Liquid for preservation or transplantation of encapsulated islet

Abstract

Provided is a means for more efficiently transplant an encapsulated islet. A liquid for preservation or transplantation of an encapsulated islet, said liquid containing trehalose, a derivative thereof or a salt of the same.

Description

Storage or transplantation solution for encapsulated pancreatic islets

[0001] Techniques relating to storage or transplantation solutions for encapsulated pancreatic islets and their uses are disclosed.

[0002] Diabetes mellitus is a disease caused by impaired pancreatic function, which controls blood sugar. There are two main types: type I diabetes and type II diabetes. Type I diabetes develops at a relatively young age and causes symptoms in which insulin-producing cells are severely affected by an autoimmune reaction. Type II diabetes develops in middle-aged and elderly people and is caused by an inability to properly utilize insulin. In Japan, 5% of diabetic patients have type I diabetes, an estimated 300,000 people. While many of these diabetic patients control their blood sugar with insulin therapy, they are not immune to the development of complications such as nephropathy, retinopathy, and neuropathy. Furthermore, there are many cases in which blood sugar cannot be controlled even with insulin therapy.

[0003] Pancreatic islet transplantation is an alternative treatment for diabetes to insulin therapy. Pancreatic islets (also known as islets of Langerhans) are endocrine cell groups, including insulin-secreting β cells, scattered in the pancreatic tissue. Pancreatic islet transplantation is a transplantation therapy in which islets are isolated from a pancreas provided by a donor, a dispersion of the islets is prepared, and the dispersion is then administered percutaneously to the patient.

[0004] Pancreatic islet transplantation has the following advantages: (1) Because it can be administered percutaneously, it does not require laparotomy or general anesthesia, and the burden on the patient during the transplant surgery is reduced. (2) The amount of immunosuppressant administered after transplantation is smaller than in the case of marrow transplantation. (3) It can also be used with the pancreas of organ donors who have died due to cardiac arrest or arteriosclerosis, which is not suitable for pancreatic transplantation. In addition, encapsulated islets, in which pancreatic islets are encapsulated in tiny microcapsules, are known as a means of further reducing the amount of immunosuppressant administered after transplantation (Patent Documents 1 and 2).

[0005] Since many of the beta cells that make up pancreatic islets become inactive over time after isolating and purifying the islets, it is desirable to properly preserve the prepared islets so as to preserve their vitality as much as possible and to provide them for transplantation as quickly as possible. However, it takes a certain amount of time (e.g., approximately 10 to 18 hours) from the surgery to remove the pancreas from the donor to the preparation of islets for transplantation. Furthermore, even when preparations for islet transplantation are complete, the transplant surgery may not be possible immediately due to reasons such as the patient's poor physical condition.

[0006] WO2021 / 256451WO2021 / 153365

[0007] One challenge is to provide a means to perform encapsulated islet transplantation more efficiently.

[0008] Through extensive research aimed at solving the above problems, it has been discovered that a common solution can be used for the preservation and transplantation of encapsulated islets. Further investigations based on this finding have led to the following representative inventions. Item 1: A solution for preserving or transplanting encapsulated islets, comprising trehalose or a derivative thereof, or a salt thereof. Item 2: The solution according to Item 1, which is an isotonic solution. Item 3: The solution according to Item 2, wherein the isotonic solution is lactated Ringer's solution. Item 4: The solution according to any one of Items 1 to 3, wherein the concentration of trehalose or a derivative thereof, or a salt thereof is 2.0 to 6.0 (w / v)%. Item 5: The solution according to any one of Items 1 to 4, which is used to prevent tissue adhesion of encapsulated islets. Item 6: A method for preserving encapsulated islets, comprising a step of preserving encapsulated islets in the solution according to any one of Items 1 to 5. Item 7: The solution according to any one of Items 1 to 5, further comprising encapsulated islets. Item 8: A method for treating diabetes, comprising administering the solution according to Item 7 to a subject in need of islet transplantation.

[0009] During encapsulated islet transplantation, the preserved encapsulated islets can be transplanted without transferring them to a transplant solution different from the preservation solution. This allows for more efficient encapsulated islet transplantation (or shortens the time required for encapsulated islet transplantation). Furthermore, this method also has the excellent function of inhibiting adhesion of the encapsulated islets to tissues after transplantation.

[0010] 1 shows changes in body weight (mean value) measured for the non-preserved group, control group, and test group. 2 shows changes in blood glucose levels (mean value) measured during fasting for the non-preserved group, control group, and test group. 3 shows DTZ-stained photographs of encapsulated islets recovered from the non-preserved group, control group, and test group. 4 shows insulin secretion and glucose responsiveness measured for encapsulated islets recovered from the non-preserved group, control group, and test group.

[0011] 1. Storage or Transplantation Solution The storage or transplantation solution for encapsulated islets preferably contains trehalose, a derivative thereof, or a salt thereof (these may be collectively referred to as "trehaloses"). Examples of trehalose include α,α-trehalose, a disaccharide formed by 1,1-glycosidic bonds between two α-glucoses; α,β-trehalose, a disaccharide formed by 1,1-glycosidic bonds between an α-glucose and a β-glucose; and β,β-trehalose, a disaccharide formed by 1,1-glycosidic bonds between two β-glucoses. Among these, α,α-trehalose is preferred. These trehaloses can be produced by any known method, such as chemical synthesis, microbial production, or enzymatic production, but commercially available products can also be used. For example, commercially available products such as α,α-trehalose (manufactured by Hayashibara Co., Ltd. or Fujifilm Wako Pure Chemical Industries, Ltd.) can be used.

[0012] Trehalose derivatives include, but are not limited to, glycosyltrehaloses in which one or more sugar units are bound to the disaccharide trehalose, such as glucosyltrehalose, maltosyltrehalose, and maltotriosyltrehalose.

[0013] The salt of trehalose or its derivatives is not particularly limited as long as it is pharmaceutically acceptable. Examples include acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; metal salts such as sodium salt, potassium salt, and calcium salt; ammonium salt; and alkylammonium salt. These salts preferably have the same effect as trehalose when used as a solution. These salts may form hydrates or solvates. Two or more types may also be used in appropriate combination. A preservation or transplantation solution may contain only one trehalose or a combination of two or more trehaloses.

[0014] The concentration of trehaloses in the preservation or transplantation solution is not particularly limited, and for example, the lower limit is 0.1 (w / v)% or more, preferably 0.3 (w / v)% or more, more preferably 0.6 (w / v)% or more, even more preferably 1.0 (w / v)% or more, and most preferably 2.0 (w / v)% or more, calculated as trehalose. The upper limit is 40 (w / v)% or less, preferably 20 (w / v)% or less, more preferably 15 (w / v)% or less, even more preferably 10 (w / v)% or less, and most preferably 6.0 (w / v)% or less, calculated as trehalose. These lower and upper limits can be combined in any manner. For example, the concentration of trehalose or its derivatives in the preservation or transplantation solution is within the range of 0.1 to 40 (w / v)%, preferably 0.3 to 20 (w / v)%, more preferably 0.6 to 15 (w / v)%, even more preferably 1.0 to 10% (w / v), and most preferably 2.0 to 6.0 (w / v)%, calculated as trehalose.

[0015] The preservation or transplantation fluid may be any fluid (e.g., isotonic, hypotonic, or hypertonic) that allows for the transplantation and preservation of encapsulated islets, and is preferably an isotonic fluid. Here, "isotonic fluid" refers to a fluid having an osmotic pressure approximately equal to that of bodily fluids or cellular fluids, specifically a fluid having an osmotic pressure in the range of 250 to 380 mOsm / L. "Hypotonic fluid" refers to a fluid having an osmotic pressure lower than that of bodily fluids or cellular fluids, specifically a fluid having an osmotic pressure less than 250 mOsm / L. A hypotonic fluid is preferably a hypotonic fluid that does not cause cell rupture (specifically, a fluid having an osmotic pressure in the range of 100 to less than 250 mOsm / L). "Hypertonic fluid" refers to a fluid having an osmotic pressure higher than that of bodily fluids or cellular fluids, specifically a fluid having an osmotic pressure greater than 380 mOsm / L (preferably greater than 380 mOsm / L to 1000 mOsm / L).

[0016] The isotonic solution is not particularly limited as long as the salt concentration, sugar concentration, etc. are adjusted using sodium ions, potassium ions, and / or calcium ions, etc., so that the osmotic pressure is approximately the same as that of body fluids or cellular fluids. Specific examples include physiological saline, buffered physiological saline (e.g., PBS, Tris-buffered saline (TBS), HEPES-buffered saline), Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, 5% aqueous glucose solution, basal medium for animal cell culture (e.g., DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199), and solutions prepared with isotonic agents (e.g., glucose, D-sorbitol, D-mannitol, lactose, sodium chloride). Among these, lactated Ringer's solution is preferred. The isotonic solution may be commercially available or may be prepared by hand. Examples of commercially available products include Otsuka Saline Injection (manufactured by Otsuka Pharmaceutical Factory, Inc.) (physiological saline), Ringer's Solution "Otsuka" (manufactured by Otsuka Pharmaceutical Factory, Inc.) (Ringer's solution), Lactec (registered trademark) Injection (manufactured by Otsuka Pharmaceutical Factory, Inc.) (lactated Ringer's solution), Veen (registered trademark) F Infusion (manufactured by Fuso Pharmaceutical Industries, Ltd.) (acetated Ringer's solution), Otsuka Sugar Solution 5% (manufactured by Otsuka Pharmaceutical Factory, Inc.) (5% aqueous glucose solution), and Bicanate (registered trademark) Infusion (manufactured by Otsuka Pharmaceutical Factory, Inc.) (bicarbonate Ringer's solution).

[0017] The pH of the storage or transplantation solution is not particularly limited as long as it is suitable for transplantation and storage of encapsulated islets, and may be, for example, within the range of 6.5 to 8.5. For example, the pH may be 6.5-8.4; 6.5-8.3; 6.5-8.2; 6.5-8.1; 6.5-8.0; 6.5-7.9; 6.5-7.8; 6.5-7.7; 6.5-7.6; 6.5-7.5; 6.5-7.4; 6.5-7.3; 6.5-7.2; 6.5-7.1; 6.5-7.0; 6.5-6.9; 6.5-6.8; 6.6-8.5; 6.7-8.5; 6.8-8.5; 6.9-8.5; 7.0-8.5; 7.1-8.5; 7.2-8.5; 7.3-8.5; 7.4-8.5; 7.5-8.5; 7.6-8.5; 7.7-8.5; 7.8-8.5; 7.9-8.5; 8.0-8.5; 8.1-8.5; 8.2-8.5; 6.6-8.4; 6.6-8.3; 6.6-8.2; 6.6-8.1; 6.6-8.0; 6.6-7.9; 6.6-7.8; 6.6-7.7; 6.6-7.6; 6.6-7.5; 6.6-7.4; 6.6-7.3; 6.6-7.2; 6.6-7.1; 6.6-7.0; 6.6-6.9; 6.7-8.4; 6.7-8.3; 6.7-8.2; 6.7-8.1; 6.7-8.0; 6.7-7.9; 6.7-7.8; 6.7-7.7; 6.7-7.6; 6.7-7.5; 6.7-7.4; 6.7-7.3; 6.7-7.2; 6.7-7.1; 6.7-7.0; 6.8-8.4; 6.8-8.3; 6.8-8.2; 6.8-8.1; 6.8-8.0; 6.8-7.9; 6.8-7.8; 6.8-7.7; 6.8-7.6; 6.8-7.5; 6.8-7.4; 6.8-7.3; 6.8-7.2; 6.8-7.1; 6.9-8.4; 6.9-8.3; 6.9-8.2; 6.9-8.1; 6.9-8.0; 6.9-7.9; 6.9-7.8; 6.9-7.7; 6.9-7.6; 6.9-7.5; 6.9-7.4; 6.9-7.3; 6.9-7.2; 7.0-8.4; 7.0-8.3; 7.0-8.2; 7.0-8.1; 7.0-8.0; 7.0-7.9; 7.0-7.8; 7.0-7.7; 7.0-7.6; 7.0-7.5; 7.0-7.4; 7.0-7.3; 7.1-8.4; 7.1-8.3; 7.1-8.2; 7.1-8.1; 7.1-8.0; 7.1-7.9; 7.1-7.8; 7.1-7.7; 7.1-7.6; 7.1-7.5; 7.1-7.4;7.2-8.4; 7.2-8.3; 7.2-8.2; 7.2-8.1; 7.2-8.0; 7.2-7.9; 7.2-7.8; 7.2-7.7; 7.2-7.6; 7.2-7.5; 7.3-8.4; 7.3-8.3; 7.3-8.2; 7.3-8.1; 7.3-8.0; 7.3-7.9; 7.3-7.8; 7.3-7.7; 7.3-7.6; 7.4-8.4; 7.4-8.3; 7.4-8.2; 7.4-8.1; 7.4-8.0; 7.4-7.9; 7.4-7.8; 7.4-7.7; 7.5-8.4; 7. Examples include 7.5 to 8.3; 7.5 to 8.2; 7.5 to 8.1; 7.5 to 8.0; 7.5 to 7.9; 7.5 to 7.8; 7.6 to 8.4; 7.6 to 8.3; 7.6 to 8.2; 7.6 to 8.1; 7.6 to 8.0; 7.6 to 7.9; 7.7 to 8.4; 7.7 to 8.3; 7.7 to 8.2; 7.7 to 8.1; 7.7 to 8.0; 7.8 to 8.4; 7.8 to 8.3; 7.8 to 8.2; 7.8 to 8.1; 7.9 to 8.4; 7.9 to 8.3; 7.9 to 8.2; 8.0 to 8.4; 8.0 to 8.3; 8.1 to 8.3; and the like.

[0018] In one embodiment, the preservation or transplantation solution preferably does not contain substances unsuitable for mammalian cell transplantation, such as biologically derived components (e.g., serum or serum-derived components (e.g., albumin)), dimethyl sulfoxide (DMSO), glycerin, ethylene glycol, trimethylene glycol, dimethylacetamide, polyethylene glycol (PEG), polyvinylpyrrolidone, etc.

[0019] The type of pancreatic islets used for the encapsulated islets to be transplanted (and preserved) using a preservation or transplantation solution is not particularly limited. The pancreatic islets preferably contain insulin-producing β cells, glucagon-containing α cells, somatostatin-secreting delta cells, and pancreatic polypeptide-containing cells (PP cells). The majority of the pancreatic islets are preferably insulin-producing β cells. The origin of the pancreatic islets can be selected depending on the purpose, and is preferably human, pig, mouse, rat, monkey, or dog. In one embodiment, the pancreatic islets are preferably derived from pigs, and are preferably islets from young pigs (e.g., 3 days to 4 weeks old or 7 days to 3 weeks old). Pancreatic islets can be obtained by any method known in the art.

[0020] The size of the pancreatic islets is preferably 50 μm or more and 400 μm or less, more preferably 50 μm or more and 350 μm or less. The size of the pancreatic islets can be measured using a micrometer under a microscope. A preferred characteristic of the pancreatic islets is that they contain 10% or more β cells. There is no particular upper limit to the proportion of β cells, but it is, for example, 80%.

[0021] In one embodiment, the encapsulated pancreatic islet is preferably an encapsulated pancreatic islet as described in Patent Document 1 or 2. In one embodiment, the encapsulated pancreatic islet has a structure in which a core is surrounded by three membranes, the core containing a pancreatic islet, the first and third membranes from the inside of the three membranes preferably containing alginate, and the second membrane from the inside preferably containing polyornithine. In one embodiment, the encapsulated pancreatic islet may be surrounded by more than three membranes (e.g., four-, five-, six-, seven-, or eight-layer membranes).

[0022] The membrane containing alginic acid means that the membrane is mainly composed of alginic acid. The membrane containing polyornithine means that the membrane is mainly composed of polyornithine. The encapsulated pancreatic islets preferably contain 1 to 5 pancreatic islets per capsule.

[0023] From the viewpoint of suppressing fibrosis, the encapsulated islets preferably have an average diameter of 400 μm or more, more preferably 420 μm or more. There is no particular upper limit to the average diameter, but it can be set to, for example, 700 μm or less, 600 μm or less, or 500 μm or less. The average diameter can be measured using the measurement method employed in the examples described below.

[0024] Encapsulated pancreatic islets can be prepared by the methods described in Patent Documents 1 and 2, for example.

[0025] The above-mentioned preservation or transplantation solution can be used for transplantation of the preserved encapsulated islets directly, without transferring them to another transplantation solution. Therefore, in one embodiment, the preservation solution or transplantation solution can serve as both a preservation solution and a transplantation solution for pancreatic islets. Furthermore, the preservation or transplantation solution has an excellent function of inhibiting adhesion of encapsulated islets to tissues (e.g., the liver) after transplantation. Therefore, the preservation or transplantation solution can be used to prevent adhesion of encapsulated islets to tissues. The storage period and storage temperature for preserving encapsulated islets using the preservation or transplantation solution of the present invention are as described below in "2. Storage Method."

[0026] 2. Preservation Method There is provided a method for preserving encapsulated islets, which comprises the step of preserving the encapsulated islets in the preservation solution or transplant solution described above.

[0027] The storage period is arbitrary and is not particularly limited. In one embodiment, the storage period can be from several hours to several days. Several hours refers to, for example, 0 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, or 22 hours. Several days refers to, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days. For example, the storage period may be 0 hours to 12 days; 0 hours to 11 days; 0 hours to 10 days; 0 hours to 9 days; 0 hours to 8 days; 0 hours to 7 days; 0 hours to 6 days; 0 hours to 5 days; 0 hours to 4 days; 0 hours to 3 days; 0 hours to 2 days; 0 hours to 1 day; 2 hours to 12 days; 2 hours to 11 days; 2 hours to 10 days; 2 hours to 9 days; 2 hours to 8 days; 2 hours to 7 days; 2 hours to 6 days; 2 hours to 5 days; 2 hours to 4 days; 2 hours to 3 days; 2 hours to 2 days; 2 hours to 1 day; 4 hours to 12 days; 4 hours to 11 days; 4 hours to 10 days. Days; 4 hours to 9 days; 4 hours to 8 days; 4 hours to 7 days; 4 hours to 6 days; 4 hours to 5 days; 4 hours to 4 days; 4 hours to 3 days; 4 hours to 2 days; 4 hours to 1 day; 6 hours to 12 days; 6 hours to 11 days; 6 hours to 10 days; 6 hours to 9 days; 6 hours to 8 days; 6 hours to 7 days; 6 hours to 6 days; 6 hours to 5 days; 6 hours to 4 days; 6 hours to 3 days; 6 hours to 2 days; 6 hours to 1 day; 8 hours to 12 days; 8 hours to 11 days; 8 hours to 10 days; 8 hours to 9 days; 8 hours to 8 days; 8 hours to 7 days; 8 hours to 6 days; 8 hours 8 hours to 5 days; 8 hours to 4 days; 8 hours to 3 days; 8 hours to 2 days; 8 hours to 1 day; 10 hours to 12 days; 10 hours to 11 days; 10 hours to 10 days; 10 hours to 9 days; 10 hours to 8 days; 10 hours to 7 days; 10 hours to 6 days; 10 hours to 5 days; 10 hours to 4 days; 10 hours to 3 days; 10 hours to 2 days; 10 hours to 1 day; 12 hours to 12 days; 12 hours to 11 days; 12 hours to 10 days; 12 hours to 9 days; 12 hours to 8 days; 12 hours to 7 days; 12 hours to 6 days; 12 hours to 5 days; 12 hours to 4 days ; 12 hours to 3 days; 12 hours to 2 days; 12 hours to 1 day; 14 hours to 12 days; 14 hours to 11 days; 14 hours to 10 days; 14 hours to 9 days; 14 hours to 8 days; 14 hours to 7 days; 14 hours to 6 days; 14 hours to 5 days; 14 hours to 4 days; 14 hours to 3 days; 14 hours to 2 days; 14 hours to 1 day; 16 hours to 12 days; 16 hours to 11 days; 16 hours to 10 days; 16 hours to 9 days; 16 hours to 8 days; 16 hours to 7 days; 16 hours to 6 days; 16 hours to 5 days; 16 hours to 4 days; 16 hours to 3 days;16 hours to 2 days; 16 hours to 1 day; 18 hours to 12 days; 18 hours to 11 days; 18 hours to 10 days; 18 hours to 9 days; 18 hours to 8 days; 18 hours to 7 days; 18 hours to 6 days; 18 hours to 5 days; 18 hours to 4 days; 18 hours to 3 days; 18 hours to 2 days; 18 hours to 1 day; 20 hours to 12 days; 20 hours to 11 days; 20 hours to 10 days; 20 hours to 9 days; 20 hours to 8 days; 20 hours Examples include 20 hours to 7 days, 20 hours to 6 days, 20 hours to 5 days, 20 hours to 4 days, 20 hours to 3 days, 20 hours to 2 days, 20 hours to 1 day, 22 hours to 12 days, 22 hours to 11 days, 22 hours to 10 days, 22 hours to 9 days, 22 hours to 8 days, 22 hours to 7 days, 22 hours to 6 days, 22 hours to 5 days, 22 hours to 4 days, 22 hours to 3 days, 22 hours to 2 days, and 22 hours to 1 day.

[0028] The storage temperature is not particularly limited and may be any temperature. In one embodiment, the storage temperature may be 0 to 40°C. For example, storage temperatures may be 0-40°C; 0-38°C; 0-36°C; 0-34°C; 0-32°C; 0-30°C; 0-28°C; 0-26°C; 0-24°C; 0-22°C; 0-20°C; 0-18°C; 0-16°C; 0-14°C; 0-12°C; 0-10°C; 0-8°C; 0-6°C; 0-4°C; 0-2°C; 2-40°C; 2-38°C; 2-36°C; 2-34°C; 2-32°C; 2-30°C; 2-28°C; 2-26°C; 2-24°C; 2-22°C; 2-20°C; 2-18°C; 2-16°C; 2-14°C; 2-12°C; 2-10°C; 2-8°C; 2-6°C; 2-4°C; 4°C; Up to 40°C; 4-38°C; 4-36°C; 4-34°C; 4-32°C; 4-30°C; 4-28°C; 4-26°C; 4-24°C; 4-22°C; 4-20°C; 4-18°C; 4-16°C; 4-14°C; 4-12°C; 4-10°C; 4-8°C; 4-6°C; 6-40°C; 6-38°C; 6-36°C; 6-34°C; 6-32°C; 6-30°C; 6-28°C; 6-26°C; 6-24°C; 6-22°C; 6-20°C; 6-18°C; 6-16°C; 6-14°C; 6-12°C; 6-10°C; 6-8°C; 8-40°C; 8-38°C; 8-36°C; 8-34°C; 8-32°C; 8- 30°C; 8-28°C; 8-26°C; 8-24°C; 8-22°C; 8-20°C; 8-18°C; 8-16°C; 8-14°C; 8-12°C; 8-10°C; 10-40°C; 10-38°C; 10-36°C; 10-34°C; 10-32°C; 10-30°C; 10-28°C; 10-26°C; 10-24°C; 10-22°C; 10-20°C; 10-18°C; 10-16°C; 10-14°C; 10-12°C; 12-40°C; 12-38°C; 12-36°C; 12-34°C; 12-32°C; 12-30°C; 12-28°C; 12-26°C; 12-24°C; 12-22°C ;12-20°C;12-18°C;12-16°C;12-14°C;14-40°C;14-38°C;14-36°C;14-34°C;14-32°C;14-30°C;14-28°C;14-26°C;14-24°C;14-22°C;14-20°C;14-18°C;14-16°C;16-40°C;16-38°C;16-36°C;16-34°C;16-32°C;16-30°C;16-28°C;16-26°C;16-24°C;16-22°C;16-20°C;16-18°C;18-40°C;18-38°C;18-36°C;18-34°C;18-32°C;18-30°C; 18-28°C; 18-26°C; 18-24°C; 18-22°C; 18-20°C; 20-40°C; 20-38°C; 20-36°C; 20-34°C; 20-32°C; 20-30°C; 20-28°C; 20-26°C; 20-24°C; 20-22°C; 22-40°C; 22-38°C; 22-36°C; 22-34°C; 22-32°C; 22-30°C; 22-28°C; 22-26°C; 22-24°C; 24-40°C; 24-38°C; 24-36°C; 24-34°C; 24-32°C; 24-30°C; 24 up to 28°C; 24-26°C; 26-40°C; 26-38°C; 26-36°C; 26-34°C; 26-32°C; 26-30°C; 26-28°C; 28-40°C; 28-38°C; 28-36°C; 28-34°C; 28-32°C; 28-30°C; 30-40°C; 30-38°C; 30-36°C; 30-34°C; 30-32°C; 32-40°C; 32-38°C; 32-36°C; 32-34°C; 34-40°C; 34-38°C; 34-36°C; 36-40°C; 36-38°C; 38-40°C; and the like.

[0029] In one embodiment, encapsulated islets preserved in a preservation solution or a transplant solution are preferably transplanted as is (after transferring to another solution). By eliminating the need to transfer from a preservation solution to another transplant solution, the transplantation of encapsulated islets can be performed efficiently or the time required for transplantation can be shortened. Furthermore, by transplanting encapsulated islets without transferring from a preservation solution to another transplant solution, the effects of transfer on the encapsulated islets can be avoided, and the functionality of the encapsulated islets to be transplanted can be maintained at a higher level.

[0030] The subject to which the encapsulated islets are transplanted may be any subject in need thereof. For example, the subject may be a mammal, specifically, a human, a dog, a cat, a horse, a cow, a pig, etc., and preferably a human. Furthermore, the subject in need of the encapsulated islet transplantation is preferably a subject suffering from diabetes, and more preferably a subject suffering from type 1 diabetes.

[0031] The transplantation site of the encapsulated islets is not particularly limited, and may be subcutaneous, intramuscular, intraomental, or intraperitoneal, with intraperitoneal transplantation being preferred. The number of islet cells per administration is preferably about 5.0 x 10 per kg of subject body weight. ６ ~1.2 × 10 ８ per kg body weight, more preferably about 8.0 x 10 ６ ~8.0 x 10 ７ cells / kg body weight, and more preferably about 1.2 x 10 ７ ~4.0 x 10 ７ The number of pancreatic islets per administration is preferably about 5,000 to 60,000 per kg of subject body weight, more preferably about 8,000 to 40,000 per kg of subject body weight, and even more preferably about 12,000 to 20,000 per kg of subject body weight.

[0032] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0033] The feasibility of using encapsulated islets as a preservation or transplantation solution was evaluated in the following test.

[0034] 1. Test Liquid A liquid composition having the properties shown in the table below was used as the test liquid.

[0035]

[0036] 2. Comparative preservative solution A comparative preservative solution was prepared by adding 50 mL of inactivated porcine serum (Gibco) and 5 mL of nicotinamide stock solution (1 M nicotinamide) to 500 mL of RPMI-1640 (Gibco) and mixing.

[0037] 3. Encapsulated Pancreatic Islets. Pancreatic islets isolated from young pigs were suspended in sodium alginate solution and encapsulated by dripping the alginate suspension into 109 mM calcium chloride solution using a peristaltic pump. The capsules were recovered and washed with saline. A 0.10% poly-L-ornithine (PLO) solution was added and mixed on a mixing rotor for 10 minutes. The supernatant was removed, washed with saline, and then a 0.05% PLO solution was added and mixed on a mixing rotor for 6 minutes. The supernatant was removed, washed with saline, and then a 10-fold diluted sodium alginate solution was added and mixed on a mixing rotor for 6 minutes. The supernatant was removed, washed with saline, and then a 1.15% sodium citrate solution was added and mixed on a mixing rotor for 2 minutes. The supernatant was removed, washed with saline, and then the supernatant was removed again to obtain encapsulated pancreatic islets. The resulting encapsulated islets were suspended in RPMI-1640 medium supplemented with porcine serum, nicotinamide, and antibiotics, and then distributed into T175 flasks, where they were cultured in a CO2 incubator at 37°C for 26 or 30 days.

[0038] 4. Test Animals B6 mice with diabetes mellitus induced by STZ administration were used as test animals. Mice with fasting blood glucose levels of 300 mg / dL or higher on both days 2 and 3 (the day of transplantation) after STZ administration and no abnormalities in visual observation of their general condition were selected as subjects. Stratified randomization was performed using the statistical analysis system EXSUS 10.0 (CAC Exicare Co., Ltd.) based on body weight and blood glucose levels on day 3.

[0039] 5. Test Setup The test was set up as shown in the table below.

[0040]

[0041] In the non-preserved group, STZ was administered to test animals at a dose of 200 mg / kg. Encapsulated juvenile porcine islets cultured until Day 30 were suspended in saline without preserving them in the test or comparative preservation solution. 8,000 IEQ (Islet EQ equivalent: 1 IEQ = one 150 μm islet) were transplanted intraperitoneally. Encapsulated juvenile porcine islets were prepared as follows: Based on the IEQ count results on the day of transplantation, the culture flasks were removed from the incubator immediately before transplantation, and each IEQ dose was dispensed into a 50 mL tube. The tubes were left to stand at room temperature until washing. The medium in the tubes was removed with a pipette, and approximately 10 mL of room-temperature HBSS (Gibco) was added. After allowing to stand, the supernatant was removed. This procedure was repeated three times, followed by the addition of approximately 10 mL of room-temperature saline, suspension, and subsequent removal of the supernatant to approximately 4 mL. The islets were then immediately transplanted.

[0042] In the control group, encapsulated juvenile porcine islets cultured until Day 26 were stored at 4°C for 96 hours in the comparative preservation solution (Fukushima Galilei, FMS-702G) described in "2. Comparative Preservation Solution" above, suspended in saline, and transplanted intraperitoneally at 8,000 IEQ. The encapsulated juvenile porcine islets were prepared as follows: Based on the IEQ count results on the first day of storage, the storage container was removed from the refrigerator immediately before transplantation, and each IEQ dose was dispensed into a 50 mL tube. The tubes were kept on ice until washing. The preservation solution in the tube was removed with a pipette, and approximately 10 mL of room-temperature HBSS (Gibco) was added. After allowing to stand, the supernatant was removed. This procedure was repeated three times, followed by resuspension in approximately 10 mL of room-temperature saline, followed by removal of the supernatant to approximately 4 mL, and the islets were immediately transplanted.

[0043] In the test group, encapsulated infant porcine islets cultured until Day 26 were stored at 4°C for 96 hours in the test solution described in "1. Test Solution" above (Fukushima Galilei, FMS-702G) and then directly transplanted into the abdominal cavity at 8,000 IEQ. The encapsulated infant porcine islets were prepared as follows: Based on the IEQ count results on the first day of storage, the storage container was removed from the refrigerator immediately before transplantation, and each IEQ dose was dispensed into a 50 mL tube. The tubes were kept on ice until the washing procedure. The storage solution in the tubes was removed to approximately 4 mL with a pipette and immediately used for transplantation.

[0044] In both groups, encapsulated islets were transplanted using the following procedure. Mice were anesthetized using an isoflurane vaporizer, and their abdomens were disinfected with a 70% ethanol spray. A prescribed dose of encapsulated islet solution was administered intraperitoneally via a 16G Surflow puncture. The skin at the puncture site was closed with surgical adhesive (Aron Alpha A "Sankyo", Daiichi Sankyo Co., Ltd.). Since body temperature dropped after surgery, the mice were kept warm with a heater until they woke up. The day of transplantation was designated Day 0.

[0045] 6. Observation, Measurement, and Examination 6-1. Observation of General Condition Every day, the general condition of the animals was observed, including confirmation of whether they were alive or dead.

[0046] 6-2. Body weight measurements Body weight was measured on the day of STZ administration, the day of transplantation, twice a week thereafter, and on the day of the oral glucose tolerance test (OGTT).

[0047] 6-3. Blood Glucose Measurement Mice were restrained in a restrainer on the day of STZ administration (just before administration), the second day of administration, the day of transplantation, and twice a week thereafter, and blood glucose levels were measured by puncturing the tail vein with a 27G needle (several μL). Niprostat Strips (NIPRO) were used for measurement. Blood glucose levels were measured in the morning.

[0048] 6-4. Oral glucose tolerance test (OGTT) This was performed four weeks after transplantation, after measuring fasting blood glucose levels. Mice were fasted for approximately 16 hours the day before and then orally administered 10% glucose solution (Otsuka Pharmaceutical Factory) at 10 μL / g (glucose 1 g / kg) using a probe, and blood glucose levels were evaluated. Blood glucose levels were measured before administration and at 0, 15, 30, 45, 60, 75, 90, 105, and 120 minutes after administration.

[0049] 6-5. Autopsy After the OGTT, the abdomen was opened under isoflurane anesthesia, and any abnormalities observed macroscopically were recorded. Blood was collected from the caudal vena cava using a syringe with a 26G needle. The blood was placed in a Microtina blood collection tube (BD, EDTA-2K), gently mixed by inversion, and then stored on ice. The blood was centrifuged at 4°C and 3000 x g for 10 minutes, and the plasma was collected and placed in a separate microtube and stored at -30°C or below as a sample for porcine C-peptide measurement.

[0050] 6-6. DTZ Staining of Recovered Encapsulated Islets DTZ solution was added to the recovered encapsulated islet suspension, and the islets were observed and photographed using an inverted microscope (CKX41, Olympus) to confirm DTZ staining. DTZ solution was prepared by adding 100 mg of Dithizone (Fujifilm Wako Pure Chemical Industries) to 5 mL of DMSO (Sigma) and 45 mL of HBSS, dissolving thoroughly, and then sterilizing with a 0.22 μm filter. Spheroids within the field of view of the resulting image were counted for those with and without dithizone staining, and purity was calculated using the following formula: "Purity (%) = (number of spheroids with dithizone staining) / (total number of spheroids) × 100."

[0051] 6-7. Measurement of porcine C-peptide The stored plasma was thawed at room temperature, and the porcine C-peptide concentration was measured using a Mercodia Porcine C-peptide ELISA kit (Mercodia, product number: 10-1256-01).

[0052] 6-8. SGS Assay The recovered capsules were subjected to an SGS assay. After incubation for 1 hour in RPMI 1640 containing 500 mg / L glucose (low glucose) or 5000 mg / L glucose (high glucose), the amount of insulin secreted was determined using Mercodia Porcine Insulin ELISA. Measurements were performed according to the kit instructions, with 1 mg = 115 U used for unit calculations. The SI (stimulation index), which is the ratio of insulin secretion levels in low glucose to high glucose, was calculated using the SGS assay.

[0053] 6-9. Statistical processing The average value and standard deviation of the obtained data were calculated and graphed as necessary. Statistical analysis was performed using EXSUS 10.0 with a Student's t-test. p<0.05 or less was considered to be a significant difference, and p<0.1 or less was considered to be a significant trend.

[0054] 7. Results 7-1. General condition After transplantation of encapsulated islets, animal number 15 (non-preserved group) and animal number 38 (test group) showed behavioral depression and were euthanized. None of the other animals showed behavioral depression due to diabetes or a weight loss of 20% or more from the time of transplantation, and none were euthanized.

[0055] Body weight increased from capsule implantation to the day of autopsy. No significant differences in mean body weight were observed between groups (Figure 1).

[0056] 7-3. Blood glucose levels during a period of fasting A decrease in blood glucose levels during a period of fasting was observed in all groups after capsule implantation. 13 days after implantation, blood glucose levels in the control group rose temporarily but began to decrease again, and ultimately no significant difference was observed between the groups (Figure 2).

[0057] 7-4. OGTT Although some individuals showed poor glucose responsiveness, there was no significant difference between the groups, demonstrating glucose responsiveness.

[0058] At autopsy one month after transplantation, adhesion of the capsule to the liver or capsule masses in the abdominal cavity was observed in 1 / 7 cases (14.3%) in the non-preserved group and 2 / 8 cases (25.0%) in the control group. In the test group, adhesion of the capsule to the liver or capsule masses in the abdominal cavity were not observed.

[0059] DTZ staining of recovered encapsulated islets (islet purity) DTZ-positive cells were confirmed in the encapsulated islets recovered one month after transplantation in all groups (Figure 3). The positive rates were 96.9% in the non-preserved group, 91.5% in the control group, and 95.1% in the test group, with no significant differences between the groups.

[0060] Porcine-derived C-peptide was detected in the plasma of all mice one month after transplantation. The mean values ​​for each group were 165.44 pmol / L in the non-preserved group, 178.72 pmol / L in the control group, and 174.75 pmol / L in the test group, with no significant differences between the groups.

[0061] 7-8. Insulin secretion and glucose responsiveness (SGS assay) of recovered encapsulated islets. Encapsulated islets recovered one month after transplantation from all individuals demonstrated glucose responsiveness (Figure 4). In Figure 4, "L1" and "L2" represent insulin secretion levels when placed in low glucose (500 mg / L), "H" represents insulin secretion levels when placed in high glucose (5000 mg / L), "SI1" represents the stimulation index of "H / L1," and "SI2" represents the stimulation index of "H / L2." In Figure 4, "11-18" represents the non-preserved group, "21-28" represents the control group, and "31-37" represents the test group.

[0062] These results demonstrate that the test solution is not only useful as a preservation solution for encapsulated islets, but also that its use as a transplant solution can prevent the adhesion of encapsulated islets to tissues after transplantation. The ability to use a solution with the same composition as the transplant and preservation solution makes it possible to omit the step of transferring preserved encapsulated islets to a transplant solution before transplantation, which not only improves work efficiency but also prevents contamination during solution exchange, dramatically improving the ease of handling of encapsulated islets.

Claims

1. A solution for preserving or transplanting encapsulated pancreatic islets, comprising trehalose or its derivatives or salts thereof.

2. The liquid according to claim 1, which is an isotonic liquid.

3. The solution according to claim 1 or 2, wherein the isotonic solution is Ringer's lactate solution.

4. The liquid according to claim 1 or 2, wherein the concentration of trehalose or its derivative or a salt thereof is 2.0 to 6.0 (w / v)%.

5. The liquid according to claim 1 or 2, used to prevent tissue adhesion of encapsulated pancreatic islets.

6. A method for preserving encapsulated pancreatic islets, comprising the step of preserving encapsulated pancreatic islets in the liquid described in claim 1 or 2.