Liquid for preservation or transplantation of encapsulated pancreatic islet

Abstract

The present invention provides a means for performing transplantation of encapsulated pancreas islets more efficiently. A liquid for preservation or transplantation of encapsulated pancreatic islets, which contains trehalose, a derivative thereof, or a salt of the trehalose or the derivative.

Description

Technical Field

[0001] Disclosed are a liquid for storage or transplantation of encapsulated pancreatic islets and a technology for use thereof. Background Art

[0002] Diabetes is a disease caused by impaired function of the pancreas that controls blood sugar. There are two main types: type I diabetes and type II diabetes. Type I diabetes develops at a younger age and causes symptoms of strong damage to insulin-producing cells due to autoimmune reactions. In contrast, type II diabetes develops in middle-aged and elderly people and is caused by the inability to properly utilize insulin. In Japan, 5% of diabetic patients have type I diabetes, which is estimated to be about 300,000 people. Most of these diabetic patients control their blood sugar through insulin therapy, but they cannot avoid complications such as nephropathy, retinopathy, and neurological disorders. In addition, even with insulin therapy, there are still many cases where blood sugar cannot be controlled.

[0003] As a diabetes treatment method that replaces insulin therapy, there is pancreatic islet transplantation. Pancreatic islets (also called Langerhans islets) are endocrine cell groups that are dispersed in the pancreatic tissue in an island shape and contain insulin-secreting β cells. Pancreatic islet transplantation is a transplantation therapy in which pancreatic islets are isolated from the pancreas provided by a donor, a dispersion of the islets is prepared, and the dispersion is administered percutaneously to the patient.

[0004] Islet transplantation has the following advantages. (1) Since it can be administered percutaneously, there is no need for laparotomy or general anesthesia, and the burden on the patient during the transplantation operation is small. (2) As for the amount of immunosuppressant administered after transplantation, a smaller amount is sufficient compared to pancreatic transplantation. (3) Even the pancreas of organ donors who are not suitable for pancreatic transplantation, such as those who died of cardiac arrest or arteriosclerosis, can be used. In addition, as a means to further reduce the amount of immunosuppressant administered after transplantation, encapsulated islets are known that are encapsulated in tiny microcapsules (Patent Documents 1 and 2).

[0005] Most of the β cells that make up the pancreatic islets will inactivate over time after being isolated and purified from the pancreatic islets. Therefore, it is desirable to appropriately store the prepared pancreatic islets and provide them for transplantation in a short period of time so as not to lose their vitality as much as possible. However, it takes a certain amount of time (e.g., about 10 to 18 hours) from the operation of removing the pancreas from the donor to the preparation of the pancreatic islets for transplantation. In addition, even if the preparations for pancreatic islet transplantation are ready, sometimes the transplantation operation cannot be performed immediately due to reasons such as the patient's poor physical condition.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: WO2021 / 256451

[0009] Patent document 2: WO2021 / 153365 Summary of the invention

[0010] Problems to be solved by the invention

[0011] One object of the present invention is to provide means for more efficiently performing encapsulated islet transplantation.

[0012] Means for solving problems

[0013] The inventors of the present application have conducted intensive studies to solve the above problems and have found that a common solution can be used for the storage and transplantation of encapsulated pancreatic islets. Based on this finding, the inventors have conducted further studies and provided the following representative inventions.

[0014] Item 1

[0015] A liquid for storage or transplantation of encapsulated pancreatic islets, comprising trehalose or a derivative thereof or a salt thereof.

[0016] Item 2

[0017] The liquid as described in item 1, which is an isotonic liquid.

[0018] Item 3

[0019] The liquid according to Item 2, wherein the isotonic liquid is lactated Ringer's solution.

[0020] Item 4

[0021] The liquid 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) %.

[0022] Item 5

[0023] The liquid according to any one of items 1 to 4, which is used for preventing tissue adhesion of encapsulated pancreatic islets.

[0024] Item 6

[0025] A method for preserving encapsulated pancreatic islets, comprising the step of preserving the encapsulated pancreatic islets in the liquid according to any one of items 1 to 5.

[0026] Item 7

[0027] The liquid according to any one of items 1 to 5, further comprising encapsulated pancreatic islets.

[0028] Item 8

[0029] A method for treating diabetes, comprising administering the liquid according to item 7 to a subject in need of pancreatic islet transplantation.

[0030] Effects of the Invention

[0031] When encapsulated islets are transplanted, the encapsulated islets can be transplanted without transferring the stored encapsulated islets to a transplantation liquid different from the storage liquid. Thus, the encapsulated islet transplantation can be performed more effectively (or the time required for encapsulated islet transplantation can be shortened). In addition, the function of inhibiting adhesion of the encapsulated islets to tissues after transplantation is also excellent. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [ Figure 1 ] shows the changes in body weight (average value) measured for the unpreserved group, control group, and test group.

[0033] [ Figure 2 ] shows the changes in blood sugar levels (average value) when full when measured for the unpreserved group, control group, and experimental group.

[0034] [ Figure 3 ] shows DTZ-stained photographs of encapsulated islets recovered from the unpreserved group, control group, and experimental group.

[0035] [ Figure 4 ] show the insulin secretion and glucose responsiveness measured for encapsulated islets recovered from the unpreserved group, the control group, and the experimental group. DETAILED DESCRIPTION

[0036] 1. Liquid for storage or transplantation

[0037] The liquid for storage or transplantation of encapsulated islets preferably contains trehalose or its derivatives or salts thereof (sometimes collectively referred to as "trehaloses"). As trehalose, there can be mentioned α,α-trehalose, a disaccharide formed by two α-glucoses bonded by 1,1-glycosidic bonds, α,β-trehalose, a disaccharide formed by α-glucose and β-glucose bonded by 1,1-glycosidic bonds, and β,β-trehalose, a disaccharide formed by two β-glucoses bonded by 1,1-glycosidic bonds. Among these, α,α-trehalose is preferred. These trehaloses can be manufactured by any known method such as chemical synthesis, microbial-based production, enzyme-based production, etc., and commercial products can also be used. For example, commercial products such as α,α-trehalose (manufactured by Hayashibara Co., Ltd. or Fujifilm Wako Pure Chemical Industries, Ltd.) can be mentioned.

[0038] The derivative of trehalose is not particularly limited, and examples thereof include glycosylated trehaloses in which one or more sugar units are bonded to trehalose of the disaccharide. Glycosylated trehaloses include glucosyl trehalose, maltosyl trehalose, maltotriosyl trehalose, and the like.

[0039] The salt of trehalose or its derivatives is not particularly limited as long as it is a pharmaceutically acceptable substance. For example, hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate and other acid addition salts, metal salts such as sodium salt, potassium salt, calcium salt, ammonium salt, alkylammonium salt, etc. can be cited. These salts are preferably used as solutions when used, and the effect is the same as that of using trehalose. These salts can form hydrates or solvates. In addition, two or more can be appropriately combined and used. The liquid for preservation or transplantation can contain only one trehalose, or it can be combined to contain two or more trehalose.

[0040] The concentration of trehalose in the storage or transplantation liquid is not particularly limited. 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, further preferably 1.0 (w / v)% or more, and most preferably 2.0 (w / v)% or more, in terms of trehalose conversion. In addition, the upper limit is, for example, 40 (w / v)% or less, preferably 20 (w / v)% or less, more preferably 15 (w / v)% or less, further preferably 10% (w / v)% or less, and most preferably 6.0 (w / v)% or less in terms of trehalose conversion. The values ​​of these lower and upper limits can be arbitrarily combined. For example, the concentration of trehalose in the liquid for storage or transplantation is in the range of 0.1 to 40 (w / v)% in terms of trehalose, preferably 0.3 to 20 (w / v)%, more preferably 0.6 to 15 (w / v)%, further preferably 1.0 to 10% (w / v)%, and most preferably 2.0 to 6.0 (w / v)%.

[0041] The liquid for preservation or transplantation can be any liquid (e.g., isotonic solution, hypotonic solution, hypertonic solution) that can be used for transplantation and preservation of encapsulated islets, preferably isotonic solution. Here, "isotonic solution" refers to a liquid having an osmotic pressure substantially the same as that of body fluids or cell fluids, specifically, a liquid having an osmotic pressure in the range of 250 to 380 mOsm / L. "Hypotonic solution" refers to a liquid having an osmotic pressure lower than that of body fluids or cell fluids, specifically, a liquid having an osmotic pressure less than 250 mOsm / L. As a hypotonic solution, a hypotonic solution to a degree that does not cause cell rupture (specifically, a liquid having an osmotic pressure in the range of 100 or more and less than 250 mOsm / L) is preferred. “Hypertonic fluid” refers to a fluid having an osmotic pressure higher than that of body fluids or cell fluids, and specifically refers to a fluid having an osmotic pressure greater than 380 mOsm / L (preferably within a range of greater than 380 mOsm / L and less than 1000 mOsm / L).

[0042] Isotonic solution is not particularly limited as long as it is an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted by sodium ions, potassium ions, and / or calcium ions in a manner that is roughly the same as the osmotic pressure of body fluids and cell fluids. Specifically, physiological saline, physiological saline with a buffering effect (e.g., PBS, Tris buffered saline [Tris Buffered Saline; TBS], HEPES buffered saline), Ringer's solution, lactated Ringer's solution, acetic acid Ringer's solution, bicarbonate Ringer's solution, 5% glucose aqueous solution, basal medium for animal cell culture (e.g., DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199), liquid prepared by isotonic agents (e.g., glucose, D-sorbitol, D-mannitol, lactose, sodium chloride), etc. Among these, lactated Ringer's solution is preferred. Isotonic solution can be a commercially available isotonic solution or a homemade isotonic solution. Examples of commercially available isotonic solutions include Otsuka Normal Saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) (physiological saline solution), Ringer's solution "Otsuka" (manufactured by Otsuka Pharmaceutical Co., Ltd.) (Ringer's solution), Lactec (registered trademark) injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) (lactic Ringer's solution), Veen (registered trademark) F injection (manufactured by Fuso Pharmaceutical Co., Ltd.) (acetic Ringer's solution), Otsuka Glucose Injection 5% (manufactured by Otsuka Pharmaceutical Co., Ltd.) (5% glucose aqueous solution), and Bicante (registered trademark) injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) (bicarbonate Ringer's solution).

[0043] The pH of the liquid for storage or transplantation is not particularly limited as long as it is suitable for transplantation and storage of encapsulated pancreatic islets, and may be, for example, in the range of 6.5 to 8.5. For example, the pH may be 6.5 to 8.4; 6.5 to 8.3; 6.5 to 8.2; 6.5 to 8.1; 6.5 to 8.0; 6.5 to 7.9; 6.5 to 7.8; 6.5 to 7.7; 6.5 to 7.6; 6.5 to 7.5; 6.5 to 7.4; 6.5 to 7.3; 6.5 to 7.2; 6.5 to 7.1; 6.5 to 7.0; 6.5 to 6.9; 6.5 to 6.8; 6.6 to 8.5; 6.7 to 8.5; 6.8 to 8.5; 6.9 to 8.5; 7.0 to 8.5; 7.1 to 8.5; 7.2 to 8.5; 7.3 to 8.5; 7.4 to 8.5; 7.5 to 8.5; 7.6 to 8.5; 7.7 to 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; 7.5~8. 3; 7.5~8.2; 7.5~8.1; 7.5~8.0; 7.5~7.9; 7.5~7.8; 7.6~8.4; 7.6~8.3; 7.6~8.2; 7.6~8.1; 7.6~8.0; 7.6~7.9; 7.7~8.4; 7.7~8.3; 7.7~8.2; 7.7~8.1; 7.7~8.0; 7.8~8.4; 7.8~8.3; 7.8~8.2; 7.8~8.1; 7.9~8.4; 7.9~8.3; 7.9~8.2; 8.0~8.4; 8.0~8.3; 8.1~8.3; etc. .

[0044] In one embodiment, the storage or transplantation liquid preferably does not contain substances that are not suitable for mammalian cell transplantation. Examples of substances that are not suitable for mammalian cell transplantation include components from organisms (e.g., serum or components from serum (e.g., albumin)), dimethyl sulfoxide [DMSO], glycerol, ethylene glycol, propylene glycol, dimethylacetamide, polyethylene glycol [PEG], polyvinyl pyrrolidone, etc.

[0045] There is no particular restriction on the type of islets used for encapsulated islets transplanted (and preserved) using a preservation or transplantation liquid. The islets preferably contain insulin-producing β cells, glucagon-containing α cells, somatostatin-secreting δ cells, and pancreatic polypeptide-containing cells (PP cells). The islets are preferably mostly insulin-producing β cells. The source of the islets can be selected according to the purpose, preferably humans, pigs, mice, rats, monkeys or dogs. In one embodiment, the islets are preferably derived from pigs, preferably islets of piglets (e.g., 3 to 4 weeks after birth or 7 to 3 weeks after birth). The islets can be obtained by arbitrarily adopting methods known in the art.

[0046] The size of the islets is preferably 50 μm to 400 μm, more preferably 50 μm to 350 μm. The size of the islets can be measured using a micrometer of a microscope. As a characteristic, it is preferred that the islets contain 10% or more of β cells. The upper limit of the ratio of β cells is not particularly limited, and is, for example, 80%.

[0047] In one embodiment, the encapsulated islets are preferably encapsulated islets as described in Patent Document 1 or 2. In one embodiment, the encapsulated islets preferably have a structure in which a core is covered with three layers of membranes, the core contains the islets, the first and third layers of membranes from the inner side of the three layers of membranes contain alginate, and the second layer of membrane from the inner side contains polyornithine. In one embodiment, the encapsulated islets may be covered with more than three layers of membranes (e.g., four layers of membranes, five layers of membranes, six layers of membranes, seven layers of membranes, or eight layers of membranes).

[0048] The phrase "the membrane contains alginic acid" means that the membrane is mainly composed of alginic acid. The phrase "the membrane contains polyornithine" means that the membrane is mainly composed of polyornithine. The encapsulated pancreatic islets preferably contain 1 to 5 pancreatic islets in one capsule.

[0049] From the viewpoint of inhibiting fibrosis, the average diameter of the encapsulated islets is preferably 400 μm or more, more preferably 420 μm or more. The upper limit of the average diameter is not particularly limited, and 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 by the measurement method used in the examples described below.

[0050] Encapsulated pancreatic islets can be produced by the methods described in Patent Documents 1 and 2, etc.

[0051] With regard to the above-mentioned storage or transplantation liquid, the encapsulated islets can be directly used for transplantation without being transferred to other transplantation liquids. Therefore, in one embodiment, the storage liquid or transplantation liquid can be a storage liquid and a transplantation liquid for the islets. In addition, the storage or transplantation liquid has an excellent function of inhibiting adhesion of the encapsulated islets after transplantation to tissues (e.g., liver). Therefore, the storage or transplantation liquid can be used to prevent adhesion of the encapsulated islets to tissues.

[0052] When encapsulated pancreatic islets are stored using the storage or transplantation liquid of the present invention, the storage time and storage temperature are as described in "2. Storage method" described later.

[0053] 2. Storage method

[0054] Provided is a method for preserving encapsulated pancreatic islets, comprising the step of preserving the encapsulated pancreatic islets in the above-mentioned preservation solution or transplantation solution.

[0055] The storage time is arbitrary and not particularly limited. In one embodiment, the storage time can be 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 time can 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; 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 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 to 7 days; 20 hours ~6 days; 20 hours ~5 days; 20 hours ~4 days; 20 hours ~3 days; 20 hours ~2 days; 20 hours ~1 day; 22 hours ~12 days; 22 hours ~11 days; 22 hours ~10 days; 22 hours ~9 days; 22 hours ~8 days; 22 hours ~7 days; 22 hours ~6 days; 22 hours ~5 days; 22 hours ~4 days; 22 hours ~3 days; 22 hours ~2 days; 22 hours ~1 day; etc. ;

[0056] The storage temperature is arbitrary and not particularly limited. In one embodiment, the storage temperature may be 0 to 40°C. For example, the storage temperature may be 0 to 40°C; 0 to 38°C; 0 to 36°C; 0 to 34°C; 0 to 32°C; 0 to 30°C; 0 to 28°C; 0 to 26°C; 0 to 24°C; 0 to 22°C; 0 to 20°C; 0 to 18°C; 0 to 16°C; 0 to 14°C; 0 to 12°C; 0 to 10°C; 0 to 8°C; 0 to 6°C; 0 to 4°C; 0 to 2°C; 2 to 40°C; 2 to 38°C; 2 to 36°C; 2 to 34°C; 2 to 32°C; 2 to 30°C; 2 to 28°C; 2 to 26°C; 2 to 24°C; 2 to 22°C; 2 to 20°C; 2 to 18°C; 2 to 16°C; 2 to 14°C; 2 to 12°C; 2 to 10°C; 2 to 8°C; 2 to 6°C; 2 to 4°C; 4 to 4 0℃;4~38℃;4~36℃;4~34℃;4~32℃;4~30℃;4~28℃;4~26℃;4~24℃;4~22℃;4~20℃;4~18℃;4~16℃;4~14℃;4~12℃;4~10℃;4~8℃;4~6℃;6~40℃;6~38℃;6~36℃;6~34℃;6~32℃;6~30℃;6~28℃;6~26℃;6~24℃;6~22℃;6~20℃;6~18℃;6~16℃;6~14℃;6~12℃;6~10℃;6~8℃;8~40℃;8~38℃;8~36℃;8~34℃;8~32℃;8~30℃; 8~28℃;8~26℃;8~24℃;8~22℃;8~20℃;8~18℃;8~16℃;8~14℃;8~12℃;8~10℃;10~40℃;10~38℃;10~36℃;10~34℃;10~32℃;10~30℃;10~28℃;10~26℃;10~24℃;10~22℃;10~20℃;10~18℃;10~16℃;10~14℃;10~12℃;12~40℃;12~38℃;12~36℃;12~34℃;12~32℃;12~30℃;12~28℃;12~26℃;12~24℃;12~22℃;12~20 ℃;12~18℃;12~16℃;12~14℃;14~40℃;14~38℃;14~36℃;14~34℃;14~32℃;14~30℃;14~28℃;14~26℃;14~24℃;14~22℃;14~20℃;14~18℃;14~16℃;16~40℃;16~38℃;16~36℃;16~34℃;16~32℃;16~30℃;16~28℃;16~26℃;16~24℃;16~22℃;16~20℃;16~18℃;18~40℃;18~38℃;18~36℃;18~34℃;18~32℃;18~30℃;18~28℃;18~26℃;18~24℃;18~22℃;18~20℃;20~40℃;20~38℃;20~36℃;20~34℃;20~32℃;20~30℃;20~28℃;20~26℃;20~24℃;20~22℃;22~40℃;22~38℃;22~36℃;22~34℃;22~32℃;22~30℃;22~28℃;22~26℃;22~24℃;24~40℃;24~38℃;24~36℃;24~34℃;24~32℃;24~30℃;2 4~28℃;24~26℃;26~40℃;26~38℃;26~36℃;26~34℃;26~32℃;26~30℃;26~28℃;28~40℃;28~38℃;28~36℃;28~34℃;28~32℃;28~30℃;30~40℃;30~38℃;30~36℃;30~34℃;30~32℃;32~40℃;32~38℃;32~36℃;32~34℃;34~40℃;34~38℃;34~36℃;36~40℃;36~38℃;38~40℃;etc.;

[0057] In one embodiment, the encapsulated islets stored in a storage solution or a transplantation solution are preferably directly (not transferred to other liquids) for transplantation. By omitting the operation of transferring from the storage solution to other transplantation solutions, the encapsulated islets can be transplanted efficiently or the time required for transplantation can be shortened. In addition, by transplanting the encapsulated islets without transferring from the storage solution to other transplantation solutions, the effect of the transfer on the encapsulated islets can be avoided, and the function of the encapsulated islets for transplantation can be maintained in a better state.

[0058] The subject to which the encapsulated islets are transplanted is any subject as long as it is in need thereof. For example, the subject is a mammal, specifically, humans, dogs, cats, horses, cattle, and pigs, preferably humans. In addition, the subject to which the encapsulated islets are transplanted is preferably a subject suffering from diabetes, preferably a subject suffering from type 1 diabetes.

[0059] The site of transplantation of encapsulated islets is not particularly limited, and examples include subcutaneous, intramuscular, intraomental, and intraperitoneal transplantation, and is preferably transplanted into the peritoneal cavity. The number of islet cells per administration is preferably about 5.0×10 per kg of the subject's body weight. 6 ~1.2×10 8 / kg body weight, more preferably about 8.0×10 6 ~8.0×10 7 / kg body weight, more preferably about 1.2×10 7 ~4.0×10 7The number of islets administered per administration is preferably about 5,000 to 60,000 per kg of the subject's body weight, more preferably about 8,000 to 40,000 per kg of the subject's body weight, and even more preferably about 12,000 to 20,000 per kg of the subject's body weight.

[0060] Example

[0061] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0062] The usability of the encapsulated pancreatic islets as a storage solution or a transplantation solution was evaluated by the following test.

[0063] 1. Test fluid

[0064] As the test liquid, a liquid composition having the following table and properties was used.

[0065] [Table 1]

[0066] <![CDATA[Na + (mEq / L)]]> 130 <![CDATA[K + (mEq / L)]]> 4 <![CDATA[Ca 2+ ((mEq / L)]]> 3 <![CDATA[Cl - (mEq / L)]]> 109 <![CDATA[L-lactic acid - (mEq / L)]]> 28 Trehalose (w / v%) 3 pH 6.5-8.5 (about 7.4) Properties (hue and clarity) Colorless clear liquid

[0067] 2. Comparison object preservation solution

[0068] To 500 mL of RPMI-1640 (manufactured by Gibco), 50 mL of inactivated porcine serum (manufactured by Gibco) and 5 mL of a nicotinamide stock solution (1 M nicotinamide) were added and mixed to prepare a storage solution as a comparative object.

[0069] 3. Encapsulation of Pancreatic Islets

[0070] The islets isolated from the piglets were suspended in a sodium alginate solution, and a peristaltic pump was used to drip the alginate suspension into a 109 mM calcium chloride solution for encapsulation. The capsules were recovered, washed with physiological saline, and 0.10% poly-L-ornithine (PLO) solution was added and mixed on a roller mixer for 10 minutes. The supernatant was removed, washed with physiological saline, and 0.05% PLO solution was added and mixed on a roller mixer for 6 minutes. The supernatant was removed, washed with physiological saline, and a 10-fold diluted sodium alginate solution was added and mixed on a roller mixer for 6 minutes. The supernatant was removed, washed with physiological saline, and 1.15% sodium citrate solution was added and mixed on a roller mixer for 2 minutes. The supernatant was removed, washed with physiological saline, and the supernatant was further removed to obtain encapsulated islets. The obtained encapsulated islets were suspended in a culture medium prepared by adding porcine serum, nicotinamide, and antibiotics to RPMI-1640, and dispensed into T175 flasks, and then cultured in a CO2 incubator at 37°C for 26 or 30 days.

[0071] 4. Experimental Animals

[0072] The B6 mouse diabetes model prepared by STZ administration was used as the test animal. Mice with a blood glucose level of 300 mg / dL or more when full on the 2nd and 3rd days (transplantation day) after STZ administration and no abnormalities in general state by visual observation were used as the subjects, and stratified random grouping was implemented using the statistical analysis system EXSUS10.0 (CAC EXICARE Co., Ltd.) based on the body weight and blood glucose level on the 3rd day.

[0073] 5. Experimental structure

[0074] The test was structured as shown in the following table.

[0075] [Table 2]

[0076]

[0077] In the unpreserved group, STZ was administered to the test animals at a dose of 200 mg / kg, and the encapsulated piglet islets cultured to day 30 were suspended in physiological saline solution without being preserved in the test solution or the comparative object preservation solution, and 8,000 IEQ (Islet Equivalent: 1 IEQ is 1 islet with a diameter of 150 μm) were transplanted into the abdominal cavity. Encapsulated piglet islets were prepared according to the following steps. Based on the IEQ counting results on the day of transplantation, the culture flask was removed from the incubator just before transplantation, and each IEQ amount was dispensed into a 50 mL tube. Let it stand at room temperature until the washing operation. Remove the culture medium in the tube with a pipette, add about 10 mL of HBSS (Gibco) at room temperature, let it stand, and remove the supernatant. After repeating these operations 3 times, add about 10 mL of physiological saline solution at room temperature, suspend and let it stand, remove the supernatant until about 4 mL, and quickly provide it for transplantation.

[0078] In the control group, encapsulated piglet islets cultured to day 26 were stored in the comparative storage solution described in the aforementioned "2. Comparative storage solution" at 4°C for 96 hours (Fukushima Galilei, FMS-702G), then suspended in physiological saline solution and 8,000 IEQ were transplanted into the abdominal cavity. Encapsulated piglet islets were prepared according to the following steps. Based on the IEQ count results on the day of storage start, the storage container was taken out of the refrigerator just before transplantation, and each IEQ amount was dispensed into a 50 mL tube. Let it stand in an ice bath until the washing operation. Remove the storage solution in the tube with a pipette, add about 10 mL of HBSS (Gibco) at room temperature, let it stand, and then remove the supernatant. After repeating these operations three times, add about 10 mL of physiological saline solution at room temperature, suspend and let it stand, remove the supernatant until about 4 mL, and quickly provide it for transplantation.

[0079] In the experimental group, the encapsulated piglet islets cultured to day 26 were stored in the test solution described in the aforementioned "1. Test solution" at 4°C for 96 hours (Fukushima Galilei, FMS-702G), and then 8,000 IEQ were directly transplanted into the abdominal cavity. Encapsulated piglet islets were prepared according to the following steps. Based on the IEQ count results on the day of storage start, the storage container was taken out of the refrigerator just before transplantation, and each IEQ amount was dispensed into a 50 mL tube. Let stand in an ice bath until the cleaning operation. Use a pipette to remove the storage solution in the tube until about 4 mL, and quickly provide it for transplantation.

[0080] In any group, the transplantation of encapsulated islets was performed according to the following steps. Anesthesia was induced and maintained in mice using an isoflurane vapor anesthesia machine, and the abdomen was disinfected using 70% ethanol spray. Puncture was performed through a 16G indwelling needle (Surflo), and a prescribed amount of encapsulated islet solution was administered into the abdominal cavity. The skin at the puncture site was closed with a surgical adhesive (AronAlpha A "Sankyo", Daiichi Sankyo Co., Ltd.). It should be noted that since the body temperature of the mice would drop after the operation, they were kept warm with a heater until they woke up. The transplantation day was set as day 0.

[0081] 6. Observation, measurement, and inspection

[0082] 6-1. Observation of general condition

[0083] Observation of the general condition including confirmation of life and death is performed daily.

[0084] 6-2. Body weight measurement

[0085] Body weight was measured on the day of STZ administration, the day of transplantation, twice a week thereafter, and on the day of oral glucose tolerance test (OGTT).

[0086] 6-3. Blood sugar level measurement

[0087] On the day of STZ administration (immediately before administration), the second day after administration, the day of transplantation, and twice a week thereafter, mice were restrained with a restrainer, and blood (several μL) was collected by puncturing the tail vein with a 27G injection needle to measure blood glucose levels. Nipro StatStrip (NIPRO) was used for measurement. Blood glucose levels were measured in the morning.

[0088] 6-4. Oral glucose tolerance test (OGTT)

[0089] The blood glucose level was measured on the day after the 4th week of transplantation. 10% glucose solution (Otsuka Pharmaceutical) was orally administered to mice that had fasted for about 16 hours from the previous day at 10 μL / g (glucose 1 g / kg) by gavage to evaluate the change in blood glucose level. The blood glucose level was measured at 0, 15, 30, 45, 60, 75, 90, 105, and 120 minutes before and after administration.

[0090] 6-5. Autopsy

[0091] After the OGTT, the abdomen was opened under isoflurane anesthesia, and abnormalities were recorded if observed with the naked eye. Blood was collected from the posterior vena cava using a syringe with a 26G needle. The blood was placed in a Microtainer blood collection tube (BD, EDTA-2K), gently inverted to mix, and then stored in an ice bath. The plasma was separated by centrifugation at 4°C and 3000×g for 10 minutes, placed in another microcentrifuge tube, and stored below -30°C as a sample for porcine C-peptide determination.

[0092] 6-6. DTZ staining of recovered encapsulated islets

[0093] DTZ solution was added to the recovered encapsulated islet suspension, and the DTZ staining was observed with an inverted microscope (CKX41, Olympus), and the image was taken. DTZ solution was prepared by the following method: 5 mL DMSO (Sigma) and 45 mL HBSS were added to 100 mg dithizone (Fujifilm Wako Pure Chemical Industries, Ltd.), and after fully dissolving, sterilized with a 0.22 μm filter. For the spheroids in the visual field of the obtained image, the spheroids with the part stained with dithizone and the spheroids without the part stained with dithizone were counted respectively, and the purity was calculated according to the following calculation formula: "Purity (%) = (the number of spheroids with the part confirmed to be stained with dithizone) / (the total number of spheroids) × 100".

[0094] 6-7. Determination of porcine C-peptide

[0095] The stored plasma was thawed at room temperature, and the porcine C-peptide concentration was measured using Mercodia Porcine C-peptide ELISA Kit (Mercodia, Product No.: 10-1256-01).

[0096] 6-8.SGS analysis

[0097] The recovered capsules were subjected to SGS analysis. Using Mercodia porcine insulin ELISA, the insulin secretion was determined when the capsules were allowed to stand for 1 hour in RPMI 1640 containing 500 mg / L glucose (low glucose) or 5000 mg / L glucose (high glucose). The measurement was performed according to the instructions of the kit, and the unit of calculation was 1 mg = 115 U. According to the SGS analysis, the SI (stimulation index) was determined as the ratio of the insulin secretion in low glucose to that in high glucose.

[0098] 6-9. Statistical analysis

[0099] The mean value and standard deviation of the obtained data were calculated and graphed as needed. Statistical analysis was performed using EXSUS10.0 for Student's t-test. p<0.05 was considered to be significantly different, and p<0.1 was considered to be a tendency toward significance.

[0100] 7. Results

[0101] 7-1. General status

[0102] After encapsulated islet transplantation, animal No. 15 (unpreserved group) and animal No. 38 (experimental group) showed behavioral inhibition and were euthanized. Other animals were not euthanized because behavioral inhibition due to diabetes and weight loss of more than 20% compared to transplantation were not observed.

[0103] 7-2.Weight

[0104] From capsule implantation to the day of autopsy, body weight increased. No significant difference was found in mean body weight between the groups ( Figure 1 ).

[0105] 7-3. Blood sugar level when full

[0106] In terms of satiation blood sugar levels, all groups showed a decrease after capsule transplantation. Thirteen days after transplantation, the control group showed a trend of increasing blood sugar levels but then decreasing again, and no significant difference was found between the groups. Figure 2 ).

[0107] 7-4.OGTT

[0108] Although poor glucose responsiveness was observed in some individuals, there was no significant difference in any of the groups, indicating glucose responsiveness.

[0109] 7-5. Autopsy

[0110] In autopsies one month after transplantation, adhesion of the capsule to the liver or capsule masses in the abdominal cavity were found in 1 / 7 cases (14.3%) of the non-preserved group and 2 / 8 cases (25.0%) of the control group. No adhesion of the capsule to the liver or capsule masses in the abdominal cavity were found in the experimental group.

[0111] 7-6. DTZ staining of recovered encapsulated islets (islet purity)

[0112] In the encapsulated islets recovered one month after transplantation, DTZ-positive cells were confirmed in all groups ( Figure 3 ). As for the positive rate, the non-preserved group was 96.9%, the control group was 91.5%, and the test group was 95.1%, and no difference was found among the groups.

[0113] 7-7. Porcine C-peptide

[0114] Porcine C-peptide was detected in mouse plasma in all individuals 1 month after transplantation. The average values ​​of 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 difference observed between the groups.

[0115] 7-8. Insulin secretion and glucose responsiveness of recovered encapsulated islets (SGS analysis)

[0116] Encapsulated islets from any individual recovered 1 month after transplantation showed glucose responsiveness ( Figure 4 ). It should be noted that Figure 4 "L1" and "L2" in the table represent the amount of insulin secreted when left at rest in low glucose (500 mg / L), "H" represents the amount of insulin secreted when left at rest in high glucose (5000 mg / L), "SI1" represents the stimulation index of "H / L1", and "SI2" represents the stimulation index of "H / L2". Figure 4 "11-18" in the table indicates the unsaved group, "21-28" indicates the subject group, and "31-37" indicates the experimental group.

[0117] The above results show that the test solution is not only useful as a storage solution for encapsulated islets, but also can inhibit the adhesion of encapsulated islets to tissues after transplantation by using the test solution as a transplantation solution. The use of liquids with the same composition as the transplantation solution and the storage solution can not only omit the operation of transferring the encapsulated islets to the transplantation solution before transplantation and improve the operation efficiency, but also inhibit contamination during the liquid change, thereby greatly improving the operability of encapsulated islets.

Claims

1. A liquid for storage or transplantation of encapsulated pancreatic islets, comprising trehalose or a derivative thereof or a salt thereof.

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

3. The liquid according to claim 1 or 2, wherein The isotonic solution was lactated Ringer's solution.

4. The liquid according to any one of claims 1 to 3, wherein The concentration of trehalose or its derivatives or salts thereof is 2.0 to 6.0 (w / v) %.

5. The liquid according to any one of claims 1 to 4, which is used for preventing tissue adhesion of encapsulated pancreatic islets.

6. A method for storing encapsulated pancreatic islets, comprising the step of storing the encapsulated pancreatic islets in the liquid according to any one of claims 1 to 5.

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