3D bioprinting platform for generation of functional primary and HPSC-derived organoids in hydrogel constructs

Abstract

The disclosed concept includes the preparation and use of biocompatible bioinks, as well as techniques and methods therein for bioprinting, preparing and 3D printing primary cells and patient-specific cells and forming functional soft tissue in a three-dimensional construct form that incorporates the cells and mimics native tissues. The biocompatible bioinks include alginate, methylcellulose, and primary or iPSC-derived cells in the form of single cells or aggregates. The bioprinted 3D construct includes one or more of the iPSC organoids, human islets, as well as TEPCs.

Description

3D BIOPRINTING PLATFORM FOR GENERATION OF FUNCTIONAL PRIMARY AND HPSC-DERIVED ORGANOIDS IN HYDROGEL CONSTRUCTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to provisional patent application U.S. Patent Application Serial No. 63 / 705,314, entitled “3D BIOPRINTING PLATFORM FOR GENERATION OF FUNCTIONAL PRIMARY AND HPSC DERIVED-ORGANOIDS IN HYDROGEL CONSTRUCTS”, filed on October 9, 2024, the contents of which are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant 2229156 awarded by the National Science Foundation and grant P200A 180097 awarded by the Department of Education. The government has certain rights in the invention.Field of the Invention

[0003] The disclosed concept includes the preparation and use of bioinks, as well as techniques and methods of bioprinting, for preparing and printing primary cells and patient- specific cells, such as induced pluripotent stem cells, and forming functional soft tissue on a three-dimensional construct that incorporates the cells and mimics native tissues.Background

[0004] One of the fastest growing biomedical fields is bioprinting, which has a current market evaluated at 1.5 billion USD and is projected to grow at an alarming rate to a 5.1 billion USD market share by 2030[l ] . Bioprinting is an additive manufacturing process where cells and biocompatible polymers arc mixed to form a printable solution called bioink that is printed to form three-dimensional (3D) constructs that can mimic the structure and the micro physiological environment of native tissues. The major attributes of 3D bioprinting over traditional 2D and 3D culture methods is that it has the potential to accurately scale up the tissue production to meet the level needed for personalized clinical demand. These 3D printed tissues are useful in a variety of downstream applications, such as drug discovery, disease modeling, transplantation, and personalized cellular therapy.

[0005] In 3D bioprinting, specifically direct extrusion bioprinting, cells and bioink can be patterned and printed in a synergistic fashion using pressure as the driving force. These printed constructs can then closely mimic physiological soft tissue environments, allowing the transport of oxygen and molecules through the 3D printed construct to the embedded cells.

[0006] The majority of bioprinting research and technology falls under the category of direct extrusion printing, where pressure drives the formation of either hard or soft tissue structures.

[0007] Currently, work in creating bioprinted soft tissue structures is still in its infancy, with primary difficulties arising in identifying and generating the hydrogels that can form stable constructs which can support the growth and functionality of the detailed cell and organ constructs. Beyond the formation of the bioink, it is also vital to develop a printing protocol that can minimize any types of various mechanical stresses that the cells may encounter while printing, which is extremely critical to preserve cell viability and subsequently functionality.

[0008] Accordingly, there is a need in the art to design, develop, fabricate, and implement biocompatible bioinks, and methods for preparation thereof, as well as bioprinting techniques, and methods thereof to form the 3D constructs having functional soft tissue that mimics the structure and micro physiological environment of native tissues. More particularly, there is a need for printing a variety of functional primary cells and patient- specific cells, e.g., induced pluripotent stem cells, for incorporation into the constructs, such that the constructs have the capability to function as a personalized therapeutic and diagnostic tool.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1E are images that show TEPC aggregate printing and maturation: FIG. 1A is a schematic that shows a process for iPSC-TEC differentiation, FIG. IB is an image that shows iPSC aggregates, FIG. 1C is an image that shows TEPCs printed and immuno stained, FIG. ID is an image that shows iPSC aggregates, FIG. IE is an image that shows TEPCs printed and immuno stained, in accordance with certain embodiments of the inventive concept; and

[0010] FIGS. 2A-2F illustrate images of single iPSC printing to TEPC differentiation: FIG. 2A shows phase images (Expansion DO / Expansion D3 / Thymus D2 / Thymus D6) of single cell TEPCs cultured to aggregation and differentiated, FIG. 2B shows an image of a TEPC embedded construct, FIG. 2C shows a Live / Dead image of decapsulated TEPCs, FIG. 2D shows a plot of average TEPC aggregate diameter over days of differentiation, FIGS. 2E and 2F show printedTEPC aggregates characterized with FCM for GP38 and EpCAM markers , in accordance with certain embodiments of the inventive concept.SUMMARY OF THE INVENTION

[0011] In one aspect, the disclosed concept provides a biocompatible bioink including alginate; methylcellulose; primary or iPSC-derived cells in the form of single cells or aggregates; and optionally, one or more additives selected from nanoparticles and growth factors.

[0012] In certain embodiments, the alginate comprises 3% w / v of the bioink.

[0013] In certain embodiments, the methylcellulose comprises 6% w / v of the bioink.

[0014] In certain embodiments, the bioink further comprises an ionic cross-linking compound. The cross-linking compound can include a divalent cation containing compound, such as, calcium chloride.

[0015] In another aspect, the disclosed concept provides a method of preparing a bioink. The method includes selecting alginate; selecting methylcellulose; mixing the alginate and methylcellulose to form an alginate / methylcellulose mixture; placing the mixture in a print cartridge; and subsequent to the placing step, adding primary or iPSC-derived cells to the bioink in the form of single cells or aggregates.

[0016] The method can further include loading the print cartridge into an extrusion bioprinter; extruding the ink through a nozzle; and printing the bioink to form a three-dimensional (3D) construct. In certain embodiments, the method also includes ionically crosslinking the 3D construct; and placing the ionically crosslinked 3D construct in a culture medium.

[0017] In certain embodiments, the alginate comprises 3% w / v of the bioink.

[0018] In certain embodiments, the methylcellulose can include 6% w / v of the bioink.

[0019] In certain embodiments, the method further includes combining with the alginate and the methylcellulose, one or more additives selected from nanoparticles and growth factors.

[0020] In certain embodiments, the iPSC-derived cells are obtained from a patient, transformed into iPSCs, differentiated, and re-introduced into the patient.

[0021] In yet another aspect, the disclosed concept provides a 3D bioprinting method. The method includes preparing a biocompatible bioink; incorporating patient-derived iPSC single cells or pre-formed aggregates into the bioink; printing the bioink; forming a 3D printed construct; printing single cell or aggregate iPSCs within the 3D printed construct; culturing theiPSCs within the 3D printed construct; and thus, forming one or more of iPSC organoids, human islets, and TEPCs within the 3D printed construct.

[0022] In certain embodiments, the islets secrete an amount of insulin in response to changing levels of glucose.

[0023] In still another aspect, the disclosed concept provides a 3D bioprinted construct including a bioprinting surface; a bioink printed on the surface, the bioink including alginate; methylcellulose; and primary or iPSC-derived cells in a form of single cells or aggregates; wherein the bioprinted 3D construct includes one or more of iPSC organoids, human islets, and TEPCs.

[0024] In certain embodiments, the 3D printed construct has a high resolution from 700-1200 |im.

[0025] In certain embodiments, the bioink is printed in a layer-by-layer fashion, with the 3D printed shape easily controlled by a CAD file provided by a user.

[0026] In certain embodiments, the 3D printed construct comprises printed 5-10 m single cells at densities between 0.5 million- 2 million cells per milliliter of bioink, and aggregates between 100-400 pm in diameter.DETAILED DESCRIPTION

[0027] The disclosed concept includes bioinks, and methods for their preparation and use in bioprinting a variety of functional primary cells, e.g., primary human cells, and patient-specific cells, e.g., induced pluripotent stem cells (iPSCs), and providing a bio-printed 3D construct.

[0028] A key aspect of cellular bioprinting is an optimal bioink which retains structural fidelity while also supporting cell viability and function, immediately after printing and long term. The immediate effect of printing on the cells are primarily governed by printing parameters, while long term effects are dictated by the cell microenvironment, influenced by the physio-chemical properties of the ink.

[0029] Alginate based bioinks are useful for printing pancreatic islets and iPSC-derived cell populations. In certain embodiments, bioinks include from 3% w / v alginate to 4% w / v alginate. In addition, in certain embodiments, the alginate is combined with gelatin, e.g., 4% w / v alginate and 4% w / v gelatin. Further, alginate has been a widely adopted biocompatible polymer for cell encapsulation and drug delivery purposes.

[0030] In accordance with the inventive concept, it has been found that the combination of mcthylccllulosc and alginate offers advantages. While the mcthylccllulosc in the 3D printed constructs does leave and reduce the stability of the structure, the crosslinked alginate can still be manipulated for potential implantation. In certain embodiments, for example, a bioink contains 3% w / v alginate and 6% w / v methylcellulose. Additionally, in certain embodiments, the bioink is crosslinked with various divalent cation containing materials such as, but not limited to, calcium chloride, magnesium chloride, barium chloride, and strontium chloride.

[0031] The inventive concept also includes the ability to bioprint both primary human and iPSC- derived cells, preferably in a xeno-free bioink, with demonstrated viability and sustained function. In certain embodiments, the 3% w / v alginate / 6% w / v methylcellulose bioink is used to encapsulate and print primary human islets, iPSC derived islets, iPSC derived TEPCs, and undifferentiated iPSCs in both single cell, and aggregate form. In certain embodiments, printing the bioink at pressures between 25-30 kPa and then crosslinking with calcium chloride produces macroporous structures that facilitate adequate transportation of oxygen and nutrients, while maintaining a stiffness comparable to the pancreatic tissue. More importantly, the primary human islets and iPSC-derived islets, according to the inventive concept, endure the stress of bioprinting without causing any significant adverse effect on the viability of the cells. The bioprinted iPSC-derived islets and the primary human islets both successfully maintain functionality, indicating that with the inventive bioink and 3D printing protocol, diffusional limitations are largely mitigated.

[0032] The iPSC-derived islets exhibit similar levels of functionality to printed primary human islets, such that iPSC-derived islets provide an alternative to primary human islets. In addition, the inventive bioink and 3D printing protocol applied to a variety of iPSC-derived tissue constructs, as printed TEPCs also maintain their viability and phenotypic expression. Thus, the inventive concept demonstrates extended functionality of both primary human cells and iPSC- derived cells upon printing and extended culture.

[0033] Additionally, with single cell RNAseq, printing according to the inventive concept has no significant effect on the metabolic functionality of the iPSC islet like cells in comparison to traditional culture methods. This serves to further solidify bioprinting as a valid alternative to traditional methods of developing and maintaining iPSC-derived cell tissue for downstream analysis and personalized patient implantation.

[0034] In general, iPSCs are generated through the reprogramming of adult somatic cells with transcriptions factors: Oct4, Klf4, Sox2, and c-myc[2]; hPSCs (including both human embryonic stem cells and induced pluripotent stem cells), have the capability for infinite self-renewal, and can be differentiated into almost any tissue type[3]. Importantly, iPSCs are derived directly from the patient, thereby reducing the potential or possibility of rejection in terms of treatment or implantation. The hPSCs are differentiated to form organoids, which are 3D cell clusters that can replicate tissue function and assembly in-vitro[4, 5].

[0035] The 3D printing protocol and the bioink developed according to the inventive concept demonstrate an ability to print hPSCs and subsequently form functional organoids within a 3D printed construct. Thus, the inventive concept provides for successful formation of functional soft tissue that incorporates hPSCs. Furthermore, the inventive concept provides a 3D bioprinting protocol and the ability to mass produce functional hPSC-derived organoids.

[0036] The 3D printing protocol includes printing and culturing functional primary cells and hPSC-derived cells. The inventive concept provides for hPSC-derived cells directly harvested from an individual patient, transformed into the desired cell type via use of biocompatible bioinks and 3D bioprinting protocols, and then re-introduced to the patient, thus reducing the need for immunosuppressants necessary after foreign body introduction. These cells are now the future of personalized cellular therapy but however, require the correct physiological microenvironment to remain functional, which is what the inventive 3D printed biocompatible structure provides.

[0037] According to the inventive concept, alginate and methylcellulose are effective to form a biocompatible 3D printable bioink. In certain embodiments, the bio ink is comprised of 3% w / v alginate / 6% w / v methylcellulose, both biocompatible polymers, and after 3D printing, the bioink is crosslinked with a divalent cation containing ionic compound, such as but not limited to calcium chloride, e.g., 100 mM CaCh. Preparation includes the alginate and methylcellulose being combined by stirring (gently) and heated to form the printable bioink. The ease of formulation allows for the potential inclusion of other additives to the bioink, such as nanoparticles and growth factors. Utilizing an alginate / methylcellulose bioink provides a 3D printed stable, high resolution (700-1200 pm) 3D construct that is printable at lower pressures and nozzle speeds, which is vital for reducing any type of various mechanical stresses the cells are placed under as they are extruded.

[0038] In certain embodiments, a multi-layer, e.g., layer-by-layer technique is useful to print the alginatc / mcthylccllulosc bioink, with the 3D printed shape easily controlled by a CAD file provided by the user.

[0039] Multiple cell / aggregate sizes and densities are incorporated into the bioink while avoiding the loss of bioink integrity. In certain embodiments, the disclosed concept includes printed 5-10 pm single cells at densities between 0.5 million-2 million cells per milliliter of bioink, and aggregates between 100-400 pm in diameter. The aggregates / single cells are easily embedded into the bioink through gentle manual mixing, rather than a complex integration process.

[0040] In certain embodiments, the methylcellulose elutes out of the printed structure, e.g., within three days of printing, as it is not crosslinked, leaving a macroporous structure^].Therefore, the inventive concept provides for successful incorporation of primary cells in the form of cadaveric human islets, undifferentiated single hPSCs and aggregate hPSCs, and differentiated hPSC aggregates comprising of different lineages such as the hPSC-derived islet, and the hPSC-derived thymus epithelial progenitor cell (TEPC). Islets are the endocrine portion of the pancreas and contribute to regulating blood glucose levels in the body through a hormone secretion feedback loop. TEPCs in conjunction with hematopoietic stem cells (HSCs) help guide the selection of T cells, i.e., primary immune cells, within the thymus. In the case of iPSC- derivation, prior to the inventive concept, the islets and TEPC have not been successfully printed as long-term functional cells capable of maturation within a printed construct.

[0041] Primary tissue, such as primary cadaveric islets, are 3D printed using the inventive bioprinting protocol and bioink, cultured long-term, and exhibit appropriate insulin secretion in response to the introduction of glucose. This functionality is importantly noted while the islets are still incorporated within the 3D printed construct, indicating that there are no diffusional limitations present in the system that can significantly impede the flow of oxygen and nutrients. The primary cells also maintain their viability and phenotypic identity, demonstrating that bioprinting has no adverse effect on the cells. This bioprinting protocol and bioink can also be adapted for the inclusion of multiple forms of hPSCs.

[0042] According to the disclosed concept, single cell hPSCs are 3D printed and successfully aggregated within the 3D printed construct. This 3D formation of cells is required to further replicate tissue function and assembly in-vitro. These 3D printed single cells also displaypluripotency, meaning that when provided with the correct external cues they can differentiate into any tissue lineage desired.

[0043] Furthermore, according to the disclosed concept, pre-formed hPSC aggregates are also incorporated into the biocompatible bioink, 3D printed, and then differentiated to various islet developmental stages, while displaying the appropriate phenotypic markers for each stage. Building on that, immature hPSC-derived islets and immature TEPCs are capable of being 3D printed and cultured within the 3D printed construct to maturation. The TEPC-embedded 3D prints display the appropriate epithelial markers after culture and maintained viability, demonstrating that the 3D printing process does not adversely affect differentiation. The hPSC- derived islets also have the capability to be 3D printed and cultured to maturation, and not only display the correct phenotypic islet markers, but demonstrate functionality in response to glucose, as seen in the 3D printing of the primary tissue. Importantly, the functionality between the 3D printed hPSC-derived islets and the 3D printed primary islets is comparable, indicating that the hPSC tissue has the potential to emerge as a competitor to the primary human tissue in the field of personalized medicine. Thus, the 3D printing protocol and the innovative biocompatible bioink developed demonstrate versatility, and the functionality achieved after 3D printing, furthermore, demonstrates the ability to create valuable and useful tissue according to the invention.

[0044] An advantage of using the disclosed method of extrusion 3D bioprinting for the culturing of hPSCs is that it offers a uniform method to scale-up the production of soft tissue-like 3D constructs that are useful in a variety of downstream applications. The disclosed concept provides an alternative method for the aggregation and differentiation of hPSCs and presents a successful use of 3D bioprinting to print and culture both functional human primary tissue and hPSC-derived organoids. Based on the disclosed concept, scientists are no longer limited to the traditional 2D and 3D culture methods, which can be difficult to employ when differentiating hPSC cell lines and are not easily conducive to the increase in cell production needed for the future of stem-cell based personalized care.EXAMPLESExample 1Method for Development of Biocompatible Alginate / Methylcellulose Bioink

[0045] The biocompatible bioink was created by dissolving 3% w / v alginate in Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) at 35 °C for 1 hour. The alginate was then cooled to room temperature, mixing continuously for two hours. Then 6% w / v methylcellulose (MilliporeSigma) was added, mixing for 3 hours at 25 °C, and 0.5 hour at 50°C to remove any air bubbles present. The bioink was then transferred to 3 mL print cartridges and stored at 4 °C until use. Before the addition of cells, the ink cartridge was placed at room temperature for 2 hours, and UV sterilized for 1 hour. Primary or hPSC-derived cells were then incorporated in the bioink in either single cell or aggregate forms. Stable 3D printing was successfully demonstrated with single cell seeding densities between 0.5- 2 million cells, and cell aggregates 100- 400 m in diameter. It should be noted that the cell aggregates maintained their morphology and structure after addition to the bioink, and through simple manual mixing, and an even distribution of cells was achieved.

[0046] After the addition of the cells to the bioink, the print cartridge was loaded into an extrusion bioprinter. The bioink was then extruded through a 410 pm nozzle at pressures of 20- 30 kPa, and nozzle speeds of 10-20 mm / s. The stage was kept at 25 °C, and the ink cartridge maintained at a temperature of 25-30 °C while printing. Post-printing, the 3D printed constructs were ionically crosslinked with 50-100 mM CaChfor 5-10 minutes, washed three times with PBS, and placed in the appropriate culture medium. If printed in single cell form, the hPSCs would be cultured in the 3D printed construct to an aggregate form, and then further differentiated. If printed as undifferentiated aggregates, the hPSCs would undergo differentiation to the desired cell type. Finally, if printed as differentiated aggregates, the hPSCs would be cultured to reach maturation and then subjected to further downstream analysis.

[0047] Initially, cadaveric primary human islets were bioprinted. However, this system according to the inventive concept, demonstrates significant success with a variety of cell types and lineages and confirmed versatility. Importantly, for the test cases, long-term viability and functionality was achieved, further establishing the impact of this 3D bioprinting protocol.Example 2Testing and Printing of Alginate, Alginatc / Gcl, and Alginatc / Mcthylccllulosc Bioinks

[0048] To test the effect of bioink composition on the resolution of the 3D printed construct, 4% w / v alginate, 4% w / v alginate / 4% w / v gelatin (alg / gel), and 3% w / v alginate / 6% w / v methylcellulose (alg / MC) were each printed at pressures between 8 and 30 kPa, and nozzle speeds between 15 and 45 mm s-1onto plastic substrates. The alg / gel bioink also required extra heating of the bioink before extrusion to prevent formation of a granular hydrogel, which requires a higher printing pressure and thereby reduces the print resolution. The alginate and alg / gel bioinks produced structures that had an average diameter of 2861 ± 665 / / m and 1532+388 «m, respectively. In the case of the alg / MC bioink, when printed at the same pressures and nozzle speeds, the 3D printed constructs were on average 1198 ± 273 L / m in diameter. The difference in printing resolution between the bioinks was also qualitatively evident, as the alginate and alg / gel bioinks exhibited spreading after printing at 15 kPa and 30 mm s-1, in comparison to the alg / MC ink.

[0049] Having determined that alg / MC bioink produces the highest resolution 3D printed construct in comparison to other bioinks tested, this bioink was next examined for its crosslinking capabilities. Ionic crosslinking of the structures increases the 3D print resolution and the 3D construct becomes structurally intact. The 3% alg / 6% MC bioink was crosslinked with either 50 mM CaCh or 100 mM CaCh, for 3, 5 or 10 min. The bioink when crosslinked with 50 mM CaCh displayed no obvious deformities when crosslinked for 5 min but was not fully formed when crosslinked for 3 min. Crosslinking with 100 mM CaCh resulted in deformities in the printed construct, with non-uniform swelling all throughout, regardless of the crosslinking time. Rheological characterization of the printed construct was accomplished using atomic force microscopy (AFM) micro-indentation with a colloidal probe. AFM in liquid contact mode utilizing force-volume measurements on the printed constructs produced a Young’s modulus of 7.86 ± 3.71 kPa and a stiffness of 11.59+4.16 mN m-1.

[0050] To quantify the potential of the 3D printed construct to efficiently transport oxygen and nutrients, it was placed on a microfluidic device membrane and monitored under constant FITC- dextran (70 kDa) flow for 24 h. The FITCdextran was used to track molecular diffusion within the 3D printed structure through measuring the fluorescence intensity. When measured at specific regions of interests (ROIs) a clear increase of intensity with time was noted, indicatingdiffusion of 70 kDa dextran into the 3D printed construct, albeit with some variations in the dynamics of diffusion based on the location of the ROI in the flow field. The initial ‘dead time’, where fluorescence intensity did not change with respect to time, was observed to be ~2 h, as this is the time required to attain the detectable FITC concentration in the system. All six of the ROI’s reached steady state, where the change in fluorescent intensity with respect to time is constant, within 1.5 h after ‘dead time’. From steady state at each of the ROIs, the average diffusion coefficient for 70 kDa dextran through the printed construct was estimated to be 7.14 x 10-6cm2s-1. Based on these conclusions, it is a reasonable assumption that 0.180 kDa glucose and 5.8 kDa insulin molecules would be able to successfully diffuse in and out of the 3D printed construct within an appropriate time frame when loaded with islets. In parallel, the 3D printed structures were also loaded with RTDP and DAPI beads to report the oxygen concentration within the printed construct. The RTDP bead fluorescence was quenched in the presence of oxygen, and the DAPI beads acted as a control, providing an intensity ratio that was proportional to oxygen concentration in the system. Representative images of bead fluorescence at 0% and 18% oxygen can be found in supplemental information. Over the course of 24 h, the printed constructs maintained an oxygen concentration of ~18%, indicating that the 3D printed structure retained ambient oxygen concentration throughout the duration of the experiment.Example 3Materials and MethodsPrimary Islet Isolation[00511 Human cadaveric pancreatic islets were procured from Prodo Labs (San Francisco, USA) and maintained in suspension using the proprietary Prodo islet media for recovery (PIM(R)) with media changes every 2-3 days, which maintains a fasting islet state at 5 " 10-3 M glucose. Islets were maintained in a 100 mm x 25 mm petri dish (Fisher Scientific) suspended in PIM(R) (7-10 mL). The islets were initially cultured for 7 days in PIM(R) under static suspension conditions to recover from isolation and transport. A 200 pL pipette with SureOne aerosol barrier pipette tips (Fisher Scientific) was used to transfer the individual islets for mixing in the bioink with the aid of a dissecting microscope.iPSC Culture and Encapsulation

[0052] All the iPSCs used in these experiments were progeny of two iPSC cell lines, Yl, denoted iPSC-1, and hFL262 iPSC, denoted iPSC-2. Undifferentiated iPSCs were encapsulated as a single cell suspension. To obtain a single cell suspension, undifferentiated iPSCs were incubated in mTeSRl (STEMCELL Technologies) with 10 x 10’6M Y-27632 (R&DSystems) overnight prior to encapsulating. To dissociate, the cells were incubated with Accutase (Life Technologies) for 7 min at 37 °C to detach the cells and pipetted up and down obtain a single cell suspension. The cells were suspended in 1.5% (w / v) low viscosity alginate (Sigma Aldrich) at room temperature at a ratio of 1 million cells: 3 mL alginate and added drop wise to a bath of 100 mM calcium chloride (MilliporeSigma) with 10 x 10’3M HEPES (MilliporeSigma) using a 22-gauge needle. Alginate capsules were incubated for 5 min in the 100 x 10’3M calcium chloride and 10 x 10’3M HEPES solution at room temperature to allow for complete crosslinking and gelation of the alginate capsules. The capsules were washed three times with PBS and suspended in mTeSRl with 10 x 10’6M Y-27632. Before starting differentiation, encapsulated cells were cultured for 4 days in mTeSRl with 10 x 10’6M Y-27632 followed by 1 day of mTeSRl. iPSC Differentiation into Pancreatic Islet Endocrine Cells

[0053] Using a modified version of a previously published protocol, islet-like cells were generated using stagewise differentiation steps. The differentiation base media was composed of 2.44 x 10’3M D-Glucose (Gibco), 1.23 g L’1NaHCCL (MilliporeSigma), 2% FAF-BSA (Fisher Scientific), 2 x 10’3M Glutamax (Gibco), 1% Pen / Strep (Lonza), and MCDB131 (490 mL) (Coming / Gibco). Media changes were completed as follows with supplements to the differentiation base media. Definitive Endoderm Media (Days 1-3): differentiation base media was supplemented to have 2.46 g L’1NaHCCE, 0.25 x 10’3M Vitamin C (MilliporeSigma), and 1:50 ITS-X (MilliporeSigma). Day 1: 100 ng mL’1Activin A (R&D Systems) and 1.4 pg mL’1Chir99021 (Stemgent). Days 2-3: 100 ng mL’1Activin A. Primitive Gut Tube (Days 4 and 6): 50 ng mL’1KGF (Peprotech), 0.25 x 10’3M Vitamin C, and 1:50 ITS-X. Pancreatic Progenitor 1 (Days 7 and 8): 50 ng mL’1KGF, 0.25 x 10’6M Santl (MilliporeSigma), 2 x 10’6M Retinoic Acid (MilliporeSigma), 500 x 10’9M PdBU (MilliporeSigma), 0.25 x 10’3M Vitamin C, 10 x 10’6M Y-27632, and 1:200 ITS-X. Pancreatic Progenitor 2 (Days 9, 11, and 13): 50 ng mL’1KGF,0.25 x 10"6M Sant 1 , 0.1 x 10"6M Retinoic Acid, 0.25 x IO"3M Vitamin C, 10 x 10"6M Y-27632, 5 ng mL'1Activin A and 1:200 ITS-X. Endocrine Progenitor (Days 14-30): increase NaHCO, to 1.75 g mL-1and increase glucose to 20 x 10"3M. Days 14 and 16: 0.25 x 10"6M Santl, 0.1 x 10"6M Retinoic Acid, 0.25 x 10"3M Vitamin C, 1 x 10"6M XXI (MilliporeSigma), 10 x 10"6M ALk5i II (Axxora), 1 x 10"6M T3 (MilliporeSigma), 20 ng mL"1Betacellulin (Fisher Scientific), 10 pg mL"1Heparin (MilliporeSigma), 1:200 ITS-X. Days 18-30: 0.025 x 10"6M Retinoic Acid, 0.25 x 10"3M Vitamin C, 1 x 10"6M XXI, 10 x 10"6M ALk5i II, 1 x 10"6M T3, 20 ng mL"1Betacellulin, 10 pg mL"1Heparin, 1: 200 ITS-X. Maturation (Days 32+): 1: 200 ITS, and 0.25 x IO"3M Vitamin C. iPSC Differentiation into Thymic Epithelial Progeniter Cells

[0054] Using a modified version of a previously published protocol thymic epithelial cells were generated using stagewise differentiation steps. Stage 1 for definitive endoderm was carried out following a beta cell differentiation protocol. The stage 1 base media was composed of 2.44 mM D-Glucose (Gibco), 2.46 g / L NaHCCL (MilliporeSigma), 2% FAF-BSA (Fisher Scientific), 2 mM Glutamax (Gibco), 1% Pen / Strep (Lonza), 0.25 mM Vitamin C (MilliporeSigma), 1:50 ITS- X (MilliporeSigma), and 490 mL MCDB131 (Coming / Gibco). Stage 2 base media for anterior foregut endoderm was Roswell Park Memorial Institute (RPMI) media supplemented with 0.5% B27 (Gibco). Stages 3 and 4 base media for ventral pharyngeal endoderm and thymus epithelial progenitor was DMEM / F12 (Gibco) media supplemented with 0.5% B27.

[0055] Media changes were completed as follows with supplements added to the appropriate base media. Stage 1 (Days 1-3): 100 ng mL"1Activin A (R&D Systems) and 3 pM Chir99021 (Stemgent) (Day 1 only). Stage 2 (Day 4): 100 ng mL"1Activin A, 0.25 pM retinoic acid (Millipore Sigma). Stage 2 (Days 5-6): 0.25 pM retinoic acid, 50 ng mL"1BMP4 (Miltenyi Biotec), and 5 pM LY364947 (MilliporeSigma). Stage 3 (Days 7-8): 0.1 pM retinoic acid, 5 pM LY364947 (Days 7-8 only), 50 ng mL"1BMP4, 50 ng mL"1Wnt3A (R&D Systems), 50 ng mL"1FGF8b (Miltenyi Biotec), and 0.5 pM KAAD-cyclopamine (MilliporeSigma). Stage 4 (Days 9- 10) supplements matched Stage 3 with the exclusion of LY364947. (Days 11+): DMEM / F12 supplemented with 0.5% B27, no additional growth factors.Alginate / Gelatin and Alginate / Methylcellulose Boink Preparation

[0056] For all the bioinks that contained an alginate component, alginic acid sodium salt derived from brown algae was used (A2158, Sigma). To prepare the alginate bioink, 4% w / v alginate was dissolved in DMEM / F12 (Gibco) at 35 °C for one hour. This protocol was also followed when developing 1.5% w / v alginate. The 4% alginate / 4% gelatin bioink was synthesized by stirring 4% w / v alginate into DMEM / F12 at 35 °C for one hour. 4% w / v gelatin derived from bovine Type B (MilliporeSigma), was then stirred into the alginate for 2.5 hours at RT. For the development of the alginate / methylcellulose (Alg / MC) bioink, 3% w / v alginate was dissolved in DMEM / F12 for one hour at 35°C. The alginate was then cooled to RT, mixing continuously for two hours. 6% w / v methylcellulose (MilliporeSigma) was added, mixing for 3 hours at 25 °C, and 0.5 hours at 50 °C to remove any air bubbles present. The bioink was then transferred to 3 mL print cartridges and stored at 4 °C until use.An Alg / MC Bioink Printing Protocol Applied to the Successful Differentiation of Other iPSC Cell Lineages

[0057] Based on the success of printing functional iPSC-derived islets, the culture and differentiation of other iPSC-derived cell types was tested. The thymus is also heavily indicated in the pathogenesis of T1D, and so it was attempted to print iPSC-derived TEPCs, to see if phenotypic expression and viability could be maintained. Differentiating to the TEPC stage from undifferentiated single iPSCs, followed the encapsulation protocol used in iPSC islet differentiation (FIG. 1A). As shown in FIG. 1A, iPSC cells were cultured and then mixed in 1.5% w / v alginate hydrogel. The resulting single cell iPSC suspension was added to 100 mM of CaCh, wherein the alginate beads were crosslinked for 5 minutes. In this study, iPSC aggregates were decapsulated at either Day 7 or Day 9 of differentiation, printed using the same parameters as the islets, and kept in culture until reaching the TEPC stage. When printed at Day 7 and maintained in the 3D printed constructs for 4 days, the aggregates could be printed and retained their structure with no significant fragmentation noted. By the end of the culture however, significant blebbing, where the aggregates balloon out and create inner void spaces, was occurring (FIG. IB, left). LiveDead image indicated that the majority of the aggregates maintained viability, but did not maintain sphericity (FIG. IB, right). Even with this loss insphericity, the TEPCs still maintained their phenotypic expression, as evidence by the positive staining for TEPC markers EpCAM and KRT8 (FIG. 1C).

[0058] When the organoids were printed at Day 9 of differentiation and also maintained in culture for 4 days, no significant blebbing was noted (FIG. ID, left). Similar to printing in Day 7 however, some dead aggregates were noted after culture, and the organoids lost their sphericity post decapsulation (FIG. ID, right). Positive immunostaining was observed for EpCAM and KRT8, demonstrating that by printing at Day 9 of differentiation phenotypic identity could be maintained (FIGS. IE ). While in both cases viability and sphericity were affected, the phenotypic expression was not, indicating that printing is a potential route for the culture and differentiation of iPSC-derived TEPCs, similar to what was seen when printing iPSC-derived islets.

[0059] Lastly, while printing in the single cell form and differentiating within the 3D construct was initially dismissed for iPSC-derived islets due to the cell debris noted after printing and the significant cell overgrowth noted when differentiating printed aggregates, we reconsidered this printing protocol for TEPCs. These cells have a significantly shorter differentiation period, meaning the cell overgrowth in the construct is reduced, and would be cultured with the intent to decapsulate and reseed with Pro-T cells, eliminating the issue of single cell debris during transplantation. Following the same single cell printing protocol described prior, 1.6 million iPSCs per mL were printed and cultured in propagation media for 4 days. Aggregates were clearly visible in the 3D printed constructs by Day 3 of expansion and grew within the construct through the duration of differentiation (FIG. 2A, “Expansion DO,” “Expansion D3”, “Thymus D2”, and “Thymus D6”). By Day 11 aggregates still remained in the construct (FIG. 2B, “Thymus Dl l”) and were spherical and highly viable post-decapsulation (FIG. 2C, “Live / Dead”). The average organoid diameter by the end of differentiation was 175 ± 30 pm, which is comparable in size to the TEPC aggregates we traditionally generate in alginate beads (FIG. 2D). Additionally, to put into perspective the number of TEPCs generated, 800,000 undifferentiated iPSCs were printed in 0.5 mL bioink, and 8.170 million cells were found to be viable at the end of differentiation, which translates to a 10-fold increase in the number of cells within the 3D printed construct. Finally, flow cytometry analysis (FCM) showed that around 58% of the cells were EpCAM-i- (FIG. 2E) and 92% of cells were GP38+ (FIG. 2F), demonstrating that the printed cells in the 3D constructs could successfully obtain the TEPCphenotypic identity. These results show the versatility of the 3D printed construct, and the capabilities it has for differentiating iPSCs.Discussion

[0060] This work demonstrated direct extrusion 3D bioprinting with a biocompatible alginate / methylcellulose bioink to be a viable option for the 3D printing and extended culture of functional primary islets and iPSC-derived cells. The 3D printed constructs formed after ionic crosslinking of the bioink provided the appropriate environment necessary for both iPSC-derived islets and iPSC-derived TEPCs to maintain their viability and phenotype, and the iPSC-derived islets also maintained their functionality. The3D printed iPSC islet function was found to be comparable to that of the 3D printed primary human islets. It was demonstrated by scRNAseq analysis that key islet specific pathways are upregulated in the 3D printed cells, and none of the major stress pathways are upregulated, indicating that there is no significant deleterious stress caused by the 3D printing process and utilizing the biocompatible bioink formulation. By 3D printing iPSC-derived cells a biocompatible 3D printed scaffold may be formed.

[0061] Primary human islets that were printed in the 3% w / v alg / 6% w / v MC bioink retained function after 7 days of culture; GSIS showed that the islets responded appropriately to low and high glucose conditions, resulting in an average SI of 2.87 ± 1.52 (first high glucose incubation / second low glucose incubation). The GSIS was conducted on the intact printed construct, meaning that no diffusional limitations were present that could significantly hinder the functionality of the primary islet, even in extended culture. The lack of a diffusional barrier was also verified through the inclusion of two rounds of low glucose level exposures followed by two rounds of high glucose exposure. If an increase in secretion was noted between the two low glucose level exposures or the two high glucose exposures, this delay in insulin secretion could be indicative of a diffusion barrier. In both the primary and iPSC-derived islet cases, no significant difference was noted between the two low exposures or two high exposures.

[0062] While alginate has been widely adopted as a biocompatible polymer for cell encapsulation and drug delivery purposes, the combination of methylcellulose and alginate offered many advantages. While the methylcellulose in the 3D printed constructs was eluted and reduced the stability of the structure, the crosslinked alginate can still be manipulated for potential implantation. While the bioink ultrastructure was not imaged, the potential effect of thebioink on the embedded cells was measured, in terms of the diffusional properties and structural attributes. While it should be noted that under continuous perfusion for 24 hours fluorescently tagged dextran (70 kDa), comparable in size to glucose and insulin, was able to penetrate even to the middle of the 3D printed 3% alginate / 6% methylcellulose construct within 1 hour. In addition, it was estimated that the diffusion coefficient of the 70 kDa dextran in the construct was 7.14 x 10-6 cm2s'1.

[0063] Testing the mechanical properties of the 3D printed alg / MC construct with AFM, an average Young’s modulus of 7.86 kPa was obtained. Thus, the elasticity of the 3D printed construct is comparable in magnitude to that of both a native thymic and pancreatic environment. The Young’s modulus of the 3D printed construct is attributed to the use of both a low viscosity alginate and calcium chloride as the crosslinker.

[0064] Cell viability during the extrusion bioprinting process is largely dependent on the printing conditions, including nozzle diameter, and printing pressure; these same parameters also govern the resolution of the print, but with the inverse relationship. Furthermore, the print resolution influences the necessary diffusion of oxygen and nutrients to and from the embedded cells. Multiple alginate-based bioinks were therefore tested, of which the 3% w / v alg / 6% w / v MC bioink produced the highest resolution structures when 3D printed at pressures below 30 kPa and nozzle speeds between 5-45 mm s'1. Each 3D printed construct was printed as a 3D single layer, with a focus on optimizing the bioink to facilitate the maintenance of phenotype and function.

[0065] In printing iPSC-derived tissues there is a need to account for the dynamics of differentiation as an additional printing parameter, since the requirement for microenvironmental niche is specific to the stage of organ maturation. Hence, printing the iPSCs at different stages of differentiation were evaluated, starting from the pluripotent stage. The first attempt was to 3D print the iPSCs in the initial propagation stage, as single cells suspended in the bioink, and continue with the process of cell aggregation and subsequent differentiation within the 3D printed construct. When single cell iPSCs were printed at a cell density of 1.6 million cells mL'1of alg / MC bioink, aggregate formation was noted by Day 4 of culture, however a significant amount of cell debris was also observed. The aggregates that formed were uniform, spherical, viable, and remained intact after decapsulation, indicating that even with the debris present, aggregation was successful. Uniform aggregation of the single cells was observed and aggregation in the more porous and softer bioink (lower hydroxypropyl chitin concentration) wasattributed to a combination of both migration and in situ proliferation. While this study indicated the feasibility of minimizing single cells by increasing cell seeding density, for islet printing, the route of pre-aggregating the iPSCs prior to printing was taken instead of printing single cells.

[0066] Printing of iPSC aggregates were tested at different stages of differentiation, which revealed interesting stage-specific dynamics, likely arising from the differential balance of proliferation and differentiation. The 3D printed undifferentiated iPSC aggregates in both lines coalesced and formed larger aggregates within the construct, but clearly maintained pluripotency and viability, demonstrating the potential for induction towards a desired lineage by specific chemical cues. Coalescence was also clearly observed with differentiation post-printing to the pancreatic progenitor or endocrine progenitor stage within the 3D printed construct, where the aggregates not only merged, but also produced a significant amount of debris around the aggregate. Although these aggregates displayed phenotypic expression of PDX1 and NKX6.1 they were not structurally stable and fragmented upon decapsulation. When printed at a later differentiation stage, coalescence and fragmentation were not observed in the 3D printed organoids. While coalescence was not observed when printing TEPCs, a similar pattern in proliferation versus differentiation was observed. When 3D printed at the earlier anterior foregut endoderm stage (Day 7), organoids formed blebbing voids, whereas 3D printing at a later stage, the ventral pharyngeal endoderm (Day 9), this phenomenon was largely avoided.

[0067] The iPSC-islets printed at the pre-mature stage followed by post-printing maturation were able to maintain structure and viability over 7 days during which they displayed a significant increase in insulin gene expression in comparison to control iPSC islets, indicating the 3D printed environment as being supportive for maturation. In the same manner as the 3D printed primary human islets, GSIS conducted after 7 days indicated that the iPSC-islets could appropriately respond to glucose, resulting in an average SI of 4.93 + / - 0.74. In comparison to the insulin secretion exhibited by the printed primary human islets under the same GSIS conditions, there was no significant difference in insulin secretion levels between the two populations, demonstrating that the iPSCs were comparatively functional. This work demonstrated successful bioprinting of both primary and iPSC-islets with preserved post-printing function. This study successfully retained islet function for both primary human islets and iPSC- derived islets when tested a week after 3D printing of the constructs.

[0068] It is still relatively unknown the extent of stress the cells tend to undergo when printed, therefore, scRNAScq was conducted to determine the alterations made to key stress and islet specific metabolic pathways in comparison to control iPSC islets that have not undergone printing. Key islet pathways such as calcium signaling and insulin secretion signaling are significantly upregulated in comparison to the control iPSC islets, supporting the results of the functionality and phenotypic assays conducted on the printed islets. In comparison to the control, stress pathways ER stress, and NRF-2 oxidative stress mediated response were not significantly altered. For cells specifically within the endocrine cell population, it was determined that the expression levels of only 1 / 7 genes of interest from both stress pathways were significantly different, highlighting that printing is not causing undue stress on the islets.

[0069] Although the insulin secretion signaling pathway of the 3D printed iPSC islets was significantly enriched in comparison to the control iPSC islets and there was no clear activation of metabolic stress pathways, there are indicators that 3D printing and culturing of iPSC islets in the 3D printed constructs may, in some ways, adversely affect key metabolic pathways involved in insulin secretion. PPARa / RXRa activation was significantly downregulated (z-score = - 1.987, p < 0.001) in the 3D printed iPSC islets in comparison to the control islets.

[0070] When testing this bioink and protocol for multiple cell fates, the same approach used in iPSC islet bioprinting was adopted, where organoids were taken and printed at later stages of differentiation, when proliferation has slowed due to the differentiation process. While success was seen in printing later stage TEPCs and maintaining phenotypic expression, demonstrating the utility of the bioink and printing protocol, additional testing was conducted to determine whether or not single iPSC printing would be feasible for cell differentiation lineages that had shorter differentiation timescales. Although significant overgrowth was noted when differentiating the 3D printed undifferentiated aggregate over 32 days to the endocrine progenitor phase, it was possible that the use of different growth factors and subsequently different aggregate dynamics would lead to the successful differentiation and culture of other iPSC organoids, like the TEPCs. To test this, single iPSCs were 3D printed, formed aggregates within the 3D construct, and were differentiated over 11 days to TEPCs. In this case, no significant overgrowth or cell death was noted, and the organoids displayed expression of key TEPC markers, suggesting that differentiation was achieved within the 3D printed construct. Theseresults indicate that a complete differentiation cycle can be carried out within the 3D printed constructs, but optimization is required for specific cell fates, such as iPSC-dcrivcd islets.

[0071] This aim expands the possibilities of personalized cellular therapy research through the successful combination of bioprinting and iPSCs. Through this work, there was developed a robust and versatile bioink and bioprinting procedure that facilitates the viability and functionality of not only primary cells, but also multiple iPSC differentiation lineages. These tissue mimetic 3D printed constructs can serve to further improve drug testing efficiency, tissue and disease modeling, and most importantly, personalized cellular therapy effectiveness. This can be accomplished through the incorporation of patient specific iPSCs into the bioprinted 3D scaffolds, where the exact nature of the disease for the patient can be modelled, and more effective treatment such as the functional printed iPSCs, can be employed.References:1. Choudhury, D., S. Anand, and M.W. Naing, The arrival of commercial bioprinters- Towards 3D bioprinting revolution! International Journal of Bioprinting, 2018. 4(2).2. Shi, Y., et al., Induced pluripotent stem cell technology: a decade of progress. Nature reviews Drug discovery, 2017. 16(2): p. 115-130.3. Medvedev, S., A. Shevchenko, and S. Zakian, Induced pluripotent stem cells: problems and advantages when applying them in regenerative medicine. Acta Naturae (aHrjios3biHHaa Bepciia), 2010. 2(2 (5)): p. 18-27.4. Turhan, A.G., et al., iPSC-Derived organoids as therapeutic models in regenerative medicine and oncology. Frontiers in Medicine, 2021. 8: p. 728543.5. Garreta, E., et al., Rethinking organoid technology through bioengineering. Nature Materials, 2021. 20(2): p. 145-155.6. Schutz, K., et al., Three-dimensional plotting of a cell-laden alginate / methylcellulose blend: towards biofabrication of tissue engineering constructs with clinically relevant dimensions. Journal of tissue engineering and regenerative medicine, 2017. 11(5): p. 1574-1587.

Claims

We claim:

1. A biocompatible bio ink, comprising: alginate; methylcellulose; primary or iPSC-derived cells in a form of single cells or aggregates; and optionally, one or more additives selected from nanoparticles and growth factors.

2. The bioink of claim 1, wherein the alginate comprises 3% w / v of the bioink.

3. The bioink of claim 1, wherein the methylcellulose comprises 6% w / v of the bioink.

4. The bioink of claim 1, further comprising an ionic cross-linking compound.

5. The bioink of claim 1, wherein the cross-linking compound comprises a divalent cation containing compound.

6. The bioink of claim 5, wherein the divalent cation containing compound is calcium chloride.

7. A method of preparing a bioink, comprising: selecting alginate; selecting methylcellulose; mixing the alginate and methylcellulose to form an alginate / methylcellulose mixture; placing the mixture in a print cartridge; and subsequent to the placing step, adding primary or iPSC-derived cells to the bioink in the form of single cells or aggregates.

8. The method of claim 7, further comprising: loading the print cartridge into an extrusion bioprinter; extruding the ink through a nozzle; and printing the bioink to form a 3D construct.

9. The method of claim 8, further comprising: ionically crosslinking the 3D construct; and placing the ionically crosslinked 3D construct in a culture medium.

10. The method of claim 7, wherein the alginate comprises 3% w / v of the bioink.

11. The method of claim 7, wherein the methylcellulose comprises 6% w / v of the bioink.

12. The method of claim 7, further comprising combining one or more additives selected from nanoparticles and growth factors with the alginate and methylcellulose.

13. The method of claim 7, wherein the iPSC-derived cells are obtained from a patient, transformed into iPSCs, differentiated, and re-introduced into the patient.

14. A 3D bioprinting method, comprising: preparing a biocompatible bioink; incorporating pre-formed or patient-derived iPSC cells or aggregates into the bioink; printing the bioink; forming a 3D printed construct; printing single cell or aggregate iPSCs within the 3D printed construct; culturing the iPSCs within the 3D printed construct; and forming one or more of iPSC organoids, human islets, and TEPCs within the 3D printed construct.

15. The method of claim 14, wherein the islets produce an amount of insulin in response to changing levels of glucose.

16. A bioprinted 3D construct, comprising: a bioprinting surface; a bioink printed on the surface, the bioink comprising: alginate;methylcellulose; and primary or iPSC-dcrivcd cells in a form of single cells or aggregates; wherein the bioprinted 3D construct includes one or more of iPSC organoids, human islets, and TEPCs.

17. The bioprinted 3D construct of claim 16, wherein the 3D printed construct has a high resolution from 700-1200 pm.

18. The bioprinted 3D construct of claim 16, wherein the bioink is printed in a layer-by-layer fashion, with the 3D printed shape easily controlled by a CAD file provided by a user.

19. The bioprinted 3D construct of claim 16, wherein the construct comprises printed 5-10 pm single cells at densities between 0.5 million- 2 million cells per milliliter of bioink, and aggregates between 100-400 pm in diameter.

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