cells;co-culturing EPCs with tissue- specific cells, creating mature tissue-specific endothelial cells.

22. The method of claim 21, comprising co-culturing EPCs during the maturation process with brain astrocytes and neural progenitors, creating endothelial cells that are phenotypically close to brain microvascular endothelial cells (BMVECs).

23. The method of claim 21, comprising culturing patient-derived EPCs with liver hepatocytes and stellate cells, creating endothelial cells with similar functional and phenotypic profiles as liver sinusoid endothelial cells (LSECs).

24. The method of claim 21, further comprising creating autologous tissue endothelial cell banks without the need of invasive biopsies, enabling creation of more physiologically relevant tissue models for drug screening and biomarker discovery applications, predicting patient-specific tissue responses.

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