Method for generating homogenous, size-defined pseudo-islets of porcine origin by RHO / rock pathway inhibition

Abstract

The invention relates to an ex vivo method for generating pseudo-islets from a porcine subject by providing single cells dissociated from islets or islet-like cell clusters (ICC) obtained from a porcine pancreas, treating the dissociated cells with a Rho / ROCK inhibitor, and obtaining pseudo-islets. The invention further relates to an in vitro population of porcine pseudo-islets obtained by treatment with a Rho / ROCK inhibitor, wherein greater than 30% of the population exhibit a defined size with an average diameter of 30-350 μm and a standard deviation (SD) of 10 or less. The invention also relates to the pseudo-islet population for use in the treatment of diabetes, preferably type 1 diabetes, in a subject, wherein the pseudo-islet population is transplanted to the subject.

Description

[0001] METHOD FOR GENERATING HOMOGENOUS, SIZE-DEFINED PSEUDO-ISLETS OF PORCINE ORIGIN BY RHO / ROCK PATHWAY INHIBITION

[0002] DESCRIPTION

[0003] The invention is in the field of molecular biology and medicine, more specifically in the field of endocrinology, metabolic diseases, and diabetes.

[0004] The invention relates to an ex vivo method for generating pseudo-islets from a porcine subject by providing single cells dissociated from islets or islet-like cell clusters (ICC) obtained from a porcine pancreas, treating the dissociated cells with a Rho / ROCK inhibitor, and obtaining pseudo-islets. The invention further relates to an in vitro population of porcine pseudo-islets obtained by treatment with a Rho / ROCK inhibitor, wherein greater than 30% of the population exhibit a defined size with an average diameter of 30-350 pm and a standard deviation (SD) of 10 or less. The invention also relates to the pseudo-islet population for use in the treatment of diabetes, preferably type 1 diabetes, in a subject, wherein the pseudo-islet population is transplanted to the subject. The invention further covers various uses or applications of the pseudo-islets described herein, for example for research applications such as drug screening or investigations into developmental biology.

[0005] BACKGROUND OF THE INVENTION

[0006] Diabetes is a chronic metabolic disease characterized by elevated blood glucose levels, leading to severe damage to vital organs. Type 2 diabetes, prevalent in adults, results from insulin resistance or inadequate insulin production, and its global prevalence has surged. Type 1 diabetes is primarily diagnosed in youth and involves insufficient insulin production. According to the World Health Organization, approximately 422 million people have diabetes worldwide, with 1.5 million deaths attributed to it annually. Therefore, early diagnosis is crucial, with prevention strategies focusing on lifestyle modifications. Effective interventions include controlling blood glucose, managing blood pressure and lipids, and screening for early complications.

[0007] Furthermore, genetic and environmental factors contribute to diabetes. Genetic variations, especially within human leukocyte antigen (HLA) and insulin genes, play a role in Type 1 diabetes, with a subset classified as idiopathic. Type 1 diabetes mellitus (T1 D or T1 DM) is characterized by insulin deficiency, typically beginning in childhood or adolescence (C. Pathak et al., Clin Med Insights Endocrinol Diabetes, 2019). Its pathogenesis involves T cell-mediated destruction of pancreatic (3-cells , with biomarkers like autoantibodies targeting (3-cell proteins aiding in early identification of at-risk individuals. T1 D progression has three stages based on hyperglycemia and symptoms, and lifelong insulin therapy is the standard treatment. Genetics, including HLA haplotypes and non-HLA variants, contribute to T1 D risk, but predicting or distinguishing T 1 D from other diabetes types with non-HLA loci is challenging. Complex interactions between (3-cells and the immune system, viral infections, immune cell dysfunction, and insulitis play key roles in the disease.

[0008] Treatment options for Type 1 diabetes primarily revolve around managing blood glucose levels, as individuals with T1 D do not produce insulin. Therefore, the primary purpose of treatment is to achieve and maintain blood glucose levels while minimizing the risk of hyper- and hypoglycemia and reducing the risk of long-term complications. The most common methods to treat T1 D are insulin injections or insulin pumps. However, both methods require ongoing and active management, as T1 D patients must monitor their blood glucose levels and calculate insulin doses. Managing insulin manually carries a risk of hyper- or hypoglycemia if insulin doses are miscalculated or if there are changes in physical activity or diet.

[0009] Pancreas transplantation and islet transplantation are advanced treatments for T 1 D, with the primary objective of reinstating endogenous insulin production and reestablishing physiological hypoglycemic counter-regulation mechanisms. Pancreas transplantations involve the surgical replacement of the entire pancreas from a deceased donor. It can provide long-term insulin independence but requires immunosuppressive medications to prevent rejection. On the other hand, the transplantation of islet cells is less invasive. It involves isolating insulin-producing islet cells from a donor pancreas and transplanting them into the liver of the patient.

[0010] Nevertheless, the production of pseudo-islets for transplantation still faces several challenges, including a limited supply of human donor pancreata for islet isolation, ischemic damage incurred during the isolation and purification processes leading to islet loss, necessitating multiple donor pancreata to attain a therapeutic transplant dose per recipient. Additionally, shortcomings in current islet purification methods contribute to reduced islet yields during isolation. Furthermore, immediate blood-mediated inflammatory responses by immunologic cells to islet grafts may result in transplant failure. Human islets exhibit high sensitivity to low oxygen levels at the transplant site, also increasing the risk of transplant failure.

[0011] WO2017214465A1 describes a method for generating beta-like cells from stem cells. This method involves cultivating said cells in a specialized cell culture medium while concurrently suppressing the activity of Rho kinase (ROCK). Furthermore, these cultured cells are subjected to the action of RNA interference molecules, specifically targeting ROCK1 , ROCK2, or a combination of both. The primary focus of this approach is to enable sustained in vitro cultivation and expansion of mature islet-like cells derived from stem cells.

[0012] Britt L. D. et al. and Honarpisheh M. et al. reveal the extent to which porcine pseudo-islets can be obtained using gyrotational tissue culture conditions; the use of a Rock inhibitor is not mentioned. The dimensions of the obtained pseudo-islets are discussed, as well as the possibility of using porcine pseudo-islets in cell transplantation to treat diabetes mellitus.

[0013] Hildernik J. et al. discloses how pancreatic cell clusters can be derived from human donor islets. After transplantation of these cell clusters into immunodeficient mice, intact endocrine function of the islets was observed. The culture conditions do not include the use of Rock inhibitors. Zaniar G. et al. discloses the use of Rock inhibitor H1152 in the culture of various human stem cells, leading to the generation of insulin-producing beta-like cells. The treatment of porcine islets or islet-like cell clusters is not mentioned.

[0014] Siehler J. et al. summarizes the current state of the art in the generation of islet cells from stem cells. Zhang Q. et al. describes the potential of different methods of encapsulating islets to treat type 1 diabetes mellitus.

[0015] Hammer E. et al. reveals the role of the Rho-ROCK signaling pathway in regulating pancreatic beta cells. The inhibition of the Rho-ROCK signaling pathway by H-1152 and Y-27632 led to an improved distribution of beta cells in the tissue and an increased short-term and prolonged glucose-induced insulin secretion. However, a significant challenge persists concerning generating size-defined pseudo-islet clusters from primary cells, particularly from porcine pancreatic tissues. It is essential that these pseudo-islets are viable, fully functional, and reproducible, rendering them suitable for potential transplantation procedures.

[0016] Considering the advantages of using pigs as an islet source for xenotransplantation, investigating pancreatic islet cells from porcine (pig) donors emerges as a promising alternative. Pigs are readily available, with their pancreas providing a relatively substantial number of islet cells compared to human donors. Moreover, porcine and human insulin exhibit similar structures, and their physiological glucose levels are comparable. This renders porcine islets a viable and promising source for transplantation material when human donor islets or stem cell-derived beta cells are scarce or fail, leading to low success in treatment.

[0017] Consequently, an unmet need exists for developing strategies to generate pseudo-islets from porcine sources that are both viable and consistent in functionality. Given the current state of the art, there remains a significant demand for advanced techniques in the field to generate fully functional porcine pseudo-islets suitable for successful transplantation.

[0018] SUMMARY OF THE INVENTION

[0019] The inventors surprisingly found that utilizing a Rho / ROCK inhibitor for generating pseudo-islets from a porcine subject resulted in improved cluster formation and pseudo-islet function without negatively affecting the viability, cell composition, and architecture of the islets.

[0020] In light of the prior art, one objective of the invention is to provide improved or alternative means for treating diabetes.

[0021] Another objective is the provision of pseudo-islets with improved clustering and / or a regular size distribution, and pseudo-islet function, without negatively affecting the viability, cell composition, and architecture of the islets.

[0022] Another objective is the provision of means that improve the generation of porcine pseudo-islets that are reliable and reproducible and can be used for xenotransplantation under various conditions, such as naked or encapsulated, and / or for research purposes, e.g., physiological studies, drug screening, etc. This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims

[0023] The invention, therefore, relates to an ex vivo method for generating pseudo-islets from a porcine subject by providing single cells dissociated from islets or islet-like cell clusters (ICC) obtained from a porcine pancreas, treating the dissociated cells with a Rho / ROCK inhibitor, and obtaining pseudo-islets.

[0024] Pigs are currently regarded as one of the most suitable candidates for organ donation in xenotransplantation as their genetic distance from humans is relatively small. Pigs have short gestation periods, large litters, ease of breeding, affordability, and adaptability to pathogen-free environments. Advances in gene editing technologies have enabled genetic modifications to combat organ rejection and zoonotic disease potential. Porcine-derived treatments, especially insulin for diabetes, have proven successful. Genetically engineered pigs may also enhance transplant success rates and reduce potential unwanted immune reactions to transplanted material due to, for example, mismatched HLA antigens. Notably, pig organs, particularly kidneys and hearts, have been successfully transplanted into humans. After transplantation, pig kidneys have maintained function for approximately two months. Both kidney and heart transplantations provide proof of concept and enable further xenotransplantation opportunities in clinical practice

[0025] With respect to the present invention, it was entirely surprising that the Rho / ROCK inhibitor treatment optimized the generation of porcine pseudo-islets, in particular with respect to achieving a pseudo-islet population with a regular and homogenous size distribution, thus enabling controlled definition of pseudo-islet size, depending on the application.

[0026] Moreover, treatment with the Rho / ROCK inhibitor resulted in higher insulin secretion after glucose challenge and a higher stimulation index, indicating improvements in function of the pseudo-islets due to the inventive treatment.

[0027] Various aspects of the invention therefore facilitate improved cluster formation and size definition of the pseudo-islets, while also achieving high viability, purity, and maintenance or even improvements in islet function. Therefore, the invention further enables the treatment of diseases such as diabetes, more particularly type 1 diabetes, by generating and administering said pseudo-islets into a subject in need thereof.

[0028] The following Table 1 comprises a non-limiting and exemplary list of the size of adult porcine pseudo-islets that were treated with or without the Rho / ROCK inhibitor Y-27632 in comparison to other species (human and rat).

[0029] Table 1 : By way of example, the table lists non-limiting sizes of adult porcine pseudo-islets (PI) in comparison to human and rat pseudo-islets (PI).

[0030] In other embodiments, the particular cell numbers and diameters of pseudo-islets may vary within the ranges disclosed herein. These ranges represent a generalization of the examples and may be obtained without undue effort by carrying out the present method.

[0031] Table 2: By way of example, the table lists non-limiting size ranges of adult porcine pseudo-islets (PI) in comparison to human and rat pseudo-islets (PI).

[0032] In embodiments, the average diameter of adult porcine pseudo-islets treated with the Rho / ROCK inhibitor Y-27632 is comparable or similar to the mean diameter observed in human or rat pseudo-islets.

[0033] This outcome was indeed surprising since the treatment of adult porcine pseudo-islets with Y- 27632 resulted in sizes similar to human or rat pseudo-islets, while the mean diameter of adult porcine pseudo-islets without treatment of Y-27632 was significantly smaller. This result allows for the potential utilization of these pseudo-islets in subsequent experiments and potential transplantation procedures, and by using Rho / ROCK inhibition, comparable pseudo-islet size can be achieved from porcine and human sources.

[0034] In embodiments, the method according to the invention further comprises seeding the dissociated cells into microwells, allowing the cells to aggregate into pseudo-islets, wherein preferably each microwell consists of a single pseudo-islet.

[0035] For the generation of pseudo-islets, the cells can be dissociated and re-aggregated by various methods. Established techniques for generating pseudo-islets include spontaneous reaggregation, the hanging drop method, and the utilization of microwells. In specific embodiments, the microwell technology is employed for the re-aggregation process.

[0036] The advantage of microwells is the ability to achieve a uniform size of pseudo-islets consistently. Moreover, this approach has been demonstrated to be efficient for large-scale experiments, rendering it a practical and scalable method with significant potential for further application in preparing material either for therapeutic treatments or other research applications.

[0037] Suitable microwells or corresponding devices or products are known to a skilled person and may be elected for use accordingly. By way of non-limiting example, commercially available microwells include but are not limited to Sphericalplate 5D, KUGELMEIERS, AggreWell Microwell Plates, STEMCELL Technologies, Bioflat, SARSTEDT; Millicell Ultra-low Attachment (ULA), Merck; Spheroid Microplates, Corning Life Sciences. However, also customized microwells from inhouse production may be used (H Kato, T Miwa, et al., Front Endocrinol, 2022; R Suenaga et al., Sci Rep, 2022). Typically, each well can generate thousands of pseudo-islets, making it an efficient platform for conducting large-scale studies or preparation of transplantation material.

[0038] In embodiments, the islet cells may be genetically manipulated during dissociation, culturing, or other stage of the inventive method. Examples of genetic manipulation methods include but are not limited to gene and genome editing techniques, gene silencing techniques, transfection, transduction, gene synthesis, mutagenesis, and RNA editing. Non-limiting examples for gene editing techniques include CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZNFs). Non-limiting gene silencing techniques include RNA interference (RNAi) and antisense oligonucleotides. Non-limiting examples for viral transduction are lentiviral transduction, adenoviral transduction, adeno-associated viral (AAV) transduction, herpes simplex viral (HSV) transduction, vesicular stomatitis virus (VSV) transduction, poxviral transduction, baculoviral transduction, and retro-transposon-based transduction. Non-limiting examples for chemical transfection methods are lipofection, calcium phosphate transfection, DEAE-Dextran transfection, polyethyleneimine (PEI) transfection, dendrimer-based transfection, hydrodynamic transfection, peptide-based transfection, and nanoparticle-based transfection. Non-limiting examples for physical transfection methods are electroporation, microinjection, biolistic particle delivery (gene gun), sonoporation, hydrodynamic injection, laser-based transfection, nanoinjection, and optoporation. The skilled person is aware of distinct genetic manipulation methods, and a suitable method may be selected when carrying out the invention.

[0039] In embodiments, greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the pseudo-islets exhibit a defined size with an average diameter of 30-350 pm. The defined size of the pseudo-islets may also fall within a size range formed between any two endpoints mentioned in the list.

[0040] In embodiments, the defined size of the pseudo-islets is an average of 50-250 pm, preferably 70- 200 pm.

[0041] It is known in the art that the optimal islet size for transplantation ranges between 100 to 150 pm. This particular size range is favored because islets of this size are more resistant to hypoxia and exhibit increased diffusion characteristics for oxygen, nutrition, and hormones. However, generation of pseudo-islets with a clearly defined size had previously proven to be a formidable challenge. Therefore, it was entirely surprising that treating the islets with a Rho / ROCK inhibitor facilitated the aggregation of cells into size-defined pseudo-islets.

[0042] In embodiments, a pseudo-islet comprises or consists of 100-2000 aggregated cells. In embodiments, a pseudo-islet comprises or consists of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000 cells. The number of cells of the pseudo-islets may also fall within a size range formed between any two endpoints mentioned in the list.

[0043] In embodiments, a population of 10 or more pseudo-islets exhibits a standard deviation (SD) of 10 or less. The standard deviation of 10 or less signifies that the method of pseudo-islet generation, according to the invention, consistently produces results with minimal variability and high reproducibility. In embodiments, a population of 20, 50, 100, or 200 or more pseudo-islets exhibits a standard deviation (SD) of 10 or less. In embodiments, a standard deviation of pseudoislet size may be 10 or less, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0, or any other value within these values.

[0044] SD ranges may also be formed using any value listed in combination with any other value to form a range.

[0045] The following Table 3 comprises a non-limiting and exemplary list of the size of adult porcine pseudo-islets that were treated with or without the Rho / ROCK inhibitor Y-27632. In embodiments, the Rho / ROCK inhibitor used herein may differ.

[0046] Table 3: By way of example, the table lists non-limiting sizes of adult porcine pseudo-islets (PI) depending on the number of cells in a microwell.

[0047] In other embodiments, the particular cell numbers per microwell, diameters of pseudo-islets and the distribution of different sizes within a pseudo-islet population (here represented by the SD) may vary within the ranges disclosed herein. These ranges represent a generalization of the examples and may be obtained without undue effort by carrying out the present method.

[0048] Table 4: By way of example, the table lists non-limiting size ranges of adult porcine pseudo-islets (PI) depending on the number of cells in a microwell.

[0049] In embodiments, the adult porcine pseudo-islet diameter depends on the number of cells seeded in a microwell.

[0050] For example, in embodiments, about 300 cells per microwell tend to form a pseudo-islet with an average of 64.7 pm diameter (55 to 65 pm range) and an SD of 6.4 (preferably 0 to 10) when not treated with Y-27632.

[0051] In another embodiment, about 300 (preferably 150-450) cells per microwell may be used to form a pseudo-islet with an average of 85.8 pm diameter (70 to 100 pm, preferably 81 to 88 pm range) and an SD of 1 .8 (preferably 0 to 10) when treated with Y-27632.

[0052] In another embodiment, about 600 cells (preferably 450-750) per microwell tend to form a pseudo-islet with an average of 71 .3 pm diameter (65 to 74 pm range, preferably 65 to 70 pm) and an SD of 15.5 when not treated with Y-27632.

[0053] In another embodiment, about 600 cells (preferably 450-750) per microwell may be used to form a pseudo-islet with an average of 104.5 pm diameter (95 to 115 pm, preferably 101 to 107 pm range) and an SD of 0.0 (preferably 0 to 10) when treated with Y-27632.

[0054] In another embodiment, about 900 cells (preferably 750-1050) per microwell may be used to form a pseudo-islet with an average of 124.4 pm diameter (110 to 125 pm, preferably 120 to 125 pm range) and an SD of 0.8 (preferably 0 to 10) when treated with Y-27632.

[0055] In another embodiment, about 1200 cells (preferably 1050-1350) per microwell may be used to form a pseudo-islet with an average of 133.9 pm diameter (125 to 145 pm, preferably 130 to 137 pm range) and an SD of 3.5 (preferably 0 to 10) when treated with Y-27632.

[0056] In another embodiment, about 1500 cells (preferably 1350-1700) per microwell may be used to form a pseudo-islet with an average of 147.2 pm diameter (135 to 160 pm, preferably 145 to 160 pm range) and an SD of 4.9 (preferably 0 to 10) when treated with Y-27632.

[0057] As can be derived from the table, different cells per microwell lead to aggregation of pseudo-islets of various sizes. The following Table 5 comprises a non-limiting and exemplary list of the size of neonatal porcine pseudo-islets (or neonatal islet-like cell clusters, NICCs) that were treated with the Rho / ROCK inhibitor Y-27632. In embodiments, the Rho / ROCK inhibitor used herein may differ.

[0058] Table 5: By way of example, the table lists non-limiting sizes of neonatal islet-like cell clusters (NICCs) depending on the number of cells in a microwell.

[0059] In other embodiments, the particular cell numbers per microwell, diameters of pseudo-islets and the distribution of different sizes within a pseudo-islet population (here represented by the SD) may vary within the ranges disclosed herein. These ranges represent a generalization of the examples and may be obtained without undue effort by carrying out the present method.

[0060] Table 6: By way of example, the table lists non-limiting sizes of neonatal islet-like cell clusters (NICCs) depending on the number of cells in a microwell.

[0061] In embodiments, the NICC diameter depends on the number of cells seeded in a microwell.

[0062] For example, 600 cells per microwell tend to form a pseudo-islet with an average of 87.2 pm diameter (84 to 90 pm range) and an SD of 1 .2 when treated with Y-27632. In another embodiment, 900 cells per microwell may be used to form a pseudo-islet with an average of 96.5 pm diameter (93 to 99 pm range) and an SD of 1 .5 when treated with Y-27632. In another embodiment, 1200 cells per microwell may be used to form a pseudo-islet with an average of 107.4 pm diameter (104 to 110 pm range) and an SD of 1.9 when treated with Y-27632.

[0063] In another embodiment, about 600 cells (preferably 450-750) per microwell may be used to form a pseudo-islet with an average of 87.2 pm diameter (80 to 95 pm, preferably 84 to 90 pm range) and an SD of 1 .2 (preferably 0 to 10) when treated with Y-27632. In another embodiment, about 900 cells (preferably 750-1050) per microwell may be used to form a pseudo-islet with an average of 96.5 pm diameter (90 to 105 pm, preferably 93 to 99 pm range) and an SD of 1 .5 (preferably 0 to 10) when treated with Y-27632.

[0064] In another embodiment, about 1200 cells (preferably 1050-1500) per microwell may be used to form a pseudo-islet with an average of 107.4 pm diameter (100 to 115 pm, preferably 104 to 111 pm range) and an SD of 1 .9 (preferably 0 to 10) when treated with Y-27632.

[0065] As can be derived from the table, different cells per microwell lead to aggregation of pseudo-islets of various sizes.

[0066] In embodiments, the pseudo-islets exhibit a stimulation index of above 1 .

[0067] As used herein, the stimulation index is a metric for quantifying islet functionality. Surprisingly and beneficially, the pseudo-islets according to the invention demonstrated a stimulation index higher than 1 , indicating the functionality of these islets, thus enabling effective therapeutic treatments.

[0068] In other embodiments, the pseudo-islets exhibit a stimulation index of above 1 , above 10, above 20, above 30, above 40, above 50, above 60, above 70, above 80, above 90, above 100, above 110, above 120, above 130. Stimulation indices may also fall within a range formed between any two endpoints mentioned in the list.ln embodiments, the treatment with the Rho / Rock inhibitor has no influence or a negligible influence on the viability of the islets.

[0069] In embodiments, the viability of the cells is measured by flow cytometry and / or a staining method suitable for determining the viability of the cells, for example by staining for dead cells and analyzing via flow cytometry.

[0070] In embodiments, the treatment with the Rho / Rock inhibitor has no influence or a negligible influence on cellular subtypes present within the pseudo-islets.

[0071] In embodiments, the formation of pseudo-islets from neonatal porcine ICC improves the viability in comparison to native ICC (control).

[0072] As shown in the examples below, the method of the present invention has a surprising lack of negative effect on the viability of the cells or functionality of the pseudo-islets. The Rho / Rock inhibitor treatment is, therefore, beneficial in generating pseudo-islets with a robust function and regular size distribution.

[0073] The preferred methods to confirm the viability of the islets are flow cytometry, e.g., “fluorescence- activated cell sorting / cell analysis” (FACS), and / or a staining method, e.g., fluorescein diacetate / propidium iodide (FDA / PI) staining, wherein dead cells are stained with propidium iodide and live cells with fluorescein diacetate. For the analysis via flow cytometry, dead cells may be stained with Zombie Violet dye. In order to assess proper islet architecture, the islets can be imaged by various microscopy methods, including but not limited to light microscopy, fluorescence microscopy, confocal microscopy, electron microscopy, live-cell microscopy, etc. The cell composition of the pseudo-islets can be determined by assessing the secretion of various hormones, such as insulin, glucagon, and / or somatostatin, which are secreted by distinct cell types within the pseudo-islets. Additionally, the functionality of the pseudo-islets can be assessed by measuring the secreted insulin and calculating the stimulation index thereof. In embodiments, the inhibitor comprises one or more of Y-27632 (dihydrochloride), Thiazovivin, Fasudil hydrochloride, H-1152 dihydrochloride, Ripasudil, HA 1100 hydrochloride (Hydroxyfasudil), N-Boc-Y-27632, GSK429286A and Narciclasine, preferably Y-27632 (dihydrochloride).

[0074] The Rho / ROCK pathway proteins belong to the RAS superfamily and exhibit GTPase activity. They play a pivotal role in diverse cellular functions, including cell adhesion, migration, cytoskeletal organization, and apoptosis regulation.

[0075] In embodiments, the treatment with the Rho / ROCK inhibitor enables the generation of intact pseudo-islets with high viability and proper architecture and functionality. As mentioned above, it was entirely surprising that the treatment with a Rho / ROCK inhibitor would result in the formation of size-defined pseudo-islets without affecting the viability or cell subtypes of said islets.

[0076] Moreover, treatment with the Rho / ROCK inhibitor also resulted in higher insulin secretion after glucose challenge and a higher stimulation index, indicating the proper functionality of these islets.

[0077] To the best of the inventors’ knowledge, this represents the first instance where the technical challenge of achieving a defined size and function of porcine pseudo-islets has been successfully addressed and resolved.

[0078] The particular Rho / Rock inhibitor to be applied is not limiting to the invention and various such inhibitors may be employed. The examples demonstrate multiple functional inhibitors, thus enabling the use of a Rho / Rock inhibitor in the method without the requirement for further limitation to specific embodiments or examples.

[0079] In embodiments, the pseudo-islets are incubated in islet cultivation media with the Rho / ROCK inhibitor at a concentration of 0.1 to 30 pM.

[0080] In embodiments, pseudo-islets are incubated in islet cultivation media with the Rho / ROCK inhibitor at a concentration of 0.1 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM. The Rho / ROCK inhibitor concentration used for generating pseudo-islets may also fall within a concentration range formed between any two endpoints mentioned in the list.

[0081] In embodiments, the pseudo-islets are incubated in islet cultivation media with the Rho / ROCK inhibitor for 1 to 14 days. In embodiments, the pseudo-islets are incubated in islet cultivation media with the Rho / ROCK inhibitor for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13, and 14 days. The Rho / ROCK treatment duration for generating pseudo-islets may also fall within a time range formed between any two endpoints mentioned in the list.ln embodiments, the islet cultivation media used for culturing of the porcine pseudo-islets may include Pancreatic islet culture media, LONZA, Corning Neonatal Porcine Islet Culture Medium, CMRL-1099 medium, Ham F10, and / or any cultivation media for culture, maturation, and / or generation of pseudo-islets.

[0082] In one embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises bovine serum albumin (BSA). In a preferred embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises 0.5% bovine serum albumin (BSA). In one embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises fetal bovine serum (FBS). In a preferred embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises 5-20% fetal bovine serum (FBS), more preferably 5-15% FBS, most preferably 10% FBS.

[0083] In some embodiments, the addition of FBS results in higher insulin secretion and / or a higher stimulation index.

[0084] In one embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises human serum (HS). In a preferred embodiment, the islet cultivation media used for culturing the porcine pseudo-islets comprises 5-20% human serum (HS), more preferably 5-15% human serum (HS), most preferably 10% human serum (HS).

[0085] The use of human serum in GMP (Good Manufacturing Practice) grade ensures the safety, quality, and efficacy of the pseudo-islets (PI) described herein. This GMP-grade serum is critical for maintaining consistent quality across batches, which is essential for the reproducibility of PI generation. By adhering to GMP standards, the method reduces the risk of contamination and variability, ensuring that the pseudo-islets meet stringent regulatory requirements for clinical applications, thereby enhancing the reliability of therapeutic outcomes.

[0086] In embodiments, the porcine subject is an adult porcine or a neonatal porcine subject, and optionally wherein the single cells dissociated from islets or islet-like cell clusters (ICC) were cryopreserved prior to step (i).

[0087] In embodiments, the porcine subject is an adult porcine. The advantages for using adult porcine islets are that they are morphologically distinct and can therefore be extracted and purified as a single unit, they are mature cells and respond immediately to hyperglycemia following transplantation without latency, they do not require culturing, and the islet yield is relatively high (R. Chinnuswami, Porcine Islet Cell Xenotransplantation, 2020).

[0088] In other embodiments, neonatal porcine subjects may be used due to several reasons: simple isolation process, no gradient purification, scalability to provide a clinical product, isolation is not dependent on the enzyme collagenase, easy maintenance of neonates, and resistance to inflammatory and hypoxia-induced injury.

[0089] In embodiments, the single cells dissociated from islets or islet-like cell clusters (ICC) were cryopreserved prior to step (i). Typical cryopreservation procedures are established in the art and may be employed. It was surprising that cryopreserved material is also susceptible to effective Rho / ROCK treatment to obtain the desired pseudo-islet sizes.

[0090] In another aspect or in embodiments of the invention described herein, the invention relates to a method of generating pseudo-islets, comprising the steps of: a) Dissociating isolated porcine islets into single cells, b) Stopping the dissociation process by adding cold culture media, c) Filtering the cell suspension through a mesh, d) Washing and counting the cell suspension, e) Re-aggregating the cells to allow pseudo-islet formation by culturing the pseudo-islets in cultivation media with a Rho / ROCK inhibitor, f) Harvesting the pseudo-islets.

[0091] In embodiments, the dissociation of the isolated adult porcine islets or islet-like cell clusters (ICC) into single cells involves washing them twice with PBS and incubating with trypsin-DNase- dissociation solution (cell dissociation buffer, 0.5 mg / ml DNAse I, Trypsin-EDTA) for 8 min. at 37°C, while gently shaking.

[0092] In embodiments, the dissociation medium is trypsin-DNase-dissociation solution or Accumax.

[0093] In embodiments, the successful dissociation of the cells can be confirmed by microscopy.

[0094] In embodiments, the cell suspension is filtered through a mesh, preferably a 10-30 pm (20 pm) or a 30-50 pm (40 pm) mesh for neonatal porcine islets and adult porcine islets.

[0095] In embodiments, the cell suspension can be counted using a Neubauer chamber.

[0096] In embodiments, the dissociated single cells are added on AggreWell 400™ Microwell Plates or the Spherical microwell plate 5D™ from Kugelmeiers™ to form pseudo-islets containing 300, 600, 900, 1200 or 1500 cells. In embodiments, the AggreWell 400™ Microwell Plates or the Spherical microwell plate 5D™ from Kugelmeiers™ are pre-coated with an anti-adherence solution or the culture medium (500 pl per well).

[0097] In embodiments, the anti-adherence solution is a surfactant solution (StemCell™, catalog number 07010) or the same culture medium used to culture the pseudo-islets. Both can be used for pretreating culture ware to reduce surface tension and prevent cell adhesion.

[0098] In embodiments, the cultivation media is HAM F10 or CMRL-1099 containing a Rho / ROCK inhibitor (e.g. Y-27632) with a final concentration of 10 pM.

[0099] In embodiments, the pseudo-islets are cultivated in a humidified chamber with 5% CO2, 20% O2, or 90% humidity at 37 °C for 1 to 14 days before harvest. In embodiments, the culture medium is changed every 48 hours. In embodiments, the pseudo-islets are cultivated for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 days before harvest, preferably 7 to 10 days. The cultivation of pseudo-islets may also fall within a time range formed between any two endpoints mentioned in the list.

[0100] In embodiments, the size-defined pseudo-islets are encapsulated after step (iii), wherein preferably the size of the encapsulated pseudo-islets exhibits an average diameter of 70-150 pm, preferably 80-120 pm.

[0101] In embodiments, the pseudo-islets are encapsulated in a microencapsulation device (preferably comprising a single pseudo-islet), macroencapsulation device (preferably comprising multiple pseudo-islets) and / or a 3D-printed scaffold.

[0102] One advantage of the invention is the possibility of using different encapsulation devices, such as micro-Zmacro-encapsulation devices and / or 3-D printed scaffolds. Encapsulation devices of various builds, types and dimensions are known to a skilled person and may be employed accordingly. In embodiments, the size of the pseudo-islets can be modified or set at a desired size depending on the intended encapsulation approach and device.

[0103] In embodiments, macroencapsulation devices may include a device as disclosed in WO2023 / 117849 A1 , or in US9132226. Microencapsulation devices may include devices according to CA2362022 or KR101401798. 3-D printed encapsulation methods may include hydrogel-based scaffolds (for example, those in Duin, et al., Adv Healthc Mater, 2019; Ghasemi A, et al., Front Bioeng Biotechnol., 2021),

[0104] In another aspect, the invention provides an in vitro population of porcine pseudo-islets obtained by treatment with a Rho / ROCK inhibitor, wherein greater than 30% of the population exhibit a defined size with an average diameter of 30-350 pm and a standard deviation (SD) of 10 or less.

[0105] Hence, the invention relates to a method and an in vitro population, preferably produced from the method as disclosed herein.

[0106] The in vitro population may be defined by any one or more of the features disclosed herein for the method of the invention. For example, the preferred size of the pseudo-islets, their size distribution, the number of cells and their physical and / or functional characteristics as disclosed above apply, in embodiments, to both the method and the in vitro population of the present invention.

[0107] In one embodiment, the pseudo-islet population according to the invention is not encapsulated and used directly (naked) for medical administration into a mammalian subject, preferably a human subject.

[0108] In one embodiment, the pseudo-islet population according to the invention is encapsulated in a device suitable for medical administration in a mammalian subject.

[0109] As described in detail herein, the pseudo-islet population of the invention leads to therapeutically active pseudo-islets suitable for administration to a subject in need thereof. By encapsulating the pseudo-islets, the therapeutic agent can be effectively separated from the immune system of the subject post-administration and any negative reactions to the pseudo-islets. The pseudo-islets of the invention may therefore be offered after preparation, already present in an encapsulation device, or provided in a kit, for example comprising pseudo-islets and an encapsulation device in proximity, for example in the same packaging, or available from the same supplier.

[0110] The invention further relates to the use of the pseudo-islet population according to the invention for the treatment of diabetes. In embodiments, the pseudo-islet population may be used for treating type 1 diabetes, wherein the pseudo-islet population is transplanted to the subject. The invention further relates to methods for treating diabetes, comprising administering the pseudoislets of the invention to a subject in need thereof. In embodiments, the pseudo-islet population may be used for treating any medical condition causing insulin deficiency, such as type 1 diabetes, cystic fibrosis, or pancreatectomy.

[0111] In another aspect or in embodiments of the invention, the ex vivo method is used for in vitro culture (such as research purposes), drug screening, toxicity screening, and / or for studying metabolic diseases, preferably diabetes. In embodiments, the in vitro culture may be used to study the effects of drugs and compounds on insulin secretion, glucose metabolism, and other processes related to diabetes or other endocrine diseases.

[0112] In another aspect the present invention relates to a method for testing a candidate compound, the method comprising: a. providing a pseudo-islet according to the invention, b. bringing into contact a candidate compound with said pseudo-islet, c. detecting a readout, characteristic and / or one or more parameter(s) of the pseudo-islet, for example the size, size distribution, stimulation index or other functional properties or developmental processes such as differentiation, dedifferentiation, or cell-fate switching, d. optionally repeating steps a-c with one or more further candidate compounds, growth factors, or small molecules.

[0113] In another aspect, the present invention relates to a method assessing toxicity of a candidate substance on pseudo-islets, comprising: a. providing a pseudo-islet according to the invention, b. bringing into contact a candidate compound with said pseudo-islet, c. identifying the candidate substance as a safe candidate substance (a substance with low or no toxic effect) when said pseudo-islet exhibits no cytotoxic effect, preferably no detectable effect.

[0114] Another aspect of the present invention relates to a system for identifying a candidate substance, comprising: a. providing a pseudo-islet according to the invention, b. optionally at least one substance or a substance library, or means for providing and / or utilizing such a substance or substance library, c. means for determining (screening) an effect of said substance or substance library on the pseudo-islet, such as one or more compound(s) or composition(s), a device and / or a system configured (optionally for high-throughput screening) for identifying a readout, characteristic and / or one or more parameter(s) of the cell culture model.

[0115] In preferred embodiments, the pseudo-islets and methods of the invention may be employed to investigate the effects of drugs and compounds on insulin secretion, pseudo-islet size or size distribution, metabolism of glucose or other nutrients, or other processes related to diabetes disease development or progression, or to study the potential characteristics of a diabetes medicament or potential diabetes medicament on the properties of the pseudo-islets. Moreover, further investigations may include the generation, differentiation, dedifferentiation, and proliferation of islet cells or islet cell subtypes, e.g., regarding potential characteristics of growth factors, hormones, compounds or small molecules, on the proliferation or differentiation of progenitor cells into endocrine or exocrine cells that also can be applied to stem cell differentiation protocols. In embodiments, the present invention relates to a kit, preferably suitable for carrying out the method of the invention, said kit comprising a culture medium and at least one supplement comprising at least one Rho / ROCK inhibitor.

[0116] In embodiments, the kit optionally comprises a dissociation solution.

[0117] In embodiments, the kit optionally comprises a mesh for filtering the cell suspension.

[0118] In embodiments, the kit optionally comprises an anti-adherence solution.

[0119] In embodiments, the kit optionally comprises microwell plates suitable for pseudo-islet formation.

[0120] Embodiments and features of the invention described with respect to the ex vivo method and the in vivo population are considered to be disclosed with respect to each and every other aspect of the disclosure, such that features characterizing the ex vivo method may be employed to characterize the in vitro population and vice-versa. The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding Rho / ROCK inhibitor treatment leads to advantageous pseudo-islets of porcine origin, for example defined by their regular size distribution and reliable and stable production without loss of functional properties.

[0121] DETAILED DESCRIPTION OF THE INVENTION

[0122] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0123] The invention is directed to an ex vivo method for generating pseudo-islets from a porcine subject by providing single cells dissociated from islets or islet-like cell clusters (ICC) obtained from a porcine pancreas, treating the dissociated cells with a Rho / ROCK inhibitor, and obtaining pseudo-islets.

[0124] A “porcine subject” refers to any mammalian subject belonging to the Artiodactyla order, Suidae family, and St / s genus, commonly referred to as “pig” or “swine.” Herein, a porcine subject includes or preferably refers to neonatal and / or adult porcine subjects.

[0125] In embodiments, the neonatal phase encompasses the first 30 days after birth, with neonatal islets typically obtained from the pancreas within the first week of life. Depending on the age of the neonatal porcine islets, they constitute approximately 25-85% of endocrine cells and 25-85% of epithelial, mesenchymal, exocrine, progenitor and immune cells, considered islet precursor cells. These islet cells exhibit correct hyperglycemia in diabetic animal models as the precursor cells also have the potential to differentiate and / or proliferate into beta cells. The cellular aggregates are usually composed of less than 75% endocrine cells (10-45% beta cells), with the majority being cytokeratin-positive epithelial ductal cells, Vimentin-positive mesenchymal cells or amylase-positive acinar cells. Around day 45 of gestational phase the islet-like cell clusters begin to form. Over time, and during the neonatal phase, they start to resemble adult islets. Such clusters are often referred to as “islet-like cell clusters” or “ICC” and can be reliably obtained from neonatal material (Korsgren O, et al., Diabetologia., 1991 ; Chinnuswami, Rajeswar, et al., Xenotranspl-Compreh Stud, 2020).

[0126] In embodiments, the adult porcine islets refer to differentiated islets that exhibit distinct and intact capsules and vasculature with few insulin-positive cells outside these islets. The optimal age for obtaining adult islets depends on the type of the breed (such as Gottingen minipig or landrace or Yorkshire), typically over one or two years, at which point they predominantly consist of islet endocrine cells.

[0127] Herein, the terms “islet,” “pancreatic islet,” or “islet of Langerhans” may be used interchangeably, unless specified to the contrary, and refer to any regions within the pancreas that consist of clusters of endocrine cells. In general, each islet consists of distinct cell types, including but not limited to alpha (a), beta (P), delta (5), epsilon (E), and pancreatic polypeptide (PP) cells, which regulate the glucose metabolism by producing and secreting hormones. These hormones may include, without limitation, insulin, glucagon, somatostatin, pancreatic polypeptide, ghrelin, amylin, and c-peptide.

[0128] The term “pseudo-islet” (also “pseudoislet” or “PI”) is used herein to refer to an artificial cellular aggregate or structure, where native islets or ICC have been isolated from the pancreas, then dissociated to single cells, and reaggregated to form said “pseudo-islets.” “Pseudo-islets” are typically composed of various cell types, including but not limited to insulin-producing beta cells and other supporting cells. These engineered structures are designed to replicate the physiological behavior of natural islets, such as the ability to respond to changes in blood glucose levels by releasing insulin and maintaining glucose homeostasis in the body.

[0129] In embodiments, the single cells dissociated from islets or islet-like cell clusters (ICC) were used immediately to generate ‘fresh’ pseudo-islets or were cryopreserved after obtaining the islets from the porcine pancreas. The process of “cryopreservation” is known to the skilled person in art. The temperature for cryopreservation can be variable, most commonly between -80°C to -196°C (Liu F. et al., J Artif Organs, 2014), and may include the process of one or several freeze and thaw periods.

[0130] As used herein, “encapsulation” is enclosing pancreatic islets within a protective “immune- isolation device,” obviating the need for immunosuppression. The fundamental concept behind encapsulation involves placing transplanted cells within an artificial compartment separated from the immune system by a semipermeable membrane. This membrane serves as a protective barrier, shielding the encapsulated cells from potential harm caused by antibodies, complement proteins, and immune cells. As a result, the encapsulation device is often referred to as an “immunoisolation device.” These devices, usually composed of semipermeable materials, facilitate the exchange of essential substances like glucose, oxygen, nutrients, metabolic waste, and insulin while preventing the entry of large molecules, such as immune cells or antibodies. This encapsulation process transforms the islets into a “bioartificial pancreas” or “in vivo pseudoislets”. “Immunoisolation devices” or “bioartificial pancreata” are typically categorized into two main types: intravascular and extravascular devices. The distinction between them is whether or not they are directly connected to the bloodstream. Furthermore, extravascular devices can be subcategorized into macroencapsulation and microencapsulation devices, depending on their specific design and application.

[0131] In embodiments, the pseudo-islets are encapsulated in a microencapsulation device (preferably comprising a single pseudo-islet), macroencapsulation device (preferably comprising multiple pseudo-islets) and / or a 3D-printed scaffold. The skilled person is aware of distinct encapsulation methods, and a suitable method may be selected when carrying out the invention.

[0132] In embodiments, the pseudo-islets are encapsulated in a microencapsulation device. Microencapsulation materials typically comprise natural or synthetic polymers or blends, including but not limited to collagen, gelatin, fibrin, polyphosphazene, poly(acrylic acids), poly (methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(alkylene oxides), poly(vinyl acetate), polyvinylpyrrolidone, polyethylene glycol (PEG), polyethersulfone, polysaccharides such as agarose, cellulose sulfate, chondroitin sulfate, chitosan, hyaluronan, and copolymers, and blends of each (Pereira MS et al., Materials, 2020), or any combination of said materials.

[0133] The most widely researched microencapsulated cells are pancreatic beta cells within alginate / poly-l-lysine-based hydrogel microcapsules. The alginate membrane is preferably a biodegradable polymer derived from brown algae and has been used in xenotransplantation procedures to generate protection for the transplanted cells against attack by the immune system, allowing for the diffusion of nutrients, oxygen, and metabolic products to maintain cellular physiology.

[0134] In embodiments, the pseudo-islets are encapsulated in a macroencapsulation device, including but not limited to tubular chambers, sealed hollow fibers, planar devices, agarose hydrogel membranes with microwell patterns, and microwell porous membranes, or any combination of said devices. Non-limiting examples of macroencapsulation devices are the Theracyte device or the Bioartificial Pancreas (BAP) (Photiadis et al., ASAIO J, 2021).

[0135] In embodiments, the pseudo-islets are encapsulated in a 3D-printed scaffold. As used herein, “3D-printed scaffold” or “3D-printed capsule” may be used interchangeably and refers to a three- dimensional structure fabricated using bioprinting technology designed to support and guide the growth of cells or tissues in biological applications. These scaffolds are typically constructed with biocompatible materials, such as polymers or hydrogels used as inks and piezoelectric or thermally driven mechanisms to process high-resolution bio-ink droplets.

[0136] It is essential to acknowledge that the research for suitable encapsulation devices is still at an early stage. Therefore, using alternative or more advanced encapsulation methods is a distinct possibility in the future as the field progresses. The Buhler group has recently published a review article on islet encapsulation, including “Design Rules for Islets Encapsulation,” which may be considered for possible encapsulation devices (Zhang et al., Front Immunol., 2022).

[0137] The delivery of encapsulation devices varies based on the specific device and the treatment strategy. In general, the implantation of these devices may be performed in the subcutaneous space - an implant site that is easily accessible but has inherently low vasculature.

[0138] Macroencapsulation devices may typically be placed in a suitable anatomical location, such as the abdominal cavity or subcutaneously. Microencapsulation methods may involve injection or implantation, depending on the size and type of capsules used.

[0139] Herein, the term “Rho / ROCK inhibitor,” "Rho / ROCK pathway inhibitor,” “ROCK inhibitor,” or “Rho inhibitor” may be used interchangeably and refers to any compound or substance that targets Rho-associated protein kinases (ROCK) and inhibits the Rho / ROCK pathway. In the context of the present invention, any Rho / ROCK inhibitor is not limited to any particular compounds, but in embodiments may be any one or more of the herein exemplarily named compounds.

[0140] Non-limiting examples of Rho / ROCK inhibitors are WAY-624704, Y-27632 (dihydrochloride), Y- 27632 2HCI, N-Boc-Y-27632, Y-39983 HCI, Y-30141 .Y-33075, Thiazovivin, Fasudil (HA-1077) HCI, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001), RKI-1447, AT13148, GSK429286A, GSK269962A HCI, GSK429256A, GSK576371 , Belumosudil (KD025), Ripasudil (K-115) hydrochloride dihydrate, Hydroxyfasudil (HA-1100) HCI, Netarsudil (AR-13324) 2HCI, Verosudil (AR-12286), Narciclasine, ZINC00881524 (ROCK inhibitor), -Elemene, Chromanl , DJ4, H- 1152, Ibuprofen, LX-7101 , TCS-7001.

[0141] Common methods to test the activity of a chemical inhibitor is known by a skilled person. In embodiments, testing the activity of the Rho / ROCK inhibitor may include cell viability assays, western blot analysis, immunofluorescence, immunohistochemistry, enzyme activity assays, phenotypic assays, cell signaling pathway assays, functional assays, animal studies, doseresponse curves, time course experiments, and / or control experiments.

[0142] As used herein, the “stimulation index” or “SI” serves as a metric to express islet functionality (Groot Nibbelink M. et al., Int J Mol Sci, 2016). It is defined as the ratio of insulin secreted under high glucose stimulation to the basal insulin secreted in low glucose conditions. For islets of Langerhans, a threshold stimulation index of above 1 defines a functional response. Generally, a higher SI is indicative of better pseudo-islet functionality. In particular embodiments, pseudo-islets were determined as functional when the stimulation index was above one.

[0143] As used herein, the “viability” of a cell is as commonly understood by a skilled person and refers to the amount and / or number of live cells within a population and is usually expressed as a percentage of the control, e.g., dead cells. Cell viability assays are used to measure the physical or physiological health of cells (how “viable” a cell is), e.g., in response to extracellular stimuli, chemical agents, therapeutic treatments, etc.

[0144] A skilled person is aware of different methods for detecting the viability and / or composition of an islet cell. Examples of methods for detecting the viability and / or composition of an islet cell include fluorescein diacetate and propidium iodide (FDA / PI) staining and / or flow cytometry, e.g., fluorescence-activated cell sorting (FACS).

[0145] Diabetes, also known as diabetes mellitus, constitutes a group of metabolic disorders characterized by chronic hyperglycemia resulting from impairments in insulin secretion, insulin function, or both (Kharroubi AT, Darwish HM. World J Diabetes. 2015 Jun 25;6(6):850-67). These metabolic irregularities extend to carbohydrates, lipids, and proteins. The root causes of these metabolic disturbances lie in insufficient insulin levels for an effective response and / or resistance to insulin in target tissues, primarily skeletal muscles and adipose tissue, with some impact on the liver. These issues stem from anomalies within insulin receptors, the signal transduction system, or effector enzymes and genes. The traditional classification into types 1 , 2, other types, and gestational diabetes remains widely accepted.

[0146] In embodiments, the pseudo-islet population, according to the invention, is used for the treatment of diabetes, preferably type 1 diabetes, in a subject, wherein the pseudo-islet population is transplanted to the subject.

[0147] Herein, "type I diabetes," "type 1 diabetes, "or"T1 D"are used interchangeably and refer to one class of diabetes (also diabetes mellitus), including but not limited to autoimmune type 1 diabetes, idiopathic type 1 diabetes, and fulminant type 1 diabetes. Type 1 diabetes results from autoimmune destruction of pancreatic p cells, driven by T-cell mediated inflammation and B-cell responses. Autoantibodies against islet cells, insulin, GAD65, IA2, IA2p, and ZnT8A are characteristic, often detected before symptoms. Genetic predisposition plays a significant role, with specific HLA-DR / DQ alleles being either predisposing or protective. Several environmental factors are implicated, including viral infections (e.g. coxsackievirus B, rubella, enteroviruses, cytomegalovirus, rotavirus), low vitamin D levels, exposure to pollutants, hygiene changes, infant nutrition, and insulin resistance due to obesity or rapid growth. Type 1 diabetes symptoms include but are not limited to extreme thirst, frequent urination, fatigue, excessive hunger, unexplained weight loss, slow wound healing, infections, and blurred vision. Children typically experience more severe symptoms. Individuals with this type are at higher risk for other autoimmune disorders like Graves' disease, Hashimoto's thyroiditis, and vitiligo.

[0148] Herein, "type II diabetes," "type 2 diabetes, "or"T2D" refer to one class of diabetes (also diabetes mellitus) and refers to a chronic metabolic disorder characterized by insulin resistance coupled with a relative deficiency of insulin production by the pancreas and thus elevated blood sugar levels. Long-term effects include damage to nerves and blood vessels. Over 95% of diabetes cases are type 2, characterized by insulin resistance often coupled with reduced insulin secretion. "Prediabetes," a condition where glucose levels are higher than average but not yet high enough to be classified as type 2 diabetes, can precede Type 2 diabetes. Contributing factors include obesity, inactivity, poor diet, stress, and genetics.

[0149] Herein, "gestational diabetes" refers to diabetes that manifests during pregnancy due to hormonal interference with insulin function, resulting in elevated blood glucose levels. Gestational diabetes shares similarities with type 2 diabetes, involving insufficient insulin secretion and responsiveness. Screening typically occurs at 24-28 weeks of pregnancy. Elevated maternal glucose levels during pregnancy pose health risks to both the mother and the baby. Gestational diabetes often reoccurs in subsequent pregnancies. Untreated, gestational diabetes can harm both the fetus and the mother. Risks to the baby include high birth weight, congenital abnormalities, respiratory distress syndrome, and increased bilirubin levels. In severe cases, perinatal death may occur due to poor placental function. Medical interventions, such as labor induction or cesarean section, may be necessary to manage complications like macrosomia and shoulder dystocia.

[0150] Other uncommon types of diabetes include maturity onset diabetes of the young (MODY), latent autoimmune diabetes in adults (LADA), ketosis-prone diabetes, and other secondary forms of diabetes such as pancreatic cancer and haemochromatosis (YN Lee and MSB Huda, Clin Med, 2021).

[0151] A pseudo-islet population comprised in a (pharmaceutical) composition can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intraocular, intranasal, intrapulmonary, or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes.

[0152] In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the pseudo-islet population may, in embodiments, be administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected condition or one or more symptom(s) thereof, wherein the condition is diabetes.

[0153] "Administration of’ and "administering" should be understood to mean providing a compound, namely the pseudo-islet population, or a pharmaceutical composition or a device comprising the pseudo-islet population as described herein.

[0154] As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve either the reduction or amelioration of symptoms of the disease or condition or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or associated symptoms thereof.

[0155] As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, preferably humans, and other organisms.

[0156] As used herein, "a subject in need thereof," refers to a subject afflicted with, or at risk of developing, a disease susceptible to treatment with the inventive pseudo-islets. The subject in need thereof may be a subject with diabetes, pre-diabetes, or suspected of having diabetes or pre-diabetes or any other related disorder.

[0157] In the present invention "treatment" or “therapy” generally means to obtain a desired pharmacological effect and / or physiological effect. The effect may be prophylactic in view of completely or partially preventing a disease and / or a symptom, for example by reducing the risk of a subject having a disease or symptom or may be therapeutic in view of partially or completely curing a disease and / or adverse effect of the disease.

[0158] In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom. The term “xenotransplantation” or “cross-species transplantation” refers to the transfer of live cells, tissues, or organs from one species to another. “Human xenotransplantation” refers to the transfer from non-human animals into humans, e.g., due to the shortage of human donor organs, and holds promise for treating organ failure and certain diseases like heart failure, kidney failure, neurodegenerative disorders and diabetes. Initially, non-human primates, such as chimpanzees and baboons, were considered suitable candidates. To date, pigs, especially genetically engineered pigs or interspecies chimeras, are regarded as the most preferred donors in xenotransplantation. Examples of organs for xenotransplantation include but are not limited to blood, blood vessels, skin, cells, corneal, kidney, heart, liver, and islet xenotransplantations.

[0159] The instant disclosure also includes kits, packages, and multi-container units containing the herein-described pseudo-islet population or pharmaceutical compositions comprising the same and / or means for administering the same for use in the prevention and treatment of conditions described herein and other conditions in human subjects.

[0160] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0161] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, where relevant, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value or the variation that exists among the study subjects.

[0162] FIGURES

[0163] The invention is demonstrated by way of example in the following figures. The figures are to provide a further description of potentially preferred embodiments that enhance the support of one or more non-limiting embodiments of the invention.

[0164] Brief description of the figures:

[0165] Figure 1 : Morphology of neonatal porcine islets and pseudo-islets with and without Y-27632 treatment. Figure 2: Diameter of neonatal porcine islets and pseudo-islets with or without Y-27632 treatment.

[0166] Figure 3: Mean diameter of isolated neonatal porcine islets and pseudo-islets.

[0167] Figure 4: Immunofluorescence images of native islets and pseudo-islets of different species.

[0168] Figure 5: Cell composition of neonatal porcine islets and pseudo-islets treated with or without Y- 27632.

[0169] Figure 6: Stimulation index of neonatal porcine islets and pseudo-islets treated with or without Y- 27632.

[0170] Figure 7: Morphology of adult porcine pseudo-islets treated with and without Y-27632 treatment.

[0171] Figure 8: Diameter of adult porcine pseudo-islets with or without Y-27632 treatment.

[0172] Figure 9: Mean diameter of isolated adult porcine native control islets and pseudo-islets composed of 300, 600, 900, 1200, and 1500 cells / islets of two separate experiments cultured with or without Y-27632.

[0173] Figure 10: Darkfield images of human, rat, and pig pseudo-islets (900 cells) treated with or without Y-27632.

[0174] Figure 11 : Brightfield images of dithizone-stained native adult porcine islets and pseudo-islets (900 cells) treated with or without Y-27632.

[0175] Figure 12: Representative immunohistochemical images of (A) neonatal pseudo-islets and (B) adult pseudo-islets. Quantification of viability staining of (C) neonatal pseudo-islets and (D) adult pseudo-islets treated with or without Y-27632.

[0176] Figure 13: Cell composition of adult porcine islets and pseudo-islets treated with or without Y- 27632.

[0177] Figure 14: Immunohistochemical image of adult porcine islets and pseudo-islets treated with or without Y-27632.

[0178] Figure 15: (A) Secreted insulin and (B) stimulation index of adult porcine pseudo-islets treated with or without Y-27632.

[0179] Figure 16: Schematic diagram of pseudo-islet generation.

[0180] Figure 17: Simplified overview of the pseudo-islet generation method.

[0181] Figure 18: Morphology of adult porcine pseudo-islets untreated or treated with various Rho / ROCK pathway inhibitors.

[0182] Figure: 19 Diameter of adult porcine pseudo-islets untreated or treated with various Rho / ROCK pathway inhibitors.

[0183] Figure 20: Viability assessment of adult porcine pseudo-islets untreated or treated with various Rho / ROCK pathway inhibitors. Figure 21 : Quantification of viability of adult porcine pseudo-islets untreated or treated with various Rho / ROCK pathway inhibitors.

[0184] Figure 22: Cell composition of native adult porcine islets under Y27632 treatment and pseudoislets with various Rho / ROCK pathway inhibitors.

[0185] Figure 23: Functionality assessment by dynamic glucose-stimulated insulin secretion assay of adult porcine pseudo-islets treated without and with various Rho / ROCK pathway inhibitors.

[0186] Figure 24: Functionality assessment by dynamic GSIS of neonatal porcine native islets and derived pseudo-islets under 0.5% BSA and 10% FBS.

[0187] Figure 25: Morphology of neonatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0188] Figure 26: Diameter of neonatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0189] Figure 27: Viability assessment of neonatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0190] Figure 28: Quantification of viability of neonatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0191] Figure 29: Cell composition of neatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0192] Figure 30: Functionality assessment by dynamic GSIS of neonatal porcine derived pseudo-islets from fresh or cryopreserved cells under the effect of Y-27632.

[0193] Figure 31 : Morphology of neonatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0194] Figure 32: Diameter of neonatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0195] Figure 33: Viability assessment of neonatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0196] Figure 34: Quantification of viability of neonatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0197] Figure 35: Cell composition of neatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0198] Figure 36: Functionality assessment by dynamic GSIS of neonatal porcine derived pseudo-islets generated with 10% FBS or 10% HS under the effect of Y-27632.

[0199] Detailed description of the figures:

[0200] Figure 1 : Morphology of neonatal porcine islets and pseudo-islets with and without Y-27632 treatment. Darkfield images of harvested pseudo-islets after 7 days of culture in microwells with and without Y-27632. Increased amounts of seeded cell numbers per microwell (600, 900, 1200) led to larger pseudo-islets. Treatment with Y-27632 improved cluster formation and resulted in clusters morphologically similar to native undissociated control islets.

[0201] Figure 2: Diameter of neonatal porcine islets and pseudo-islets with or without Y-27632 treatment. Size distribution (mean + / - SD) of isolated neonatal porcine native control islets and pseudo-islets composed of 600, 900, 1200 cells / islet of three separate experiments cultured with or without Y-27632. Reassociation of islet cells to spheroid clusters was accomplished after 7 days. Treatment with Y-27632 resulted in homogenous and size-defined pseudo-islets.

[0202] Figure 3: Mean diameter of isolated neonatal porcine islets and pseudo-islets. Mean diameter of isolated neonatal porcine native control islets and Pseudo-Islets composed 600, 900 and 1200, cells / islets of three separate experiments cultured with or without Y-27632. Reassociation of islet cells to spheroid clusters was accomplished after 7 days.

[0203] Figure 4: Immunofluorescence images of native islets and pseudo-islets of different species. Native islets and pseudo-islets of different species (human, rat, adult pig, neonatal pig) are compared regarding to the location of distinct cell subtypes. The formation of pseudo-islets does not affect the location of distinct cell subtypes.

[0204] Figure 5: Cell composition of neonatal porcine islets and pseudo-islets treated with or without Y- 27632. Percentage of beta-, alpha- and delta-cells of harvested native control islets and pseudoislets treated with or without Y-27632 measured by flow cytometry analyses. Treatment with Y- 27632 does not influence islet cells subtypes composition.

[0205] Figure 6: Stimulation index of neonatal porcine islets and pseudo-islets treated with or without Y- 27632. After equilibration at 5 mM glucose (pre-incubation), islets were incubated on PluriStrainer mini 10 pm in 1 ml of 5 mM glucose for 1 hr (basal secretion) followed by incubation in 11 mM for 1 hr (stimulatory secretion). Insulin measurement was performed using cisbio HTRF© insulin ultra-sensitive kit according to manufacturer’s instructions. Stimulation index is expressed as stimulation index (SI) (mean - / + SD) from three biological replicates. Islets treated with Y27632 tend to have a significantly higher stimulation index (stimulatory secretion over basal secretion) in comparison to untreated ones of respective size.

[0206] Figure 7: Morphology of adult porcine pseudo-islets treated with and without Y-27632 treatment. Darkfield images of harvested pseudo-islets after 7 days of culture in microwells with and without Y-27632. Increased amounts of seeded cell numbers per microwell (300, 900, 1500) led to larger pseudo-islets. Treatment with Y-27632 improved cluster formation and resulted in clusters morphologically similar to native undissociated control islets.

[0207] Figure 8: Diameter of adult porcine pseudo-islets with or without Y-27632 treatment. Size distribution (mean + / - SD) of isolated adult pig native control islets and pseudo-islets composed of 300, 600, 900, 1200, and 1500 cells / islet of two separate experiments cultured with or without Y-27632. Reassociation of islet cells to spheroid clusters was accomplished after 7 days. Treatment with Y-27632 resulted in homogenous and size-defined pseudo-islets within and between experiments. Figure 9: Mean diameter of isolated adult porcine native control islets and pseudo-islets composed of 300, 600, 900, 1200, and 1500 cells / islets of two separate experiments cultured with or without Y-27632. Reassociation of islet cells to spheroid clusters was accomplished after 7 days.

[0208] Figure 10: Darkfield images of human, rat, and pig pseudo-islets (900 cells) treated with or without Y-27632. Porcine pseudo-islets treated with Y-27632 exhibit similar sizes to human or rat pseudo-islets. Porcine pseudo-islets not treated with Y-27632 result in a smaller and inhomogeneous diameter.

[0209] Figure 11 : Brightfield images of dithizone-stained native adult porcine islets and pseudo-islets (900 cells) treated with or without Y-27632. Treatment with Y-27632 improves cluster formation and results in clusters morphologically similar to pseudo-islets derived from human and rat.

[0210] Figure 12: Representative immunohistochemical images of (A) neonatal pseudo-islets and (B) adult pseudo-islets. Quantification of viability staining of (C) neonatal pseudo-islets and (D) adult pseudo-islets treated with or without Y-27632. Treatment with Y-27632 does not influence the viability of the islets, as shown by (A, B) fluorescein diacetate (green) I propidium iodide (red) (FDA / PI) staining and (C, D) Zombie UVTMby Flow Cytometry analyses.

[0211] Figure 13: Cell composition of adult porcine islets and pseudo-islets treated with or without Y- 27632. Percentage of beta-, alpha- and delta-cells of harvested native control islets and pseudoislets treated with or without Y-27632 were measured by flow cytometry analyses. Treatment with Y-27632 does not influence islet cell subtype composition.

[0212] Figure 14: Immunohistochemical image of adult porcine islets and pseudo-islets treated with or without Y-27632. Representative images (400x) stained for insulin (green) and glucagon (red). Architecture and cell composition of pseudo-islets are comparable to native islets treated with or without Y-27632. Treatment with Y-27632 has no influence on islet architecture and cell composition.

[0213] Figure 15: (A) Secreted insulin and (B) stimulation index of adult porcine pseudo-islets treated with or without Y-27632. Islets were perifused in a closed column at a flow rate of 200 pl / min using a perifusion system. After equilibration at 3.3 mM glucose, islets were stimulated with high glucose (16.7 mM for 40 min), followed by 30 min of 3.3 mM glucose and 20 min of 60 mM KCI + 16.7 mM glucose. Insulin measurement was performed using cisbio HTRF kit for Insulin according to manufacturer’s instructions. Insulin release is expressed as secreted insulin and stimulation index (SI) (mean - / + SD) over mean basal secretion (0-10 min) and area under the curve (AUC) (mean - / + SEM). (A) Islets dynamically respond to glucose and start to secret insulin after glucose challenge. (B) Stimulation index of adult porcine pseudo-islets treated with or without Y-27632. Treatment with Y-27632 resulted in higher insulin secretion after glucose challenge and higher stimulation index (SI) for smaller pseudo-islets.

[0214] Figure 16: Schematic diagram of pseudo-islet generation. Native islets are dissociated into single cells and then re-aggregated into pseudo-islets. The generated pseudo-islets exhibit similar cell numbers, identical sizes, uniform cellular compositions, and similar functionality. Figure 17: Simplified overview of the pseudo-islet generation method. Native islets first undergo enzymatic dissociation. Dissociated single cells are then seeded in microwells to generate pseudo-islets of different sizes by culturing the islets for 7 days with or without the Rho / ROCK inhibitor, e.g., Y-27632.

[0215] Figure 18: Representative dark-field microscopic images depicting the impact of various Rho / ROCK pathway inhibitors (Y-27632, Fasudil, Thiazovivin, H-1152, Ripasudil) on pseudo-islet formation containing 900 cells (P900) from adult pig after 7 days of culture compared to untreated group. Scale bars represent 200 pm.

[0216] Figure 19: Quantitative analysis of the diameter of adult pig derived pseudo-islet of 900 cells (P900) formed with different Rho / ROCK pathway inhibitors (Y-27632, Fasudil, Thiazovivin, H- 1152, Ripasudil) relative to untreated group across three independent experiments highlighting significant differences between treatments. Statistical significance was deretemined by one-way ANOVA followed by Tukey's post hoc test with ns indicating not significant (p > 0.05), ****p < 0.0001.

[0217] Figure 20: Representative fluorescent microscopic images depicting the effect of different Rho / ROCK pathway inhibitors (Y-27632, Fasudil, Thiazovivin, H-1152, Ripasudil) on the viability of pseudo-islets (P900) compared to untreated. Cells are stained with Fluorescein diacetate (FDA-green) for viable cells and Prodium iodide (Pl-red) for dead cells. Scale bars represent 100 pm.

[0218] Figure 21 : Quantitative analysis of the viability assessment by FDA / PI staining for adult pig derived pseudo-islets of 900 cells (P900) untreated or treated with different Rho / ROCK pathway inhibitors (Y-27632, Fasudil, Thiazovivin, H-1152, Ripasudil) from three independent experiments. Statistical significance was deretemined by one-way ANOVA followed by Tukey's post hoc test with ns indicating not significant (p > 0.05), *p < 0.05, **p < 0.01 .

[0219] Figure 22: Percentage of delta cells (Somatostatin+), beta cells (lnsulin+), and alpha cells (glucagon+) in native islets treated with Y-27632 (gray), and pseudo-islets treated with various Rho / ROCK pathway inhibitors: Y-27632 (blue), Fasudil (orange), Thiazovivin (yellow), H-1152 (green), Ripasudil (purple). Data are from three independent experiments (N=3) and are presented as mean ± standard deviation (SD). Statistical significance was determined by oneway ANOVA followed by Tukey's post hoc test with ns indicating not significant (p > 0.05).

[0220] Figure 23: Dynamic glucose-stimulated insulin secretion (GSIS) profile of adult pig pseudo-islets (P900) untreated (black) or treated with Y-27632 (blue), Fasudil (orange), Thiazovivin (yellow), H- 1152 (green) and Ripasudil (purple). (A) Line plots show the changes in insulin secretion under basal (3 mM) and stimulatory (16.7 mM) glucose and KCL (60 mM) over time. (B) Bar graphs represent the total insulin content at the end of the experiment in ng / mL. (C) Line plots depict the changes in secreted insulin normalized to total insulin content over time. (D) Bar graphs represent the average of the normalized secreted insulin under basal glucose concentration (3mM) in the first 10 minutes (basal insulin secretion). (E) Line plots show the fold change in secreted insulin relative to basal insulin secretion(stimulation index). (F) Bar graphs represent the area under the curve (AUC) of the fold change of secreted insulin (0-80 min). Data points represent means, shaded areas and error bars represent the standard error of the mean (SEM). Statistical significance between conditions was assessed by one-way ANOVA followed by Dunnett's multiple comparisons test, with ‘ns’ marks non-significant differences (p > 0.05) and ****p < 0.0001 . Data is derived from three independent isolations (N=3).

[0221] Figure 24: Dynamic GSIS profile of native NICCs under 10% FBS (purple) in comparison to pseudo-islets (900 cells) under 0.5% BSA (black) and 10% FBS (blue) after 10 days in culture. All are treated with Y-27632 (A) Line plots show the changes in insulin secretion under basal (3 mM) and stimulatory (16.7 mM) glucose and KCL (60 mM) over time. (B) Bar graphs represent the total insulin content at the end of the experiment in ng / ml. (C) Line plots depict the changes in secreted insulin normalized to total insulin content over time. (D) Bar graphs represent the average of the normalized secreted insulin under basal glucose concentration (3mM) in the first 10 minutes (basal insulin secretion). (E) Line plots show the fold change in secreted insulin relative to basal insulin secretion (stimulation index). (F) Bar graphs represent the area under the curve (AUC) of the fold change of secreted insulin (0-80 min). Data points represent means, shaded areas and error bars represent the standard error of the mean (SEM). Statistical significance between conditions was assessed by one-way ANOVA followed by Dunnett's multiple comparisons test, with ‘ns’ marks non-significant differences (p > 0.05), and ****p < 0.0001 . Data is derived from three independent isolations (N=3).

[0222] Figure 25: Representative dark-field microscopy images of neonatal piglet derived pseudo-islets (P900) generated from cryopreserved (left) and fresh (right) cells. Scale bars represent 200 pm.

[0223] Figure 26: Diameter measurements of neonatal piglet derived pseudo-islets from three independent experiments (two experiment with 900 cells and one experiment with 600 cells) comparing pseudo-islets generated from cryopreserved (blue) and fresh (brown) cell. Data are presented as mean ± standard deviation (SD), N=3. Statistical significance was determined by Student's t-test, with ns indicating not significant (p > 0.05).

[0224] Figure 27: Representative fluorescent microscopic images of pseudo-islets (P900) generated from cryopreserved (left) and fresh (right) cells under Y-27632 treatment for 10 days in culture stained with FDA (green) for viable cells and PI (red) for dead cells. Scale bars represent 100 pm.

[0225] Figure 28: Quantitative analysis of the viability assessment by FDA / PI staining, showing the percentage of viable cells in cryopreserved (blue) and fresh (brown) pseudo-islets. Data are presented as mean ± standard deviation (SD) from three independent experiments Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test with ns indicating not significant (p > 0.05).

[0226] Figure 29: Bar plots showing the percentage of delta cells (somatostatin+), beta cells (lnsulin+) and alpha cells (glucagon+) in neonatal pseudo-islets generated from fresh cells (brown) or cryoperserved cells (blue). N=3, Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined by Student's t-test, with ns indicating not significant (p > 0.05).

[0227] Figure 30: Dynamic GSIS profile of pseudo-islets (900 cells) dervied from neonatal islet like cell clusters (NICCs) before (brown) and after (blue) cryopreservation of the cells, all treated with Y- 27632. (A) Line plots show the fold change in secreted insulin relative to the average normalized insulin in the first 10 minutes (basal insulin secretion). (B) Bar graphs represent the area under the curve (AUC) of the fold change of secreted insulin (0-80 min). Data points represent means, and shaded areas and error bars represent the standard error of the mean (SEM). Statistical significance between conditions was assessed using two-tailed t-test, with ‘ns’ marks nonsignificant differences (p > 0.05). Data is derived from three independent isolations (N=3).

[0228] Figure 31 : Representative dark-field microscopy images of neonatal piglet derived pseudo-islets (P900) generated under 10% of Fetal bovine serum ‘FBS’ (left) or under 10% human serum ‘HS’ (right) cells. Both conditions were treated with 10pm Y-27632 for 10 days in culture before imaging. Scale bars represent 200 pm.

[0229] Figure 32: Diameter measurements of neonatal piglet derived pseudo-islets (P900) generated under 10% of Fetal bovine serum ‘FBS’ (brown) or under 10% human serum ‘HS’ (blue). Data are presented as mean ± standard deviation (SD), N=3. Statistical significance was determined by Student's t-test, with ns indicating not significant (p > 0.05).

[0230] Figure 33: Representative fluorescent microscopic images of pseudo-islets (P900) generated under 10% of Fetal bovine serum ‘FBS’ (left) or under 10% human serum ‘HS’ (right). Both conditions were treated with 10pM Y-27632 for 10 days in culture before staining with FDA (green) for viable cells and PI (red) for dead cells. Scale bars represent 100 pm.

[0231] Figure 34: Quantitative analysis of the viability assessment by FDA / PI staining, showing the percentage of viable cells in pseudo-islets generated under 10% HS (blue) and 10% FBS (brown) after 10 days of culutre with 10pM of Y-27632. Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined by one-way ANOVA followed by T ukey's post hoc test with ‘ns’ indicating not significant (p > 0.05).

[0232] Figure 35: Bar plots showing the percentage of delta cells (somatostatin+), beta cells (lnsulin+) and alpha cells (glucagon+) in neonatal pseudo-islets generated under 10% FBS (brown) or 10% HS (blue) after 10 day in culture with 10 pM of Y-27632. N=3, Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined by Student's t-test, with ns indicating not significant (p > 0.05).

[0233] Figure 36: Dynamic GSIS profile of pseudo-islets (900 cells) dervied from neonatal islet like cell clusters (NICCs) under 10% FBS (brown) and 10% HS (blue) after 10 day of culture with 10pM of Y27632 ). (A) Line plots show the changes in insulin secretion under basal (3 mM) and stimulatory (16.7 mM) glucose and KCL (60 mM) over time. (B) Bar graphs represent the total insulin content at the end of the experiment in ng / mL. (C) Line plots depict the changes in secreted insulin normalized to total insulin content over time. (D) Bar graphs represent the average of the normalized secreted insulin under basal glucose concentration (3mM) in the first 10 minutes (basal insulin secretion). (E) Line plots show the fold change in secreted insulin relative to basal insulin secretion(stimulation index). (F) Bar graphs represent the area under the curve (AUC) of the fold change of secreted insulin (0-80 min). Data points represent means, shaded areas and error bars represent the standard error of the mean (SEM). Statistical significance between conditions was assessed by one-way ANOVA followed by Dunnett's multiple comparisons test, with ‘ns’ marks non-significant differences (p > 0.05). EXAMPLES

[0234] The following examples further describe the invention. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for a greater illustration of the invention described herein.

[0235] In order to demonstrate the functionality and beneficial properties of the pseudo-islet generation described herein, the following examples are to be considered:

[0236] General procedure for generating porcine pseudo-islets.

[0237] Functional analysis of porcine pseudo-islets from NICCs.

[0238] Example 1: General procedure for generating porcine pseudo-islets

[0239] In general, for the dissociation into single cells, isolated porcine islets were washed twice with PBS. 15-20% of the isolation is kept as native (control) and cultured in 10-cm suspension Petridish (1000 IEQ in each dish) and the rest of the islets (75-80%) are dissociated with either trypsin-DNase-dissociation solution (cell dissociation buffer (gibco) 10.5 mg / ml Dnase I (Roche) I Trypsin-EDTA (gibco)) or Accumax for 8 min at 37°C with gentle shaking.

[0240] After pipetting up and down several times, the proper dissociation was confirmed using a microscope, and the dissociation process was stopped by adding cold corresponding culture media (see tables below). The cell suspension was filtered through a 20 pm or 40 pm mesh, washed twice with culture media, and the cell number was counted using a Neubauer chamber.

[0241] For reaggregation, single cells were added onto AggreWell 400™ Microwell Plates or Spherical microwell plates 5D™ from Kugelmeiers™ to form pseudo-islets containing 300, 600, 900, 1200 or 1500 cells.

[0242] Non-limiting example of number of cells required for generation of pseudo-islets in Aggrewell 400™ (1200 microwells per well)

[0243] Pseudo-islets and native controls were incubated in corresponding islet cultivation media with or without 10 pM Y-27632 (TargetMol) in a humidified chamber with 5% CO2 at 37 °C for 7 days before harvest. Half of the medium was changed every 48 hr. The pseudo-islets and the native islets were then used for further validation and experimentation.

[0244] Adult Culture medium: NICCs Culture medium:

[0245] Results for pseudo-islets generated from adult porcine islets:

[0246] The morphology of adult porcine pseudo-islets is dependent on the cells seeded per microwell. As shown in Figure 7, an increased amount of seeded cell numbers per microwell (300, 900, 1500) leads to an increased size of pseudo-islets. Moreover, the morphology of the pseudo-islets treated with the Rho / ROCK inhibitor is comparable to those of the native control islets. Quantification of the pseudo-islet sizes confirms the results (Fig. 8 and 9); treatment of the islets with the inhibitor results in size-defined spherical pseudo-islets.

[0247] Example 2: Functional analysis of porcine pseudo-islets from NICCs In brief, islets were perifused in a closed column at 200 pl / min flow rate using a perifusion system (Biorep). After equilibration at 3.3 mM glucose, the islets were stimulated with high glucose (16.7 mM for 40 min), followed by 30 min of 3.3 mM glucose and 20 min of 60 mM KCI + 16.7 mM glucose. Insulin measurement was performed using cisbio HTRF kit for insulin according to the manufacturer’s instructions. Detailed methods of the insulin secretion forNICCs:

[0248] This is an exemplary protocol of the insulin secretion assay of neonatal pseudo-islets. Notably, this protocol also applies to pseudo-islets derived from adult porcine islets.

[0249] Static glucose-stimulated insulin secretion (GSIS):

[0250] Collect the pseudo-islets from the microwells: o Collect the pseudo-islets from the well into a 15 ml tube. o Wash the well twice, each time with 500 pl of the basal glucose medium. o Centrifuge the pseudo-islets at 100g 4 / 6 for 1 min at RT. Discard the supernatant. o Wash the pseudo-islets with 5 ml of pre-warmed basal glucose medium. o Centrifuge the pseudo-islets at 100g 4 / 6 for 1 min at RT. Discard the supernatant. o Resuspend the pseudo-islets in 1 ml of pre-warmed basal glucose medium.

[0251] Prepare the millicell insert 12 pm PC or PluriStrainer mini 10 pm: o Put the millicell insert into a 24-well plate. o Pre-hydrate the millicell insert or PluriStrainer mini 10 pm with 1 ml of basal glucose medium.

[0252] Pre-incubation: o Pipette the volume which contains pseudo-islets of 120,000 cells in total onto millicell insert or PluriStrainer mini. o Incubate at 37°C 90% humidity 5 % CO2 for two hrs.

[0253] Basal incubation: o After two hrs. Carefully aspirate the medium from outside the millicell insert I PluriStrainer mini. o Move the millicell insert / PluriStrainer mini into a new well; wash with 1 ml of basal glucose medium as the following: 500 pl inside the millicell I PluriStrainer mini and 500 pl outside the millicell I PluriStrainer mini. o Carefully aspirate the medium from outside the millicell insert I PluriStrainer mini. o Move the millicell insert I PluriStrainer mini into a new well containing 1 ml of basal glucose medium. o Incubate at 37°C 90% humidity 5% C02for one hr. o After 1 hr., collect the supernatant from outside the millicell insert I PluriStrainer mini into 1.5 ml tubes. Store immediately on ice. o Centrifuge the sample tubes at 400g 9 / 9 at 4°C for 5 mins. o Collect aliquot of 200 pl into a 96-well plate. o Store at -20°C

[0254] Stimulatory incubation: o Move the millicell I PluriStrainer mini into a new well; wash with 1 ml of Stimulatory glucose medium as the following: 500 pl inside the millicell I PluriStrainer mini and 500 pl outside the millicell I PluriStrainer mini. o Carefully aspirate the medium from outside the millicell insert I PluriStrainer mini. o Move the millicell insert I PluriStrainer mini into a new well containing 1 ml of Stimulatory glucose medium. o Incubate at 37°C 90% humidity 5% CO2 for one hrs. o After 1 hr., collect the supernatant from outside the millicell insert I PluriStrainer mini into 1.5 ml tubes. Store immediately on ice. o Centrifuge the sample tubes at 400g 9 / 9 at 4°C for 5 mins. o Collect aliquot of 200 pl into a 96-well plate. o Store at -20°C.

[0255] Total insulin extraction: o Move the millicell I PluriStrainer mini into a new well, wash with 1 ml of cold PBS as the following: 500 pl inside the millicell and 500 pl outside the millicell I PluriStrainer mini. o Carefully aspirate the PBS from outside the millicell insert I PluriStrainer mini. o Add 1 ml of RIPA buffer supplemented with protease inhibitor cocktail, pipette 10x with 1 ml pipette. o Incubate overnight at 4°C o Next morning, collect all the volume into the 1 .5 ml tube. o Centrifuge at 8000g for 10 mins at 4°C o Collect 200 pl aliquot from the middle of the tube. o Dilute the total insulin 1 :100 into a basal glucose medium in a 96-well plate. o Store at -20°C.

[0256] Results for pseudo-islets generated from NICCs: Islets respond to glucose and start to secrete insulin after glucose challenge (Fig. 6). Insulin release is expressed as absolute amount of secreted insulin (Fig. 6A) and normalized to total insulin content (Fig. 6B) and stimulation index (Fig. 6C) (mean - / + SD). Treatment with Y-27632 resulted in higher insulin secretion after glucose challenge (Fig. 6A and 6B) and higher stimulation index (SI) for smaller pseudo-islets (Fig. 6C)

[0257] Results for pseudo-islet generated from adult porcine islets:

[0258] Islets dynamically respond to glucose and start to secrete insulin after glucose challenge (Fig. 15A). Insulin release is expressed as secreted insulin and stimulation index (SI) (mean - / + SD) over mean basal secretion (0-10 min) and area under the curve (AUC) (mean - / + SEM). Treatment with Y-27632 resulted in higher insulin secretion after glucose challenge (Fig. 15A) and higher stimulation index (SI) for smaller pseudo-islets (Fig. 15B).

[0259] Treatment of pseudo-islets with various Rho / ROCK pathway inhibitors:

[0260] Porcine pseudo-islets were cultured in the presence of several Rho / ROCK pathway inhibitors, including Y27632, Fasudil, Thiazovivin, H-1152, and Ripasudil. As illustrated in Figures 18 to 23, treatment with these Rho / ROCK pathway inhibitors resulted in a notable enhancement of pseudo-islet formation, characterized by distinct sizes compared to the untreated control groups. Additionally, the data indicate improved viability and cell composition of the pseudo-islets following treatment with the Rho / ROCK pathway inhibitors. Furthermore, there was a significant increase in insulin release from the pseudo-islets in response to these treatments, suggesting that modulation of the Rho / ROCK signaling pathway positively influences the functional attributes of porcine pseudo-islets.

[0261] Cryopreservation:

[0262] Pseudo-islets generated from both fresh and cryopreserved cells were analyzed. The data demonstrate that cryopreservation, when followed by Y-27632 treatment, effectively preserves islet morphology and size. Additionally, Y-27632 treatment significantly enhances cell viability post-cryopreservation, ensuring consistency in the distribution of cell types essential for islet functionality. Furthermore, Y-27632 treatment plays a critical role in maintaining functional insulin release after cryopreservation. Refer to Fig. 25-30.

[0263] Assessment of different serum conditions:

[0264] Neonatal pseudo-islets were cultured in the presence of 10% Fetal Bovine Serum (FBS) and 10% Human Serum (HS) with Y-27632 treatment. The described methods and compositions demonstrate significant improvements in culturing neonatal porcine-derived pseudo-islets, optimizing their viability and functional performance. Incorporating human serum, particularly in GMP (Good Manufacturing Practice) grade, ensures the pseudo-islets' safety, quality, and efficacy for potential therapeutic applications, further enhancing their suitability for clinical use. Refer to Fig. 31-36. REFERENCES

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Claims

CLAIMS1 . An ex vivo method for generating pseudo-islets from a porcine subject, comprising: i. providing single cells dissociated from islets or islet-like cell clusters (ICC) obtained from a porcine pancreas, ii. treating the dissociated cells with a Rho / ROCK inhibitor, and iii. obtaining pseudo-islets.

2. The method according to any one of the preceding claims, wherein step ii) further comprises seeding the dissociated cells into microwells, allowing the cells to aggregate into pseudo-islets, wherein preferably each microwell comprises a single pseudo-islet.

3. The method according to the preceding claim, wherein greater than 30% of the pseudo-islets exhibit a defined size with an average diameter of 30-350 pm, wherein preferably greater than 50%, more preferably greater than 70% exhibit said defined size.

4. The method according to any one of the preceding claims, wherein the defined size of the pseudo-islets is an average diameter of 50-250 pm, preferably 70-200 pm.

5. The method according to any one of the preceding claims, wherein a pseudo-islet exhibits 100-2000 aggregated cells.

6. The method according to any one of the preceding claims, wherein a population of said pseudo-islets exhibits a standard deviation (SD) of 10 or less.

7. The method according to any one of the preceding claims, wherein the pseudo-islets exhibit a stimulation index of above 1 .

8. The method according to any one of the preceding claims, wherein the treatment with the Rho / Rock inhibitor: i. has no influence or a negligible influence on the viability of the islets, preferably wherein the viability of the cells is measured by flow cytometry and / or a staining method, and / or ii. has no influence or a negligible influence on cellular subtypes.

9. The method according to any one of the preceding claims, wherein the inhibitor comprises one or more of Y-27632 (dihydrochloride), Thiazovivin, Fasudil hydrochloride, H-1152 dihydrochloride, Ripasudil, HA 1100 hydrochloride (Hydroxyfasudil), N-Boc-Y-27632, GSK429286A and Narciclasine, preferably Y-27632 (dihydrochloride).

10. The method according to any one of the preceding claims, wherein the porcine subject is an adult porcine or a neonatal porcine subject, and optionally wherein thesingle cells dissociated from islets or islet-like cell clusters (ICC) were cryopreserved prior to step (i).11 . The method according to any one of the preceding claims, wherein the size-defined pseudo-islets are encapsulated after step (iii), preferably in a microencapsulation device (preferably comprising a single pseudo-islet), a macroencapsulation device (preferably comprising multiple pseudo-islets) or a 3D-printed scaffold or naked (without encapsulation), and wherein preferably the size of the encapsulated pseudoislets exhibits an average diameter of 70-150 pm, preferably 80-120 pm.

12. Use of the ex vivo methods according to one of claims 1 to 11 for in vitro culture (such as research purposes), drug screening, toxicity screening, and / or for studying metabolic diseases, preferably diabetes.

13. An in vitro population of porcine pseudo-islets obtained by treatment with a Rho / ROCK inhibitor, wherein greater than 30% of the population exhibit a defined size with an average diameter of 30-350 pm with a standard deviation (SD) of 10 or less.

14. The pseudo-islet population according to the preceding claim, wherein the pseudoislet population is encapsulated in a device suitable for medical administration in a mammalian subject.

15. The pseudo-islet population produced by the method of claim 1 or according to claims 13 and 14 for use in the treatment of diabetes, preferably type I diabetes in a subject, wherein the pseudo-islet population is transplanted to the subject.

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