3D bioprinted hydrogel microfluidic systems and perfusion bioreactors for engineering vascularized tissue constructs

Abstract

3D bioprinted hydrogel microfluidic systems and perfusion bioreactors for engineering vascularized tissue constructs are provided. An example of an internally perfusable scaffold comprises an exterior surface, a body, a first port, and an interior wall. The first port comprises a first coupling feature configured to engage a fitting and form a fluid tight seal therewith. The first interior wall defines a fluid cavity extending into the body from the first port. The scaffold comprises a hydrogel.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICEPCT APPLICATION FOR3D BIOPRINTED HYDROGEL MICROFLUIDIC SYSTEMS AND PERFUSION BIOREACTORS FOR ENGINEERING VASCULARIZED TISSUE CONSTRUCTSInventors: Daniel J. Shiwarski, Andrew Hudson, and Adam Walter FeinbergRELATED APPLICATIONS

[0001] The present application claims priority to United States provisional patent application Serial No. 63 / 625,061, filed January 25, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] This invention was made with United States government support under HL155777 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.BACKGROUND

[0003] Existing organ-on-chips and microfluidic devices may not be remodeled rapidly by cells. There are challenges with the manufacture and remodeling of organ-on-chips and microfluidic devices.SUMMARY

[0004] According to general aspects, the present disclosure provides an internally perfusable scaffold comprising an exterior surface, a body, a first port, and an interior wall. The first port comprises a first coupling feature configured to engage a fitting and form a fluid tight seal therewith. The first interior wall defines a fluid cavity extending into the body from the first port. The scaffold comprises a hydrogel.

[0005] In other general aspects, the present disclosure provides an internally perfusable scaffold comprising an exterior surface, a body comprising a first portion, a first port, a second port, a third port, a fourth port, a first interior wall, and a second interior wall. The first port, second port, third port, and fourth port extend through the exterior surface. The first interior wall defines a first fluid cavity extending through the body from the first port to the second port. The second interior wall defines a second fluid cavity extending through thebody from the third port to the fourth port. The first portion is intermediate the first interior wall and the second interior wall and the first portion comprises cells, spheroids, organoids, or a combination thereof. The scaffold comprises a hydrogel.

[0006] In yet other general aspects, the present disclosure provides an internally perfusable scaffold comprising an exterior surface, a body comprising a first portion, a first port, a second port, a third port, a fourth port, a first interior wall, and a second interior wall. The first port, second port, third port, and fourth port extend through the exterior surface. The first interior wall defines a first fluid cavity extending through the body from the first port to the second port. The second interior wall defines a second fluid cavity extending through the body from the third port to the fourth port. The first portion is intermediate the first interior wall and the second interior wall. The first portion comprises a third infill density less than a first infill density of the first interior wall and a second interior density of the second wall. The scaffold comprises a hydrogel.

[0007] In yet other general aspects, the present disclosure provides a method for vascularizing the scaffold according to the present disclosure. The method comprises perfusing a medium through the first fluid cavity. The method comprises growing vasculature from at least one of the first interior wall and the second interior wall into the first portion of the body.

[0008] In yet other general aspects, the present disclosure provides a system comprising a bioreactor, a scaffold according to the present disclosure, and a pump. The bioreactor comprises a reactor wall defining a reactor cavity, a first fitting extending into the reactor cavity, and a second fitting extending into the reactor cavity. The scaffold is in the reactor cavity. The first port is engaged with the first fitting and the second port is engaged with the second fitting. The pump is capable to urge fluid through the first fitting and thereby the first fluid cavity.

[0009] In yet other general aspects, the present disclosure provides an additive manufacturing method for manufacturing the scaffold according to the present disclosure. The method comprises depositing a structure material, by a nozzle, into a support material by applying a force to the structure material such that the structure material flows through the nozzle. The method comprises repeating the depositing of the structure material as necessary to create the scaffold. The method comprises at least partially removing the support material from scaffold.

[0010] Various embodiments and implementations of the present invention provide many benefits and improvements relative to prior additive printing techniques, such as, forexample, techniques related to embedded printing. For example, the methods according to the present disclosure can enhance filament resolution, prevents formation agglomerates on the needle, control diffusion of structure material, control gelation of structure material, control of reaction rate, and enable a wider range of reactant concentrations to be utilized in the support material. These and other benefits that are potentially realizable through various implementations of the present invention will be apparent from the description that follows.

[0011] It will be understood that the invention disclosed and described in this specification is not limited to the aspects summarized in this Summary. The reader will appreciate the foregoing details, as well as others, upon considering the following detailed description of various non-limiting and non-exhaustive aspects according to this specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Features and advantages of the examples presented herein, and the manner of attaining them, will become more apparent, and the examples will be better understood, by reference to the following description taken in conjunction with the accompanying drawings, wherein:

[0013] FIG. 1 A is a schematic diagram illustrating the FRESH bioprinting and release process.

[0014] FIG. IB is a schematic diagram illustrating the design, fabrication, and fidelity assessment of FRESH CHIPS CAD design (i), FRESH printing using a collagen bioink (ii), volumetric OCT imaging revealing patent lumens (dark regions, (iii), and 3D gauging of the luminal region for quantitative fidelity assessment of Serpentine channel internal network designs.

[0015] FIG. 1C is a schematic diagram illustrating the design, fabrication, and fidelity assessment of FRESH CHIPS CAD design (i), FRESH printing using a collagen bioink (ii), volumetric OCT imaging revealing patent lumens (dark regions, (iii), and 3D gauging of the luminal region for quantitative fidelity assessment of Triple channel internal network designs.

[0016] FIG. ID is a schematic diagram illustrating the design, fabrication, and fidelity assessment of FRESH CHIPS CAD design (i), FRESH printing using a collagen bioink (ii), volumetric OCT imaging revealing patent lumens (dark regions, (iii), and 3D gauging of the luminal region for quantitative fidelity assessment of Stacked channel internal network designs.

[0017] FIG. IE is a schematic diagram illustrating the multi-scale vascular bed design with open lumens from 1 mm to 250 pm.

[0018] FIG. IF is an image illustrating a Multi-scale vascular bed FRESH printed from collagen I.

[0019] FIG. 1G is an image illustrating perfusion of dye through the multi-scale vascular scaffold.

[0020] FIG. 1H is a volumetric OCT imaging and cross-sectional analysis illustrating lumen patency and circular fidelity of the internal fluidic network.

[0021] FIG. II is a 3D model illustrating a 3D gauging of the multi-scale vascular lumen volume reveals high-fidelity printing with average deviations < 11 pm.

[0022] FIG. 2A is a CAD design illustrating a dual inlet single outlet serpentine network CHIPS with barb-shaped cutouts for mating to the VAPOR inlets and outlets.

[0023] FIG. 2B is a stereomicroscope image illustrating a FRESH printed serpentine CHIPS.

[0024] FIG. 2C is an OCT volumetric imaging and cross-sectional analysis illustrating patent 500 pm lumens (dark regions).

[0025] FIG. 2D is an image illustrating a VAPOR perfusion of Serpentine CHIPS with two dyes.

[0026] FIG. 2E is a graphic illustration illustrating a laminar flow perfusion experiment to demonstrate balanced laminar flow of two fluids within a serpentine CHIPS.

[0027] FIG. 2F is an image illustrating serpentine CHIPS perfused with two dyes demonstrating laminar perfusion and minimal interfacial mixing.

[0028] FIG. 2G is a graphic illustration illustrating a pulsatile flow perfusion experiment to demonstrate fluidic mixing within a serpentine network using an acidic phenol indicator dye and basic NaOH solution.

[0029] FIG. 2H is an image illustrating pulsatile flow in Serpentine CHIPS produces a mixing gradient along the fluidic path.

[0030] FIG. 21 is a quantitative colorimetric analysis illustrating the pH-based mixing profile achieved within the Serpentine channel as the phenol red is neutralized.

[0031] FIG. 2J is a CAD design and adaptation illustrating Stacked channel CHIPS for interfacing with VAPOR.

[0032] FIG. 2K is a stereomicroscope image illustrating a FRESH printed Stacked CHIPS.

[0033] FIG. 2L is a zoomed in portion of the stereomicroscope image of FIG. 2K illustrating an OCT XZ cross-sectional image of the stacked channels highlighting the fidelity and resolution achieved via FRESH bioprinting collagen.

[0034] FIG. 2M is images illustrating initiation of Stacked CHIPS perfusion of two dyes in VAPOR.

[0035] FIG. 2N is an overview image illustrating the perfused Stacked CHIPS within VAPOR.

[0036] FIG. 20 is a CAD model illustrating the 3D Helical CHIPS network.

[0037] FIG. 2P is a stereomicroscope image illustrating a FRESH-printed 3D Helical CHIPS.

[0038] FIG. 2Q is an OCT volumetric imaging and cross-sectional analysis illustrating patent circular lumens (XY) and a YZ projection image to view the full helical network.

[0039] FIG. 2R is images illustrating VAPOR perfusion of 3D Helical CHIPS for 210 min resulting in complete volumetric diffusion of low molecular weight dyes throughout the CHIPS.

[0040] FIG. 3 A is a CAD model illustrating dual parallel channel CHIPS with the channels separated by a collagen wall.

[0041] FIG. 3B is a model illustrating OCT cross-sectional views of dual parallel channel CHIPS.

[0042] FIG. 3C is an overview image illustrating VAPOR perfusion of dual parallel channel CHIPS with two dyes.

[0043] FIG. 3D is a graph illustrating perfusion and tracking of fluorescent microbeads to measure velocity profiles and mean bead speed compared to the theoretical estimation (dotted line).

[0044] FIG. 3E is a schematic illustrating the perfusion of dual parallel channel CHIPS where one channel is always perfused with PBS while the other channel is perfused with different sizes of FITC-conjugated dextran with 6 ROIs being selected for diffusivity analysis.

[0045] FIG. 3F is time lapse images illustrating fluorescence with fire intensity look up table of dual parallel channel CHIPS undergoing VAPOR perfusion with FITC-conjugated dextrans ranging from 3 to 70 kDa.

[0046] FIG. 3G is a series of graphs illustrating ROI-based Analysis of dextran fluorescence intensity over time beginning proximal to the dextran source channel (i) and ending distal to the PBS source channel (vi).

[0047] FIG. 3H is a schematic illustrating increased pressure (+5 mmHg) within the dextran source channel driving diffusion into the perfusate from the PBS circulation that was sampled for spectrophotometric analysis.

[0048] FIG. 31 is a graph illustrating an analysis of 40 kDa FITC-conjugated dextran fluorescence intensity within the PBS circulation after 24 hours of either normal (NP), or increased pressure (+5 mmHg, +10 mmHg) within the dextran source channel.

[0049] FIG. 3 J is a series of timelapse images illustrating fluorescence intensity at 0, 6 and 12 hours calculated as a ratio of the high pressure (HP) image divided by the normal pressure (NP) image to reveal pressure-dependent regions of increased diffusion.

[0050] FIG. 3K is a graph illustrating a line profile analysis of fluorescence intensity at 0, 6 and 12 hours calculated as a ratio of the high pressure (HP) image divided by the normal pressure (NP) image to reveal pressure-dependent regions of increased diffusion.

[0051] FIG. 4A is a schematic and 3D printer machine pathing (G-code) illustrating a dual parallel channel multi-material CHIPS with an internal fibrin-based vascular cell bioink (HUVEC and MSC) region containing 1 mm2fluidic channels.

[0052] FIG. 4B is a 3D confocal fluorescence image illustrating optically cleared high- fidelity collagen and vascular cellular bioink printing with XY and XZ plane views revealing internal structure, open channels, and high-fidelity volumetric registration between the collagen and vascular bioink.

[0053] FIG. 4C is a schematic design and pathing G-code illustrating dual channel CHIPS with collagen walls between the cellular channels.

[0054] FIG. 4D is an XY mid-plane view illustrating whole mount fluorescence images of optically cleared 1-day statically cultured vascular CHIPS with collagen walls. Combined multiphoton fluorescence and second harmonic imaging of the vascular bioink and collagen shows the spatial alignment between the cellular biomaterial and collagen walls.

[0055] FIG. 4E is a depiction of both XY and XZ midplane slice views illustrating 3D confocal imaging of optically cleared dual parallel channel cellular CHIPS with fibrin walls statically cultured for 8 days and stained for actin, nuclei, and the endothelial marker VE- Cadherin (VE-Cad).

[0056] FIG. 4F is a confocal image illustrating regions within revealing microvascular cell spreading and migration with an inset image displaying magnified views of the VE-Cad only channel.

[0057] FIG. 4G is the inset image displaying magnified views of the VE-Cad only channel of FIG. 4F.

[0058] FIG. 4H is a depiction of both XY and XZ slice views illustrating the channel bottom surface from 3D confocal imaging of optically cleared dual parallel channel cellular CHIPSwith collagen walls following 8 days of perfusion culture within VAPOR. *Indicates air bubble artifacts introduced into the channels during CHIPS optical clearing and imaging.

[0059] FIG. 41 is confocal images illustrating strong lumenal expression of VE-Cadherin with an inset image displaying magnified views of the VE-Cad and Actin only channels respectively, indicates air bubble artifacts introduced into the channels during CHIPS optical clearing and imaging.

[0060] FIG. 4J is confocal images illustrating evidence of cellular remodeling and vascular- like network maturation with an inset image displaying magnified views of the VE-Cad and Actin only channels respectively, indicates air bubble artifacts introduced into the channels during CHIPS optical clearing and imaging.

[0061] FIG. 5A is a schematic design illustrating a 3D printer machine pathing G-code of a dual parallel channel multi -material CHIPS with pancreatic vascular cell bioink (MIN6, HUVEC, and MSC) regions surrounding both sides of the channels.

[0062] FIG. 5B is a 3D confocal fluorescence image illustrating pancreatic CHIPS FRESH printed and visualized via brightfield stereomicroscope (inset) and 3D confocal fluorescence imaging of the optically cleared pancreatic scaffold after 12 days of static culture.

[0063] FIG. 5C is a confocal fluorescence image illustrating the XY midplane view from the image of the 12-day statically cultured pancreatic CHIPS following 3D vascular network segmentation for quantification of network diameter and density within the migratory zones.

[0064] FIG. 5D is an example confocal fluorescence image illustrating additional cell migration into the acellular regions of the CHIPS beneath the cellular regions guided by the printed collagen filaments.

[0065] FIG. 5E is a 3D confocal fluorescence image illustrating XY midplane projection view from 3D confocal fluorescence imaging of the optically cleared 12-day VAPOR perfused pancreatic CHIPS.

[0066] FIG. 5F is a graphic illustration illustrating example regions of interest (ROIs) depicting evidence of early MIN6 pancreatic bud and microlumen formation with actin and insulin fluorescence images from ROI 2 and 3 in (E).

[0067] FIG. 5G is a graphic illustration illustrating quantification for insulin secretion ELISA assay from 1.5-hour glucose-stimulated (ratio of high glucose-HG to low glucose-LG) insulin secretion experiment between 12-day static and perfusion cultured pancreatic CHIPS (mean ± STDEV; **P < 0.01 for n= 2 static tissues; n = 2 perfused tissues).

[0068] FIG. 6A is a CAD model and schematic illustrating the (i) VAPOR base, (ii) lid and gasket assembly, (iii) CHIPS and VAPOR barb interface for watertight seal, (iv) dual independent flow paths through VAPOR and CHIPS.

[0069] FIG. 6B is a diagram illustrating the dimensions of the assembled VAPOR system.

[0070] FIG. 6C is an overview image illustrating VAPOR perfused with two dyes to visualize the internal fluidic networks.

[0071] FIG. 6D is an overview image illustrating a fully assembled VAPOR and inserted serpentine CHIPS prior to perfusion.

[0072] FIG. 6E is a schematic illustrating the perfusion setup utilized in VAPOR.

[0073] FIG. 6F is an image illustrating the assembled VAPOR platform and perfusion system prior to installation into a cell culture incubator.

[0074] FIG. 7A is an image illustrating a CHIPS model midway through FRESH printing within a gelatin support bath.

[0075] FIG. 7B is an image illustrating a top (helical) view of CHIPS immediately after FRESH printing.

[0076] FIG. 7C is an image illustrating a bottom (stacked) view of CHIPS immediately after FRESH printing.

[0077] FIG. 7D is a stereomicroscope image illustrating a Stacked CHIPS model after release from the FRESH support bath.

[0078] FIG. 7E is a graph illustrating OCT quantification of measured versus expected channel dimensions for Serpentine CHIPS.

[0079] FIG. 7F is a graph illustrating OCT quantification of measured versus expected channel dimensions for Stacked (H) CHIPS.

[0080] FIG. 7G is a graph illustrating OCT quantification of measured versus expected channel dimensions for 3D Helical CHIPS.

[0081] FIG. 8A is a series of time lapse images illustrating dual parallel channel CHIPS undergoing VAPOR perfusion with FITC-conjugated 10 kDa dextran.

[0082] FIG. 8B is a series of time lapse images illustrating dual parallel channel CHIPS undergoing VAPOR perfusion with FITC-conjugated 40 kDa dextran under normal pressure.

[0083] FIG. 8C is a series of time lapse images illustrating dual parallel channel CHIPS undergoing high pressure (+10 mmHg) VAPOR perfusion of 40 kDa FITC-conjugated dextran over 24 hours.

[0084] FIG. 9A is an image illustrating a custom high-performance 3D bioprinter based on a commercial Aerotech motion control platform and open-source Replistruder 5 syringe pumps.

[0085] FIG. 9B is an image illustrating how three Replistruder 5 syringe pumps are utilized for material multi-material FRESH printing of CHIPS. An onboard OCT system allows for volumetric imaging and in-process monitoring of FRESH printed CHIPS.

[0086] FIG. 10A is a schematic diagram illustrating a parallel plate style CHIPS design with a collagen frame and uniform single layer of fluorescently-tagged fibronectin (Fn) containing collagen displayed as (i) CAD design and stereomicroscopy image, and (ii) quantified for multi-material printing registration with 3D confocal microscopy (iii-v, dotted line indicates cut plane, inset scale bar in v = 250 pm).

[0087] FIG. 10B is a schematic diagram illustrating a parallel plate style CHIPS design with a collagen frame and vertical lines of fluorescently-tagged fibronectin (Fn) containing collagen displayed as (i) CAD design and stereomicroscopy image, and (ii) quantified for multi-material printing registration with 3D confocal microscopy (iii-v, dotted line indicates cut plane, inset scale bar in v = 250 pm).

[0088] FIG. 10C is a schematic diagram illustrating a parallel plate style CHIPS design with a collagen frame and branching network of fluorescently-tagged fibronectin (Fn) containing collagen displayed as (i) CAD design and stereomicroscopy image, and (ii) quantified for multi-material printing registration with 3D confocal microscopy (iii-v, dotted line indicates cut plane, inset scale bar in v = 250 pm).

[0089] FIG. 10D is a schematic diagram illustrating a collagen stiffness gradient generated by multi-material printing of 6, 12, and 23 mg / mL collagen in adjacent regions within the parallel plate CHIPS (i), stereomicroscopy image (ii) of the 3-material print demonstrates high-fidelity and multi-material registration upon fluorescence image analysis (iii-v).

[0090] FIG. 11 A is a Dual channel CHIPS CAD design illustrating internally lined channels containing 12 mg / mL collagen + Fn with a central dividing region between the fluidic channels containing 23 mg / mL collagen + VEGF growth factor.

[0091] FIG. 1 IB is a confocal fluorescence image illustrating maximum Z-proj ection of the 12 mg / mL collagen channel lining and VEGF patterning.

[0092] FIG. 11C. is an XZ slice plane image of FIG. 1 IB illustrating the Fn channel lining and VEGF patterning.

[0093] FIG. 1 ID is a confocal fluorescence image illustrating central channel regions from (B) seeded with HUVECs and stained for nuclei distribution at day 1 after seeding.

[0094] FIG. 1 IE is a volumetric confocal fluorescence image illustrating optically cleared multi-material dual parallel channel CHIPS stained for endothelial cell marker CD-31 and actin.

[0095] FIG. 1 IF is a confocal fluorescence image illustrating a 3D view with XZ crosssection of endothelial channel lining.

[0096] FIG. 11G is a confocal fluorescence image illustrating an XY mid-plane view of open channel lumen and cell seeding.

[0097] FIG. 11H is a confocal fluorescence image illustrating an XY top surface of channel view of endothelial monolayer coating the channel lumen.

[0098] FIG. 1 II is a confocal fluorescence image illustrating magnified XY images of endothelial lining within channels and increased zoom (i).

[0099] FIG. 12A is a schematic diagram illustrating a dimensional comparison of the vascular CHIPS with complete cellular + fibrin walls CAD model (i) to the vascular CHIPS after 8-days of static culture (ii).

[0100] FIG. 12B is a schematic diagram illustrating a dimensional comparison of the vascular CHIPS with collagen walls CAD model (i) to vascular CHIPS after 1 (ii) and 8-days of static culture (iii).

[0101] FIG. 13 A is a confocal fluorescence image illustrating both XY and XZ perspective slice views of the channel bottom surface from 3D confocal imaging of optically cleared dual parallel channel cellular CHIPS with collagen walls statically cultured for 8 days.

[0102] FIG. 13B. is a zoomed in portion of the confocal fluorescence image of FIG. 13 A illustrating cellular alignment along the length of the channels, lumenal VE-Cadherin expression.

[0103] FIG. 13C is a zoomed in portion of the confocal fluorescence image of FIG. 13A illustrating cellular alignment along the length of the channels, and evidence of cell migration following the printed collagen infill of distances exceeding 1.5 mm from the channel outside edge.

[0104] FIG. 13D is a confocal fluorescence image illustrating both XY and XZ perspective midplane slice views from 3D confocal imaging of optically cleared dual parallel channel fibrin walls cellular CHIPS following 8 days of perfusion culture within VAPOR, indicates air bubble artifacts introduced into the channels during CHIPS optical clearing and imaging.

[0105] FIG. 13E. is a zoomed in portion of the confocal fluorescence image of FIG. 13D illustrating enhanced VE-Cadherin expression around the flow channels.

[0106] FIG. 13F. is a zoomed in portion of the confocal fluorescence image of FIG. 13D illustrating extensive cell spreading throughout the central cellular region between channels.

[0107]

[0108] FIG. 14A is a schematic design illustrating pancreatic CHIPS with fibrin walls.

[0109] FIG. 14B is a schematic design illustrating Machine pathing G-code of pancreatic CHIPS with fibrin walls.

[0110] FIG. 14C is a stereomicroscope image illustrating a pancreatic CHIPS after 8 days of static culture.[OHl] FIG. 14D is a confocal fluorescence image illustrating an XY plane view from whole mount confocal fluorescence imaging of optically cleared 8-day statically cultured pancreatic CHIPS.

[0112] FIG. 14E is a zoomed in portion of the confocal fluorescence image of FIG. 14D illustrating the central cellularized area within the CHIPS.

[0113] FIG. 14F is a zoomed in portion of the printed channel wall in theconfocal fluorescence image of FIG. 14E illustrating the high cellularization achieved via 60 million cell / mL bioink and presence of insulin expression.

[0114] FIG. 14G. is a zoomed in portion of the confocal fluorescence image of FIG. 14F illustrating showing the cellular distribution and protein expression within the fibrin printed wall after 8 days of static culture.

[0115] FIG. 14H is a series of confocal fluorescence images illustrating CD-31 colocalization with Actin within CHIPS resulting in a Pearson’s Correlation Coefficient of 0.63 ± 0.04.

[0116] FIG. 141 is a graphic illustration illustrating the FRESH printing pattern at the surface of the cellular region.

[0117] FIG. 14J is a confocal fluorescence image illustrating the cellular alignment (actin) along the direction of the original print path (-45° angle) shown in (I).

[0118] FIG. 14K is a graphic illustration illustrating the FRESH printed volume infill pattern (alternating ±45° angle) at a depth 200 pm from the surface of the cellular region.

[0119] FIG. 14L is a confocal fluorescence image illustrating cellular migration (actin) along the printed collagen filaments 200 pm below the original printed cellular region.

[0120] FIG. 15A is a confocal fluorescence image illustrating XY, YZ, and XZ plane views revealing the intricate cell network and migration between the patent flow channels.

[0121] FIG. 15B is a confocal fluorescence image illustrating XY max intensity Z-proj ection image demonstrating the range of cell migration (actin) outward from the printed regions.

[0122] FIG. 15C is a confocal fluorescence image illustrating a zoomed in image to the outer migration zone.

[0123] FIG. 15D is a confocal fluorescence image illustrating an XZ cross sectional analysis expression profile of the cellular (actin) markers lining the original acellular channels, in addition to a YZ projection showing a dense luminal cell monolayer along the side walls.

[0124] FIG. 16A is a confocal fluorescence image illustrating from ROI 1 in Figure 5 (E) illustrating a (A) branched vessel-like structure 100 pm in diameter within the CHIPS.

[0125] FIG. 16B is a confocal fluorescence image from ROI 1 in Figure 5 (E) illustrating a vascular-like bundle of 50 pm in diameter bridging and following the infill lattice structure within the CHIPS.

[0126] FIG. 16C is a confocal fluorescence image from ROI 1 in Figure 5 (E) illustrating a25 pm Y-branched vascular-like structure bridging across the collagen infill pattern (outline with dotted white lines) within the CHIPS.

[0127] FIG. 16D is a 3D perspective view illustrating a 24 pm vessel with visible open lumen expressing actin and VE-cadherin (VE-Cad).

[0128] FIG. 17A is a schematic diagram illustrating a top view of a scaffold according to the present disclosure.

[0129] FIG. 17B is a schematic diagram illustrating a side view of the scaffold of FIG. 17A.

[0130] FIG. 17C is a schematic diagram illustrating an isometric perspective view of the scaffold of FIG. 17 A.

[0131] FIG. 18A is a schematic diagram illustrating a perspective view of a scaffold according to the present disclosure.

[0132] FIG. 18B is a schematic diagram illustrating a top view of the scaffold of FIG. 18 A.

[0133] FIG. 19A is a schematic diagram illustrating a perspective view of a system comprising a bioreactor and scaffold according to the present disclosure.

[0134] FIG. 19B is a schematic diagram illustrating an isolated top view of the bioreactor of FIG. 19 A.

[0135] FIG. 20 is a block diagram of an example of an additive manufacturing FRE system according to the present disclosure, the X-axis is coming out of the page.

[0136] FIG. 21 is flow chart of an example of an additive manufacturing FRE method according to the present disclosure.

[0137] Corresponding reference characters indicate corresponding parts throughout the several views. The exemplifications set out herein illustrate certain embodiments, in oneform, and such exemplifications are not to be construed as limiting the scope of the appended claims in any manner.DETAILED DESCRIPTION

[0138] Various examples are described and illustrated herein to provide an overall understanding of the structure, function, and use of the disclosed scaffolds, systems, and methods. The various examples described and illustrated herein are non-limiting and non- exhaustive. Thus, the invention is not limited by the description of the various non-limiting and non-exhaustive examples disclosed herein. Rather, the invention is defined solely by the claims. The features and characteristics illustrated and / or described in connection with various examples may be combined with the features and characteristics of other examples. Such modifications and variations are intended to be included within the scope of this specification. As such, the claims may be amended to recite any features or characteristics expressly or inherently described in, or otherwise expressly or inherently supported by, this specification. Further, Applicant reserves the right to amend the claims to affirmatively disclaim features or characteristics that may be present in the prior art. The various examples disclosed and described in this specification can comprise, consist of, or consist essentially of the features and characteristics as variously described herein.

[0139] Existing organ-on-chips and microfluidics devices typically have perfusion ports either in the top surface or in the sides and cannot be remodeled rapidly by cells. The present inventors determined that ports suitable for fluid tight connections, such as, for example, undercut or tubular connections may be incorporated into a scaffold to create a snap fit system for fluid flow perfusion and / or nutrient delivery.

[0140] Existing organ-on-chips and microfluidics device may require extensive perfusion and / or may not be able to direct growth within the device as desired. Existing devices may have limited cell migration within the body of the device and slow nutrient diffusivity which may be caused by a lack of microporosity and monolithic, solid construction. Existing devices have not been able to achieve complex 3D geometries or desirable multi-material patterned biomaterials, leading to simplistic designs, such as, for example, rectangular channels. The inventors have determined there are challenges with manufacture and remodeling of organ-on-chips and microfluidic devices using materials and cells that can remodel, vascularize, and form into new tissue-like materials that have improved physiologic relevance.

[0141] The present inventors have adapted additive manufacturing techniques to create hydrogels with structures previously not obtainable that enable control of growth within scaffolds. The scaffolds according to the present disclosure may facilitate installation of engineered organ-on-chips into bioreactors for sterile culture and perfusion. The scaffolds may promote rapid cellular infiltration, microvascular network formation, and / or remodeling, which may result in functional tissue. The scaffolds according to the present disclosure can direct growth beyond simply fluid channels and within the body of the scaffold itself.

[0142] Referring to FIGs. 18A-18B, an internally perfusable scaffold 100 is provided. The scaffold 100 can enable perfusion of fluids and / or nutrient delivery to enable cell survival, vascular network formation, remodeling, and / or tissue maturation for various tissue and / or organ systems. These systems may exceed the 200 pm passive diffusion limits.

[0143] The scaffold 100 can be formed by various methods, such as, for example, additive manufacturing. The additive manufacturing can comprise an extrusion based additive manufacturing technique, such as, for example, Freeform Reversible Embedded (FRE) additive manufacturing technique and / or a FRE of Suspended Hydrogel (FRESH) additive manufacturing technique as described herein, and / or other additive manufacturing technique.

[0144] The scaffold 100 can comprise a hydrogel, cells, organoids, spheroids, and / or other additives (e.g., xanthan gum, proteins). In various examples, the solid portions (e.g., nonvoid space) of the scaffold 100 can comprise at least 80% by weight of the hydrogel based on the total weight of the scaffold, such as, for example, at least 90% by weight of the hydrogel, at least 95% by weight of the hydrogel, or at least 99% by weight of the hydrogel.

[0145] The hydrogel can comprise a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, a Matrigel, or a combination thereof.

[0146] In various examples, the hydrogel can comprise a collagen concentration in a range of 1 mg collagen per milliliter to 150 mg collagen per milliliter, such as, for example, 6 mg collagen per milliliter to 150 mg collagen per milliliter, 10 mg collagen per milliliter to 150 mg collagen per milliliter, 11 mg collagen per milliliter to 150 mg collagen per milliliter, 15 mg collagen per milliliter to 150 mg collagen per milliliter, 20 mg collagen per milliliter to 150 mg collagen per milliliter, 20 mg collagen per milliliter to 100 mg collagen per milliliter, 10 mg collagen per milliliter to 70 mg collagen per milliliter, 15 mg collagen per milliliter to 70 mg collagen per milliliter, or 20 mg collagen per milliliter to 80 mg collagen per milliliter.

[0147] The scaffold 100 can comprise at least two different materials. For example, the scaffold 100 can comprise a first material, a second material, and optionally additional materials. Both the first material and the second material can comprise a hydrogel or only one of the first material and the second material may comprise a hydrogel. The first material can comprise a collagen material having a first collagen concentration and the second material can comprise a collagen material having a second collagen concentration. The first collagen concentration and the second collagen concentration can be different. In certain examples, the first material can comprise a collagen material and the second material can comprise a fibrinogen material and cells.

[0148] The cells can comprise eukaryotic cells derived from an animal. The cells can be obtained from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), primary tissues, a cell line, or a combination thereof. The cells can comprise human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells (MSCs), MIN6 (Mouse insulinoma cell line), and / or other cell type.

[0149] In certain examples, the scaffold 100 can comprise, consist essentially of, or consist of biologically derived materials. The biologically derived materials can enable the biological compatibility of the scaffold 100 for in vitro and / or in vivo use.

[0150] The scaffold 100 can be a soft material. For example, the scaffold 100 can comprise an elastic modulus in a range of 1 kPa to 100 kPa, such as, for example, 1 kPa to 50 kPa or 1 kPa to 10 kPa.

[0151] The scaffold 100 can comprise an exterior surface 102, a body 104, a first port 106a, and an interior wall 114a. The scaffold 100 can enable perfusion through the exterior surface 102 and / or interior wall 114a. The scaffold 100 can comprise various fluid cavities (e.g., channels) that enable both active and passive fluid transport into and out of the body 104. The fluid transport can be enabled by the microporosity and / or otherwise design of the scaffold 100. For example, void space within an infill pattern of the body 104 can enable fluid transport within the body 104.

[0152] The body 104 can be porous. For example, the body 104 may comprises microporosity and / or voids. In various examples, the voids can be produced by additive manufacturing processes. For example, the voids may be a result of an infill pattern.

[0153] The scaffold 100 can comprise various ports as desired, such as, for example, at least two ports (e.g., first port 106a and second port 106b), at least three ports (e.g., first port 106a, second port 106b, and third port 106c), at least four ports (e.g., first port 106a, second port 106b, third port 106c, and fourth port 106d). Each port 106a-106d can extend through theexterior surface 102 (e.g., FIGs. 18A-18B) or from the exterior surface 102 (e.g., FIGs. 17A- 17C) as the application may require. Each port 106a-106d can be individually configured and may be the same or at least one port may be differently configured than the rest. The scaffold 100 can be desired with a quantity of ports as desired for the application. For example, the scaffold 100 can be configured as a blood circuit, a urine circuit, a kidney model, an air circuit, and / or other scaffold type.

[0154] Each port 106a-106d can comprise a coupling feature configured to engage a fitting and form a fluid tight seal therewith (e.g., fitting 256a-256d attached to bioreactor 250 in FIGs. 19A-19B).

[0155] Each coupling feature can be the same or different. Each coupling feature can be a female feature or a male feature. For example, the coupling feature can comprise an undercut 110 as shown in FIGs. 18A-18B. The coupling feature can comprise a frustoconical shape. For example, each coupling feature can be a complementary shape to a barbed fitting.

[0156] Referring to FIGs. 17A-17C, each coupling feature can be a tubular protrusion 112 extending a distance from the exterior surface 102. A fitting may be slide within the tubular protrusion 112 and the tubular protrusion 112 can be clamped to the fitting. In various examples, the tubular protrusion 112 can be capable to be anastomosed to a blood vessel.

[0157] The scaffold 100 can comprise an interior wall 114a defining a fluid cavity 116a extending into the body 104 from the first port 106a. For example, the fluid cavity 116a can extend from the first port 106a through the body 104 to the second port 106b. The scaffold 100 can define additional fluid cavities as desired for the application, such as, for example, at least two fluid cavities, at least three fluid cavities, at least four fluid cavities, or at least five fluid cavities. For example, the scaffold 100 can comprise an interior wall 114b defining a fluid cavity 116b extending from the third port 106c through the body 104 to the fourth port 106d.

[0158] Although FIGs. 18A-18B illustrates a single inlet and a single outlet for each fluid cavity 114a-l 14b, it is understood that a fluid cavity can comprise multiple inlets and / or multiple outlets as desired (e.g., FIG. 2A). It is also understood that a fluid cavity 114a-l 14b may only have an inlet (e.g., no outlet) or may only have an outlet (e.g., no inlet). Fluid flow can be introduced through one of fluid cavities 114a-l 14b, diffuse through the first portion 118, and exit through a different one of fluid cavities 114a-l 14b.

[0159] Referring yet again to FIGs. 18A-18C, the fluid cavity 116a and / or 116b can comprise various shapes as desired for the application. For example, the fluid cavity 116a and / or 116b can comprise a substantially straight shape, a curved shape, a serpentine shape, a helicalshape, or other shape as desired. The fluid cavity 116a and / or 116b may remain a single channel throughout the body 104 or the fluid cavity 116a and / or 116b can split into multiple fluid channels that can recombine into a single channel (e.g., FIG. IE) and / or at least partially stay split into multiple fluid channels.

[0160] Referring yet again to FIGs. 18A-18C, the body 104 can comprise various portions. For example, the body 104 can comprise a first portion 118 that can be intermediate the first interior wall 114a and the second interior wall 114b. For example, the first portion 118 can be a diffusion zone intermediate adjacent fluid cavities. The first portion 118 can act as a barrier for large molecule diffusion and / or separate the fluid flow between adjacent fluid cavities.

[0161] The first portion 118 can be the same as the remainder of the body 104 and / or different than other portions of the body 104. The first portion 118 can comprise a different material than interior walls 114a-l 14b, a different concentration of a material than interior walls 114a-l 14b, and / or a different infill density than the interior walls 114a-l 14b.

[0162] For example, the first portion 118 can comprise a hydrogel, cells, spheroids, organoids, additives, or a combination thereof. In various examples, the first portion 118 comprises at least one of cells, spheroids, organoids, or a combination thereof. In various examples, other portions of the body 104 than the first portion 118 may be acellular (e.g., may not comprise cells) or they may comprise cells, which are different and / or the same.

[0163] The first portion 118 can comprise a cell concentration of at least 10 million cells per milliliter, such as, for example, at least 20 million cells per milliliter, at least 30 million cells per milliliter, at least 40 million cells per milliliter, at least 50 million cells per milliliter, at least 10 million cells per milliliter, or at least 200 million cells per milliliter. The first portion 118 can comprise 10 million cells per milliliter to at least 500 million cells per milliliter, such as, for example, 10 million cells per milliliter to at least 300 million cells per milliliter, 10 million cells per milliliter to at least 100 million cells per milliliter, 60 million cells per milliliter to at least 300 million cells per milliliter, or 60 million cells per milliliter to at least 100 million cells per milliliter.

[0164] A region of one or both of the interior walls 114a and 114b adjacent to the first portion 118 can comprise cells, such as, for example, at least 10 million cells per milliliter, at least 20 million cells per milliliter, at least 30 million cells per milliliter, at least 40 million cells per milliliter, at least 50 million cells per milliliter, at least 10 million cells per milliliter, or at least 200 million cells per milliliter.

[0165] The first portion 118 can comprise a first material and one or both of the interior walls 114a and 114b can comprise a second material. The first material can comprise fibrinogen and the second material can comprise collagen. For example, the first material can comprise at least 10 milligrams per milliliter of fibrinogen and the second material can comprise at least 6 milligrams per milliliter of collagen.

[0166] The scaffold 100 can comprise a variable infill density.

[0167] For example, the first portion 118 can comprise a third infill density less than a first interior density of the first interior wall 114a and a second interior density of the second interior wall 114b. For example, the third infill density can be no greater than 85% by volume, such as, for example, no greater than 80% by volume, no greater than 70% by volume, no greater than 60% by volume, no greater than 50% by volume, no greater than 40% by volume, no greater than 30% by volume, or no greater than 20% by volume. The third infill density can be in a range of 5% by volume to 85% by volume, such as, for example, the third infill density can be in a range of 10% by volume to 50% by volume or 10% by volume to 40% by volume. The void space in the infill pattern can enable formation of new vasculature. The solid portions of the infill pattern can direct vasculature growth such that the vasculature substantially grows in the direction of the infill pattern.

[0168] The first and second infill densities, individually, can be at least 90% by volume, such as, for example, at least 95% by volume, at least 96% by volume, at least 97% by volume, at least 98% by volume, at least 99% by volume, or 100% by volume. For example, the first and second infill densities can be solid.

[0169] The first portion 118 can comprise various infill patterns as desired for the application. For example, the first portion 118 can comprise an infill pattern selected from the group consisting of gyroid infill, cubic, adaptive cubic, support cubic, rectilinear, aligned rectilinear, grid, triangles, stars, line, concentric, honeycomb, 3D honeycomb, Hilbert curve, Archimedean chords, octagram spiral, and lightning. In various examples, the first portion 118 can comprise a rectilinear infill pattern. In certain examples, the infill pattern can be configured similar to a biological feature (e.g., microvascular network) and / or anatomical structure.

[0170] The infill pattern in the first portion 118 can be capable to promote vascular network formation with or without perfusion through the fluid cavities 116a and / or 116b. For example, microporosity of the interior walls 114a and 114b and certain printed filaments (e.g., collagen filaments) can facilitate sprouting vascular network formation. Perfusionthrough the fluid cavities 116a and / or 116b can enable mechanical cues that enhance the vascular maturation and result in vessel remodeling and pruning.

[0171] The microporosity in the interior walls 114a and 114b and / or solid portions of the infill pattern can be in a range of 0% to 80% by volume, such as, for example, 1% to 80% by volume, 10% to 80% by volume, 20% to 80% by volume, 30% to 80% by volume, 40% to 80% by volume, 50% to 80% by volume, 60% to 80% by volume, or 65% to 75% by volume. In various examples, the average diameter of pores in the interior walls 114a and 114b and / or solid portions of the infill pattern can be in a range of 10 microns to 40 microns, as measured with microscopy. The microporosity of the interior walls 114a and 114b and / or infill pattern, which may be printed by FRESH, can enhance vascular network formation.

[0172] The scaffold 100 can be used with a bioreactor, implanted in a subject, or both. For example, referring to FIGs. 19A-19B, a system 200 is provided that comprises the scaffold 100 and a bioreactor 250. The bioreactor 250 can be capable to enable the growth of cells, organoids, spheroids, vasculature, and / or other biological structures within and / or on the scaffold 100.

[0173] The bioreactor 250 can comprise a reactor wall 252 defining a reactor cavity 254.The scaffold 100 can be in the reactor cavity 254.

[0174] The bioreactor 250 can be configured to facilitate fluid communication with the scaffold and enable cell growth The bioreactor 250 can comprise a first fitting 256a extending into the reactor cavity 254. The first fitting 256a can be engaged with the first port 106a of the scaffold 100. The engagement can enable fluid communication between the bioreactor 250 and the scaffold 100.

[0175] The bioreactor 250 can comprise additional fittings as the application may require. For example, the reactor can comprise a second fitting 256b, a third fitting 256c, and a fourth fitting 256d and each fitting 256b-256d can extend into the reactor cavity 254. Each fitting 256b-256c can be engaged with a respective port 106b-106d of the scaffold 100 to enable fluid communication between the bioreactor 250 and the scaffold 100.

[0176] The bioreactor 250 can comprise various components to enable perfusion in the scaffold 100. For example, the bioreactor 250 can comprise a pump, a bubble trap, a valve, a tube, a reservoir, and / or other components as desired. For example, referring to FIG. 19A, the bioreactor 250 can comprise a pump 258 capable to urge fluid through the first fitting 256a and thereby the first fluid cavity 116a via one or more tubes 260. Each fitting 256a- 256d can be in fluid communication with a pump, a bubble trap, a valve, a tube, a reservoir,fittings (e.g., barbed, luer lock), drainage, pressure regulation, and / or other component as desired. In various examples, each fitting 256a-256d can be a barbed fitting.

[0177] The pump 258 can be a mechanical pump (e.g., piston, cavity, peristaltic), a gravity pump, and / or a gravity well.

[0178] The bioreactor 250 can comprise a lid 262 disposed over the reactor cavity 254 and removably connected to the reactor wall 252. The lid 262 can urge the scaffold 100 towards the reactor wall 252 and maintain engagement between the port 106a-106d and the fittings 114a-l 14d. With the lid 262 in place, the reactor cavity 254 can be a closed system with fluid exchange only through the fittings 256a-256d.

[0179] The bioreactor 250 can comprise an elastomeric seal 264 intermediate the lid 262 and the reactor wall 252. The elastomeric seal 264 can inhibit fluid from being expelled from the reactor cavity 254. In certain examples, the elastomeric seal 264 can comprise silicone.

[0180] The reactor cavity 254 can be filled with a fluid to surround the scaffold 100 and / or float the scaffold 100. For example, the fluid in the reactor cavity 254 can comprise water and a buffer. In various non-limiting embodiments, the bioreactor 250 can comprise drainage to regulate pressure and / or remove bubbles from a respective fluid cavity and / or the reactor cavity 254.

[0181] The scaffold 100 can be vascularized and / or other subject to cell growth. For example, the scaffold 100 can be vascularized with the system 200. Vascularizing can comprise perfusing a medium through the a respective fluid cavity 116a-l 16d of the scaffold 100. For example, the pump 258 can urge fluid through the tube 260 into the first fitting 256a through the first port 106a and through the first fluid cavity 116a. The fluid can contact the first interior wall 114a and permeate into the body 104 of the scaffold 100, such as, for example, the first portion 118.

[0182] The medium can comprise cells, water, blood, plasma, a buffer, an antibiotic, glucose, growth factor, and / or other components as the application may require. The medium can comprise a growth medium, a vascularization medium, a differentiation medium, or a combination thereof.

[0183] Vasculature can be grown from the first interior wall 114a and / or the second interior wall 114b into the first portion 118 of the body 104. In various examples, cell growth can be guided along filaments within the first portion 118.

[0184] In certain examples, the first portion 118 may comprise pancreatic cells forming a pancreatic niche. The developmental structure of embryonic islets may form within 2 weeks of growth of the pancreatic niche. In various examples, the pancreatic niche can be capableof insulin secretion in response to glucose. In certain examples, the scaffold 100 can comprise at least 1 mm of tissue viable and / or perfused. In various examples, the scaffold 100 can comprise volumetric vascularized functional tissues.

[0185] As used herein, “additive manufacturing” means a process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies. For example, additive manufacturing can comprise fused deposition modeling (FDM) and Freeform Reversible Embedding (FRE). FDM can comprise extruding a material by heating it to a temperature above its melting temperature and depositing the extruded material in a pattern to form a layer of an object. Subsequent layers can be deposited on top of the previous layer as necessary to form an object.

[0186] FRE is similar to FDM, but instead of depositing a material on top of previous depositions or supports, FRE embeds structure material near other embedded deposits inside a support material and relies on the triggered assembly or reorganization of the material using targeted heating, photopolymerization, crosslinking, slow reaction kinetics, application of binders, and / or other curing technique. For example, the support material may provide divalent cations for crosslinking, such that when the structure material contacts the support material, the structure material begins to cure.

[0187] For additive manufacturing techniques such as FDM, support materials are usually as stiff as the printed material, printed as part of the previous layer, and placed only underneath or neighboring the print layers to prevent deformations. In FRE, the support material can surround the extrusion nozzle and the print material can be deposited inside the support. The support material can be a non-newtonian fluid that allows for deposition of various materials while maintaining a buoyant, physical support for already embedded deposits of print material. When two embedded deposits of print material with a predetermined distance inside of the support material, they can fuse. After printing, the support material can be removed from the deposited print material to form a fully assembled object from the deposited print material.

[0188] In FRE, an object can be printed in any direction in 3D space and is not limited to layer-by-layer printing. For example, a structure can also be printed layer by layer in an X-Y plane, or a non-X-Y plane, such as the X-Z plane, or in a plane at any angle offset from the X-Y Plane. An object can also be printed utilizing FRE in a non-planar fashion, such as, for example, in a curved path such as a helix. Utilizing FRE can enable printing of objects with mechanical properties that are different in the plane of printing versus orthogonal to the plane of printing or other angle to the plane of printing. Additional details regarding the FRE andsimilar processes can be found in U.S. Patent No. 10,150,258 filed January 29, 2016, International Patent Application No. PCT / US2019 / 026787 filed April 10, 2019, International Patent Application No. PCT / US2020 / 056338 filed October 19, 2020, International Patent Application No. PCT / US2021 / 051856 filed September 24, 2021, International Patent Application No. PCT / US2023 / 035131 filed October 13, 2023, International Patent Application No. PCT / US2024 / 042464 filed August 15, 2024, International Patent Application No. PCT / US2024 / 042473 filed August 15, 2024, and International Patent Application No. PCT / US2024 / 024897 filed April 17, 2024, each of which are hereby incorporated by reference herein.

[0189] As the demand for donor tissue and organs continues to outpace the supply, clinicians are turning to regenerative medicine and tissue engineering strategies to create tissue de novo. 3D bioprinting has emerged as a way to build these tissues using robotic control to precisely pattern cells and biological hydrogels in a layer-by-layer process. However, this technology has been slowed by the difficulty of printing these soft, deformable materials into complex 3D architectures that recapitulate anatomic structure from the micro to macro length scale. Embedded 3D bioprinting, such as FRE or a FRE of Suspended Hydrogels (FRESH), can addresses this challenge by providing a sacrificial support that prevents tissue deformation during bioprinting to produce highly relevant constructs such as heart valves, cardiac tissue scaffolds, and perfusable vascular networks. These sophisticated bioprinted constructs could prove useful as clinical diagnostic tools or direct tissue replacements.

[0190] Referring to FIG. 20, a block diagram illustrating an example of an additive manufacturing system 2000 for additive manufacturing, such as, for example, FRE, according to the present disclosure is provided. The system 2000 comprises an extruder assembly 2002, a computer system 2004, and a material deposition region 2006. In various examples, a detector, additional extruders, and / or nozzles may be added to the additive manufacturing system to increase the printing capabilities of the additive manufacturing system. For example, the system 2000 can comprise at least two extruders and at least two nozzles, one extruder and nozzle assembly can be used for various different structure materials to be printed. Each extruder and nozzle assembly can be individually controlled. For simplicity, only one extruder and nozzle assembly is shown in FIG. 20.

[0191] The system 2000 can be capable to print structure material in the support material 2008 in the material deposition region 2006 using the extruder assembly 2002 to form an object 2014. The object 2014 can be a scaffold, such as, for example, scaffold 100.

[0192] The support material 2008 can mechanically support at least a portion of the embedded structure material (i.e., object 2014), maintain the intended geometry of the embedded structure material, and inhibit deformation of the structure material during the FRE additive manufacturing process. For example, the embedded structure material can be held in position within the support material 2008 until the structure material is cured. The support material 2008 can be stationary at an applied stress level below a threshold stress level and can flow at an applied stress level at or above the threshold stress level during the FRE additive manufacturing process.

[0193] The support material 2008 can be a viscoplastic material with Bingham plastic-like rheological behavior. The support material 2008 may demonstrate a significant shear thinning behavior such that the support material 2008 acts like a solid material during deposition of the structure material and then acts like a fluid when the nozzle 2010 is moved through the support material 2008 such that the movement of the nozzle 2010 does not disturb deposited structure material. A drop in viscosity of the support material 2008 under dynamic loading can make the support material 2008 suitable for FRE. For example, in FRE, the dynamic loading can be caused by the force of the nozzle 2010 through the support material 2008, affecting the support material 2008 in a number of ways. The extruder assembly 2002 can be configured to change the support material 2008 by imposing a mechanical load via shear, pressure, or vibration. The extruder assembly 2002 can be configured to irradiate or heat the support material 2008 to thin it. In various examples, the support material 2008 can reduce viscosity under vibration, heating, or irradiation that occurs locally to the extruder assembly 2002.

[0194] The support material 2008 can comprise other materials with viscoplastic behavior, such as Herschel-Bulkley fluid. Bingham plastics and Herschel-Bulkley fluids are viscoplastic materials included in the “shear-thinning” or “yield-stress fluid” category. Below a specific shear stress, these materials appear as a solid material. Above a threshold shear force, these materials behave as a fluid. A Bingham plastic may not necessarily “shear thin,” but rather may act much like a Newtonian fluid once it begins to flow. In contrast, the Herschel-Buckley fluid undergoes shear thinning once it begins to flow.

[0195] The support material 2008 can comprise at least two phases. For example, the support material 2008 can comprise particles (e.g., microparticles) dispersed in a diluent. In various examples, the support material 2008 can be a hydrogel. The at least two phases can enable the Bingham plastic-like rheological behavior.

[0196] The diluent can be aqueous or non-aqueous depending on the desired properties of the support material.

[0197] The particles can comprise gelatin, alginate, soy lecithin, Carbopol, acrylamide, agarose, alginate, a cell spheroid, a cell organoid, gellan gum, hyaluronic acid, laponite nanoclay, nanoclay, pluronic F127, poly(ethylene oxide), oxidized bacterial cellulose, xanthan gum methacrylate, fumed silica, hyaluronic acid-norbornenefibronectin & hyaluronic acid, K-Carrageenan, decellularizaed extracellular matrix, other suitable particle forming compound, or a combination thereof. For example, the particles can comprise gelatin. Gelatin may act as a porogen and enhance the microporosity of the formed objected.

[0198] The particles can various sizes. In certain examples, the particles can comprise an average particle size in a range of 1 micron to 100 microns, such as, for example, 5 microns to 50 microns, 10 microns to 30 microns, or 15 microns to 20 microns, all as measured with optical microscopy.

[0199] Depending on the printing technique, the support material 2008 can be clear or opaque. The support material 2008 can further comprise other components, such as, for example, a surfactant, a thickening agent, a buffer, a reactant (e.g., thrombin), or a combination thereof. In various examples, the support material 2008 does not comprise a thickening agent. In various examples, the support material 2008 can comprise 4-(2- hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES) buffer, such as, for example, at least 10 mM HEPES, at least 50 mM HEPES, or at least 100 mM HEPES.

[0200] The structure material can comprise a yield stress material that transitions between a fluid (e.g., liquid) state to a solid or semi-solid state by application of a pressure. For example, the structure material can be in a solid or semi-solid state in the extruder assembly 2002, a pressure can be applied to the structure material to transition the structure material to a fluid state such that the structure material can flow through the nozzle 2010 and can be deposited into the support material 2008. After leaving the nozzle 2010, the applied pressure to the structure material is removed and the structure material can transition into a solid or semi-solid state and thereby resisting deformation while in the material deposition region 2006. The density of the support material 2008 can enhance the structure material’s resistance to deformation in the support material 2008 and / or decrease diffusion of the structure material into the support material 2008.

[0201] The structure material can comprise various materials that can be used to form the object 2014 (e.g, a scaffold). The structure material can comprise a hydrogel (e.g., hydrogel precursor / water and bio compatible polymer) and optionally cells and / or an additive, such as,for example, a curing agent, a contrast agent, and / or other additives. In various examples, gelatin may be added to the structure material, which may act as a porogen and enhance the microporosity of the formed objected.

[0202] The structure material can comprise at least 70% hydrogel based on the total weight of the structure material, such as, for example, at least 80% hydrogel based on the total weight of the structure material or at least 90% hydrogel based on the total weight of the structure material. In various examples, the structure material comprises a fluid that transitions to a solid or semi-solid state after deposition.

[0203] The structure material can comprise at least 10 million cells per milliliter of structure material, such as, for example, at least 20 million cells per milliliter of structure material, at least 25 million cells per milliliter of structure material, at least 30 million cells per milliliter of structure material, at least 40 million cells per milliliter of structure material, at least 50 million cells per milliliter of structure material, at least 75 million cells per milliliter of structure material, or at least 100 million cells per milliliter of structure material. In various examples, the structure material can comprise a concentration of cells in a range of 10 million cells per milliliter of structure material to 1,000 million cells per milliliter of structure material or a range of 75 million cells per milliliter of structure material to 500 million cells per milliliter of structure material.

[0204] The structure material and / or support material 2008 can comprise a contrast agent.

[0205] The structure material can exhibit a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap, such as, for example, 1 cP to 4,000 cP, 1 cP to 3,000 cP, 1 cP to 2,000 cP, or 1 cP to 1,000 cP as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap.

[0206] In various examples, the structure material can include pancreatic bioink to stimulate islet formation and / or insulin secretion in response to glucose. The structure material with the pancreatic bioink can form a pancreatic niche by printing into an infill region. In various examples, the structure material can be an angiogenic bioink.

[0207] The extruder assembly 2002 may be a syringe-based extruder, which can include a reservoir 2012 (e.g., a barrel of a syringe) for receiving and storing structure material or support material, and a nozzle 2010 (e.g., a needle) which can be in fluid communication with the reservoir 2012 and can receive the structure material or the support material from the reservoir 2012. For example, the reservoir 2012 can comprise structure material and thestructure material can be extruded through the nozzle 2010 can be configured to deposit the extruded structure material in the support material 2008 disposed in the material deposition region 2006.

[0208] In various examples, the first extruder assembly 2002 and / or additional components can comprise a gantry or other robotic device to support and / or move the extruder assembly 2002 relative to the material deposition region 2006. Optionally, the extruder assembly 2002 can comprise a motor assembly or other movement assembly configured to translate and / or rotate the gantry and / or robotic device. In various examples, the extruder assembly comprises an actuator (e.g., a motor) configured to depress a plunger into the reservoir 2012 to extrude material through the nozzle 2010 into material deposition region 2006 as nozzle 2010 is translated through the material deposition region 2006 to additively form an object 2014.

[0209] The computer system 2004 is in signal / data communication with the extruder assembly 2002 (such as via a wired and / or wireless data bus or link). The computer system 2004 can be configured through programming to control the operation of the extruder assembly 2002. The computer system 2004 can also receive data from and send data (e.g. control data) to the extruder assembly 2002a. The components in the additive manufacturing system 2000 may be in communication with the computer system 2004 via any suitable type of data bus (e.g., parallel or bit serial connections).

[0210] The computer system 2004 comprises one or more processors 2020 operatively coupled to one or more non-transitory memories 2022 (only one processor 2020 and one memory 2022 are shown in FIG. 20 for simplicity). The processor 2020 may comprise one or multiple processing cores. The memory 2022 can comprise primary storage (e.g., main memory that is directly accessible by the processor 2020, such as RAM, ROM processor registers or processor cache); secondary storage (e.g., SSDs or HDDs that are not directly accessible by the processor); and / or off-line storage. The memory 2022 stores computer instructions (e.g., software) that are executed by the processor 2020. The processor 2020 can be configured (through execution of the software stored in the memory 2022) to control operation of the extruder assembly 2002 to thereby control the deposition of the structure material through the nozzle 2010. For example, the processor 2020 can control the flow rate of material through the nozzle 2010 (e.g., by the actuation rate of a plunger in the extruder assembly 2002) and / or the pose of the extruder assembly 2002 relative to the material deposition region 2006.

[0211] The memory 2022 can store a digital or electronic computer model 2024 of the object 2014 to be manufactured by the additive manufacturing process. The computer model 2024 can be loaded locally into the memory 2022 or can be downloaded from another device (e.g., another computer device, cloud) that is in data communication with the computer system 2004. To that end, the computer system 2004 may comprise a network interface controller (NIC) that connects the computer system 2004 to a computer network. The computer model 2024 can be in a variety of different digital or electronic formats, such as an STL file, a OBJ file, a FBS file, a COLLADA file, a 3DS file, an IGES file, a STEP file, a VRML / X3D file, a point cloud, or another 3D model file format type. The computer model 2024 can be generated from image data of a biological structure, an engineered structure, a computationally derived structure, or a combination thereof. In various examples, the biological structure can be generated from the image data of a patient.

[0212] The optional detector can comprise a light-based camera, a brightfield microscope, a fluorescence microscope, CT scanner, a MRI scanner, an OCT scan, a laser scan, an ultrasound scan, or a combination thereof.

[0213] The processor 2020 can be configured to separate (e.g., slice (utilizing Slic3r, Cura, Simplify3D, Skeinforge, KISSlicer software, etc.)) the computer model 2024 into different segments 2026, each segment containing a portion of the computer model 2024. In various examples, the processor 2020 can be configured to convert the computer model 2024 to a different 3D model file format prior to separating.

[0214] Each segment 2026 can be a layer, 2014a and 2014b, of the object 2014 to be deposited, a portion of a layer, 2014a and 2014b, of the object 2014 to be deposited, or other geometry of the object 2014. The segments 2026 can be created based on a design of the computer model 2024. For example, a segment of segments 2026 can comprise an overlapping region, an overhang region, an infill region, a perimeter region, another region of the object 2014, or a combination thereof. Each segment 2026 may or may not be in the X-Y plan and a segment can be in a non-X-Y plane, such as the X-Z plane, the Y-Z plane, other plane offset from the X-Y plane, or a non-planar segment, such as, for example, a curve. Utilizing various segments 2026 for different regions of the object 2014 can enable variations of machine path instructions and / or print parameters for each segment 2026. Therefore, the machine path instructions and / or print parameters can be selected to suit the particular geometry to be printed in the respective segment 2026.

[0215] From the segments 2026, the processor 2020 can be configured to create machine path instructions (e.g., G-code instructions) 2032 for the segments 2026 based on the design of theportion of the computer model 2024 in the respective segment 2026. The machine path instructions 2032 can be stored in the memory 2022. The machine path instructions 2032 can comprise print parameters 2034 and can be executed by the processor 2020 to cause the processor 2020 to control the operation (e.g., pose, extrusion, suction, cure) of the extruder assembly 2002 or other device (e.g., structure material removal device such as a suction tube).

[0216] The nozzle 2010 can be configured to deposit a structure material into the support material 2008 by applying a force to the structure material in the reservoir 2012 such that the structure material can flow from the reservoir 2012 through the nozzle 2010. The structure material can comprise a yield stress, a thixotropic property, an increased viscosity, or a combination thereof. In examples where the structure material comprises a yield stress, the force applied can be at least the yield stress. In certain examples, applying the force to the structure material can cause the structure material to flow through the nozzle 2010. For example, with an increase viscosity, the force can overcome the increased viscosity and cause the material to flow through the nozzle 12010. In examples wherein the structure material comprises a thixotropic property, the thixotropic property can cause the time scale to start flow of the structure material to be longer than the printing process.

[0217] In various examples, a plunger can be translated through the reservoir 2012. In various examples, the force can be pneumatically applied or the deposition can be controlled by a cavity pump. The application of the force can cause the material in the reservoir 2012 to change form a solid or semi-solid state into fluid state (e.g., liquid), so that the material can be deposited into the material deposition region 2006. The structure material can be suspended in the support material 2008 at a location where the structure material was deposited by the nozzle 2010 within the support material 2008. Since the processor 2020 can control the extruder assembly 2002 and nozzle 2010, the deposition of the structure material by the nozzle 2010 can be based on the machine path instructions 2032 and associated print parameters 2034 as executed by the processor 2020.

[0218] The extruder assembly 2002 can move the nozzle 2010 in two-dimensions when depositing structure material similar to FDM or in three-dimensions when depositing material, i.e., simultaneously in the X, Y, and Z directions. Further, the extruder assembly 2002, nozzle 2010, and / or material deposition region 2006 can be rotatable. The machine pathing instructions 2032 can be defined according to both Cartesian and polar coordinates, which can allow for the production of objects having complex geometries or very specific mechanical properties. 3D movement of the nozzle 2010 during deposition of the structurematerial can enable, for example, additive manufacture of a helical spring in one constant motion. In various examples, other complex geometries are achievable with robotic arm assemblies capable of simultaneously controlling movement with six degrees of freedom (i.e., in any Cartesian or rotational direction).

[0219] The depositing of the structure material can be repeated as necessary to additively form an object. For example, the processor 2020 can control the nozzle 2010 to deposit the structure material in layers, such as layers 2014a and 2014b, in order to additively form the object 2014 in the support material 2008 based on the computer model 2024, another plane, and / or non-planar movement. In some examples, layer 2014a can be deposited prior to layer 2014b. Layer 2014a may not be partially and / or fully cured prior to deposition of layer 2014b. Thus, the processor 2020 can control the nozzle 2010a to deposit layer 2014b proximal to (e.g., adjacent, in contact with, directly on top of) the layer 2014a such that the deposition of the layer 2014b contacts the layer 2014a.

[0220] The material deposition region 2006 can be configured for mechanically supporting and / or holding the support material 2008 during FRE additive manufacturing. For example, the material deposition region 2006 can comprise a vessel in which the support material 2008 is disposed and a platform on which the vessel is supported. The material deposition region can comprise a motor and / or actuator that can move the platform in 3D space as needed.

[0221] The structure material can be curable and after curing, the structure material can be considered cured. The object 2014 can be at least partially cured in the support material 2008 after deposition of the structure material. In various examples, the structure material can be at least partially cured prior to removing the support material 2008. In some examples, the structure material may not be cured until after removing the support material 2008. As used in this specification, the terms “cure” and “curing” refer to the chemical crosslinking of components in the structure material. Accordingly, the terms “cure” and “curing” do not encompass solely physical drying of structure material through solvent or carrier evaporation. In this regard, the term “cured,” as used in this specification, refers to the condition of the structure material in which a component of the structure material forming the object 2014 has chemically reacted to form new covalent bonds in the structure material (e.g., new covalent bonds formed between a polymeric resin and a curing agent), new ionic bonds, new hydrogen bonds, new Vander walls bonds, or combinations thereof.

[0222] For example, curing of the object 2014 can comprise cross-linking. The object 2014 can be treated through various cross-linking techniques to selectively increase the rigidity of the overall object 2014 or portions thereof. Cross-linking can be induced by variousmechanisms such as, for example, photo mechanisms (e.g., exposing the structure material to UV light), ionic mechanism, enzymatic mechanism, pH mechanisms (e.g., exposing the structure material to a different pH) or thermally driven mechanisms (e.g., cooling, heating). In various examples, the support material 2008 can include a cross-linking agent or pH suitable for curing the structure material as it is deposited into the support material 2008.

[0223] The mechanical properties of the object 2014 can be controlled by controlling the amount of curing that occurs within the object 2014. For example, the machine pathing instructions 2032 can be modified to control the amount of crosslinking that occurs within the object 2014. For example, the extruder assembly 2002 and / or other assembly can comprise a UV light and can selectively subject the embedded structure material to the UV light as desired.

[0224] The object 2014 can be at least partially removed from the support material 2008. Removing the support material 2008 may include heating the support material 2008, cooling the support material 2008, removing cations to disrupt crosslinking of the support material 2008, physically removing the support material 2008, vibration, irradiation with ultraviolet, infrared, or visible light, application of a constant or oscillating electric or magnetic field, other mechanism, or a combination thereof. For example, the support material can comprise a thermoreversible material and removing the support material can comprise heating the support material to a threshold temperature at which the support material transitions from a solid or semi-solid state to a liquid state.

[0225] The system 2000 can comprise nanoliter level control over fluid flow through the nozzle 2010. Each nozzle 2010 can be fortified in strength and reduced in diameter to enhance resistance to deflections and printing of small features. In various examples, the extruder assembly 2002 can be rigid and high performance to control fluid better. In certain examples, the nozzle 2010 can be aligned in 3-axis. In various examples, the object 2014 can comprise dovetail joints. In certain examples, the system 2000 can be capable to utilizes overlap slicing parameters.

[0226] The methods for additive manufacturing herein, such as those illustrated in described in FIG. 21 below, can be implemented in whole or in part as computer-executable instructions stored in the memory 2022 of the computer system 2004 that, when executed by a processor 2020 of the computer system 2004, cause the computer system 2004 to perform the enumerated steps. The computer instructions can be implemented as one or more software modules 2016 stored in the memory 2022 that are each programmed to cause the processor 2020 to execute one or more discrete steps of the processes described herein orother functions. For example, the software modules 2016 can comprise a separation module programmed to convert the computer model 2024 into segments; a conversion module programmed to convert the segments 2026 into computer instructions (e.g., G-code) for controlling the movement of the extruder assembly 2002 to fabricate the object 2014; an imaging module for controlling imaging parameters and movement of a detector; a modeling module programmed to receive, store, create, and / or modify part files of objects to be fabricated; and a robotic control module programmed to control the extruder assembly 2002 according to the instructions generated by the conversion module to fabricate the object 2014. Various other modules can be implemented in addition to or in lieu of the aforementioned modules. In certain examples, the processes described herein can be executed across multiple computer systems that are communicably connected together in a network, a computer system communicably connected to a cloud computing system configured to execute one or more of the described steps, and so on.

[0227] Referring to FIG. 21, a flow chart illustrating an additive manufacturing method according to certain implementations of the present disclosure is provided. The method can comprise receiving, by the processor 2020, a computer model 2024 of an object 2014 at step 2102. At step 2104, the processor 2020, executing the separation module software, can separate (e.g., slice) the computer model into different part segments and the processor 2020, executing the conversion module, can create machine path instructions (e.g., G-code instructions) based on the design computer model. The machine path instructions can be stored in memory 2022. The support material 2008 can be placed in the material deposition region at step 2106 prior to printing of the object 2014 and / or during printing of the object 2014.

[0228] The method can comprise, at step 2108, depositing a structure material, by the nozzle 2010, into the support material 2008 such as, for example, a first portion (e.g., the first layer 2014a) of the object 2014 can be deposited in the support material 2008a. The structure material and the support material together are also referred to herein as an assembly.

[0229] The depositing of structure material at step 2108 can be repeated over as many iterations as necessary to additively form the object 2014. Each iteration can deposit portions (e.g., the second layer 2014b) of the structure material and the iterations can be repeated until additive formation of the object 2014 is complete (if not aborted earlier).

[0230] Thereafter, at step 2110, the structure material can be at least partially cured after depositing and then, at step 2112, the support material can be at least partially removed fromthe object 2014. The curing can occur prior to, during, after, or a combination thereof, removal of the support material at step 2112.

[0231] At step 2114, the object 2014 can be perfused. The perfusion can comprise vascularizing and / or other culturing the object 2014. Perfusion can be performed under desirable conditions for the cells. For example, the object 2014 can be maintained at a temperature proximal to normal human body temperature (e.g., 37 °C + / - 2°C) , maintained with humidity, maintained with suitable carbon dioxide levels, and optionally supplied with suitable nutrients and / or additives to facilitate cell growth, vascularization, islet growth, etc.

[0232] The methods for additive manufacturing and systems for additive manufacturing described herein can be used to create various products. The products can be various product types, such as, for example, a soft structure, a bioprosthetic, a scaffold, a medical device, an implantable device, a gasket, a tube, a seal, an aerospace part, an automotive part, a building component, or other structures that may be additively manufactured. In various examples, the product (e.g., object 2014) can be surgically fit into a patient.EXAMPLES

[0233] Various aspects, benefits and features that are potentially realizable through implementation of the present invention will be more fully understood by reference to the following examples, which provide illustrative non-limiting aspects of the invention. It is understood that the invention described in this specification is not necessarily limited to the examples described in this section.

[0234] Microfluidics have rapidly advanced into cellularized and perfused organ-on-a-chip and microphysiological systems that can model increasingly complex biological processes. Advances include lung-on-a-chip, miniature cardiac pumps, multi-organ systems, and 3D vascular networks. The fluidic control can mimics aspects of the capillary network in native tissue to provide nutrient transport and control complex interactions between different cell and tissue types. However, the polydimethylsiloxane (PDMS) silicone elastomer, thermoplastics, and photoresins used in current microfluidics inherently limit the biological relevance and translation potential of these devices. Specifically, PDMS and thermoplastics are orders-of-magnitude stiffer than native tissue, typically absorb lipophilic biomolecules out of the media, cannot be remodeled by cells into more complex structures, and can only be used in vitro. Fabricating microfluidics from other materials such as hydrogels can overcome many of these challenges, with the ability to be cellularized and better mimic the native extracellular matrix (ECM). However, hydrogels remain difficult to form into complex 3D structures and the soft lithography techniques used by most studies produce fairly simple2.5D tissue designs. Thus, a primary obstacle is developing a fabrication process for microfluidic devices which can expand both the biomaterials and cells that can be used, as well as the 3D complexity of the model systems that can be built.

[0235] The examples provide direct 3D bioprinting of collagen-based hydrogels, ECM and cells into fully-biologic microfluidic devices with high-fidelity control of structure and composition. Advantages include rapid fabrication and design iteration, high spatial resolution, true 3D control of device architecture, integration of multiple cell types through direct printing and perfusion post-fabrication, and integration with custom-designed vasculature and perfusion organ-on-a-chip reactor (VAPOR) bioreactors. These microfluidic devices remodel into cellularized constructs with multi-scale vascular-like networks that can develop physiologic function, demonstrated here for perfusion culture of a glucoseresponsive, insulin-secreting microphysiological system. To do this we leverage freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting to combine additive manufacturing of microfluidics with cell-mediated ECM remodeling and morphogenesis to bridge the macro (>1 mm) and micro (<1 mm) length scales. These devices are FRESH 3D bioprinted from cells, collagen type I, fibrin, and other ECM components and growth factors into complex 3D geometries termed collagen-based high-resolution internally perfusable scaffolds (CHIPS). Though high-resolution 3D printing of microfluidics using photoresins such as polyacrylates and biocompatible hydrogels like PEG-diacrylate and gelatin methacryloyl are established, these approaches are typically limited to a single material and at least some of the cells must be perfusion seeded after device fabrication. Similarly, extrusion 3D bioprinting of vascular-like networks using sacrificial polymers can produce perfusable tissue constructs, however, the spatial resolution and design complexity are limited. Our approach can have the advantages of multi-material extrusion-based 3D printing to combine multiple cell-laden and ECM hydrogel bioinks into integrated 3D structures together with the high-resolution that previously could only be achieved with light-based 3D printing. Further, as the primary structural protein in the body providing mechanical strength as well as defining vascular and tissue compartments, collagen type I can be an ideal material to serve as the structural bioink for CHIPS. Together, the microfluidic CHIPS and VAPOR bioreactor form an integrated system that can recreate established microfluidic capabilities while enabling a new class of devices that combine cells with ECM proteins such as collagen and fibrin into functional, tissue-scale systems.Results & Discussion:Fabrication of CHIPS via FRESH 3D Bioprinting

[0236] The CHIPS are fabricated using FRESH 3D bioprinting of collagen-based bioinks as the main structural component, replacing PDMS, photoresins or thermoplastics as the main material traditionally used in microfluidics. Collagen hydrogels used in the literature typically range in concentration from 3-10 mg / mL, which are quite soft and well below the concentration of 40-80 mg / mL found in human tissues. Using FRESH we overcome this limitation to 3D bioprint collagen bioinks with concentrations up to and exceeding 35 mg / mL, resulting in constructs that can be handled and maintain shape fidelity. In FRESH, bioinks are printed in a thermo-reversible support bath consisting of a gelatin microparticle slurry designed to trigger rapid gelation during extrusion (FIG. 1 A). The aqueous fluid phase of the support bath is pH-buffered to rapidly neutralize the acidified collagen bioink and drive the self-assembly of a fibrillar network, while the Bingham-plastic rheology of the support bath provides mechanical support for the embedded collagen filaments. Cell-laden fibrinogen bioinks (e.g., structure material) are FRESH printed in a similar manner, where thrombin in the support bath enzymatically triggers fibrin gelation during extrusion. Upon print completion, the support bath is melted by raising the temperature to 37°C, allowing for nondestructive print retrieval.

[0237] To demonstrate the ability to fabricate collagen-based microfluidic devices, we FRESH printed a range of designs from the literature commonly made from PDMS. First, we designed a serpentine mixing network using computer-aided design (CAD) software, similar to designs reported by Whitesides and co-workers, with channel dimensions 500 pm wide and 1,000 pm tall (FIG. IB). The translucent nature of the FRESH printed collagen enabled visualization of the microfluidic serpentine network, while 3D imaging using optical coherence tomography (OCT) provided detailed structural data throughout the 3D volume of the device. Quantitative gauging comparing the dimensions of the FRESH printed serpentine channel to the original CAD model demonstrated the excellent print fidelity achieved, with average overprint and underprint root mean squared (RMS) errors of less than 20 pm. Next, we recreated a more complicated microfluidic design by Kamm and co-workers with 3 inlets and 3 outlets used to study vasculogenesis. This design highlights the ability to fabricate well-defined channels within a construct separated by narrow collagen walls (FIG. 1C). We also fabricated a stacked channel, lung-on-chip device based on a design by Ingber and coworkers, showing the ability to create a multi-layer design with channels in different Z-planes using a one-step FRESH printing process (FIG. ID). Finally, we designed a vascular-like network with circular lumens branching from 1 mm inner diameter (I.D.) inlets down to 250 pm I.D. (FIG. IE). This was FRESH printed with high fidelity (FIG. IF) and manuallyperfused with a blue dye to demonstrate network patency and integrity of the channel walls (FIG. 1G). Quantitative OCT-based 3D analysis confirmed patent channels, clearly-defined individual collagen infill features outside the channels, and circular channel cross-sections (FIG. 1H). The average overprint and underprint RMS error of the channels was 11 pm (FIG. II), showing that FRESH printed CHIPS have excellent fidelity for channels that have both square (FIG. IB to ID) and circular (FIG. IE to II) cross-sections. Together, these examples demonstrate that fully biologic microfluidic CHIPS can be designed and FRESH 3D bioprinted in a one-step process, whereas traditional microfluidics require photomask creation, sequential photolithographic mold fabrication and assembly, specialized clean room facilities, and a multi-day fabrication process.Microfluidic Flow within CHIPS using VAPOR Bioreactors

[0238] Microfluidic chips made from PDMS, thermoplastics and photoresins are rigid enough to support direct inlet and outlet cannulation for perfusion, but the softer FRESH 3D bioprinted collagen desired for a different approach to prevent leaking when pressurized. To overcome this, we developed the VAPOR bioreactor consisting of two components: (i) a main body 3D printed from UV-curable biocompatible resin with Luer lock connection fittings and internal fluidic channels, and (ii) a removable lid containing a silicone gasket and glass imaging window (FIGs. 6A to 6D). The internal fluidic channels terminate in barbed fittings, a design feature, as CHIPS for VAPOR have the negative of the barb incorporated into the inlet and outlet geometry to form an interlocking connection when assembled (FIG. 2A). A peristaltic pump, media reservoir, and bubble trap complete the perfusion circuit (FIG. 6E) and the entire setup is placed in a cell culture incubator for cellular perfusion studies (FIG. 6F). Since both the CHIPS and VAPOR are 3D printed, the overall dimensions and placement of inlets and outlets, as well as the internal perfusable network are easily modified and iterated upon to meet specific experimental desires.

[0239] To assess microfluidic function, a CHIPS design containing an internal serpentine channel 500 pm wide and 1 mm tall (FIG. 2A) was FRESH printed (FIG. 2B, FIGs. 7A to 7D), verified for lumen patency by OCT (Fig. 2C, FIG. 7E), placed in the VAPOR bioreactor, and then perfused (FIG. 2D). For validation of laminar flow within the CHIPS device, we simultaneously perfused red and blue dyed solutions into the inlets (FIG. 2E, Reynolds Number, Re = 0.37). Video time-lapse imaging revealed two distinct parallel dye streams throughout the serpentine channel with minimal mixing at the dye interface due to the relatively short path length (FIG. 2F). Next, we designed a pulsatile perfusion experiment to drive non-laminar mixing between an acidic phenol red solution (starting as a visibleyellow solution, pH 6.8) and a clear basic NaOH solution (pH 11) (FIG. 2G). Pulsatile flow resulted in mixing and a pH-dependent color change from yellow to magenta along the length of the serpentine path (FIG. 2H). Colorimetric analysis of the phenol red pH indicator confirmed a pH change from 6.8 to 9 along the channel, with a wave-like pH profile likely due to changes in the fluid dynamics that occur at turns within serpentine networks matching observations similarly described in the field (FIG. 21).

[0240] We sought to further demonstrate the versatility of CHIPS by fabricating and perfusing multi-layered and helical microfluidic designs. A multi-layered CHIPS with stacked channels based on the lung-on-chip (FIG. ID) was designed with two 1000 x 600 pm channels separated in the Z-axis by a 400 pm collagen barrier (FIG. 2J to 2L, FIG. 7F). Perfusion of red and blue dye within the stacked channels resulted in two independent fluid streams separated by the collagen barrier (e.g., first portion) (FIG. 2M and 2N). This demonstrates that the collagen walls can prevent the mixing between different fluidic channels, though at longer time scales it is expected that there will be diffusion out of the channels. For this purpose, we designed a CHIPS containing two circular channels in a double helix configuration (FIG. 20), a design that previously could only be created in hydrogels using DLP 3D bioprinting. This was FRESH printed with high fidelity (FIGs. 2P and 2Q, FIG. 7F) and perfused to demonstrate time-dependent diffusion of low molecular weight (-700 Da) red and blue dyes into the bulk collagen scaffold over 3.5 hours (FIG. 2R). The diffusion observed throughout the helical CHIPS is due to a combination of diffusion through the collagen walls of the channels and diffusion through the internal open lattice structure defined by the percentage of infill during the G-code slicing. Regardless of whether CHIPS contain planar, 2.5D, or fully 3D internal channel networks, the single-step FRESH printing approach allows a wide range of CHIPS designs to be fabricated to support perfusion.Molecular Weight-Dependent Diffusion Within Perfused CHIPS

[0241] To further assess molecular diffusion within CHIPS, a dual parallel channel design separated by a collagen wall (FIG. 3 A) was FRESH printed, verified by OCT (FIG. 3B), and then perfused within the VAPOR bioreactor (FIG. 3C). Laminar flow (Re = 0.55) and channel integrity were confirmed by perfusion and tracking of fluorescent microbeads within the channels (FIG. 3D). We observed the expected laminar flow, with a mean speed of 0.91 ± 0.30 mm / s (theoretical value calculated as 1.2 mm / s for the center of the channel). Next, we mimicked diffusion of biomolecules of different molecular weights using FITC-conjugated fluorescent dextrans from 3 to 70 kDa in one channel as a source with the opposing channelperfused with IX phosphate buffered saline (PBS) as a sink (FIG. 3E). Time-lapse fluorescence imaging of defined regions of interest (ROIs) expanding outward from the dextran-perfused channel (FIG. 3F, FIGs. 8A and 8B) established that diffusion throughout the CHIPS was molecular weight-dependent (FIG. 3G). The 3 kDa dextran reached steady state diffusion 1 mm (ROI 1) from the source channel within 3 hours, while the 70 kDa dextran reached a steady state rate at ~20 hours. The diffusivity of larger 40 and 70 kDa molecules is notable compared to cast solid hydrogel scaffolds, where diffusion has been reported to be much lower. To increase molecular diffusion and mimic the interstitial pressure found in tissues, we introduced a 5 or 10 mmHg pressure differential between the dextran source and PBS sink channels (Fig. 3H). Spectrophotometric analysis of the PBS sink perfusate after 24 hours showed significantly more 40 kDa dextran present in the perfused PBS sink following a pressure increase of either 5 or 10 mmHg (FIG. 31). Comparing the ratio of fluorescence intensity between high pressure (HP, + 10 mmHg) and normal pressure (NP) over time showed that increasing pressure drove the dextran into the PBS channel as well as throughout the CHIPS and into the furthest distal regions (FIG. 3 J and 3K, FIG. 8C). Together, these results establish that molecules with a wide range of molecular weights can diffuse out of the CHIPS channels and into the bulk of the construct, something that is not possible with the vast majority of microfluidic devices made from PDMS or plastics. Multi-material FRESH Printing to Create Spatially Patterned and Cellularized CHIPS

[0242] The spatial patterning of cells and ECM within internal regions of microfluidic devices is one of the major challenges in engineering more sophisticated microphy si ologi cal systems. To address this, we implemented multi-material FRESH printing using a custom 3D bioprinter with three Replistruder syringe pump extruders for bioink deposition (FIGs. 9A- 9B). For printing validation, we designed a parallel plate CHIPS device containing an open rectangular channel to facilitate confocal imaging of the patterned biomaterials (FIGs. 10A- 10D). Collagen bioinks doped with fluorescently-labeled fibronectin (Fn) were printed along the channel lumen to form regions -200 pm thick to demonstrate control over spatial patterning of ECM composition. Using multi -material FRESH printing, we created a uniform layer across the entire channel (FIG. 10 A), a pattern of lines 1 mm wide running the length of the channel (FIG. 10B), or a defined branching pattern with segments down to 500 pm in width within the channel (FIG. 10C). Finally, we patterned softer and stiffer regions along the length of the channel by printing collagen bioinks with concentrations of 6, 12, and 23 mg / mL, each labeled with different fluorescent dyes (FIG. 10D).

[0243] Next, we sought to implement multi-material FRESH printing to pattern cells and ECM in 3D, with the goal to support endothelial cell attachment within our dual channel CHIPS. The ability of vascular cells to attach, proliferate, and form a network within our devices is one of the advantages of CHIPS as a way to provide nutrient delivery and waste removal to a larger tissue volume. For our design, we choose to line the channels with a layer of 12 mg / mL collagen with fluorescently labeled Fn to improve endothelial cell attachment combined with a central region between the two channels consisting of 23 mg / mL collagen with fluorescently labeled Fn and vascular endothelial growth factor (VEGF), intended to promote cell infiltration (FIG. 6A). The CHIPS was FRESH printed, optically cleared, and imaged via confocal fluorescence microscopy for visualization of the internally patterned structures (FIG. 1 IB). The 12 mg / mL collagen and Fn channel linings were confirmed to be patterned as intended, while the central region containing VEGF was spatially restricted between the channels as designed (FIG. 11C). To test cell adhesion and growth within the multi-material CHIPS, human umbilical vein endothelial cells (HUVECs) were perfusion seeded into the dual channel CHIPS at a high cell density (FIG. 6D). Following 5 days of static culture, the HUVECs spread along the channel lumen, formed a visible monolayer, and stained positive for CD-31 (FIGs. 1 IE to 1 II). While there was some evidence of cell migration into the bulk scaffold, the HUVECs were primarily restricted to the channels into which they were seeded, potentially due to the absence of a stromal cell population present within our CHIPS.

[0244] To expand the complexity of the CHIPS we can engineer, we sought to fully leverage our multi-material printing by printing cell-laden bioinks and avoid the laborious process of perfusion seeding of cells that is desired for organ-on-chip and microphy si ologi cal systems. To do this, we directly printed a vascular bioink within the channel walls to encourage HUVEC vasculogenesis followed by angiogenic sprouting. The process for printing cells with FRESH is similar to that of collagen, except rather than using a pH-change to initiate bioink gelation, thrombin is used to enzymatically trigger the cross-linking of fibrinogen into fibrin. Both collagen and cell-laden fibrinogen bioinks are FRESH printed into the same support bath as their gelation mechanisms are orthogonal to one another, enabling the 3D bioprinting of fully integrated, multi-material cellularized CHIPS. Our cell-laden fibrinogen vascular bioink contained HUVECs and human bone marrow-derived mesenchymal stem cells (MSCs) as the pericyte-like cell to support microvessel formation and stability. We evaluated multiple designs for these cellularized CHIPS, starting with a vascular bioink that was printed as both the channel lining and dividing region between dual parallel fluidicchannels, while the remaining structure was collagen (FIG. 4A). Confocal imaging of the CHIPS immediately after printing confirmed our ability to volumetrically pattern both the cellular and collagen bioinks in 3D while maintaining patent fluidic channels (FIG. 4B). However, with this CHIPS design after static culture for 8 days, we observed buckling due to cell-driven compaction forces (FIG. 12 A). To prevent these large-scale deformations and maintain proper fit for use in the VAPOR bioreactor, we added collagen walls between the channel linings and central region as mechanical reinforcement (FIG. 4C). Multiphoton and second harmonic generation imaging of the collagen-reinforced CHIPS revealed high fidelity cellular channel linings and the presence of the extra collagen walls (FIG. 4D). After 8 days of static culture, the mechanical reinforcement provided by the collagen walls was sufficient to prevent the CHIPS from buckling compared to the fibrin-wall CHIPS (FIG. 12B). These results highlight the effects of both CAD design and material properties for long-term success and performance of cellularized CHIPS and our ability to rapidly make such changes.

[0245] Having demonstrated that vascular cells can be directly FRESH printed within CHIPS, we investigated the importance of culture time and perfusion on cell behavior. After 8 days of static culture in CHIPS with fibrin walls, large areas of vascular endothelial cadherin (VE-Cad)-expressing microvascular-like structures were observed within and adjacent to the cellular printed regions (FIG. 4E). Despite the absence of perfusion, the cells appeared to form networks spanning distances >2 mm, migrate from the ends of the printed cellular regions into the acellular collagen (FIG. 4F), and established dense, microvascular- like networks throughout the central dividing region (FIG. 4G). We observed similar results after 8 days of static culture in CHIPS with collagen walls (FIG. 13 A), with VE-Cad expression along the open channel lumen (FIG. 13B), and migration of cells (>1 mm) beyond the edge of the printed cellular regions from where the cells originated (FIG. 13C). This was the first evidence we found that cells can preferentially migrate along collagen filaments. While static culture conditions can result in capillary -like network formation, the addition of perfusion has been shown to increase endothelial cell-specific protein expression, and promote angiogenesis. After 8 days of VAPOR perfusion culture in CHIPS with fibrin walls (FIG. 13D) we observed high levels of VE-Cad expression encircling the printed channels (FIG. 13E) and cell elongation within the central dividing region (FIG. 13F). The results in the CHIPS with collagen walls after 8-day perfusion were comparable, with extensive cell migration throughout the device and formation of capillary -like structures far from where the HUVECs and MSCs were printed (FIG. 4H). Vascular CHIPS with collagen walls were more stable in the VAPOR bioreactors during perfusion, maintaining larger channel openings thatremained lined with cells (FIG. 41). Furthermore, evidence of perfusion-stimulated changes of the HUVECs and MSCs into a vascular-like morphology reminiscent of larger diameter microvessels was observed within the VE-Cad-rich zones all around the fluidic channels (FIG. 4J). These results demonstrate that VAPOR perfusion of vascular CHIPS promotes cell assembly into capillary-like networks spanning 8-100 pm in diameter by supporting initial vasculogenesis followed by angiogenesis throughout the scaffold.Pancreatic-like CHIPS Demonstrate Glucose Stimulated Insulin Response

[0246] Secretory function is a role of many organs in the body, and we focused on integrating additional cell types into our perfused CHIPS to demonstrate the complexity and physiology that can be achieved. Specifically, we focused on glucose stimulated insulin secretion (GSIS) by islets of the pancreas, the failure of which leads to type I diabetes. The engineering of pancreatic tissue has made a number of advances, including hydrogel-based encapsulation to evade the immune system and microphy si ologi cal systems containing isolated islets, showing that implantation of insulin-producing engineered tissues has therapeutic potential. However, there has yet to be a perfusable tissue construct created entirely out of ECM and cells with any of these approaches. To model the GSIS of pancreatic tissue, mouse MIN6 cells were chosen as they exhibit beta-like cell secretion of insulin in response to glucose. MIN6 cells were incorporated into the vascular bioink to form a high-concentration (60 million cells / mL total) pancreatic-like bioink for multi-material FRESH printing into the parallel channel CHIPS design. The first pancreatic-like CHIPS design we evaluated resembled the dual channel fibrin-wall CHIPS (FIG. 4A-J) with the cell-laden bioink lining the fluidic channels and within the central dividing region (FIGs. 14A and 14B). The pancreatic-like CHIPS were FRESH printed and statically cultured for 8 days where construct size remained relatively unchanged (FIG. 14C). After fixing and imaging, we observed dense cellularization in the regions where cells were printed as well as migration of cells into the surrounding collagen scaffold (FIG. 14D). In addition, the CHIPS exhibited patent fluidic channels and stained positive for insulin expression within both the central dividing region and adjacent channel walls (FIG. 14E to 14G). Within the cellularized regions, we also observed a dense, capillarylike network staining positive for actin and CD-31 (FIG. 14H). There was also further evidence that we can guide cells to align along the direction of the printed collagen filaments, as cells appeared to follow the 45° surface filament pattern of the printed collagen (FIG. 141 and 14J). Cells were also seen migrating up to 200 pm from the cellular regions into the collagen scaffold along the printed collagen infill filaments (FIGs. 14K and 14L), indicatingthat the topology of printed filaments can serve as a potential pathway to define network density and branching.

[0247] While the static pancreatic-like CHIPS did express insulin, a large volume of our construct remained acellular. In principle, since our entire scaffold is fabricated from ECM, we can increase the total cellular volume within the CHIPS by printing additional cellularized regions. A modified pancreatic-like CHIPS was designed with three distinct cellularized regions between and adjacent to each side of the channels to increase the printed cell volume by 50%, and included printed collagen channel linings to strengthen the device and prevent cell-mediated deformation (FIG. 5A). The CHIPS were successfully FRESH printed and after static culture for 12 days displayed extensive cell migration into adjacent acellular regions (FIG. 5B). Notably, the channel lining that was initially acellular collagen now had a lumen with dense capillary-like networks that bridged the original printed regions (FIG. 5C, FIG. 15 A). In addition to the cell migration into and around the fluidic channels, extensive cell migration outward into the surrounding collagen scaffold was observed a full millimeter from the initial printed regions (FIGs. 15B and 15C). The channel lumens remained patent and the originally acellular walls were now densely populated with cells (FIG. 15D). Analysis of the segmented branching network growing into the channels revealed a mean diameter of 9.3 ± 2.7 pm (FIG. 5C). Cells were also observed migrating hundreds of microns into the scaffold along the printed collagen filaments (FIG. 5D).

[0248] To improve nutrient delivery, increase cell proliferation and growth, and enhance MIN6 insulin secretion, we perfused the pancreatic-like CHIPS in the VAPOR bioreactor for 12 days. Perfusion resulted in increased cellularization throughout the device, with vascular- like structures ranging in diameter from 25-100 pm originating from the printed cellular regions and following the printed collagen filaments (FIG. 5E, FIGs. 16A to 16D). We also found extensive insulin expression within all printed cellular regions along with insulinpositive structures far from the printed cellular regions, suggesting co-migration of the MIN6 cells as well as the HUVECs and MSCs that formed the capillary-like structures (FIG. 5E). Indeed, one of the most exciting observations was the morphogenesis of looping structures that contained insulin-positive MIN6 cells and actin-positive HUVECs and / or MSCs, reminiscent of early branching morphogenesis observed during pancreatic islet development (FIG. 5F). To confirm that the MIN6 positive insulin staining was indicative of functionally secreted insulin in response to glucose stimulation, we performed a GSIS ELISA assay comparing the static and perfusion-cultured pancreatic-like CHIPS. Note that due to the thickness of the pancreatic-like CHIPS (~6 mm) and resulting insulin diffusion time, we wererequired to restrict our experiment to one low and one high glucose stimulation per device without a second low glucose stimulation. Results showed that pancreatic-like CHIPS cultured statically produced a 2-fold increase in insulin secretion following glucose stimulation whereas those cultured under perfusion exhibited a statistically significant 4.6- fold increase (FIG. 5G). This places the performance of pancreatic-like CHIPS between that reported for MIN6 pseudo-islet spheroids and primary human islets, which frequently produce stimulation indices of approximately 1.5 to 3-fold and 10-fold, respectively. Further, we quantified the amount of insulin secreted over 24 hours of perfusion after a fresh media exchange. We detected >8 ng of additional insulin present within the media perfused through the pancreatic-like CHIPS compared to the control media sample. This result demonstrates that placing beta cell-like MIN6 in a fully biologic 3D ECM environment with appropriate vascular cell types and perfusion can drive the formation of a tissue construct with sustained secretory function. Beyond these proof-of-concept studies for the pancreatic-like CHIPS, further functional improvement is likely achievable through multiple approaches including increasing the beta-like cell volume within the construct, extending the culture time to promote more mature cell phenotypes, and incorporating fully differentiated human iPSC- derived beta-like cell clusters or human primary islets.Conclusion

[0249] In summary, we have FRESH 3D bioprinted microfluidic CHIPS made entirely from cells and ECM proteins instead of traditional materials such as PDMS, opening up a range of new capabilities for organ-on-a-chip and microphy si ologi cal systems. While PDMS, photoresins and thermoplastics are staples for microfluidic chip production, there are material limitations that restrict their use in developing more advanced applications that require a fully biologic environment. The inability of PDMS and these other polymers to undergo cell- driven remodeling restricts the complexity to casting cellularized hydrogels within channels or seeding them on luminal surfaces. In clear contrast, we show that by FRESH printing CHIPS from ECM proteins, we allow for cell-driven remodeling and migration broadly throughout the device into regions that would typically be inaccessible within traditional microfluidics. Further, perfusion with the VAPOR bioreactor supports tissue viability, longdistance cell migration within the CHIPS, self-assembly of capillary-like networks, and the emergence of tissue-scale function as demonstrated by insulin secretion in the pancreatic-like CHIPS. We also believe this approach will create a new type of engineered tissue construct that blurs the line between in vitro and in vivo systems. While microfluidics has provided a powerful platform for observing tissue morphogenesis, disease mechanisms, andpharmaceutical response, the inert nature of PDMS and other polymers precludes their suitability for long-term in vivo implantation. This is where in vitro maturation of fully biologic CHIPS via VAPOR bioreactor perfusion provides a path forward for generating larger, pre-vascularized tissues with improved viability, functionality, and therapeutic potential upon implantation. For example, the human pancreas contains ~1 billion beta cells, which using our current pancreatic-like bioink of 30 million MIN6 / mL would require an ~33 cm3construct to match cell number. However, we have previously demonstrated FRESH printed bioinks containing >200 million cells / mL, which would reduce the pancreatic-like bioink volume to ~5 mL, only ~3 times larger than our current CHIPS. This suggests that in the future it may be possible to engineer CHIPS that can recreate at least some aspects of full organ-scale function, whether as in vitro devices for disease modeling and pharmacology, or as in vivo devices designed for implantation as a cell replacement therapy.

[0250] Materials and Methods

[0251] 3D Bioprinter Setup

[0252] All FRESH printing was executed on custom-built 3D bioprinters utilizing our open- source Replistruder 5 syringe pumps (parts files available upon publication for download under a CC-BY-SA license found at https: / / 3dprint.nih.gov / users / awfeinberg). Single material printing utilized a custom gantry 3D printer configuration driven by four Parker Hannifin 404 * R 100 mm travel precision stages (8 pm travel accuracy) as previously described. For multi-material bioprinting a high-performance motion control system was designed utilizing the AGS 1000 platform (Aerotech Inc.). Three Replistruder 5 syringe pumps and a dovetail mounted OCT scanhead (Thorlabs) were each mounted to TU-30 linear ball screw stages (IKO) for independent actuation of each extruder. The custom bioprinter platform resulted in a build volume of 100 mm (X), 300 mm (Y), 100 mm (Z), and 80 mm for each TU-30 substage. A 500 pL, 1 mL, or 2.5 mL syringe (Hamilton) containing bioink with a stainless-steel needle of either 80 (Jensen Global, JG34-0.25HPX) or 150 (Jensen Global, JG30-0.5HPX) pm inner diameter (ID) was used.Collagen Bioink Preparation

[0253] Unless stated otherwise, a 23 mg / mL acidified collagen bioink was utilized for all prints and prepared as previously described. Briefly, sterile 35 mg / mL neutral collagen bioink (Lifeink 200, Advanced Biomatrix, 5278) was diluted in a 2: 1 volume ratio with 0.24M acetic acid (VWR, 97064-482) or sterile 35 mg / mL acidified collagen bioink (Lifeink 240, Advanced Biomatrix, 5267) was diluted in a 2: 1 volume ratio with sterile DI H2O and mixed back and forth 40 times between two mated syringes. For the preparation of fluorescentbioinks, acidified collagen bioinks were mixed with 10-20 pL of 500 pg / mL human fibronectin (Corning, 356009) fluorescently conjugated to either Alexa-Fluor 405, 488, 555, and 633 NHS Esters. In multi-material printing experiments, the final concentration of the fibronectin within the collagen bioink was 50 ug / mL. In all cases, syringes containing acellular bioink were centrifuged at 3000 g for 5 min at room temperature to remove any air bubbles generated during the preparation process. The bioink was then transferred to a Hamilton glass syringe for printing.Cell Culture

[0254] Unless otherwise stated, cells were cultured at 37°C under 5% CO2 with media supplemented with 1% (v / v) penicillin-streptomycin being exchanged every 2 days. Pooled human umbilical vein endothelial cells (HUVEC) (Lonza, CC-2519) were cultured in endothelial cell media (Lonza, EGM-2, CC-3162). Bone marrow-derived MSCs (ATCC, PCS-500-012) were cultured on flasks coated with quick coating solution with growth media (ATCC, PCS-500-041). MIN6 (Mouse insulinoma cell line) (Addexbio C0018008) were cultured in high-glucose DMEM (Gibco, 11-965-092) with 15% FBS (v / v), 2 mM sodium pyruvate, 20 mM HEPES, and 0.05 mM P-mercaptoethanol, using passages 9-15. MIN6 cells were seeded at a density of 2 X 104cells / cm2and passaged when they reached 80-90% confluency.Cellular Bioink Preparation

[0255] Vascular and pancreatic bioinks were prepared for cellular bioprinting experiments following similar protocols. For the vascular bioink, HUVECS (passage 4-6) and MSCs (passage 2-4) were cultured following the previously described procedure and lifted using a trypsin-EDTA solution. Trypsin was neutralized using trypsin neutralizing solution with 7.5 pM bivalirudin (Cayman Chemical, 23035) as a thrombin inhibitor at a 1 :2 ratio. The cells were pelleted at 200 g for 5 minutes and then resuspended in 1 mL Hank’s Balanced Salt Solution (HBSS, Gibco, 14175-095). 1 X 106MSCs and 9 X 106HUVECs were transferred into a 1 mL BD syringe. This syringe was then centrifuged at 190 g for 5 minutes and the supernatant was aspirated until approximately 100 pL remained. 165 pL of 120 mg / mL fibrinogen (Millipore Sigma, 341573) and 65 pL 5% xanthan gum (dissolved in HBSS) were loaded into a separate 500 pL Hamilton gastight syringe. The 1 mL and 500 pL syringes were connected with a female luer lock adapter and the fibrinogen, xanthan gum, and cells were mixed 50 times. The vascular bioink was centrifuged at 300 g for 3 minutes in the 1 mL syringe to remove bubbles and transferred to the 500 pL syringe. The final vascular bioink consisted of 30 X 106cells / mL, 60 mg / mL fibrinogen, and 1.0% (w / v) xanthan gum. Thepancreatic bioink was prepared similarly to the vascular bioink with the following adaptations. MIN6 cells (passage 9-15) were lifted using trypsin solution for 5 min, and 10 X 106MIN6 cells were added to the 1 X 106MSCs and 9 X 106HUVECs in a 1 mL syringe. The final pancreatic bioink consisted of 60 X 106cells / mL (30 X 106MIN6 / mL, 27 X 106HUVEC / mL, 3 X 106MSC / mL), 60 mg / mL fibrinogen, and 1.0% (w / v) xanthan gum. FRESH Support Bath Generation

[0256] Cellularized CHIPS were printed using a sterile support bath (LifeSupport, FluidForm) prepared according to the manufacturer's instructions. When printing collagen- based bioinks the support bath was rehydrated with a 2:1 mixture of cold 100 mM 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), pH 7.4 (Corning, 60-034-RO) and serum free DMEM (Gibco, 11-965-092). When printing fibrinogen-based bioinks, the support bath was rehydrated with cold 100 mM HEPES, pH 7.4, and 1 U / mL thrombin (Millipore Sigma, T4648). Acellular CHIPS were printed using a FRESH support bath generated using a complex coacervation method as previously described. Briefly, FRESH v2.0 support bath was made by dissolving 3.0% (w / v) gelatin type B (Fisher Scientific, G7- 500), 0.3% (w / v) gum arabic (Sigma Aldrich, G9752), and 0.125% (w / v) Pluronic® F-127 (Sigma Aldrich, P2443) in a 50% (v / v) ethanol solution at 45°C. Soon after dissolving, the pH of the solution was adjusted to 5.65 using IM hydrochloric acid. The solution was sealed and stirred overnight at room temperature. The following morning the slurry was centrifuged at 300 g for 2 min, the supernatant discarded, replaced with DI H2O and shaken to wash the particles. The slurry was recompacted by centrifuging at 750 g for 3 min, the supernatant discarded, and replaced with DI H2O. This washing step was repeated a total of 3 times. After the final round of washing the slurry was resuspended to a final concentration of 100 mM HEPES, pH 7.4 and stored at 4°C. Prior to printing, the slurry was placed in a vacuum chamber at room temperature for 30 min followed by centrifugation at 2000 g for 5 min. The supernatant was discarded, and the slurry was transferred into the print container of choice. CHIPS Design

[0257] CHIPS were created in computer-aided design (CAD) software (Autodesk Inventor; Autodesk Fusion 360). All models were exported as STL files prior to printing. Overall design features and dimensions varied between experimental conditions and requirements. Perfusable networks were designed as void space within the printed CHIPS. All STL files used in the manuscript can be found at https: / / 3dprint.nih.gov / users / awfeinberg. 3D renders for manuscript preparation were generated within the rendering environment and exported asPNG files. Assembly videos of components were made within the animation environment and exported as .avi or .mp4 files.FRESH 3D Bioprinting of Collagen Type I

[0258] Collagen type I was FRESH printed as previously described (75). All STL files were sliced using slicing software (Ultimaker, Cura; PrusaResearch, PrusaSlic3r; Slic3r) to produce G-code files. For 80 and 150 pm ID needles, a layer height of 32 and 60 pm was used, respectively. Chips were printed at 23-70 mm / s with 2 perimeters, 4 top and bottom layers, and 35% infill. All constructs were printed at room temperature (22°C). Upon completion, constructs were incubated at 37°C for at least 30 min to melt the support bath. The molten support bath was exchanged with warm print storage solution (PSS) consisting of consisting of IX PBS, 50 mM HEPES, pH 7.4, and 1% (v / v) penicillin-streptomycin (Life Technologies, 15140-122). Acellular CHIPS were optionally sterilized by 10 min UV ozone treatment followed by overnight incubation in PSS to remove residual gelatin.

[0259] Brightfield and Stereoscopic Imaging

[0260] For all brightfield and stereoscopic images we utilized a Leica M165FC microscope with a IX Plan Apo lens, fully adjustable base with darkfield capability, and a Prime 95B CMOS camera. Additionally, images of printed CHIPS and various equipment utilized were taken with either a Soney A5 camera equipped with a Laowa 24mm f / 14 probe lens, or an iPhone 14 pro. Image contrast adjustment and resizing was performed in FIJI ImageJ or Adobe Photoshop.

[0261] OCT Imaging and 3D Gauging of CHIPS

[0262] The full 3D structure of CHIPS were imaged using optical coherence tomography (OCT) to non-invasively assess lumen patency for quality control and in-process monitoring as previously described. Briefly, OCT images were acquired using a Vega 1300 nm OCT system (Thorlabs, VEG210C1) mounted onto the bioprinter using an objective (OCT-LK4 objective) with an imaging depth of 11 mm and 13 pm lateral resolution. XYZ voxel sizes were acquired at 16.22 x 16.22 x 8.11 pm, respectively. Once scanned, XY, XZ, and YZ planes of the 3D images were analyzed to qualitatively assess the print for patency, significant defects, and potential blockages within the printed networks that would compromise flow through the CHIPS. Computational 3D gauging was performed using a combination of FIJI ImageJ, Imaris (Bitplane v9.5), 3D Slicer, and Cloud Compare software. Raw OCT images were denoised and scaled to account for refractive index of the imaging medium. Processed images were exported as TIFF stacks and imported to either Imaris or 3D slicer for segmentation of the internal perfusable networks. The segmented internal networkswere exported as .STL files in 3D Slicer and imported into 3D builder to center the model at the origin. Both the segmented .STL and original .STL used for printing were imported into Cloud Compare 3D point cloud registration software. A standardized process was implemented to align the models to their bounding box centers and perform fine registration. A custom LUT centered around 0 was implemented to map positive (red) and negative (blue) deviations from the intended .STL onto the segmented .STL model. Gauging data and rendered 3D images of the deviations were exported for further analysis, display, and graphing.

[0263] Manual Perfusion of Branching Vascular Bed CHIPS

[0264] Perfusion of the branching vascular bed CHIPS was demonstrated by manually perfusing a concentrated ddH2O solution of blue food coloring through the top inlet of the construct with a 10 mL plastic BD syringe and a 20-gauge needle. The perfusion rate was modulated to achieve filling of the branching network and exit from the bottom outlet. Micromanipulators and helping hands devices were implemented to stabilize the perfusion without moving the printed construct. Perfusion was performed with the vascular bed construct submerged in a 50 mM HEPES buffer, pH 7.4.

[0265] VAPOR Assembly

[0266] Vasculature and perfusion organ-on-a-chip reactor (VAPOR) was designed with computer-aided design (CAD) software (Autodesk, Inventor; Autodesk, Fusion 360) and printed from Biomed Clear (Formlabs, RS-F2-BMCL-01) resin on a Form 3B (Formlabs, RS- F2-BMCL-01). Stainless steel M3 hex nuts (McMaster Carr, 94150A325) were inserted into the nut cutouts in the bioreactor main body. A glass coverslip was sealed into the lid by pipetting on 100 pL of resin followed by pressing a 22 x 22 mm glass coverslip (VWR, 48366-227) into the lid which was then sealed in place by baking in a UV oven. Custom gaskets were cut from 1.5 mm thick silicone sheeting (McMaster-Carr, 5787T93) and pressed into a cutout in the lid.

[0267] Bioreactor Perfusion System Assembly

[0268] A 100 mL glass bottle (Cole Parmer, EW-34523-00) was used as a media reservoir. Holes for tubing lines were drilled into the cap and 1 / 16” ID silicone tubing (Cole Parmer, EW-95802-02) was pulled through to ensure airtightness. Two additional holes were drilled for air filtration and media exchange. A peristaltic pump (Ismatec, EW-95663-34) using 1.42 mm ID peristaltic tubing (Cole Parmer, EW-95663-34) was connected to the media reservoir tubing. Autoclavable bubble traps (Darwin Microfluidics, LVF-KBT-L-A) with 1 / 16” barb adapters (Darwin Microfluidics, CIL-D-646) were placed after the pump and then connectedto stopcocks on the reactor. Tubing exiting the reactor’s perfusion channels and lymph outlet returned to the media reservoir.

[0269] Bioreactor Perfusion of CHIPS

[0270] For sterile operation 3D printed parts were sonicated for 30 min in sterile filtered 70% ethanol, dried for 1 hour in a biosafety cabinet, and sterilized by 15 min UV ozone treatment. All remaining parts such as peristaltic tubing, media reservoir and bubble traps were autoclaved. For air filtration, a 0.2 pm pore size filter (VWR, 28145-501) was screwed onto the media reservoir. The system was assembled in a biosafety cabinet on an incubator shelf. All flow paths were purged with media to avoid entrapment or perfusion of air bubbles on or in the tissue. CHIPS were then transferred into the VAPOR chamber, gently pressed onto the barbs and sealed by bolting on the lid. CHIPS were perfused from 60 - 1000 pL / min. The system was then inserted into a 37°C incubator for perfusion culture. Cellularized CHIPS containing vascular bioink (HUVEC and MSC) were perfused with endothelial cell media while pancreatic CHIPS were perfused with a 50:50 ratio of endothelial cell and MIN6 media.

[0271] pH-Sensitive Dye Perfusion

[0272] Serpentine CHIPS were placed in VAPOR reactors and perfused at 100 pL / min at each inlet. Colorimetric video acquisition was performed with a Sony A5 camera equipped with a Laowa 24mm f / 14 probe lens. During perfusion, one flow path consisted of an acidic phenol red solution adjusted to pH 6.5 using hydrochloric acid (HC1). The second flow path consisted of a basic PBS buffer solution adjusted to pH 11 using sodium hydroxide (NaOH). Initial perfusion was performed with a pulsatile roller pump (Masterflex 77202-60) to stimulate mixing along the serpentine network length. The change in phenol red color from the acidic yellow to basic magenta was quantified using the Color Profiler (ImageJ, downloaded and installed from https: / / imagej.net / ij / plugins / color-profiler.html) for a segmented line then traversed the length of the serpentine network. Values for Magenta (White-Green) and Yellow (White-Blue) were extrapolated from RGB intensity to determine the ratio of Yellow:Magenta along the path during different perfusion states. The ratio was graphed as a function of path length along the serpentine network and color coded to match the yellow and magenta values according to the pH indicator values for phenol red.Color Dye Perfusion

[0273] Laminar flow within Serpentine CHIPS was demonstrated by perfusing either red or blue food coloring while maintaining equal flow rates of 100 pL / min in both channels. Vessel patency and dye diffusion into the bulk of stacked or 3D helical channel CHIPS wasdemonstrated by perfusing red and blue food coloring through separate channels. Colorimetric video acquisition was performed with a Sony A5 camera equipped with a Laowa 24mm f / 14 probe lens. Extended time lapse imaging was acquired with a GoPro Hero 5 camera mounted to a tripod.FITC-Conjugated Dextran Perfusion

[0274] Dual parallel channel CHIPS were perfused at 100 pL / min with 0.1 mg / mL 3, 10, 40 or 70 kDa dextran. Dextrans were conjugated with fluorescein isothiocyanate (FITC) (Thermofisher Scientific, D3305; D1821; D1844; D1823). Dual parallel channel CHIPS’ second vessel was perfused with IX PBS. CHIPS were perfused from 1 - 72 hours. Time lapse images were recorded on an epifluorescent stereomicroscope (either Nikon SMZ1000; or Leica M165FC) using a FITC filter, an X-Cite lamp (Excelitas), and a Prime 95B Scientific CMOS camera (Photometries) with an image being taken every minute. To quantify dextran diffusion, fluorescence intensity over time was measured using ImageJ (National Institutes of Health). Six regions of interest (ROIs) were selected at increasing distances away from the FITC and PBS channels and fluorescence intensity over time was calculated relative to the intensity at the initiation of perfusion while accounting for background signal.

[0275] Microbead Perfusion

[0276] Fluorescent polystyrene microbeads 10 pm in diameter were perfused at 100 pL / min at a concentration of 3.6 X 103beads / mL. Beads had either 580 / 605 (red) (Thermofisher Scientific, F8838) or 505 / 515 (yellow-green) (Thermofisher Scientific, F8836) excitation / emission wavelengths. Beads were perfused through dual parallel CHIPS in either the same or opposite directions, and videos were recorded on stereofluorescence microscopes with a TexasRed filter set similar to dextran perfusions. Particle tracking and bead velocimetry were performed in Imaris 9.5.1 (Bitplane) using spot detection and tracking algorithms.Perfusion of Dual Parallel Channel CHIPS with Afterload Pressure

[0277] Dual parallel channel CHIPS were perfused as previously described at 100 pL / min with 0.1 mg / mL 40 kDa FITC-conjugated dextran and IX PBS. Each reservoir contained 20 mL of solution. Pressure within the CHIPS’ dextran channel was increased by raising the height of the dextran reservoir to produce an additional 5 or 10 mmHg of afterload. To assess the diffusion of dextran from the source channel into the systemic PBS circulation, 50 pL samples were taken from the PBS reservoir bottle at 0 and 24 hours. The relativeconcentration of FITC-conjugated dextran compared to the source reservoir was then assessed by spectrophotometric analysis (Molecular Devices, SpectraMax i3x).

[0278] To assess the effect of afterload on molecular diffusion through CHIPS, time lapse images of perfusion with 5 mmHg of afterload (HP) were recorded as previously described and compared to perfusion with no additional afterload pressure (NP). The recordings were overlaid, and the fluorescence signal of the HP time lapse was divided by the NP time lapse after accounting for background signal. A vertical profile analysis was performed down the center of the HP / NP time lapse in ImageJ at various time points to further visualize the impact of increased afterload on diffusion into the peripheral regions of CHIPS.

[0279] Multi -Material Needle Alignment

[0280] In order to align multiple needles, we created a custom dual camera optical alignment system. Briefly, two IX, 40mm WD CompactTL™ Telecentric C-mount Lens (Edmund Optics #63-745) were mounted to Alvium 1800 U-500 (Allied Vision) USB cameras. A custom 3D printed alignment plate and XY positioning system allowed for focus adjustment to achieve parfocality. To image the bottom needle tip to obtain the XY position and needle diameter, a mirror (Thorlabs ME2S-G01) was mounted at a 45° angle. The second camera was mounted perpendicular to the XY camera to view the side profile of the needle tip for Z- height alignment. A custom Lab View program was written to simultaneously view the XY and Z positions. Each extruder was then moved to the center of the field of view for each camera to measure the relative XYZ offsets between each needle. The offset positions were stored as additional global software coordinate systems for use during multi-material printing using the Aerotech CNC operator’s interface.Multi -Material FRESH Printing

[0281] 3D models were prepared using Fusion 360 (Autodesk) for multi-material printing by creating individual nested components for each material within the desired location of the CHIPS. Each component was exported as an STL part file and imported into Cura 5.2 (Ultimaker) slicing software. The main components were centered around the XYZ origin and offset according to their designed spacing based on the original 3D model location. A separate material profile was created for each bioink within Cura to permit the assignment and indexing of the respective bioinks to one of the three extruders. Additionally, the creation of individual bioink specific profiles enabled component-specific color visualization of the CHIPS within the slicing software. Custom start and end G-code was specified for each extruder tool profile to recall the stored position offsets determined during the alignment process and prime the extruder between tool changes.Immunofluorescence Staining

[0282] Cellularized CHIPS were fixed via incubation in 10% neutral buffered formalin (Sigma Aldrich, HT501128) supplemented to a final molarity of 630 pM MgCh and 108 pM CaCh. Tissues were then incubated in blocking buffer overnight. Blocking buffer consisted of 90% (v / v) IX PBS supplemented to a final molarity of 1 mM CaCh and MgCh each, 5% (v / v) IM Glycine (Fisher Scientific, BP381), 5% (v / v) goat serum (Thermo Fisher Scientific, 16210072), and 0.1% (v / v) Triton X-100 (Thermo Fisher Scientific, 85112). Samples were then immediately incubated with primary antibodies for a week at 4°C. Primary antibodies are diluted in antibody dilution buffer consisting of IX PBS supplemented to a final molarity of 1 mM CaCh and MgCh each, 0.1% (w / v) bovine serum albumin (BSA) (Sigma Aldrich, A2153), and 0.1% (v / v) Triton X-100. Primary antibodies and their dilutions included VE- Cadherin rabbit mAb (Cell Signaling Technology, 2500S) at 1 :400, CD-31 mouse mAb (Cell Signaling Technology, 3528S) at 1 :800, insulin mouse mAb (Cell Signaling Technology, 8138S) at 1 :400, and insulin rabbit polyclonal (Abeam, abl81547) at 1 :400. Samples were then washed 3 times for 1 hour in antibody buffer without triton followed by an overnight wash at 4°C. The following day, samples were incubated in secondary antibodies for a week at 4°C. All secondary antibodies were diluted in antibody dilution buffer. Secondary antibodies and their dilutions included 4',6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, D9542) at 1 :400, phalloidin conjugated to Alexa-Fluor 488 (Life Technologies, A12379) at 1 :400, goat anti-rabbit IgG 555 (Thermofisher Scientific, A- 21428) at 1 : 1000 dilution, and goat anti-mouse IgG 633 (Thermofisher Scientific, A-21050) at 1 : 1000. Samples were then washed 3 times for 1 hour in antibody buffer without triton followed by an overnight wash at 4°C.Tissue Clearing

[0283] After immunofluorescent staining, tissues were optically cleared using Benzyl Alcohol / Benzyl Benzoate (BABB). Samples were first serially dehydrated by 1 hour incubation each in 10%, 25%, 50%, 75%, 90%, and 100% (v / v) ethanol solutions. Samples were then transferred into fresh 100% ethanol solution and incubated overnight at 4°C. Samples were then optically cleared by incubation in BABB for at least 1 hour prior to imaging.Confocal Imaging

[0284] All fluorescence confocal imaging was performed on a Nikon AIR HD MP multiphoton microscope equipped with a 4* (NA = 0.20) plan apochromat objective, 16x (NA = 0.80) long working distance water immersion objective, a 25* (NA = 1.10) planapochromat water immersion objective, 4 visible light internal detectors, 4 visible laser lines (405, 488, 561, 633 nm), a motorized Prior Z-deck stage, Piezo Z Nosepiece, and Insight X3 DeepSee multiphoton laser (Spectra Physics). Large overview tile scans and 3D z-stack images of tissues were acquired using the 4* (NA = 0.20) plan apochromat (Nikon) objective with NIS Elements software. 3D rendering and image processing was performed in Imaris (v9.5, Bitplane). For cleared tissues, a custom machined aluminum chamber was constructed to permit imaging through a large coverslip window and immobilization of the tissue within the BABB solution.Fluorescence Image Analysis

[0285] Advanced 3D fluorescence image analysis and animations were generated in Imaris 10.0 (Oxford Instruments). Specifically, the machine learning based vascular segmentation wizard was utilized to quantify the migratory network density and diameter within the vascular CHIPS. Colocalization analysis for the Actin channel with CD-31 channel in the pancreatic CHIPS was performed on regions of interest using the Coloc-2 plugin within FIJI Image- J. Pearson’s correlation coefficients and manders Ml and M2 values were recorded. Glucose-Stimulated Insulin Secretion

[0286] Glucose stimulated insulin secretion (GSIS) was conducted using a static incubation approach in which FRESH bioprinted pancreatic CHIPS were removed from the bioreactor. The MIN6-containing pancreatic CHIPS were exposed to serial incubations in 4 mL low glucose (LG) (1.67 mM) and high glucose (HG) (16.7 mM) Krebs Buffer containing. A preincubation period in LG Krebs buffer was followed by serial incubations in fresh LG followed by HG Krebs buffer for 1.5 hours each. GSIS was performed in duplicate. Samples collected from each incubation phase were stored at -80°C for subsequent analysis of insulin concentration. Insulin content was analyzed using a mouse insulin ELISA (Mercodia) with each sample assayed in duplicate.Statistics and Data Analysis

[0287] Statistical analysis was performed with Prism 10 (Graphpad) using appropriate tests based on experimental conditions and data. For comparison of GSIS insulin concentrations, an unpaired t-test was performed. Statistical significance was based on a P < 0.05 (*) with lower P-values being denoted as P < 0.01 (**). Non-significant P-values were denoted as ns.

[0288] Figures and visuals were constructed using Illustrator version 28.1 and Photoshop version 25.3.1 (Adobe). Supplemental videos and time-lapse images were edited in FIJI ImageJ and compiled in Premiere Pro version 24.1 (Adobe).

[0289] Various aspects of the invention include, but are not limited to, the aspects listed in the following numbered clauses.

[0290] Clause 1. An internally perfusable scaffold comprising: an exterior surface; a body; a first port comprising a first coupling feature configured to engage a fitting and form a fluid tight seal therewith; and a first interior wall defining a fluid cavity extending into the body from the first port, wherein the scaffold comprises a hydrogel.

[0291] Clause 2. The scaffold of clause 1, wherein the scaffold further comprises a second port comprising a second coupling feature configured to engage a fitting and form a fluid tight seal therewith.

[0292] Clause 3. The scaffold of clause 2, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise a tubular protrusion extending from the exterior surface.

[0293] Clause 4. The scaffold of clause 3, wherein the tubular protrusion is capable to be anastomosed to a blood vessel.

[0294] Clause 5. The scaffold of any of clauses 2-4, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise an undercut.

[0295] Clause 6. The scaffold of clause 5, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise a frustoconical shape.

[0296] Clause 7. The scaffold of any of clauses 2-6, wherein the a fluid cavity extends from the first port to the second port, the first port extends through the exterior surface, and the second port extends through the exterior surface.

[0297] Clause 8. An internally perfusable scaffold comprising: an exterior surface; a body comprising a first portion; a first port extending through the exterior surface; a second port extending through the exterior surface; a first interior wall defining a first fluid cavity extending through the body from the first port; and a second interior wall defining a second fluid cavity extending through the body from the second port; wherein the first portion is intermediate the first interior wall and the second interior wall and the first portion comprises cells, spheroids, organoids, or a combination thereof, and wherein the scaffold comprises a hydrogel.

[0298] Clause 9. The scaffold of any of clauses 2-7, further comprising: a second interior wall defining a second fluid cavity extending through the body from the second port; wherein the body comprises a first portion intermediate the first interior wall and the second interior wall.

[0299] Clause 10. The scaffold of any of clauses 8-9, wherein the first portion comprises cells, spheroids, organoids, or a combination thereof.

[0300] Clause 11. The scaffold of claim any of clause 10, wherein the first portion comprises a cell concentration in a range of 10 million cells per milliliter to 500 million cells per milliliter.

[0301] Clause 12. The scaffold of any of clauses 10-11, wherein a region of the first interior wall adjacent to the first portion comprises at least 10 million cells per milliliter.

[0302] Clause 13. The scaffold of any of clauses 10-12, wherein the first portion comprises fibrinogen and the first interior wall comprises collagen.

[0303] Clause 14. The scaffold of any of clauses 10-13, wherein the first portion comprises at least 10 milligrams fibrinogen per milliliter and the first interior wall comprises at least 6 milligrams collagen per milliliter.

[0304] Clause 15. The scaffold of any of clauses 10-14, wherein the first interior wall comprises a miscroporosity in a range of 1% to 80% by volume.

[0305] Clause 16. An internally perfusable scaffold comprising: an exterior surface; a body comprising a first portion; a first port extending through the exterior surface; a second port extending through the exterior surface; a first interior wall defining a first fluid cavity extending through the body from the first port; and a second interior wall defining a second fluid cavity extending through the body from the second port; the first interior wall and the second interior wall and the first portion comprises a third infill density less than a first infill density of the first interior wall and a second interior density of the second wall, and wherein the scaffold comprises a hydrogel.

[0306] Clause 17. The scaffold of any of clauses 8-15, wherein the first portion comprises a third infill density less than a first infill density of the first interior wall and a second infill density of the second wall.

[0307] Clause 18. The scaffold of any of clauses 16-17, wherein the third infill density is no greater than 85% by volume and the first infill density and the second infill density are individually at least 90% by volume.

[0308] Clause 19. The scaffold of any of clauses 16-18, wherein the third infill density is no greater than 50% by volume and the first infill density and the second infill density are individually at least 95% by volume.

[0309] Clause 20. The scaffold of any of clauses 16-19, wherein the first portion comprises at least one infill pattern selected from the group consisting of gyroid infill, cubic, adaptive cubic, support cubic, rectilinear, aligned rectilinear, grid, triangles, stars, line, concentric,honeycomb, 3D honeycomb, Hilbert curve, Archimedean chords, octagram spiral, and lightning.

[0310] Clause 21. A method for vascularizing the scaffold of any of clauses 8-20, the method comprising: perfusing a medium through the first fluid cavity; and growing vasculature from at least one of the first interior wall and the second interior wall into the first portion of the body.

[0311] Clause 22. The method of clause 21, wherein the medium comprises cells, water, a buffer, an antibiotic, glucose, growth factor, or a combination thereof.

[0312] Clause 23. The method of any of clauses 21-22, further comprising guiding cell growth along filaments within the first portion.

[0313] Clause 24. The scaffold of any of clauses 1-20, wherein the hydrogel comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, a Matrigel, or a combination thereof.

[0314] Clause 25. The scaffold of any of clauses 1-20 and 24, wherein the hydrogel comprises a collagen material.

[0315] Clause 26. The scaffold of any of clauses 1-20 and 24-25, wherein the hydrogel comprises a collagen concentration in a range of 1 mg collagen per milliliter to 150 mg collagen per milliliter.

[0316] Clause 27. The scaffold of any of clauses 1-20 and 24-26, wherein the hydrogel comprises a collagen concentration in a range of 10 mg collagen per milliliter to 70 mg collagen per milliliter.

[0317] Clause 28. The scaffold of any of clauses 1-20 and 24-27, wherein the scaffold consists essentially of biological derived materials.

[0318] Clause 29. The scaffold of any of clauses 1-20 and 24-28, wherein the scaffold comprises a variable infill density.

[0319] Clause 30. The scaffold of any of clauses 1-20 and 24-29, wherein the scaffold comprises at least two different materials including a first material and a second material.

[0320] Clause 31. The scaffold of clause 30, wherein the first material comprises a collagen material having a first collagen concentration and the second material comprises a collagen material having a second collagen concentration, wherein the first collagen concentration and the second collagen concentration are different.

[0321] Clause 32. The scaffold of any of clauses 30-31, wherein the first material comprises a collagen material and the second material comprises a fibrinogen material and cells.

[0322] Clause 33. The scaffold of any of clauses 1-20 and 24-32, wherein the body has a yield strength in a range of 1 kPa to 100 kPa.

[0323] Clause 34. The scaffold of any of clauses 1-20 and 24-33, wherein the scaffold is manufactured by an extrusion based additive manufacturing technique

[0324] Clause 35. The scaffold of clause 34, wherein the scaffold is manufactured by a freeform reversible embedding of suspended hydrogel additive manufacturing technique.

[0325] Clause 36. A system comprising: a bioreactor comprising a reactor wall defining a reactor cavity, a first fitting extending into the reactor cavity, and a second fitting extending into the reactor cavity; the scaffold of any of clauses 1-20 and 24-35 in the reactor cavity, wherein the first port is engaged with the first fitting and the second port is engaged with the second fitting; and a first pump capable to urge fluid through the first fitting and thereby the first fluid cavity.

[0326] Clause 37. The system of clause 36, further comprising a lid disposed over the reactor cavity and removably connected to the reactor wall.

[0327] Clause 38. The system of clause 37, wherein the lid is configured to urge the scaffold towards the reactor wall and maintain engagement between the first port and the first fitting and the second port and the second fitting.

[0328] Clause 39. The system of any of clauses 37-38, further comprising an elastomeric seal intermediate the lid and the reactor wall.

[0329] Clause 40. An additive manufacturing method for manufacturing the scaffold of any of clauses 1-20 and 24-35, the method comprising: depositing a structure material, by a nozzle, into a support material by applying a force to the structure material such that the structure material flows through the nozzle, wherein the structure material comprises a polymer and a rheological modifier; repeating the depositing of the structure material as necessary to create the scaffold; and at least partially removing the support material from object.

[0330] Any patent, publication, or other disclosure material identified herein is incorporated herein by reference in its entirety unless otherwise indicated but only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material expressly set forth in this specification. As such, and to the extent necessary, the express disclosure as set forth in this specification supersedes any conflicting material incorporated by reference herein. Any material, or portion thereof, that is said to beincorporated by reference into this specification, but which conflicts with existing definitions, statements, or other disclosure material set forth herein, is only incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. Applicant reserves the right to amend this specification to expressly recite any subject matter, or portion thereof, incorporated by reference herein.

[0331] In this specification, unless otherwise indicated, all numerical parameters are to be understood as being prefaced and modified in all instances by the term “about,” in which the numerical parameters possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of the parameter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter described herein should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0332] Also, any numerical range recited herein includes all sub-ranges subsumed within the recited range. For example, a range of “1 to 10” includes all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10. Any maximum numerical limitation recited in this specification is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited. All such ranges are inherently described in this specification.

[0333] Any references herein to “various examples”, “some examples”, “one example”, “an example”, or like phrases mean that a particular feature, structure, or characteristic described in connection with the example is included in at least one embodiment. Thus, appearances of the phrases “in various examples”, “in some examples”, “in one example”, “in an example”, or like phrases in the specification do not necessarily refer to the same examle. Furthermore, the particular described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, the particular features, structures, or characteristics illustrated or described in connection with one embodiment may be combined, in whole or in part, with the features, structures, or characteristics of one or more other embodiments without limitation. Such modifications and variations are intended to be included within the scope of the present embodiments.

[0334] As used herein, “at least one of’ A and B, means only A, only B, or both A and B. Additionally, there may be multiple As and / or multiple Bs.

[0335] One skilled in the art will recognize that the herein described articles and methods, and the discussion accompanying them, are used as examples for the sake of conceptual clarity and that various configuration modifications are contemplated. Consequently, as used herein, the specific examples / embodiments set forth and the accompanying discussions are intended to be representative of their more general classes. In general, use of any specific exemplar is intended to be representative of its class, and the non-inclusion of specific components, devices, operations / actions, and objects should not be taken to be limiting.While the present disclosure provides descriptions of various specific aspects for the purpose of illustrating various aspects of the present disclosure and / or its potential applications, it is understood that variations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should be understood to be at least as broad as they are claimed and not as more narrowly defined by particular illustrative aspects provided herein.

Claims

CLAIMSWhat is claimed is:

1. An internally perfusable scaffold comprising: an exterior surface; a body; a first port comprising a first coupling feature configured to engage a fitting and form a fluid tight seal therewith; and a first interior wall defining a fluid cavity extending into the body from the first port, wherein the scaffold comprises a hydrogel.

2. The scaffold of claim 1, wherein the scaffold further comprises a second port comprising a second coupling feature configured to engage a fitting and form a fluid tight seal therewith.

3. The scaffold of claim 2, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise a tubular protrusion extending from the exterior surface.

4. The scaffold of claim 3, wherein the tubular protrusion is capable to be anastomosed to a blood vessel.

5. The scaffold of any of claims 2-4, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise an undercut.

6. The scaffold of claim 5, wherein the first coupling feature, the second coupling feature, or both the first and second coupling feature comprise a frustoconical shape.

7. The scaffold of any of claims 2-6, wherein the fluid cavity extends from the first port to the second port, the first port extends through the exterior surface, and the second port extends through the exterior surface.

8. An internally perfusable scaffold comprising:an exterior surface; a body comprising a first portion; a first port extending through the exterior surface; a second port extending through the exterior surface; a first interior wall defining a first fluid cavity extending through the body from the first port; and a second interior wall defining a second fluid cavity extending through the body from the second port; wherein the first portion is intermediate the first interior wall and the second interior wall and the first portion comprises cells, spheroids, organoids, or a combination thereof, and wherein the scaffold comprises a hydrogel.

9. The scaffold of any of claims 2-7, further comprising: a second interior wall defining a second fluid cavity extending through the body from the second port, wherein the body comprises a first portion intermediate the first interior wall and the second interior wall.

10. The scaffold of any of claims 8-9, wherein the first portion comprises cells, spheroids, organoids, or a combination thereof.

11. The scaffold of claim any of claim 10, wherein the first portion comprises a cell concentration in a range of 10 million cells per milliliter to 500 million cells per milliliter.

12. The scaffold of any of claims 10-11, wherein a region of the first interior wall adjacent to the first portion comprises at least 10 million cells per milliliter.

13. The scaffold of any of claims 10-12, wherein the first portion comprises fibrinogen and the first interior wall comprises collagen.

14. The scaffold of any of claims 10-13, wherein the first portion comprises at least 10 milligrams fibrinogen per milliliter and the first interior wall comprises at least 6 milligrams collagen per milliliter.

15. The scaffold of any of claims 10-14, wherein the first interior wall comprises a miscroporosity in a range of 1% to 80% by volume.

16. An internally perfusable scaffold comprising: an exterior surface; a body comprising a first portion; a first port extending through the exterior surface; a second port extending through the exterior surface; a first interior wall defining a first fluid cavity extending through the body from the first port; and a second interior wall defining a second fluid cavity extending through the body from the second port; wherein the first portion is intermediate the first interior wall and the second interior wall and the first portion comprises a third infill density less than a first infill density of the first interior wall and a second interior density of the second wall, and wherein the scaffold comprises a hydrogel.

17. The scaffold of any of claims 8-15, wherein the first portion comprises a third infill density less than a first infill density of the first interior wall and a second infill density of the second wall.

18. The scaffold of any of claims 16-17, wherein the third infill density is no greater than 85% by volume and the first infill density and the second infill density are individually at least 90% by volume.

19. The scaffold of any of claims 16-18, wherein the third infill density is no greater than 50% by volume and the first infill density and the second infill density are individually at least 95% by volume.

20. The scaffold of any of claims 16-19, wherein the first portion comprises at least one infill pattern selected from the group consisting of gyroid infill, cubic, adaptive cubic, support cubic, rectilinear, aligned rectilinear, grid, triangles, stars, line, concentric, honeycomb, 3D honeycomb, Hilbert curve, Archimedean chords, octagram spiral, and lightning.

21. A method for vascularizing the scaffold of any of claims 8-20, the method comprising: perfusing a medium through the first fluid cavity; and growing vasculature from at least one of the first interior wall and the second interior wall into the first portion of the body.

22. The method of claim 21, wherein the medium comprises cells, water, a buffer, an antibiotic, glucose, growth factor, or a combination thereof.

23. The method of any of claims 21-22, further comprising guiding cell growth along filaments within the first portion.

24. The scaffold of any of claims 1-20, wherein the hydrogel comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, a Matrigel, or a combination thereof.

25. The scaffold of any of claims 1-20 and 24, wherein the hydrogel comprises a collagen material.

26. The scaffold of any of claims 1-20 and 24-25, wherein the hydrogel comprises a collagen concentration in a range of 1 mg collagen per milliliter to 150 mg collagen per milliliter.

27. The scaffold of any of claims 1-20 and 24-26, wherein the hydrogel comprises a collagen concentration in a range of 10 mg collagen per milliliter to 70 mg collagen per milliliter.

28. The scaffold of any of claims 1-20 and 24-27, wherein the scaffold consists essentially of biological derived materials.

29. The scaffold of any of claims 1-20 and 24-28, wherein the scaffold comprises a variable infill density.

30. The scaffold of any of claims 1-20 and 24-29, wherein the scaffold comprises at least two different materials including a first material and a second material.

31. The scaffold of claim 30, wherein the first material comprises a collagen material having a first collagen concentration and the second material comprises a collagen material having a second collagen concentration, wherein the first collagen concentration and the second collagen concentration are different.

32. The scaffold of any of claims 30-31, wherein the first material comprises a collagen material and the second material comprises a fibrinogen material and cells.

33. The scaffold of any of claims 1-20 and 24-32, wherein the body has an elastic modulus in a range of 1 kPa to 100 kPa.

34. The scaffold of any of claims 1-20 and 24-33, wherein the scaffold is manufactured by an extrusion based additive manufacturing technique35. The scaffold of claim 34, wherein the scaffold is manufactured by a freeform reversible embedding of suspended hydrogel additive manufacturing technique.

36. A system comprising: a bioreactor comprising a reactor wall defining a reactor cavity, a first fitting extending into the reactor cavity, and a second fitting extending into the reactor cavity; the scaffold of any of claims 1-20 and 24-35 in the reactor cavity, wherein the first port is engaged with the first fitting and the second port is engaged with the second fitting; and a first pump capable to urge fluid through the first fitting and thereby the first fluid cavity.

37. The system of claim 36, further comprising a lid disposed over the reactor cavity and removably connected to the reactor wall.

38. The system of claim 37, wherein the lid is configured to urge the scaffold towards the reactor wall and maintain engagement between the first port and the first fitting and the second port and the second fitting.

39. The system of any of claims 37-38, further comprising an elastomeric seal intermediate the lid and the reactor wall.

40. An additive manufacturing method for manufacturing the scaffold of any of claims 1- 20 and 24-35, the method comprising: depositing a structure material, by a nozzle, into a support material by applying a force to the structure material such that the structure material flows through the nozzle; repeating the depositing of the structure material as necessary to create the scaffold; and at least partially removing the support material from scaffold.

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