Hydrogel-extracelllar matrix granular composites with viscous interstitium

Abstract

Composite matrix materials including a plurality of microgel particles having a mean particle size in a range of about 1 μm to about 100 μpm, and an interstitial matrix, where the composite matrix is characterized by reversible yield-stress behavior at 4°C; methods of preparation of the composite matrix materials; and methods of preparing organoids from the composite matrix materials are disclosed.

Description

HYDROGEL-EXTRACELLLAR MATRIX GRANULAR COMPOSITES WITH VISCOUS INTERSTITIUMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 605,710, filed on December 4, 2023, the entirety of which is incorporated by reference herein.FIELD

[0002] The present disclosure relates generally to yield-stress composite matrix formulations for organoid printing and methods for making and using the yield-stress matrix formulations.BACKGROUND

[0003] Organoids are attractive models of health and disease as they recapture many important structural and physiological aspects of the mature organ. They achieve this while consisting of relatively fewer cell types, minimal extracellular matrix (ECM), and little stromal support. While this makes them powerful engineering targets, the lack of developmental and anatomical context leads to a high degree of heterogeneity in traditional 3D cultures. Though some of this heterogeneity is biologically intrinsic, there are significant external factors such as starting composition, microenvironment, and neighboring organoids that contribute to morphogenetic disparity. This manifests as difference in organoid size, structural features, and most importantly, response to treatments. Thus, a substantial number of organoids are required to uncover statistically significant phenotypes.

[0004] Many engineering platforms have sought to reproducibly build tissues from organoids, ranging from microphysiological systems to microwell arrays to 3D bioprinting. In microphysiologcial systems, typically highly controlled geometries are lithographically patterned on chips, which are then coated with cells and extracellular matrix (ECM) to generate the microtissue. This allows for a high degree of flexibility in 2D geometry and incorporation of multiple cell types. However, the pre-defined geometry precludes 3D patterning or dynamic transformations such as morphogenesis. Microwell arrays capitalize on high-density cell aggregation, composition control, and spatial arrangement for imaging, but generally limit the eventual size and geometry of the resulting tissue. Of these, 3D bioprinting is the most flexible platform for generating on-demand, arbitrary tissues of defined composition. While sorting for proliferative cell types has been shown to improve organoid formation efficiency, the most successful methods for generating cell-dense tissues like epithelial organoids generally rely on patterned cell slurries that circumvent challengesassociated with differences in cell state and growth during self-organization. In addition, these approaches leverage soft, viscoelastic basement membrane materials (typically Matrigel®) that have ideal physicochemical properties for promoting growth and morphogenesis. For example, DNA-programmed assembly of cells (DPAC) offers the highest resolution (on the order of a single cell) method for printing microtissues, but lacks scalability and has limited height resolution. In contrast, bioprinting-assisted tissue emergence (BATE) relies on a very limited print window for construction of large 3D tissues. Combined, these limitations highlight a need for better materials that enable high-fidelity bioprinting while also supporting cell growth and morphogenesis using soft, viscoelastic extracellular matrices.

[0005] Matrigel® remains the gold-standard basement membrane material (BMM) for 3D organoid culture. However, Matrigel® is a poor bioprinting material due to its steep transition from low viscosity fluid to predominately elastic solid as a function of temperature. While it is possible to print into Matrigel® during this cross-linking, the narrow window precludes seeding many organoids or complex architectures over long print times. As a result, embedded bioprinting materials have been explored, such as Carbopol or gelatin microgel slurries, that provide ideal bioprinting mechanics. Embedded bioprinting uses yielding matrices for printhead entry and translocation that simultaneously support the extruded bioink once the nozzle is removed. However, most of these materials are not optimal for long-term cell growth. Many embedded bioprinting methods employ microgels that range on the order of 100-1000 pm in diameter. To date, embedded bioprinting materials have largely relied on collagen I as the primary interstitial matrix (IM) due to its ease-of-use and ubiquity in a variety of tissues. However, collagen I matrices are fibrous and strain-stiffen, which can limit epithelial expansion and morphogenesis. In contrast, Matrigel® is largely successful due to its softness and high viscosity over large time scales that promote organoid anchoring, polarization, basement membrane deformation, and subsequent morphogenesis such as luminal expansion or budding. In fact, many organoids grown in collagen I show inhibited growth and structural expansion compared to pure Matrigel®.

[0006] The background description provided herein is for the purposes of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of the filing, are neither expressly or not impliedly admitted as prior art against the present disclosure.SUMMARY

[0007] The disclosure provides composite matrix materials comprising a plurality of microgel particles having a mean particle size in a range of about 1 pm to about 100 pm, wherein the plurality of microgel particles is characterized by a storage modulus no greater than about 200 Pa; and an interstitial matrix, wherein the composite matrix is characterized by: (a) a storage modulus of no greater than about 200 Pa; and (b) reversible yield-stress behavior at 4°C.

[0008] Accordingly, in a first aspect, the present disclosure encompasses a composite matrix material. In some embodiments, the composite matrix material includes a plurality of microgel particles having a mean particle size in a range of about 1 pm to about 100 pm; and an interstitial matrix, wherein the composite matrix is characterized by reversible yieldstress behavior at 4°C.

[0009] In some embodiments, the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.

[0010] In some embodiments, the composite matrix maintains a viscosity for a time in a range of 2 minutes to 14 days at 37°C, 1 day to 10 days, or 3 days to 7 days.

[0011] In some embodiments, the microgel mean particle size range is from about 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, or about 15 pm to about 25 pm.

[0012] In some embodiments, the storage modulus of the composite matrix is no greater than about 200 Pa.

[0013] In some embodiments, the storage modulus of the plurality of microgel particles is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, about 5 Pa to about 75 Pa, or about 10 Pa to about 50 Pa.

[0014] In some embodiments, the storage modulus of the microgel particles is no greater than about 50 Pa.

[0015] In some embodiments, the storage modulus of the interstitial matrix is no greater than 200 Pa.

[0016] In some embodiments, the storage modulus of the interstitial matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

[0017] In some embodiments, the storage modulus of the composite matrix is no greater than about 150 Pa, or no greater than about 100 Pa.

[0018] In some embodiments, the storage modulus of the composite matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

[0019] In some embodiments, the composite matrix is characterized by a yield-stress of less than or equal to 2 Pa or less than or equal to 1 Pa, for example in a range of about 0.05 Pa to about 2 Pa, about 0.1 Pa to about 1 .75 Pa, about 0.5 Pa to about 1 .5 Pa, about 0.75 Pa to about 1 .25 Pa.

[0020] In some embodiments, the microgel particles and interstitial matrix are admixed at a ratio in a range of 20%:80% by volume, respectively, to 99%:1 % by volume, respectively.

[0021] In some embodiments, the microgel includes alginate, cross-linked alginate, polyethylene glycol, agarose, hyaluronic acid, collagen IV, laminin, fibronectin, peptide-linked hydrogels, and a combination thereof.

[0022] In some embodiments, the composite matrix also includes at least one cell.

[0023] In some embodiments, the material is substantially free of collagen I.

[0024] In some embodiments, the interstitial matrix includes a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, basement membrane extract, or a combination thereof.

[0025] In some embodiments, the interstitial matrix includes (a) structural proteins selected from the group of laminin, nidogen, collagen IV, and a combination thereof and (b) adhesive peptide sequences.

[0026] In a second aspect, the present disclosure encompasses a composite matrix material. In some embodiments, the composite matrix material includes a mixture of: a plurality of cross-linked alginate microgel particles having a mean particle size in a range of about 1 pm to about 100 pm; and an interstitial matrix comprising a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, wherein the composite matrix is characterized by reversible yield-stress behavior at 4°C.

[0027] In some embodiments, the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.

[0028] In some embodiments, the composite matrix maintains a viscosity for a time in a range of 2 minutes to 14 days, 1 day to 10 days, or 3 days to 7 days, at 37°C.

[0029] In some embodiments, the alginate microgel mean particle size range is from about 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, or about 15 pm to about 25 pm.

[0030] In some embodiments, the storage modulus of the composite matrix is no greater than about 200 Pa.

[0031] In some embodiments, the storage modulus of the plurality of alginate microgel particles is in a range of about 1 Pa to about 100 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, about 5 Pa to about 75 Pa, or about 10 Pa to about 50 Pa.

[0032] In some embodiments, the storage modulus of the alginate microgel particles is no greater than about 50 Pa.

[0033] In some embodiments, the storage modulus of the interstitial matrix is no greater than 200 Pa.

[0034] In some embodiments, the storage modulus of the interstitial matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

[0035] In some embodiments, the storage modulus of the composite matrix is no greater than bout 150 Pa, or no greater than about 100 Pa.

[0036] In some embodiments, the storage modulus of the composite matrix is in a range of about 1 Pa to about 100 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

[0037] In some embodiments, the composite matrix is characterized by a yield-stress of less than or equal to 2 Pa or less than or equal to 1 Pa, for example in a range of about 0.05 Pa to about 2 Pa, about 0.1 Pa to about 1 .75 Pa, about 0.5 Pa to about 1 .5 Pa, about 0.75 Pa to about 1 .25 Pa.

[0038] In some embodiments, the alginate microgel particles and interstitial matrix are admixed at a ratio in a range of 30%:70% by volume, respectively, to 90%:10% by volume, respectively.

[0039] In a third embodiment, the present disclosure encompasses a method for preparing a composite matrix material. In some embodiments, the method includes crosslinking a polysaccharide in an aqueous solution; shearing the cross-linked-polysaccharide tocreate a microgel mixture; suspending the microgel mixture in a basal medium with a combination of antibacterial and antifungal compounds to provide a microgel slurry; and admixing the microgel slurry with an interstitial matrix.

[0040] In some embodiments, the polysaccharide includes alginate.

[0041] In some embodiments, the interstitial matrix includes a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells.

[0042] In some embodiments, admixing includes diluting the microgel slurry with the interstitial matrix.

[0043] In some embodiments, the interstitial matrix is provided in an amount in a range of about 1 part by volume based on 100 parts total volume of microgel slurry and interstitial matrix to about 70 parts by volume based on 100 parts total volume of microgel slurry and interstitial matrix.

[0044] In some embodiments, the microgel slurry and interstitial matrix are admixed at a temperature in a range of about 0°C to about 8°C.

[0045] In a fourth aspect, the present disclosure encompasses a method for preparing a composite matrix material. In some embodiments the method includes admixing a microgel slurry comprising a plurality of particles of a polysaccharide, a protein, a polyethylene glycol, or a combination thereof; and an interstitial matrix to provide a composite matrix characterized by: (a) a storage modulus of no greater than about 200 Pa; and (b) reversible yield-stress behavior at 4°C.

[0046] In some embodiments, the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.

[0047] In some embodiments, admixing includes diluting the microgel slurry with the interstitial matrix.

[0048] In some embodiments, the interstitial matrix is provided in an amount in a range of about 1 part by volume based on 100 parts total volume of microgel slurry and interstitial matrix to about 70 parts by volume based on 100 parts total volume of microgel slurry and interstitial matrix.

[0049] In some embodiments, the microgel slurry and interstitial matrix are admixed at a temperature in a range of 0°C to 10°C.

[0050] In a fifth aspect, the present disclosure encompasses a method for preparing an organoid. In some embodiments, the method includes providing a composite matrix material of any one of claims 1 to 31 at a first temperature in a range of 0°C to 8°C, preferably 4°C; depositing in the composite matrix at the first temperature a cell slurry bioink to form a cell- seeded composite matrix; and warming the cell-seeded composite matrix to a temperature in a range of about 34°C to about 40°C, for example, about 37°C.

[0051] In some embodiments, the method also includes adding cell-culture media to the cell-seeded composite matrix at the temperature in the range of about 34°C to about 40°C.

[0052] In a sixth aspect, the present disclosure encompasses another method for preparing an organoid. In some embodiments, the method includes providing a composite matrix material of any one of claims 1 to 31 at a first temperature in a range of about 0°C to about 8°C; depositing a cell slurry bioink in the composite matrix at the first temperature to form a cell-seeded composite matrix comprising a first array element; and warming the cell- seeded composite matrix to a second temperature in a range of about 34°C to about 40°C.

[0053] In some embodiments, the first temperature is in a range of about 0°C to about 8°C, about 2°C to about 6°C, about 3°C to about 5°C, or about 4°C.

[0054] In some embodiments, the second temperature is in a range of about 36°C to about 38°C, or about 37°C.

[0055] In some embodiments, the cell slurry bioink is deposited by an extruder.

[0056] In some embodiments, the extruder is a syringe pump, or a printhead.

[0057] In some embodiments, the printhead is an inkjet printhead, a pneumatic printhead, or a piezoelectric printhead.

[0058] In some embodiments, the cell slurry bioink may be deposited along any of a horizontal-x axis, vertical-y axis, angular-z axis, or any combination thereof to form the cellular array.

[0059] In some embodiments, the method also includes depositing additional cell slurry bioink in the composite matrix at the first temperature to form a plurality of array elements.

[0060] In some embodiments, the array element develops into an organoid during a period of time from about 2 minutes to about 14 days, 1 day to 10 days, or 3 days to 7 days.

[0061] In some embodiments, the deposition of the cell slurry bioink is computer controlled.

[0062] In some embodiments, the method also includes programming the computer to deposit an array element having a predetermined size along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof.

[0063] In some embodiments, the method also includes programming the computer controller to print an organoid having a predetermined shape along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof.

[0064] In some embodiments, the method also includes preparing the composite matrix material.

[0065] In some embodiments, the method also includes preparing the cell slurry bioink.

[0066] In some embodiments, the method also includes loading the extruder with the cell slurry bioink.

[0067] In some embodiments, the method also includes adding a cell-culture medium to the cell-seeded composite matrix at the second temperature.

[0068] In some embodiments, the cell-culture medium is Epidermal Growth Factor, Noggin, R Spondin (ENR) medium.

[0069] In some embodiments, the method also includes allowing the cell-seeded composite matrix to stand at the second temperature for a period of time to allow the organoid to form.

[0070] In some embodiments, the period of time is in a range from about 2 minutes to about 14 days, 1 day to 10 days, or 3 days to 7 days.

[0071] In some embodiments, the cell-culture media is changed every 2 to 4 days, every 2 to 3 days, every 3 to 4 days, every 2 days, every 3 days, or every 4 days.

[0072] The disclosure further provides composite matrix materials comprising a mixture of a plurality of cross-linked alginate microgel particles having a mean particle size in a range of about 1 pm to about 100 pm, wherein the plurality of microgel particles is characterized by a storage modulus no greater than about 200 Pa; and an interstitial matrix comprising a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells,wherein the composite matrix is characterized by (a) a storage modulus of no greater than about 200 Pa; and (b) reversible yield-stress behavior at 4°C.

[0073] The disclosure further provides methods for preparing composite matrix materials comprising cross-linking a polysaccharide in an aqueous solution; shearing the cross-linked- polysaccharide to create a microgel mixture; suspending the microgel mixture in a basal medium with a combination of antibacterial and antifungal compounds to provide a microgel slurry; and admixing the microgel slurry with an interstitial matrix.

[0074] The disclosure further provides methods for preparing a composite matrix material, comprising admixing a microgel slurry comprising a plurality of particles of a polysaccharide, a protein, a polyethylene glycol, or a combination thereof; and an interstitial matrix to provide a composite matrix characterized by (a) a storage modulus of no greater than about 200 Pa; and (b) reversible yield-stress behavior at 4°C.

[0075] The disclosure further provides method for preparing an organoid, comprising providing a composite matrix material of any one of claims 1 to 31 at a first temperature in a range of about 0°C to about 8°C; depositing a cell slurry bioink in the composite matrix at the first temperature to form a cell-seeded composite matrix comprising a first array element; and warming the cell-seeded composite matrix to a second temperature in a range of about 34°C to about 40°C.

[0076] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description. While the compositions and methods are susceptible of embodiments in various forms, the description hereafter includes specific embodiments with the understanding that the disclosure is illustrative and is not intended to limit the disclosure to the specific embodiments described herein.

[0077] These and other aspects are described further below with reference to the drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0078] While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter which is regarded as forming the present disclosure, it is believed that the disclosure will be better understood from the following description taken in conjunction with the accompanying drawings.

[0079] FIG. 1 A is a schematic comparison of a manual organoid seeding process to a composite matrix bioprinting process.

[0080] FIG. 1 B is a schematic of a bioprinting system of the present disclosure and its advantageous components.

[0081] FIG. 1C is a schematic and confocal fluorescent microscopy image of an extracellular matrix of the present disclosure. Scale bars 100 pm (left) and 40 pm (inset, right).

[0082] FIG. 1 D is a schematic of a comparison of organoid bioink printed into liquid Matrigel® having dispersed in the matrix and formed an amorphous shape with organoid bioink printed into an extracellular matrix of the present disclosure, showing an array element printed into a matrix of the disclosure having a size and shape corresponding to the size and shape of the printhead.

[0083] FIG. 1 E is a schematic of a bioprinting process of the present disclosure, and associated images.

[0084] FIG. 2A shows a plot of shear modulus (Pa) vs. temperature for composite matrices of the disclosure including 0.5 wt.% alginate particles and comparative samples of alginate microgels alone and Matrigel® alone. At temperatures below about 22°C, all materials except Matrigel® have are characterized by G’ > G”, indicating the composite materials behave as viscoelastic solids at all temperatures while Matrigel® behaves as a liquid at temperatures below about 22°C. At temperatures above about 22°C, Matrigel® contains significant cross-linking which effects the rheological behavior such that it behaves as a viscoelastic solid.

[0085] FIG. 2B shows a plot of shear modulus (Pa) vs. shear stress (Pa) at 4°C for composite matrices of the disclosure and a comparative sample of alginate microgel alone. G’ is greater than G” for all samples indicating the composite matrices have a higher storage modulus than loss modulus and are viscoelastic solids until a certain shear stress is applied, at which they yield, indicated by the drop in G’.

[0086] FIG. 2C shows a plot of modulus at 37°C (Pa) vs. Matrigel® dilution for composite matrices of the disclosure (30% microgel dilution; 50% microgel dilution) and comparative samples of alginate microgel alone and Matrigel® alone; the composite matrices of the disclosure have similar moduli at 37°C as Matrigel® alone.

[0087] FIG. 2D shows a plot of shear modulus (Pa) over time for composite matrices of the disclosure (30% microgel dilution; 50% microgel dilution) and a comparative sample of alginate microgel alone for applied strains of 1% and 100%, alternated every 60 s at 4°C. The shear modulus recovery indicates reversible yield-stress behavior.

[0088] FIG. 2E shows a plot of shear stress (Pa) vs. Shear Rate (s-1) for composite matrices of the disclosure (30% microgel dilution; 50% microgel dilution) and a comparative sample of alginate microgel alone. From this plot yield-stress (Pa) can be calculated. As the amount of Matrigel® present in the composite matrix increases, the yield-stress of the matrixdecreases indicating the material will yield more easily when the printhead is extruding bioink / cell slurry. The yield-stress values represent one replicate; the yield-stress for a given composition was calculated as an average of three trials.

[0089] FIG. 2F shows a plot of calculated yield-stress (Pa) vs. amount of Matrigel® in the composite matrix, calculated as in FIG. 2E.

[0090] FIGS. 3A-3F show plots of viscoelasticity and reversible shear stress behavior.

[0091] FIG. 4A shows a plot of shear modulus (Pa) vs. shear stress (Pa). At 4 °C, oscillatory amplitude sweeps at 1 Hz reveal that various MAGIC matrix compositions behave as yield-stress materials, indicated by G’ and G” cross-over.

[0092] FIG. 4B shows a plot of calculated yield stress. MAGIC matrices behave as Herschel-Bulkley fluids at 4 °C with yield-stresses calculated using a power law model.

[0093] FIG. 4C shows a plot of storage and loss moduli at 1 Hz and 1% strain of MAGIC matrices at 37 °C prepared from 1 wt% or 0.5 wt% AMGs.

[0094] FIG. 4D shows creep experiments quantified as stress relaxation curves at constant applied stress.

[0095] FIG. 4E shows mouse intestinal organoids grown in pure Matrigel® (top row) and MAGIC matrices of several compositions (lower rows) five days after seeding. Scale bars = 200 pm.

[0096] FIG. 4F shows quantification of organoid crypt width as a function of matrix composition.

[0097] FIG. 4G shows quantification of crypt length as a function of matrix composition. For all rheological experiments, data shown are mean ± SD from n = 3 independently prepared replicates. For crypt length measurements, data shown are mean ± SD on the median n = 15 crypts from >10 organoids per matrix condition. ** = p < 0.01 ; **** = p < 0.0001 ; ns = not significant determined by one-way ANOVA with Tukey’s multiple comparisons (4B, 4C) or Dunnett’s multiple comparisons (4F).

[0098] FIGS. 5A-5F show graphs of data to measure creep response.

[0099] FIGS. 6A -6C show images of organoid growth assays and associated data analysis.

[0100] FIG. 7 shows plots of shear modulus (Pa) vs. shear stress (Pa) and shear modulus (Pa) vs. shear rate (s-1) for composite matrices including Matrigel® or Dulbecco’s Modified Eagle Medium (DMEM) as the interstitial matrix material at 4°C, demonstrating the effect of the viscosity of the interstitial matrix material on the yield-stress behavior of the composite matrix.

[0101] FIG. 8A is a schematic of the process to prepare alginate microparticles of the present disclosure.

[0102] FIGS. 8B and 8C show images of organoids which exhibit standard morphology in diluted Matrigel®.

[0103] FIG. 8D shows determination of the mean particle size of the alginate by confocal fluorescence microscopy of stained microgels.

[0104] FIG. 9 is a schematic of preparing an organoid and / or array of organoids.

[0105] FIG. 10A shows a piezoelectric printhead of the present disclosure and precise xyz-control tolerate rapid pressure ramps and print plate movement, enabling scripts such as tail-breaking to improve print fidelity of viscous cell slurry bioinks.

[0106] FIG. 10B shows representative fluorescent images of bioprinted spheroids with and without a tail-breaking script enabled by piezoelectric bioprinting through rapid changes in applied voltage and printhead position. Scale bars = 200 pm.

[0107] FIG. 10C shows representative brightfield images of bioprinted Caco-2 tissues at day 0 and day 3 post-print as a function of extrusion step size controlled via applied voltage. Data are representative of at least n = 9 individual tissues per extrusion step condition, scale bars = 200 pm.

[0108] FIG. 10D shows a plot of organoid areas.

[0109] FIG. 10E shows a plot of circularity measured using max intensity projections of confocal z-stack images of GFP-expressing Caco-2 cell slurries. At both day 0 and day 3, organoid area significantly depends on extrusion step size until beyond 1 .0 pm as determined by one-way ANOVA with Tukey’s multiple comparisons. Data shown are mean ± SEM of n > 9 individual tissues per extrusion condition. At day 3, organoid circularity does not depend significantly on extrusion step size as determined by one-way ANOVA.

[0110] FIG. 10F shows bioprinted mouse intestinal organoid pairs printed with 250, 500, or 1000 pm center-to-center organoid spacing. Tissues printed close together (~75 pm edge- to-edge) fuse (i), whereas tissues printed far enough apart (>~300 pm edge-to-edge) don’t fuse (ii, iii). Scale bars = 500 pm.

[0111] FIG. 10G shows organoids printed in arrays with 500 pm pitch lack crypts between day 6 and 11 . Carrots indicate crypts formed close to the neighboring organoids that are gone by day 11 . Scale bars = 500 pm.

[0112] FIG. 10H shows maximum intensity projections of intestinal organoid arrays bioprinted at different depths within the MAGIC matrix (500 pm, 1000 pm, or 1500 pm from the cover glass). Scale bars = 500 pm.

[0113] FIG. 101 shows organoids printed deeper in the matrix (i) do not significantly grow between days 5 and 7 post-print compared to organoids printed closer to the media interface (ii, iii). Data shown are mean ± SD of n = 12 organoids per condition; ns = not significant, **** = p < 0.0001 determined by non-parametric t-test between day 5 and day 7.

[0114] FIG. 11 A shows quantification of bioprinted tube diameter at day 0 and day 3 post-printing as a function of both stage translation speed and extrusion step speed. Fit demonstrates that initial and final tube diameter are approximately linear functions of extrusion step speed for a given stage translation speed. Insets show representative brightfield images of tubes from that day; scale bar = 500 pm. Data shown are n = 3 bioprinted tubes per condition.

[0115] FIG. 11 B shows representative live images of bioprinted mCherry-expressing HUVEC tubes during printing (day 0) and following self-organization (day 7). Tubes over 2 mm long could be printed, with signs of vascular sprouting. Scale bars = 200 pm (day 0) and 1 mm (day 7).

[0116] FIG. 11C shows representative live images of bioprinted intestinal organoid tubes during printing (day 0) and after self-organization (day 3), showing lumenization, crypt formation, and epithelial shedding. Scale bars = 200 pm (day 0) and 500 pm (day 3).

[0117] FIG. 11 D shows brightfield images of bioprinted intestinal organoid tubes that are manually perfused with a glass capillary attached to a micromanipulator and flushed to get rid of cell debris and access the lumen. Scale bars = 200 pm.

[0118] FIG. 11 E shows quantification of tube diameter and resulting strain upon application and removal of fluid flow. Gray bars correspond to times when fluid flow was applied.

[0119] FIG. 12A shows a schematic comparison of cell growth assays under different conditions.

[0120] FIGS. 12B - 12D show microscopic images of mouse duodenal organoids.

[0121] FIGS. 12E - 12F show microscopic images of mouse submandibular salivary gland organoids.

[0122] FIG. 13A shows maximum intensity projections of DAP I- (left) or ECAD- and GFP-stained (middle) intestinal organoid arrays 3 days after bioprinting. Scale bar = 1 mm. 3D rendering of one bioprinted organoid stained for DAPI, ECAD, and Paneth cells (LYZ) demonstrating radial extension of crypts in 3D. Scale bar = 100 pm.

[0123] FIG. 13B shows staining as in FIG. 13A of bioprinted intestinal organoid tubes 3 days after bioprinting.

[0124] FIG. 13C shows maximum intensity projections of bioprinted spheroid arrays and tubes of human mammary epithelial cell (HMEC) organoids of different luminal and myoepithelial compositions. Organoids were allowed to sort for one day following printing. Scale bars = 500 pm for arrays and tubes; scale bars = 200 pm for individual spheroids.

[0125] FIG. 13D shows representative live images and quantification of luminal cell boundary occupation in bioprinted organoids as a function of composition. Dashed linesrepresent expected boundary occupancy for mechanically equivalent cells22. Data shown are mean ± SD for n > 20 organoids analyzed per composition. Scale bars = 200 pm.

[0126] FIG. 13E shows a comparison of manually seeded and bioprinted induced pluripotent stem cell-derived human cortical brain organoids. Brightfield images of manually seeded cortical brain organoids in 96 or ultra-low attachment (ULA) well plates (top) or bioprinted arrays (bottom) over time. Scale bars = 1 mm (array) or 200 pm (manually seeded or individual bioprinted organoids).

[0127] FIG. 13F shows at left, 20 pm maximum intensity projections of bioprinted cortical organoids stained for cortical identity (top) and neuronal differentiation (bottom). Scale bars = 50 pm, and at right, quantification of cortical identity and neuronal differentiation compared to manually seeded cortical brain organoids. Data shown are mean ± SD of n = 2 or 3 organoids per marker; ns = not significant, ** = p < 0.01 as determined by non-parametric t- test.

[0128] FIGS. 14A - 14D show microscopic images of human cortical brain cells printed in the composite matrix in comparison to cells printed in an AMG slurry.

[0129] FIG. 15A shows live images of triple-negative breast cancer (TNBC) patient- derived organoids transduced overnight with GFP-expressing lentivirus while seeded in Matrigel (top) or MAGIC matrix (bottom). Scale bars = 200 pm.

[0130] FIG. 15B shows fractions of GFP+ organoids transduced in suspension before seeding or transduced after seeding for either ECM composition. Data shown are mean ± SD of n = 3 replicate ECM conditions; ns = not significant, **** = p < 0.001 determined by non-parametric t-test between ECM conditions.

[0131] FIG. 15C shows live images of bioprinted TNBC organoids transduced with GFP- expressing lentivirus overnight after printing (top) or transfected using Lipofectamine and Cy3-conjugated single-stranded non-coding small RNA for 24 h, 3 days after printing (bottom). Scale bars = 500 pm.

[0132] FIG. 15D shows live imaging of bioprinted intestinal organoid arrays following printing and after 2 and 5 days in culture at left and live imaging of manually seeded organoids after 5 days of culture at right. Scale bars = 500 pm.

[0133] FIG. 15E shows organoid area over time for manual or bioprinted intestinal organoids; data shown are mean ± SD of n > 190 organoids per time point.

[0134] FIG. 15F shows quantification of crypts per organoid for those manually seeded in Matrigel® or MAGIC matrix, or bioprinted. Data shown are mean ± SD of n > 30 organoids; ns = not significant; **** = p < 0.0001 determined by one-way ANOVA with Dunnett’s multiple comparisons.

[0135] FIG. 15G shows an experimental outline of phenotypic assay for inhibition of gamma-secretase.

[0136] FIGS. 15H and 151 show live imaging of manually seeded (FIG. 15H) and bioprinted (FIG. 151) organoids treated with and without gamma-secretase inhibitor. Red fluorescence indicates Atoh1 + secretory progenitors. Scale bars = 500 pm.

[0137] FIG. 15J shows total red fluorescence volume per organoid in treated and untreated conditions. For bioprinted arrays, data shown are mean ± SD of n = 45 organoids per condition. For manually seeded organoids, data shown are mean ± SD of n > 135 organoids per condition. **** = p < 0.0001 determined by non-parametric t-test.

[0138] FIG. 15K shows a bootstrapping analysis of statistical significance between treated and untreated conditions for either bioprinted or manually seeded organoids as a function of number of paired comparisons. Inset shows statistical significance approaches zero (< 10-9) for bioprinted organoids using an equivalent number of comparisons as it takes manually seeded organoids to approach p = 0.05.

[0139] FIG. 16 shows images of transfected bioprinted organoid arrays.DETAILED DESCRIPTION

[0140] The disclosure provides a matrix material that provides advantageous yield-stress properties in combination with soft, viscoelastic properties to facilitate, for example, homogeneous organoid preparation. The matrix material of the disclosure can provide one or more advantages, including, but not limited to, having a reversible yield-stress behavior to allow a printhead to enter and extrude a bioink such as a cell slurry, then recovery of the matrix material to provide elastic support of the extruded bioink, and / or promoting long-term cell growth for example, by providing a matrix material that is soft and highly viscous over time scales of cell growth and / or by providing a matrix material with particle sizes on the order of the size of cells to allow for yielding behavior of the matrix to promote morphogenesis and organoid expansion.Introduction and Context

[0141] In vitro tissue models that reproducibly and scalably recapitulate complex tissue physiology are required for applications in regenerative medicine, disease modeling, and drug testing. Organoids have the potential to satisfy these requirements. Organoids are selforganizing tissues derived from stem and progenitor cells that incorporate multiple mature cell types and simple morphological features. In order to self-organize, organoids must be derived from the appropriate cellular progenitors and cultured within the appropriate 3D microenvironments, typically laminin-rich extracellular matrix (ECM) gels like Matrigel®.Even when these requirements are satisfied, however, organoids lack developmental and anatomical contexts that support, constrain, and guide their morphogenesis in vivo. Consequently, they lack much of the complex morphology of the tissue from which they arederived and generally suffer from a high degree of structural heterogeneity. Though some of this heterogeneity is intrinsic to the stochastic nature of cell and tissue growth and morphogenesis, there are significant external factors such as initial tissue size, composition, media access, and neighboring interfaces that contribute to morphological heterogeneity. These manifest as differences in organoid mass, structural features, and cellular composition, all of which lead to variability in the response to drug, microenvironmental, or genetic perturbations (FIG. 1 A). FIG. 1 A illustrates that traditional manual methods of seeding organoids lead to heterogeneity in organoid growth and morphogenesis due to heterogeneity in starting tissue size, composition, and microenvironment. By controlling for initial conditions such as cell number, media access, and organoid spacing, bioprinting platforms facilitate rapid generation of reproducible organoid arrays or freeform 3D microphysiological systems. This variability in turn decreases statistical precision between experimental conditions, requiring many replicates to elucidate phenotypes.

[0142] To address these challenges, engineering platforms have sought to better control the initial conditions from which organoids emerge. Such platforms include microwell arrays, microphysiological systems (or organs-on-a-chip), and 3D bioprinting. Microwell arrays are screening platforms that emerge from high-density cell aggregates, generally of a composition defined by Poisson statistics, and arranged spatially for straightforward imaging. Microwells can improve organoid homogeneity but are generally limited in the eventual size and geometry of the resulting tissue. In microphysiologcial systems, controlled geometries are lithographically patterned on polymeric chips, which are then coated with cells and ECM to generate the microtissue. These powerful tools allow for complex tissue geometries, incorporation of multiple cell types, and microfluidic plumbing for exchange of metabolites between tissue and organ compartments. However, the pre-defined geometry and artificial interfaces such as PDMS impose many constraints on the tissue that may impact normal morphogenesis. Additionally, the top-down constraints on geometry imposed by the workflow can incorporate biases on cell and tissue function. Finally, the chip format requires complete reconfiguration for each build iteration, slowing the design-build-test cycle. In contrast, 3D bioprinting comprises a suite of rapid prototyping tools that provides a potentially more flexible platform for generating tissues of defined size, composition, and geometry on- demand and in arbitrary microenvironments. However, most applications of bioprinting remain in their infancy and have typically focused on printing non-living biomaterials or composites of hydrogels and cells. Moreover, these efforts mostly focus on controlling the printed geometry of cells and materials in 3D space, rather than the critical importance of the subsequent morphogenesis of these living materials in time. Consequently, little emphasis has been placed on the interaction between printed structures and the spatial, mechanical, and molecular details of the microenvironment that support their morphogenesis.

[0143] Recently, several applications of printing and patterning technology have attempted to better program cell and organoid morphogenesis by fabricating tissues from cell slurries at tissue-like densities, then embedding these seeds directly into gold-standard ECMs like Matrigel®. For example, DNA-programmed assembly of cells (DPAC) is a bottom- up method for patterning dense cell slurries in Matrigel® and Matrigel®-Collagen-I mixtures that has been applied to fibroblast clusters, mammary organoids, and vasculature. Cells are first directed to self-assemble on a 2D template and then released into Matrigel® as it polymerizes. They can then undergo morphogenesis in 3D. While this method is among the highest in resolution (on the order of a single cell) for printing microtissues, it lacks scalability, has limited height resolution, and can be technically arduous. Embedded bioprinting methods such as bioprinting-assisted tissue emergence (BATE) extrude dense cell slurries directly into liquid Matrigel® or collagen to construct large 3D patterns that undergo morphogenesis48. These methods potentially address many of the scalability issues presented by DPAC. However, BATE remains limited by Matrigel®s steep transition from fluid to solid as a function of temperature, which provides only a very narrow time window in which extruded cell slurries conform to the geometry prescribed by the printer. Consequently, the capacity of BATE to support automation and extended printing times has not been explored.

[0144] To extend available print times, freeform bioprinting has largely turned to granular media such as Carbopol or gelatin microgel slurries which provide improved bioprinting mechanics. One central property of these materials is reversible yield-stress behavior, in which the slurry yields in response to the printhead entering the bath and extruding material, then recovers to provide elastic support to the extruded bioink once the nozzle is removed. However, most of these materials are not optimal for long-term cell growth and morphogenesis. Several groups have introduced interstitial matrices derived from natural ECM like collagen I with the goal of improving cell survival and dynamics. However, the mechanics and composition of the resulting materials elicit cell behaviors that can be challenging to predict. For example, collagen l-containing ECM strain-stiffens and is not optimized to support epithelial growth and morphogenesis compared to basement membrane extracts. Matrigel® in particular has several properties that make it unique as a biomaterial for tissue morphogenesis, but poor as a biomaterial for freeform bioprinting. It is rich in laminin, collagen IV and nidogen — the major components of the basement membrane that provide critical polarity signals to tissue supporting their early morphogenesis. Matrigel® is also degradable, undergoes plastic deformations at long times scales, and is a viscoelastic biomaterial with a storage modulus significantly lower than typical synthetic materials used in 3D cell culture and bioprinting applications. However, it is a poor support for embedded bioprinting because it behaves as a viscous fluid at 4 °C, while quicklytransitioning to a soft hydrogel at 37 °C for cell culture. Therefore, a freeform bioprinting material that combines the advantageous yield-stress properties of microgels with the mechanical and biochemical properties of basement membrane extracts could revolutionize bioprinting for generation of reproducible organoid arrays or advanced 3D microphysiological systems (FIG. 1 B). MAGIC matrix bioprinting combines two major advances shown in FIG. 1 B: (1) a soft embedded bioprinting material that supports gold-standard morphogenesis and (2) a piezoelectric printhead with fast pressure ramps and direct bioink aspiration / extrusion.

[0145] Hence, an embedded bioprinting material that comprises alginate microgels and interstitial Matrigel®, termed Matrigel®-Alginate Granular-Interstitial Composite (MAGIC) matrix (FIG. 1C) was designed and optimized. MAGIC matrix comprises of an inert alginate microgel granular support and a viscous basement membrane interstitium as illustrated in FIG. 1C. This compositionally simple material acts as a yield-stress embedded bioprinting medium at 4 °C while supporting complex tissue morphogenesis at 37 °C. Confocal microscopy images showing a standard MAGIC matrix composition using FITC-alginate microgels and NHS-labeled Matrigel®. Scale bar = 100 pm. Zoomed view shows white pixels where fluorescent signals overlap, suggesting ECM in alginate microgels. Scale bar = 40 pm.

[0146] MAGIC matrices employ alginate microgels that are optically transparent and approximately cell-sized, which facilitated yielding behavior, high print fidelity, imaging, and organoid expansion. Matrigel® was concurrently used as the interstitial material to create a switchable composite matrix that remains liquid at 4 °C to allow for long print times (>3 h), but cross-links at 37 °C to create a soft, viscoelastic, and viscoplastic environment that is mechanically similar to pure Matrigel® across several metrics. To capitalize on the reproducibility, scalability, and automation afforded by 3D printing, a piezoelectric printhead was designed that is linked to a microscope stage and robotic arms that allow for full xyz- control, real-time imaging, and flexible scripted print geometries (FIG. 1 B). This printhead enabled direct aspiration and extrusion of saturated cell slurries exceeding 108cells / mL (FIG. 1 D), minimizing dead volume and required biomass, with a theoretical limit of 0.1 nL delivered volume. Organoid slurries printed into liquid Matrigel® at 4 °C distort and settle to the bottom of the dish due to insufficient mechanical support. Organoid bioink printed into MAGIC matrix at 4 °C conforms to the desired morphology due to the support’s reversible yield-stress properties.

[0147] This led to orders-of-magnitude improvements in inter-organoid homogeneity and statistical power with approximately 100% organoid formation efficiency (FIG. 1 E). The combined result is a flexible 3D bioprinting platform using a simple but powerful biomaterial that can generate reproducible yet complex in vitro tissues. FIG. 1 E shows sequential brightfield images demonstrating MAGIC matrix bioprinting, in which the printhead enterscold MAGIC matrix, extrudes organoid slurry, and is then removed from the matrix, leaving a geometrically and spatially controlled feature behind. These organoid bioinks subsequently undergo self-organization at 37 °C to form mature organoids. Scale bars = 500 pm.

[0148] An embedded bioprinting material that combines the advantageous yield-stress properties of microgels with the soft and viscoelastic properties of Matrigel® will facilitate long sought applications of organoids in research and medicine. The composite material of the disclosure can advantageously exhibit recoverable yield-stress behavior to support long printing times (>2 h) at 4 °C without compromising mechanical integrity or organoid viability. Further, the material can exhibit identical bulk viscoelasticity to Matrigel®. Conventionally, a material for (i) printing a cell slurry into and (ii) facilitating organoid growth would need to be optimized for either the printing or cell growth and morphogenesis. It was surprisingly found that a composite matrix material of the disclosure including a microgel and an interstitial matrix can demonstrate good morphogenesis and cell growth across multiple printing parameters. For example, the yield-stress of a composite matrix material including a microgel and an interstitial matrix can be tuned at 4°C while maintaining G' and G" equivalent to Matrigel® at 37°C, which advantageously allows tuning of the print-relevant parameters while ultimately not impacting the bulk viscoelastic properties of the composite matrix material at 37°C that allow normal cell growth and morphogenesis.

[0149] A piezoelectric printhead design and ECM formulation can be used to provide homogeneity in organoid-to-organoid structures, for example, in organoid size, shape, maturation time, and crypt budding efficiency.

[0150] A piezoelectric printhead can be used to control direct aspiration and deposition of dense cellular bioinks for fast and high-throughput generation of organoid arrays. The homogeneity of these arrays reduces the number of observations necessary to achieve statistical significance, thereby requiring fewer printed tissues and shorter print times while accelerating assay completion. Combined, these advances lay the foundation for long- envisioned applications of organoids in personalized medicine, drug screening, and disease modeling.

[0151] The disclosure provides a composite matrix material comprising a plurality of microgel particles having a mean particle size in a range of about 1 pm to about 100 pm, wherein the plurality of microgel particles is characterized by a storage modulus of no greater than about 200 Pa and an interstitial matrix, wherein the composite matrix is characterized by (a) a storage modulus of no greater than about 200 Pa, and (b) reversible yield-stress behavior at 4°C.

[0152] The term “about” is used according to its ordinary meaning, for example, to mean approximately or around. In one embodiment, the term “about” means ±10% of a stated value or range of values. In another embodiment, the term “about” means ±5% of a statedvalue or range of values. A value or range described in combination with the term “about” expressly includes the specific value and / or range as well (e.g., for a value described as “about 40,” “40” is also expressly contemplated).

[0153] The composited matrix material of the disclosure generally includes an interstitial matrix that is a liquid at a temperature in a range of about 0°C to about 8°C, for example 4°C and plurality of microgel particles that exhibit reversible yield-stress behavior at a temperature in a range of about 0°C to about 8°C. As used herein, “reversible yield-stress” refers to a rheological behavior of a material wherein the material is a viscoelastic solid (G' > G") until a certain shear stress is applied to the material at which point G' will drop below G" and the material will flow until the shear stress is removed and / or a lower shear stress is applied at which point G' recovers to within about 20% of the original G' value and the material is again a viscoelastic solid wherein G' is greater than G". For example, G' can recover to within about 20% of the original value (e.g., at least 80% of the original value), within about 15% of the original value (e.g., at least 85% of the original value), within about 10% of the original value, within about 5% of the original value, or within about 2.5% of the original value. It will be readily understood that the certain shear stress required for G' to drop below G" is material dependent and such values are routinely determined by persons of ordinary skill in the art. As used throughout the description, and unless indicated otherwise, the temperature of 4°C is representative of a low temperature in a range of about 0°C to about 8°C, for example, greater than 0°C and greater than or equal to 8°C, which is known in the art to be a suitable temperature for cell seeding into an extracellular matrix. As used throughout the description, and unless indicated otherwise, the temperature of 37°C is representative of a physiological temperature in a range of about 34°C to about 40°C, and is known in the art to be a suitable temperature for cell growth and morphogenesis.

[0154] Without intending to be bound by theory, it is believed that this combination of a liquid interstitial matrix and reversible yield-stress microgel particles at 4°C provide a composite matrix that can facilitate a printed bioink / cel I slurry holding the shape in which it was printed into the matrix and can leverage the reversible yield-stress behavior of the composite matrix at 4°C, allowing the microgel particles and composite matrix to yield (deform) and reform as a printhead moves through it while depositing the bioink / cell slurry. Further, without intending to be bound by theory, it is believed that because the microgel particles are solid when at rest (and until a certain stress is applied), the extent of displacement of the particles in the matrix is partially limited by the concentration of particles (for example, a particle can only move in the matrix until it bumps into another particle) and such limited movement of the microgel particles can help facilitate the printed bioink / cell slurry maintaining its printed shape. In contrast, as shown in FIG. 4B, a matrix consisting only of a liquid interstitial matrix material at 4°C, such as Matrigel®, cannot hold the shape ofa printed bioionk / cell slurry and the bioink / cell slurry takes an amorphous shape as it displaces the Matrigel®. As shown in FIG. 4B, a printed structure can maintain the shape of a dot / sphere when printed into a composite matrix of the disclosure whereas depositing the same material into a Matrigel® only matrix results in diffusion of the printed material into an amorphous structure.

[0155] In general, the storage modulus of the composite matrix materials of the disclosure can be any storage modulus suitable to provide a soft material which allows normal growth of cells and morphogenesis of cells deposited into the composite matrix. The storage modulus represents the elastic portion of the viscoelastic behavior of the composite matrix and is a measure of how much energy must be put into the sample in order to distort it. Thus, as the storage modulus of the composite matrix increases, the amount of energy required to distort the matrix increases. Without intending to be bound by theory, it is believed that at 4°C, as the storage modulus of the composite matrix decreases, the force required to move a printhead through the composite matrix decreases and that at 37°C, as the storage modulus of the composite matrix decreases cells can more readily expand into the matrix facilitating normal growth and morphogenesis. In contrast, without intending to be bound by theory, as the storage modulus of the composite matrix increases, for example, above 200 Pa, the composite matrix can inhibit cell growth and morphogenesis. Methods of measuring storage modulus are well known in the art. Storage modulus was determined by shear oscillatory rheology of composite material samples. Amplitude sweeps ranging 0.1% to 100% strain and frequency sweeps ranging 0.1 Hz to 100 Hz were performed at 1 Hz and 1% strain, respectively. Temperature sweeps were performed at 1 Hz and 1% strain with 60s allowed for temperature equilibration at each measurement. In some embodiments, the storage modulus of the composite matrix is up to 1 kPa. The storage modulus of the composite matrix at 4°C and / or 37°C can be no greater than about 200 Pa, no greater than 150 Pa, no greater than 100 Pa, or no greater than 50 Pa, for example, in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, or about 10 Pa to about 50 Pa. Without intending to be bound by theory, it is believed that below about 1 Pa, the storage modulus of the composite matrix is too close to liquid to support the shape of the printed material. In general, storage modulus is temperature independent unless a physical or chemical change occurs in the material, e.g., cross-linking, as the temperature changes.

[0156] In general, the composite matrix materials of the disclosure are characterized by a reversible yield-stress behavior at 4°C. Advantageously, the composite matrices of the disclosure having a reversible yield-stress behavior at 4°C allows the composite matrix to yield and reform as a printhead moves through it while depositing the bioink / cell slurry.

[0157] The composite matrix materials of the disclosure can also be characterized by a yield-stress at 4°C that is lower than the force exerted by a printhead moving through the matrix and / or a yield-stress at 37°C that is lower than the forces required for normal tissue growth or morphogenesis. As described in Phillips, Rob etal., Physical Biology of the Cell, 2ndEdition, New York, Garland Science, October 29, 2012 and Janmey etal., “The Mechanical Cell” Curr. Biol. 19 (17), 2009, it is known in the art that tissue growth and morphogenesis generate physical forces that can act upon the surrounding environment, e.g., blood and lymphatic vessels, other tissues, etc. in vivo, and growth media, extracellular matrix, etc. in vitro. To promote organoid growth with size and morphogenetic homogeneity, the physical forces generated by the bioprinted cell slurry during tissue growth should be able to displace the composite matrix. Without intending to be bound by theory, it is believed that if the yield-stress of the composite matrix is greater than the value of the force exerted during tissue growth, the composite matrix will act as a barrier, preventing tissue from growing normally and morphogenesis. The yield-stress of the composite matrix can be an order of magnitude smaller than the force exerted during tissue growth or morphogenesis. The yield-stress of the composite matrix can be less than or equal to about 2 Pa or less than or equal to about 1 Pa, for example, in a range of about 0.05 Pa to about 2 Pa, about 0.1 Pa to about 1 .75 Pa, about 0.5 Pa to about 1 .5 Pa, about 0.75 Pa to about 1 .25 Pa, as determined from unidirectional shear measurements at 1% strain and filling to a Herschel- Bulkley Power Law model, using the y-intercept to determine the yield-stress. The yieldstress of the composite matrix can be less than or equal to the yield-stress of cross-linked Matrigel® at 37°C.

[0158] In general, the composite matrix material of the disclosure at 37°C can maintain its viscosity for a time suitable for cell growth and morphogenesis, for example, in a rage of 2 minutes to 14 days as determined by measuring the viscosity with a 25 mm cone-and-plate geometry on an Anton-Parr rheometer. The composite matrix materials can maintain the viscosity against applied strain and for timescales of applied strain that are relevant to tissue morphogenesis, as are known in the art. A cell slurry bioink can develop into an organoid on a timescale of minutes to days. The composite matrix can maintain its viscosity for a time in a range of about 2 minutes to about 14 days, about 1 day to about 10 days, about 3 days to about 7 days, for example, 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, or 14 days. The composite matrix materials of the disclosure can advantageously maintain rheological properties necessary to accommodate organoid growth.

[0159] The microgel particles of the composite matrix material of the disclosure can be a material that is a soft viscoelastic solid at 37°C, such that cell growth and morphogenesis can occur in the composite matrix. In some embodiments, the storage modulus of the microgel particles is up to 1 kPa. The microgel particles can have a storage modulus at 4°Cand / or at 37°C of no greater than about 200 Pa, no greater than 150 Pa, no greater than 100 Pa, or no greater than 50 Pa, for example, in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, or about 10 Pa to about 50 Pa. The microgel particles can have a storage modulus of no greater than 200 Pa. The microgel particles can have a storage modulus of no greater than 50 Pa.

[0160] The microgel particles can be a viscoelastic solid at temperatures in a range of about 0°C to about 40°C when at rest and characterized by a reversible yield-stress behavior, for example, at 4°C to allow yielding and reforming of the matrix in response to an extruder (e.g., print head) moving through the matrix.

[0161] The microgel particles can have any mean particle size suitable to allow cell growth and morphogenesis. Without intending to be bound by theory, it is believed that as the mean particle size of the microgel increases relative to the size of the cell printed within the composite matrix material, the ability of the cell to displace a microgel particle to allow normal growth decreases and the quality of the resulting organoid decreases. Further without intending to be bound by theory, it is believed that as the mean particle size increases above about 100 pm and / or above about 5 times the size of the cell, the ability of the cell to displace a microgel particle to allow normal growth decreases and the quality of the resulting organoid decreases. The mean particle size of the microgels of the disclosure can be in a range of 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, or about 15 pm to about 25 pm, for example, about 15 pm, about 18 pm, about 20 pm, about 22 pm, or about 25 pm. The mean particle size of the microgel particles was determined by confocal fluorescence microscopy of stained microgels, as shown in FIG. 12D.

[0162] In general, the microgel particles can include a polysaccharide, proteins, synthetic polymers, or a combination thereof. The microgel particles can include a polysaccharide, a protein, or a combination thereof. The microgel particles can include a polysaccharide. The microgel particles can include a protein. The microgel particles can include a synthetic polymer. Suitable polysaccharides include, but are not limited to alginate, agarose, hyaluronic acid, and a combination thereof. Suitable proteins include, but are not limited to, collagen IV, laminin, fibronectin, or a combination thereof. Suitable synthetic polymers include, but are not limited to polyethylene glycols, modified polyethylene glycols, poly(acrylic acid), and a combination thereof. The microgel particles can include alginate, crosslinked alginate, polyethylene glycols, agarose, hyaluronic acid, collagen IV, laminin, fibronectin, or a combination thereof. The microgels can be cross-linked to provide mechanical support. The microgel particles are substantially free of fibrous materials. As use herein, and unless specified otherwise, “substantially free of” refers to less than 5 wt.%, for example, less than 2.5 wt.% or less than 1 wt.% of the identified component, based onthe total weight. Thus, a microgel particle that is substantially free of fibrous materials can include less than 5 wt.% of a fibrous material, based on the total weight of the microgel particle. The composite matrix material can be substantially free of fibrous materials. The composite matrix material and the microgel particles can be substantially free of collagen I. The microgel particles can include alginate. The microgel particles can include cross-linked alginate. The microgel material can include non-fibrous or globular materials, such as proteins including laminin and collagen IV, provided that the microgel material has a mean particle size and a reversible yield-stress behavior as described herein.

[0163] The amount of polysaccharide, protein, and / or synthetic polymer in the microgels can be provided in any amount suitable to provide a soft, viscoelastic solid having a reversible yield-stress at 4°C. It will be understood that the amount of polysaccharide, protein, and / or synthetic polymer will vary depending on the specific polysaccharide, protein, and / or synthetic polymer used. The amount of polysaccharide, protein, and / or synthetic polymer in the microgels can be in a range of about 0.10 wt.% to about 20 wt.%, about 0.1 wt.% to about 15%, about 0.1 wt.% to about 10 wt.%, about 0.10 wt.% to about 5 wt.%, based on the total weight of the microgel, for example, about 0.15 wt.% to about 2.5 wt.%, about 0.20 wt.% to about 1 wt.%, or about 0.25 wt.% to about 0.75 wt.%, for example, about 0.25 wt.%, about 0.50 wt.%, about 0.75 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, or about 5 wt.%, based on the total weight of the microgel. The amount of polysaccharide and / or protein in the microgels can be in a range of about 0.10 wt.% to about 5 wt.%, based on the total weight of the microgel, for example, about 0.15 wt.% to about 2.5 wt.%, about 0.20 wt.% to about 1 wt.%, or about 0.25 wt.% to about 0.75 wt.%, for example, about 0.25 wt.%, about 0.50 wt.%, about 0.75 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, or about 5 wt.%, based on the total weight of the microgel. When the polysaccharide is a cross-linked alginate, the amount of alginate in the microgel can be in a range of about 0.15 wt.% to about 1 wt.%, for example, about 0.2 wt.% to about 0.8 wt.%, about 0.25 wt.% to about 0.75 wt.%, or about 0.25 wt.%, about 0.3 wt.%, about 0.35 wt.%, about 0.4 wt.%, about 0.45 wt.%, about 0.5 wt.%, about 0.55 wt.%, about 0.6 wt.%, about 0.65 wt.%, about 0.7 wt.%, or about 0.75 wt.%, based on the total weight of the microgel.

[0164] The interstitial matrix can generally be any material that is a liquid at 4°C and can promote cell adhesion. The interstitial matrix can include (a) structural proteins and (b) adhesive peptide sequences. Suitable structural proteins include, but are not limited to laminin, nidogen, collagen IV, or a combination thereof. The interstitial matrix can be a basement membrane extract, a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, for example, Matrigel®, or a combination thereof. The interstitial matrix can be a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells.

[0165] In some embodiments, the storage modulus of the interstitial matrix is up to 1 kPa. The interstitial matrix can be characterized by a storage modulus at 4°C and / or 37°C can be no greater than about 200 Pa, no greater than 150 Pa, no greater than 100 Pa, or no greater than 50 Pa, for example, in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, about 1 Pa to about 100 Pa, or about 1 Pa to about 50 Pa. The storage modulus of the interstitial matrix at 4°C and / or 37°C can be no greater than 200 Pa. The storage modulus of the interstitial matrix at 4°C and / or 37°C can be no greater than 150 Pa. The storage modulus of the interstitial matrix at 4°C and / or 37°C can be no greater than 100 Pa. The storage modulus of the interstitial matrix at 4°C and / or 37°C can be no greater than 50 Pa. In general, storage modulus is temperature independent unless a physical change occurs in the material, e.g., cross-linking, as the temperature changes. The interstitial matrix can undergo temperature dependent cross-linking at temperatures between about 10°C and about 37°C.

[0166] The viscosity of the interstitial matrix is not particularly limiting. As shown in FIG. 10, the viscosity of the interstitial matrix at 4°C decreases, the yield-stress of the resulting composite matrix decreases and the G' and G" values of the composite matrix decrease for all microgel dilution levels. Accordingly, the rheological properties of the composite matrix can be controlled by tuning the viscosity of the interstitial matrix.

[0167] The ratio of microgel particles and interstitial matrix in the composite matrix can be any ratio provided that the composite matrix material is characterized by a storage modulus of no greater than about 200 Pa and a reversible yield-stress behavior at 4°C. The ratio of microgel particles and interstitial matrix are volumetric ratios at the time of admixing the microgel particles and the interstitial matrix to form the composite matrix. The relative amount of interstitial matrix can be any amount suitable to promote cell adhesion / anchoring to the composite matrix. The interstitial matrix can be provided in an amount of at least about 1% by volume, based on the total volume of interstitial matrix and microgel particles admixed. For example, the interstitial matrix can be provided in an amount of at least about 1%, at least about 5%, or at least about 10% and up to about 60%, up to about 70%, or up to about 80%, by volume based on the total volume of interstitial matrix and microgel particles admixed.

[0168] The microgel particles can be provided in any amount to provide a sufficient concentration of solid particles to help facilitate a printed bioink / cell slurry maintaining its printed shape in the composite matrix. The microgel particles can be provided in an amount of at least about 20% by volume, based on the total volume of interstitial matrix and microgel particles admixed. For example, the microgel particles can be provided in an amount of at least 20%, at least 30%, at least 40% and up to about 99%, about 95%, or about 90%, by volume based on the total volume of interstitial matrix and microgel particles admixed. Theratio of the microgel particles and interstitial matrix at the time of admixing can be in a range of about 20%:80% by volume to about 99%: 1% by volume, respectively, for example, about 20%:80% to about 95%:5%, about 30%:70% to about 90%:10%, or about 30%:70%, about 40%:60%, about 50%:50%, about 60%:46%, about 70%:30%, about 80%:20%, or about 90%:10%, respectively, by volume.

[0169] As the amount of interstitial matrix increases, the yield-stress of the composite matrix generally decreases. At very low yield-stresses, a printhead can easily move through the composite material; however, as the yield-stress becomes very low, e.g., 0.1 Pa or less, there may be a loss of print quality.

[0170] The composite matrix material of the disclosure can comprise a mixture of a plurality of cross-linked alginate microgel particles having a mean particle size in a range of about 10 pm to about 30 pm, wherein the plurality of microgel particles is characterized by a storage modulus no greater than about 150 Pa; and an interstitial matrix comprising a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, wherein the composite matrix is characterized by a storage modulus of no greater than about 150 Pa and reversible yield-stress behavior at 4°C. The composite matrix comprising cross-linked alginate microgel particles and an interstitial matrix comprising a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells can be characterized by a yield-stress of less than or equal to Pa. The alginate microgel particles and interstitial matrix can be provided in a ratio of about 30%:70% to about 9%:10%, by volume.

[0171] The disclosure provides methods of preparing a composite matrix material of the disclosure comprising admixing a microgel slurry comprising a plurality of particles of a polysaccharide, a protein, a synthetic polymer, or a combination thereof and an interstitial matrix to provide a composite matrix characterized by a storage modulus of no greater than about 200 Pa and reversible yield-stress behavior at 4°C, a schematic of which is shown in FIG. 1C. The polysaccharide, protein, and synthetic polymer can be any polysaccharide, protein, or synthetic polymer disclosed herein for the microgel slurry. The interstitial matrix can be any interstitial matrix disclosed herein. The composite matrix material can have any storage modulus disclosed herein for composite matrix materials. The composite matrix can further be characterized by an of the rheological properties disclosed herein for composite matrix materials.

[0172] The admixing of the microgel slurry and interstitial matrix can include diluting a microgel slurry with an interstitial matrix. The interstitial matrix can be provided in an amount in a range of about 1 part by volume based on 100 parts total volume of microgel slurry and interstitial matrix to about 70 parts by volume based on 100 parts total volume of microgelslurry and interstitial matrix. The interstitial matrix can be provided in an amount of at least about 1% by volume, based on the total volume of interstitial matrix and microgel particles admixed. For example, the interstitial matrix can be provided in an amount of at least about 1%, at least about 5%, or at least about 10% and up to about 60%, up to about 70%, or up to about 80%, by volume based on the total volume of interstitial matrix and microgel particles admixed. The microgel particles can be provided in an amount of at least about 20% by volume, based on the total volume of interstitial matrix and microgel particles admixed. For example, the microgel particles can be provided in an amount of at least 20%, at least 30%, at least 40% and up to about 99%, about 95%, or about 90%, by volume based on the total volume of interstitial matrix and microgel particles admixed. The ratio of the microgel particles and interstitial matrix at the time of admixing can be in a range of about 20%:80% by volume to about 99%:1 % by volume, respectively, for example, about 20%:80% to about 95%:5%, about 30%:70% to about 90%:10%, or about 30%:70%, about 40%:60%, about 50%:50%, about 60%:46%, about 70%:30%, about 80%:20%, or about 90%:10%, respectively, by volume.

[0173] The microgel slurry and interstitial matrix can be admixed at any temperature suitable to maintain any cells that will be printed into the composite matrix in a dormant state and / or to avoid cross-linking of any cross-linkable interstitial matrix materials. The microgel slurry and interstitial matrix can be admixed at a temperature in a range of about 0°C to about 8°C, about 0°C to about 6°C, about 2°C to about 6°C, or about 4°C.

[0174] The methods of the disclosure can further comprise preparing a microgel slurry. Preparing the microgel slurry can include cross-linking a polysaccharide in aqueous solution and shearing the cross-linked polysaccharide to create a microgel mixture. The microgel mixture can be strained to remove non-sheared particles. Any supernatant liquid can be aspirated. The microgel mixture can be suspended in a basal medium to form the microgel slurry. The basal medium can include an antibacterial compound, an antifungal compound, or a combination thereof.

[0175] The disclosure further provides methods for preparing an organoid. The methods can include providing a composite matrix material of the disclosure at a first temperature in a range of about 0°C to about 8°C, depositing a cell slurry bioink in the composite matrix at the first temperature to form a cell-seeded composite matrix, and warming the cell-seeded composite matrix to a second temperature in a range of about 35°C to about 40°C. The methods can include providing a composite matrix material of the disclosure at a first temperature in a range of about 0°C to about 8°C, depositing a cell slurry bioink in the composite matrix at the first temperature to form a cell-seeded composite matrix comprisinga first array element, and warming the cell-seeded composite matrix to a second temperature in a range of about 34°C to about 40°C

[0176] The cell slurry bioink can be deposited into the composite matrix material at a first temperature from about 0°C to about 8°C, for example, greater than 0°C and less than or equal to 8°C, or about 2°C to about 6°C, about 3°C to about 5°C, or about 4°C. The cell- seeded composite matrix can be warmed to a second temperature from about 34°C to about 40°C, about 36°C to about 38°C, or about 37°C.

[0177] The cell slurry bioink is deposited by an extruder, such as a syringe pump or a printhead, as shown in FIGS. 4 and 9. The printhead can be an inkjet printhead, a pneumatic printhead, or a piezoelectric printhead. The cell slurry bioink may be deposited a long any of a horizontal-x axis, vertical-y axis, angular-z axis, or any combination thereof to form the cellular array. Cell slurry extrusion in the bioprinting head can be accomplished in numerous ways, each of which include: (1) a reservoir containing the cell slurry to be printed, (2) a printing nozzle fluidically connected to the reservoir (the size and shape of the nozzle will depend on the size and shape of the structures to be printed, but most often will take the form of a thin and long tube), and (3) a mechanism for applying pressure to the reservoir to force the slurry to extrude out of the nozzle at a very well controlled rate. The mechanism for applying pressure to the slurry in the reservoir can take many forms, and the most common is to have a plunger that moves by the action of a motor, such that the reservoir functions like, or is, a syringe. Another mechanism would be to connect the reservoir to a controlled source of air pressure, although this solution has several disadvantages, such as excessive compliance, which makes it very difficult to make fast and accurate changes to the extrusion rate. In another mechanism, pressure is applied to the slurry reservoir by a flexible diaphragm (which forms part of one of the walls of the reservoir) that is deflected into the reservoir by a piezoelectric actuator. The methods of the disclosure can include the use of a piezoelectric actuator. The actuator pushes on the diaphragm to extrude the slurry through the nozzle. One advantage of this configuration is that piezoelectric actuators can be extremely precise, moving by increments as small as nanometers, which in turn results in extremely precise control of the extrusion volumes. The piezoelectric actuator can more as little as tens of picometers, resulting in high print resolution and precision. The piezoelectric actuator used in the Examples herein had a resolution of about 0.23 nm, which translates to an extrusion volume resolution of about 10 pL. The volume resolution can be made even smaller by changing the size of the diaphragm that the piezo actuator pushes against to extrude. Additionally, the piezoelectric actuator can move at a very wide range of speeds, which allows for a wide range of extrusion rates, and the rate can be very precisely controlled. Suitable piezoelectric tools include those described by Anis et al., "Diaphragm pico-liter pump for single-cell manipulation.” Biomedical Microdevices 13, no. 4 (2011) pp651-59. Anis et al. describes the tool for use as a suction device, while the present disclosure utilizes the piezoelectric printhead as a printing device.

[0178] The methods of the disclosure can further comprise depositing additional cell slurry bioink in the composite matrix at the first temperature to form a plurality of array elements. The array elements can have any desired shape or configuration. The array elements can be spheroids, tubes, or a combination thereof. The array elements can be spheroids. The array elements can be tubes. Deposition of the cell slurry bioink can be computer controlled to allow precise control of the amount and location of array elements in the composite matrix. The computer can be programmed to print the array element in a predetermined size and / or shape along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof to provide an organoid having a predetermined size and morphology. The computer can be programmed to print an organoid having a predetermined shape along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof. The method can further comprise loading the extruder with the cell slurry bioink.

[0179] The methods can further comprise preparing the composite matrix material of the disclosure. The methods can further comprise preparing the cell slurry bioink.

[0180] The array elements each develop into an organoid during a period of time from about 2 minutes to about 14 days, about 1 day to about 10 days, about 3 days to about 7 days, for example, 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, or 14 days. A cell-culture medium can be added to the cell-seeded composite matrix at the second temperature to help facilitate organoid growth. The cell-culture medium can include Epidermal Growth Factor, Noggin, R Spondin (ENR), or a combination thereof. The cell- seeded composite matrix can be allowed to stand for a period of time sufficient to allow the organoid to develop. The cell-seeded composite matrix can be allowed to stand at the second temperature for a period of time in a range from about 2 minutes to about 14 days, about 1 day to about 10 days, about 3 days to about 7 days, for example, 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, or 14 days, to allow the organoid to form. When cellculture media is included, the cell-culture media can be changed every 2 to 4 days, every 2 to 3 days, every 3 to 4 days, every 2 days, every 3 days, or every 4 days.EXAMPLES

[0181] All rheological measurements were performed using a 25mm cone-and-plate geometry on an Anton-Parr rheometer. For shear oscillatory measurements (A-D), both frequency and amplitude sweeps were performed, with constant 1% strain and 1 Hz frequency, respectively. Temperature sweeps were also performed at 1% strain and 1 Hzfrequency, with 60 s for temperature equilibration before each measurement. For reversible shear experiments (D), 1% or 100% strain was applied at 1 Hz for 1 min each. To calculate the specific yield-stress of each composition (E,F), unidirectional shear measurements were performed at 1% strain and the data were fit to a Herschel-Bulkley Power Law model. The y- intercept was used to determine the yield-stress.EXAMPLE 1 : PREPARATION OF COMPOSITE MATRIX

[0182] Alginate microgels of 0.5 wt.% were prepared as follows. 1 gram sodium alginate (Sigma, 9005-38-3) was dissolved in 100 mL sterilized, distilled, deionized water (“SDD water”), that had been pre-heated to 60 °C, and stirred until the solution was homogeneous, approximately 2-4 h. In a separate flask, 200 mg of calcium carbonate was mixed into 100 mL SDD water. The dispersed calcium carbonate suspension was added to the alginate solution and cooled to room temperature while stirring for 1 h, yielding a mixture of 0.5 wt% alginate and 0.1 wt% CaCOs. 400 pL Acetic acid (Sigma, 64-19-7) was added drop-wise, with stirring at 1000 RPM. The solution increased in viscosity, which indicated the release of Ca2+ions and alginate cross-linking. The solution was stirred overnight (-12-16 hours) at 1000 RPM to shear the mixture, which led to microgel formation. The resulting mixture was blended for 60 s on the High setting using a Hamilton Beach commercial blender (BioSpec 908) (or equivalent). The microgel mixture was then strained through a 100 pm filter to remove large particles. The pH of the filtered microgels was increased to about neutral and the microgels were buffered as follows. The filtered microgels were centrifuged at 18,500xg for 20 min at 4 °C and the supernatant aspirated, followed by resuspension in 1x volume of Dulbecco’s Modified Eagle Medium (DMEM):F-12 (UCSF Cell Culture Facility) medium supplemented with penn / strep, Primocin (Invivogen), and 2 mM NaOH, and stored at 4 °C overnight (-12-16 hours) or until the day before use. The day before use, the mixture was again centrifuged under the same conditions and the media replaced with 2x volume DMEM:F-12 medium supplemented with penn / strep, Primocin, 4% v / v 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (1 M, UCSF Cell Culture Facility), and 4% v / v NaHCOs (37 g / L in water, pH = 9.5 with NaOH). This mixture was left overnight (-12-16 hours), centrifuged the next day, and the supernatant aspirated. The 2X volume in the last step allows the microgels to swell to maximum swelling capacity and soften. The resulting, swollen microgels had a mean particle size of about 20 pm. The resulting packed microgels were considered an undiluted microgel slurry and were stored at 4 °C until use.

[0183] Alginate microgels having an alginate concentration of 0.25 wt% or 1 .0 wt.% were prepared in the same way except 0.5 grams or 2 grams, respectively, of sodium alginate was dissolved in the initial 100 mL SDD water. In addition, 100 mg or 400 mg, respectively, of calcium carbonate was dissolved in the second 100 mL of SSD water.

[0184] Just before seeding or printing, the undiluted slurry was mixed in various ratios with Growth Factor-Reduced Matrigel®, Corning 354230 at 4 °C. This mixture yielded the desired dilution of microgels in interstitial matrix, i.e., the composite matrix, for seeding or printing. The composite matrix was kept cold (greater than 0°C - 8°C) throughout seeding or printing, and then moved to a CO2 incubator at 37 °C for 5-10 min after seeding or printing to allow for interstitial matrix cross-linking.EXAMPLE 2: VISCOELASTIC PROPERTIES OF COMPOSITE MATRIX

[0185] Composite matrices of alginate microgels diluted with Matrigel® were prepared as described in Example 1 , at alginate concentration of 0.5 wt.% and microgel dilutions of 10%, 30%, 50%, and 70% as follows. A total volume of alginate microgels and Matrigel® was predetermined. Alginate microgels as prepared in Example 1 were micropipetted into a receiving container at 90% of the total predetermined volume. Matrigel®, at an amount of 10% of the total predetermined volume, was micropipetted into the receiving container including the alginate microgels to provide a 10% microgel dilution of composite matrix. A similar procedure was carried out for each of the remaining dilutions. The relative volumes of the alginate microgels and Matrigel® used to prepare the dilutions are shown in the table below. The % microgel dilution refers to the level of dilution of the alginate microgels by the Matrigel®, as a percentage of the total volume:

[0186] The shear modulus over various temperatures and times, shear modulus vs shear stress, and shear stress vs. shear rates, and modulus and yield-stress were determined and plotted as shown in FIGS. 2 and 3.

[0187] As shown in FIG. 2, the composite matrix of the disclosure behaves as a reversible yield-stress matrix at 4°C and cross-links at 37°C. The bulk viscoelastic properties are identical to Matrigel®. The specific yield-stress of the composite matrices of the disclosure is tunable, which may be advantageous for controlling anisotropic tissue growth or designing mesenchymal-epithelial interfaces. FIG. 2A shows a plot of shear modulus (Pa) vs. temperature for composite matrices of the disclosure including 0.5 wt.%alginate microgels and comparative samples of alginate microgels alone and Matrigel® alone. As shown in FIG. 2A, the composite matrices all have similar storage and loss modulus. At temperatures below about 22°C, all materials except Matrigel® have are characterized by G’ > G”, indicating the composite materials behave as viscoelastic solids at all temperatures while Matrigel® behaves as a viscoelastic liquid at temperatures below about 22°C. At temperatures above about 22°C, Matrigel® contains significant cross-linking which effects the rheological behavior such that it behaves as a viscoelastic solid. FIG. 3A shows a plot of shear modulus (Pa) vs. temperature for composite matrices including 1 .0 wt.% alginate microgels and comparative samples of alginate microgels alone and Matrigel® alone. FIG. 2B and FIG. 3C show G’ is greater than G” for all samples indicating the composite matrices have a higher storage modulus than loss modulus and are viscoelastic solids until a certain shear stress is applied, at which they yield, indicated by the drop in G’. FIG. 2C shows a plot of modulus at 37°C (Pa) vs. Matrigel® dilution for composite matrices of the disclosure (30% microgel dilution; 50% microgel dilution) and comparative samples of alginate microgel alone and Matrigel® alone; the composite matrices of the disclosure have similar moduli at 37°C as Matrigel® alone. FIG. 2D shows the reversible yield-stress behavior for the composite matrix material of the disclosure. Applied strains of 1% and 100% were alternated every 60 s at 4°C; after applied strain of 100%, the G' value decreased below G" and when applied strain was decreased to 1% G' recovered to near its original value. FIGS. 2E and 2F show that as the amount of interstitial matrix increases, the yield-stress of the matrix decreases. After cross-linking of Matrigel®, cells experience similar bulk viscoelasticity. It was advantageously found that the bulk viscoelasticity of the composite matrix can be designed to be the same as for bulk Matrigel®, for example, by controlling the amount of alginate in the microgel.

[0188] MAGIC matrices were characterized by shear rheology under conditions relevant for bioprinting (4 °C) and organoid culture (37 °C). Matrix compositions having mechanical properties spanning those likely to support both bioprinting and morphogenesis were explored. In addition to pure formulations of alginate and Matrigel®, six different matrix compositions in total: three different ratios of Matrigel and alginate microgel (AMG) slurry spanning 2:1 , 1 :1 , and 1 :2 by volume; and two different polymer weight fractions in the AMG preparation, 0.5 and 1 wt% were tested. As expected, pure AMG slurries exhibited temperature-independent, but shear stress-dependent viscoelasticity, including yielding at ~10 Pa shear stress for 0.5 wt% AMGs (FIGS. 3A-3F). In contrast, pure Matrigel® exhibits significant temperature-dependence as it cross-links to form a hydrogel at physiological temperature (FIGS. 3A-3F).

[0189] All compositions of MAGIC matrix demonstrated reversible yield-stress behavior at 4 °C, confirming their utility as embedded bioprinting materials (FIGS. 3A-3F, 4A). At rest, they behaved as viscoelastic solids, with G’ greater than G”, in contrast to pure Matrigel® (FIGS. 3A-3F, 4A). However, as shear stress was increased, G” overtook G’, indicating microgel yielding and rearrangement (FIG. 4A). These materials remained as reversible yield-stress fluids for as long as they were kept cold, suggesting their ability to support long print times. The specific yield-stress of each composition was measured by unidirectional shear tests and fit well to a Herschel-Bulkley exponential model (FIGS. 3A-3F). Despite their molecular similarity, varying MAGIC matrix composition tuned yield-stress values over an order of magnitude (FIGS. 3A-3F, 4B). This implies that the material can be engineered to fit the needs of a particular print or tissue type.

[0190] To assess MAGIC matrix behavior under physiological conditions, viscoelastic properties following cross-linking at 37 °C were measured. At 0.5 wt% alginate, the storage and loss modulus of the composite matrix approached equivalent values to pure Matrigel® (FIG. 4C). This was true across a range of MAGIC matrix compositions using the lower alginate weight fraction (FIGS. 3A-3F), implying that for these formulations, composite mechanics are dominated by cross-linked Matrigel®. However, this was not the case with MAGIC matrices composed of 1 wt% AMGs, where viscoelastic properties were dominated by the microgel slurry (FIGS. 3A-3F, 4C). Given their similarity to pure Matrigel®, the 0.5 wt% MAGIC matrix formulations were notably softer than many traditional bioprinting and biomaterial scaffolds, suggesting their utility for supporting tissue growth and morphogenesis.

[0191] Matrigel® and other basement membrane extracts also exhibit complex mechanical behaviors such as stress relaxation and viscoplasticity over the time scales of tissue growth. Thus, it was hypothesized that these time-dependent behaviors might play a role in bioprinted organoid morphogenesis. The creep response of MAGIC matrices at 37 °C using 10 Pa applied shear stress over 10 minutes was measured to simulate the forces and timescales of processes such as lumen expansion and crypt budding. Indeed, the creep response was a strong function of material composition, with Matrigel® exhibiting the greatest strain rate and highest plasticity (FIGS. 5A-5F). MAGIC matrices comprising soft (0.5 wt%) AMGs, regardless of composition ratio, exhibited similar strain rates that were less than pure Matrigel®, but greater than MAGIC matrices comprising stiff (1 wt%) microgels (FIGS. 5A-5F). The corresponding relaxation modulus was nearly identical to Matrigel® for MAGIC matrices using soft AMGs (FIG. 4D). In contrast, stress relaxation was substantially impeded compared to Matrigel® for MAGIC matrices using stiff AMGs (FIG. 4D).

[0192] To arrive at an ideal MAGIC matrix formulation for both bioprinting and morphogenesis, we applied these ECM compositions to organoid growth assays. Organoids isolated from mouse duodenum were passaged by mechanical dissociation and plated in Matrigel® or MAGIC matrix domes. Given the sensitivity of crypt budding and lumen expansion to matrix mechanics, we measured crypt width and length as representative signatures of a permissive ECM and healthy morphogenesis. After five days in culture, we found that MAGIC matrix compositions employing 0.5 wt% alginate gels at low enough packing density yielded organoids that were indistinguishable from those grown in pure Matrigel® (FIG. 4E). However, all compositions of MAGIC matrix using 1 wt% AMGs yielded organoids with shorter and wider crypts (FIG. 4F). We also observed a negative impact on crypt length for MAGIC matrices composed of 0.5 wt% soft microgels for a high microgelfraction composition, namely 1 :2 Matrigel®:AMG by added volume (FIG. 4G). Given that these materials have similar viscoelasticity at 37 °C (FIG. 4C), and that organoids exhibit standard morphology in diluted Matrigel® (FIGS. 6A-6C), it is possible that microgel packing may also play a role in organoid phenotype. In sum, the formulations that were most mechanically similar to pure Matrigel® showed no significant impact on crypt morphogenesis (FIGS. 6A-6C). These results support the need for soft, viscoelastic materials for embedded bioprinting to promote certain aspects of morphogenesis. In addition, they suggest that for MAGIC matrices of a given storage and loss moduli, other material properties such as timedependent viscoelasticity, plasticity, and microgel packing may independently inform intestinal organoid morphogenesis. For most bioprinting experiments, unless otherwise noted, a standard MAGIC matrix composite with a 1 :1 ratio by added volume of Matrigel® and 0.5 wt% AMGs was used.EXAMPLE 3: ORGANOID PRINTING

[0193] Organoids were bioprinted into composite matrices prepared according to Example 2. FIG. 1 shows organoids that were bioprinted into composite matrices of the disclosure prepared according to Example 2, having microgel dilutions of 30%, 50%, and 70% and 0.5 wt.% and 1 wt.% alginate concentrations in the microgel, and as a comparison, an organoid from manually seeding a Matrigel® matrix. The crypt lengths of the organoids shown in FIG. 1 were determined and plotted in FIG. 3. Organoids were bioprinted into composite matrices prepared according to Example 2, having 1 :1 ratios of alginate microgel and Matrigel® (a 50% microgel dilution). As a control, manual seeding of organoids was also performed into Matrigel®. The results are shown in FIG. 12. As shown in FIG. 12, organoids bioprinted into composite matrices of the disclosure show improved size and morphogenetic homogeneity over traditionally seeded organoids. FIG. 12A shows an array of organoids bioprinted into a composite matrix of the disclosure over 5 days; the bioprinted organoidsdemonstrate size and morphogenetic homogeneity. FIG. 12B shows an array of organoids manually seeded into Matrigel® according to known methods over 5 days; the organoids do not display size or morphogenetic homogeneity. FIG. 12C shows a plot of organoid area (pm2) for the bioprinted and manual seeded organoids of FIG. 12A and 12B on day 0, day 2, and day 5. FIG. 12D shows a plot of the number of crypts per organoid for the bioprinted and manual seeded organoids of FIG. 11A and 11 B on day 2.

[0194] When organoids are more homogeneous, a smaller sample size is required to arrive at statistically significant phenotypes. This was shown by biasing for secretory cell types in FIG. 13.

[0195] Thus, Example 3 demonstrates an improvement in preparing uniform, morphogenetically homogenous organoids using the methods and materials of the disclosure relative to current, known, methods and materials.

[0196] EXAMPLE 4: BIOPRINTING WORKFLOW

[0197] Cells or organoids were cultured according to standard protocols specific to that cell type. For mouse proximal small intestinal organoids (gastrointestinal organoids), tissues were cultured according to published protocols (Mahe et al., “Establishment of Gastrointestinal Epithelial Organoids,” Current Protocols in Mouse Biology 3:217 -240, (2013). Briefly, gastrointestinal organoids were cultured in 3D Matrigel® domes on 24-well plates with 1 mL of Epidermal Growth Factor (EGF), Noggin (N), R. Spondin (R) medium (ENR) for 4-7 days before passaging, with media changes every 2-4 days, depending on desired growth rate. For passaging, gastrointestinal organoids were collected by mechanical dissociation of the Matrigel® domes using 1 mL cold basal medium (BM) and centrifuging. All centrifugation steps were performed at 160 xg and 4 °C for 4 min unless stated otherwise. The supernatant was aspirated, and the pellet was fragmented by pipetting up and down in 1 mL BM using a non-filtered 10-100 pL pipette tip attached to the end of a 1000 pL pipette tip to gently increase shear. The crypt fragments were collected by again centrifuging and the supernatant was aspirated. These fragments were resuspended in Matrigel®, generally at a 1 :4 or 1 :6 split ratio (e.g., 100 or 150 pL of Matrigel® for one 25 pL dome) and plated onto the bottom of 24-well plates. The plate was then inverted to prevent gastrointestinal organoid settling, and the Matrigel® allowed to polymerize in a CO2 incubator at 37 °C for 5-10 min before adding 1 mL of warm ENR to each well.Gastrointestinal organoids that were to be used for bioprinting were instead cultured in 1 mL EGF-N-R-CHIR99021 -Valproic acid (ENRCV) (with added 3 pM CHIR99021 and 1 mM valproic acid) to promote stem cell expansion.

[0198] Gastrointestinal organoids were collected for bioprinting 3 days after passaging, when the culture consists mostly of large clear cysts without significant internal dead cell debris. For most bioprinting experiments, either 10 or 20 Matrigel® domes were collected. As during passaging, gastrointestinal organoids were collected by mechanical dissociation of the Matrigel® domes. After centrifugation, the pellet was gently resuspended in dissociation medium consisting of TrypLE Express (Gibco) supplemented with 2000 U*mL'1DNase I (STEMCELL Technologies), 1 mM N-acetylcysteine (Sigma), and 10 pM Y-27632 (R&D Systems). 1 mL of this mixture was used for every ~5 wells used for printing, so generally 2 or 4 mL. The resuspended cells were incubated for 10 min in a water bath at 37 °C, with gentle shaking to agitate the pellet every 5 min. This mixture was neutralized using 8 mL or 16 mL BM supplemented with 10% FBS and gently pipetting up and down. Using a serological pipette, the cells were extruded dropwise through a 40 pm strainer to ensure a roughly single-cell suspension and filter debris. This mixture was centrifuged and the supernatant aspirated. The pellet was resuspended in 1 mL ENR supplemented with 2.5 pM thiazovivin (Stemgent) and 2 mM EDTA (Gibco) and centrifuged again. This pellet was resuspended in -30 pL of ENR with thiazovivin and EDTA and transferred to the 384-well collection plate on the bioprinter.

[0199] A composite matrix material was prepared according to Example 2, having 1 :1 pipetted volume ratios of alginate microgel and Matrigel® (a 50% microgel dilution) at 4 °C. 100 pL of the composite matrix was then deposited into each well of a chilled 96-well plate (printbed plate) that was expected to receive cell slurry using a positive-displacement pipette to minimize bubbles. The printbed plate was kept cold by blowing dry, chilled compressed air on its bottom side. The printhead was then equipped with a 75 or 125 pm ID plastic denudation micropipette (CooperSurgical, EZ-Tip) to use as the print nozzle. To prevent clogs, a blocking solution comprised of 10 mg*mL-1BSA, 5 mM EDTA, and 10 mg*mL-1Tetronic 90R4 in DPBS was aspirated into the tip and allowed to sit for 10 minutes before purging and printing. The 384-well collection plate was centrifuged at 100xg for 1 min to pellet the cell slurry bioink at the bottom of the well. This slurry was aspirated directly into the printhead nozzle to reduce dead volume and minimize required cell slurry volume for printing. The motorized stage and print dimensions were calibrated manually, and print parameters set using a custom MATLAB script controlling both the microscope stage and the printhead. This allowed for printing of spheroids or tubes in custom arrays with defined inter-organoid spacing, number of organoids in an array, depth of printed organoid in the matrix, and extruded volume of cell slurry at 4 °C into composite matrices. Most frequently, 3x3 or 4x4 arrays of -200 pm-wide organoid spheroids were deposited with 750 pm interorganoid spacing.

[0200] After printing, the printbed plate was carefully moved to a 37 °C CO2 incubator and allowed to sit for 5-10 minutes to allow for basement membrane cross-linking. 200 pL of warm ENR media with 2.5 pM thiazovivin was added to each well. Media was changed every 2-4 days for spheroids, every 2 days for tubes.

[0201] When cultured at 37 °C, these organoids rapidly self-organized into the expected 3D architectures. Live imaging and immunofluorescence revealed that these organoids were morphologically indistinguishable from those grown in pure Matrigel®, but exhibit improved homogeneity in organoid size, shape, maturation time, and crypt budding efficiency, as shown in FIGS. 14B-14D.

[0202] Cell slurry bioink including mouse salivary gland cells was prepared and printed similarly. The bioprinting of mouse salivary gland organoids similarly revealed expected morphogenetic programs and improved homogeneity, relative to traditional seeding, highlighting the broad utility of the claimed ECM and printhead. As shown in FIG. 14, bioprinted salivary gland organoids showed characteristic structure, differentiation, and selforganization (FIG. 14).

[0203] Organoid morphogenesis can be highly stereotyped when cells are embedded in physiologically relevant matrices at tissue-like cell densities. Therefore, efforts were focused on bioinks comprising single-cell suspensions of dissociated organoids centrifuged to produce saturated slurries at approximately 108cells / mL. Rheological modifiers were omitted and generally excluded divalent cations to temporarily prevent cell adhesion through calcium-dependent adhesion molecules. In addition, we employed microgels that were approximately cell-sized to facilitate self-organization, migration, and diffusion (FIG. 8). However, reproducible and controlled printing of cell slurries at these densities required a printhead that could handle small volumes with high precision, fast pressure ramps, and without excess loss due to tubing and fluidics (i.e. “dead volume”). A printhead was for direct aspiration and extrusion that employs a piezoelectric actuator that is mechanically coupled to a fluid-filled cavity via a polyether ether ketone (PEEK) diaphragm (FIG. 1 B). This allowed voltage applied to the piezoelectric material to control its expansion or contraction, and thus both aspirate and dispense precise volumes. The chosen piezoelectric actuator, PEEK diaphragm diameter and diaphragm thickness result in a volume displacement resolution of approximately 10 pL, a maximum aspiration / extrusion volume of approximately 660 nL, and a maximum theoretical aspiration / extrusion rate of approximately 300 pL / s (the actual rate will be highly dependent on the bioink rheological properties).

[0204] The piezoelectric printhead was mounted on a cantilevered beam, with motorized Z-control but a fixed XY-position. The printhead can be aligned to the optical axis of thewidefield microscope with manual centering screws. Micron-scale resolution of xy-position and shape was achieved using the microscope stage, which held the print plate. Microscope integration provided real-time imaging during the printing process, which allowed rapid identification and diagnosis of printing issues should they occur. Bioinks were loaded by directly aspirating a user-specified volume from a 384-well sample plate, so that only the printed volume was loaded - this is especially beneficial for precious samples such as biopsies or cell populations with insufficient bioink volume for loading typical commercial bioprinting syringes. A solenoid valve toggled fluidic connection of the printhead cavity to a fluid reservoir, allowing the printhead to be filled, cleaned, or purged, and then sealed for single-ended printing operation. The entire process, including microscope, printer motion, and fluidics, were controlled using MATLAB. Custom printing protocols were scripted for maximum repeatability, efficiency, and iterative troubleshooting.

[0205] High-viscosity ink droplets are challenging to print in yield-stress fluids due to inertial, cohesive, and shear forces between the ink and matrix. For example, cohesive interactions between cells in the slurry causes the ink to be pulled as a continuous tail from the tip as it is removed from the bath, generating a structure reminiscent of capillary bridging. To combat this, we leveraged a rapid voltage switch on the piezo printhead to “break” this tail by applying a small negative pressure and xz-displacement, generally attenuating this effect (FIGS. 10A-10B). We then applied this aspiration, extrusion, and tail-breaking program to script an automated spheroid bioprinting array. Using commercially available plastic micropipettes with 1 mm OD and 125 pm ID mounted onto the printhead, we first demonstrated that a Caco-2 cell slurry bioink could be delivered to create spheroids (FIG. 10C) of customizable dimensions. Spheroid area was an approximately linear function of extrusion step, which was controlled through changes in voltage applied to the piezoelectric actuator (FIG. 10D).

[0206] In general, around 90% of cells were viable after dissociation and slurry preparation as measured by trypan blue exclusion. In addition, Caco-2 spheroids underwent lumenization, as is expected for these tissues in 3D culture, implying the dissociation and printing process did not substantially impact morphogenesis. Notably, the high viscosity of cell slurry bioinks negatively impacted print circularity for larger extrusion steps; however, the active rheological behavior of these living inks can overcome imperfections in initial geometry, highlighting the benefits of self-organizing systems as living inks (FIG. 10E). Cell slurry could also be printed using 75 or 200 pm ID micropipettes, highlighting the flexibly of the platform for various print resolutions. Thus, the piezoelectric printhead affords high volume precision, working with small volumes of precious cellular inks, and flexible control over bioink behavior by rapidly changing applied voltage. This is the first demonstration ofapplying a piezoelectric material to control direct aspiration and extrusion of dense cell slurries.

[0207] Having established conditions for the automated preparation of 3D tissues using Caco-2 cells, mouse small intestine organoid arrays were bioprinted. Primary cells were harvested, cultured, passaged, and dissociated into a cell slurry as previously described. A significant contributor to organoid heterogeneity in traditional ECM dome cultures may be the disparity in extrinsic conditions such as media access or inter-organoid spacing. To assess the impact of initial spatial conditions on organoid growth, either inter-organoid spacing or z- depth were systematically varied within the MAGIC matrix support bath. Organoids were printed with an average diameter of 200 pm. Organoid seed pairs printed with 250 pm interbolus spacing fused over time, leading to one large organoid (FIG. 10F). This fusion is known to occur in manually seeded ECM dome culture. Organoids spaced 500 or 1000 pm apart remained distinct and appeared to undergo normal morphogenesis through at least 7 days of culture (FIG. 10F). To test denser organoid seeding densities, 3 x 3 arrays with 500 pm spacing were printed. Notably, after 11 days in culture, crypts only appeared at the periphery of the organoid array, while the central organoid remained compacted and showed signs of cell death (FIG. 10G). We hypothesize that this could be caused either by nutrient depletion by outermost organoids or autocrine gradients. Printing depth also had a profound effect on organoid health, with organoids printed closer to the ECM-media interface growing the largest and forming the most crypts (FIG. 10H). Organoids printed too far from the ECM- media interface did not significantly grow over the same time (FIG. 101). Together, these results highlight the importance of initial conditions on organoid morphogenesis and imply that these effects could impact experimental results when working with organoids in manual 3D culture. Ultimately, the automation and standardization afforded using the piezoelectric printhead and MAGIC matrices allows rapid exploration of a wide parameter space and initial culture conditions tailored for a desired assay or application.

[0208] Thus, Example 4 demonstrates successful bioprinting of organoid arrays in a composite matrix of the disclosure.

[0209] EXAMPLE 5: COMPOSITE MATRIX FOR 3D PERFUSABLE ORGANOID TUBES

[0210] In addition to enabling more rapid and homogeneous formation of organoids in a standard array format, MAGIC matrices and the piezoelectric printhead present opportunities to create more structurally complex and functional 3D tissues from organoid slurries. One architecture common to many tissue types is a tubular geometry. Many tissues spontaneously lumenize to form tubes from aninitially cylindrical morphology when placed in laminin-rich ECM. We therefore optimized process parameters for bioprinting organoid cylinders, with the hypothesis that MAGIC matrices would support their spontaneous lumenization, and therefore, their perfusion. Using Caco-2 cell slurries, cylinder diameter could be tuned using either or both extrusion speed and stage speed, providing multiple engineering controls (FIG. 11 A). Tube diameter was an approximately linear function of extrusion speed for the two stage speeds tested. Bioprinted Caco-2 cylinders underwent similar self-organization and compaction followed by lumenization as observed in spheroid arrays (FIG. 11 A, insets).

[0211] Organoids and cell-dense tissues in vitro are generally limited to about 1 mm3in volume without vasculature. Thus, generating de novo vasculature is a focus within the bioprinting community. We validated MAGIC matrix bioprinting for generating vascular cords by preparing cell slurry bioinks from human umbilical vein endothelial cells (HUVECs) and printing them in tubular geometries. Similar to our previous work preparing vasculature from cell-dense HUVEC patterns, after 7 days in culture, these vascular cords exhibited signs of tube formation and microvessel sprouting when printed into MAGIC matrix compositions that additionally included 1 mg / mL Collagen I (FIG. 11 B). However, in contrast to this previous work, MAGIC matrix bioprinted vessels could be tuned in length and width over a greater range. These results highlight the utility of printing into MAGIC matrices having flexible composition including additives like collagen or viscosity modifiers including, but not limited to methylcellulose, xanthan gum, or linear polymers like PEG or alginate.

[0212] MAGIC matrix bioprinting was applied to form perfusable tubular geometries with more complex local architectures, such as intestinal tubes prepared from mouse duodenal organoid bioinks. These tubes lumenized and began forming crypts radially around the long axis of the intestinal tube 2-3 days after printing (FIG. 11C). The emergence of patent lumens in printed tissues provides an opportunity to interface these complex culture models with perfusion systems. Using glass capillaries piercing the tissue lumen, fluid was pushed through the tubes, clearing internal cellular debris (FIG. 11 D). Radial expansion of the perfused tubes when pressurized was observed, indicating that the boundaries of the tissue remained flexible, unlike cultures in materials with non-physiological stiffness such as PDMS (FIG. 11E). By applying oscillatory fluid flow through the tube, cyclic expansion andcontraction of the tubes as might occur during peristalsis could be simulated. Thus, MAGIC matrix tube bioprinting enables access to the apical lumen, a key feature of microphysiological systems, while maintaining a free basal surface that ultimately can be interfaced with other tissue types.

[0213] EXAMPLE 6: STUDY OF VARIOUS DEVELOPMENTAL LINEAGES CULTURED IN COMPOSITE MATRIX

[0214] Having established initial conditions supporting controlled organoid growth, we next assessed whether organoid arrays from various developmental lineages cultured in MAGIC matrix exhibited characteristic features of their self-organization. We assessed the morphology of organoids using confocal microscopy, taking advantage of the optical transparency of the matrix and the consistent z-position of all organoids in a printed array. For mouse duodenal organoids, live imaging of a membrane-localized tdTomato reporter revealed crypt budding and protrusion within 2-3 days after printing in 96-well microplates (FIG. 12). In these folded outgrowths of the epithelium, Lgr5+ stem cells were effectively positioned toward the base of the folded structures (FIG. 13A). Fixed intestinal organoids stained positive for mature epithelial cell types, including Paneth cells (lysozyme) and enteroendocrine cells (chromogranin-A) (FIGS. 12, 13A). Cohesive and contiguous cell borders were marked with E-cadherin. Bioprinted intestinal organoid tubes showed similar selforganization and were positive for Paneth cells (FIG. 13B). 3D reconstruction of organoids in MAGIC matrix revealed that crypts radiated in all directions from the organoid, suggestive of 3D morphogenesis unencumbered by neighboring interfaces (FIG. 13A, FIG. 12).

[0215] To establish the generality of MAGIC matrix for bioprinting and morphogenesis of tissues from different developmental lineages, we derived organoids from mouse submandibular salivary gland, an ectoderm-derived tissue. These organoids were cultured in Matrigel® domes and then harvested as a singlecell slurry in an identical fashion to the small intestine organoids (FIG. 12). Bioprinted salivary gland organoid arrays self-organized and exhibited characteristic multilobular structures within 3 days of printing. These organoids expressed ductal and basal epithelial markers, keratins 8 and 14, respectively.

[0216] Self-organization occurs in a variety of contexts with different mechanisms, including sorting based on differences in cell-cell and cell-ECM interfacial interactions. Given that organoid bioinks are dissociated and mixed before bioprinting, it was decided to test whether cells retain the ability to sort after printing into the MAGIC matrix. To test this, spheroid arrays of patient-derived human mammary epithelial cells (HMECs) given their robust cell sorting into bilamellar structures comprised of a core of luminal cells (LEP) and an outer layer of myoepithelial cells (MEP) were printed (FIG. 13C). It was confirmed that the printed tissues robustly sort to form bilamellar structures within one day, and the extent of MEP coverage was dependent on the proportion of the two cell types (FIG. 13D). Additionally, it was also confirmed that HMEC printed as tubes also sort robustly while maintaining the initial tissue geometry. In contrast, MEP-only spheroids showed no evidence of sorting (FIG. 13D). Thus, MAGIC matrix bioprinting is compatible with a variety of self-organization mechanisms, including sorting, lumenization, and tissue folding.

[0217] The most common approach for preparing many organoid types is to aggregate dissociated tissue or stem cells using low attachment wells. However, these methods frequently lead to uncontrolled spheroid formation and a field of unincorporated or dead cells at the periphery of the organoids. Furthermore, while these organoid seeds appear to coarsen over time, it is unclear how these dynamics impact tissue organization (FIG. 13E). It is postulated that MAGIC matrix bioprinting could attenuate this effect by providing a mechanical support during tissue formation.

[0218] To test this idea, the feasibility of bioprinting human induced pluripotent stem cell (hiPSC)-derived forebrain organoids was explored. Specifically, brain organoids at 7 weeks of in vitro differentiation were dissociated into a cell slurry bioink and printed into both MAGIC matrix or a pure AMG slurry and assessed for rosette formation and neural identity. Cortical brain organoids printed into MAGIC matrix exhibited sprouting behavior that is known to occur in basement membrane gels, as well as neuroepithelial bud formation, but lacked neuroectoderm.

[0219] In contrast, cortical brain organoids printed in pure AMG slurry formed dense spheroids without surrounding cell debris that exhibited radially organized neuroectoderm (FIG. 13E). These organoids were positive for the dorsal forebrain marker FOXG1 and negative for the ventral forebrain marker DLX2, with about 40%dorsal identity, comparable to hiPSC-derived cortical organoids aggregated with low attachment wells and not bioprinted (FIG. 13F). Furthermore, the bioprinted cortical organoids demonstrated characteristic self-organization with neural progenitors surrounding ventricular zone-like neuroepithelium regions (PAX6+, -30%), and intermediate progenitor cells (EOMES+, -4%) and deep layer excitatory neurons (CTIP2+, -15%) extending radially out from this apical surface (FIG. 13F). Bioprinted organoids showed similar cell proportions to manually seeded organoids, except for a decreased PAX6+ population, likely due to increased organoid age in bioprinted organoids (63 d) compared to manually seeded (39 d). Together, these results validate organoid bioprinting for hiPSC-derived tissues and lay the groundwork for generating more complex organoid interfaces such as neural assembloids.

[0220] EXAMPLE 7: STUDY OF HIGH THROUGHPUT BIOPRINTED ORGANOID ARRAYS

[0221] Similar to aggrewell or microwell methods, bioprinted organoid arrays hold great promise for high-throughput assay development, CRISPR libraries, and drug screens. However, there is the added benefit of flexibility in tissue geometry and initial conditions. To explore the utility of MAGIC matrix for assays requiring genetic perturbations, like CRISPR screens, triple-negative breast cancer (TNBC) patient- derived organoids using lentivirus expressing H2B-GFP were transduced (FIG. 15A). TNBC organoids showed no difference in infection efficiency when transduced in suspension and then plated in Matrigel® or MAGIC matrix domes (FIG. 15B). However, when transducing TNBC organoids through the ECM domes by including lentivirus in the media after plating and gel cross-linking, infection efficiency was significantly greater in MAGIC matrix, with nearly 90% GFP+ organoids. In contrast, there was minimal-to-no infection in Matrigel®. This may be due to improved diffusivity afforded by low wt% (and therefore large pore size) alginate microgels in the composite support. These results implied that TNBC organoids could be transduced after printing in arrays, presenting opportunities to multiplex combinatorial libraries in a single well plate. To this end, surface cells on TNBC organoids could be successfully transduced by including lentivirus in the media after bioprinting (FIG. 15C). Bioprinted TNBC organoid arrays were also highly amenable to transfection using Lipofectamine, showing strong RNA uptake as a function of both transfection time and amount of RNA delivered (FIGS. 15C and 16). Whiletransduction efficiency in bioprinted arrays could be improved, this provides an important proof-of-concept for future assays using patient-derived organoids in this format.

[0222] Achieving regularity for organoids with complex morphogenetic features is a key challenge in developing organoid-based assays, as it effects the response of the organoids to genetic, mechanical and chemical perturbations. For examples, crypt morphogenesis in mouse small intestinal organoids is highly chemo- and mechanosensitive. The timing and regularity of crypt morphogenesis after bioprinting in MAGIC matrix compared to manual culture methods were quantified. Over the course of 5 days, bioprinted organoids synchronously self-organized, initially into lumenized cysts that further underwent budding morphogenesis to form crypts (FIG. 15D). By contrast, manually seeded organoids showed far more heterogeneous sizes and morphologies over the same timeframe (FIG. 15E). Furthermore, for a given time post-seeding, bioprinted organoid arrays underwent more extensive and uniform morphogenesis, exhibiting a greater number of crypts with decreased variance compared to manually seeded organoids (FIG. 15F). Organoids grown in manually seeded cultures using MAGIC matrix as opposed to pure Matrigel® showed no difference in crypt number, indicating that the controlled initial conditions afforded by bioprinting leads to this improved maturity.

[0223] The heterogeneity of manually seeded organoid culture limits their potential in a variety of phenotypic assays. Differences in organoid size, morphological features such as number of crypts, and position across multiple z- planes obscures subtle phenotypes that are only revealed after analyzing dozens to hundreds of individual organoids. The regularity of bioprinted organoid arrays in MAGIC matrix could dramatically improve the sensitivity of such assays while reducing input tissue. To test this idea, intestinal organoids were treated with a gamma-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which is known to increase the number of Atoh1 + secretory progenitors. Using an Atoh1 CreERT2:Rosa26tdTomato reporter line, we treated bioprinted and manually seeded organoid cultures with DAPT for 2 days after seeding and imaged after 3 days (FIG. 15G). In manually seeded organoids, differences in overall tdTomato signal acquired by confocal microscopy were obscured by heterogeneity in organoid position and size (FIG. 15H). In contrast,there was a clear increase in tdTomato signal for DAPT-treated organoids in bioprinted arrays (FIG. 151). Quantifying the volume of tdTomato-positive signal in each condition revealed that while there was a statistically significant increase in signal for both bioprinted and manually seeded organoids in treated vs. untreated conditions, the effect size was substantially improved using bioprinted arrays (~3.7- fold increased difference between treated and untreated means) (FIG. 15J). Signalpositive volumes were also not normally distributed when manually seeded (p < 0.0001 as determined by D’Agostino & Pearson test), whereas they were normally distributed when bioprinted, resulting in more normally distributed variance in bioprinted organoids and an order-of-magnitude decreased coefficient of variance (printed CoV = 48% for treated and 58% for untreated; manual CoV = 127% for treated and 174% for untreated). Computing a post-hoc power analysis (a = 0.05; [3 = 0.2) with the given effect sizes and variances in each condition recommended n = 12 printed organoids compared to n = 100 manually seeded organoids. This order- of-magnitude decrease in required comparisons emphasizes the attractiveness of MAGIC matrix bioprinting for rare tissues or subtle phenotypes.

[0224] To more precisely quantify how bioprinted arrays improved assay sensitivity, bootstrapping was performed on the bioprinted and manually seeded populations to calculate p-value as a function of the number of paired organoid comparisons (treated vs. untreated). In the bioprinted arrays, p-values below 0.05 were achieved after comparing only 5 organoids; in manually seeded organoid cultures, 45 comparisons were required to reach the same statistical significance (FIG. 15K). P-values for bioprinted organoids continued to decrease with additional comparisons approaching p~ 10-10 after 45 comparisons - the same number of organoids necessary to reach a p-value of 0.05 in manually seeded organoids. This 108-fold improvement in assay sensitivity highlights the potential of organoid arrays in chemical, microenvironmental, and genetic screens.Conclusion

[0225] Although the foregoing embodiments have been described in some detail for the purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. Itshould be noted that there are many alternative ways of implementing the processes, systems and apparatus of the present embodiments. Accordingly, the present embodiments are considered as illustrative and not restrictive, and the embodiments are not to be limited to the details given herein.

[0226] Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0227] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

Claims

What is claimed is:1 . A composite matrix material, the material comprising: a plurality of microgel particles having a mean particle size in a range of about 1 pm to about 100 pm; and an interstitial matrix, wherein the composite matrix is characterized by reversible yield-stress behavior at 4°C.

2. The material of claim 1 , wherein the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.

3. The material of claim 1 or claim 2, wherein the composite matrix maintains a viscosity for a time in a range of 2 minutes to 14 days at 37°C, 1 day to 10 days, or 3 days to 7 days.

4. The material of any one of claims 1 to 3, wherein the microgel mean particle size range is from about 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, or about 15 pm to about 25 pm.

5. The material of any one of claims 1 -4, wherein the storage modulus of the composite matrix is no greater than about 200 Pa.

6. The material of any one of claims 1 -5, wherein the storage modulus of the plurality of microgel particles is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, about 5 Pa to about 75 Pa, or about 10 Pa to about 50 Pa.

7. The material of any one of claims 1 -6, wherein the storage modulus of the microgel particles is no greater than about 50 Pa.

8. The material of any one of clams 1-7, wherein the storage modulus of the interstitial matrix is no greater than 200 Pa.

9. The material of any one of claims 1 -8, wherein the storage modulus of the interstitial matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

10. The material of any one of claims 1 -9, wherein the storage modulus of the composite matrix is no greater than about 150 Pa, or no greater than about 100 Pa.11 . The material of any one of claims 1 -10, wherein the storage modulus of the composite matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

12. The material of anyone of the preceding claims, wherein the composite matrix is characterized by a yield-stress of less than or equal to 2 Pa or less than or equal to 1 Pa,for example in a range of about 0.05 Pa to about 2 Pa, about 0.1 Pa to about 1 .75 Pa, about 0.5 Pa to about 1 .5 Pa, about 0.75 Pa to about 1 .25 Pa.

13. The material of any one of the preceding claims, wherein the microgel particles and interstitial matrix are admixed at a ratio in a range of 20%:80% by volume, respectively, to 99%: 1% by volume, respectively.

14. The material of any one of the preceding claims, wherein the microgel comprises alginate, cross-linked alginate, polyethylene glycol, agarose, hyaluronic acid, collagen IV, laminin, fibronectin, peptide-linked hydrogels, and a combination thereof.

15. The material of any one of the preceding claims, further comprising at least one cell.

16. The material of any one of the preceding claims, wherein the material is substantially free of collagen I.

17. The material of any one of the preceding claims, wherein the interstitial matrix comprises a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, basement membrane extract, or a combination thereof.

18. The material of any one of the preceding claims, wherein the interstitial matrix comprises (a) structural proteins selected from the group of laminin, nidogen, collagen IV, and a combination thereof and (b) adhesive peptide sequences.

19. A composite matrix material, the material comprising: a mixture of: a plurality of cross-linked alginate microgel particles having a mean particle size in a range of about 1 pm to about 100 pm; and an interstitial matrix comprising a basement membrane secreted byEngelberth-Holm-Swarm (EHS) mouse sarcoma cells, wherein the composite matrix is characterized by reversible yield-stress behavior at 4°C.

20. The material of claim 19, wherein the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.21 . The material of claim 19 or claim 20, wherein the composite matrix maintains a viscosity for a time in a range of 2 minutes to 14 days, 1 day to 10 days, or 3 days to 7 days, at 37°C.

22. The material of any one of claims 19-21 , wherein the alginate microgel mean particle size range is from about 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, or about 15 pm to about 25 pm.

23. The material of any one of claims 19-22, wherein the storage modulus of the composite matrix is no greater than about 200 Pa.

24. The material of any one of claims 19-23, wherein the storage modulus of the plurality of alginate microgel particles is in a range of about 1 Pa to about 100 Pa, about 1 Pa to about 150 Pa, about 5 Pa to about 100 Pa, about 5 Pa to about 75 Pa, or about 10 Pa to about 50 Pa.

25. The material of any one of claims 19-24, wherein the storage modulus of the alginate microgel particles is no greater than about 50 Pa.

26. The material of any one of clams 19-25, wherein the storage modulus of the interstitial matrix is no greater than 200 Pa.

27. The material of any one of claims 19-26, wherein the storage modulus of the interstitial matrix is in a range of about 1 Pa to about 200 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

28. The material of any one of claims 19-27, wherein the storage modulus of the composite matrix is no greater than bout 150 Pa, or no greater than about 100 Pa.

29. The material of any one of claims 19-28, wherein the storage modulus of the composite matrix is in a range of about 1 Pa to about 100 Pa, about 1 Pa to about 150 Pa, or about 5 Pa to about 100 Pa.

30. The material of any one of claims 19-29, wherein the composite matrix is characterized by a yield-stress of less than or equal to 2 Pa or less than or equal to 1 Pa, for example in a range of about 0.05 Pa to about 2 Pa, about 0.1 Pa to about 1 .75 Pa, about 0.5 Pa to about 1 .5 Pa, about 0.75 Pa to about 1 .25 Pa.31 . The material of any one of claims 19-29, wherein the alginate microgel particles and interstitial matrix are admixed at a ratio in a range of 30%:70% by volume, respectively, to 90%:10% by volume, respectively.

32. A method for preparing a composite matrix material, the method comprising: cross-linking a polysaccharide in an aqueous solution; shearing the cross-linked-polysaccharide to create a microgel mixture; suspending the microgel mixture in a basal medium with a combination of antibacterial and antifungal compounds to provide a microgel slurry; and admixing the microgel slurry with an interstitial matrix.

33. The method of claim 32, wherein the polysaccharide comprises alginate.

34. The method of claim 32 or claim 33, wherein the interstitial matrix comprises a basement membrane secreted by Engelberth-Holm-Swarm (EHS) mouse sarcoma cells.

35. The method of any one of claims 32-34, wherein admixing comprises diluting the microgel slurry with the interstitial matrix.

36. The method of claim 35, wherein the interstitial matrix is provided in an amount in a range of about 1 part by volume based on 100 parts total volume of microgelslurry and interstitial matrix to about 70 parts by volume based on 100 parts total volume of microgel slurry and interstitial matrix.

37. The method of any one of claims 32 to 36, wherein the microgel slurry and interstitial matrix are admixed at a temperature in a range of about 0°C to about 8°C.

38. A method for preparing a composite matrix material, the method comprising: admixing a microgel slurry comprising a plurality of particles of a polysaccharide, a protein, a polyethylene glycol, or a combination thereof; and an interstitial matrix to provide a composite matrix characterized by:(a) a storage modulus of no greater than about 200 Pa; and(b) reversible yield-stress behavior at 4°C.

39. The method of claim 38, wherein the composite matrix is characterized by a yield-stress having a value less than forces required for normal tissue growth or morphogenesis.

40. The method of claim 38 or claim 39, wherein admixing comprises diluting the microgel slurry with the interstitial matrix.41 . The method of anyone of claims 38-40, wherein the interstitial matrix is provided in an amount in a range of about 1 part by volume based on 100 parts total volume of microgel slurry and interstitial matrix to about 70 parts by volume based on 100 parts total volume of microgel slurry and interstitial matrix.

42. The method of any one of claims 38 to 41 , wherein the microgel slurry and interstitial matrix are admixed at a temperature in a range of 0°C to 10°C.

43. A method for preparing an organoid, the method comprising: providing a composite matrix material of any one of claims 1 to 31 at a first temperature in a range of 0°C to 8°C, preferably 4°C; depositing in the composite matrix at the first temperature a cell slurry bioink to form a cell-seeded composite matrix; and warming the cell-seeded composite matrix to a temperature in a range of about 34°C to about 40°C, for example, about 37°C.

44. The method of claim 43, further comprising adding cell-culture media to the cell-seeded composite matrix at the temperature in the range of about 34°C to about 40°C.

45. A method for preparing an organoid, the method comprising: providing a composite matrix material of any one of claims 1 to 31 at a first temperature in a range of about 0°C to about 8°C; depositing a cell slurry bioink in the composite matrix at the first temperature to form a cell-seeded composite matrix comprising a first array element; andwarming the cell-seeded composite matrix to a second temperature in a range of about 34°C to about 40°C.

46. The method of claim 45, wherein the first temperature is in a range of about 0°C to about 8°C, about 2°C to about 6°C, about 3°C to about 5°C, or about 4°C.

47. The method of any one of claims 45 or 46, wherein the second temperature is in a range of about 36°C to about 38°C, or about 37°C.

48. The method of any one of claims 45 to 47, wherein the cell slurry bioink is deposited by an extruder.

49. The method of any one of claims 45 to 48, wherein the extruder is a syringe pump, or a printhead.

50. The method of claim 49, wherein the printhead is an inkjet printhead, a pneumatic printhead, or a piezoelectric printhead.51 . The method of any one of claims 45 to 50, wherein the cell slurry bioink may be deposited along any of a horizontal-x axis, vertical-y axis, angular-z axis, or any combination thereof to form the cellular array.

52. The method of any one of claims 45 to 51 , further comprising depositing additional cell slurry bioink in the composite matrix at the first temperature to form a plurality of array elements.

53. The method of any one of claims 45 to 52, wherein the array element develops into an organoid during a period of time from about 2 minutes to about 14 days, 1 day to 10 days, or 3 days to 7 days.

54. The method of any one of claims 45 to 53, wherein the deposition of the cell slurry bioink is computer controlled.

55. The method of claim 54, further comprising programming the computer to deposit an array element having a predetermined size along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof.

56. The method of claim 54, further comprising programming the computer controller to print an organoid having a predetermined shape along any of the horizontal-x axis, the vertical-y axis, the angular-z axis, or any combination thereof.

57. The method of any one of claims 45 to 56, further comprising preparing the composite matrix material.

58. The method of any one of claims 45 to 57, further comprising preparing the cell slurry bioink.

59. The method of any one of claims 48 to 58, further comprising loading the extruder with the cell slurry bioink.

60. The method of any one of claims 45 to 59, further comprising adding a cellculture medium to the cell-seeded composite matrix at the second temperature.61 . The method of claim 60, wherein the cell-culture medium is Epidermal Growth Factor, Noggin, R Spondin (ENR) medium.

62. The method of claim 61 , further comprising allowing the cell-seeded composite matrix to stand at the second temperature for a period of time to allow the organoid to form.

63. The method of claim 62, wherein the period of time is in a range from about 2 minutes to about 14 days, 1 day to 10 days, or 3 days to 7 days.

64. The method of any one of claims 60 to 63, wherein the cell-culture media is changed every 2 to 4 days, every 2 to 3 days, every 3 to 4 days, every 2 days, every 3 days, or every 4 days.

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