A human pluripotent stem cell-derived pancreatic islet cell preparation and a method for preparing the same

Abstract

The present application provides a human pluripotent stem cell-derived pancreatic islet cell preparation which can maintain the structure and biological activity of human pluripotent stem cell-derived pancreatic islet cells during transportation, can be used for preparing injections, and the finished product meets the requirements of sterile, non-pyrogenic, safety, stability and other injection requirements.

Description

Technical Field

[0001] This invention relates to the field of stem cells. Specifically, it provides a pancreatic islet cell preparation derived from human pluripotent stem cells and its preparation method, which can be used to treat insulin deficiency-related diseases via portal vein or subrectal muscle sheath injection. Background Technology

[0002] Pancreatic beta cell (BC) transplantation may offer a potential cure for type 1 diabetes. However, the limited availability of donor BC cells restricts the application of this treatment as a clinical therapy. Pluripotent stem cells (PS cells) can proliferate indefinitely and differentiate into various cell types. Therefore, PS cells are a promising source of BC cells. Human pluripotent stem cell-derived islet cells are produced by directionally inducing human pluripotent stem cells to differentiate into cell spheroids with a diameter greater than 50 μm. Currently, there are no reports on the formulation of human pluripotent stem cell-derived islet cells into preparations. After the stem cell-derived islet cells complete their directional differentiation in a cell factory, they need to be transported to medical institutions for transplantation. During transportation, the cell viability may decrease. Summary of the Invention

[0003] The pancreatic islet cell preparation derived from human pluripotent stem cells provided by this invention is easy to transport over long distances. During long-distance transport, the structure and bioactivity of the pancreatic islet cells derived from human pluripotent stem cells can be maintained, making it suitable for the preparation of injectable formulations. It is particularly suitable for treating insulin deficiency-related diseases via injection through the portal vein or subrectal sheath after long-distance transport. After being prepared into an injectable form, the finished product meets the requirements for sterility, pyrogen-free properties, safety, and stability required for injectable formulations.

[0004] This invention provides a pancreatic islet cell preparation derived from human pluripotent stem cells, which can maintain the proportion of NKX6.1+ and C-PEP+ double-positive cells in the pancreatic islet cells derived from human pluripotent stem cells and / or maintain a high level of biological activity.

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

[0006] In some embodiments, the human pluripotent stem cell-derived islet cell preparation comprises pluripotent stem cell (hPSC)-derived islet cells, human serum albumin, and a base solvent, wherein the mass-volume concentration (m / v) of human serum albumin is 0%-5% (i.e., 0-5 g / 100 mL). The applicant found that when the added human serum albumin concentration was 0%, cell adhesion was severe, and when the human serum albumin concentration was higher than 5%, the bioactivity of the preserved human pluripotent stem cell-derived islet cells significantly decreased.

[0007] In some embodiments, the base solvent in the human pluripotent stem cell-derived islet cell preparation is selected from 0.9% physiological saline, water for injection, 5% glucose, or a mixture thereof, with water for injection being preferred. The inventors have found that when the base solvent is selected from phosphate buffer, the morphology and activity of the cells are significantly reduced.

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

[0009] In some embodiments, the number of human pluripotent stem cell (hPSC)-derived islet cell spheres per milliliter of the formulation is equivalent to 1-20 million single cells of human pluripotent stem cell-derived islet cells. Preferably, the number of hPSC-derived islet cell spheres per milliliter of the formulation is equivalent to 2-16 million single cells of human pluripotent stem cell-derived islet cells.

[0010] In some embodiments, each milliliter of the human pluripotent stem cell-derived islet cell preparation contains 1,000-10,000 IEQs, preferably 2,000-8,000 IEQs, and more preferably 2,500-7,000 IEQs. The applicant has found that when the content of human pluripotent stem cell-derived islet cells exceeds 9,000 IEQs, cell activity and morphology are poor. One IEQ of human pluripotent stem cell-derived islet cells is equivalent to 1,000-2,000 single cells.

[0011] In some implementations, the mass-volume concentration (m / v) of human serum albumin is 0%-5% (i.e., 0-5 g / 100 mL), 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%, 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%, 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 0.65%, 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%, 5%.

[0012] In some implementations, the pancreatic islet cell preparation derived from human pluripotent stem cells further includes osmotic pressure regulators and pH regulators.

[0013] In some embodiments, the osmotic pressure regulator is selected from: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc sulfate, glucose, trehalose, or combinations thereof. In some embodiments, the osmotic pressure regulator 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 implementations, human pluripotent stem cell-derived islet cell preparations further include a pH adjuster to control the pH of the preparation in the range of 6.5-8.0.

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

[0016] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0017] The pancreatic islet cells are pluripotent stem cell (hPSC) derived from human pluripotent stem cell (HPSC) cells, which exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 1-20 million single cells equivalent to human pluripotent stem cell (HPSC) derived pancreatic islet cells; or the density of HPSC derived pancreatic islet cells per milliliter of the formulation is 1,000-10,000 IEQ cells.

[0018] 0%-5% human serum albumin;

[0019] Osmotic pressure regulator, which is selected from a combination of sodium chloride, potassium chloride and calcium chloride, or a combination with the addition of magnesium chloride;

[0020] pH adjuster, selected from sodium lactate, or a combination of sodium bicarbonate, sodium citrate and citric acid;

[0021] Optional additional stabilizers, selected from histidine, sodium gluconate, or combinations thereof;

[0022] An optional antioxidant, selected from one or more of pyruvate and nicotinamide.

[0023] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0024] The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ.

[0025] 0%-5% human serum albumin;

[0026] Osmotic pressure regulators, selected from sodium chloride at 1.0-9.0 mg / ml, potassium chloride at 0.1-3.0 mg / ml, calcium chloride at 0.01-2.0 mg / ml, and magnesium chloride at 0-1.0 mg / ml;

[0027] Preferably, the combination of sodium chloride at 6.0 mg / ml, potassium chloride at 0.3 mg / ml, and calcium chloride at 0.2 mg / ml, or a combination of sodium chloride at 6.0 mg / ml, potassium chloride at 0.3 mg / ml, calcium chloride at 0.2 mg / ml, and magnesium chloride at 0.3 mg / ml;

[0028] The pH adjuster is selected from a combination of 1.5-5.0 mg / ml sodium lactate, 20-30 mM HEPES, or 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.

[0029] Optional additional stabilizers, selected from histidine at 0-6.0 mg / ml; preferably histidine at 0-2.0 mg / ml;

[0030] An optional antioxidant, selected from 0-10 mM nicotinamide.

[0031] In some embodiments, the amount of sodium chloride, an osmotic pressure regulator, is selected from 1.0-9.0 mg / ml, specifically 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 potassium chloride, an osmotic pressure regulator, is selected from 0.1-3.0 mg / ml, specifically including 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg / ml. In some embodiments, the amount of calcium chloride, an osmotic pressure regulator, is selected from 0.01-2.0 mg / ml, specifically 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 implementations, the amount of magnesium chloride, the osmotic pressure regulator, is selected from 0-1.0 mg / ml, specifically including 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.

[0032] In some implementations, the dosage of the pH adjuster sodium lactate is 1.5-5.0 mg / ml. Specific values ​​include 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, 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, 3.7, 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, 5mg / ml.

[0033] In some embodiments, the dosage of histidine is 0-6.0 mg / ml; preferably 0-2.0 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.

[0034] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0035] The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ.

[0036] 0%-5% human serum albumin;

[0037] 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;

[0038] 3.1 mg / ml sodium lactate;

[0039] Histidine at concentrations of 0-6.0 mg / ml, preferably 0-2.0 mg / ml;

[0040] 0-10mM nicotinamide.

[0041] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0042] The pancreatic islet cells are pluripotent stem cells (hPSCs) present in the form of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSCs)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQs.

[0043] 0%-5% human serum albumin;

[0044] 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;

[0045] 25 mM HEPES or a combination of 25 mM HEPES and 3.1 mg / ml sodium lactate;

[0046] Histidine at concentrations of 0-2.0 mg / ml;

[0047] 0-10mM nicotinamide.

[0048] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0049] The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ.

[0050] 0%-5% human serum albumin;

[0051] 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;

[0052] A combination of 2.35 mg / ml sodium bicarbonate, 0.2 mg / ml sodium citrate, and 0.14 mg / ml citric acid;

[0053] Optional additional stabilizers, selected from histidine at 0-2.0 mg / ml;

[0054] An optional antioxidant, selected from 0-10 mM nicotinamide.

[0055] In some implementation schemes, human pluripotent stem cell-derived islet cell preparations include:

[0056] The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ.

[0057] 0%-5% human serum albumin;

[0058] 9.0 mg / ml sodium chloride (equivalent to 0.9% physiological saline);

[0059] Optional additional stabilizers, selected from histidine at 0-2.0 mg / ml;

[0060] An optional antioxidant, selected from 0-10 mM nicotinamide.

[0061] In some embodiments, pancreatic islet cells derived from human pluripotent stem cells express one or more of PDX1, NKX6.1, C-PEP, ISL1, ARX, GCG, SST, HHEX, MAFA, VMAT1, and CHGA. In some embodiments, pancreatic islet cells derived from human pluripotent stem cells simultaneously express NKX6.1 and C-PEP.

[0062] In some implementations, the pancreatic islet cells derived from human pluripotent stem cells are differentiated from human pluripotent stem cells. The human pluripotent stem cells are selected from: non-gene-edited or optionally gene-edited human embryonic stem cells, human induced pluripotent stem cells (hiPSCs), or combinations thereof.

[0063] In some embodiments, the human induced pluripotent stem cells (hiPSCs) are derived from human adipose tissue, human pancreas, human epidermis, blood, and urine. The gene editing is selected from CRISPR / Cas technology, zinc finger nucleases (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas technology. The preferred CRISPR / Cas technologies are CRISPR-Cas3, CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-CasX, and CRISPR-IscB systems.

[0064] In some implementation schemes, the human pluripotent stem cell-derived islet cells in the human pluripotent stem cell-derived islet cell preparation are double-positive for NKX6.1+ and C-PEP+. Preferably, after the formulation is stored at 4°C to 28°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+; preferably, at least 30% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 35% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 40% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+. Specific data can be selected from any integer or decimal value between at least 20% and 95%. More preferably, after the formulation is stored at 16°C to 22°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+; preferably, at least 30% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 35% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 40% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+. Specific data can be selected from any integer or decimal value between at least 20% and 95%.

[0065] In some embodiments, the pluripotent stem cell-derived islet cells in the human pluripotent stem cell-derived islet cell preparation are cell spheroids larger than 50 μm; preferably, cell spheroids between 50 and 350 μm. Specific values ​​are selected from any integer or decimal value between 50 μm and 350 μm, such as 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, and 350 μm. In some embodiments, 95% of the cell spheroids in the human pluripotent stem cell-derived islet cell preparation have a diameter between 50 and 350 μm, between 75 and 300 μm, or between 75 and 275 μm.

[0066] In some embodiments, the pancreatic islet cell preparation derived from human pluripotent stem cells, when stored at 4°C to 28°C (±3°C) for 24 hours, preferably at 4°C to 22°C (±3°C) for 24 hours, maintains a cell viability (bioactivity) of more than 85%; preferably maintaining a cell viability of 85%, 90%, 92.5%, 95%, 97.5%, or 98%.

[0067] In some embodiments, the pancreatic islet cell preparation derived from human pluripotent stem cells, when stored at 4°C to 28°C (±3°C) for 24 hours, preferably 4°C to 22°C (±3°C), maintains good cell morphology. Good cell morphology is characterized by smooth cell surfaces, uniform size, and good refractive properties. In some embodiments, the islet cell preparation derived from human pluripotent stem cells, when stored at 4°C to 28°C (±3°C) for 24 hours, exhibits an islet score of 6.0-10 in the cell spheres. Specifically, the islet score of the cell spheres is selected from 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.

[0068] In some implementations, human pluripotent stem cell-derived islet cell preparations, when stored at 4°C to 22°C (±3°C) for 24 hours, can maintain a good ratio of NKX6.1+ and C-PEP+ double-positive cells, bioactivity, and cell spheroid morphology.

[0069] In some embodiments, the temperatures of 4°C to 37°C, 4°C to 28°C, 4°C to 22°C, and 16°C to 22°C in this invention include any numerical value using one decimal place or any integer value within the above temperature range.

[0070] In some embodiments, the pancreatic islet cell preparation derived from human pluripotent stem cells is an injectable formulation. In some embodiments, the injectable formulation is preferably administered via portal vein injection or anterior rectus abdominis sheath injection, more preferably via sub-rectus abdominis sheath injection.

[0071] On the other hand, the present invention provides a method for preparing the pancreatic islet cell preparation derived from human pluripotent stem cells according to any of the foregoing embodiments, comprising the following steps:

[0072] (1) Preparation of cell preservation composition: Add human serum albumin and other components to the base solvent, mix evenly and prepare human pluripotent stem cell-derived pancreatic islet cell preservation composition;

[0073] (2) The cell preservation composition and pancreatic islet cells derived from human pluripotent stem cells are mixed and filled into a packaging container.

[0074] On the other hand, the present invention provides the use of the human pluripotent stem cell-derived islet cell preparation described in any of the foregoing embodiments in the preparation of a medicament for treating insulin deficiency. In some embodiments, the medicament is preferably a portal vein injection preparation or a subrectus sheath injection preparation, more preferably an anterior rectus sheath injection preparation.

[0075] On the other hand, the present invention provides the use of a human pluripotent stem cell-derived islet cell preservation composition in long-distance transportation, wherein the preservation composition is the component of the preparation described in any of the foregoing embodiments other than human pluripotent stem cell-derived islet cells.

[0076] In some implementations, the long-distance transport temperature is between 4°C and 37°C; the transport time does not exceed 96 hours. In some implementations, the preferred long-distance transport temperature is 4°C, 10°C, 16°C, 22°C, or 28°C. In some implementations, the preferred transport time is no more than 96 hours, 48 ​​hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 6 hours, or 4 hours.

[0077] The differentiation and preparation methods of pancreatic islet cells derived from human pluripotent stem cells in this invention are described in WO2023 / 097513A1 and PCT / CN2023 / 096328. The pancreatic islet cells derived from human pluripotent stem cells in this invention also include islet cell spheres obtained after treatment with resuscitation culture medium and / or suspension culture medium as described in CN118726236A. The entire contents of WO2023 / 097513A1, PCT / CN2023 / 096328, and CN118726236A are incorporated herein by reference, particularly concerning the differentiation and preparation methods of pancreatic islet cells derived from human pluripotent stem cells and the culture methods of resuscitated islet cell spheres.

[0078] definition

[0079] In this application, unless otherwise specified, the use of the singular includes the plural. It should be noted that, as used in the specification, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural forms.

[0080] The terms “some implementation schemes,” “implementation scheme,” “one implementation scheme,” or “other implementation schemes” used in this specification refer to specific features, structures, or characteristics described in connection with the implementation schemes, which are included in at least some of the implementation schemes of this disclosure, but not necessarily in all of them.

[0081] As used in this specification and claims, the word "comprising" (and any form of inclusion, such as "having," "including," or "containing") is inclusive or open-ended and does not exclude other elements or method steps not mentioned. This is to be taken into consideration that any embodiment discussed in this specification relates to any method or composition implementation of this disclosure, and vice versa. Furthermore, combinations or compositions of this disclosure can be used to implement methods of this disclosure. In some embodiments, "having," "including," or "containing" may be replaced with closed-ended expressions, such as "composes of," "consisting of," or "comprises of."

[0082] As used herein, the term "about" and its grammatical equivalents relating to reference values ​​can include the value itself and a range of values ​​plus or minus 10%. Where a particular value is described in this application and claims, unless otherwise stated, it should be assumed that the specific value is included within an acceptable range of error for that particular value. In the absence of a particular definition, an acceptable range of error includes values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0083] As used herein, “optional” includes situations where “optional” or “not optional”, “use” or “not use”. For example, optional additional stabilizers include both “using additional stabilizers” and “not using additional stabilizers”.

[0084] As used in this article, the unit of human serum albumin mass / volume concentration (m / v) is g / 100mL, and it can be used interchangeably with %. For example, a human serum albumin mass / volume concentration (m / v) of 0%-0.5% is equivalent to 0-0.5g / 100mL; a human serum albumin mass / volume concentration of 20% is equivalent to 20g / 100mL; and a human serum albumin mass / volume concentration of 0.25% is equivalent to 0.25g / 100mL.

[0085] As used herein, the terms “diabetes,” “insulin deficiency,” and their grammatical equivalents can refer to a disease characterized by prolonged high blood glucose levels. For example, as used herein, the term “diabetes” and its grammatical equivalents can refer to all or any type of diabetes, including, but not limited to, type 1 diabetes, type 2 diabetes, prediabetes, cystic fibrosis-associated diabetes, surgically induced diabetes, gestational diabetes, mitochondrial diabetes, or any combination thereof, and the risk of, for example, requiring islet transplantation into a subject who needs such treatment. In some cases, diabetes can be a form of hereditary diabetes.

[0086] Unless otherwise specified, the terms "islet," "islet cell," "islet equivalent," "islet-like cell," and "pancreaticislet" can refer to hormone-producing cells present in the pancreas of an organism. In embodiments, islet cells can be distinguished from pancreatic progenitor cells or precursors. Islet cells can include different types of cells, including but not limited to pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, and / or pancreatic ε cells. Islet cells can also refer to a group of cells, a cluster of cells, etc.

[0087] As used herein, the term "stem cell" can refer to an undifferentiated cell capable of proliferating and producing more progenitor cells with the ability to generate a large number of parent cells, which in turn can produce differentiated or potentially differentiated daughter cells. These daughter cells themselves can be induced to proliferate and produce offspring that subsequently differentiate into one or more mature cell types while retaining one or more cells with parental developmental potential. In a formal sense, cells that begin as stem cells may proceed toward a differentiated phenotype and then "reverse" and re-express the stem cell phenotype, a process often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "anti-differentiation." As used herein, the term "pluripotent stem cell" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and so on.

[0088] As used herein, the term "pluripotent" can refer to a cell that, under different conditions, has the ability to differentiate into more than one differentiated cell type, and preferably into cell types characteristic of all three germ layers. Pluripotent cells are characterized primarily by their ability to differentiate into more than one cell type, preferably into all three germ layers, as demonstrated, for example, using a nude mouse teratoma formation assay. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is demonstrating the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not inherently make them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as the term is defined herein) also possess the ability to prolong passages without losing growth potential, compared to primitive cell parents which typically exhibit only a limited number of divisions in culture.

[0089] The terms "human pluripotent stem cell-derived islet cells," "hPSC-islets," or "hPSC-islet cells" refer to hormone-producing cells obtained through differentiation of human pluripotent stem cells, similar to those found in the pancreas, 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.

[0090] 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 exhibiting at least one marker indicating pancreatic β cells (e.g., NKX6.1, C-PEP, or PDX1), expressing insulin, and exhibiting glucose-stimulated insulin secretion (GSIS) response characteristics characteristic of endogenous mature β cells (e.g., non-native pancreatic 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. It should be understood that SC-β cells do not need to be derived from stem cells (e.g., directly), because the methods of this disclosure can use 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 that have been partially reprogrammed into an intermediate state between induced pluripotent stem cells and their derived somatic cells), multipotent cells, pluripotent cells, and any transdifferentiated form of the aforementioned cells).

[0091] The percentage of pancreatic islet spheroids NKX6.1 & C-PEP double-positive cells refers to the proportion of NKX6.1 and C-PEP double-positive cells detected by flow cytometry after digesting pancreatic islet spheroids into single cells.

[0092] The term "subrectus sheath transplant" refers to the placement of a graft or implant in a location below and within the rectus sheath, preferably in the space between the rectus sheath and the rectus abdominis muscle. "Subrectus anterior sheath transplant" specifically refers to the placement of a graft or implant in a location below and anterior to the rectus sheath, preferably in the space between the anterior rectus sheath and the anterior rectus abdominis muscle, reaching a position below the anterior rectus sheath, as shown in WO2023 / 227068A1 and its accompanying diagram.

[0093] The terms “rectus abdominis”, “musculus rectus abdominis”, or “abdominal muscle” are used interchangeably in the context of this invention and refer to two rows of parallel, flat muscles located on either side of the linea alba and extending along the entire length of the anterior abdomen.

[0094] The term “islet equivalent” or “IEQ” is a standardized measure of the quality of islets to be transplanted. For example, see Lembert N et al. for measuring IEQ (Lembert N, et al. A real 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).

[0095] Beneficial effects

[0096] 1. In the screening of stabilizers, this invention unexpectedly discovered that human serum albumin, within an appropriate concentration range, can maintain the biological activity of pancreatic islet cells and prevent the physical adhesion of islet cells to culture dishes or packaging containers. Specifically, in some embodiments, when the added human serum albumin concentration is 0%, cell aggregation and adhesion are severe, resulting in significant loss during collection and affecting the yield. When the human serum albumin concentration is higher than 5%, the biological activity of preserved human pluripotent stem cell-derived islet cells is less than 90%, and cell viability, β-cell expression, and islet scores are all significantly reduced. Furthermore, this application also found that low concentrations of histidine can be used as an auxiliary component. Adding low concentrations of histidine (0-2.0 mg / mL) increases β-cell expression, while adding high concentrations of histidine (6.0 mg / mL) results in severe cell adhesion, and both cell viability and β-cell expression decrease.

[0097] 2. In the screening of osmolarity regulators, this invention found that using only physiological saline as the base solution resulted in poor maintenance of pancreatic islet cell morphology. When using water for injection as the base solvent, sodium chloride, potassium chloride, and calcium chloride are essential components for maintaining osmolarity. If the osmolarity is too low, islet cells exhibit edema defects. Calcium chloride helps maintain the biological activity of islet cells, with a suitable concentration range of 0.01 mg / ml-2.0 mg / ml. However, at a high concentration of 5.0 mg / ml, the osmolarity reaches as high as 372 mOsmol / kg, causing islet cells to become compact and prone to dehydration, which may affect their normal biological activity. Magnesium chloride, as a dominant component, helps maintain the proportion of pancreatic β cells, cell morphology, and biological activity. Low concentrations of glucose (0-10 mM) can be used as auxiliary osmolarity regulators. However, when high concentrations of glucose were added (27.7 mM), defects were observed, including cell darkening, loss of viability, and inability to maintain cell functional markers.

[0098] 3. In the screening of pH adjusters, this invention found that phosphate buffer could not maintain the morphology of pancreatic islet cells. This application also found that sodium lactate is an essential component for maintaining the pH of the formulation solution, and can effectively maintain the biological activity of pancreatic islet cells at a concentration range of 1.5 mg / ml–5.0 mg / ml. HEPES is a non-essential component and can help maintain the pH of the formulation solution (7.2-8.0).

[0099] 4. In the screening of cell density in the formulation, this invention found that when the formulation density was high (9000 IEQ / mL), the viability of islet cells, β-cell expression, and islet scores all decreased significantly over 72 hours as the formulation was stored, and cell adhesion was significantly aggravated. These defects were improved after reducing the formulation density (below 8000 IEQ / mL).

[0100] 5. In the screening of stable formulation solutions, the present invention found that the formulation solution provided by the present invention can maintain the levels of islet score, cell viability and NKX6.1 / C-PEP in a temperature range of 4℃-28℃. Detailed Implementation

[0101] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. The reagent kits and their applications mentioned in this invention shall be operated in accordance with the kit instructions. Where operating methods are not mentioned, they shall be operated in accordance with the instructions for commercially available instruments or according to conventional operating methods known to those skilled in the art.

[0102] Detection methods

[0103] The morphology and size of pancreatic islet cells derived from human pluripotent stem cells were observed under a microscope 0–72 h after preparation, and the diameter distribution was analyzed using CellView.

[0104] pass NC-200 TM Cell viability was detected using a cell counter, specifically by digesting pancreatic islet cells derived from human pluripotent stem cells into single cells using Accutase, and then aspirating samples from Via1-Cassette cells. TM Insert into the device card slot, use NC-200 TM The cell counter was used for testing.

[0105] The expression of bioactivity-related markers in pancreatic islet cells derived from human pluripotent stem cells was detected by flow cytometry, including the proportion of β cells (NKX6.1+ and C-PEP+), the proportion of endocrine cells (CHGA+), and the proportion of α cells (ARX+ / GCG+). Specifically, pancreatic islet cells derived from human pluripotent stem cells were digested into single cells using Accutase, and then analyzed using BD Cytofix / Cytoperm. TM After fixing the solution overnight, the solution was permeabilized with Wash Buffer and then incubated with antibody. After washing, the solution was filtered through a cell sieve and analyzed by flow cytometry.

[0106] The islet scores were evaluated according to the islet scoring rules in Table 1, and the cumulative scores for each group were calculated.

[0107] Table 1. Detailed Scoring Rules for Pancreatic Islet Cells (Maximum Score: 10 points)

[0108]

[0109] Preparation of cell preparation solutions

[0110] Human pluripotent stem cell-derived islet cell preparations were prepared according to the respective implementation formulations and comparative formulations.

[0111] The pancreatic islet cells derived from human pluripotent stem cells used in the embodiments of this invention were all prepared using the method described in Stages 1-6 on pages 36-37 of WO2023 / 097513A, with cell spheroids having a diameter of 50-350 μm.

[0112] Using water for injection as the base solvent, each component was added according to the formulations in the tables, mixed thoroughly, and sterilized using a 0.22 μm filter to prepare a human pluripotent stem cell-derived islet cell preservation solution. Subsequently, the cell preservation solutions and human pluripotent stem cell-derived islet cells were prepared into formulations at a concentration of 1000-9000 IEQ / ml, filled into formulation bags, and stored at 22℃ (±3℃). Unless otherwise specified (e.g., in Example 3), the content of human pluripotent stem cell-derived islet cells per milliliter of the formulation is 6000 IEQ.

[0113] Example 1: Screening of human serum albumin concentration as a stabilizer

[0114] According to the formulations in Table 2, pancreatic islet cell preparations containing 0%, 0.5%, and 5% (i.e., 50 mg / ml) of human pluripotent stem cells were prepared respectively. The pH value and osmolarity mOsmol / kg, as well as the islet scores, cell viability (%), and the proportions (i.e., efficiency %) of cell surface markers NKX6.1+ and C-PEP+ at 0H, 24H, 48H, and 72 hours were measured respectively.

[0115] Table 2. Formulation screening of stabilizers at different concentrations

[0116]

[0117]

[0118] Table 2-1 Test results of HSA stabilizer at different concentrations

[0119]

[0120] Table 2-1 shows that when 5% human serum albumin was added to the formulation solution, compared to 0H, the islet score, β-cell expression (efficiency, NKX6.1 / C-PEP ratio), and pancreatic cell viability of the formulation solution all decreased significantly within 72H. When 0% and 0.5% human serum albumin were added to the formulation solution, compared to 0H, the cell formulation solution could effectively maintain the islet score, β-cell expression, and pancreatic cell viability within 72H, with no significant difference between the 0% and 0.5% human serum albumin groups; however, in the 0% human serum albumin group, cell spheres were observed to settle at the bottom of the culture dish or packaging container, exhibiting severe adhesion and significant loss during collection, while no adhesion defect was 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 scores, β-cell expression, and pancreatic cell viability, without any observed adhesion defects.

[0121] Table 2-2 Test results of histidine stabilizer at different concentrations

[0122]

[0123] Table 2-2 shows that low concentrations of histidine can be used as an auxiliary component of stabilizers. Adding low concentrations of histidine (0-2.0 mg / mL) increased β-cell expression. Adding high concentrations of histidine (6.0 mg / mL) resulted in severe cell adhesion, decreased cell viability, and reduced β-cell expression.

[0124] Example 2: Formulation screening of osmotic pressure regulators and pH adjusters

[0125] According to the formulation in Table 4-1, pancreatic islet cell preparation solutions containing different types and concentrations of osmoregulators were prepared, and the islet scores, cell viability, and the ratio of cell surface marker NKX6.1 / C-PEP were measured at 0H, 24H, 48H, and 72 hours.

[0126] Test results show that using only physiological saline as the osmotic pressure regulating solution has a poor ability to maintain the morphology of pancreatic islet cells. When using water for injection as the base solvent, sodium chloride, potassium chloride, and calcium chloride are necessary components for maintaining osmotic pressure. When the osmotic pressure is too low, pancreatic islet cells exhibit edema defects.

[0127] Table 4-1 Formulation screening of basic osmotic pressure regulators

[0128]

[0129] Table 4-2 Test data for screening basic osmotic pressure regulators

[0130]

[0131] Calcium chloride helps maintain the biological activity of pancreatic islet cells, with a suitable concentration range of 0.01 mg / ml to 2 mg / ml. However, at high concentrations of 5 mg / ml, the cell spheroids become compacted and difficult to digest.

[0132] Table 5-1 Screening of Calcium Chloride Concentration in Osmotic Pressure Conditioners

[0133]

[0134] Table 5-2 Test data for screening the concentration of calcium chloride, an osmotic pressure regulator.

[0135]

[0136] Note: " / " indicates that the cell state is abnormal and data collection of quality parameters is not possible.

[0137] The test results in Tables 6-1 and 6-2 indicate that potassium chloride is an essential component for maintaining osmotic pressure, and its suitable concentration range is 0-3.0 mg / ml.

[0138] Table 6-1 Screening of potassium chloride concentration in osmotic pressure regulators

[0139]

[0140] Table 6-2 Test data for screening potassium chloride concentration as an osmotic pressure regulator

[0141]

[0142] The test results in Tables 7-1 and 2-2 indicate that sodium chloride is an essential component for maintaining osmotic pressure, thus maintaining the morphology of islet cells. Too low a concentration of sodium chloride is detrimental to maintaining osmotic pressure; when osmotic pressure is too low, islet cells exhibit edema defects. Both excessively high and excessively low concentrations of sodium chloride lead to a significant decrease in islet scores at 24 hours.

[0143] Table 7-1 Screening of Sodium Chloride Concentration in Osmotic Pressure Conditioners

[0144]

[0145] Table 7-2 Test data for screening sodium chloride concentration as an osmotic pressure regulator

[0146]

[0147] The test results in Tables 8-1 and 8-2 indicate that magnesium chloride, as an auxiliary component (dominant component) of the osmotic pressure regulator, helps maintain the proportion of pancreatic β cells, maintain cell spheroid morphology, and maintain biological activity.

[0148] Table 8-1 Screening of Magnesium Chloride Concentration in Osmotic Pressure Conditioners

[0149]

[0150] Table 8-2 Test data for screening magnesium chloride concentration as an osmotic pressure regulator

[0151]

[0152] The test results in Tables 9-1 and 9-2 show that low concentrations of glucose (0-10 mM) can be used as an auxiliary osmotic pressure regulator; when high concentrations of glucose (27.7 mM) are added, cell spheroids turn black, and cell viability and functional markers cannot be maintained.

[0153] Table 9-1 Screening of Glucose Concentration in Osmoregulators

[0154]

[0155] Table 9-2 Test data for screening glucose concentration as an osmolarity regulator

[0156]

[0157] The test results in Tables 10-1 and 10-2 indicate that sodium lactate is an essential component of pancreatic islet cell preparation solutions. It regulates the pH value of the preparation solution and can effectively maintain the biological activity of pancreatic islet cells within a concentration range of 1.5 mg / ml–5.0 mg / ml.

[0158] Table 10-1 Screening of Sodium Lactate Concentration as a pH Adjuster

[0159]

[0160] Table 10-2 Test data for screening pH adjuster sodium lactate concentration

[0161]

[0162] Example 3: Screening of cell density in formulation

[0163] Pancreatic islet cell formulations with cell densities of 1000 IEQ / mL, 6000 IEQ / mL, and 9000 IEQ / mL were prepared using formulation B1 of Example 2. The islet scores, cell viability (%), and the proportion (efficiency%) of the cell surface marker NKX6.1 / C-PEP were determined using the same method at 0H, 24H, 48H, and 72H.

[0164] Test results showed that when the formulation density was high (9000 IEQ / mL), the viability of islet cells, β-cell expression, and islet scores all decreased significantly over 72 hours as the formulation was stored, and cell adhesion was significantly aggravated. However, these defects were mitigated when the formulation density was reduced (below 8000 IEQ / mL).

[0165] Table 10 Test data for cell density screening

[0166]

[0167] Example 4: Screening of Prescription Solution Storage Temperature

[0168] Pancreatic islet cell formulation solutions were prepared according to formulation B1 of Example 2. Islet scores, cell viability (%), and the proportion of the cell surface marker NKX6.1 / C-PEP (efficiency %) were measured at 4°C, 22°C, and 37°C at 0H, 24H, 48H, and 72 hours. The test results show that the formulation solutions of the examples can maintain islet scores, cell viability, and NKX6.1 / C-PEP levels at temperatures ranging from 4°C to 22°C, which is beneficial for formulation transport under temperature control.

[0169] Table 11 Test data for storage temperature

[0170]

[0171] Example 5: Cell viability and pancreatic islet cell ratio in different cell preparation solutions

[0172] Pancreatic islet cell spheres derived from human pluripotent stem cells were collected, the culture supernatant was discarded, and the formulation solution was added. Equal amounts of cell spheres were collected at time points of 0H, 24H, 48H, and 72H of the formulation solution. Single-cell suspensions were obtained by digestion with Accutase enzyme solution for 15-25 minutes. Cell viability was assessed using an NC200 cell counter. Flow cytometry was used to detect the expression of functional markers: the proportion of pancreatic β cells was determined by the double positivity rate of NKX6.1 and C-PEP; the proportion of pancreatic α cells was determined by the double positivity rate of ARX and GCG; the proportion of pancreatic δ cells was determined by the double positivity rate of SST and HHEX; and the proportion of endocrine cells was determined by the CHGA positivity rate. The sampling method, sample volume, digestion method, and final detection procedures were maintained consistently each day.

[0173] Table 12 Formulas of Compound Preparations

[0174]

[0175] Table 13 shows that at 24 hours, the cell viability of compound preparations 1, 2, and 3 was relatively high, above 90%. At 48 hours, the cell viability of compound preparations 1 and 2 remained stable above 90%, while the cell viability of compound preparation 3 decreased significantly. At 72 hours, compound preparation 1 had the highest cell viability, which remained stable above 90%.

[0176] Table 13 Results of pancreatic islet cell viability test

[0177] Preparation liquid name 0H 24H 48H 72H Compound preparation liquid 1 96.1％ 95.1％ 92.9％ 91.6％ Compound preparation liquid 2 96.1％ 91.4％ 90.1％ 88.0％ Compound preparation liquid 3 96.1％ 94.4％ 85.4％ 68.2％ FRS 96.1％ 86.3％ 78.7％ 75.5％

[0178] Table 14 shows that the expression ratio of pancreatic α cells in each group remained relatively stable within 72 hours of the formulation, with no significant differences.

[0179] Table 14 Comparison of pancreatic α-cell percentages among different types of formulations

[0180] Preparation liquid name 0H 24H 48H 72H Compound preparation liquid 1 21.6％ 24.8％ 25.2％ 25.5％ Compound preparation liquid 2 21.6％ 26.7％ 27.2％ 27.0％ Compound preparation liquid 3 21.6％ 24.0％ 24.0％ 23.9％ FRS 21.6％ 26.1％ 25.5％ 25.8％

[0181] Table 15 shows that within 24 hours of the formulation, the expression rate of pancreatic β cells in all other groups was above 40%, with the highest rate in compound preparation group 1. Within 72 hours of the formulation, compound preparation group 1 showed significantly better maintenance of the pancreatic β cell proportion than the other groups.

[0182] Table 15 Comparison of pancreatic β-cell percentages among different types of formulations

[0183] Preparation liquid name 0H 24H 48H 72H Compound preparation liquid 1 42.4％ 45.6％ 46.4％ 41.3％ Compound preparation liquid 2 42.4％ 42.5％ 40.9％ 31.6％ Compound preparation liquid 3 42.4％ 41.8％ 39.5％ 32.5％ FRS 42.4％ 41.5％ 40.7％ 35.6％

[0184] Table 16 shows that within 24 hours of preparation, the expression ratio of pancreatic δ cells in each group did not differ significantly, with the highest ratio observed in Compound Preparation 1 and Compound Preparation 2. Between 48 and 72 hours of preparation, the expression ratio of pancreatic δ cells in Compound Preparation 3 and the FRS group decreased significantly; while the expression of pancreatic δ cells in Compound Preparation 1 and Compound Preparation 2 remained relatively stable.

[0185] Table 16 Comparison of pancreatic delta cell percentage among different types of formulations

[0186] Preparation liquid name 0H 24H 48H 72H Compound preparation liquid 1 8.18％ 10.10％ 9.80％ 9.21％ Compound preparation liquid 2 8.18％ 10.40％ 9.50％ 9.10％ Compound preparation liquid 3 8.18％ 8.50％ 3.70％ 3.60％ FRS 8.18％ 7.50％ 5.00％ 3.90％

[0187] Table 17 shows that within 24 hours of preparation, there was no significant difference in the expression ratio of pancreatic islet endocrine cells among the different preparation groups; within 72 hours of preparation, the expression of endocrine cells in compound preparation liquid 1 and compound preparation liquid 2 groups was more stable and significantly better than that in other pancreatic islet cell preparation liquid groups.

[0188] Table 17 Comparison of Endocrine Cell Proportion in Different Types of Liquid Formulations

[0189] Preparation liquid name 0H 24H 48H 72H Compound preparation liquid 1 85.1％ 88.8％ 89.9％ 89.1％ Compound preparation liquid 2 85.1％ 88.3％ 88.3％ 88.5％ Compound preparation liquid 3 85.1％ 80.5％ 80.6％ 81.9％ FRS 85.1％ 87.3％ 86.1％ 85.5％

Claims

1. A human pluripotent stem cell-derived islet cell preparation comprising pluripotent stem cell (hPSC)-derived islet cells existing in the form of cell spheres larger than 50 μm, 0%-5% human serum albumin, and a base solvent.

2. The human pluripotent stem cell-derived islet cell preparation as described in claim 1, wherein the pluripotent stem cell (hPSC)-derived islet cell spheres in each milliliter of the preparation are equivalent to 1-20 million single cells of human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cell spheres in each milliliter of the preparation is 1,000-10,000 IEQs; Preferably, the number of pluripotent stem cell (hPSC)-derived islet cell spheres per milliliter of the formulation is equivalent to 2-16 million single cells of human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cell spheres per milliliter of the formulation is 2,000-8,000 IEQ.

3. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1 or 2, further comprising an osmotic pressure regulator and a pH regulator.

4. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 3, wherein the osmotic pressure regulator is selected from: Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc sulfate, glucose, trehalose, or combinations thereof; Preferably, it is 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 pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 3, wherein the pH adjuster controls the pH of the preparation within the range of 6.5-8.0; The pH adjuster is selected from sodium lactate, sodium acetate, HEPES, sodium bicarbonate, sodium citrate, citric acid, or combinations thereof; preferably sodium lactate, HEPES, or a combination of sodium bicarbonate, sodium citrate, and citric acid.

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

7. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 6, wherein the additional stabilizer is selected from one or more of histidine, sodium gluconate, pyruvate, nicotinamide ITS (1X), linoleic acid, and vitamin E.

8. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pancreatic islet cells are pluripotent stem cell (hPSC) derived from human pluripotent stem cell (HPSC) cells, which exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 1-20 million single cells equivalent to human pluripotent stem cell (HPSC) derived pancreatic islet cells; or the density of HPSC derived pancreatic islet cells per milliliter of the formulation is 1,000-10,000 IEQ cells. 0%-5% human serum albumin; Osmotic pressure regulator, which is selected from a combination of sodium chloride, potassium chloride and calcium chloride, or a combination with the addition of magnesium chloride; pH adjuster, selected from sodium lactate, or a combination of sodium bicarbonate, sodium citrate and citric acid; Optional additional stabilizers, selected from histidine, sodium gluconate, or combinations thereof; An optional antioxidant, selected from one or more of pyruvate and nicotinamide.

9. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ. 0%-5% human serum albumin; Osmotic pressure regulators are selected from sodium chloride at concentrations of 1.0-9.0 mg / ml, potassium chloride at concentrations of 0.1-3.0 mg / ml, calcium chloride at concentrations of 0.01-2.0 mg / ml, and magnesium chloride at concentrations of 0-1.0 mg / ml. Preferably, the combination of sodium chloride at 6.0 mg / ml, potassium chloride at 0.3 mg / ml, and calcium chloride at 0.2 mg / ml, or a combination of sodium chloride at 6.0 mg / ml, potassium chloride at 0.3 mg / ml, calcium chloride at 0.2 mg / ml, and magnesium chloride at 0.3 mg / ml; The pH adjuster is selected from a combination of 1.5-5.0 mg / ml sodium lactate, 20-30 mM HEPES, or 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. Optional additional stabilizers, selected from histidine at 0-6.0 mg / ml; An optional antioxidant, selected from 0-10 mM nicotinamide.

10. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ. 0%-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 at concentrations of 0-6.0 mg / ml, preferably 0-2.0 mg / ml; 0-10mM nicotinamide.

11. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pancreatic islet cells are pluripotent stem cells (hPSCs) present in the form of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSCs)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQs. 0%-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 concentrations of 0-2.0 mg / ml; 0-10mM nicotinamide.

12. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ. 0%-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; Optional additional stabilizers, selected from histidine at 0-2.0 mg / ml; An optional antioxidant, selected from 0-10 mM nicotinamide.

13. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in claim 1, comprising: The pluripotent stem cell (hPSC)-derived islet cells exist in the form of cell spheres of 50-350 μm, wherein each milliliter of the formulation contains 2-16 million single cells equivalent to human pluripotent stem cell-derived islet cells; or the density of pluripotent stem cell (hPSC)-derived islet cells per milliliter of the formulation is 2,000-8,000 IEQ. 0%-5% human serum albumin; 9.0 mg / ml sodium chloride (equivalent to 0.9% physiological saline); Optional additional stabilizers, selected from histidine at 0-2.0 mg / ml; An optional antioxidant, selected from 0-10 mM nicotinamide.

14. The human pluripotent stem cell-derived islet cell preparation according to any one of claims 1 to 13, wherein the human pluripotent stem cell-derived islet cells are NKX6.1+ and C-PEP+ double positive; Preferably, after the preparation is stored at 4°C to 28°C (±3°C) for 24 hours, at least 20% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+; preferably, at least 30% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 35% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 40% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 45% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, at least 50% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+, and at least 55% of the human pluripotent stem cell-derived islet cells are double-positive for NKX6.1+ and C-PEP+.

15. The human pluripotent stem cell-derived islet cell preparation according to any one of claims 1 to 14, wherein the pluripotent stem cell-derived islet cells are cell spheres of 50-350 μm.

16. The pancreatic islet cell preparation derived from human pluripotent stem cells according to any one of claims 1 to 7, when stored at 4°C to 28°C (±3°C) for 24 hours, preferably at 4°C to 24°C (±3°C) for 24 hours, maintains a cell viability (bioactivity) of more than 85%; preferably, it maintains a bioactivity of more than 90%, 92.5%, or 95%.

17. The pancreatic islet cell preparation derived from human pluripotent stem cells as described in any one of claims 1 to 16, wherein the preparation is an injectable preparation, preferably a portal vein injection preparation or a subrectus sheath injection preparation, more preferably a subrectus sheath injection preparation.

18. A method for preparing the pancreatic islet cell preparation derived from human pluripotent stem cells according to any one of claims 1-17, comprising the following steps: (1) Preparation of cell preservation composition: Add human serum albumin and other components to the base solvent, mix evenly and prepare human pluripotent stem cell-derived pancreatic islet cell preservation composition; (2) The cell preservation composition and pancreatic islet cells derived from human pluripotent stem cells are mixed and filled into a packaging container.

19. The pancreatic islet cell preparation derived from human pluripotent stem cells according to any one of claims 1-17, for use in the preparation of a medicament for treating insulin deficiency or for the treatment of insulin deficiency diseases; Preferably, the drug is a portal vein injection preparation or a subsheath injection preparation for the rectus abdominis muscle, and more preferably, the drug is a subsheath injection preparation for the anterior rectus abdominis muscle.

20. Use of a human pluripotent stem cell-derived pancreatic islet cell preservation composition in long-distance transportation, wherein the preservation composition is the component of the preparation according to any one of claims 1-17 other than human pluripotent stem cell-derived pancreatic islet cells, the long-distance transportation temperature is 4°C to 37°C, and the transportation time does not exceed 96 hours; The preferred temperatures for long-distance transportation are 4℃, 10℃, 16℃, 22℃, and 28℃. The preferred transportation time is no 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.

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