Recovery medium for human pluripotent stem cell-derived pancreatic islet cell stock solution and application thereof

Abstract

The application discloses a recovery culture solution for human pluripotent stem cell-derived pancreatic islet cell stock in a frozen state, which can improve the spheroidization rate of cells in a single cell form frozen and reaggregated after recovery. The application also provides a culture method for pancreatic islet cell spheroids, which uses the recovery culture solution and a suspension culture solution in sequence, improves the islet score of the suspension cultured spheroid product, and maintains or improves the differentiation efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell biology, and particularly relates to a resuscitation culture solution for human pluripotent stem cell-derived islet cell stock solution and application thereof. BACKGROUND

[0002] Diabetes mellitus (DM) is a chronic metabolic disease caused by insufficient insulin secretion or impaired biological action of the human body. Long-term hyperglycemia will cause blindness, kidney failure, heart attack, stroke and other complications, seriously endangering global public health and causing serious social and economic burden.

[0003] For type 1 diabetes and type 2 diabetes patients with severely impaired islet function, although insulin intensive treatment or insulin pump therapy can regulate blood glucose to some extent, the efficacy of these methods is limited and cannot effectively prevent the occurrence and development of diabetes and its complications. The damaged islet tissue of diabetic patients is difficult to repair, and islet transplantation can fundamentally cure diabetes. However, there is a serious shortage of islet donors at present, and how to obtain effective and sufficient islet cells is a research difficulty in stem cell regenerative medicine. Human pluripotent stem cells (hPSCs) in vitro induced differentiation islet organoids (IO) are similar in structure and function to in vivo islet organs, and can be cultured on a large scale, which is a very promising cell replacement therapy for diabetes.

[0004] Cell cryopreservation is an important means of preserving cells, which can make cells in a certain period of time out of the growth state and preserve their cell characteristics, and can be recovered for experimental research or clinical application when needed. WO2017 / 222879A1, WO2019 / 169351A1, WO2023097513A1 all describe the steps of cryopreserving human pluripotent stem cell-derived pancreatic differentiation-related cell types.

[0005] WO2017 / 222879A1 describes a method of differentiating human pluripotent stem cells and deriving functional beta cells, by cryopreserving pancreatic endoderm cells, using S4D3 medium to treat thawed cells, forming spheres through Aggrewell plates, and continuing to use S4D3 medium to suspend culture cell spheres, which can be further differentiated into pancreatic beta cells.

[0006] WO2019 / 169351A1 describes a composition and method for enhancing differentiation of stem cells into beta cells using one or more epigenetic modification compounds, and the cryopreserved cells are thawed and recovered at the end of stage 5 to further differentiate into pancreatic beta cells.

[0007] WO2023097513A1 provides a method for in vitro generation of functional hPSC pancreatic islet cells, describes the ability of efficient cryopreservation makes hPSC pancreatic islet cells a continuously available, ready-to-use cell source, which is particularly important for clinical applications, providing flexibility for transplantation into humans. However, WO2023097513A1 does not describe how to recover the cryopreserved cells. The content disclosed in WO2023097513A1 is incorporated herein by reference in its entirety.

[0008] WO2023227068A1 (ANEWSITE FOR TRANSPLANTATION) describes the recovery step after freezing of hPSC pancreatic islet cells, using Gibco TM BASIC DMEM, High Glucose (DMEM-basic Cat#C11965 500BT) for recovery of cryopreserved hPSC pancreatic islet cells, and resuspension of recovered cells in Gibco TM BASIC DMEM, High Glucose. After verifying viability and yield, the cell clusters are used for transplantation after suspension culture in culture medium containing Gibco TM BASIC DMEM, High Glucose (containing 1% B27). The content disclosed in WO2023227068A1 is incorporated herein by reference in its entirety.

[0009] The recovery culture technique restores the viability of cryopreserved hPSC pancreatic islet cells and has an important influence on the recovery of dispersed cell spheres. How to recover the single-cell-state dispersed cryopreserved human pluripotent stem cell-derived pancreatic islet cell stock into uniform cell spheres with high differentiation efficiency and activity that can be reinfused into the human body is an important problem that needs to be solved. SUMMARY

[0010] The present application provides a recovery culture medium for human pluripotent stem cell-derived pancreatic islet cell stock. The recovery culture medium can improve the sphere formation rate of cryopreserved pancreatic islet cells in single-cell state. The present application also provides a culture method for pancreatic islet cell spheres, which uses recovery culture medium and suspension culture medium in sequence, improves the islet score of the suspension cultured cell sphere product, and maintains or improves its differentiation efficiency.

[0011] In one aspect, the present application provides a resuscitation medium for human pluripotent stem cell-derived pancreatic islet cells.

[0012] In one embodiment, the present application provides a resuscitation medium for human pluripotent stem cell-derived pancreatic islet cells, comprising or consisting of a basal medium and an additive; wherein the additive consists of a Rho-associated kinase (ROCK) inhibitor and human blood albumin; and the human pluripotent stem cell-derived pancreatic islet cells are human pluripotent stem cell-derived islet cells (hPSC-islet cells) cryopreserved in single cell form.

[0013] In some embodiments, the basal medium is selected from one or more of:

[0014] 1) CMRL 1066,

[0015] 2) Miami medium #1A with HSA and sodium bicarbonate,

[0016] 3) DMEM, low glucose, pyruvate, no glutamine, no phenol red,

[0017] 4) BASIC DMEM, High Glucose,

[0018] 5) MCDB 131 Medium, no glutamine,

[0019] 6) RPMI 1640 Medium,

[0020] 7) MEM alpha, nucleosides,

[0021] 8) DMEM / F-12.

[0022] In some embodiments, the basal medium is selected from one or more of:

[0023] 1) CMRL 1066,

[0024] 2) Miami medium #1A with HSA and sodium bicarbonate,

[0025] 3) DMEM, low glucose, pyruvate, no glutamine, no phenol red,

[0026] 4) BASIC DMEM, High Glucose,

[0027] 5) MEM alpha, nucleosides.

[0028] In some embodiments, the basal medium CMRL 1066 is selected from 500 mL CMRL 1066 (composition see Table 2), Sigma CMRL 1066 Cat# C0422 (composition see Table 3), Pan Biotech Cat# P04-84600 (composition see Table 4), basal medium Miami medium #1A with HSA and sodium bicarbonate is selected from Miami medium #1A with HSA and sodium bicarbonate, basal medium DMEM, low glucose, pyruvate, no glutamine, no phenol red is selected from Gibco TM DMEM, low glucose, pyruvate, no glutamine, no phenol red, basal medium BASIC DMEM, High Glucose is selected from Gibco TM BASIC DMEM, High Glucose, basal medium MCDB 131 Medium, no glutamine is selected from Gibco TM MCDB 131 Medium, no glutamine, basal medium RPMI 1640 Medium is selected from Gibco TM RPMI 1640 Medium, basal medium MEM alpha, nucleosides is selected from Gibco TM MEM alpha, nucleosides, basal medium DMEM / F-12 is selected from Gibco TM DMEM / F-12.

[0029] In some embodiments, the Rho-associated kinase (ROCK) inhibitor is selected from one or more of Y27632, Thiazovivin, GSK269962, H1152, SR-3677, or a pharmaceutically acceptable salt thereof. In some embodiments, the Rho-associated kinase (ROCK) inhibitor is selected from Y27632 or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the concentration of the Rho-associated kinase (ROCK) inhibitor is 5-20 µM.

[0031] In some embodiments, the concentration of Y27632 or a pharmaceutically acceptable salt thereof (e.g., Y27632 2HC1) is 5-20 μΜ. In some embodiments, the Y27632 or a pharmaceutically acceptable salt thereof is Y27632 2HC1. In some embodiments, the concentration of Y27632 or a pharmaceutically acceptable salt thereof is specifically selected from 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ.

[0032] In some embodiments, the mass by volume concentration (m / v) of human blood albumin is 0-2% (i.e., 0-2 g / 100 mL). In some embodiments, the mass by volume concentration of human blood albumin is 0-0.5% (i.e., 0-0.5 g / 100 mL). In some embodiments, the mass by volume concentration of human blood albumin is specifically selected from 0%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%; most preferably, the mass by volume concentration of human blood albumin is 0.25%.

[0033] In some embodiments, when the mass by volume concentration (m / v) of human blood albumin is 0%, the basal medium is Miami medium #1A with HSA and sodium bicarbonate.

[0034] In some embodiments, in the resuscitation medium for human pluripotent stem cell-derived pancreatic islet cell stock solution, the Rho-associated kinase (ROCK) inhibitor is 5-20 μΜ of Y27632 2HC1, and the mass by volume concentration (m / v) of human blood albumin is 0-0.5% (i.e., 0-0.5 g / 100 mL).

[0035] In some embodiments, the population of human pluripotent stem cell-derived pancreatic islet cells comprising non-native pancreatic beta cells is dissociated into a cell suspension and cryopreserved to obtain a stock of human pluripotent stem cell-derived pancreatic islet cells. In some embodiments, the cryopreservation temperature of the stock of human pluripotent stem cell-derived pancreatic islet cells is below -80°C, in some embodiments, the cryopreservation temperature of the stock of human pluripotent stem cell-derived pancreatic islet cells is below -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, -200°C. In some cases, the cells are frozen at a temperature of about -196°C. Any cooling method can be used to provide the low temperature required for cryopreservation, including but not limited to, electrical freezing equipment, solid carbon dioxide or liquid nitrogen. In some cases, any cryopreservation solution available to one of skill in the art can be used to cryopreserve the dissociated human pluripotent stem cell-derived pancreatic islet cells, including custom solutions and commercial solutions.

[0036] In some embodiments, the human pluripotent stem cell-derived pancreatic islet cells after resuscitation express one or several of PDX1, NKX6.1, C-PEP, ISL1, ARX, GCG, SST, HHEX, MAFA, VMAT1, CHGA. In some embodiments, the human pluripotent stem cell-derived pancreatic islet cells after resuscitation express both NKX6.1 and C-PEP.

[0037] In some embodiments, the human pluripotent stem cell-derived pancreatic islet cells are differentiated from human pluripotent stem cells. The human pluripotent stem cells are selected from the group consisting of non-genetically edited or optionally genetically edited human embryonic stem cells, human induced pluripotent stem cells (hiPSCs), or a combination thereof.

[0038] In some embodiments, the human induced pluripotent stem cells (hiPSCs) are human adipose-derived, human pancreatic-derived, human epidermis-derived, and the like. The genetic editing is selected from the group consisting of CRISPR / Cas technology, Zinc Finger Nucleases (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas technology. The CRISPR / Cas technology is preferably CRISPR-Cas3, CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-CasX, CRISPR-IscB system.

[0039] In another aspect, the present application provides use of the resuscitation medium for a stock of human pluripotent stem cell-derived pancreatic islet cells as described in any of the preceding embodiments for resuscitating cryopreserved human pluripotent stem cell-derived pancreatic islet cells.

[0040] In another aspect, the present application provides a method for culturing islet cell spheroids.

[0041] In some embodiments, the method for culturing islet cell spheroids comprises the following steps:

[0042] (1) preparing the resuscitation medium for human pluripotent stem cell-derived islet cell stock solution as described in any of the preceding embodiments, and preheating;

[0043] (2) measuring the basal medium according to the amount of resuscitated cells, and preheating;

[0044] (3) taking out the cryopreservation container containing the human pluripotent stem cell-derived islet cell stock solution, thawing in a water bath, collecting the thawed human pluripotent stem cell-derived islet cell stock solution into a centrifuge container, and adding the basal medium of step (2), and discarding the supernatant after centrifugation;

[0045] (4) resuspending the cell pellet obtained in step (3) using the resuscitation medium of step (1);

[0046] (5) inoculating the cell suspension obtained in step (4) into a culture container to culture into spheroids;

[0047] (6) preparing a suspension culture medium, which consists of the basal medium and the human blood albumin as described in any of the preceding embodiments, or which is the same as the resuscitation medium used in step (4);

[0048] (7) collecting the cell spheroids, centrifuging, and discarding the supernatant;

[0049] (8) resuspending the cell spheroids with the suspension culture medium, and culturing.

[0050] In some embodiments, the operations of steps (1)-(8) in the method for culturing islet cell spheroids can be adjusted, for example, including sequential, simultaneous or partially overlapping operations.

[0051] In some embodiments, the cell spheroid time of step (5) is 0.5-3 days, specifically selected from 0.5, 1, 1.5, 2, 2.5, 3 days. In some embodiments, the suspension culture time of step (8) is 0.5-7 days, specifically selected from 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 days.

[0052] In some embodiments, the resuscitation medium for human pluripotent stem cell-derived islet cell stock solution is used in step (5) to adjust the cell density to 0.5x10 6 to 2x10 7 , specifically selected from 0.5x10 6 , 1x10 6 , 2x106 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , 1 x 10 7 , 1.25 x 10 7 , 1.5 x 10 7 , 1.75 x 10 7 , 2 x 10 7 .

[0053] In some embodiments, the culture vessel of step (5) is Aggrewell plate, which is incubated into a sphere at 37°C, 5% CO2.

[0054] In some embodiments, the sphere formation rate of the cell spheres obtained in step (5) is greater than 30%. In some embodiments, the sphere formation rate of the cell spheres obtained in step (5) is greater than 50%, 55%, 60%, 65%, 70%. The sphere formation rate is calculated as follows:

[0055] Sphere formation rate = (1 - the amount of single cells that do not form spheres / the total amount of cells inoculated for sphere formation) x 100%.

[0056] In some embodiments, the pancreatic islet cell spheres obtained in step (8) comprise at least 20% of NKX6.1&C-PEP double positive cell population; preferably the pancreatic islet cell spheres obtained in step (8) comprise at least 50% of NKX6.1&C-PEP double positive cell population. The proportion of NKX6.1&C-PEP double positive cell population in the pancreatic islet cell spheres obtained in step (8) is specifically selected from at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0057] In some embodiments, the highest value of the pancreatic islet score of the cell spheres obtained in step (8) is greater than 6 according to the scoring table in Table 7. In some embodiments, preferably the highest value of the pancreatic islet score of the cell spheres obtained in step (8) is greater than 7 on the first to third day of culture. In some embodiments, more preferably the highest value of the pancreatic islet score of the cell spheres obtained in step (8) is greater than 8 on the first to third day of culture.

[0058] In some embodiments, the islet score of the cell spheroids obtained in step (8) on day 2-3 of culture is 6-10 according to the Table 7 scoring table, in some embodiments, it is preferred that the islet score of the cell spheroids obtained in step (8) on day 2-3 of culture is 6.5-10. Specifically, the islet score of the cell spheroids obtained in step (8) on day 2-3 of culture is selected from 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10.

[0059] In some embodiments, the cell spheroids obtained in step (8) have a diameter of 95% of the cell spheroids between 50-350 μm, 75-300 μm or 75-275 μm.

[0060] In some embodiments, the cell spheroids obtained in step (8) have a cell viability of greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%.

[0061] In some embodiments, the spherogenesis rate of the cell spheroids obtained in step (5) is greater than 30%, the highest value of the islet score of the cell spheroids obtained in step (8) on day 1-3 of culture is greater than 7, and the cell population comprises at least 20% of NKX6.1&C-PEP double positive ratio cells.

[0062] In some embodiments, the Rho-associated kinase (ROCK) inhibitor is contacted with the islet cells for a period of 0.5-10 days, preferably 0.5-4 days, 1-3 days, 1-2 days or 1 day, specifically selected from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embodiments, the Rho-associated kinase (ROCK) inhibitor is contacted with the islet cells at least in step (5) and / or step (8). In some embodiments, the Rho-associated kinase (ROCK) inhibitor is contacted with the islet cells in steps (4) to (5) and steps (7) to (8).

[0063] In another aspect, the present application provides a combination of a resuscitation medium and a suspension medium for human pluripotent stem cell-derived islet cell stock.

[0064] In some embodiments, the combination of a resuscitation medium and a suspension medium for human pluripotent stem cell-derived islet cell stock comprises,

[0065] (1) the resuscitation medium is the resuscitation medium for human pluripotent stem cell-derived islet cell stock as described in any of the preceding embodiments;

[0066] (2) the suspension culture medium consists of the basal medium of any of the preceding embodiments and the human blood albumin (i.e. the thawing recovery culture medium for human pluripotent stem cell-derived pancreatic islet cells of any of the preceding embodiments after removal of the Rho-associated kinase (ROCK) inhibitor), or the suspension culture medium is the same as the recovery culture medium.

[0067] In some embodiments, the recovery culture medium and the suspension culture medium are separately packaged. The separately packaged means that the recovery culture medium and the suspension culture medium are not in direct contact, there is a physical separation. For example, the recovery culture medium and the suspension culture medium are separately packaged in different containers, or packaged in a joint container with a physical separation. In some embodiments, the recovery culture medium and the suspension culture medium are packaged in combination.

[0068] In another aspect, the present application provides a human pluripotent stem cell-derived pancreatic islet cell spheroid prepared by any of the preceding methods. The human pluripotent stem cell-derived pancreatic islet cell spheroid prepared by any of the preceding methods has any one parameter or a combination of multiple parameters of step (8) obtaining the product.

[0069] In another aspect, the present application provides a pharmaceutical composition comprising the human pluripotent stem cell-derived pancreatic islet cell spheroid prepared by any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0070] In some embodiments, the pharmaceutical composition is an injection composition. In some embodiments, the pharmaceutical composition is an injection. In some embodiments, the pharmaceutical composition is a hepatic portal vein injection composition or an injection composition under the rectus abdominis sheath. In some embodiments, the pharmaceutical composition is an injection composition under the anterior rectus abdominis sheath. In another aspect, the present application provides the use of the human pluripotent stem cell-derived pancreatic islet cell spheroid or the pharmaceutical composition prepared by any of the preceding embodiments.

[0071] In some embodiments, the present application provides the use of the human pluripotent stem cell-derived pancreatic islet cell spheroid or the pharmaceutical composition prepared by any of the preceding embodiments in the preparation of a medicament for treating insulin deficiency.

[0072] In some embodiments, the present application provides the use of the human pluripotent stem cell-derived pancreatic islet cell spheroid or the pharmaceutical composition prepared by any of the preceding embodiments in the treatment of insulin deficiency.

[0073] In some embodiments, the present application provides the human pluripotent stem cell-derived pancreatic islet cell spheroid or the pharmaceutical composition prepared by any of the preceding embodiments for use in the treatment of insulin deficiency.

[0074] Definitions

[0075] In this application, the use of the singular includes the plural, unless specifically stated otherwise. It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0076] References in the specification to "some embodiments", "an embodiment”, "one embodiment” or "other embodiments” mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments, but not necessarily all embodiments, of the disclosure.

[0077] As used in this specification and claims, the terms "comprising", "having", "including” or "containing" and any grammatical variations thereof are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any embodiment discussed in relation to any aspect of the disclosure discussed in the specification applies to any method or composition of the disclosure, and vice versa. Furthermore, combinations or compositions of the disclosure can be used to practice the methods of the disclosure. In some embodiments, "having”, "including” or "containing” can be replaced with a closed-ended recitation, such as "consisting”, "consisting essentially of”.

[0078] As used herein, the term "about” in relation to a reference numerical value, and grammatical equivalents thereof, can include the numerical value itself and a range of values that is plus or minus 10% of the numerical value. Where a particular value is described in the application and claims, it should be assumed that the specific numerical value is included within an acceptable error range of the particular value, unless otherwise indicated. In the absence of a specific definition, an acceptable error range includes a range of values that is plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the numerical value.

[0079] As used herein, the mass volume concentration (m / v) units for human blood albumin are g / 100 mL and are interchangeable with %. For example: a mass volume concentration (m / v) of human blood albumin of 0-0.5% is equivalent to 0-0.5 g / 100 mL; a mass volume concentration of human blood albumin of 20% is equivalent to 20 g / 100 mL; a mass volume concentration of human blood albumin of 0.25% is equivalent to 0.25 g / 100 mL.

[0080] As used herein, the terms "diabetes," "insulin deficiency," and grammatical equivalents thereof can refer to a disease characterized by chronically high blood glucose levels. For example, as used herein, the terms "diabetes" and grammatical equivalents thereof can refer to all or any type of diabetes, including but not limited to, type 1 diabetes, type 2 diabetes, prediabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes, mitochondrial diabetes, or a risk of any combination thereof, e.g., a need to transplant islets into a subject in need of such treatment. In some cases, the diabetes can be a form of inherited diabetes.

[0081] The terms "islet," "islet cell," "islet equivalent," "islet-like cell," "pancreatic islet" can refer to hormone-producing cells found in the pancreas of an organism, if not otherwise specified. In embodiments, islet cells can be distinguished from pancreatic progenitor or precursor cells. Islet cells can include different types of cells, including but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Islet cells can also refer to a group of cells, a cluster of cells, and the like.

[0082] As used herein, the term "stem cell" can refer to an undifferentiated cell that is capable of proliferating and giving rise to more progenitor cells with the ability to give rise to a large number of mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and give rise to progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with the developmental potential of the parent. Formally, cells that begin as stem cells can go to a differentiated phenotype, then "revert" and re-express the stem cell phenotype, which is generally referred to in the art by the terms "de-differentiation" or "reprogramming" or "transdifferentiation." As used herein, the term "pluripotent stem cell" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0083] As used herein, the term "pluripotent" can refer to a cell that has the ability to differentiate into more than one differentiated cell type, and preferably into cell types characteristic of all three germ layers, under different conditions. Pluripotency is characterized primarily by the ability of a cell to differentiate into more than one cell type, preferably into all three germ layers, for example using the nude mouse teratoma formation assay. Pluripotency can also be evidenced by expression of embryonic stem (ES) cell markers, although the preferred test of pluripotency is the ability to demonstrate cells that differentiate into each of the three germ layers. It should be noted that simply culturing such a cell by itself does not render it pluripotent. A reprogrammed pluripotent cell (e.g., an iPS cell, as that term is defined herein) is also characterized by the ability to extend passaging without loss of growth potential relative to the original cell parent, which typically has only a limited number of divisions in culture.

[0084] The term "human pluripotent stem cell-derived pancreatic islet cell," "hPSC- islet," or "hPSC-islet cell" refers to a hormone-producing cell similar to that found in the pancreas of an organism that is obtained by differentiation from a human pluripotent stem cell, which has a mature insulin secreting function. Flow cytometry analysis shows that hPSC-islets contain, on average, about 30-60% pancreatic beta cells, 5-20% pancreatic alpha cells, and 1-10% pancreatic delta cells.

[0085] The terms "stem cell-derived beta cell," "SC-beta cell," "functional beta cell," "functional pancreatic beta cell," "mature SC-beta cell," and their grammatical equivalents can refer to a cell (e.g., a non-native pancreatic beta cell) that displays at least one marker indicative of a pancreatic beta cell (e.g., NKX6.1, C-PEP, or PDX1), expresses insulin, and displays the glucose-stimulated insulin secretion (GSIS) response characteristic of an endogenous mature beta cell. In some embodiments, the terms "SC-beta cell" and "non-native beta cell" are used interchangeably herein. In some embodiments, an "SC-beta cell" includes a mature pancreatic cell. It will be appreciated that an SC-beta cell need not be derived from a stem cell (e.g., directly), as the methods of the present disclosure are capable of using any cell as a starting point (e.g., an embryonic stem cell, an induced pluripotent stem cell, a progenitor cell, a partially reprogrammed somatic cell (e.g., a somatic cell that has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), a multipotent cell, a totipotent cell, a transdifferentiated form of any of the foregoing can be used.

[0086] The term “human pluripotent stem cell-derived islet cell stock” refers to islet cells obtained from differentiation of pancreatic endocrine progenitor cells cryopreserved in single cell format. An exemplary “human pluripotent stem cell-derived islet cell stock” is WO2023097513A1 Stage 6 (2-4 days) functional hPSC- islets cryopreserved in single cell format; or WO2019169351A1 cells cryopreserved at the end of Stage 5 cryopreserved in single cell format.

[0087] The percentage of NKX6.1&C-PEP double positive cell population in islet cell spheroid refers to the percentage of NKX6.1&C-PEP double positive cell population in islet cell spheroid detected by flow cytometry after digestion of islet cell spheroid into single cells. DETAILED DESCRIPTION

[0088] The schemes of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the examples below are only for illustration of the present disclosure and should not be regarded as limiting the scope of the present disclosure. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of reagent or instrument is specified, it is a conventional product that can be commercially available.

[0089] The kits mentioned in the present disclosure and the use thereof are operated according to the instructions of the kits. If no operation method is mentioned, it is operated according to the instructions of the commercially available instrument or according to the conventional operation method of those skilled in the art.

[0090] Table 1 Commercial reagent information

[0091]

[0092] For The introduction of CMRL 1066 can be found in Table 2 or https: / / ecatalog.corning.com / life-sciences / b2c / US / en / Media,-Sera,-and-Reagents / Classical-Media / CMRL-1066 / Corning®-500-mL-CMRL-1066 / p / 15-110-CV.

[0093] Table 2

[0094]

[0095]

[0096] For The introduction of CMRL 1066 can be found in Table 3.

[0097] Table 3

[0098]

[0099]

[0100] For an introduction to Pan Biotech CMRL 1066, please refer to Table 4.

[0101] Table 4

[0102]

[0103]

[0104] right For information on Miami Medium #1A[+]HSA and sodium bicarbonate, please see https: / / ecatalog.corning.com / life-sciences / b2b / US / en / Media,-Sera,-and-Reagents / Islet-Solutions / Islet-Solutions-and-Reagents / Corning%C2%AE-Miami-Medium-%231A-%5B+%5D-HSA-and-sodium-bicarbonate / p / 98-021-CV.

[0105] For Gibco TM For information on DMEM, low glucose, pyruvate, no glutamine, and no phenol red, please see https: / / www.thermofisher.cn / order / catalog / product / 11054020.

[0106] For Gibco TM For information on BASIC DMEM and High Glucose, please see https: / / www.thermofisher.cn / order / catalog / product / cn / en / C11965500BT.

[0107] For Gibco TM For information on MCDB 131 Medium, no glutamine, please see https: / / www.thermofisher.cn / order / catalog / product / 10372019.

[0108] Gibco TM For RPMI 1640 Medium, see https: / / www.thermofisher.cn / order / catalog / product / 11875093.

[0109] Gibco TM For MEMα, nucleosides, see https: / / www.thermofisher.cn / order / catalog / product / 12571063.

[0110] Gibco TM For DMEM / F-12, see https: / / www.thermofisher.cn / order / catalog / product / 11320033?SID=srch-srp-11320033.

[0111] Gibco TM B-27 TM For B-27 Supplement (50X), minus vitamin A, see https: / / www.thermofisher.cn / order / catalog / product / 12587010.

[0112] Detection method

[0113] The morphology and size of human pluripotent stem cell-derived pancreatic islet cells were observed under a microscope, and the diameter size distribution was analyzed using CellView software. The scores of each suspension-cultured islet cells were recorded according to Table 7.

[0114] By Nucleo NC-200 TM The cell viability was detected by a cell counter. Specifically, human pluripotent stem cell-derived pancreatic islet cells were digested into single cells using Accutase, and the Via1-Cassette TM was inserted into the device card slot, and the Nucleo NC-200 TM cell counter was used for detection.

[0115] Spheroid rate determination method: the cell sphere suspension collected in step (7) was mixed, and single cell suspension was taken to avoid sucking into cell spheres. The Via1-Cassette TM was inserted into the device card slot, and the Nucleo NC-200 TM The total amount of non-sphere-forming single cells was detected by cell counter, and the sphere-forming rate was (1-the total amount of non-sphere-forming single cells / the total amount of sphere-forming inoculated cells) x 100%, with a measurement error within 5%.

[0116] The expression of human pluripotent stem cell-derived pancreatic islet cell biological activity-related markers, such as the proportion of pancreatic beta cells (NKX6.1&C-PEP), the proportion of endocrine cells (CHGA), and the proportion of pancreatic alpha cells (ARX&GCG), was detected by flow cytometry. Specifically, the human pluripotent stem cell-derived pancreatic islet cells were digested into single cells using Accutase, fixed with BD Cytofix / Cytoperm solution, permeated with Wash Buffer, and then incubated with antibodies. After washing, the cells were filtered with a cell strainer and detected by flow cytometry. TM solution fixed, permeated with Wash Buffer, and then incubated with antibodies. After washing, the cells were filtered with a cell strainer and detected by flow cytometry.

[0117] Testing of resuscitation culture solution and suspension culture solution in Example 1

[0118] The resuscitation culture solution was prepared according to the components shown in Table 5. Except for 2# resuscitation culture solution, 0.25% (mass / volume concentration) HSA and 10 μM (molar concentration) Y-27632·2HCl were added to the other resuscitation culture solutions. The resuscitation culture solution comparative example 1 was Gibco TM BASIC DMEM, High Glucose.

[0119] After mixing the components uniformly, the resuscitation culture solution was prepared, and was preheated at 37°C before use.

[0120] Table 5 Composition of resuscitation culture solution

[0121]

[0122] N / A means not added

[0123] The mass / volume concentration of HSA 0.25% is equivalent to 0.25 g / 100 mL, and the same below

[0124] The suspension culture solution was obtained by removing Y-27632·2HCl from the resuscitation culture solution. The composition of each suspension culture solution is shown in Table 6, and was preheated at 37°C before use.

[0125] Table 6 Composition of suspension culture solution

[0126]

[0127] N / A means not added

[0128] Using chemical reprogramming strategy, human adipose-derived cells were induced to prepare hPSC-derived islets (hPSC-islet cells) using the differentiation protocol described in pages 36-37 of WO2023 / 097513A, stage 1-6. The hPSC-derived islet cells (hPSC-islet cells) can be dissociated into a cell suspension, for example a single cell suspension, the particle size of individual islet cells is mainly distributed between 10-20 pm, flow cytometry analysis shows that the hPSC-islet cell population contains about 30-60% beta cells, 5-20% alpha cells and 1-10% delta cells on average. The hPSC-derived islets (hPSC-islets) are stored at a temperature below -130°C to obtain a stock solution of human pluripotent stem cell-derived islet cells.

[0129] The medium formula of stage 1-6 is as follows (percentages are calculated by volume, unless otherwise specified):

[0130] Stage 1 (4 days) . Only day 1: MCDB131 (Gibco, Cat# 10372-019) supplemented with 4.5 mM glucose (Sigma, Cat# G7021), 1% Glutamax (Gibco, Cat# 35050-061), 1% penicillin / streptomycin, 1% B27 (Gibco, Cat# 12587-010), 100 ng / mL Activin A, 0.25 mM Vitamin C, 6 µM Chir99021, 50 nM I103 and 10 µM Y27632. Days 2-4, media was refreshed daily in MCDB131 containing 4.5 mM glucose, 1% Glutamax, 1% penicillin / streptomycin, 1% B27, 50 ng / mL Activin A and 0.25 mM Vitamin C.

[0131] Stage 2 (2 days) . MCDB131 supplemented with 4.5 mM glucose, 1% Glutamax, 1% penicillin / streptomycin, 0.5% BSA (Sigma, Cat# A4612) or 1% B27, 50 ng / mL KGF, 0.25 mM Vitamin C, 5 µM SB431542 and 100 nM Wnt-C59.

[0132] Stage 3 (4 days)DMEM-Base medium supplemented with 1% Penicillin / Streptomycin, 1% B27, 2 mM Y27632, 0.1 mM LDN193189, 0.25 mM Sant1, and 100 nM Wnt-C59. At the end of stage 3, cells were dispersed by exposure to Accutase. Released cells were rinsed with DMEM-Base medium and centrifuged at 300 g for 3 minutes. Cells were then seeded into 6-well AggreWell 400 plates (Stem Cell, Cat. No. 27940) supplemented with 10 mM Y27632 and the plate was centrifuged at 300 g for 5 minutes to settle the cells to the bottom of the microwells. Cells were then incubated at 37°C, 5% CO2 for 20 hours and the generated cell aggregates were transferred to ultra-low attachment 6-well plates (Beaverbio, Cat. No. 40406) with stage 4 medium. Suspended aggregates were cultured in an incubator shaker (Infors-HT, Multitron) at 90 rpm, 37°C, 5% CO2, and 85% humidity. TM DMEM-Base medium supplemented with 1% Penicillin / Streptomycin, 1% B27, 2 mM Y27632, 0.1 mM LDN193189, 0.25 mM Sant1, and 100 nM Wnt-C59. At the end of stage 3, cells were dispersed by exposure to Accutase. Released cells were rinsed with DMEM-Base medium and centrifuged at 300 g for 3 minutes. Cells were then seeded into 6-well AggreWell 400 plates (Stem Cell, Cat. No. 27940) supplemented with 10 mM Y27632 and the plate was centrifuged at 300 g for 5 minutes to settle the cells to the bottom of the microwells. Cells were then incubated at 37°C, 5% CO2 for 20 hours and the generated cell aggregates were transferred to ultra-low attachment 6-well plates (Beaverbio, Cat. No. 40406) with stage 4 medium. Suspended aggregates were cultured in an incubator shaker (Infors-HT, Multitron) at 90 rpm, 37°C, 5% CO2, and 85% humidity.

[0133] Stage 4 (5-6 days) DMEM-Base medium supplemented with 1% Penicillin / Streptomycin, 1% Glutamax, 1% B27, 100 ng / mL EGF, 0.2 mM TPB, 10 mM Nicotinamide, 0.25 mM Sant1, and 0.25 mM Vitamin C.

[0134] Stage 5 (6 days) DMEM-Base medium supplemented with 1% Penicillin / Streptomycin, 1% Glutamax, 1% B27, 10 mM ALK5 Inhibitor II, 0.3 mM LDN193189, 1 mM T3, 10 mM ISX9, 10 pg / mL Heparin, 0.1 mM Gamma-secretase inhibitor Xxi, 100 nM Wnt-C59, 10 mM Y27632, and 0.25 mM Vitamin C.

[0135] Stage 6 (2-4 days) DMEM-Base medium supplemented with 1% Penicillin / Streptomycin, 1% B27, 10 mM ALK5 Inhibitor II, 0.5 mM R428, 1 mM T3, 10 mM forskolin, 10 pg / mL Heparin, 10 mM Zinc sulfate, 2 mM N-acetyl cysteine, and 0.25 mM Vitamin C.

[0136] 1% Penicillin / Streptomycin non-essential addition in stage 1-6 medium formulations.

[0137] The human pluripotent stem cell-derived pancreatic islet cell stock solution is subjected to cell recovery according to the following steps (1)-(5), and the suspended culture human pluripotent stem cell-derived pancreatic islet cell sphere product is obtained through steps (6)-(8).

[0138] (1) According to the amount of recovered cells, prepare the recovery culture solution of the human pluripotent stem cell-derived pancreatic islet cell stock solution described in Table 5, which contains a Rho-associated kinase (ROCK) inhibitor and human blood albumin, and preheat;

[0139] (2) Prepare the basal medium, the volume ratio of the recovery cell stock solution to the basal medium is 1:5-1:10, and preheat;

[0140] (3) Take out the cryopreservation tube / bag, thaw in a 37°C water bath for 2-5 min, collect the thawed cell suspension into a centrifuge tube containing the basal medium preheated to 37°C, mix gently and centrifuge, discard the supernatant;

[0141] (4) Resuspend the cell pellet with the recovery culture solution of the human pluripotent stem cell-derived pancreatic islet cell stock solution described in Table 5, and take a sample for counting;

[0142] (5) Adjust the cell density to an appropriate density and inoculate into Aggrewell plates, and culture into spheres at 37°C, 5% CO2;

[0143] (6) Prepare the suspension culture solution according to the amount of sphere-forming cells, which consists of the basal medium and the human blood albumin;

[0144] (7) Collect the cell spheres into a low-adsorption centrifuge tube and centrifuge, discard the supernatant;

[0145] (8) Resuspend the cell spheres with the suspension culture solution and culture at 37°C, 5% CO2.

[0146] The recovery culture solution number used in the above steps corresponds to the suspension culture solution number. For example, when 1# recovery culture solution is used in steps (1)-(5), 1# suspension culture solution is used in steps (6)-(8).

[0147] Take the cell spheres obtained in step (5) and measure their sphere-forming rate according to the following formula, which is used to evaluate the recovery effect of the recovery culture solution, and the test results are summarized in Table 5.

[0148] Sphere-forming rate = (1 - amount of single cells that do not form spheres / total cell amount inoculated for sphere formation) x 100%.

[0149] Take the suspended culture cell spheres obtained in step (8) and evaluate them according to the islet scoring rules in Table 7, calculate the cumulative score of each group, and the test results are summarized in Table 8.

[0150] The suspension-cultured cell spheroids obtained in step (8) were used to determine the proportion of NKX6.1 / C-PEP double-positive cell spheroids using flow cytometry, and the test results are summarized in Table 9.

[0151] N / A in Table 8 and Table 9 represents that there are no cell spheroids or samples available for scoring and flow detection at the culture days, indicating that the cell loss is severe.

[0152] Suspension-cultured islet cell spheroids were prepared using the combination of the recovery culture solution of Comparative Example 1 and the suspension culture solution of Comparative Example 1.

[0153] Table 7: Suspension-cultured islet cell spheroid scoring details (full score 10)

[0154]

[0155] Table 8: Suspension-cultured islet cell scoring table

[0156]

[0157] Note: The suspension culture 1-7 days in the table header represents the culture of islet cells in the suspension culture solution for 1-7 days, and the same applies below.

[0158] Table 9: Proportion of NKX6.1&C-PEP double-positive cells

[0159]

[0160] Test of the mass concentration of HSA in the recovery culture solution of Example 2

[0161] The recovery culture solution was prepared according to the components shown in Table 10. After mixing the components uniformly, the recovery culture solution was prepared, and was preheated at 37°C before use.

[0162] Table 10: Composition of the recovery culture solution

[0163]

[0164] The suspension culture solution was obtained by removing Y-27632·2HCl from the recovery culture solution. The composition of each suspension culture solution is shown in Table 11, and was preheated at 37°C before use.

[0165] Table 11: Composition of the suspension culture solution

[0166]

[0167] The human pluripotent stem cell-derived islet cell stock solution was prepared using the same method as in Example 1. The human pluripotent stem cell-derived islet cell stock solution was subjected to cell recovery according to steps (1)-(5) of Example 1, and the suspension-cultured human pluripotent stem cell-derived islet cell spheroid product was obtained after steps (6)-(8).

[0168] The numbers of the recovery culture solution used in the above steps correspond to the numbers of the suspension cell solution. For example, when 9# recovery culture solution is used in steps (1)-(5), 9# suspension cell sphere culture solution is used in steps (6)-(8).

[0169] The cell spheres obtained in step (5) were used to determine the sphere formation rate according to the following formula, which was used to evaluate the recovery effect of the recovery culture solution. The test results are summarized in Table 10.

[0170] Sphere formation rate = (1 - amount of un-sphere-formed single cells / total amount of cells inoculated for sphere formation) x 100%.

[0171] The suspension culture cell spheres obtained in step (8) were subjected to AO / PI staining to analyze the cell viability, and the test results are summarized in Table 12.

[0172] The suspension culture cell spheres obtained in step (8) were subjected to flow cytometry to determine the proportion of NKX6.1&C-PEP double-positive cell spheres, and the test results are summarized in Table 13.

[0173] Suspension culture islet cell spheres were prepared using the combination of the recovery culture solution of Comparative Example 1 and the suspension sphere culture medium of Comparative Example 1.

[0174] Table 12 Viability of suspension culture islet cells

[0175]

[0176] Note: The header of the table "Sphere culture for 1 day" represents the culture of islet cells in the recovery culture solution for 1 day, and "Suspension culture for 1-5 days" represents the culture of islet cells in the suspension culture solution for 1-5 days, and so on.

[0177] Table 13 Proportion of NKX6.1&C-PEP double-positive cells

[0178]

[0179] Example 3 Test of the molar concentration of Y-27632·2HCl in the recovery culture solution

[0180] The recovery culture solution was prepared according to the components shown in Table 14. After the components were mixed uniformly, the recovery culture solution was prepared, and was preheated at 37°C before use.

[0181] Table 14 Composition of the recovery culture solution

[0182]

[0183] The suspension culture solution was obtained by removing Y-27632·2HCl from the recovery culture solution. The composition of each suspension culture solution is shown in Table 15, and was preheated at 37°C before use.

[0184] Table 15 Composition of suspension culture medium

[0185]

[0186] Human pluripotent stem cell-derived pancreatic islet cell stock was prepared using the same method as in Example 1. The human pluripotent stem cell-derived pancreatic islet cell stock was subjected to cell recovery according to steps (1)-(5) of Example 1, and the suspension-cultured human pluripotent stem cell-derived pancreatic islet cell spheroid product was obtained through steps (6)-(8).

[0187] The recovery culture medium number used in the above steps corresponds to the suspension culture medium number. For example, when 14# recovery culture medium is used in steps (1)-(5), 14# suspension culture medium is used in steps (6)-(8).

[0188] The spheroids obtained in step (5) were used to determine the spheroid formation rate according to the following formula, which was used to evaluate the recovery effect of the recovery culture medium, and the test results are summarized in Table 14.

[0189] Spheroid formation rate = (1 - amount of non-spheroidized single cells / total amount of spheroid formation inoculated cells) x 100%.

[0190] The suspension-cultured spheroids obtained in step (8) were used to determine the proportion of NKX6.1&C-PEP double-positive spheroids using flow cytometry, and the test results are summarized in Table 16.

[0191] Table 16 Proportion of NKX6.1&C-PEP double-positive spheroids

[0192]

[0193] Example 4 Test of Y-27632·2HCl treatment duration

[0194] The recovery culture medium was prepared according to the components shown in Table 17. After mixing the components uniformly, the recovery culture medium was prepared, and was preheated at 37°C before use.

[0195] Table 17 Composition of recovery culture medium

[0196]

[0197] The suspension culture medium was prepared with reference to the components shown in Table 18, and was preheated at 37°C before use.

[0198] Table 18 Composition of suspension culture medium

[0199]

[0200] + indicates that the medium contains 10 μM Y27632 on this day; - indicates that the medium does not contain 10 μM Y27632 on this day

[0201] The human pluripotent stem cell-derived pancreatic islet cell stock solution was prepared using the same method as in Example 1. The human pluripotent stem cell-derived pancreatic islet cell stock solution was subjected to cell resuscitation according to steps (1)-(5) of Example 1, and the suspended culture human pluripotent stem cell-derived pancreatic islet cell sphere product was obtained after steps (6)-(8).

[0202] The resuscitation culture solution number used in the above steps corresponds to the suspended culture solution number. For example, when 18# resuscitation culture solution is used in steps (1)-(5), 18# suspended culture solution is used in steps (6)-(8).

[0203] The cell spheres obtained in step (8) were taken to count the IEQ (islet equivalent) amount for evaluating the resuscitation effect of different Y27632 2HCl treatment durations of the resuscitation culture solution, and the test results are summarized in Table 20.

[0204] The IEQ (islet equivalent) amount was calculated according to the Chinese Technical Operation Specification for Clinical Islet Preparation (2023 Edition), and the pancreatic islet cells with a diameter of 150 μm were taken as one IEQ. According to the different diameters, each group of IEQ = number of pancreatic islet cells x conversion index, and the total IEQ amount of the pancreatic islet cells obtained in this test was obtained by adding the IEQ of each group. Different pancreatic islet cell diameters and their corresponding IEQ conversion indices are shown in Table 19.

[0205] Table 19 Pancreatic islet cell diameter and corresponding IEQ conversion index

[0206] Islet cell diameter (pm) IEQ Conversion index 50～100 0.167 101～150 0.648 151～200 1.685 201～250 3.500 251～300 6.315 301～350 10.352 351～400 15.833 ＞400 22.750

[0207] The suspended culture cell spheres obtained in step (8) were taken for DAPI / AOPI staining analysis of cell viability, and the test results are summarized in Table 20.

[0208] The suspended culture cell spheres obtained in step (8) were taken for DAPI / AOPI staining analysis of cell viability, and the test results are summarized in Table 20.

[0209] Table 20 Suspended culture solution Y27632 2HCl treatment duration data statistics

[0210]

Claims

1. The use of resuscitation culture medium in the resuscitation of cryopreserved human pluripotent stem cell-derived islet cells, wherein the resuscitation culture medium comprises a basal culture medium and additives; wherein, The additive consists of a Rho-associated kinase (ROCK) inhibitor and human serum albumin; the human pluripotent stem cell-derived islet cell stock solution is human pluripotent stem cell-derived islet cells cryopreserved in single-cell form; The Rho-associated kinase (ROCK) inhibitor is selected from Y27632 or a pharmaceutically acceptable salt thereof; The basal culture medium is selected from one or more of the following: 1) CMRL 1066, 2) Miami medium #1A with HSA and sodium bicarbonate, 3) DMEM, low glucose, pyruvate, no glutamine, no phenol red, 4) BASIC DMEM, High Glucose, 5) MEM α, nucleosides.

2. The use according to claim 1, wherein the concentration of Y27632 or a pharmaceutically acceptable salt thereof is 5-20 μM.

3. The use according to claim 2, wherein the concentration of Y27632 or a pharmaceutically acceptable salt thereof is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM or 20 μM.

4. The use according to claim 2, wherein the pharmaceutically acceptable salt of Y27632 in the resuscitation culture medium is selected from Y27632·2HCl.

5. The use according to any one of claims 1-4, wherein, The mass-volume concentration of human serum albumin in the resuscitation culture medium is 0.05%-2%.

6. The use according to claim 5, wherein the mass-volume concentration of human serum albumin is 0.05%-0.5%.

7. The use according to claim 5, wherein the mass-volume concentration of human serum albumin is 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, or 2.0%.

8. The use as described in claim 1, wherein, The Rho-associated kinase (ROCK) inhibitor in the resuscitation medium was 5-20 μM Y27632·2HCl, and the mass-volume concentration of human serum albumin was 0.05%-0.5%.

9. The use according to any one of claims 1-4 or 6-8, wherein the human pluripotent stem cell-derived pancreatic islet cell stock solution is cryopreserved at a temperature below -80°C.

10. The use according to claim 9, wherein the human pluripotent stem cell-derived pancreatic islet cell stock solution is cryopreserved at a temperature below -130°C.

11. The use according to any one of claims 1-4, 6-8 or 10, wherein the pancreatic islet cells derived from resuscitated human pluripotent stem cells express one or more of PDX1, NKX6.1, C-PEP, ISL1, ARX, GCG, SST, HHEX, MAFA, VMAT1, and CHGA.

12. The use according to any one of claims 1-4, 6-8 or 10, wherein the human pluripotent stem cells are selected from: non-gene-edited or gene-edited human embryonic stem cells, human induced pluripotent stem cells (hiPSCs) or combinations thereof.

13. Use of resuscitation culture medium in resuscitating cryopreserved human pluripotent stem cell-derived islet cells, wherein the resuscitation culture medium comprises basal culture medium and additives; The human pluripotent stem cell-derived islet cell stock solution is human pluripotent stem cell-derived islet cells cryopreserved in single-cell form; The additive is selected from Y27632 or its pharmaceutically acceptable salt; The basal culture medium is Miami medium #1A with HSA and sodium bicarbonate.

14. The use as claimed in claim 13, wherein the concentration of Y27632 or a pharmaceutically acceptable salt thereof is 5-20 μM.

15. A method for culturing pancreatic islet cell spheres, comprising the following steps: (1) Prepare the resuscitation culture medium for pancreatic islet cell stock solution of human pluripotent stem cells as described in any of the preceding items, and preheat it; (2) Measure the basal culture medium according to the amount of revived cells and preheat it; (3) Take out the cryopreservation container containing the original solution of human pluripotent stem cell-derived pancreatic islet cells, thaw it in a water bath, collect the thawed original solution of human pluripotent stem cell-derived pancreatic islet cells into a centrifuge container, add the basal culture medium obtained in step (2), and discard the supernatant after centrifugation. (4) Resuspend the cell pellet obtained in step (3) using the resuscitation culture medium obtained in step (1); (5) Inoculate the cell suspension obtained in step (4) into a culture container and culture it into spheres; (6) Prepare a suspension culture medium, wherein the suspension culture medium is composed of the basic culture medium described in any of the preceding items and the human serum albumin, or the suspension culture medium is the same as the resuscitation culture medium used in step (4); (7) Collect the cell spheres, centrifuge or allow them to settle naturally, and discard the supernatant; (8) Add suspension culture medium to resuspend the cell spheres and inoculate them into a culture container for suspension culture.

16. The method for culturing pancreatic islet cell spheres according to claim 15, The cell spheroidization time in step (5) is 0.5-3 days; The suspension culture time in step (8) is 0.5-7 days.

17. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein, In step (4), the cell density is adjusted to 1 × 10⁶ cells per milliliter using the resuscitation culture medium for human pluripotent stem cell-derived pancreatic islet cell stock solution. 6 Up to 2×10 7 .

18. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein, In step (5), the spheroidization rate of the obtained cell spheroids is greater than 30%, and the spheroidization rate is calculated as (1 - number of unspheroidized single cells / total number of spheroidized cells inoculated) × 100%.

19. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein, In step (5), the spheroidization rate of the cell spheres obtained is greater than 50%.

20. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein, The pancreatic islet cell spheres obtained in step (8) are digested into single cells for flow cytometry detection. The proportion of NKX6.1 and C-PEP double positive cell populations is at least 20%.

21. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein, The pancreatic islet cell spheres obtained in step (8) are digested into single cells for flow cytometry detection. The proportion of NKX6.1 and C-PEP double positive cell populations is at least 40%.

22. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein in step (8), 95% of the cell spheres obtained are between 50 and 350 µm in size.

23. The method for culturing pancreatic islet cell spheres according to claim 15 or 16, wherein the cell viability of all cell spheres obtained in step (8) is greater than 70%.

24. The method for culturing islet cell spheres according to claim 15 or 16, wherein the Rho-associated kinase (ROCK) inhibitor is in contact with the islet cells for 0.5-10 days.

25. The method for culturing islet cell spheres according to claim 15 or 16, wherein the Rho-associated kinase (ROCK) inhibitor is in contact with the islet cells for 0.5-4 days.

26. The method for culturing islet cell spheres according to claim 15 or 16, wherein the Rho-associated kinase (ROCK) inhibitor is in contact with the islet cells for 1-3 days.

27. The method for culturing islet cell spheres according to claim 15 or 16, wherein the Rho-associated kinase (ROCK) inhibitor is in contact with the islet cells for 2 days.

28. A combination of resuscitation and suspension culture media for use in the production of pancreatic islet cells derived from human pluripotent stem cells, comprising, (1) The resuscitation culture medium is the resuscitation culture medium for pancreatic islet cell stock solution derived from human pluripotent stem cells as described in any of the preceding claims; (2) The suspension culture medium is composed of the basic culture medium described in any of the preceding items and the human serum albumin, or the suspension culture medium is the same as the resuscitation culture medium.

29. The combination of resuscitation culture medium and suspension culture medium for pancreatic islet cell stock solution derived from human pluripotent stem cells as described in claim 28, wherein, The resuscitation culture medium and the suspension culture medium are packaged separately.

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