Derivation of stem cells and organoids from nonhuman primate gastrointestinal tract tissue

Abstract

Methods are provided for isolating and expanding gastrointestinal (GI) tract stem cells from nonhuman primates (NHP)s. The method includes recovering a portion of GI tissue from a NHP, enzymatically digesting the tissue in a digestion medium comprising a tissue dissociation enzyme, and isolating cells through filtration and centrifugation. The isolated cells are cultured in a two-dimensional or three-dimensional (3D) culture system using an expansion medium comprising a basal formulation supplemented with one or more growth factors, vitamins, amino acids, and pathway modulators. In some embodiments, the cells are suspended in a basement membrane extract and plated as a 3D structure for organoid formation. The resulting stem cells or organoids may be analyzed using microscopy, flow cytometry, or transcriptomic profiling, and may be used for preclinical drug testing, disease modeling, or toxicity screening. The methods reduce reliance on live animal testing and improve translational relevance of in vitro gastrointestinal models.

Description

DERIVATION OF STEM CELLS AND ORGANOIDS FROM NONHUMAN PRIMATE GASTROINTESTINAL TRACT TISSUECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 656,733, filed June 6, 2024, the contents of which are herein incorporated by reference in their entirety.TECHNICAL FIELD

[0002] This disclosure relates generally to the field of molecular biology, and more specifically to the field of stem cell science and in vitro modeling. Described herein are systems and methods for isolating and deriving stem cells from nonhuman primate (NHP) gastrointestinal (GI) tract tissues and for generating corresponding organoid models.BACKGROUND

[0003] NHPs, such as, cynomolgus macaques (Macaca fascicularis), are typically utilized as an animal model in nonclinical and / or preclinical safety assessments of human pharmaceutical compounds. Their close phylogenetic relationship to humans, including similarities in immune response, physiology, metabolism, and / or genetic structure, makes them a standard model for evaluating drug toxicity, pharmacokinetics, and / or efficacy prior to initiating many human clinical trials. Some regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) often use data from one or more NHP studies, especially for biologies, vaccines, and / or advanced therapeutics where rodent models are typically inadequate. However, ethical concerns, high costs, and / or limited availability of live NHPs have intensified efforts to develop alternative testing systems.SUMMARY

[0004] In some aspects, the techniques described herein relate to a method of deriving stem cells from nonhuman primate (NHP) gastrointestinal (GI) tract tissue, including: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tract tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissuein a digestion medium including a tissue dissociation enzyme; filtering the digested GI tract tissue to isolate one or more cells; and culturing the one or more isolated cells in an expansion medium to derive one or more stem cells.

[0005] In some aspects, the techniques described herein relate to a method of deriving organoids from gastrointestinal (GI) tract tissue of a nonhuman primate (NHP), including: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissue in a digestion medium including a tissue dissociation enzyme; filtering the digested tissue of the one or more regions of the at least a portion of gastrointestinal tract tissue to isolate one or more cells; suspending the one or more isolated cells in a culture including; a basement membrane extract (BME); plating the one or more suspended isolated cells into a three-dimensional (3D) structure on a culture surface of the culture including extracellular matrix (ECM) proteins; incubating the 3D structure to form an organoid; and culturing the 3D structure in an expansion medium to promote organoid growth.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The foregoing is a summary, and thus, necessarily limited in detail. The above-mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings.

[0007] FIG. 1 illustrates an embodiment of a method of deriving stem cells from NHP GI tract issue, in accordance with some embodiments.

[0008] FIG. 2 illustrates an embodiment of a method of deriving organoids from NHP GI tract tissue, in accordance with some embodiments.

[0009] FIG. 3A is a 4X magnification of a gut organoid derived from culturing an ileum of a Macaca fascicularis, in accordance with some embodiments.

[0010] FIG. 3B is a 10X magnification of a gut organoid derived from culturing an ileum of a Macaco, fascicularis, in accordance with some embodiments.

[0011] FIG. 3C is a 20X magnification of a gut organoid derived from culturing an ileum of a Macaca fascicularis, in accordance with some embodiments.

[0012] FIG. 3D is a 40X magnification of a gut organoid derived from culturing an ileum of aMacaca fascicularis, in accordance with some embodiments.

[0013] FIG. 4A is a 10X magnification of a gut organoid derived from culturing a jejunum of a Macaca fascicularis, in accordance with some embodiments.

[0014] FIG. 4B is a 1 OX magnification of a gut organoid derived from culturing a caecum of a Macaca fascicularis, in accordance with some embodiments.

[0015] FIG. 4C is a 10X magnification of a gut organoid derived from culturing a colon of a Macaca fascicularis, in accordance with some embodiments.

[0016] FIG. 5A is a 4X magnification of a gut organoid derived from culturing an ileum of a Macaca fascicularis, in accordance with some embodiments.

[0017] FIG. 5B is a 4X magnification of a gut organoid derived from culturing an ileum of a Macaca fascicularis, in accordance with some embodiments.

[0018] FIG. 6A is a 4X magnification of a gut organoid derived from culturing a colon of a Macaca fascicularis, in accordance with some embodiments.

[0019] FIG. 6B is a 4X magnification of a gut organoid derived from culturing a colon of a Macaca fascicularis, in accordance with some embodiments.

[0020] FIG. 7 is a schematic illustration of portions of the gastrointestinal tract, showing the duodenumjejunum, ileum, cecum, and colon, in accordance with some embodiments.

[0021] FIG. 8 shows an example tissue processing step for a NHP GI tract sample in a sterile laboratory setting in which a 6-well tissue culture plate with Matrigel domes are plated in multiple wells for culturing NHP GI organoids, in accordance with some embodiments.

[0022] FIG. 9 is a flowchart illustrating an example method of deriving stem cells, in accordance with some embodiments.

[0023] FIG. 10 is a flowchart further illustrating the method of deriving stem cells from FIG. 9, in accordance with some embodiments.

[0024] FIG. 11 is a flowchart further illustrating the method of deriving stem cells from FIG. 9, in accordance with some embodiments.

[0025] FIG. 12 is a flowchart illustrating an example method of deriving organoids, in accordance with some embodiments.

[0026] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale.DETAILED DESCRIPTION

[0027] The foregoing is a summary, and thus, necessarily limited in detail. The above-mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0028] The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0029] In recent years, there has been reduced availability of live NHPs for testing safety and efficacy of human drugs. The technical solution to this availability problem is in vitro models of NHPs. In vitro models of NHPs may play a role in human drug efficacy and safety assessment prior to testing such drugs in live NHPs. In vitro models may save cost and time in human drug development, reduce the use of live NHPs in the human drug development process, and / or improve the accuracy of nonclinical safety by complementing human in vitro models. In some embodiments, GI tract stem cells may be isolated from including, but not limited to, one or more regions, such as a plurality of regions (e.g., one region, two regions, three regions, four regions, five regions, one to five regions, one to ten regions, etc.) of an intestine, including, but not limited to, the duodenumjejunum, ileum, cecum, small intestine, large intestine, rectum, stomach, and / or colon of NHPs. In some embodiments, the NHP is a cynomolgus macaque (Macaco fascicularis), rhesus macaques (Macaca mulatto), and / or pig-tailed macaque (Macaco nemestrina) or other similar species of NHPs (that are used as NHP models for human diseases). For example, nonhuman primates may include northern pig-tailed macaque (Macaco leonina),barbary macaque (Macaca sylvanus), baboons, African green monkeys, chimpanzees, and the like.

[0030] At least one practical application of deriving stem cells and organoids from NHP GI tract tissue includes testing, validating, and / or assaying one or more preclinical drugs on the derived NHP stems cells and / or organoids and correlating the testing, validation, and / or assay results with one or more in vivo NHP tests results.

[0031] Another practical application of deriving stem cells and organoids from NHP GI tract tissue includes deriving GI stem cells and differentiating the derived GI stem cells to specific gut cells to evaluate drug safety and / or efficacy (e g., side effects of the drugs). Another practical application of deriving stem cells and organoids from NHP GI tract tissue includes executing one or more experiments and / or or assays to determine a pathophysiology of a disease. Exemplary, non-limiting examples of diseases include: IBD (Inflammatory bowel disease), Ulcerative Colitis, Chronic idiopathic diarrhea (CID), and / or various types of gut tumors including, but not limited to, ileocecal adenocarcinoma. Another practical application of deriving stem cells and organoids from NHP GI tract tissue includes, but is not limited to, predicting, using in vitro methods using the derived stem cells and / or organoids, an outcome of in vivo human experiments or therapy testing and / or an NHP experiment or testing.

[0032] The derived stem cells and / or organoids may serve as physiologically relevant proxies for live animal and / or human systems for researchers to evaluate including, but not limited to, drug efficacy, safety, metabolism, and / or toxicity in a controlled and reproducible environment. This approach can be particularly valuable in cases where ethical and / or practical constraints preclude the direct testing of drugs in live human subjects. As noted, in some embodiments, human gut stem cells are not readily obtainable for experimental use in parallel in vitro and in vivo settings due to ethical and / or logistical limitations. As a result, no laboratory can ethically and / or legally perform drug testing in humans using matched in vitro and in vivo systems. In contrast, NHP-derived stem cells and organoids provide a species-appropriate alternative, allowing for mechanistic studies, hypothesis testing, and / or preclinical validation that closely mirror human physiological responses. The use of such in vitro NHP models thus bridges a translational gap, enhancing the predictive value of preclinical studies while reducing reliance on live animal experimentation.

[0033] Another practical application of deriving stem cells and organoids from NHP GI tract tissue includes deriving a specific differentiated cell type (e g., epithelial cells) from a primarytissue. For example, the methods and compositions described herein may be optimized to derive such cells and maintain viable cells in culture conditions. For example, the percentage of viability may be greater than about 90% or between about 90% to about 95%.

[0034] As shown in FIG. 1, an embodiment of a method 100 of isolating GI tract stem cells from one or more regions of a GI tract 120 of a NHP 110 includes recovering at least a portion of a GI tract 120 from the NHP 110. In some embodiments, the term “recovering” refers to obtaining, isolating, and / or retrieving biological tissue, such as a portion of the GI tract 120, from the NHP 110 in a manner that substantially preserves tissue viability for downstream processing. In some embodiments, this process may include surgically excising one or more regions of the GI tract 120, such as the duodenum, jejunum, ileum, cecum, small intestine, large intestine, rectum, stomach, and / or colon, under sterile conditions during a necropsy and / or a surgical procedure.

[0035] An embodiment of the method 100, as shown in FIG. 1, may include sectioning 130 the at least a portion of the GI tract 120 into one or more regions of the GI tract 120. In some embodiments, a weight of each gastrointestinal (GI) tract region may be about 0.1 g to about 5.0 g. In some embodiments, a weight of each region may be about 0.5 g to about 5.2 g. In some embodiments, the weight may range from about 1.0 g to about 5.5 g, or from about 1.5 g to about 6.0 g. In some embodiments, the weight of each region may be about 2.0 g to about 6.5 g, or about 3.0 g to about 7.0 g. In some embodiments, one or more regions of the GI tract 120 having a substantially equal weight are used in additional processing.

[0036] An embodiment of a method 100, as shown in FIG. 1, of isolating GI tract stem cells may include digesting 140 the one or more regions of the GI tract in a digestion medium. The digestion medium may be supplemented with one or more non-essential amino acids, (e.g., L-glutamine, alanyl-L-glutamine, glycyl-L-glutamine, or an L-glutamine substitute), which supports cellular metabolism and viability during tissue dissociation. The digestion medium may include one or more basal formulations such as Advanced Dulbecco’s Modified Eagle Medium (AdvDMEM+++) and / or another Dulbecco Modified Eagle Medium (DMEM), for example DMEM / F-12, MEM (Minimum Essential Medium), L-15 Leibovitz Medium (L-15), Roswell Park Memorial Institute 1640 (RPMI 940), and / or similar. The one or more basal formulations may facilitate a buffered, nutrient-rich environment that can sustain cell health, reduce mechanical damage, and / or improve enzymatic activity during the breakdown of extracellular matrices. Basal formulations refer to standardized cell culture media that provide nutrients, salts, and / or buffering agents formaintaining basic cellular functions, such as energy production, pH balance, and / or osmotic stability. Basal formulations serve as the foundational environment in which cells can survive, grow, and, in some cases, differentiate. Basal formulations are often used as the base medium to which other supplements (e.g., growth factors, amino acids, hormones, or antibiotics) may be added depending on the specific application or cell type.

[0037] In some embodiments, the digestion medium may include a tissue dissociation enzyme. For example, the tissue dissociation enzyme may include a collagenase, a dispase, and / or a liberase, thereby enhancing the efficiency and reproducibility of cell release.

[0038] In some embodiments, the dissociation enzyme is a collagenase, for example a type II collagenase. Collagenase enzymes degrade collagen, a primary structural component of the extracellular matrix (ECM), thereby providing the release of viable cells from dense and / or fibrous tissues such as those found in the GI tract. Type II collagenase may be effective due to its balanced enzymatic profile, which includes both collagenolytic and proteolytic activity. This dual action facilitates more complete tissue dissociation without excessive degradation of sensitive cell surface proteins or stem cell markers. The use of type II collagenase can improve cell yield, viability, and / or purity by minimizing mechanical shear forces and preserving the functional integrity of the isolated stem cells. Additionally, its efficacy at relatively low concentrations and compatibility with physiological pH and temperature conditions make it well-suited for controlled enzymatic digestion in preclinical or translational applications involving NHP GI tissue. In some embodiments, Collagenase Type I may be used. In some embodiments, Collagenase Type IV may be used. In some embodiments, any combination of collagenase (Type I, II, and / or IV), a dispase, and / or a liberase may be used for cellular dissociation. The dissociation enzyme(s) may be used at a concentration of about 1 mg / mL to about 10 mg / mL; about 2.5 mg / mL to about 7.5 mg / mL; about 4 mg / mL to about 6 mg / mL; etc.

[0039] In some embodiments, the digestion medium may include one or more inhibitors, which may improve cell survival and / or prevent apoptosis of sensitive epithelial or stem cell populations during and after digestion. One example of such an inhibitor is a Rho-associated protein kinase (ROCK) inhibitor, which acts by suppressing the Rho / ROCK signaling pathway, an intracellular pathway for regulating cytoskeletal dynamics, contractility, and / or apoptosis. Inhibition of ROCK can prevent detachment-induced cell death (anoikis) and support cell-cell junction integrity during dissociation. In some embodiments, the ROCK inhibitor may be used at a concentration of about5 pM to about 20 pM, including example ranges such as about 5 pM to about 15 pM, about 7 pM to about 13 pM, about 8 pM to about 12 pM, or about 9 pM to about 11 pM, depending on the enzyme formulation and tissue type. The inclusion of such inhibitors may improve the viability of the isolated cell population and contribute to expansion and differentiation in downstream culture steps.

[0040] In some embodiments, digesting the one or more regions may include mechanically dissociating the one or more regions of tissue prior to enzymatic treatment. Mechanically dissociating may refer to mincing, mechanical fragmentation, or mechanical disruption of tissue into smaller pieces, ranging in size from about 1 mm3to about 5 mm3, such as about 1.5 mm3to about 4.5 mm3, about 2 mm3to about 4 mm3, or about 2.5 mm3to about 3.5 mm3, to increase the surface area exposed to the digestion medium and enzymatic agents. This step facilitates more efficient and uniform penetration of tissue dissociation enzymes throughout the dense extracellular matrix and cellular compartments. Mechanical dissociation may be performed using sterile scissors, scalpels, and / or automated tissue choppers under aseptic conditions. In some embodiments, mechanical dissociation may be carried out in the digestion vessel, such as a conical tube or a digestion chamber, to streamline the workflow and minimize tissue handling. For example, intestinal segments from the ileum or colon of a NHP may be cut longitudinally, rinsed to remove luminal contents, and then mechanically dissociated into small fragments before incubation with a digestion solution containing one or more tissue dissociation enzymes and / or one or more inhibitors. This approach enhances cell yield, reduces enzymatic incubation time, and preserves the viability of epithelial and stem cell populations by promoting more rapid and controlled tissue dissociation.

[0041] In some embodiments, digesting the one or more regions may include vibrating the one or more regions (e.g., before or after mechanically dissociating). For example, vibrating may include positioning a receptacle containing the one or more regions on an orbital shaker or other vibration apparatus. In some embodiments, the receptacle may be positioned at an angle, for example, an angle (relative to normal) of about 0 degrees to about 30 degrees; about 15 degrees to about 45 degrees; or about 20 degrees to about 90 degrees; etc. The vibrating may occur for about 15 minutes to about 60 minutes; about 20 minutes to about 40 minutes; about 25 minutes to about 35 minutes; about 30 minutes to about 50 minutes; about 40 minutes to about 50 minutes; about 35 minutes to about 55 minutes; etc. The vibrating may occur at a temperature of about 30°C toabout 42°C; about 35°C to about 40°C; about 36°C to about 38°C; or about 37°C. The vibrating may occur at about 100 rpm to about 250 rpm; about 110 rpm to about 180 rpm; about 120 rpm to about 150 rpm; about 130 rpm to about 160 rpm; etc.

[0042] In some embodiments, the method optionally includes visually inspecting a turbidity of the digestion medium in the receptacle. In some embodiments, the method optionally includes spectroscopically inspecting a turbidity of the digestion medium in the receptacle. In some embodiments, when the turbidity meets or exceeds a threshold, the method may continue; in contrast; when the turbidity is lower than a threshold, further vibrating may continue. Alternatively, the turbidity may be visually or spectroscopically inspected without impacting progression of the method. A threshold turbidity for progressing the method may range from about 0.2 to about 1.0 optical density units (OD) at 600 nm, for example about 0.3 to about 0.8 OD, about 0.4 to about 0.7 OD, or about 0.5 OD as a representative cutoff value. These ranges may correspond to the visually or spectroscopically assessed cloudiness of the digestion medium indicative of adequate tissue dissociation.

[0043] In some embodiments, a method of isolating GI tract stem cells may include filtering the digested one or more regions in the medium to isolate one or more cells. The filter may have a pore size of about 70 pm, about 100 pm, or about 200 pm, or about 70 pm to about 200 pm, including example ranges such as about 10 pm to about 500 pm, including example ranges such as about 70 pm to about 200 pm, about 80 pm to about 180 pm, about 90 pm to about 150 pm, or about 100 pm to about 130 pm.

[0044] In some embodiments, a method of isolating GI tract stem cells may include centrifuging the filtered media including the one or more cells. Centrifugation may occur for about 2 minutes to about 10 minutes; about 4 minutes to about 6 minutes; about 5 minutes to about 8 minutes; etc. The centrifugation may occur at a temperature of greater than about 0°C; or between about 2°C to about 6°C. The centrifugation may occur at about 250 x g to about 500 x g; about 300 x g to about 500 x g; about 400 x g to about 500 x g; etc.

[0045] In some embodiments, when red blood cells (RBCs) are present in the digested tissue suspension, the method may optionally include a step of selectively lysing the RBCs to prevent contamination and improve the purity of the isolated stem cell population. The presence of RBCs, which may result from tissue vascularization and / or handling during dissection and / or digestion, may interfere with downstream processing, obscure flow cytometry or microscopy analysis, and / orcompromise the quality of organoid culture. To address this, a lysis solution may be applied that includes ammonium chloride (or alternatively, Veralyse® or other lysis buffers), which induces osmotic lysis by disrupting the RBC membrane, in combination with disodium ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations and prevent cell aggregation, and optionally potassium bicarbonate as a buffering agent to maintain near-neutral pH during the lysis reaction. The lysis buffer may be formulated at a working concentration comprising, for example, ammonium chloride in a concentration range of about 140 mM to about 220 mM, about 155 mM to about 200 mM, or about 170 mM to about 190 mM; potassium bicarbonate in a concentration range of about 5 mM to about 70 mM, about 10 mM to about 60 mM, or about 20 mM to about 50 mM; and disodium ethylenediaminetetraacetic acid (EDTA) in a concentration range of about 0.05 mM to about 2.0 mM, about 0.1 mM to about 1.5 mM, or about 0.3 mM to about 1.0 mM, each in sterile water, and may be diluted at a ratio of approximately 1 :10 with the cell suspension prior to application. In some embodiments, the cell pellet may be resuspended in the lysis buffer and incubated at room temperature (e.g., about 20°C to about 25°C) for a duration ranging from approximately 1 minute to approximately 15 minutes, for example from about 2 minutes to about 10 minutes, from about 3 minutes to about 8 minutes, or from about 4 minutes to about 6 minutes, with intermittent or occasional mixing during the incubation period. After optional lysis, the sample may be diluted with a balanced salt solution (e.g., PBS) and centrifuged to remove lysed debris, thereby enriching the cell population for viable epithelial and stem cells. This step enhances the clarity and interpretability of downstream cell analysis and promotes growth in organoid or expansion culture conditions.

[0046] In some embodiments, a method of isolating GI tract stem cells may optionally include washing the one or more cells one or more times.

[0047] In some embodiments, as shown in FIG. 1, a method 100 of isolating GI tract stem cells may include adding expansion medium to the one or more cells obtained following digestion, filtration, and centrifugation and maintaining the culture in an incubator 150. In some embodiments, the incubator may be maintained at a temperature ranging from about 33°C to about 42°C, for example from about 35°C to about 40°C, from about 36°C to about 38°C, or substantially about 37°C. The carbon dioxide (CO?) concentration within the incubator may range from about 3% to about 7%, for example from about 4% to about 6%, from about 4.5% to about 5.5%, or substantially about 5%.

[0048] As noted above, in some embodiments, the expansion medium may include a cell culture formulation supporting the survival, proliferation, and maintenance of stem cells isolated from NHP GI tissue. In some embodiments, the expansion medium includes a basal medium, such as Advanced DMEM / F-12, (L-15), RPMI 940, etc., which provides one or more nutrients, salts, and / or buffering capacity. In some embodiments, to promote stem cell renewal and / or organoid formation, the expansion medium may be supplemented with one or more and / or a combination of one or more growth factors and / or one or more small-molecule signaling modulators, including but not limited to, a Wnt surrogate, R-spondin 1 conditioned medium (Rspol-CM), Noggin, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and hepatocyte growth factor (HGF).

[0049] In some embodiments, additional additives may include but not be limited to, a B-27 supplement, nicotinamide, N-acetylcysteine, prostaglandin E2, and / or p38 or ALK5 inhibitors, which support epithelial lineage maintenance and / or inhibit undesired differentiation or apoptosis. In some embodiments, the expansion medium may also contain amino acids and vitamins, such as L-glutamine, thiamine, biotin, and / or folic acid, to optimize cell metabolism and / or support longterm culture viability. In some embodiments, the expansion medium may be refreshed at intervals ranging from about 1 day to about 5 days, for example, about every 1 to 3 days, about every 2 to 4 days, or about every other day. The cultures may be maintained at a temperature ranging from about 33°C to about 42°C, for example about 35°C to about 40°C, or substantially about 37°C, in a humidified incubator. The carbon dioxide (CO2) concentration may range from about 3% to about 7%, for example from about 4% to about 6%, or substantially about 5%. The medium formulation enables the expansion of GI tract-derived stem cells and promotes the formation and maintenance of physiologically relevant organoid structures suitable for downstream drug screening or disease modeling applications.

[0050] In some embodiments, a method of isolating GI tract stem cells may include refreshing the medium every about one day to about 4 days; about one day to about 3 days; about 1 day to about 2 days; about 2 days to about 3 days; etc.

[0051] After a predefined time period (e g., about 3 days to about 10 days; about 4 days to about 8 days; about 6 days to about 8 days; about 7 days; etc.), one or more organoids may be expanded and / or split 160, as shown in FIG. 1. Expanding and / or splitting may include mechanical disruption (e g., repetitive pipetting; vibration; etc.). The expansion medium may include one or more cellularproliferation, growth, and / or differentiation factors. The expansion medium is formulated to support the survival, proliferation, and maintenance of sternness in the isolated cell population. In some embodiments, the expansion medium includes a basal formulation such as Advanced DMEM / F-12, supplemented with one or more growth and / or differentiation factors. For example, the expansion medium may include a B-27 supplement, a Wnt surrogate, R-spondin 1 conditioned medium (Rspol-CM), Noggin, epidermal growth factor, fibroblast growth factor 2 (FGF2), EGF, hepatocyte growth factor (HGF), tumor-growth factor-beta (TGF-b), bone morphogenetic protein (BMP), Notch signaling pathway activators, Notch signaling pathway inhibitors, and / or prostaglandin E2. In some embodiments, prostaglandin E2 is included to enhance epithelial regeneration and stem cell self-renewal.

[0052] In some embodiments, one or more of the cellular proliferation, growth, and / or differentiation factors (e.g., Wnt Surrogate) may be present at a concentration of about 0.1 nM to about 1 nM, about 0.25 nM to about 0.75 nM, about 0.05 nM to about 2.0 nM, about 0.15 nM to about 0.85 nM, about 0.25 nM to about 0.75 nM, or about 0.3 nM to about 0.6 nM. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., Rspol-CM) may be present at a concentration ranging from about 0.1 pg / mL to about 1.5 pg / mL, about 0.5 pg / mL to about 1.5 pg / mL, about 0.2 pg / mL to about 1.2 pg / mL, about 0.3 pg / mL to about 1.0 pg / mL, about 0.6 pg / mL to about 1.4 pg / mL, about 0.75 pg / mL to about 1.25 pg / mL, or about 0.9 pg / mL to about 1.1 pg / mL. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., Noggin) may be present at a concentration of about 50 ng / mL to about 150 ng / mL, about 60 ng / mL to about 140 ng / mL, about 70 ng / mL to about 130 ng / mL, about 80 ng / mL to about 120 ng / mL, about 90 ng / mL to about 110 ng / mL, or about 95 ng / mL to about 105 ng / mL. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., A-acety I cysteine) may be present at a concentration of about 0.5 mM to about 2 mM, about 0.75 mM to about 1.75 mM, about 1 mM to about 1.5 mM, or about 1.1 mM to about 1.4 mM.

[0053] One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., nicotinamide) may be present at a concentration of about 5 mM to about 15 mM; about 7 mM to about 13 mM; or about 8 mM to about 12 mM. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., p38 inhibitor) may be present at a concentration of about 1 pM to about 15 pM; about 5 pM to about 15 pM; or about 8 pM to about 12 pM. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., EGF) may be present at aconcentration of about 30 ng / mL to about 70 ng / mL; about 40 ng / mL to about 60 ng / mL; etc. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., ALK5 inhibitor) may be present at a concentration of about 0.1 pM to about 1 pM; about 0.25 pM to about 0.75 pM; etc. One or more of the cellular proliferation, growth, and / or differentiation factors (e.g., prostaglandin E2) may be present at a concentration of about 0.1 pM to about 1 pM; about 0.25 pM to about 0.75 pM; etc. For example, the expansion medium may include one or more inhibitors. For example, the expansion medium may include an ALK5 inhibitor A83-01, a p38 inhibitor, XAV939 Wnt inhibitor, and / or another Wnt inhibitor.

[0054] In some embodiments, the expansion medium may include one or more amino acids and / or vitamins that function to support cellular metabolism, promote antioxidant protection, and / or enhance proliferation and differentiation of GI stem cells. For example, the expansion medium may include A-acetylcysteine, nicotinamide, retinol, ascorbic acid, folic acid, biotin, thiamine, L- glutamine, L-alanine, L- arginine, or the like.

[0055] As noted above, in some embodiments, N-acetylcysteine acts as a potent antioxidant and a precursor to glutathione, which can reduce oxidative stress during in vitro culture and improve stem cell viability. Nicotinamide, a form of vitamin B3, supports cellular energy metabolism and has been shown to enhance organoid-forming efficiency and epithelial cell differentiation. Retinol (vitamin A) and ascorbic acid (vitamin C) may be included to support epithelial barrier integrity and collagen synthesis, respectively. Folic acid, biotin, and thiamine are cofactors in nucleotide synthesis, mitochondrial function, and energy production. These vitamins are used for rapidly dividing stem cell populations. The expansion medium may also include a panel of one or more amino acids, such as L-glutamine, L-alanine, and L-arginine. L-glutamine may be included to generate carbon and nitrogen for nucleic acid and / or amino acid biosynthesis and to support proliferation. L-alanine may enhance osmotic stability and protein synthesis, while L-arginine may signal cells and produce nitric oxide, which can affect epithelial regeneration. These components may be used at concentrations optimized for organoid growth, for example ranging from about 0.1 mM to about 4 mM, about 0.05 mM to about 5 mM, from about 0.1 mM to about 4 mM, from about 0.25 mM to about 3 mM, or from about 0.5 mM to about 2 mM, depending on the specific amino acid or vitamin. By providing a balanced mix of metabolic substrates and cofactors, the expansion medium supports long-term culture stability, promotes physiological relevance of the organoid model, and enhances reproducibility across experimental conditions.

[0056] In some embodiments, as shown in FIG. 2, a method 200 of isolating GI tract stem cells may include blocks 210-240 that are similar to blocks 110-140 that are described above.

[0057] In some embodiments, as shown in FIG. 2, a method 200 of isolating GI tract stem cells may include suspending the one or more cells in a two-dimensional culture 250 and / or in a three- dimensional (3D) culture 260. The method 200 may further include suspending, at block 270, the one or more cells in basement membrane extract (BME) (e.g., Matrigel, Geltrex, Cultrex, MaxGel® ECM, synthetic matrices (e.g., collagen, fibrin, vitronectin, etc.), decellularized dermal fibroblast ECM, dry human amniotic membrane, etc.) and plating the one or more cells in a 3D structure, such as a dome, including one or more extracellular matrix proteins, as shown at block 260. The 3D structure may include one or more of extracellular matrix proteins, for example, laminin, type IV collagen, and / or entactin. The 3D structure may further include one or more growth factors and / or proteoglycans to support stem cell viability and differentiation. For example, the 3D structure may comprise Matrigel®, Geltrex, Cultrex, MaxGel® ECM, or a synthetic matrix (e.g., collagen, fibrin, vitronectin, etc.) for the BME.

[0058] In some embodiments, the receptacle containing the 3D structure and suspended cells may be positioned upside in an incubator to solidify the 3D structures. The 3D structure and suspended cells may be positioned upside down for about 10 minutes to about 30 minutes; about 15 minutes to about 25 minutes; etc. to form a uniform gelation and encapsulation of the cells. After solidification, the 3D structure and receptacle are inverted to the upright position, and the culture is maintained under standard incubation conditions. The incubator may be at a temperature of about 35°C to about 40°C, or substantially about 37°C. The percent CO2 in the incubator may be about 4% to about 6%, or substantially about 5%. The 3D structure may be used to form one or more organoids from the cell suspension.

[0059] In some embodiments, a method of isolating GI tract stem cells may include adding expansion medium to the 3D structure and maintaining the culture in the incubator.

[0060] In some embodiments, a method of isolating GI tract stem cells may include refreshing the medium every about one day to about 4 days; about one day to about 3 days; about 1 day to about 2 days; about 2 days to about 3 days; etc.

[0061] After a predefined time period (e.g., about 3 days to about 10 days; about 4 days to about 8 days; about 6 days to about 8 days; about 7 days; etc.), one or more organoids or a plurality of cells may be expanded and / or split 160, as shown in FIG. 1. Expanding and / or splitting mayinclude mechanical disruption (e.g., repetitive pipetting; vibration; etc.). The expansion medium may include one or more cellular proliferation, growth, and / or differentiation factors. For example, the expansion medium may include a Wnt surrogate, Rspol-CM, Noggin, epidermal growth factor, EGF, HGF, TGF-b, BMP, Notch signaling pathway activators, Notch signaling pathway inhibitors, and / or prostaglandin E2. For example, the expansion medium may include one or more inhibitors. For example, the expansion medium may include an ALK5 inhibitor A83-01, a p38 inhibitor, XAV939 Wnt inhibitor, or another Wnt inhibitor. The expansion medium may include one or more amino acids or vitamins. For example, the expansion medium may include N- acetylcysteine, nicotinamide, ascorbic acid, essential and branded amino acids including, but not limited to, leucine, isoleucine, and valine.

[0062] In some embodiments, as shown at block 280 in FIG. 2, the one or more organoids may be analyzed by microscopy, flow cytometry, or other cellular assay. The one or more organoids may undergo transcriptomic analysis, for example.

[0063] As noted above, in some embodiments, the one or more organoids derived from NHP GI tissue may be analyzed using a variety of morphological, functional, and / or molecular assays to assess cell composition, viability, and / or physiological relevance. Such analyses may include brightfield or confocal microscopy to evaluate structural features of the organoids, including, but not limited to, lumen formation, epithelial polarity, and / or crypt-like domains. Immunofluorescence microscopy may also be used to localize one or more lineage-specific markers (e.g., LGR5 for stem cells, MUC2 for goblet cells, or CHGA for enteroendocrine cells), providing insight into differentiation status. In some embodiments, one or more organoids may be dissociated into single cells and subjected to flow cytometry to quantify subpopulations of cells based on surface markers, cell cycle profiles, and / or apoptosis indicators. Additional cellular assays may include viability staining (e g., 4',6-diamidino-2-phenylindole (DAPI), Calcein AM, or propidium iodide), barrier function assays, and / or drug response testing using pharmacologic compounds, for example human therapies prior to clinical studies or in parallel to in vivo and / or clinical studies.

[0064] In further embodiments, the organoids may undergo transcriptomic analysis, such as bulk RNA sequencing or single-cell RNA sequencing (scRNA-seq), to generate high-resolution profiles of gene expression. These molecular analyses allow for the identification of specific cell types present within the organoid (e.g., Paneth cells, enterocytes, tuft cells) and the characterization ofgene expression patterns associated with intestinal development, homeostasis, disease modeling, and / or drug response. The transcriptomic data may be compared to reference datasets from in vivo NHP or human GI tissue to assess fidelity and / or translational relevance. Together, these analytical techniques provide comprehensive validation of organoid identity and function and may be used to inform experimental design, quality control, and / or regulatory submissions.

[0065] Although FIGs. 1-2 show a sequence of steps, one of skill in the art will appreciate that the steps may be performed in alternative sequences.

[0066] WORKING EXAMPLES

[0067] Working Example 1

[0068] FIGs. 3A-3D present brightfield microscopy images acquired at increasing magnifications (4X, 10X, 20X, and 40X, respectively) to illustrate the morphological characteristics of a gut organoid derived from the ileum tissue of a Cynomolgus macaque (Macaca fascicular is). The organoid was cultured using a 3D Matrigel matrix and maintained under standard physiological conditions in a humidified incubator at 37°C with 5% CO2. Imaging was performed on day seven post-seeding using a Keyence brightfield microscope to assess early organoid development and structural organization.

[0069] At low magnification (FIG. 3A, 4X magnification), the gut organoid appears as a well- defined, spherical structure suspended within the Matrigel matrix, indicating successful initial organoid formation. FIG. 3B (10X magnification) provides a clearer view of the organoid’s morphology, revealing the presence of a distinct, closed luminal cavity characteristic of early-stage gut organoid development. In FIG. 3C (20X magnification), the image demonstrates the formation of a thin epithelial wall or lining enclosing the lumen, suggestive of initial cellular organization but limited structural complexity. FIG. 3D (40X magnification) provides a detailed visualization of the organoid wall, confirming the presence of a uniformly thin epithelial layer with no evidence of advanced differentiation features such as villus-like projections and / or crypt-like invaginations. Across the magnifications, the organoid remains in an undifferentiated state, as evidenced by the absence of complex tissue architecture and specialized structural features observed in more mature gut organoids. The thinness of the epithelial lining and the lack of morphological markers of differentiation indicate that the organoid has not yet progressed beyond the early developmental phase, providing a baseline for assessing future maturation under modified culture conditions or differentiation protocols.

[0070] Working Example 2

[0071] FIGs. 4A- 4C present brightfield microscopy images depicting gut organoids derived from different intestinal regions of a Cynomolgus macaque Macaca fascicularis . The organoids were cultured using isolated tissues from the jejunum, caecum, and colon, respectively, embedded in a 3D Matrigel matrix, and maintained under standard physiological conditions in a humidified incubator at 37°C with 5% CO2. Imaging was performed using a Keyence brightfield microscope at 10X magnification, seven days after the initial seeding, to assess early organoid formation and regional morphological variations.

[0072] FIG. 4A illustrates organoids derived from jejunal tissue, which appear as small, isolated spherical structures with clearly defined lumens. The low number of organoids and their uniform morphology suggest limited proliferation and early-stage development without structural differentiation. FIG. 4B shows organoids cultured from caecum tissue, displaying similar spherical morphologies with distinct luminal spaces; however, a greater variance in organoid size is observed, which may indicate region-specific differences in progenitor cell proliferation or viability. FIG. 4C presents organoids derived from colonic tissue, where a higher density of organoids is observed across the field of view. The colonic organoids display a wider size distribution and more frequent formation of multiple small spheroids, suggesting increased cellular proliferation or higher organoid-forming efficiency from this tissue source under identical culture conditions. Across the images, the organoids exhibit thin epithelial linings and lack complex features such as crypt-like invaginations and / or villus-like projections, indicating that they remain in an undifferentiated state. These comparative observations provide insights into the distinct organoid-forming capacities of different intestinal regions and establish a baseline for evaluating future differentiation protocols or tissue-specific functional analyses.

[0073] Working Example 3

[0074] FIGs. 5A and 5B illustrate brightfield microscopy images demonstrating the formation of gut organoids following the first passage of cultures derived from the ileum of a Cynomolgus macaque (Macaca fascicularis). The images were captured five days after the first passage using a Keyence brightfield microscope at 4X magnification, providing an overview of early organoid reformation and cellular reorganization post-passaging.

[0075] In FIG. 5A, a 4X magnification view of the formation of newly developing gut organoids derived from culturing an ileum of a Cynomolgus macaque is observed, with a representativeorganoid indicated by the arrow. The organoid exhibits a spherical morphology, characterized by a thin epithelial wall surrounding a developing luminal cavity. This indicates the initiation of organoid self-organization following enzymatic or mechanical dissociation during the passaging process. Surrounding the highlighted structure, multiple smaller cell clusters and spheroids can be seen, suggesting active aggregation and early morphogenesis within the culture environment.

[0076] FIG. 5B similarly presents a 4X magnification view of organoid development, derived from culturing an ileum of a Cynomolgus macaque, post-passage, with the arrow marking a representative gut organoid. The observed organoid shows characteristics consistent with early- stage formation, including a defined spherical structure and the initiation of lumen development. The presence of multiple small aggregates and partially formed spheroids across the field suggests ongoing cellular sorting and organization processes that are critical for re-establishing organoid architecture after passaging. Together, these images confirm the regenerative capacity of the ileum-derived stem and progenitor cells to reform gut organoids following subculture, highlighting the technical benefits of the culture system. The thin epithelial lining and / or absence of one or more complex differentiated structures at this stage further indicate that the organoids remain in an undifferentiated or early developmental state, suitable for subsequent experimental manipulation or differentiation protocols.

[0077] Working Example 4

[0078] FIGs. 6A and 6B present brightfi eld microscopy images showing gut organoids derived from colonic tissue of a Cynomolgus macaque (Macaca fascicularis). Both images were acquired five days after culturing, at 4X magnification, using a Keyence microscope. The images provide representative views of early-stage organoid formation within a Matrigel matrix environment following isolation and seeding of colon-derived epithelial and stem cell populations. In FIG. 6A, a single gut organoid is indicated by the white arrow. The organoid exhibits a well-defined spherical morphology with a developing central lumen. The relatively thin outer epithelial lining suggests that the organoid is in an undifferentiated or early proliferative state. Surrounding the highlighted structure, additional smaller cell clusters and spheroidal formations are visible, indicative of active colony formation and ongoing tissue self-organization.

[0079] FIG. 6B shows the same field of view at a slightly different focal plane or orientation, again highlighting the same organoid marked by the white arrow. The organoid retains its characteristic spherical morphology, with slight increases in edge contrast and internal complexity,possibly reflecting further maturation or lumen expansion over time. The surrounding matrix continues to support multiple micro-colonies at different stages of morphogenesis, reinforcing the viability and organoid-forming capacity of colon-derived cells under the applied culture conditions. Together, FIGs. 6A and 6B demonstrate successful initiation and progression of organoid formation from Cynomolgus macaque colon tissue using in vitro 3D culture techniques. These images establish visual benchmarks for assessing early morphogenesis and may serve as a reference point for evaluating differentiation protocols, growth factor responses, or comparative studies across intestinal regions.

[0080] ISOLATION OF NHP GASTROINTESTINAL TRACT ORGANOIDS FROM FIVE GI TRACK SECTIONS: DUODENUM, JEJUNUM, ILEUM, CECUM, & COLON.

[0081] The morphological data presented in FIGs. 5A through 6B underscore the reproducibility and regional applicability of the organoid formation approach across distinct segments of the NHP GI tract. To support these imaging observations and provide ex vivo modeling, the following section details the experimental workflow for isolating, culturing, and expanding gut organoids from various NHP GI tissue sources, including the duodenumjejunum, ileum, cecum, and colon. This protocol encompasses tissue handling, enzymatic digestion, cell isolation, Matrigel 3D structure preparation, and the use of defined expansion media containing Wnt surrogate, R- spondinl, Noggin, and additional supplements. The described methodology provides a reproducible framework for deriving region-specific GI organoids suitable for downstream applications in regenerative biology, disease modeling, and pharmacological testing.

[0082] FIG. 7 illustrates a representation of a portion of the human gastrointestinal tract 700, focusing on the small and large intestinal regions to better emphasize the digestive tract components relevant to tissue sampling, device placement, and / or gastrointestinal analysis. The duodenum 702 is depicted as the proximal segment of the small intestine that receives partially digested food from the stomach. It is continuous with the jejunum 704, which forms the middle segment of the small intestine and is generally involved in the absorption of nutrients. The ileum 706 follows the jejunum and terminates at the cecum 708, forming the distal portion of the small intestine. The cecum 708 is shown as a pouch-like structure that marks the transition between the small and large intestines. The colon 710, a component of the large intestine, extends from the cecum and is responsible for water reabsorption and fecal consolidation.

[0083] TISSUE PROCESSING METHODS FOR A NHP GI TRACT SAMPLE IN A STERILELABORATORY SETTING-TISSUE RECOVERY.

[0084] In an example tissue processing step for a NHP GI tract sample in a sterile laboratory setting in which one or more tubes containing GI tract tissue from an NHP are submerged in a cold washing buffer solution. Following tissue recovery, the GI tract samples were transferred into sterile conical tubes and placed on ice to maintain viability and prevent enzymatic degradation. In some embodiments, GI tract tissues are recovered from a euthanized NHP, such as a Cynomolgus macaque, at the collaborating facility. In some embodiments, a segment of the gastrointestinal (GI) tract tissue measuring approximately 2.5 cm, or alternatively within a range of about 1.5 cm to about 4 cm, is excised from each of five anatomical regions: the duodenum, jejunum, ileum, cecum, and colon. Each excised segment is subjected to two sequential rinses with a sterile saline buffer, or in some cases three or more rinses, to remove residual luminal contents and tissue debris. Following the rinsing step, the tissue is transferred into a 50 mL conical tube, which may contain between about 20 mL and about 50 mL of a preservation medium. The sample is then shipped under controlled temperature conditions, using cold packs maintained at approximately 0°C to 8°C, and in some embodiments between about 1°C and about 5°C, to preserve tissue integrity during overnight transport. Upon arrival, the samples are unpacked and immediately placed on ice and maintained at low temperature, for example about 0°C to about 4°C, during subsequent processing steps.

[0085] In some embodiments, the conical tubes are partially filled, for example with a volume of about 10 mL to about 40 mL of a washing buffer comprising phosphate-buffered saline (PBS; Corning Cat# 21-040-CV), about 0.05% to about 0.2% fetal bovine serum (FBS; Sigma-Aldrich Cat# F7524), and about 2.5 mL to about 10 mL of antibiotic-antimycotic solution at 100X concentration (Gibco Cat# 15240062). The washing buffer is formulated to reduce microbial contamination while maintaining physiological osmolarity and pH stability. In some embodiments, the preparation of an optimized expansion medium may include a media base supplemented with one or more essential growth factors, signaling molecules, and / or metabolic stabilizers. This medium may be formulated to promote epithelial stem cell proliferation and / or preserve the undifferentiated state necessary for establishing and / or expanding GI organoids in vitro.

[0086] EXPANSION MEDIUM COMPOSITIONS

[0087] In some embodiments, the composition of the expansion medium may be prepared using amedia base for the culture and maintenance of NHP GI organoids. This medium is formulated to support stem cell viability, proliferation, and early organoid formation by incorporating a set of supplements at specified working and final concentrations. In some embodiments, the medium includes approximately 2 mb of B-27 supplement (serum-free, 50* stock) to achieve a final concentration of about lx, or optionally from about 0.5* to about 2*, about 0.75* to about 1.25x, or about 0.8* to about 1.5*. The Wnt surrogate (2.04 mg / mL, equivalent to 17.1 pM) may be added in an amount of approximately 2.9 pL to yield a final concentration of about 0.5 nM, or optionally within a range of about 0.05 nM to about 2.0 nM, about 0.1 nM to about 1.0 nM, about 0.25 nM to about 0.75 nM, or about 0.3 nM to about 0.6 nM.

[0088] Approximately 400 pL of R-spondin 1-conditioned medium (0.25 mg / mL) may be added to reach a final concentration of about 0.001 mg / mL, or optionally from about 0.0001 mg / mL to about 0.005 mg / mL, about 0.0005 mg / mL to about 0.002 mg / mL, or about 0.0008 mg / mL to about 0.0012 mg / mL. Approximately 100 pL of Noggin-conditioned medium (100 pg / mL) may be included to yield a final concentration of about 100 ng / mL, or within a range of about 25 ng / mL to about 200 ng / mL, about 50 ng / mL to about 150 ng / mL, or about 90 ng / mL to about 110 ng / mL. To support redox balance, the medium may include approximately 100 pL of N-acetylcysteine (1.25 M) to achieve a final concentration of about 1.25 mM, or alternatively from about 0.5 mM to about 2.0 mM, about 0.75 mM to about 1.75 mM, or about 1.0 mM to about 1.5 mM. Nicotinamide (5 M) may be added at approximately 200 pL to reach a final concentration of about 10 mM, or within a range of about 2.5 mM to about 15 mM, about 5 mM to about 12.5 mM, or about 8 mM to about 12 mM.

[0089] The medium may further include about 40 pL of the p38 MAPK inhibitor SB202190 (25 mM) to yield a final concentration of about 10 pM, or optionally from about 5 pM to about 20 pM, about 7.5 pM to about 15 pM, or about 9 pM to about 11 pM. Epidermal growth factor (EGF; 50 pg / mL) may be added in an amount of approximately 100 pL to achieve a final concentration of about 50 ng / mL, or within a range of about 10 ng / mL to about 100 ng / mL, about 25 ng / mL to about 75 ng / mL, or about 40 ng / mL to about 60 ng / mL. To modulate TGF-P signaling, the medium may include about 5 pL of ALK5 inhibitor A83-01 (10 mM) to achieve a concentration of about 0.5 pM, or optionally from about 0.1 pM to about 1.0 pM, about 0.25 pM to about 0.75 pM, or about 0.4 pM to about 0.6 pM. Furthermore, about 1 pL of prostaglandin E2 (100 mM) may be added to yield a concentration of about 1 pM, or within a range of about 0.25 pM to about 2.0 pM,about 0.5 pM to about 1.5 pM, or about 0.8 pM to about 1.2 pM. The final medium may be prepared and maintained on ice to preserve the integrity of temperature-sensitive factors and must be assembled under sterile conditions using a Biosafety Cabinet (BSC) Type 2 to provide for an aseptic technique. This expansion medium provides a biochemical environment optimized for sustaining the undifferentiated, proliferative state of GI organoids and may be utilized for downstream applications including passaging, cryopreservation, and / or experimental manipulation.

[0090] Following the cold-chain transport and initial stabilization of the NHP GI tract tissue and the transferring the preserved tissue samples to a sterile laboratory setting for dissection and downstream processing, the tissue segments are carefully unpacked, rinsed with cold supplemented buffer, and prepared for region-specific handling.

[0091] TISSUE PROCESSING METHODS FOR A NHP GI TRACT SAMPLE IN A STERILE LABORATORY SETTING-DISSECTION AND DOWNSTREAM PROCESSING.

[0092] In some embodiments, blunt-end tweezers are employed to transfer gastrointestinal (GI) tissue to a tissue culture dish having a diameter of approximately 10 cm, or optionally from about 6 cm to about 12 cm, about 8 cm to about 11 cm, or about 9 cm to about 10.5 cm. The tissue may be trimmed laterally by about 1 mm to about 5 mm, about 2 mm to about 4 mm, or about 2.5 mm to about 3.5 mm on each side and inspected to identify and remove fat deposits or visibly damaged portions. In some embodiments, terminal ends are excised to a length of about 1 mm to about 10 mm, about 3 mm to about 8 mm, or about 5 mm to about 7 mm and embedded in an optimal cutting temperature (OCT) compound for histological and / or immunological analysis.

[0093] The remaining trimmed tissue portions may be divided into three substantially equal parts, each ranging in volume from about 0.2 mL to about 0.6 mL, about 0.3 mL to about 0.5 mL, or about 0.4 mL to about 0.45 mL, and each placed into an individual sterile microcentrifuge tube having a working volume of approximately 1.5 mL, or alternatively from about 1.0 mL to about 2.0 mL, about 1.2 mL to about 1.7 mL, or about 1.4 mL to about 1.6 mL. Each sample is then designated for molecular extraction of DNA, RNA, or protein, respectively. In some embodiments, a tissue processing step for the NHP GI tract samples may be implemented in a sterile laboratory setting. Using blunt-end tweezers, the NHP GI tissue is carefully removed from the approximately 50 mL conical tube and placed into a sterile approximately 10 mm tissue culture dish. Immediately, 10 mL of cold washing buffer is added over the tissue to prevent desiccation, as the GI tissueremains hydrated. The tissue is then examined for structural integrity, and any visible fat is trimmed away using blunt-end scissors (tijeras). Once cleaned, the tissue is transferred to a fresh approximately 10 mm dish for further handling. A second 10 mL aliquot of washing buffer is used to rinse the internal lumen of the tissue by flushing the buffer through the GI tract. The cleaned tissue is then moved to a sterile approximately 10 cm tissue culture dish. At this point, the ends of the GI tissue are removed with scissors or a sterile blade; one end may be discarded, while another may be preserved by embedding in optimal cutting temperature (OCT) compound for future histological or immunological analysis. Following this, two additional transverse cuts are made along the GI tube, and those tissue pieces are transferred to approximately 1.5 mL microcentrifuge tubes for subsequent genomic DNA, RNA, and protein extraction.

[0094] To complete the rinsing process, the opened gastrointestinal (GI) tract tissue is washed in one or more conical tubes, each containing approximately 20 mL to approximately 40 mL, such as approximately 25 mL to approximately 30 mL of cold washing buffer. The tissue may be opened longitudinally, for example along its anti-mesenteric border, using blunt-end scissors to expose the internal surface. Using forceps or tweezers, the opened tissue is submerged and agitated, such as by stirring or gentle inversion, in a first conical tube, then transferred to a second conical tube for a final rinse. In some embodiments, three or more sequential washes may be used to ensure adequate removal of debris. Following the rinsing process, the tissue is transferred to a clean tissue culture dish, such as a standard polystyrene culture dish having a diameter of approximately 9 cm to approximately 12 cm, preferably about 10 cm. The rinsed tissue is then weighed, and in certain embodiments, tissue samples falling within a target weight range of approximately 75 mg to approximately 1,000 mg, such as approximately 100 mg to approximately 800 mg, or more preferably approximately 150 mg to approximately 700 mg, may be selected to proceed with organoid processing. This workflow provides the intestinal region, duodenumjejunum, ileum, cecum, and / or colon, is processed under consistent, sterile conditions to maintain tissue integrity and biological relevance for future organoid derivation and comparative studies.

[0095] In some embodiments, an example tissue processing step for a NHP GI tract sample in a sterile laboratory setting after rinsing, includes the tissue being weighed and visually inspected for integrity, and any visible fat is removed. The tissue is then trimmed laterally to isolate clean, uniform segments. The terminal ends of the trimmed GI tissue are collected and transferred to anembedding mold, where they are covered with optimal cutting temperature (OCT) compound for future histological and immunological analyses. The remaining trimmed portions are subdivided into three separate fragments and placed into individual approximately 1.5 mL, such as about 1.0 mL to about 2.0 mL, about 1.2 mL to about 1.8 mL, or about 1.4 mL to about 1.6 mL, microcentrifuge tubes for DNA, RNA, and protein extraction. This step provides both the preservation of anatomical material for structural analysis and the availability of matched molecular samples from each GI region. Upon completion of the tissue trimming and molecular allocation steps, the remaining viable segments of the NHP GI tract tissue are prepared for organoid derivation.

[0096] In some embodiments, an example tissue processing step for a NHP GI tract sample in a sterile laboratory setting includes the mechanically disassociated NHP GI tract tissue being retained in an approximately 10 cm, such as from about 8 cm to about 12 cm, about 9 cm to about 11 cm, or about 9.5 cm to about 10.5 cm, tissue culture dish during enzymatic processing. The remaining trimmed NHP GI tract tissue is placed into a sterile approximately 10 cm tissue culture dish and submerged in approximately 5 mL of digestive tissue buffer. Using a sterile scalpel or scissors, the tissue is mechanically disassociated until reduced to small fragments of approximately 1 mm3in size. Additional digestive buffer may be added during this process to ensure the tissue remains immersed and to facilitate thorough mechanically dissociating. The mechanically dissociated tissue is then transferred into a conical tube, for example an approximately 50 mL conical tube, using a serological pipette, for example an approximately 5 mL serological pipette. To maximize tissue recovery, the tissue culture dish may be rinsed approximately two to five times, for example three times, with an aliquot of digestive buffer, such as about 3 mL to about 7 mL per rinse, and the rinsates are combined into the same conical tube. The tube may then be incubated at a temperature ranging from about 35°C to about 39°C, preferably about 37°C, in an orbital shaker. The incubation period may range from about 20 minutes to about 60 minutes, such as from about 25 minutes to about 50 minutes, or about 30 minutes to about 45 minutes. During this incubation, the orbital shaker may operate at a speed of about 50 rpm to about 150 rpm, optionally about 80 rpm to about 120 rpm, providing gentle agitation to ensure uniform exposure of the tissue fragments to the digestive buffer. This promotes enzymatic dissociation of the tissue, enhancing the yield and consistency of the dissociated cellular material suitable for downstream organoid culture or isolation procedures. Upon completion of the mechanically dissociating and enzymaticdigestion steps, the resulting tissue suspension is prepared for cell isolation and purification. The digested tissue, now in suspension within the digestive buffer, contains a heterogeneous mix of dissociated epithelial fragments, stem cells, and residual debris. To separate viable organoidforming cells from undigested tissue and larger particulates, the sample is subjected to a filtration process. This next stage utilizes conical tubes equipped with one or more cell strainers to facilitate the passage of dissociated cells while retaining larger tissue fragments. This transition from enzymatic digestion to mechanical filtration ensures that the cell suspension is enriched for viable, single-cell and small-cluster populations suitable for organoid culture.

[0097] In some embodiments, an example tissue filtration and cell isolation step may be implemented during the preparation of organoids derived from NHP GI tract tissue. Two conical centrifuge tubes, each having a capacity of about 45 mL to about 55 mL (e.g., approximately 50 mL), and each fitted with a cell strainer having a mesh size ranging from about 70 pm to about 120 pm (e.g., approximately 100 pm), are pre-positioned to facilitate filtration of digested tissue samples. A volume of enzymatically digested gastrointestinal (GI) tissue ranging from about 8 mL to about 20 mL (e.g., approximately 12 mL, or alternatively about 10 mL to about 15 mL) is withdrawn from the digestion vessel and transferred into the first strainer-equipped tube using a sterile serological pipette, such as a pipette ranging from about 5 mL to about 15 mL (e.g., approximately 10 mL), attached to a calibrated pipet aid. During transfer, the mixture may be subjected to a gentle orbital motion with a speed ranging from about 20 rpm to about 100 rpm (e.g., approximately 60 rpm) to promote consistent flow through the strainer. Residual tissue in the original digestion container is then rinsed with about 5 mL to about 15 mL (e.g., approximately 10 mL) of a pre-warmed base medium (e.g., A medium) supplemented with about 10 pM to about 30 pM (e.g., approximately 20 pM) of TZV or a comparable ROCK inhibitor. This rinse solution is likewise passed through the initial strainer. Optionally, one to three wash cycles may be performed, wherein each cycle consists of aspirating and dispensing about 5 mL to about 12 mL of fresh A medium supplemented with about 15 pM to about 25 pM TZV over the partially digested tissue fragments to enhance disaggregation and maximize cell release.

[0098] Following the digestion and wash steps, the combined digestion mixture may be allowed to settle undisturbed for a period ranging from about 30 seconds to about 5 minutes (e.g., approximately 1 to 3 minutes) to permit separation of larger debris. The supernatant, enriched with dissociated cells, is carefully decanted or aspirated while avoiding large residual fragments. Thisclarified supernatant is then passed through a second conical tube fitted with a strainer having a mesh size of about 80 pm to about 110 pm (e.g., 100 pm) using an orbital pipetting technique as previously described. The filtrates from both tubes are optionally pooled into a final 50 mL conical centrifuge tube (e.g., ranging from about 45 mL to about 55 mL in capacity), securely capped, and maintained at a temperature of about 0°C to about 8°C (e.g., on ice) pending centrifugation or further downstream processing.

[0099] The pooled cell suspension may be centrifuged at a relative centrifugal force (RCF) ranging from about 300 x g to about 600 x g, such as about 350xg to about 550 x g;or about 400 x g to about 500 x g, for a duration ranging from about 3 minutes to about 10 minutes, such as about 4 minutes to about 8 minutes, or about 5 minutes to about 7 minutes, in a refrigerated centrifuge maintained at a temperature ranging from about 2°C to about 8°C, such as about 3 °C to about 6°C, or approximately 4°C. Following centrifugation, the supernatant is aspirated, leaving a concentrated cell pellet at the bottom of the conical tube. In some embodiments, the cell pellet exhibits a reddish coloration, indicative of residual erythrocyte contamination when a red blood cell lysis procedure is initiated. The pellet is resuspended in a volume of red blood cell lysis buffer ranging from about 1 mL to about 5 mL, such as about 2 mL to about 4 mL, or approximately 3 mL, and incubated at ambient temperature ranging from about 18°C to about 25°C, such as about 20°C to about 24°C, or approximately 22°C, for a duration ranging from about 2 minutes to about 10 minutes, such as about 3 minutes to about 7 minutes, or approximately 5 minutes.

[0100] Following the lysis step, a volume of Media base supplemented with 20 pM TZV is added, wherein the volume may range from about 3 mL to about 7 mL, such as about 4 mL to about 6 mL, or approximately 5 mL. The tube is centrifuged again under previously described conditions (e g., about 450 x for 5 minutes at 4°C). The supernatant is discarded, and the cell pellet is subjected to a wash step. The washing process is repeated one or more times, such as two to four times, or preferably three times, each with a volume of fresh Media base supplemented with 20 pM TZV, where the volume of wash medium may range from about 8 mL to about 15 mL, such as about 9 mL to about 12 mL, or approximately 10 mL per wash. Each wash step may include centrifugation at a force ranging from about 350 x g to about 500 x g, such as about 400xg to about 475zg, for a time period ranging from about 3 minutes to about 8 minutes, such as about 4 minutes to about 6 minutes, at a temperature ranging from about 2°C to about 6°C. The pellet isinspected after each spin, and if red discoloration persists, additional lysis treatment is performed before continuing with downstream organoid culture steps. Following the fdtration, centrifugation, and red blood cell lysis steps, the resulting cell pellet, enriched for viable epithelial and stem cell populations, is prepared for embedding in extracellular matrix for organoid culture. The pellet is washed multiple times with cold Media base supplemented with approximately 20 pM TZV, and a small residual volume of medium (approximately 20 pL to approximately 30 pL) is retained to keep the cells hydrated. The washed pellet is then transferred to an approximately 1.5 mL microcentrifuge tube and kept on ice. Cold Matrigel, in a volume ranging from approximately 300 pL to approximately 600 pL depending on the original tissue input, is added to the tube to form a cell-Matrigel suspension. This step marks the transition from preparative tissue processing to the final stage of plating and initiating 3D culture for organoid generation.

[0101] In some embodiments, an example tissue processing step for a NHP GI tract sample in a sterile laboratory setting may be implemented in which one or more conical tubes retain postcentrifugation pellets of dissociated NHP GI tract cells from one or more regions, including but not limited to, a colon, an ileum, a jejunum, a cecum, and / or a duodenum, and are kept on ice prior to mixing with Matrigel for organoid culture. The preparation of resuspended organoid-forming cells derived from different anatomical regions of the NHP gastrointestinal tract follows cell isolation and washing steps. Following the final wash step using Media base supplemented with 20 pM TZV, care is exercised to avoid disruption or aspiration of the cell pellet. A residual volume of wash medium ranging from about 15 pL to about 40 pL, such as from about 18 pL to about 35 pL, or from about 20 pL to about 30 pL, may be intentionally retained in the conical tube to ensure adequate hydration and cohesion of the pellet. The hydrated pellet is then transferred to a sterile microcentrifuge tube having a nominal volume of approximately 1.5 mL, using a low-retention pipette tip to minimize sample loss and preserve cell integrity. The microcentrifuge tube is maintained on ice, or at a temperature ranging from about 0°C to about 4°C, such as about 1°C to about 3 °C, or approximately 2°C, to preserve cellular viability and inhibit premature gelation.

[0102] To embed the cells for subsequent plating or culture, a volume of cold basement membrane matrix material, such as Matrigel, may be added to the microcentrifuge tube. The volume of Matrigel may range from about 200 pL to about 800 pL, such as about 250 pL to about 700 pL, or about 300 pL to about 600 pL, depending on the mass of starting tissue. In some embodiments, when the initial gastrointestinal (GI) tissue mass is approximately 300 mg toapproximately 500 mg, such as about 350 mg to about 450 mg, or approximately 400 mg, the Matrigel volume added may be about 1,000 pL to about 1,400 pL, such as about 1,100 pL to about 1,300 pL, or approximately 1,200 pL. The resulting mixture is gently pipetted to achieve a homogeneous suspension while minimizing bubble formation or shear-induced damage to the cells. The cell pellet is resuspended with the Matrigel, providing uniform mixing while avoiding air bubbles, which can interfere with downstream plating and organoid formation. The steps are performed on ice to maintain the gel in a liquid state and preserve the physiological integrity of the cells. Once the cell-Matrigel suspension is prepared, it is ready for seeding into 3D structures on culture plates for in vitro organoid development. After mixing the NHP GI tract cell pellet with cold Matrigel, the resulting suspension is ready for plating into culture wells to initiate organoid formation. This cell-Matrigel mixture must be handled carefully to maintain homogeneity and prevent premature polymerization. As illustrated in FIG. 8, the suspension is dispensed as discrete approximately 15 pL 3D structures onto the surface of a pre-warmed 6-well tissue culture plate. The even spacing of 3D structures within each well facilitates uniform growth conditions and minimizes the risk of 3D structure merging. This plating step marks the beginning of the 3D culture phase during which the embedded cells begin to self-organize into NHP GI organoids under controlled incubation conditions.

[0103] FIG. 8 illustrates an example tissue processing step 800 for a NHP GI tract sample in a sterile laboratory setting in which the plating stage for initiating organoid cultures using resuspended NHP GI tract stem cells embedded in Matrigel. Prior to plating, a tissue culture plate having six wells is pre-incubated at a temperature ranging from about 35°C to about 40°C, such as about 36°C to about 38°C, or approximately 37°C, to optimize the surface temperature for Matrigel polymerization. A precision pipettor (e.g., a P20 pipette) is configured to dispense volumes ranging from about 10 pL to about 20 pL, such as about 12 pL to about 18 pL, or approximately 15 pL. The pipettor is used to deposit dome-shaped drops of a cell-Matrigel suspension onto the surface of the pre-warmed plate.

[0104] The dome-shaped structures may be spaced at a minimum distance of about 2 mm to about 10 mm, such as about 3 mm to about 8 mm, or about 5 mm apart, to prevent fusion of adjacent domes, which could compromise organoid integrity and viability. The cell-Matrigel suspension is mixed to a uniform consistency prior to deposition to minimize heterogeneity in cell distribution and structural collapse. In some embodiments, the mixing is performed on ice for aduration ranging from about 30 seconds to about 2 minutes to maintain Matrigel in a liquid state. Following plating, the 6-well culture plate is incubated in an upright orientation at a temperature ranging from about 35°C to about 40°C, such as approximately 37°C, and under a controlled carbon dioxide environment ranging from about 4% CO? to about 6% CO2, or approximately 5% CO2. The incubation duration may range from about 8 minutes to about 15 minutes, such as about 10 minutes to promote initial Matrigel gelation. Thereafter, the culture plate is inverted and incubated for an additional period ranging from about 8 minutes to about 15 minutes, such as about 10 minutes, to facilitate uniform 3D structural stabilization.

[0105] Once polymerization is complete, an organoid expansion medium is added to each well. In some embodiments, the medium volume may range from about 1.5 mL to about 2.5 m , such as about 1.8 mL to about 2.2 mL, or approximately 2 mL per well. The expansion medium includes NHP Organoid Medium (e.g., Method A orB) supplemented with a concentration of TZV ranging from about 15 pM to about 25 pM, such as about 18 pM to about 22 pM, or approximately 20 pM. The culture is returned to the incubator and maintained under the aforementioned temperature and CO2 conditions. The medium may be refreshed every 1 to 3 days, or about every other day. Organoids are monitored daily for viability, growth, and morphological characteristics. Passaging is performed after about 5 to 9 days, such as about 6 to 8 days, or approximately 7 days, or upon observation of exponential growth.

[0106] For passaging, the existing organoid expansion medium is aspirated, and each well is rinsed with a chilled Media base supplemented with TZV at a concentration of about 8 pM to about 12 pM, such as about 10 pM. A volume ranging from about 4 mL to about 6 mL, such as approximately 5 mL, is used for the initial rinse and collection. Mechanical disruption is performed using either a pipette or a sterile glass Pasteur pipette. The disrupted suspension is transferred into a conical tube having a nominal volume capacity of about 15 mL. An additional rinse of the original well is performed using a volume of Media base ranging from about 1 mL to about 3 mL, such as about 2 mL, which is combined with the primary suspension. The conical tube is incubated on ice for a period ranging from about 5 minutes to about 15 minutes, such as approximately 10 minutes. The suspension is then centrifuged at a force ranging from about 400xg to about 500 x g, such as approximately 450 x g, for a duration of about 4 minutes to about 6 minutes, such as approximately 5 minutes, in a refrigerated centrifuge maintained at a temperature of about 2°C to about 6°C, such as about 4°C.

[0107] Following centrifugation, the supernatant and any residual Matrigel are removed. When Matrigel remnants persist, one or more additional wash steps may be performed using Media base supplemented with TZV. The cell pellet is retained in a residual medium volume ranging from about 15 pL to about 35 pL, such as about 20 pL to about 30 pL, and transferred to a microcentrifuge tube having a capacity of approximately 1.5 mL, maintained on ice. The cells are then resuspended in a volume of cold Matrigel ranging from about 250 pL to about 700 pL, such as about 300 pL to about 600 pL, and the suspension is mixed to homogeneity. Subsequent plating includes reapplying one or more dome structures as described above, using volumes of approximately 15 pL per dome, spaced evenly across a pre-warmed 6-well plate. The plate undergoes a two-stage incubation protocol: approximately 10 minutes in the upright position followed by approximately 10 minutes in the inverted position. Following polymerization, approximately 2 mL of NHP Organoid Expansion Medium is added per well, and the plate is returned to the incubator for continued organoid propagation. This protocol enables reproducible expansion of NHP GI organoids across successive passages while maintaining structural integrity and cellular viability.

[0108] In some embodiments, a tissue processing step for a NHP GI tract sample is conducted in a sterile laboratory setting, wherein a conical tube having a capacity of approximately 15 mL to approximately 50 mL, such as about 20 mL to about 30 mL, retains a cryopreservation medium and a suspension of NHP GI organoids during a cryogenic preservation stage of the organoid culture protocol. The cryopreservation protocol is initiated following an organoid expansion phase in a 3D matrix environment, such as Matrigel or a functionally equivalent ECM substitute. Freezing is preferably carried out when the organoids reach the exponential growth phase, observed within a window of approximately 2 days to approximately 4 days post-passaging, such as about 2.5 days to about 3.5 days. At this growth stage, the organoids are harvested by mechanical disruption of the 3D matrix using pipetting with low-retention tips or a sterile glass Pasteur pipette. The disrupted suspension is transferred into a conical tube and subjected to centrifugation at a relative centrifugal force (RCF) of approximately 400 x g to approximately 500 x g, such as about 450 x g, for a duration of about 3 minutes to about 7 minutes, for example about 5 minutes, at a temperature ranging from about 2°C to about 6°C, such as about 4°C, to pellet the organoids.

[0109] After centrifugation, the supernatant is removed using vacuum aspiration orpipetting, taking care not to disturb the organoid pellet. The pellet is resuspended in a chilled media base, such as a base medium, supplemented with a neuroprotective or stability-enhancing agent, such as TZV (Tanzisertib) at a concentration of approximately 8 pM to approximately 12 pM, or about 10 pM. The volume of media base used for resuspension may range from about 100 pL to about 1,000 pL, such as about 300 pL to about 600 pL, depending on pellet size and desired concentration. Subsequently, an equal volume of a cryopreservation medium is added to the cell suspension. The cry opreservation medium may include about 8% to about 12% dimethyl sulfoxide (DMSO), such as about 10% DMSO, in a base solution of fetal bovine serum (FBS), bovine serum albumin (BSA), or a chemically defined serum substitute. The combined volume, about 600 pL to about 1,200 pL, is gently mixed to achieve a homogenous 1 :1 ratio of media base to cryopreservation medium. This formulation provides a cryoprotective environment to mitigate osmotic shock and reduce ice crystal formation during the controlled rate freezing process.

[0110] The mixture is aliquoted into cryovials having a volume of about 1 mL to about 2 mL, such as about 1.5 mL, and loaded into a freezing container designed to achieve a temperature decrease rate of approximately -0.5°C / min to -1.5°C / min, such as approximately -1.0°C / min, until a final temperature of approximately -80°C is reached. Following an overnight incubation at - 80°C, the cryovials are transferred to liquid nitrogen storage tanks maintained at temperatures below approximately -150°C for long-term preservation of organoid viability and phenotype. This method supports standardized cryopreservation of NHP GI organoids for downstream recovery and continued culture, including applications in regenerative medicine, disease modeling, and pharmacological screening.

[0111] The resuspended NHP GI organoids are aliquoted into sterile cryogenic vials, each having a volume capacity of approximately 1 mL to approximately 2 mL, such as about 1.5 mL. The cryovials are then placed into a controlled-rate freezing container, such as a passive cooling device configured to achieve a temperature descent rate of approximately -0.5°C per minute to approximately -1.5°C per minute, for example approximately -1.0°C per minute. In some embodiments, the freezing container may be a commercially available device such as a Mr. Frosty™ or a functionally equivalent passive cooling chamber filled with an isopropanol-based thermal buffering solution. The freezing container is subsequently transferred into a mechanical freezer set at a temperature ranging from approximately -70°C to approximately -90°C, such as approximately -80°C. The cryovials are maintained in this environment for a duration ofapproximately 16 hours to approximately 36 hours, such as about 20 hours to about 28 hours, and preferably about 24 hours, to allow gradual cooling and prevent intracellular ice formation that could compromise organoid viability. Following the initial freezing phase, the cryovials are transferred to a cryogenic storage system, such as a vapor-phase or liquid-phase liquid nitrogen (LN2) tank, maintained at temperatures below approximately -135°C, such as from approximately -140°C to approximately -196°C, to enable long-term biopreservation.

[0112] This staged cooling protocol ensures the structural and functional integrity of the organoids is preserved for subsequent thawing and re-culture applications. After the initial freezing step, the cryovials are transferred to a liquid nitrogen (approximately- 196°C) cryo-storage tank for long-term preservation. This cry opreservation strategy ensures high viability upon thawing and enables future expansion, banking, and / or downstream experimental applications of the NHP GI organoids. Following the plating, expansion, and observation of NHP GI organoids, the resulting cell pellets are obtained after centrifugation and undergo a preparatory step for cryopreservation. Once the organoids reach exponential growth, about 2 days to about 3 days post-passaging or initial plating, this pellet indicates readiness for long-term storage. The transition from expansion to cryostorage is a phase for maintaining viability and regenerative capacity of the organoids. As shown, the pellet is resuspended in cryoprotective medium, and the following protocol details the specific steps for freezing and preserving the organoids under controlled conditions to support subsequent recovery and downstream applications.

[0113] FREEZE ORGANOIDS PROTOCOL.

[0114] In some embodiments, NP GI organoids may be cryopreserved using a defined freezing protocol that maximizes recovery, viability, and preservation of stem-like cellular characteristics. A 2* freezing medium is prepared by combining approximately 5 mL to approximately 15 mL of dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, Cat# D2650) with approximately 35 mL to approximately 45 mL of fetal bovine serum (FBS) (e.g., Sigma-Aldrich, Cat# F7524), yielding a total volume of approximately 40 mL to approximately 60 mL. The prepared freezing medium may be stored at a temperature ranging from approximately 2°C to approximately 8°C, such as at approximately 4°C, for a duration of up to approximately 30 days, preferably between approximately 7 days to approximately 28 days.

[0115] Cryopreservation may occur at one of two distinct stages. In one embodiment,organoids are cryopreserved during an early exponential growth phase, between approximately 1.5 days and approximately 4 days post-initial plating, such as about 2 to 3 days, when the organoids remain small and morphologically compact, reflecting a high proportion of stem-like cells. Alternatively, organoids may be frozen approximately 2 to approximately 4 days following passaging, when re-entering exponential growth and prior to lumen expansion or cellular differentiation. To initiate freezing, the organoid culture medium is aspirated from the well plate. A P1000 pipette is then used to flush the Matrigel 3D structures with approximately 0.75 mb to approximately 1.5 mb of cold organoid media base supplemented with approximately 10 pM to approximately 30 pM of the ROCK inhibitor TZV, such as about 20 pM. Pipetting is repeated approximately 5 to 10 times to mechanically dissociate the Matrigel matrix and release organoids. The resulting suspension is transferred into a sterile conical tube (e.g., approximately 10 mL to approximately 15 mL capacity) prefilled with approximately 4 mL to approximately 6 mL of cold Media base with TZV. The well is rinsed with an additional approximately 4 mL to approximately 6 mL of Media base with TZV to ensure maximal cell recovery, and the rinse is combined into the same conical tube.

[0116] To enhance matrix dissociation, the suspension is further disrupted using a sterile glass Pasteur pipette and incubated on ice for approximately 8 to approximately 12 minutes. The conical tube is then centrifuged at a relative centrifugal force (RCF) of approximately 400 x g to approximately 500 x g, such as about 450 x g, for a duration of approximately 4 minutes to approximately 6 minutes, such as about 5 minutes, at a temperature of approximately 2°C to approximately 8°C, preferably about 4°C. The supernatant and visible Matrigel residues, identifiable as a cloudy upper layer, are aspirated without disturbing the organoid pellet. If residual Matrigel remains, a secondary wash step may be performed using approximately 15 mL to approximately 25 mL of cold media base supplemented with about 5 pM to about 15 pM TZV, followed by another centrifugation under identical conditions. After the final wash, the pellet is resuspended in a 1 : 1 ratio of media base with TZV and 2 freezing medium. For instance, if approximately 450 pL to approximately 550 pL of media base with TZV is used, an equal volume (e g., about 450 pL to about 550 pL) of the 2 freezing medium is added, yielding a final suspension volume of approximately 0.9 mL to approximately 1.1 mL. The mixture is gently pipetted to ensure homogeneity without introducing bubbles, and aliquots of approximately 0.8mL to approximately 1.2 mL are dispensed into individually labeled cryogenic storage vials having a volume of approximately 1.0 mL to approximately 2.0 mL.

[0117] The cryovials are then transferred into a passive-rate freezing container, such as a controlled-rate passive cooling device configured to achieve a cooling rate of approximately -0.5°C to approximately -1.5°C per minute, preferably about -1.0°C per minute. The container is placed into a mechanical freezer maintained at approximately -75°C to approximately -85°C, such as -80°C, for a duration of approximately 18 hours to approximately 30 hours, preferably about 24 hours. Following this freezing interval, the vials are transferred to long-term storage in a vapor-phase or liquid-phase liquid nitrogen tank, maintained at a temperature of approximately -135°C to approximately -196°C, such as about -150°C. This protocol facilitates the preservation of proliferative and stem-like NHP GI organoids suitable for future expansion, differentiation, and downstream functional analysis. Subsequent sections of the disclosure describe techniques for passaging and expanding the organoids, including conditions for 3D structure dissociation, pellet recovery, Matrigel recombination, and replating in a defined organoid expansion medium.

[0118] PASSAGE & EXPANSION OF NHP GI ORGANOIDS.

[0119] The following section describes, in accordance with some embodiments, a technical process for the passage and expansion of NHP GI organoids. Passage of organoid cultures is performed to maintain viability, support expansion, and preserve the stem / progenitor phenotype of the organoids. In some embodiments, the timing of passage is determined based on morphological cues such as organoid confluency, density, and exponential growth phase, observed approximately seven days following initial seeding or previous passage, subject to donor-specific variability. The passage process includes mechanical dissociation and enzymatic disruption of organoid-containing Matrigel 3D structures using ice-cold Advanced Dulbecco’s Modified Eagle Medium (AdvDMEM+++) supplemented with 20 pM TZV. Following 3D structure disruption, the organoid suspension is collected into about 15 mL conical tubes, washed to remove residual ECM components, and centrifuged at 450 x g at 4°C for about 5 minutes. The resulting pellet is resuspended in a minimal volume (e.g., 20-30 pL) of cold Media base+ 20 pM TZV, and combined with about 300 pL to about 600 pL of ice-cold Matrigel to form a homogenous mixture. In some embodiments, about 15 pL aliquots of the Matrigel -organoid suspension are plated as discrete 3D structures into a 6-well plate pre-equilibrated to about 37°C. 3D structures are spatially separated to avoid fusion during polymerization. Plates are incubated about at 37°C for about 10minutes in an upright orientation followed by about 10 minutes in an inverted orientation to promote uniform Matrigel solidification. Subsequently, about 2 mL of NHP organoid expansion medium (Method A or Method B) supplemented with about 20 pM TZV is added to each well, and the cultures are returned to a humidified incubator set to 37°C and 5% CO2. Organoid cultures are maintained by feeding every other day. The protocol described herein enables reliable expansion and maintenance of viable, proliferative organoids suitable for downstream applications such as cryopreservation, differentiation, or experimental assays.

[0120] When the NHP GI organoids are ready for passage and expansion depends on donor-to-donor variability, but on average, the process occurs approximately every 7 days. The indicator for initiating passage is confluency, when the organoid density reaches a level that limits further expansion within the existing Matrigel 3D structures. To begin, aspirate the organoid expansion medium and flush the 3D structures using 1 mL of cold Media base containing 20 pM TZV with a Pl 000 pipette. Disrupt the 3D structures by pipetting up and down and transfer the suspension to a 15 mL conical tube containing 5 mL of the same cold medium. Rinse the original well with an additional 5 mL of cold Media base+ 20 pM TZV and add it to the same tube. Using a glass Pasteur pipette, further disrupt the Matrigel. Incubate the mixture on ice for 10 minutes before centrifuging at 450 * g for 5 minutes at 4°C to prevent Matrigel solidification. After centrifugation, remove the supernatant and any remaining Matrigel from the top. If substantial Matrigel remains, perform an additional wash using 10 mL of cold Media base+ 20 pM TZV. Carefully remove the cloudy Matrigel without disturbing the organoid pellet. Leave approximately 20-30 pL of medium to resuspend the organoid pellet before adding 300-600 pL of cold Matrigel. For instance, 400 mg of GI tissue uses around 1200 pL of Matrigel for embedding. Resuspend the pellet in Matrigel thoroughly but gently to avoid bubbles and keep the suspension on ice. Using a pre-warmed (37°C) 6-well tissue culture dish, plate 15 pL drops of the cell-Matrigel suspension to create uniform 3D structures. Ensure even spacing to prevent 3D structure merging. If 3D structures collapse, this may indicate an improper ratio of Matrigel to cell suspension or insufficient mixing. Once plating is complete, incubate the plate at 37°C with 5% CO2 for 10 minutes upright, then invert the plate and incubate for another 10 minutes to allow proper 3D structure solidification. After the incubation period, add 2 mL of organoid expansion medium (Method A or B) containing 20 pM TZV to each well and return the plate to the incubator. Feed the organoids every other day to support healthy growth. For long-term storage, freeze theorganoids when they are in the exponential growth phase, about 2 days to about 3 days post -plating, for viability and recovery.

[0121] FIG. 9 illustrates a flowchart 900 depicting an example method for deriving stem cells from GI tract tissue of a NHP. The method is designed to enable the isolation, processing, and expansion of viable stem cells from GI regions including, but not limited to, a duodenum, a jejunum, an ileum, a cecum, small intestine, rectum, large intestine, stomach, and / or a colon of an NHP, such as a Macaco, fascicularis (Cynomolgus macaque). At block 910, the method includes recovering at least a portion of GI tract tissue from the NHP. In some embodiments, this includes excising a segment approximately 2.5 cm in length from a specific region of the GI tract under sterile conditions. The tissue is placed in a preservation or transport medium and maintained at low temperature to inhibit enzymatic degradation prior to further processing, such as at a temperature ranging from about 0 °C to about 10 °C, about 1 °C to about 8 °C, about 2 °C to about 6 °C, or about 3 °C to about 5 °C. At block 920, the recovered tissue is sectioned into one or more discrete regions. This may involve gross dissection to isolate anatomically distinct portions (e.g., small intestine versus large intestine) or to subdivide longer segments for parallel processing. The sectioning is performed using sterile instruments in a biosafety cabinet to ensure contamination-free handling.

[0122] At block 930, the sectioned GI tract tissue is subjected to enzymatic digestion. In some embodiments, the digestion medium includes a tissue dissociation enzyme including, but not limited to, a collagenase, a dispase, a liberase, and / or a combination thereof. The tissue is mechanically disassociated into small fragments (e.g., about 1 mm3) and incubated in the digestion buffer at approximately 37°C for a defined period (e.g., about 30 minutes to about 45 minutes) with gentle agitation, enabling the breakdown of ECM components and facilitating cellular release. At block 940, the digested tissue suspension is filtered to isolate one or more cells. This may be accomplished using one or more sequential cell strainers (e.g., 100 pm mesh size) to remove undigested debris and retain a single-cell or small-cluster population. The filtrate is centrifuged to collect the cellular pellet, and red blood cell lysis may be performed if erythrocyte contamination is observed. At block 950, the isolated cells are cultured in an expansion medium formulated to promote stem cell survival and proliferation. In some embodiments, the expansion medium includes components such as B-27 supplement, Wnt surrogate, R-spondin-1 conditioned medium (Rspol-CM), Noggin, N-acetylcysteine, nicotinamide, EGF, SB202190, A83-01 (ALK5inhibitor), and prostaglandin E2 (PGE2), prepared in a media base, as described elsewhere herein. The cells are embedded in a 3D ECM (e.g., Matrigel) and plated into 3D structures on a tissue culture plate. The culture is maintained under standard conditions (37°C, 5% CO2), and organoid formation is monitored over several days. The method outlined in FIG. 9 provides a reproducible derivation of stem cell populations from primary GI tissue of NHPs, providing a foundational step for downstream applications such as organoid generation, disease modeling, and / or therapeutic screening.

[0123] FIG. 10 illustrates a flowchart 1000 that further refines the method for deriving stem cells as depicted in FIG. 9, by detailing tissue pre-processing techniques that may improve the efficiency of subsequent digestion and cell isolation, in accordance with some embodiments. At block 1010, the method includes mechanically dissociating one or more regions of at least a portion of GI tract tissue. Mechanically dissociating refers to the mechanical fragmentation or mechanically dissociating of the tissue into smaller sections, ranging from about 0.2 mm3to about 5 mm3, for example about 0.2-0.5 mm3, about 0.5—1 mm3, about 1-2 mm3, about 2-3 mm3, or up to about 4-5 mm3, depending on the tissue type and intended application. This process enhances surface area exposure and facilitates more uniform enzymatic digestion. This can be performed using sterile surgical scissors or scalpels under a biosafety cabinet (BSC) to preserve tissue sterility. At block 1020, the method optionally includes vibrating the one or more regions of the GI tract tissue prior to the mechanical dissociation step. Vibrational agitation, such as exposure to low-frequency oscillatory motion or placement on an orbital shaker, is applied to disrupt residual mucus layers, dissociate loosely adherent debris, and / or pre-condition the ECM. This step may be performed while the tissue is submerged in a pre-warmed or chilled buffer solution (e.g., PBS supplemented with antibiotics and serum), depending on the preservation objective. By applying vibration prior to mechanical dissociation, tissue pliability may be enhanced, and enzymatic penetration during digestion may be improved, thereby increasing the yield and viability of the isolated stem cells. Together, the steps of FIG. 10 provide pre-digestion enhancement procedures that, when integrated with the protocol of FIG. 9, can increase the reproducibility, efficiency, and fidelity of stem cell derivation from NHP GI tract tissue. These pre-processing operations are particularly useful in standardizing outcomes across donor variability and regional tissue heterogeneity.

[0124] FIG. 11 illustrates a flowchart 1100 that further elaborates the method of derivingstem cells from NHP GI tissue, as introduced in FIG. 9, by providing an alternative order of mechanical processing steps that may be implemented to improve tissue dissociation efficiency, in accordance with some embodiments. At block 1110, the method includes mechanically dissociating one or more regions of the at least a portion of the GI tract tissue, performed in a sterile tissue culture dish using for example, scissors or a scalpel under aseptic conditions within a biosafety cabinet (BSC) and may be conducted in the presence of a digestive buffer or phosphate- buffered saline (PBS). At block 1120, the method includes vibrating the previously mechanically dissociated GI tract tissue to facilitate further disruption of the ECM and to enhance the release of crypt- or gland-containing fragments. Vibrational treatment may include orbital shaking or exposure to low-frequency mechanical agitation while the tissue fragments are suspended in a digestion-compatible buffer. This post-mechanically dissociating vibrational step serves to agitate the mechanically disassociated tissue, improving enzyme penetration and promoting mechanical shearing of residual cellular aggregates, ultimately increasing the release and viability of stem cell populations. The ordering of vibrational processing after mechanically dissociating, as depicted in FIG. 11, contrasts with the order presented in FIG. 10, and may be preferential in specific donor conditions or tissue types. Together, these pre-digestion steps provide tunable variables that can be optimized to accommodate anatomical heterogeneity or procedural throughput requirements, thereby refining the derivation of high-fidelity NHP GI stem cell populations.

[0125] FIG. 12 is a flowchart 1200 illustrating an example method for deriving GI organoids from NHP tissue, in accordance with some embodiments. At block 1202, at least a portion of GI tract tissue is recovered from the NHP, such as a Cynomolgus macaque. The recovered tissue may originate from one or more anatomical regions including the duodenum, jejunum, ileum, cecum, small intestine, large intestine, rectum, stomach, or colon, is sectioned into one or more discrete regions to facilitate localized or segment-specific downstream processing. At block 1204, the sectioning process may include trimming the proximal and distal ends of the tissue sample using sterile surgical scissors or scalpels to remove excess or damaged portions. The remaining tissue is then subdivided into defined segments having a length ranging from approximately 1.0 cm to approximately 5.0 cm, such as about 2.0 cm to about 3.0 cm, or more specifically about 2.5 cm, depending on the anatomical region and experimental application.

[0126] In some embodiments, the GI tissue is divided into at least three to fiveanatomically distinct regions, including but not limited to the duodenumjejunum, ileum, cecum, and colon. Each isolated region may be individually identified and processed in parallel or separately to preserve spatial and physiological distinctions. Section widths may also vary, with cross-sectional diameters ranging from approximately 0.5 cm to approximately 2.0 cm, and thicknesses ranging from approximately 1 mm to approximately 5 mm, such as about 2 mm to about 4 mm, depending on the donor species, tissue elasticity, and target organoid application. Following gross sectioning, each tissue region may be rinsed in a washing buffer (e.g., phosphate- buffered saline or another isotonic solution) to remove luminal contents or surface debris prior to enzymatic or mechanical dissociation. The sectioning may be performed using manual tools or automated microtome devices depending on precision and reproducibility requirements. The resulting sections are transferred into sterile receptacles for further tissue processing, such as mincing, digestion, or preservation. At block 1206, the one or more regions of the sectioned GI tissue are digested in a digestion medium comprising a tissue dissociation enzyme. The digestion medium facilitates enzymatic breakdown of ECM components, thereby releasing epithelial and progenitor cells into suspension. At block 1208, the digested tissue is filtered to isolate one or more cells. This step includes passing the tissue suspension through one or more mesh strainers (e.g., about 50 pm to about 150 pm) to remove residual undigested fragments and to generate a purified single-cell or small-cluster suspension. At block 1210, the one or more isolated cells are suspended in a culture medium comprising a basement membrane extract (BME), such as Matrigel. This mixture provides a 3D scaffold that mimics the native ECM and supports organoid formation.

[0127] At block 1212, the one or more suspended cells are plated into a 3D structure on a culture surface. The 3D structure includes ECM proteins that encapsulate the suspended cells in a 3D configuration suitable for organoid development. At block 1214, the 3D structures are incubated under controlled conditions (e.g., about 37°C, about 5% CO2) to promote the formation of organoids. During this period, cellular self-organization and lumen development are initiated within the matrix 3D structures. At block 1216, the 3D structures are cultured in an expansion medium formulated to support stem cell viability and proliferation. The expansion medium may include components such as Wnt surrogate, Rspol-CM, Noggin, nicotinamide, N-acetylcysteine, EGF, and small-molecule inhibitors (e.g., A83-01, SB202190, prostaglandin E2). Organoids are maintained in this medium to enable growth and to preserve their undifferentiated or early-stage phenotype for subsequent analysis or applications. This method enables the derivation andexpansion of GI organoids from NHP tissue with high fidelity to region-specific epithelial and stem cell populations. Following the organoid derivation protocol illustrated in FIG. 12, which outlines the method steps for generating organoids from tissue samples, various formulations of the basal medium may be used to support the growth and maintenance of the derived organoids. In some embodiments, an example composition of an Advanced Dulbecco’s Modified Eagle Medium / F12+++ (AdvDMEM+++) Basal Medium A may be prepared according to Method A. This formulation includes one or more supplements and / or concentration parameters for maintaining organoid viability and / or proliferative capacity during culture expansion.

[0128] ORGANIOD MEDIUM PREPARATION AND MATERIALS

[0129] Example 1: Media Base Basal Medium A

[0130] In some embodiments, an example composition of an Advanced Dulbecco’s Modified Eagle Medium / F12+++ (AdvDMEM+++) Basal Medium A 2000 may be used for culturing GI organoids. In some embodiments, the formulation, herein referred to as "Example 1," is prepared as a basal medium composition for supporting organoid expansion, comprising a total volume of approximately 500 mb. The principal component includes Advanced Dulbecco’s Modified Eagle Medium / Nutrient Mixture F- 12 (Advanced DMEM / F 12), which provides the base formulation. To the base medium, a buffering agent, such as 1 M HEPES, is added in a volume ranging from approximately 4 mb to approximately 6 mb, such as about 5 mb, from a 1000 mM stock solution to achieve a final HEPES concentration ranging from approximately 8 mM to approximately 12 mM, preferably about 10 mM. Further, a glutamine-stabilized supplement, such as 100X GlutaMAX Supplement is incorporated at a volume ranging from approximately 4 mb to approximately 6 mL, more preferably about 5 mL, to yield a final working concentration ranging from approximately 0.8X to approximately 1.2X, about IX. Antibiotic-antimycotic protection is provided by the addition of Penicillin-Streptomycin (about 5,000 U / mL to about 15,000 U / mL) at a volume of approximately 4 mL to approximately 6 mL, more preferably about 5 mL, from a 100X stock solution to achieve a final antibiotic concentration ranging from approximately 80 U / mL to approximately 120 U / mL, and / or about 100 U / mL.

[0131] The prepared Example 1 medium may be stored in sterile containers at a refrigeration temperature ranging from approximately 2°C to approximately 8°C, preferably at about 4°C, and may remain chemically stable and biologically active for a duration ranging from approximately 2 weeks to approximately 6 weeks, about one month from the date of preparation,provided aseptic handling procedures are maintained. This basal medium serves as a foundational nutrient solution to support the viability, proliferation, and maintenance of epithelial-derived organoids, and may be adapted for use in a range of tissue-derived organoid systems across species. Building on the formulation described in Example 1, which outlines an example composition of Media Base Basal Medium A, Example 2 presents an alternative embodiment identified as Media Base Basal Medium B. This formulation introduces a variation in supplement concentration and content while maintaining the core framework of the basal medium. The modified concentrations are intended to support different culture requirements, experimental conditions, or tissue-specific organoid responses, as described in the following example.

[0132] Example 2: Media Base Basal Medium B

[0133] Example 2 describes an example composition of a modified organoid culture medium referred to as Media Base Basal Medium B 2100, prepared according to Example 2. This formulation builds upon the formulation shown in Example 1 by incorporating additional supplements and concentration modifications to enhance medium performance for one or more organoid culture conditions. As described in Example 2, a medium formulation referred to herein as “Example 2” includes a total volume of approximately 500 mL and includes Advanced Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (Advanced DMEM / F12) as the base medium. The base medium volume may range from approximately 400 mL to approximately 600 mL, more preferably about 500 mL. A buffering agent, such as 1 M HEPES, is incorporated at a volume ranging from approximately 4 mL to approximately 6 mL, about 5 mL, from a 1000 mM stock solution to yield a final concentration of HEPES ranging from approximately 8 mM to approximately 12 mM, preferably about 10 mM.

[0134] The formulation additionally includes GlutaMAX Supplement (100X), which is included at a volume of approximately 4 mL to approximately 6 mL, such as about 5 mL, to achieve a final concentration ranging from approximately 1.5 nM to approximately 2.5 nM, preferably about 2.0 nM, representing a deviation from the standard IX GlutaMAX concentration utilized in Method A. Antimicrobial protection is provided by Penicillin-Streptomycin (about 5,000 U / mL to about about 15,000 U / mL), added at a volume of approximately 4 mL to approximately 6 mL, more preferably about 5 mL, to achieve a final antibiotic concentration ranging from approximately 80 U / mL to approximately 120 U / mL, and / or about 100 U / mL.

[0135] In some embodiments, Example 2 further incorporates B-27 Supplement (50Xworking concentration), added in a volume ranging from approximately 8 mL to approximately 12 mL, more preferably about 10 mL per 500 mL of total medium, to achieve a final concentration of B-27 ranging from approximately 0.8X to approximately 1.2X, and / or about IX. The B-27 supplement is a serum-free formulation comprising essential nutrients, vitamins, antioxidants, trace elements, and hormones designed to support neuronal and epithelial cell maintenance, differentiation, and survival in organoid cultures. The prepared Example 2 medium is stored at a refrigeration temperature ranging from approximately 2°C to approximately 8°C, most preferably about 4°C, and may retain stability for a period ranging from approximately 2 weeks to approximately 6 weeks, and / or up to about one month from the date of preparation when maintained under sterile conditions. The addition of B27 and the adjustment of GlutaMAX concentration may provide improved support for certain tissue types or organoid subtypes compared to the formulation of Example 1. Following the preparation of the basal medium described in Example 2, which establishes the foundational nutrient environment for organoid cultures, Example 3 describes the composition of an expansion medium to support organoid growth, viability, and proliferation. This expansion formulation builds upon the basal medium by incorporating additional signaling proteins, growth factors, and biochemical supplements that simulate elements of the in vivo stem cell niche. The resulting medium, referred to as Media Base Expansion Medium A, is optimized for maintaining sternness and / or promoting epithelial regeneration in gastrointestinal organoid cultures.

[0136] Example 3: Media Base Expansion Medium A

[0137] Example 3 describes an example composition of an expansion medium referred to as Media Base Expansion Medium A 2200, which promotes the proliferation and maintenance of GI organoids derived from epithelial tissues. This formulation is used after culturing in a basal medium and incorporates a range of growth factors, signaling molecules, and / or chemical agents that collectively support organoid expansion and viability. In some embodiments, an expansion medium formulated to support organoid growth and viability includes a combination of supplements, proteins, and / or small molecule additives, included within various concentration ranges to achieve biological activity. The medium formulation may be prepared in a total volume ranging from approximately 50 mL to approximately 500 mL, about 100 mL, and includes the following components: A B-27 Supplement (50X) is included at a concentration ranging from approximately 1.5 mL to approximately 2.5 mL per 100 mL of medium, preferably about 2 mL,to yield a final concentration between approximately 0.75X and approximately 1.25X, about IX. The B-27 Supplement includes one or more essential vitamins, hormones, antioxidants, and / or other micronutrients that promote survival and / or differentiation of epithelial and / or stem-like cells in organoid cultures.

[0138] A Wnt surrogate protein is added at a volume ranging from approximately 2.5 pL to approximately 3.5 pL, and / or about 2.9 pL, from a stock concentration ranging from approximately 1.8 mg / mL to approximately 2.2 mg / mL, preferably 2.04 mg / mL (equivalent to approximately 17.1 pM), to achieve a final concentration ranging from approximately 0.4 nM to approximately 0.6 nM, and / or about 0.5 nM. R-spondinl -conditioned medium may be added in an amount ranging from approximately 350 pL to approximately 450 pL, more preferably about 400 pL, from a source stock concentration ranging from approximately 0.2 mg / mL to approximately 0.3 mg / mL, and / or about 0.25 mg / mL. This addition yields a final concentration of R-spondinl in the range of approximately 0.0008 mg / mL to approximately 0.0012 mg / mL, preferably 0.001 mg / mL. The completed medium is either used fresh or stored according to conditions that preserve the stability and activity of its components. This specific combination of Wnt pathway agonists, niche mimetics, and biochemical supplements enables epithelial stem cell maintenance, enhances survival, and supports appropriate tissue-specific differentiation of organoids during in vitro expansion. While Example 3 illustrates an expansion medium composition (Media Base Expansion Medium A) utilizing conditioned media and signaling pathway modulators to support epithelial organoid proliferation, Example 4 provides an alternative embodiment designated as media base Expansion Medium B. This formulation introduces a more defined and recombinantbased supplement profile, reducing variability by incorporating purified human proteins and serum-free conditioned media. The revised composition is suited for applications requiring enhanced reproducibility, controlled differentiation, or customized growth conditions tailored to specific organoid subtypes or tissue sources.

[0139] Example 4: Media Base Expansion Medium B

[0140] Example 4 describes an example composition for media base Expansion Medium B 2300, an alternative to Expansion Medium A, prepared according to Example 4. This formulation supports GI organoid growth with a modified set of components, particularly emphasizing recombinant human proteins and serum-free conditioned media. The formulation is optimized to mimic one or more aspects of the in vivo stem cell niche while supporting sustainedproliferation and differentiation. In some embodiments, an expansion medium is used for the growth and maintenance of organoid cultures and includes a formulation of one or more bioactive components, including one or more growth factors, one or more signaling proteins, one or more small molecule inhibitors, and / or a conditioned media. Each component may be added at a concentration within defined ranges to optimize cellular proliferation, differentiation, and structural maintenance. For example, the formulation includes Human Gastric I peptide, which may be added in a volume ranging from approximately 80 pL to approximately 120 pL, and / or about 100 pL, from a stock solution having a concentration ranging from approximately 8 mM to approximately 12 mM, preferably about 10 mM. This results in a final concentration of Human Gastric I in the medium ranging from approximately 8 nM to approximately 12 nM, and / or about 10 nM. N-acetylcysteine may be added at a volume ranging from approximately 90 pL to approximately 110 pL, and / or about 100 pL, from a 1.25 M stock solution to achieve a final concentration ranging from approximately 0.9 mM to approximately 1.1 mM, more preferably about 1 mM. Noggin-conditioned medium is included at a volume ranging from approximately 90 pL to approximately 110 pL, and / or about 100 pL, from a stock concentration ranging from approximately 90 pg / mL to approximately 110 pg / mL, more preferably about 100 pg / mL, to yield a final concentration in the range of approximately 90 ng / mL to approximately 110 ng / mL, preferably 100 ng / mL. Epidermal growth factor (EGF) is added in a volume ranging from approximately 90 pL to approximately 110 pL, and / or about 100 pL, from a stock concentration of approximately 45 pg / mL to approximately 55 pg / mL, about 50 pg / mL, to reach a final concentration ranging from approximately 450 ng / mL to approximately 550 ng / mL, about 500 ng / mL. Recombinant human insulin-like growth factor 1 (IGF-1) is incorporated at a volume ranging from approximately 90 pL to approximately 110 pL, more preferably about 100 pL, from a stock concentration of approximately 480 pg / mL to approximately 520 pg / mL, more preferably about 500 pg / mL, resulting in a final concentration ranging from approximately 480 ng / mL to approximately 520 ng / mL, and / or about 500 ng / mL. Recombinant human fibroblast growth factor- basic (FGF-2) may be added at a volume of approximately 90 pL to approximately 110 pL, amd / or about 100 pL, from a stock of approximately 45 pg / mL to approximately 55 pg / mL, preferably about 50 pg / mL, to yield a final concentration ranging from approximately 45 ng / mL to approximately 55 ng / mL, about 50 ng / mL.

[0141] Recombinant human R-spondinl protein is included at a volume of approximately90 JJ.L to approximately 110 pL, preferably 100 pL, from a stock solution having a concentration ranging from approximately 140 pg / mL to approximately 160 pg / mL, preferably 150 pg / mL, to yield a final concentration in the range of approximately 140 ng / mL to approximately 160 ng / mL, preferably 150 ng / mL. The ALK5 inhibitor A83-01 is included at a volume ranging from approximately 90 pL to approximately 110 pL, more preferably about 100 pL, from a stock solution of approximately 9 mM to approximately 11 mM, preferably 10 mM, to provide a final concentration of about 9 pM to about 11 pM, and / or about 10 pM. In some embodiments, the formulation includes Afamin-Wnt-3A serum-free conditioned medium at a volume ranging from approximately 40 pL to approximately 60 pL, more preferably about 50 pL, per 100 mL of expansion medium, thereby providing approximately 40% to approximately 60% conditioned medium by volume, preferably about 50%. Additionally, a Wnt surrogate protein may be incorporated at a volume of approximately 2.5 pL to approximately 3.5 pL, more preferably about 2.9 pL, from a stock solution of approximately 2.0 mg / mL to approximately 2.1 mg / mL (equivalent to approximately 17.1 pM), to yield a final concentration ranging from approximately 0.4 nM to approximately 0.6 nM, and / or about 0.5 nM. One or more components may be combined under sterile conditions using aseptic technique, and the resulting expansion medium may be stored at a temperature ranging from approximately 2°C to approximately 8°C and used within a time period ranging from approximately 7 days to approximately 30 days. In some embodiment, one or more components maybe combined under aseptic conditions, and the medium is either used fresh or stored under appropriate conditions to preserve biological activity. This formulation provides enhanced reproducibility through the use of recombinant components and defined media supplements and is suitable for organoid culture systems requiring consistent growth and controlled differentiation pathways. As noted above, Example 4 describes the composition of media base Expansion Medium B using defined reagents and recombinant proteins. Example 5 provides a corresponding reagent index listing the sources and catalog numbers of one or more materials referenced in the disclosed medium formulations and culture methods.

[0142] Example 5: Organoid Reagents

[0143] Example 5 describes an example listing of reagents used throughout the development, maintenance, and / or expansion of one or more organoid cultures. One or more reagents form the biochemical basis for multiple formulations as disclosed herein, including basalmedia, tissue processing solutions, and defined expansion media. In some embodiments, the list includes one or more small molecule inhibitors, recombinant growth factors, cytokines, conditioned media, antibiotics, cryoprotectants, and support components. Small molecule inhibitors may include, but are not limited to, Rho-associated kinase (ROCK) inhibitors such as Y-27632 dihydrochloride, glycogen synthase kinase 3 (GSK3) inhibitors such as CHIR 99021, mitogen-activated protein kinase (MAPK) inhibitors such as SB202190, transforming growth factor-beta (TGF-P) signaling inhibitors such as A83-01, and tankyrase inhibitors such as XAV939. These may be supplied in powder or solution form at concentrations ranging from about 1 mM to about 50 mM and used in working concentrations of about 0.1 pM to about 10 pM.

[0144] Media supplements may include nutrient and antioxidant formulations such as B- supplement, added from a stock solution of 50X to achieve a working concentration of about IX, and GlutaMAX, added from about a 100X stock at about 5 mL per 500 mL of base medium. Buffering agents such as HEPES may be included from 1 M stock solutions to achieve working concentrations ranging from about 5 mM to about 25 mM. The basal medium may include advanced Dulbecco's Modified Eagle Medium / Nutri ent Mixture F-12 (AdvDMEMZF12), provided in volumes ranging from about 100 mL to about 500 mL per batch. Sterility is maintained using antibiotics and antimicrobials such as penicillin-streptomycin, added at about 5 mL per 500 mL of medium from a 100X stock to yield 100 U / mL working concentrations. Additional antimicrobial agents may include, but not be limited to, Normocin and / or Primocin, used in concentrations ranging from about 50 pg / mL to about 150 pg / mL.

[0145] In some embodiments, recombinant growth factors and cytokines may include, but are not limited to, epidermal growth factor (EGF), prostaglandin E2 (PGE2), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-basic (FGF-2), fibroblast growth factor-10 (FGF-10), R- spondinl, Noggin, and Wnt signaling surrogates. These may be reconstituted from lyophilized form or provided as conditioned media. Volumes may range from about 50 pL to about 500 pL per 100 mL of final medium, resulting in working concentrations such as 10 ng / mL to 500 ng / mL for EGF, 0.5 nM for Wnt surrogate, 50 ng / mL for FGF-2, and 100 ng / mL for Noggin. Conditioned media alternatives, such as Afamin-Wnt-3 A and / or other Wnt ligand-based solutions, may be used at concentrations ranging from about 10% to about 75% by volume of the final formulation, depending on the application.

[0146] In some embodiments, one or more culture support reagents may further includeabout 5% to about 15% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), ammonium-chloride potassium (ACK) red blood cell lysis buffer, and cryopreservation agents such as dimethyl sulfoxide (DMSO), used at final concentrations of about 10% v / v. Serum supplements such as fetal bovine serum (FBS) may be used at concentrations ranging from about 5% to about 20% v / v. Tissue digestion may involve collagenase Type II, provided as powder or solution and used in concentrations ranging from about 0.05 mg / mL to about 2 mg / mL. Embedding reagents such as optimal cutting temperature (OCT) compound may be included for histological embedding at cryogenic temperatures.

[0147] In some embodiments, optional supplements may include antioxidants such as N- acetylcysteine, are added from 1.25 M stock to yield final concentrations of about 1.25 mM, and vitamin-related additives such as nicotinamide, added from a 5 M stock to yield final concentrations of about 10 mM. These reagents may be assembled and filtered under aseptic conditions using standard laboratory procedures and may be stored between about 2°C and about 8°C for durations ranging from about 7 days to about 30 days, depending on stability.. In some embodiments, one or more reagents listed in Example 5 may be sourced from established suppliers and identified by catalog number to ensure reproducibility across laboratories. The selected reagents are compatible with defined medium compositions and facilitate consistent organoid derivation, propagation, and characterization. Following the listing of reagents in Example 5, which includes one or more of the chemical and / or biological inputs used across the organoid culture protocol, Example 6 identifies the corresponding equipment necessary to support these procedures. This includes instrumentation for sterile handling, temperature regulation, centrifugation, and precision liquid transfer, each of which is integral to maintaining the consistency, viability, and / or reproducibility of the organoid systems described herein.

[0148] Example 6: Equipment

[0149] Example 6 illustrates an example of equipment that may be implemented to carry out the organoid preparation, culturing, and / or maintenance procedures disclosed in the present application. The listed equipment supports sterile technique, precise environmental control, and / or accurate liquid handling for one or more parameters to provide the reproducibility and quality of organoid-based workflows. In some embodiments, the laboratory setup may include, but is not limited to, equipment and conditions suitable for aseptic handling, organoid culture, processing, and cry opreservation. The setup may include a biosafety cabinet (BSC), such as a Class II TypeA2 cabinet, configured to provide laminar airflow and HEPA-filtered containment for performing sterile manipulations. The working area within the BSC may be maintained under positive pressure and cleaned using sterilizing agents prior to and following each use.

[0150] In some embodiments, the system may further include an incubator configured to maintain a temperature ranging from approximately 35°C to approximately 39°C, such as about 36.5°C to about 37.5°C, and a controlled carbon dioxide (CO2) atmosphere of about 4% to about 6%, such as about 5%. The incubator may additionally maintain humidity levels of approximately 85% to approximately 95% relative humidity to support physiological osmotic balance and gas exchange in organoid cultures. In some embodiments, the system includes a refrigerated centrifuge capable of maintaining a temperature of approximately 2°C to approximately 8°C, such as about 4°C. The centrifuge may be configured to operate at forces ranging from approximately 300*g to approximately 600*g, such as from about 400*g to about 500*g, with cycle durations of about 3 minutes to about 10 minutes, depending on the application. This equipment is used for gentle pelleting of dissociated cells, tissue fragments, and 3D organoid structures during isolation, washing, and media exchange procedures.

[0151] In some embodiments, for cryogenic storage, the setup may include one or more ultra-low temperature freezers configured to maintain temperatures of approximately -70°C to approximately -90°C, such as about -80°C. In some embodiments, the organoid samples may be further stored in vapor-phase or liquid-phase nitrogen cryogenic storage tanks, maintained at approximately -196°C for long-term preservation of viability and genomic integrity. To support uniform sample mixing, reagent distribution, and enzymatic digestion, an orbital shaker or orbital platform may be incorporated. The platform may provide agitation rates ranging from approximately 50 revolutions per minute (RPM) to approximately 300 RPM, depending on vessel type and contents. One or more precision pipetting tools may also be included to facilitate accurate reagent addition and sample transfer. These may include manual or electronic pipettes configured for volume ranges of approximately 0.1 pL to approximately 1000 pL. For example, a system may include pipettes capable of delivering volumes such as 2 pL to 20 pL, 20 pL to 200 pL, and 200 pL to 1000 pL. Larger-volume dispensing may be conducted using serological pipettes ranging from approximately 1 mb to approximately 50 mL, operated in conjunction with a pipette aid. Use of such instrumentation promotes reproducibility, minimizes contamination risk, and supports scalable workflows for organoid expansion, passaging, differentiation, compound screening, andrelated downstream applications. Example 6 details the equipment used to maintain appropriate environmental and sterile handling conditions for organoid culture. To complement these operational systems, Example 7 illustrates the materials employed for processing gastrointestinal tract organoids. These materials include dissection tools, culture vessels, pipettes, and cryogenic storage components used throughout the tissue preparation, plating, and preservation workflows described herein.

[0152] Example 7: Materials

[0153] Example 7 describes example materials used in the preparation and / or handling of one or more GI tract organoids. These materials facilitate tissue dissection, sample processing, organoid plating, and cryopreservation. In some embodiments, materials for tissue dissection and sample preparation include surgical -grade instruments configured for precise manipulation of soft tissue. Dissection tools may include, but are not limited to, curved dissection scissors having a blade length of approximately 2 cm to approximately 4 cm and an overall instrument length ranging from approximately 10 cm to approximately 16 cm. The scissors may include blunt or rounded tips to minimize accidental puncture or tissue damage during dissection of gastrointestinal (GI) segments.

[0154] Dissecting forceps may include blunt-pointed configurations with a tip width ranging from approximately 0.5 mm to approximately 2 mm, and an overall length ranging from approximately 10 cm to approximately 14 cm, configured to gently grasp and manipulate tissue sections during gross and fine dissection stages. Disposable surgical scalpels may be included, for example #10 and #11 blades, having blade lengths of approximately 2 cm to approximately 3 cm and a cutting edge geometry configured for either curved incisions or fine-point entry incisions, respectively. Culture vessels for organoid maintenance and expansion may include sterile, flatbottom tissue culture-treated plasticware such as multi-well culture plates. In some embodiments, 6-well plates may be used, each well having a diameter of approximately 34 mm to approximately 36 mm and a capacity ranging from approximately 3 mL to approximately 5 mL per well. In other embodiments, 24-well or 96-well plates may be employed for scale-up or miniaturized screening applications.

[0155] In some embodiments, for sample transfer, processing, and / or centrifugation steps, conical centrifuge tubes in various volumes may be utilized. For example, approximately 15 mL to approximately 50 mL sterile polypropylene conical tubes may be used, featuring screw caps andgraduations for sample volume tracking. Filters such as approximately 100 pm cell strainers may be fitted into one or more tubes for dissociated tissue filtration, with mesh pore sizes ranging from approximately 70 pm to approximately 100 pm, depending on the desired exclusion cutoff for undigested material.

[0156] In some embodiments, one or more liquid handling components may include serological pipettes in volumes of approximately 5 mL, approximately 10 mb, and / or approximately 25 mL, configured with sterile individually wrapped packaging and / or designed for use with one or more handheld pipette aids. Pasteur pipettes may also be employed for gentle mechanical disruption of ECM materials or for transferring small volumes. Pasteur pipettes may range in length from approximately 145 mm to approximately 230 mm and may be either plastic or glass. Additional sample handling consumables may include macro tubes with a volume capacity of approximately 5 mL to approximately 15 mL, cryogenic vials with internal or external threading and nominal volumes of approximately 1 mL to approximately 2 mL, and screw-cap storage containers. Freezing containers may include controlled-rate freezing modules configured to reduce sample temperature at a rate of approximately -0.5°C / min to approximately -1.5°C / min until reaching -80°C.

[0157] In some embodiments, for general cell culture workflows, a sterile tissue culture dish may be used, with a growth surface area of approximately 55 cm2to approximately 60 cm2and a liquid handling capacity ranging from approximately 8 mL to approximately 12 mL. These dishes may support plating, washing, or expansion of organoid cultures at intermediate stages. One or more instruments and / or consumables are intended to be used in accordance with sterile technique protocols in a biosafety cabinet under aseptic conditions. These tools collectively support consistent tissue handling, organoid transfer, media exchange, and cryopreservation in organoid culture workflows. Example 7 identifies the instruments and consumables used during the physical processing of gastrointestinal tissues. To support preservation of tissue viability during interim handling and transport, Example 8 illustrates the composition of a tissue preservation medium formulated to stabilize non-human primate GI tissues prior to organoid isolation and culture.

[0158] Example 8: Tissue Preservation Medium

[0159] Example 8 depicts an example formulation of a preservation medium used tomaintain the viability and structural integrity of NHP GI tissues prior to organoid derivation or processing. In some embodiments, the formulation is referred to as the NHP GI Tract Tissue Preservation Medium and is configured for short-term storage of biological tissue samples at a temperature ranging from approximately 2°C to approximately 8°C, or at approximately 4°C. The formulation maintains cellular and molecular integrity of GI tract tissue during temporary storage and transport prior to downstream processing. The base medium may include a chemically defined nutrient-rich formulation such as an advanced formulation of DMEM / F12, included in an amount of approximately 400 mb to approximately 600 mb, for example approximately 500 mL, and configured to support viability under non-culture conditions. A buffering agent such as 1 M HEPES solution may be added in an amount ranging from approximately 0.5 mL to approximately 2 mL, for example approximately 1 mL, to yield a final concentration of approximately 5 mM to approximately 15 mM, for example about 10 mM, to maintain pH stability during storage.

[0160] A metabolic stabilizer such as a glutamine dipeptide substitute (e.g., GlutaMAX) may be included at a concentration sufficient to yield a final working concentration of approximately IX, by adding approximately 5 mL of a 100X stock solution. Antimicrobial protection may be provided by the addition of a penicillin-streptomycin mixture at a concentration of approximately 100 units / mL to 100 pg / mL, obtained by incorporating approximately 5 mL of a 100X stock solution of a 10,000 U / mL antibiotic mixture. In some embodiments, a broad-spectrum antibiotic formulation may be included to prevent contamination by bacteria, mycoplasma, and / or fungi. For example, a solution containing approximately 500 mg / L of antimicrobial compound may be added in an amount of approximately 4 mL to approximately 6 mL, or more specifically about 5 mL, to yield a final concentration ranging from approximately 80 pg / mL to approximately 120 pg / mL, for example approximately 100 pg / mL.

[0161] One or more of the aforementioned components may be combined under aseptic conditions in a biosafety cabinet, and the final tissue preservation medium may be filtered through a sterile 0.22 pm membrane and aliquoted into sterile botles for storage. The completed formulation may be stored at approximately 4°C and may remain chemically and functionally stable for a period ranging from approximately 2 weeks to approximately 5 weeks, for example up to about one month. The medium is suitable for use in preclinical organoid workflows using NHP GI tissues or other mammalian tissues requiring short-term preservation prior to enzymatic digestion, cryopreservation, or histological embedding. This medium may be used to hold freshGI tissue following dissection and / or prior to enzymatic digestion and / or cryopreservation, preserving tissue quality and reducing microbial risk during downstream organoid culture workflows. Following the formulation of the preservation medium detailed in Example 8, which maintains tissue viability during short-term storage, Example 9 describes the composition of a digestion medium used to enzymatically dissociate gastrointestinal tissue. This formulation enables the release of viable crypts and epithelial cell clusters for subsequent organoid culture.

[0162] Example 9: Digestion Medium

[0163] Example 9 describes an example formulation of a digestion medium used for enzymatic dissociation of GI tissue during the preparation of one or more organoid cultures. This medium breaks down one or more ECM components and / or facilitate the release of one or more epithelial crypts and / or one or more stem cell-rich cell clusters from a tissue sample. In some embodiments, the digestion medium enzymatically dissociates GI tract tissue for the isolation of viable cells and / or organoid precursors. The medium includes a nutrient-rich basal formulation, such as an advanced version of Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (Advanced DMEM / F12+++), used in a volume ranging from approximately 40 mL to approximately 60 mL, or more specifically about 50 mL. This base formulation provides one or more amino acids, glucose, vitamins, and / or buffering capacity to support tissue viability during enzymatic digestion.

[0164] In some embodiments, a matrix-degrading enzyme, such as Collagenase Type II, is added to the medium in an amount sufficient to yield a working concentration ranging from approximately 2 mg / mL to approximately 10 mg / mL. For example, approximately 40 mg to approximately 60 mg of lyophilized Collagenase Type II may be reconstituted in the 50 mL of Advanced DMEM / F12+++ to achieve a target concentration of approximately 5 mg / mL. Collagenase Type II facilitates the breakdown of ECM proteins, including collagen, enabling mechanical and enzymatic separation of epithelial crypts and stromal elements while preserving cellular integrity. In some embodiments, the digestion medium is further supplemented with a Rho-associated protein kinase (ROCK) inhibitor, such as Thiazovivin (TZV), to enhance postdissociation cell viability, particularly for epithelial or progenitor cell populations that exhibit high sensitivity to mechanical and enzymatic stress. Thiazovivin may be added from a concentrated stock solution (e.g., 10 mM to 20 mM in DMSO) in a volume of approximately 2 pL toapproximately 10 pL, for example approximately 5 pL, to achieve a final concentration of approximately 5 pM to approximately 20 pM, such as 10 pM.

[0165] In some embodiments, one or more components are combined under sterile conditions, and the medium may be pre-warmed to approximately 37°C prior to application. The digestion medium is suitable for use in tissue processing protocols involving organoid culture, epithelial isolation, and regenerative medicine applications, with optimization parameters including tissue type, sample mass (e.g., 100 mg to 800 mg), and desired digestion duration (e.g., 20 minutes to 60 minutes). The complete formulation may be freshly prepared or stored at approximately 4°C for up to 24 hours prior to use. One or more components may be combined under sterile conditions. The digestion medium is t prepared fresh prior to use and maintained at about 37°C during the dissociation procedure. This enzymatic formulation enables efficient mechanical and biochemical separation of viable organoid-forming units from GI tissue specimens while preserving cell integrity for downstream culture and expansion.

[0166] EXAMPLES

[0167] Embodiment 1. A method of deriving stem cells from nonhuman primate (NHP) gastrointestinal (GI) tract tissue, comprising: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tract tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissue in a digestion medium comprising a tissue dissociation enzyme; filtering the digested GI tract tissue to isolate one or more cells; and culturing the one or more isolated cells in an expansion medium to derive one or more stem cells.

[0168] Embodiment 2. The method of embodiment 1, wherein the at least a portion of the GI tract tissue comprising one or more of: a duodenum, a jejunum, an ileum, a cecum, or a colon.

[0169] Embodiment 3. The method of embodiment 1, wherein the NHP is a Macaca fascicularis.

[0170] Embodiment 4. The method of embodiment 1, wherein the at least a portion of the GI tissue comprises a GI tract region with a weight of about 0.1 g to about 5.0 g.

[0171] Embodiment 5. The method of embodiment 1, wherein the digesting further comprises mechanically dissociating the one or more regions of the at least a portion of the GI tract tissue.

[0172] Embodiment 6. The method of embodiment 5, wherein the digesting further comprises vibrating the one or more regions of the at least a portion of the GI tract tissue before the mechanically dissociating of the one or more regions of the at least a portion of the GI tract tissue.

[0173] Embodiment 7. The method of embodiment 5, wherein the digesting further comprises vibrating the one or more regions of the at least a portion of the GI tract tissue after the mechanically dissociating of the one or more regions of the at least a portion of the GI tract tissue.

[0174] Embodiment 8. The method of embodiment 1, wherein the tissue dissociation enzyme comprises at least one of a collagenase, a dispase, or a liberase.

[0175] Embodiment 9. The method of embodiment 8, wherein the collagenase is a type II collagenase.

[0176] Embodiment 10. The method of embodiment 1, wherein the digestion medium further comprises L-glutamine.

[0177] Embodiment 11. The method of embodiment 1, wherein the digestion medium further comprises one or more basal formulations.

[0178] Embodiment 12. The method of embodiment 1, wherein the digestion medium further comprises one or more inhibitors.

[0179] Embodiment 13. The method of embodiment 12, wherein the one or more inhibitors is a Rho-associated protein kinase (ROCK) inhibitor.

[0180] Embodiment 14. The method of embodiment 1, wherein the expansion medium comprises one or more factors selected from the group consisting of: Wnt surrogate, Rspol- CM, Noggin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), or tumor growth factor-beta (TGF-P).

[0181] Embodiment 15. A method of deriving organoids from gastrointestinal (GI) tract tissue of a nonhuman primate (NHP), comprising: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissue in a digestion medium comprising a tissue dissociation enzyme; fdtering the digested tissue of the one or more regions of the at least a portion of gastrointestinal tract tissue to isolate one or more cells; suspending the one or more isolated cells in a culture comprising; a basementmembrane extract (BME); plating the one or more suspended isolated cells into a three- dimensional (3D) structure on a culture surface of the culture comprising extracellular matrix (ECM) proteins; incubating the 3D structure to form an organoid; and culturing the 3D structure in an expansion medium to promote organoid growth.

[0182] Embodiment 16. The method of embodiment 15, wherein the one or more regions of the at least a portion of the G1 tract tissue comprise tissue selected from a duodenum, a jejunum, an ileum, a cecum, or a colon.

[0183] Embodiment 17. The method of embodiment 15, The method of claim 15, wherein the digesting the one or more regions comprises contacting the one or more regions with the digestion medium comprising a collagenase at a concentration ranging from about 1 mg / mL to about 10 mg / mL.

[0184] Embodiment 18. The method of embodiment 15, wherein the 3D structure comprises one or more ECM proteins selected from the group consisting of laminin, collagen IV, entactin, or fibronectin.

[0185] Embodiment 19. The method of embodiment 15, wherein the 3D structure comprises one or more growth factors selected from the group consisting of epidermal growth factor (EGF), Noggin, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), or R-spondinl.

[0186] Embodiment 20. The method of embodiment 15, wherein the incubating of the 3D structure comprises positioning the 3D structure in an inverted orientation for a period of time to facilitate solidification of the 3D structure.

[0187] Embodiment 21. The method of embodiment 15, wherein digesting the one or more regions further comprises mechanically dissociating the one or more regions into fragments having a size ranging from about 1 mm3to about 5 mm3prior to enzymatic digestion.

[0188] Embodiment 22. The method of embodiment 15, wherein filtering the digested tissue comprises passing the digested tissue through a mesh strainer having a pore size ranging from about 70 pm to about 120 pm.

[0189] Embodiment 23. The method of embodiment 15, wherein suspending the one or more isolated cells comprises resuspending the one or more isolated cells in a medium comprising a ROCK inhibitor at a concentration ranging from about 5 pM to about 20 pM.

[0190] Embodiment 24. The method of embodiment 15, wherein the basement membrane extract comprises one or more materials selected from Matrigel, Geltrex, Cultrex, MaxGel, or a synthetic extracellular matrix comprising collagen, fibrin, or vitronectin.

[0191] Embodiment 25. The method of embodiment 15, wherein plating the one or more suspended isolated cells into the 3D structure comprises dispensing the one or more suspended isolated cells in a dome shape on a warmed culture surface to promote gelation.

[0192] Embodiment 26. The method of embodiment 15, wherein incubating the 3D structure comprises: placing the culture surface upright in an incubator at approximately 37°C and approximately 5% CO2 for about 10 minutes; and inverting the culture surface for an additional 10 minutes to enhance gelation.

[0193] Embodiment 27. The method of embodiment 15, wherein the expansion medium comprises one or more growth factors selected from a Wnt surrogate, R-spondinl, Noggin, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), or hepatocyte growth factor (HGF).

[0194] Embodiment 28. The method of embodiment 15, wherein the expansion medium further comprises one or more small molecules selected from nicotinamide, N-acetylcysteine, prostaglandin E2 (PGE2), a p38 MAPK inhibitor, or an ALK5 inhibitor.

[0195] Embodiment 29. The method of embodiment 15, wherein culturing the 3D structure comprises refreshing the expansion medium every 1 to 3 days and maintaining the 3D structure at approximately 37°C and approximately 5% CO2.

[0196] Embodiment 30. The method of embodiment 15, further comprising monitoring the 3D structure for a period of about 3 days to about 10 days to detect exponential organoid growth.

[0197] Embodiment 31. The method of embodiment 15, further comprising expanding the organoid by disrupting the 3D structure to generate a cell suspension, centrifuging the cell suspension to form a pellet, and re-plating the pellet in a fresh volume of the basement membrane extract.

[0198] Embodiment 32. The method of embodiment 15, wherein the GI tract tissue is obtained from the NHP selected from the group consisting of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina.

[0199] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” “some embodiments,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0200] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “cell” may include, and is contemplated to include, a plurality of cells. At times, the claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0201] The term “about” or “approximately,” when used before a numerical designation or range (e g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a method, substance, or composition.

[0202] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements and may additionally include any other elements. “Consisting essentially of’ shall mean that the compositions and methods include the recited elements and exclude other elements of essential significance to the combination for the stated purpose. Thus, a composition or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the compositions and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0203] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Otherembodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMS1. A method of deriving stem cells from nonhuman primate (NHP) gastrointestinal (GI) tract tissue, comprising: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tract tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissue in a digestion medium comprising a tissue dissociation enzyme; filtering the digested GI tract tissue to isolate one or more cells; and culturing the one or more isolated cells in an expansion medium to derive one or more stem cells.

2. The method of claim 1, wherein the at least a portion of the GI tract tissue comprising one or more of a duodenum, a jejunum, an ileum, a cecum, or a colon.

3. The method of claim 1, wherein the NHP is a Macaca fascicularis.

4. The method of claim 1, wherein the at least a portion of the GI tissue comprises a GI tract region with a weight of about 0.1 g to about 5.0 g.

5. The method of claim 1, wherein the digesting further comprises mechanically dissociating the one or more regions of the at least a portion of the GI tract tissue.

6. The method of claim 5, wherein the digesting further comprises vibrating the one or more regions of the at least a portion of the GI tract tissue before the mechanically dissociating of the one or more regions of the at least a portion of the GI tract tissue.

7. The method of claim 5, wherein the digesting further comprises vibrating the one or more regions of the at least a portion of the GI tract tissue after the mechanically dissociating of the one or more regions of the at least a portion of the GI tract tissue.

8. The method of claim 1, wherein the tissue dissociation enzyme comprises at least one of a collagenase, a dispase, or a liberase.

9. The method of claim 8, wherein the collagenase is a type II collagenase.

10. The method of claim 1, wherein the digestion medium further comprises L-glutamine.

11. The method of claim 1, wherein the digestion medium further comprises one or more basal formulations.

12. The method of claim 1, wherein the digestion medium further comprises one or more inhibitors.

13. The method of claim 12, wherein the one or more inhibitors is a Rho-associated protein kinase (ROCK) inhibitor.

14. The method of claim 1, wherein the expansion medium comprises one or more factors selected from the group consisting of: Wnt surrogate, Rspol-CM, Noggin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), or tumor growth factor-beta (TGF-P).

15. A method of deriving organoids from gastrointestinal (GI) tract tissue of a nonhuman primate (NHP), comprising: recovering at least a portion of GI tract tissue from the NHP; sectioning the at least a portion of the GI tissue into one or more regions; digesting the one or more regions of the at least a portion of the GI tract tissue in a digestion medium comprising a tissue dissociation enzyme; filtering the digested tissue of the one or more regions of the at least a portion of gastrointestinal tract tissue to isolate one or more cells; suspending the one or more isolated cells in a culture comprising; a basement membrane extract (BME);plating the one or more suspended isolated cells into a three-dimensional (3D) structure on a culture surface of the culture comprising extracellular matrix (ECM) proteins; incubating the 3D structure to form an organoid; and culturing the 3D structure in an expansion medium to promote organoid growth.

16. The method of claim 15, wherein the one or more regions of the at least a portion of the GI tract tissue comprise tissue selected from a duodenum, a jejunum, an ileum, a cecum, or a colon.

17. The method of claim 15, The method of claim 15, wherein the digesting the one or more regions comprises contacting the one or more regions with the digestion medium comprising a collagenase at a concentration ranging from about 1 mg / mL to about 10 mg / mL.

18. The method of claim 15, wherein the 3D structure comprises one or more ECM proteins selected from the group consisting of laminin, collagen IV, entactin, or fibronectin.

19. The method of claim 15, wherein the 3D structure comprises one or more growth factors selected from the group consisting of epidermal growth factor (EGF), Noggin, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), or R-spondinl.

20. The method of claim 15, wherein the incubating of the 3D structure comprises positioning the 3D structure in an inverted orientation for a period of time to facilitate solidification of the 3D structure.

21. The method of claim 15, wherein digesting the one or more regions further comprises mechanically dissociating the one or more regions into fragments having a size ranging from about 1 mm3to about 5 mm3prior to enzymatic digestion.

22. The method of claim 15, wherein filtering the digested tissue comprises passing the digested tissue through a mesh strainer having a pore size ranging from about 70 pm to about 120 pm.

23. The method of claim 15, wherein suspending the one or more isolated cells comprises resuspending the one or more isolated cells in a medium comprising a ROCK inhibitor at a concentration ranging from about 5 pM to about 20 pM.

24. The method of claim 15, wherein the basement membrane extract comprises one or more materials selected from Matrigel, Geltrex, Cultrex, MaxGel, or a synthetic extracellular matrix comprising collagen, fibrin, or vitronectin.

25. The method of claim 15, wherein plating the one or more suspended isolated cells into the 3D structure comprises dispensing the one or more suspended isolated cells in a dome shape on a warmed culture surface to promote gelation.

26. The method of claim 15, wherein incubating the 3D structure comprises: placing the culture surface upright in an incubator at approximately 37°C and approximately 5% CO2 for about 10 minutes; and inverting the culture surface for an additional 10 minutes to enhance gelation.

27. The method of claim 15, wherein the expansion medium comprises one or more growth factors selected from a Wnt surrogate, R-spondinl, Noggin, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), or hepatocyte growth factor (HGF).

28. The method of claim 15, wherein the expansion medium further comprises one or more small molecules selected from nicotinamide, N-acetylcysteine, prostaglandin E2 (PGE2), a p38 MAPK inhibitor, or an ALK5 inhibitor.

29. The method of claim 15, wherein culturing the 3D structure comprises refreshing the expansion medium every 1 to 3 days and maintaining the 3D structure at approximately 37°C and approximately 5% CO2.

30. The method of claim 15, further comprising monitoring the 3D structure for a period of about 3 days to about 10 days to detect exponential organoid growth.

31. The method of claim 15, further comprising expanding the organoid by disrupting the 3D structure to generate a cell suspension, centrifuging the cell suspension to form a pellet, and re-plating the pellet in a fresh volume of the basement membrane extract.

32. The method of claim 15, wherein the GI tract tissue is obtained from the NHP selected from the group consisting of Macaco, fascicularis, Macaca mulatto, and Macaco nemestrina.

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