Preparations of pancreatic islets derived from human pluripotent stem cells and processes for preparing them.

Abstract

The present invention provides a human pluripotent stem cell-derived pancreatic islet preparation that is suitable for long-distance transport, can maintain the structure and biological activity of human pluripotent stem cell-derived pancreatic islets during long-distance transport, can be used to prepare injectable formulations, and is particularly suitable for treating insulin deficiency-related diseases via the hepatic portal vein or under rectus abdominis sheath injection after long-distance transport. [Solution] A preparation of human pluripotent stem cell-derived pancreatic islets is provided, comprising pluripotent stem cell-derived pancreatic islets in the form of cell aggregates having a diameter of more than 50 μm, 0% to 5% human serum albumin, and a basal culture medium.

Description

Technical Field

[0003] , ,

[0004]

[0001] The present invention relates to the field of stem cells. Specifically, the present invention provides a preparation of human pluripotent stem cell-derived islets useful for the treatment of insulin deficiency-related diseases via the portal vein or under rectus sheath injection, and a process for preparing the same.

Background Art

[0002] Islet beta cell (BC) transplantation can bring about a definitive cure for type I diabetes. However, due to the limited availability of donor beta cells, the application of this treatment as a clinical therapy is restricted. Pluripotent stem (PS) cells are capable of unlimited replication and differentiation into various types of cells. Therefore, PS cells are a promising source of beta cells. Human pluripotent stem cell-derived islets are generated by directing the differentiation of human pluripotent stem cells to differentiate into cell aggregates (cell spheroids) having a diameter exceeding 50 μm. At present, there are no reports on formulating human pluripotent stem cell-derived islets into a preparation. For stem cell-derived islets, after directed differentiation in a cell factory, it is necessary to transport them to a medical institution for transplantation of human pluripotent stem cell-derived islets, and it faces the problem of a decrease in cell viability during transportation.

Summary of the Invention

Problems to be Solved by the Invention

[0003] [[ID=2I]] The preparation of human pluripotent stem cell-derived islets provided by the present invention is suitable for long-distance transportation, can maintain the structure and biological activity of human pluripotent stem cell-derived islets during long-distance transportation, can be used for preparing an injection, and is particularly suitable for treating insulin deficiency-related diseases via the portal vein or under rectus sheath injection after long-distance transportation. In the case of an injection prepared from the preparation, the final product of the injection meets the requirements including sterility, non-pyrogenicity, safety and stability.

Means for Solving the Problems

[0004] The present invention provides a formulation of human pluripotent stem cell-derived pancreatic islets, thereby maintaining the proportion of NKX6.1+C-PEP+ cells in the formulation and / or high levels of biological activity in human pluripotent stem cell-derived pancreatic islets.

[0005] In a first aspect, the present invention provides a preparation of pancreatic islets derived from human pluripotent stem cells.

[0006] In some embodiments, the human pluripotent stem cell-derived islet preparation comprises pluripotent stem cell (hPSC)-derived islets, human serum albumin, and basal medium, with a mass / volume concentration (m / v) of human serum albumin ranging from 0% to 5% (i.e., 0 to 5 g / 100 mL). The applicant has found that a concentration of 0% of added human serum albumin results in more severe cell adhesion, while human serum albumin concentrations exceeding 5% significantly reduce the biological activity of the conserved human pluripotent stem cell-derived islets.

[0007] In some embodiments, the basal medium in the human pluripotent stem cell-derived pancreatic islet preparation is selected from the group consisting of 0.9% physiological saline, water for injection, 5% glucose, or a mixture thereof, preferably water for injection. The inventors have found that when the basal medium is selected from the group consisting of phosphate-buffered physiological saline, cell morphology and activity are significantly reduced.

[0008] In some embodiments, the human pluripotent stem cell-derived islet content per milliliter of the human pluripotent stem cell-derived islet formulation is 10 to 20 million single cells, preferably 4 to 12 million single cells. The applicant found that while the concentration of human pluripotent stem cell-derived islets at 1 million single cells does not affect cell viability and cell morphology, it requires a large volume of formulation solution, which is uneconomical and inconvenient for concentration operations before clinical use.

[0009] In some embodiments, the islet aggregates derived from pluripotent stem cells (hPSCs) in a per milliliter formulation of human pluripotent stem cell-derived islets correspond to 1 million to 20 million single cells derived from human pluripotent stem cell-derived islets. Preferably, the islet aggregates derived from pluripotent stem cells (hPSCs) in a per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell-derived islets.

[0010] In some embodiments, the human pluripotent stem cell-derived islet content in a per milliliter formulation is 1,000 to 10,000 IEQ, preferably 2,000 to 8,000 IEQ, and more preferably 2,500 to 7,000 IEQ. The applicant has found that a human pluripotent stem cell-derived islet content exceeding 9,000 IEQ results in poor cell activity and cell morphology. One IEQ of human pluripotent stem cell-derived islets corresponds to 1,000 to 2,000 single cells.

[0011] In some embodiments, the mass / volume concentration (m / v) of human serum albumin is 0% to 5% (i.e., 0 to 5 g / 100 mL), specifically 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%, and 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%, 2.05%, 2.1%, 2.15%, 2.2%, 2.2 5%, 2.3%, 2.35%, 2.4%, 2.45%, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.8%, 2.85%, 2.9%, 2.95%, 3.0%, 3.05%, 3.1%, 3.15%, 3.2%, 3.25%, 3.3%, 3.35%, 3.4%, 3.45%, 3.5%, 3.55%, 3.6%, 3.65%, 3 Selected from the groups consisting of 0.7%, 3.75%, 3.8%, 3.85%, 3.9%, 3.95%, 4.0%, 4.05%, 4.1%, 4.15%, 4.2%, 4.25%, 4.3%, 4.35%, 4.4%, 4.45%, 4.5%, 4.55%, 4.6%, 4.65%, 4.7%, 4.75%, 4.8%, 4.85%, 4.9%, 4.95%, or 5%.

[0012] In some embodiments, the human pluripotent stem cell-derived pancreatic islet preparation further comprises an osmotic regulator and a pH regulator.

[0013] In some embodiments, the osmoregulator is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc sulfate, glucose, trehalose, or combinations thereof. In some embodiments, the osmoregulator is preferably a combination of sodium chloride, potassium chloride, and calcium chloride; or a combination of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride; or a combination of sodium chloride, potassium chloride, calcium chloride, and glucose.

[0014] In some embodiments, the human pluripotent stem cell-derived pancreatic islet formulation further comprises a pH adjuster that controls the pH of the formulation within the range of 6.5 to 8.0.

[0015] In some embodiments, the human pluripotent stem cell-derived pancreatic islet preparation further comprises an antioxidant or an additional stabilizer, or a combination thereof. The additional stabilizer is selected from one or more of histidine, sodium gluconate, Pirvate (sodium pyruvate), nicotinamide ITS(1X), linoleic acid, and vitamin E.

[0016] In some embodiments, the preparation of human pluripotent stem cell-derived pancreatic islets includes: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates measuring 50-350 μm, where the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 1 million to 20 million single cells derived from human pluripotent stem cell islets, or the density of pluripotent stem cell (hPSC)-derived islet aggregates is 1,000 to 10,000 IEQ. 0% to 5% human serum albumin, Osmotic pressure regulators selected from a combination of sodium chloride, potassium chloride, and calcium chloride, or a combination of the same with the addition of magnesium chloride, A pH adjuster selected from sodium lactate, or a combination of sodium bicarbonate, sodium citrate, and citric acid. Optionally, additional stabilizers selected from histidine, sodium gluconate, or a combination thereof, as well as Optionally, an antioxidant selected from one or more of the following: pirubate and nicotinamide.

[0017] In some embodiments, the preparation of human pluripotent stem cell-derived pancreatic islets includes: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates measuring 50-350 μm, where the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell islets, or the density of pluripotent stem cell (hPSC)-derived islet aggregates is 2,000-8,000 IEQ. 0% to 5% human serum albumin, 1.0-9.0 mg / ml sodium chloride, 0.1-3.0 mg / ml potassium chloride, 0.01-2.0 mg / ml calcium chloride, and 0-1.0 mg / ml magnesium chloride. Preferably, an osmotic regulator selected from a combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, and 0.2 mg / ml calcium chloride, or a combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. A pH adjusting agent selected from 1.5-5.0 mg / ml sodium lactate, 20-30 mM HEPES, or a combination of 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid, preferably a combination of 3.1 mg / ml sodium lactate, 25 mM HEPES, or 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid. Optionally, an additional stabilizer selected from histidine at 0-6.0 mg / ml, Optionally, an antioxidant selected from 0-10 mM nicotinamide.

[0018] In some embodiments, the amount of the osmoregulator sodium chloride is selected from 1.0 to 9.0 mg / ml, with specific values ​​including 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 mg / ml. In some embodiments, the amount of the osmoregulator potassium chloride is selected from 0.1 to 3.0 mg / ml, with specific values ​​including 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg / ml. In some embodiments, the amount of the osmoregulator calcium chloride is selected from 0.01 to 2.0 mg / ml, with specific values ​​including 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 mg / ml. In some embodiments, the amount of the osmotic regulator magnesium chloride is selected from 0 to 1.0 mg / ml, and the specific values ​​include 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, and 1.0 mg / ml.

[0019] In some embodiments, the amount of the pH adjusting agent sodium lactate is 1.5 to 5.0 mg / ml, with specific values ​​including 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, and 3. It includes 0, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4.0, 4.05, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95 and 5 mg / ml.

[0020] In some embodiments, the amount of histidine is 0 to 6.0 mg / ml, preferably 0 to 2.0 mg / ml. Specific values are 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 and 2.0.

[0021] In some embodiments, a formulation of human pluripotent stem cell-derived islets comprises: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates of 50 to 350 μm, where the islet aggregates derived from pluripotent stem cells (hPSC) per milliliter of formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell-derived islets, or the density of the islet aggregates derived from pluripotent stem cells (hPSC) is 2,000 to 8,000 IEQ, 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride and 0.3 mg / ml magnesium chloride, 3.1 mg / ml sodium lactate, 0 to 6.0 mg / ml, preferably 0 to 2.0 mg / ml histidine, and 0 to 10 mM nicotinamide.

[0022] In some embodiments, a formulation of human pluripotent stem cell-derived islets comprises: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates of 50 to 350 μm, where the islet aggregates derived from pluripotent stem cells (hPSC) per milliliter of formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell-derived islets, or the density of the islet aggregates derived from pluripotent stem cells (hPSC) is 2,000 to 8,000 IEQ, 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. 25 mM HEPES, or a combination of 25 mM HEPES and 3.1 mg / ml sodium lactate. Histidine at 0-2.0 mg / ml, and Nicotinamide at 0-10 mM.

[0023] In some embodiments, the preparation of human pluripotent stem cell-derived pancreatic islets includes: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates measuring 50-350 μm, where the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell islets, or the density of pluripotent stem cell (hPSC)-derived islet aggregates is 2,000-8,000 IEQ. 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. A combination of 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid. Optionally, an additional stabilizer selected from histidine at a dose of 0-2.0 mg / ml. Optionally, an antioxidant selected from 0-10 mM nicotinamide.

[0024] In some embodiments, the preparation of human pluripotent stem cell-derived pancreatic islets includes: Pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates measuring 50-350 μm, where the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cell islets, or the density of pluripotent stem cell (hPSC)-derived islet aggregates is 2,000-8,000 IEQ. 0% to 5% human serum albumin, 9.0 mg / ml sodium chloride (equivalent to 0.9% physiological saline), Optionally, an additional stabilizer selected from histidine at a dose of 0-2.0 mg / ml. Optionally, an antioxidant selected from 0-10 mM nicotinamide.

[0025] In some embodiments, human pluripotent stem cell-derived pancreatic islets express one or more of the following: PDX1, NKX6.1, C-PEP, ISL1, ARX, GCG, SST, HHEX, MAFA, VMAT1, and CHGA. In some embodiments, human pluripotent stem cell-derived pancreatic islets express both NKX6.1 and C-PEP.

[0026] In some embodiments, human pluripotent stem cell-derived pancreatic islets are differentiated from human pluripotent stem cells. Human pluripotent stem cells are selected from the group consisting of unedited or optionally edited human embryonic stem cells, human induced pluripotent stem cells (hiPSCs), or combinations thereof.

[0027] In some embodiments, human induced pluripotent stem cells (hiPSCs) are derived from human adipose tissue, human pancreas, human epidermis, blood, or urine, etc. Gene editing is selected from the group consisting of CRISPR / Cas technology, engineered zinc finger nuclease (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas technology. Preferably, the CRISPR / Cas technology is the CRISPR-Cas3, CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-CasX, or CRISPR-IscB system.

[0028] In some embodiments, the human pluripotent stem cell-derived pancreatic islets in the human pluripotent stem cell-derived islet preparations are double-positive for NKX6.1+ and C-PEP+. Preferably, after storing the formulation at 4°C to 28°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, preferably at least 30% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 35% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 40% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+. You can select specific data from any integer or decimal value within a range of at least 20% to 95%. More preferably, after storing the formulation at 16°C to 22°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, preferably at least 30% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 35% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 40% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+. You can select specific data from any integer or decimal value within a range of at least 20% to 95%.

[0029] In some embodiments, the pluripotent stem cell-derived islets in the human pluripotent stem cell-derived islet preparation are cell aggregates having a diameter greater than 50 μm, preferably cell aggregates having a diameter greater than 50 to 350 μm. The specific value is any integer or decimal value between 50 μm and 350 μm, for example, selected from 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, and 350 μm. In some embodiments, 95% of the cell aggregates in the human pluripotent stem cell-derived islet preparation have a diameter of 50 to 350 μm, 75 to 300 μm, or 75 to 275 μm.

[0030] In some embodiments, the human pluripotent stem cell-derived pancreatic islet preparation has human pluripotent stem cell-derived pancreatic islets that maintain a cell viability (biological activity) of more than 85%, preferably more than 85%, 90%, 92.5%, 95%, 97.5%, or 98%, after being stored at 4°C to 28°C (±3°C) for 24 hours, preferably at 4°C to 22°C (±3°C).

[0031] In some embodiments, the human pluripotent stem cell-derived islet preparation maintains good cell morphology after storage at 4°C to 28°C (±3°C) for 24 hours, preferably at 4°C to 22°C (±3°C) for 24 hours. This good cell morphology manifests as a smooth cell aggregate surface, uniform size, and good refractive index. In some embodiments, the islet score of the cell aggregates of the human pluripotent stem cell-derived islet preparation is 6.0 to 10 after storage at 4°C to 28°C (±3°C) for 24 hours. The specific islet scores of the cell aggregates are selected from the group consisting of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.

[0032] In some embodiments, the human pluripotent stem cell-derived pancreatic islet preparations maintain a good proportion of NKX6.1-positive and C-PEP-positive double-positive cells, good biological activity, and good cell aggregate morphology after being stored at 4°C to 22°C (±3°C) for 24 hours.

[0033] In some embodiments, the temperatures in the present invention include any integer or first decimal value within the ranges of 4°C to 37°C, 4°C to 28°C, 4°C to 22°C, and 16°C to 22°C.

[0034] In some embodiments, the human pluripotent stem cell-derived pancreatic islet preparation is an injectable preparation. In some embodiments, the injectable preparation is an injectable preparation for the hepatic portal vein, or for subabdominal injection, more preferably for subabdominal injection.

[0035] In another embodiment, the present invention relates to a process for preparing a human pluripotent stem cell-derived pancreatic islet preparation according to any one of the prior embodiments, (1) Cell preservation composition preparation: A step of preparing a cell preservation composition for pancreatic islets derived from human pluripotent stem cells by adding human serum albumin and other components to a basal culture medium and mixing them uniformly, and (2) The present invention provides a process that includes the step of mixing a cell preservation composition with pancreatic islets derived from human pluripotent stem cells and filling it into a packaging container.

[0036] In another embodiment, the present invention provides the use of a human pluripotent stem cell-derived pancreatic islet preparation according to any one of the prior embodiments in the manufacture of a pharmaceutical product for the treatment of insulin deficiency. In some embodiments, preferably, the pharmaceutical product is an injectable preparation for the hepatic portal vein, or for the sub-rectus sheath, and more preferably for the sub-anterior rectus sheath.

[0037] In another embodiment, the present invention provides a cell preservation composition for human pluripotent stem cell-derived pancreatic islets in long-distance transport, comprising components of a formulation according to any one of the prior embodiments, which does not contain human pluripotent stem cell-derived pancreatic islets.

[0038] In some embodiments, the long-distance transport temperature is preferably 4°C, 10°C, 16°C, 22°C, or 28°C. In some embodiments, the transport time is preferably not more than 96 hours, 72 hours, 48 ​​hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 6 hours, or 4 hours.

[0039] For processes for the differentiation and preparation of human pluripotent stem cell-derived islets in the present invention, refer to International Publication No. 2023 / 097513 and PCT / CN2023 / 096328. Human pluripotent stem cell-derived islets in the present invention also include islet aggregates obtained after treatment with the resuscitation culture medium and / or suspension culture medium described in Chinese Patent No. 118726236. The disclosures of International Publication No. 2023 / 097513, PCT / CN2023 / 096328 and Chinese Patent No. 118726236, in particular, the disclosures relating to processes for the differentiation and preparation of human pluripotent stem cell-derived islets, and processes for culturing resuscitation islet aggregates, are incorporated herein by reference in their entirety.

[0040] definition In this application, unless otherwise specifically indicated, the use of the singular form includes the plural form. Note that, as used herein, the singular forms "a," "an," and "the" include multiple referents unless the context clearly indicates otherwise.

[0041] References to “several embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in relation to an embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments.

[0042] As used herein and in the claims, the word “comprising” (and any form of “comprising,” e.g., “having,” “including,” or “containing”) is either comprehensive or non-exclusive and does not exclude additional unlisted elements or method steps. Any embodiment described herein may be carried out with respect to any method or composition of the Disclosure, and vice versa. Furthermore, compositions of the Disclosure may be used to achieve the methods of the Disclosure. In some embodiments, the words “having,” “including,” or “containing” may be replaced with more restrictive expressions such as “consists of” or “consisting of.”

[0043] As used herein, the term “about” and its grammatical equivalents, when referring to a numerical value, may include the specific value itself and a range of values ​​that are 10% less or greater than that value.

[0044] Where used herein, the term “about,” or its grammatical equivalent relating to a reference number, and its grammatical equivalent as used herein, may include the number itself and a range of values ​​plus or minus 10% from that number. Where a specific value is given in this application and claims, unless otherwise indicated, the specific value should be assumed to include an acceptable margin of error. Where there is no specific definition, an acceptable margin of error includes a range of values ​​from ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that number.

[0045] As used herein, the term “optionally” includes the situations of “choosing” or “not choosing,” and “using” or “not using.” For example, “optionally additional stabilizers” includes both the situations of “using additional stabilizers” and “not using additional stabilizers.”

[0046] As used herein, the mass / volume concentration (m / v) of human serum albumin has the unit g / 100mL and is interchangeable with %. For example, a mass / volume concentration (m / v) of 0% to 0.5% of human serum albumin corresponds to 0 to 0.5 g / 100mL, a mass / volume concentration of 20% of human serum albumin corresponds to 20 g / 100mL, and a mass / volume concentration of 0.25% of human serum albumin corresponds to 0.25 g / 100mL.

[0047] As used herein, the terms “diabetes,” “insulin deficiency,” and their grammatical equivalents may refer to a disease characterized by prolonged high blood glucose levels. For example, as used herein, the terms “diabetes” and their grammatical equivalents may refer to any or any type of diabetes, including, but are not limited to, type 1 diabetes, type 2 diabetes, prediabetes, cystic fibrosis-associated diabetes, surgically induced diabetes, gestational diabetes, mitochondrial diabetes, and the risk of any combination thereof, including, for example, the need for islet transplantation to a subject requiring such treatment. In some cases, diabetes may be a form of monogenic diabetes.

[0048] Unless otherwise specified, the terms “islet,” “islets,” “islet equivalent,” “islet-like cell,” and “pancreatic islet” may refer to hormone-producing cells present in the pancreas of an organism. In embodiments, islets may be distinguished from pancreatic progenitor cells. Islets may include, but are not limited to, various cell types, including pancreatic α-cells, pancreatic β-cells, pancreatic δ-cells, pancreatic F-cells, and / or pancreatic ε-cells. Islets may also refer to groups of cells, cell clusters, etc.

[0049] As used herein, the term “stem cell” can refer to an undifferentiated cell capable of generating numerous progenitor cells that have the ability to proliferate and produce a number of mother cells that can give rise to differentiated or differentiateable daughter cells. The daughter cells themselves may be induced to proliferate and produce offspring that subsequently differentiate into one or more mature cell types, but also retain one or more cells that possess parental potential. Formally, cells that begin as stem cells may progress toward a differentiated phenotype, but can subsequently “reverse” and re-express the stem cell phenotype, a term often referred to by those skilled in the art as “dedifferentiation,” “reprogramming,” or “retro-differentiation.” As used herein, the term “pluripotent stem cell” includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0050] As used herein, the term “pluripotency” can refer to cells that, under various conditions, possess the ability to differentiate into multiple differentiated cell types, preferably into cell types characteristic of all three germ layers. Pluripotent cells are primarily characterized by their ability to differentiate into multiple cell types, preferably into all three germ layers, for example, using a nude mouse teratoma formation assay. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but a preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not make them pluripotent in itself. Reprogrammed pluripotent cells (e.g., iPS cells, as the term is defined herein) also possess the ability to be passed through for long periods without loss of proliferative capacity, compared to primary cell parents, which are generally only capable of a limited number of divisions during culture.

[0051] The terms "human pluripotent stem cell-derived islets," "hPSC-islets," or "hPSC-islet cells" refer to hormone-producing cells similar to those found in the pancreas of living organisms, obtained through the differentiation of human pluripotent stem cells, and possessing mature insulin-secreting function. Flow cytometry analysis shows that hPSC-islets contain, on average, approximately 30-60% pancreatic β-cells, 5-20% pancreatic α-cells, and 1-10% pancreatic δ-cells.

[0052] The terms “stem cell-derived β-cells,” “SC-β-cells,” “functional β-cells,” “functional pancreatic β-cells,” “mature SC-β-cells,” and their grammatical equivalents may refer to cells (e.g., non-native pancreatic β-cells) that exhibit at least one marker indicating pancreatic β-cells (e.g., NKX6.1, C-PEP, or PDX-1), express insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous mature β-cells. In some embodiments, the terms “SC-β-cells” and “non-native β-cells” as used herein are interchangeable. In some embodiments, “SC-β-cells” include mature pancreatic cells. Since the method of this disclosure can induce SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they originate)), pluripotent cells, totipotent cells, or any of the transdifferentiated versions of the aforementioned cells, it should be understood that SC-β cells do not need to be induced from stem cells (e.g., directly).

[0053] The NKX6.1 and C-PEP double-positive cell population (%) of pancreatic islet aggregates refers to the percentage of cells that are double-positive for NKX6.1 and C-PEP as detected by flow cytometry after digestion of pancreatic islet aggregates into single cells.

[0054] "Sub-rectus sheath grafting" refers to introducing graft tissue or a graft into a location beneath and within the rectus sheath, preferably in the space between the rectus sheath and the rectus abdominis muscle. "Anterior rectus sheath grafting" specifically refers to administering graft tissue or a graft into a location beneath the anterior rectus sheath, preferably in the space between the anterior rectus sheath and the rectus abdominis muscle, as shown in International Publication No. 2023 / 227068 and its drawings, thereby reaching a location beneath the anterior rectus sheath.

[0055] The terms “rectus abdominis,” “abdominis rectus,” “musculus rectus abdominis,” or “abdominal muscles” may be used interchangeably in the context of this invention and refer to two parallel, flat muscles located on either side of the linea alba, extending along the entire length of the anterior surface of the abdomen.

[0056] The term "islet equivalent" or "IEQ" is a standardized measure used to represent the islet mass to be transplanted. For IEQ determination, see, for example, Lembert N, et al. (Lembert N, et al. Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets. Cell Transplant. 2003;12(1):33-41).

[0057] Beneficial effects 1. In the screening of stabilizers, the present invention unexpectedly found that human serum albumin, within an appropriate concentration range, can maintain the biological activity of pancreatic islets and prevent their physical adhesion to culture dishes or packaging containers. Specifically, in some embodiments, a concentration of 0% of added human serum albumin resulted in cell aggregation and even more severe adhesion, leading to significant loss during collection and affecting yield. Human serum albumin concentrations above 5% resulted in less than 90% biological activity of stored human pluripotent stem cell-derived pancreatic islets, as well as a significant decrease in cell viability, β-cell expression, and pancreatic islet score. Furthermore, the present application further found that low concentrations of histidine can be used as an auxiliary component by observing that the addition of low concentrations (0-2.0 mg / mL) of histidine increases β-cell expression, while the addition of high concentrations (6.0 mg / mL) of histidine results in severe cell adhesion, as well as a decrease in cell viability and β-cell expression.

[0058] 2. In screening osmotic regulators, the present invention found that using only physiological saline as the basal solution resulted in insufficient maintenance of islet aggregate morphology. When using sterile water for injection as the basal medium, sodium chloride, potassium chloride, and calcium chloride are necessary components to maintain osmotic pressure. If the osmotic pressure is too low, the islets exhibit swelling defects. Calcium chloride, in its preferred concentration range of 0.01 mg / ml to 2.0 mg / ml, helps maintain the biological activity of the islets, but at its high concentration of 5.0 mg / ml, the osmotic pressure reaches 372 mOsmol / kg, causing the islets to become dense and prone to dehydration, potentially affecting normal biological activity. Magnesium chloride, as a beneficial component, helps maintain the proportion of islet β cells, cell aggregate morphology, and biological activity. Low concentrations of glucose (0–10 mM) can function as an auxiliary osmoregulator, but the addition of high glucose concentrations (27.7 mM) was observed to cause darkening of cell aggregates, resulting in a failure to maintain cell viability and functional markers.

[0059] 3. In screening of pH adjusters, the present invention found that phosphate-buffered saline was selected but could not maintain the morphology of pancreatic islet aggregates. The present application also found that sodium lactate is a necessary component for maintaining the pH of the formulation and can effectively maintain the biological activity of pancreatic islets within a concentration range of 1.5 mg / ml to 5.0 mg / ml. HEPES is a non-essential component that can help maintain the pH of the formulation (7.2 to 8.0).

[0060] 4. In screening of formulation cell density, the present invention found that at high formulation densities (9,000 IEQ / mL), islet viability, β-cell expression, and islet score all showed a significant decrease over 72 hours of storage, accompanied by significantly worsened cell adhesion. These drawbacks were mitigated by reducing the formulation density (less than 8,000 IEQ / mL).

[0061] 5. In screening for formulation stability, the present invention found that the formulations provided herein can maintain islet score, cell viability, and NKX6.1 / C-PEP levels within a temperature range of 4°C to 28°C. [Modes for carrying out the invention]

[0062] In relation to the examples, the solutions of this disclosure are described below. It will be understood by those skilled in the art that the following examples are merely illustrative of this disclosure and should not be construed as limiting the scope of this disclosure. Any techniques or conditions not specified in the examples shall be carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Any reagents or instruments whose manufacturers are not indicated are common commercially available products. For the kits and their uses referred to in this invention, the operation shall be carried out by referring to the kit instructions. Where there is no mentioned procedure, the operation shall be carried out in accordance with the instructions for the commercially available instrument or in accordance with the general procedures of those skilled in the art.

[0063] Test method The morphology and size of pancreatic islets derived from human pluripotent stem cells were observed under a microscope from 0 to 72 hours after formulation preparation, and the diameter size distribution was analyzed using CellView.

[0064] Cell viability was detected using NucleoCounter® NC-200®. Specifically, human pluripotent stem cell-derived pancreatic islet aggregates were digested into single cells using Accutase, and a Via1-Cassette® containing the pipette-collected sample was inserted into the instrument slot for detection using the NucleoCounter® NC-200® cell counter.

[0065] The expression of markers related to the biological activity of human pluripotent stem cell-derived pancreatic islets, such as the proportion of β cells (NKX6.1+ and C-PEP+), the proportion of endocrine cells (CHGA+), and the proportion of α cells (ARX+ / GCG+), was detected by flow cytometry. Specifically, human pluripotent stem cell-derived pancreatic islet aggregates were digested into single cells using Accutase, fixed overnight with BD Cytofix / Cytoperm® solution, permeabilized with Wash Buffer, and then incubated with antibodies. After washing, the cells were filtered through a cell strainer and detected using a flow cytometer.

[0066] The islet score was evaluated according to the detailed criteria in Table 1 and calculated by summing the scores across all groups. Table 1. Detailed criteria for the islet aggregate score (maximum score: 10 points)

[0067] [Table 1]

[0068] Preparation of cell preparation solution Preparations of pancreatic islets derived from human pluripotent stem cells were prepared according to the formulation compositions of each example and the comparative formulation.

[0069] The human pluripotent stem cell-derived pancreatic islets used in the examples of the present invention were all prepared using the methods described in steps 1 to 6 on pages 36 to 37 of International Publication No. 2023 / 097513, and the diameter of the cell aggregates was 50 to 350 μm.

[0070] Water for injection was used as the basal culture medium. Components were added according to the respective formulations shown in the table, and after homogeneous mixing, the mixture was sterile filtered through a 0.22 μm filter to prepare human pluripotent stem cell-derived islet preservation solutions. Subsequently, each cell preservation composition solution and human pluripotent stem cell-derived islets were prepared into formulations at concentrations of 1,000 to 9,000 IEQ / ml, filled into formulation bags, and stored at 22°C (±3°C). Unless otherwise specified (as in Example 3), the per milliliter content of human pluripotent stem cell-derived islets in the human pluripotent stem cell-derived islet formulation was 6,000 IEQ.

[0071] Example 1. Screening of human serum albumin concentration with stabilizers Human pluripotent stem cell-derived pancreatic islet preparations containing 0%, 0.5%, and 5% (where % means 50 mg / ml) human serum albumin were prepared according to the compositions shown in Table 2 to measure the pH value and osmotic pressure (mOsmol / kg), as well as the islet score, cell viability (%), and the percentage of cell surface markers NKX6.1+ and C-PEP+ (i.e., efficiency %) at 0H, 24H, 48H, and 72H. Table 2. Formulation screening using stabilizers at different concentrations.

[0072] [Table 2-0] Table 2-1. Test results of different concentrations of stabilizer HSA

[0073] [Table 2-1]

[0074] The test results in Table 2-1 show that when 5% human serum albumin was added to the formulation, the islet score, β-cell expression (efficiency, NKX6.1 / C-PEP ratio), and islet survival rate of the formulation solution decreased significantly within 72 hours compared to the 0H formulation. When 0% and 0.5% human serum albumin were added to the formulation, the formulation solution effectively maintained the islet score, β-cell expression, and islet survival rate within 72 hours compared to the 0H formulation, and there was no significant difference between the 0% human serum albumin group and the 0.5% human serum albumin group. However, defects in cell aggregate sedimentation at the bottom of the culture dish or packaging container, as well as severe adhesion and significant loss during collection, were observed in the 0% human serum albumin group, whereas no adhesion defects were observed in the 0.5% human serum albumin group. Furthermore, formulations containing 0.10%, 0.25%, 0.625%, 1%, or 2.5% human serum albumin effectively maintained islet score, β-cell expression, and islet viability, with no adhesion defects observed. Table 2-2. Test results of histidine stabilizer at different concentrations

[0075] [Table 2-2]

[0076] The test results in Table 2-2 indicate that low concentrations of histidine can be used as an auxiliary component of stabilizers. Addition of low concentrations (0-2.0 mg / mL) of histidine resulted in a corresponding increase in β-cell expression. Addition of high concentrations (6.0 mg / mL) of histidine led to severe cell adhesion, as well as decreased cell viability and β-cell expression.

[0077] Example 2. Formulation screening of osmotic pressure regulators and pH regulators To measure islet scores, cell viability, and the percentage of the cell surface marker NKX6.1 / C-PEP at 0H, 24H, 48H, and 72H, islet preparations containing different types and concentrations of osmotic regulators were prepared according to the compositions shown in Table 4-1.

[0078] The test results showed that using only physiological saline as the basal solution resulted in insufficient maintenance of the pancreatic islet aggregate morphology. When using sterile water for injection as the basal culture medium, sodium chloride, potassium chloride, and calcium chloride are necessary components to maintain osmotic pressure. If the osmotic pressure is too low, the pancreatic islets exhibit swelling defects. Table 4-1. Screening of formulations for basal osmotic pressure regulators.

[0079] [Table 4-1] Table 4-2 Screening data for basal osmotic pressure regulators

[0080] [Table 4-2]

[0081] Calcium chloride contributes to maintaining the biological activity of pancreatic islets, and its optimal concentration range was 0.01 mg / ml to 2.0 mg / ml. However, at a high concentration of 5.0 mg / ml, cell aggregates became dense and difficult to digest. Table 5-1. Screening of calcium chloride concentration in osmotic regulators.

[0082] [Table 5-1] Table 5-2. Screening test data for calcium chloride concentration in osmotic regulators.

[0083] [Table 5-2] Note: " / " indicates that the cell condition was abnormal and quality parameter data could not be collected.

[0084] The test results from Tables 6-1 and 6-2 indicate that potassium chloride is a necessary component for maintaining osmotic pressure, and that the optimal concentration range is 0 to 3.0 mg / ml. Table 6-1. Screening of potassium chloride concentration in osmotic regulators.

[0085] [Table 6-1] Table 6-2. Screening test data for potassium chloride concentration in osmotic regulators.

[0086] [Table 6-2]

[0087] The test results from Tables 7-1 and 7-2 indicate that sodium chloride is a necessary component for maintaining osmotic pressure, and that maintaining osmotic pressure helps maintain islet morphology. If the sodium chloride concentration is excessively low, osmotic pressure cannot be maintained, and if the osmotic pressure is relatively low, the islets exhibit edema defects. Both excessively high and excessively low sodium chloride concentrations result in a significant decrease in the islet score over 24 hours. Table 7-1 Screening of sodium chloride concentration in osmotic regulators

[0088] [Table 7-1] Table 7-2 Screening test data for sodium chloride concentration in osmotic regulators

[0089] [Table 7-2]

[0090] The test results from Tables 8-1 and 8-2 indicate that magnesium chloride, as an adjunct (advantageous component) of osmotic regulators, helps maintain the proportion of pancreatic islet β-cells, cell aggregate morphology, and biological activity. Table 8-1 Screening of magnesium chloride concentration in osmotic regulators

[0091] [Table 8-1] Table 8-2 Screening test data for magnesium chloride concentration in osmotic regulators

[0092] [Table 8-2]

[0093] The test results from Tables 9-1 and 9-2 indicate that low concentrations of glucose (0-10 mM) can function as an auxiliary osmoregulator, and that the addition of high glucose concentrations (27.7 mM) causes darkening of cell aggregates, resulting in a failure to maintain cell viability and functional markers. Table 9-1 Screening of glucose concentration in osmotic regulators

[0094] [Table 9-1] Table 9-2 Screening test data for glucose concentration in osmotic regulators

[0095] [Table 9-2]

[0096] The test results from Tables 10-1 and 10-2 indicate that sodium lactate is an essential component in the pancreatic islet preparation solution, and that it adjusts the pH value of the solution and maintains the biological activity of the pancreatic islets well within a concentration range of 1.5 mg / ml to 5.0 mg / ml. Table 10-1. Screening of pH adjusting agent sodium lactate concentration

[0097] [Table 10-1] Table 10-2 Screening test data for pH adjuster sodium lactate concentration

[0098] [Table 10-2]

[0099] Example 3. Screening of cell density in formulations To measure the islet score, cell viability (%), and percentage of the cell surface marker NKX6.1 / C-PEP (efficiency %) at 0H, 24H, 48H, and 72H, islet preparations with cell densities of 1,000 IEQ / mL, 6,000 IEQ / mL, and 9,000 IEQ / mL were prepared using Formulation B1 of Example 2.

[0100] The test results showed that at high formulation densities (9,000 IEQ / mL), islet survival, β-cell expression, and islet score all showed a significant decline over 72 hours of storage, accompanied by markedly deteriorated cell adhesion. These drawbacks were mitigated by reducing the formulation density (less than 8,000 IEQ / mL). Table 10 Test data for cell density screening

[0101] [Table 10-3]

[0102] Example 4. Screening of storage temperature of pharmaceutical formulations Islet preparations were prepared using Formulation B1 from Example 2 to measure islet scores, cell viability (%), and the percentage of the cell surface marker NKX6.1 / C-PEP (efficiency %) at 0H, 24H, 48H, and 72H under conditions of 4°C, 22°C, and 37°C, respectively. The test results showed that the formulations of the examples were able to maintain islet scores, cell viability, and NKX6.1 / C-PEP levels under conditions of 4°C to 22°C, which is beneficial for temperature-controlled transport of the formulation. Table 11. Test data on storage temperature

[0103] [Table 11]

[0104] Example 5. Cell viability and islet percentage in different cell preparation solutions Human pluripotent stem cell-derived islet aggregates were collected, the culture supernatant was discarded, and the formulation solution was added. Equal volumes of cell aggregates were taken at 0H, 24H, 48H, and 72H after formulation. Single-cell suspensions were obtained by digestion with Accutase enzyme solution for 15-25 minutes. Samples were collected and cell viability was detected using an NC200 cell counter. Expression of functional markers was detected by flow cytometry. The proportion of islet β cells was determined by the double-positive ratio of NKX6.1 and C-PEP. The proportion of islet α cells was determined by the double-positive ratio of ARX and GCG. The proportion of islet δ cells was determined by the double-positive ratio of SST and HHEX. The proportion of endocrine cells was determined by the CHGA positive ratio. The daily sampling method, sample volume, digestion method, and experimental procedure for final measurement were kept constant. Table 12. Formulations of compound formulations

[0105] [Table 12]

[0106] The test results in Table 13 show that at 24 hours after formulation, the cell viability of compound formulation 1, compound formulation 2, and compound formulation 3 was relatively high, exceeding 90%. At 48 hours after formulation, when compound formulation 1 and compound formulation 2 were used, the cell viability remained stable and was still maintained above 90%, but the cell viability of compound formulation 3 decreased significantly. At 72 hours after formulation, the cell viability of compound formulation 1 was the highest and remained stable above 90%. Table 13. Results of the pancreatic islet survival rate study.

[0107] [Table 13]

[0108] The test results from Table 14 show that the expression rate of pancreatic islet α cells remained relatively stable across all groups within 72 hours after formulation, with no significant differences. Table 14. Comparison of pancreatic islet α-cell percentages across different formulation types.

[0109] [Table 14]

[0110] The test results from Table 15 show that within 24 hours of formulation, the expression rate of islet β-cells exceeded 40% in all other groups, with compound formulation group 1 showing the highest rate. Within 72 hours of formulation, compound formulation group 1 was significantly superior to the other groups in maintaining the proportion of islet β-cells. Table 15. Comparison of pancreatic islet β-cell percentages across different formulation types.

[0111] [Table 15]

[0112] The test results from Table 16 show that within 24 hours after formulation, the difference in the expression rate of islet δ cells between the groups was small, with compound formulation group 1 and compound formulation group 2 showing relatively high rates. At 48 to 72 hours after formulation, the expression rate of islet δ cells decreased significantly in compound formulation group 3 and the FRS group, while the expression of islet δ cells in compound formulation group 1 and compound formulation group 2 remained relatively stable. Table 16. Comparison of pancreatic islet δ cell percentages across different formulation types.

[0113] [Table 16]

[0114] The test results from Table 17 show that within 24 hours after formulation, there was no significant difference in the expression rate of pancreatic islet endocrine cells between the groups, and within 72 hours after formulation, the expression of endocrine cells in compound formulation group 1 and compound formulation group 2 was relatively stable and significantly superior to the other pancreatic islet formulation groups. Table 17: Statistical table comparing the percentage of endocrine cells across different formulation types.

[0115] [Table 17]

Claims

1. A preparation of human pluripotent stem cell-derived pancreatic islets, comprising pluripotent stem cell-derived pancreatic islets in the form of cell aggregates having a diameter greater than 50 μm, 0% to 5% human serum albumin, and a basal culture medium.

2. The pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 1 million to 20 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation is 1,000 to 10,000 IEQ. Preferably, the human pluripotent stem cell-derived islet preparation according to claim 1, wherein the pluripotent stem cell-derived islet aggregates in the per milliliter preparation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell-derived islet aggregates in the per milliliter preparation is 2,000 to 8,000 IEQ.

3. A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1 or 2, further comprising an osmotic pressure regulator and a pH regulator.

4. The aforementioned osmotic pressure regulator is Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc sulfate, glucose, trehalose, or combinations thereof, Preferably, the preparation of human pluripotent stem cell-derived pancreatic islets according to claim 3, selected from the group consisting of a combination of sodium chloride, potassium chloride and calcium chloride, or a combination of sodium chloride, potassium chloride, calcium chloride and magnesium chloride, or a combination of sodium chloride, potassium chloride, calcium chloride and glucose.

5. The pH adjusting agent controls the pH of the formulation to a range of 6.5 to 8.

0. The preparation for human pluripotent stem cell-derived pancreatic islets according to claim 3, wherein the pH adjusting agent is selected from the group consisting of sodium lactate, sodium acetate, HEPES, sodium bicarbonate, sodium citrate, citric acid, or a combination thereof, preferably sodium lactate, HEPES, or a combination of sodium bicarbonate, sodium citrate, and citric acid.

6. A preparation of human pluripotent stem cell-derived pancreatic islets according to any one of claims 1 to 5, further comprising an antioxidant or an additional stabilizer, or a combination thereof.

7. The preparation of human pluripotent stem cell-derived pancreatic islets according to claim 6, wherein the additional stabilizer is selected from one or more of histidine, sodium gluconate, pirubate, nicotinamide ITS (1X), linoleic acid, and vitamin E.

8. The pluripotent stem cell (hPSC)-derived islets in the form of cell aggregates having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation correspond to 1 million to 20 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived islet aggregates in the per milliliter formulation is 1,000 to 10,000 IEQ. 0% to 5% human serum albumin, Osmotic pressure regulators selected from a combination of sodium chloride, potassium chloride, and calcium chloride, or the same combination with the addition of magnesium chloride, A pH adjuster selected from sodium lactate, or a combination of sodium bicarbonate, sodium citrate, and citric acid. Optionally, additional stabilizers selected from histidine, sodium gluconate, or a combination thereof, as well as Optionally, an antioxidant selected from one or more of the following: pirubate and nicotinamide. A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

9. The pluripotent stem cell (hPSC)-derived pancreatic islets having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation is 2,000 to 8,000 IEQ. 0% to 5% human serum albumin, 1.0–9.0 mg / ml sodium chloride, 0.1–3.0 mg / ml potassium chloride, 0.01–2.0 mg / ml calcium chloride, and 0–1.0 mg / ml magnesium chloride, Preferably, an osmotic pressure regulator selected from the group consisting of a combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, and 0.2 mg / ml calcium chloride, or a combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. A pH adjusting agent selected from 1.5 to 5.0 mg / ml sodium lactate, 20 to 30 mM HEPES, or a combination of 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid, preferably a combination of 3.1 mg / ml sodium lactate, 25 mM HEPES, or 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid. Optionally, an additional stabilizer selected from histidine at a dose of 0 to 6.0 mg / ml, Optionally, an antioxidant selected from 0-10 mM nicotinamide. A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

10. The pluripotent stem cell (hPSC)-derived pancreatic islets having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation is 2,000 to 8,000 IEQ. 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. 3.1 mg / ml sodium lactate, Histidine in an amount of 0 to 6.0 mg / ml, preferably 0 to 2.0 mg / ml, and Nicotinamide 0-10 mM A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

11. The pluripotent stem cell (hPSC)-derived pancreatic islets having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation is 2,000 to 8,000 IEQ. 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. 25 mM HEPES, or a combination of 25 mM HEPES and 3.1 mg / ml sodium lactate. Histidine in concentrations of 0-2.0 mg / ml, and Nicotinamide 0-10 mM A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

12. The pluripotent stem cell (hPSC)-derived pancreatic islets having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation is 2,000 to 8,000 IEQ. 0% to 5% human serum albumin, A combination of 6.0 mg / ml sodium chloride, 0.3 mg / ml potassium chloride, 0.2 mg / ml calcium chloride, and 0.3 mg / ml magnesium chloride. A combination of 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid. Optionally, an additional stabilizer selected from histidine at a dose of 0-2.0 mg / ml. Optionally, an antioxidant selected from 0-10 mM nicotinamide. A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

13. The pluripotent stem cell (hPSC)-derived pancreatic islets having a diameter of 50 to 350 μm, wherein the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation correspond to 2 million to 16 million single cells derived from human pluripotent stem cells, or the density of the pluripotent stem cell (hPSC)-derived pancreatic islet aggregates in the per milliliter formulation is 2,000 to 8,000 IEQ. 0% to 5% human serum albumin, 9.0 mg / ml sodium chloride (equivalent to 0.9% physiological saline), Optionally, an additional stabilizer selected from histidine at a dose of 0-2.0 mg / ml. Optionally, an antioxidant selected from 0-10 mM nicotinamide. A preparation of human pluripotent stem cell-derived pancreatic islets according to claim 1, comprising the above.

14. The aforementioned human pluripotent stem cell-derived pancreatic islets were double-positive for NKX6.1+ and C-PEP+. Preferably, after storing the preparation at 4°C to 28°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, preferably at least 30% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, and at least 35% of the human pluripotent stem cell-derived islets are double-positive for NKX6.1+ and C-PEP+, and at least 4 A preparation of human pluripotent stem cell-derived pancreatic islets according to any one of claims 1 to 13, wherein 0% are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived pancreatic islets are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived pancreatic islets are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived pancreatic islets are double-positive for NKX6.1+ and C-PEP+.

15. The preparation of human pluripotent stem cell-derived pancreatic islets according to any one of claims 1 to 14, wherein the pluripotent stem cell-derived pancreatic islets are cell aggregates having a diameter of 50 to 350 μm.

16. A preparation of human pluripotent stem cell-derived pancreatic islets according to any one of claims 1 to 7, wherein after storage at 4°C to 28°C (±3°C) for 24 hours, preferably at 4°C to 24°C (±3°C) for 24 hours, the human pluripotent stem cell-derived pancreatic islets maintain a cell viability rate (biological activity) of more than 85%, and preferably maintain a biological activity of more than 90%, 92.5%, or 95%.

17. The preparation of human pluripotent stem cell-derived pancreatic islets according to any one of claims 1 to 16, wherein the preparation is an injectable preparation, preferably an injectable preparation administered intrahepatic portal vein or subabdominal sheath, more preferably subanterior subabdominal sheath.

18. (1) Steps for preparing a cell preservation composition: Adding human serum albumin and other components to the basal culture medium and mixing them uniformly to prepare a cell preservation composition for pancreatic islets derived from human pluripotent stem cells, and (2) A step of mixing the cell preservation composition with pancreatic islets derived from human pluripotent stem cells and filling it into a packaging container. A process for preparing a human pluripotent stem cell-derived pancreatic islet preparation according to any one of claims 1 to 17, comprising:

19. The use of a human pluripotent stem cell-derived pancreatic islet preparation according to any one of claims 1 to 17 in the manufacture of a pharmaceutical product for the treatment of insulin deficiency, or in the treatment of an insulin deficiency disease, Preferably, the pharmaceutical product is an injectable preparation administered intrahepatic portal vein or subabdominal sheath, and more preferably, the pharmaceutical product is administered subanterior subabdominal sheath.

20. The cell preservation composition consists of components of the formulation according to any one of claims 1 to 17, which does not contain pancreatic islets derived from human pluripotent stem cells, and the long-distance transport temperature is 4°C to 37°C, and the transport time does not exceed 96 hours. Preferably, the long-distance transport temperature is 4°C, 10°C, 16°C, 22°C, or 28°C. Preferably, use of a cell preservation composition for human pluripotent stem cell-derived pancreatic islets in long-distance transport, where the transport time does not exceed 96 hours, 72 hours, 48 ​​hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 6 hours, or 4 hours.

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