High concentration cell preservation liquid and high concentration cell preservation method

Abstract

Provided are a novel high-concentration cell preservation liquid and a high-concentration cell preservation method.　The cell preservation liquid is for preserving cells in a non-frozen state. The cell preservation liquid contains a basal medium and an additive added to the basal medium. The basal medium is a culture medium used for cell culture. The additive contains at least genetically modified albumin and ascorbic acid (ascorbic acid derivative). The cell preservation liquid stores the cells at a cell concentration of at least 1 × 107 cells / mL. For example, when cells are preserved in a cell preservation liquid at 4°C at a cell concentration of 1 × 108 cells / mL, the cell survival rate when 72 hours has elapsed is 80% or more.

Description

High-concentration cell preservation solution and high-concentration cell preservation method

[0001] The present invention relates to a high-concentration cell preservation solution and a high-concentration cell preservation method, and more particularly to a high-concentration cell preservation solution and a high-concentration cell preservation method for preserving cells in an unfrozen state.

[0002] In recent years, regenerative medicine using cells collected from patients has been used. When directly administering cell medicines (cell preparations) to patients, it is preferable to use cell preparations prepared at a high cell concentration to reduce the burden on the patient. However, storing cells at a high cell concentration poses the problem of reduced cell viability. Therefore, current cell therapies require that highly concentrated cell preparations be administered to patients as soon as possible after preparation, and require treatment with cell preparations at university hospitals with advanced cell processing facilities.

[0003] Patent Document 1 discloses an invention of a liquid composition for preserving cells in an unfrozen state, but the liquid composition is not intended to preserve cells at a high cell concentration. Patent Document 2 discloses an invention of a cryopreservation solution for preserving stem cells in a frozen state, for example, when the stem cells are frozen at 1×10 ４ ~1 x 10 ８ It is disclosed that the cells are stored at a cell concentration of 1000 cells / mL. However, since this is a cell preservation solution that stores cells in a frozen state, a separate thawing operation is required, and the survival rate of viable cells decreases during freezing and thawing. Therefore, it is preferable to store cells in an unfrozen state.

[0004] International Publication No. 2019 / 49985 Japanese Patent Application Laid-Open No. 2019-154329

[0005] For example, if there were a cell preservation solution capable of preserving cells at a high cell concentration and maintaining a high viability, cell therapy would become possible not only at university hospitals with cell processing facilities, but also at medical institutions far from cell processing facilities. When introducing unfrozen cell medicines (cell preparations) to the market, a cell preservation solution capable of preserving cells for 72 hours or more is preferable, taking into consideration transportation conditions and patient circumstances. Furthermore, the cell preservation solution described above can be used not only for cell therapy purposes, but also for a wide range of other purposes, such as as a research reagent, making it a valuable tool.

[0006] An object of the present invention is to provide a novel high-concentration cell preservation solution and a high-concentration cell preservation method. Another object of the present invention is to provide a high-concentration cell preservation solution and a high-concentration cell preservation method that can preserve cells at a high cell concentration without freezing and maintain a high cell viability.

[0007] As a result of intensive research, the present inventors have found that it is possible to produce a cell preservation solution that allows cells to be preserved at a high cell concentration without freezing and maintain a high viability, and have thus completed the present invention. Specifically, as a result of intensive research into a suitable combination of a basal medium and additives to be added to the basal medium, it has been found that a cell preservation solution can be produced that allows cells to be preserved at a high cell concentration without freezing and maintain a high viability, even when the cells are not frozen, and maintains a high viability. ７ It was revealed that it is possible to produce a cell preservation solution that can preserve cells at a cell concentration of 1 × 10 cells / mL. ８ It was revealed that when stored at 4°C at a cell concentration of 1000 cells / mL, the cell viability after 72 hours was 80% or more. It was also revealed that the cells can be manufactured using pharmaceutically implantable components.

[0008] Therefore, the above-mentioned problem is solved by the high-concentration cell preservation solution of the present invention, which is a cell preservation solution for preserving cells in an unfrozen state, comprising a basal medium and an additive added to the basal medium, the basal medium being a medium used for cell culture, the additive containing at least recombinant albumin and ascorbic acid or an ascorbic acid derivative, and the cells being at least 1 × 10 ７The above-described configuration allows cells to be preserved at a high cell concentration without freezing, and realizes a high-concentration cell preservation solution and a high-concentration cell preservation method that can maintain a high cell viability.

[0009] In this case, the cells are preferably mammalian-derived cells, the recombinant albumin is preferably recombinant human serum albumin, and the cell preservation solution is preferably used for cell transplantation. ８ ~1 x 10 ９ The cells are preferably stored at a cell concentration of 1×10 cells / mL. The cells are preferably human-derived somatic cells or human-derived stem cells, and the basal medium is preferably at least one medium selected from the group consisting of RPMI1640 and DMEM. The recombinant albumin has a concentration of 0.1 to 2.0 (w / v)%. The ascorbic acid or ascorbic acid derivative has a concentration of 0.1 to 10 mg / mL. The cells are preferably stored at a cell concentration of 1×10 ８ When stored at 4°C at a cell concentration of 10 ...

[0010] Furthermore, the above-mentioned problem is solved by the high-concentration cell preservation method of the present invention, which involves suspending cells in a cell preservation solution and preserving the cells in an unfrozen state to a concentration of at least 1 × 10 ７ The above-mentioned problem can also be solved by a method for preserving cells at a cell concentration of 1000 cells / mL, wherein the cell preservation solution contains a basal medium and an additive added to the basal medium, the basal medium being a medium used for cell culture, and the additive containing at least recombinant albumin and ascorbic acid or an ascorbic acid derivative.

[0011] According to the high-concentration cell preservation solution and high-concentration cell preservation method of the present invention, cells can be preserved at a high cell concentration without freezing, and can be maintained at a high viability.

[0012] 1 is a graph showing the change over time in cell viability (cell survival rate) in Test Example 1. FIG. 2 is a graph showing the results of cell viability evaluation in Test Example 2. FIG. 3 is a graph showing the results of cytotoxicity evaluation in Test Example 3. FIG. 4 is a graph showing the change over time in cell viability in Test Example 4-1. FIG. 5 is a graph showing the change over time in cell viability in Test Example 4-2. FIG. 6 is a graph showing the change over time in cell viability in Test Example 5. FIG. 7 is a graph showing the change over time in cell viability in Test Example 6. FIG. 8 is a graph showing the change over time in cell viability in Test Example 7. FIG. 9 is a graph showing the change over time in cell viability in Test Example 8.

[0013] Hereinafter, embodiments of the present invention will be described with reference to Figures 1 to 9. The present embodiments relate to a "high-concentration cell preservation solution" that enables cells to be preserved at a high cell concentration in an unfrozen state and to maintain a high viability. The present embodiments also relate to a "high-concentration cell preservation method."

[0014] <Overview of Cell Preservation Solution> A "cell preservation solution" is a liquid composition for preserving cells, and is used to preserve cells for cell therapy (gene therapy). In this embodiment, it is a liquid composition for preserving cells in an unfrozen state (non-frozen state). The "non-frozen state" refers to a state at 1 to 20°C, preferably 1 to 15°C, more preferably 1 to 10°C, more preferably 1 to 5°C, and more preferably 4°C. Note that the cell preservation solution of this embodiment is not limited to use as a cell preparation, and can be used for a wide range of purposes such as a research reagent.

[0015] A "high-concentration cell preservation solution" is a cell preservation solution that preserves cells at a high cell concentration. For example, a cell preservation solution that preserves cells at a high cell concentration of at least 1 x 10 ７ cells / mL (1×10 ７ cells / mL or more), preferably 5 x 10 ７ cells / mL or more, more preferably 1 x 10 ８ Cells should be stored at a cell concentration of 1 x 10 cells / mL or more.７ ~1 x 10 ９ cells / mL, more preferably 5 x 10 ７ ~1 x 10 ９ cells / mL, more preferably 1 x 10 ８ ~1 x 10 ９ cells / mL, more preferably 1 x 10 ８ The cells are stored at a cell concentration of 1 x 10 cells / mL. ７ cells / mL or more (preferably 1 x 10 ８ By storing (non-cryopreserving) cells at a cell concentration of 1 × 10 cells / mL or more, the transplant volume can be reduced when directly transplanting a cell preparation (non-frozen cell preparation) into a patient, thereby reducing the burden on the patient. ９ By storing the cells at a cell concentration of 1000 cells / mL or less, a high cell viability can be maintained.

[0016] "High cell viability" means that the cells were 1×10 ７ ~1 x 10 ９ cells / mL (preferably 1 x 10 ８When stored at 4°C at a cell concentration of 1000kJ / mL (1000kcal / mL), the cell viability after 72 hours is preferably 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Alternatively, when stored at 4°C at the above cell concentration, the cell viability after 96 hours is preferably 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Alternatively, when stored at 4°C at the above cell concentration, the cell viability after 120 hours is preferably 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Alternatively, when stored at 4°C at the above cell concentration, the cell viability after 170 hours is preferably 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Alternatively, the cell viability after 144 hours and 168 hours is preferably the same as above. The "cell viability" may be calculated using the method used in Test Example 1 described below. When the cell preparation is introduced to the market in an unfrozen state, it is desirable that the cell preservation solution be capable of preserving cells for 72 hours or more, with the aim of transporting it to medical institutions nationwide. It is also desirable that the solution be composed of components that can be directly transplanted into patients.

[0017] The "cells" preserved in the cell preservation solution are not particularly limited, and various types of cells can be used. Examples include somatic cells and stem cells. "Somatic cells" include fibroblasts, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, keratinocytes, adipocytes, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nerve cells (neurons), glial cells (oligodendrocytes, microglia, astrocytes), cardiac myocytes, muscle cells, pancreatic beta cells, melanocytes, and hematopoietic progenitor cells. Adipocytes are preferred, and preadipocytes (adipocytes that have acquired proliferative properties and can be harvested by primary culture), mammalian-derived preadipocytes, and human-derived preadipocytes are even more preferred. Adipocytes (preadipocytes) are suitable for cell therapy because they have no administration or age restrictions and a low risk of tumorigenesis. These somatic cells may have exogenous genes incorporated into their genomic DNA for the purpose of gene therapy. "Stem cells" include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Mesenchymal stem cells are preferred, and human-derived mesenchymal stem cells are more preferred. These stem cells may have exogenous genes incorporated into their genomic DNA for the purpose of gene therapy.

[0018] <Composition of cell preservation solution> The high-concentration cell preservation solution contains a basal medium and additives added to the basal medium. By mainly combining the basal medium and additives, it is possible to produce a cell preservation solution that can preserve cells at a high concentration while maintaining a high viability, even without freezing the cells.

[0019] (Basal medium) The "basal medium" is a medium used for cell culture and is preferably a liquid medium. In other words, buffered salt solutions such as conventional Ringer's solution (lactated Ringer's solution) or physiological saline are not used as the basal medium. The "basal medium" is preferably at least one culture medium selected from the group consisting of RPMI1640 (Roswell Park Memorial Institute 1640), DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimum Essential Medium), αMEM, EMEM, and F-12 (Nutrient Mixture F-12 Medium). Preferably, it is RPMI1640 or DMEM, and more preferably RPMI1640. By using these media as the basal medium, it has been confirmed that cells can be preserved at a high cell concentration for a specified period of time because they contain amino acids and vitamins necessary for cell survival. In particular, the use of RPMI 1640 and DMEM maintains an appropriate metabolic state, improving cell preservation. In particular, the use of RPMI 1640 further improves cell preservation by containing glutathione, which has antioxidant properties.

[0020] When RPMI 1640 is used as the basal medium, it is preferably RPMI 1640 (containing L-glutamine).When DMEM is used as the basal medium, it is preferably low-glucose DMEM.

[0021] The medium used for the cell culture may be a "buffered salt solution" containing essential components of the medium. Examples of essential components of the medium include "amino acids," "vitamins," and "other components." Examples of "buffered salt solutions" include Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, physiological saline, Hank's balanced salt solution (HBSS), and Dulbecco's phosphate buffer solution. Examples of "amino acids" include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-hydroxyproline, L-isoleucine, L-lysine, L-methionine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-phenylalanine, and L-leucine. "Vitamins" include ascorbic acid (ascorbic acid derivatives), inositol, biotin, pantothenic acid, choline, folic acid, niacin, pyridoxine, riboflavin, thiamine, vitamin B12, etc. "Other components" include sugars, reduced glutathione, sodium salts, potassium salts, inorganic salts other than sodium salts and potassium salts, antibiotics, serum, fatty acids, etc. The above "amino acids," "vitamins," and "other additives" may be added as appropriate as additives.

[0022] The basal medium is preferably prepared so that it contains 50 (v / v)% or more of the high-concentration cell preservation solution, preferably 70 (v / v)% or more, and more preferably 80 (v / v)% or more of the total volume of the high-concentration cell preservation solution.

[0023] The "additives" have the function of maintaining cell viability when added to the basal medium. The main "additives" that can be added to the basal medium are recombinant albumin and ascorbic acid.

[0024] (Recombinant Albumin) Albumin has antioxidant properties, inhibits cell aggregation, and transports nutrients in the basal medium to cells. It also inhibits cell adsorption to storage containers, preventing the cells from becoming difficult to use. Albumin can be extracted from blood, but contains many unwanted impurities. Recombinant albumin is albumin produced using genetic engineering technology and contains very little impurities or exogenous factors. The albumin added is preferably recombinant albumin, and more preferably recombinant human serum albumin (rHA). Recombinant albumin is albumin produced using genetic engineering technology from which xenogenic components have been removed. Instead of recombinant albumin, a recombinant protein may be added to the basal medium. In this case, a protein with a molecular weight of 20,000 to 100,000 (low molecular weight protein), such as albumin, is preferably used.

[0025] The recombinant albumin is preferably prepared so that it is contained in an amount of 0.01 to 20 (w / v)% of the total volume of the high-concentration cell preservation solution, preferably 0.01 to 10 (w / v)%, more preferably 0.01 to 5.0 (w / v)%, and even more preferably 0.1 to 2.0 (w / v)%. A concentration of less than 0.01 (w / v)% has been confirmed to result in a decrease in viability. Furthermore, a concentration of more than 20 (w / v)% increases the viscosity of the high-concentration cell preservation solution, significantly reducing operability.

[0026] (Ascorbic Acid, Ascorbic Acid Derivatives) Ascorbic acid has antioxidant properties and functions to maintain cell viability. Ascorbic acid derivatives also have antioxidant properties. Preferably, ascorbic acid is used. Ascorbic acid derivatives include sodium ascorbate, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl glucoside, glyceryl ascorbate, bisglyceryl ascorbate, L-ascorbyl palmitate, ascorbyl tetrahexyldecanoate, ascorbic acid diphosphate, and the like. It has been confirmed that adding these additives to a basal medium enables cells to be preserved at a high cell concentration for a predetermined period of time. In particular, adding both recombinant albumin and ascorbic acid enables cells to be preserved at a higher cell concentration for a predetermined period of time.

[0027] The high-concentration cell preservation solution may be prepared so that it contains 0.1 to 10 mg / mL of ascorbic acid (ascorbic acid derivative) relative to the total volume of the solution, preferably 0.1 to 5 mg / mL, more preferably 0.1 to 2 mg / mL, and even more preferably 0.1 to 1 mg / mL. It has been confirmed that when the ascorbic acid concentration is less than 0.1 mg / mL or more than 10 mg / mL, the cell viability decreases.

[0028] (Other Additives) Other "additives" that may be added to the basal medium as appropriate include sodium salts, potassium salts, inorganic salts other than sodium salts and potassium salts, amino acids, vitamins, antibiotics, serum, fatty acids, sugars, etc. Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-hydroxyproline, L-isoleucine, L-lysine, L-methionine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-phenylalanine, and L-leucine. Examples of vitamins include ascorbic acid, as described above, and biotin, pantothenic acid, choline, folic acid, niacin, pyridoxine, riboflavin, thiamine, and vitamin B12.

[0029] High-concentration cell suspensions obtained by suspending cells in a cell preservation solution (high-concentration cell preservation solution) may be stored in any volume of 0.05 mL or more. Storage is preferably 0.05 to 500 mL, more preferably 0.05 to 100 mL, and even more preferably 0.05 to 50 mL. There are no particular limitations on the storage containers used for storage in an unfrozen state, but general-purpose syringes, bags, tubes, vials, ampoules, and the like that can ensure these volumes may be used. Furthermore, it is preferable to store the cell preparation in these storage containers in a sterile manner. This allows the preparation to be used directly for cell transplantation even in medical institutions or research facilities that do not have advanced sterile facilities.

[0030] <High-concentration cell preservation method> The high-concentration cell preservation method involves suspending cells in a cell preservation solution containing the above-mentioned basal medium and the above-mentioned additives, and preserving the cells in an unfrozen state at least 1 × 10 ７ This is a method of preserving cells at a cell concentration of 1000 cells / mL.

[0031] <Method for producing a high-concentration cell suspension using a cell preservation solution> As a method for producing a high-concentration cell suspension, for example, when producing a "high-concentration cell suspension" for preserving "LCAT gene-introduced human preadipocytes," the following steps are mainly carried out. In this production method, the "high-concentration cell suspension" can be produced by carrying out the first step of preparing human preadipocytes into which the LCAT gene is introduced, the second step of producing LCAT gene-introduced human preadipocytes, the third step of preparing a cell preservation solution, and the fourth step of recovering, washing, and concentrating the cells, and storing the concentrated cells in a storage container. More details are as shown in the examples below.

[0032] Examples of the present invention are described in detail below. However, the present invention is not limited to these examples. Five types of cells (LCAT gene-transduced human preadipocytes, human preadipocytes, human adipose tissue-derived mesenchymal stem cells, FVIII gene-transduced human preadipocytes, and human bone marrow-derived mesenchymal stem cells) were stored at high concentrations at 4°C using the high-concentration cell preservation solution of the present invention, and the cell viability after a certain period of time was examined. For the above cell preservation solutions, the "basal medium" was RPMI 1640 medium or DMEM medium, and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). As a comparative example, a commercially available product mainly composed of Ringer's solution was used.

[0033] Example 1 A high-concentration cell preservation solution was prepared as in the following steps (1-1) to (1-4), and a high-concentration cell suspension was prepared by suspending "LCAT gene-transduced human preadipocytes" in the cell preservation solution and preserving it. Step (1-1) Preparation of LCAT gene-transduced human preadipocytes Adipose tissue (10 g) obtained from a healthy subject was dispensed into 50 mL centrifuge tubes in 1 g portions, and 3 mL of HBSS (Sigma-Aldrich) containing 2 mg / mL collagenase (Nordmark), 40 μg / mL gentamicin (Takada Pharmaceutical), and 4 μg / mL vancomycin (Pfizer) was added to each tube, followed by shaking at 37°C for 1 hour. Next, 10 mL of "DMEM / Nutrient Mixture F-12 Ham (DMEM-HAM / 20%FBS (GM-VCM), Sigma-Aldrich)" containing 20% ​​FBS (Nichirei Biosciences), 40 μg / mL gentamicin, and 4 μg / mL vancomycin was added and stirred, followed by centrifugation at 400 × g for 1 minute to remove the solution containing the precipitated fraction. Furthermore, 10 mL of DMEM-HAM / 20%FBS (GM-VCM) was added and stirred, followed by centrifugation at 400 × g for 1 minute to remove the solution containing the precipitated fraction. This series of operations was repeated twice to obtain a supernatant fraction containing human preadipocytes. The supernatant fraction containing human preadipocytes thus obtained was filtered through a 440 μm mesh, and then 10 mL of DMEM-HAM / 20% FBS (GM-VCM) was added to the original 50 mL centrifuge tube for washing, and the washings were also filtered. The filtrate thus obtained was transferred to a 150 cm ２ After adding the medium to the flask, DMEM-HAM / 20% FBS (GM-VCM) was added so as to minimize air bubbles remaining in the flask, and the flask was then sealed. ２ The flask was placed in an incubator with the bottom (culture surface) facing the ceiling, and ceiling culture was performed for 7 days. After 7 days, the culture medium in the flask was removed, the culture surface was washed with D-PBS, and then human preadipocytes were collected by trypsinization (4.5 × 10 ７ cells).

[0034] Step (1-2) Preparation of LCAT Gene-Transduced Human Preadipocytes For gene transfer, a retroviral vector was prepared as follows. The LCAT gene cloned from a cDNA library of a human hepatoma cell line was inserted into a pDON-AI vector plasmid (manufactured by Takara Bio Inc.). A Kozak sequence was added upstream of the 5'-end start codon (ATG) of the inserted LCAT gene, and the 3'-end stop codon was modified to remove the polyA signal, thereby preparing a retroviral vector plasmid carrying the human LCAT gene. From this plasmid, a packaging system (manufactured by Takara Bio Inc.) was used to obtain a retroviral vector solution carrying the human LCAT gene that could be introduced into human cells.

[0035] Six days after the start of ceiling culture in step (1-1) (the day before the harvesting of human preadipocytes), a 20 μg / mL RetroNectin (Takara Bio Inc.) solution diluted with D-PBS was added to 225 cm ２ 45 mL of the solution was added to the flask and allowed to stand overnight at 4°C. After removing the RetroNectin solution, the flask was washed once with D-PBS to obtain a RetroNectin-coated flask. The human LCAT gene-carrying retroviral vector solution was diluted to 5 x 10 with DMEM-HAM / 20% FBS (GM) containing 20% ​​FBS and 40 μg / mL gentamicin. ８ After dilution to RNAcopies / mL, 45 mL was added to a RetroNectin-coated flask and allowed to stand for 5 hours at 32° C. After 5 hours, the vector solution was removed to prepare a viral vector-coated flask.

[0036] The human preadipocytes collected in the above step (1-1) were collected at 225 cm ２ 2.25 x 10 per flask ６The cells were seeded into a viral vector-coated flask, and gene transfer was initiated. Two days after gene transfer, the medium was replaced with a cell culture medium "MSF-BM / MSF-supplement A (Shimadzu Diagnostics)" containing 40 μg / mL gentamicin. After another two days, the cells in the flask were collected and used as LCAT gene-transduced human preadipocytes. Until the start of the test, the cells were precipitated by centrifugation at 300 × g, 4°C, for 5 minutes, suspended in a cell cryopreservation solution (CELLBANKER1: Nippon Zenyaku Kogyo Co., Ltd.), and cryopreserved at -80°C.

[0037] Step (1-3) Preparation of Cell Preservation Solution To 500 mL of RPMI1640 (L-glutamine-containing) medium (Nacalai Tesque) serving as the "basal medium," 10 mL of Recombumin Elite (Albumedix) serving as "recombinant human serum albumin" was added and mixed. Furthermore, 1 mL of Vitamin C Injection "Fuso" 500 mg (Fuso Pharmaceutical Industries) serving as "ascorbic acid" was added and mixed. The mixture was sterilized by filtration to prepare a cell preservation solution (preservation solution before cell suspension). Prepared as described above, the albumin concentration was 0.2 (w / v)% and the ascorbic acid concentration was 0.5 mg / mL. The concentrations in Examples 2 and 3 were the same.

[0038] Step (1-4): Cell Recovery, Washing, Concentration, and Preservation The LCAT gene-introduced human preadipocytes cryopreserved in step (1-2) were thawed and then cultured at 37°C and 5% CO using a Nunc Easy Fill Cell Factory (Thermo Fisher Scientific). ２ The cells were grown in an incubator. The culture medium was removed, the culture surface was washed with D-PBS, and then trypsinized. The cells were recovered using the cell preservation solution prepared in step (1-3) above, and then centrifuged at 3,000 x g for 3 minutes at 4°C, and the supernatant was removed. The cells were then suspended in the cell preservation solution, centrifuged at 3,000 x g for 3 minutes at 4°C, and the supernatant was removed three times, and the cells were washed. After measuring the cell concentration, the cells were centrifuged at 3,000 x g for 5 minutes at 4°C, and the cells were collected at 1 x 10 ８The cells were suspended in a cell preservation solution to a concentration of 1000 cells / mL. The cell suspension was dispensed into storage containers, and storage was initiated at 4°C. Through the above steps (1-1) to (1-4), the "LCAT gene-introduced human preadipocytes" were suspended in a high-concentration cell preservation solution, thereby obtaining the high-concentration cell suspension of Example 1.

[0039] Example 2 A high-concentration cell preservation solution was prepared as described below, and a high-concentration cell suspension was prepared by suspending "human preadipocytes" without gene introduction in the cell preservation solution, and then the suspension was preserved. Human preadipocytes were recovered from adipose tissue obtained from a healthy individual according to the method in step (1-1) of Example 1. The cells were suspended in a cell freezing preservation solution (CELLBANKER1) and frozen and preserved at -80°C until the start of the test. A cell preservation solution was prepared according to the method in step (1-3) of Example 1, and the cells were recovered, washed, concentrated, and preserved according to the method in step (1-4). Through the above process, the high-concentration cell suspension of Example 2 was obtained by suspending "human preadipocytes" in the high-concentration cell preservation solution.

[0040] Example 3 A high-concentration cell preservation solution was prepared as described below, and a high-concentration cell suspension was prepared by suspending "human adipose tissue-derived mesenchymal stem cells" in the cell preservation solution and storing the cells. Human adipose tissue-derived mesenchymal stem cells (manufactured by Thermo Fisher Scientific) were cultured and allowed to proliferate. The cells were suspended in a cell cryopreservation solution (CELLBANKER1) and stored frozen at -80°C until the start of testing. A cell preservation solution was prepared according to the method in step (1-3) of Example 1, and cells were recovered, washed, concentrated, and stored according to the method in step (1-4). Through the above process, the high-concentration cell suspension of Example 3 was obtained, in which "human adipose tissue-derived mesenchymal stem cells (human adipose-derived stem cells)" were suspended in the high-concentration cell preservation solution.

[0041] Comparative Examples 1 and 2: Dextran 40 and trehalose-containing lactated Ringer's solution, which is commercially available as a cell suspension preservation solution, was used as the cell preservation solution in Comparative Example 1. Trehalose-containing lactated Ringer's solution, which is commercially available as a cell washing preservation solution, was used as the cell preservation solution in Comparative Example 2. Specifically, it was prepared according to the following steps (2-1) to (2-4).

[0042] Step (2-1) Preparation of Human Preadipocytes Human preadipocytes were obtained by the method of step (1-1) of Example 1, except that HBSS containing 2 mg / mL collagenase and 400 μg / mL vancomycin was used instead of HBSS containing 2 mg / mL collagenase, 40 μg / mL gentamicin, and 4 μg / mL vancomycin. Furthermore, DMEM-HAM / 20% FBS (GM) was used instead of DMEM-HAM / 20% FBS (GM-VCM).

[0043] Step (2-2) Preparation of LCAT gene-transfected human preadipocytes LCAT gene-transfected human preadipocytes were prepared according to the method of step (1-2) of Example 1, using MesenPRO RS Medium (manufactured by Thermo Fisher Scientific) instead of MSF-BM / MSF-supplement A. The cells were suspended in a cell cryopreservation medium (CELLBANKER1) and cryopreserved at −80° C. until the start of the test.

[0044] Step (2-3) Preparation of comparative cell washing solution 10 mL of 25% donated blood albumin (Japan Blood Products Organization) as an "additive" was added to 500 mL of Ringer's solution (Otsuka Pharmaceutical Factory Co., Ltd.) as a "cell washing solution" instead of a "basal medium" and mixed. The mixture was filtered to prepare a comparative cell washing solution.

[0045] Step (2-4) Recovery, washing, concentration and storage of cells According to the method of step (1-4) of Example 1, cells were recovered and washed using the cell washing solution prepared in the above step (2-3), and 0.5 × 10 ８ In Comparative Examples 1 and 2, the cells were suspended to a concentration of 1000 cells / mL, and storage was initiated at 4° C. Through the above steps (2-1) to (2-4), high-concentration cell suspensions of Comparative Examples 1 and 2 were obtained by suspending "LCAT gene-introduced human preadipocytes" in a ready-made cell preservation solution mainly containing Ringer's solution.

[0046] Test Example 1: Calculation of cell viability based on cell morphology (different cell types) The cell viability was evaluated based on the cell morphology immediately after storage for the high-concentration cell suspensions of Examples 1, 2, and 3 and Comparative Examples 1 and 2. The high-concentration cell suspensions (Example: 1 × 10 ８ cells / mL, comparative example: 0.5×10 ８ cells / mL) was extracted and serially diluted with each cell culture medium to obtain 5 × 10 ５ The cell suspension was adjusted to 1000 cells / mL. The "total cell concentration" and "dead cell concentration" of these cell suspensions were measured using a NucleoCounter NC-100 (manufactured by Chemometec). The "dead cell concentration" was calculated based on the number of cells stained with propidium iodide. The "viable cell rate (%)" was calculated based on both measured values. The "viable cell rate (%)" corresponds to the "cell survival rate (%)."

[0047] (Results and Discussion of Test Example 1) Figure 1 shows a summary of the changes over time in the cell viability (cell survival rate) in each Example and Comparative Example. Recombinant human serum albumin is abbreviated as "rHA" and ascorbic acid as "AA." From the results of Test Example 1, it was found that the high-concentration cell suspensions (1 x 10 ８ In the case of high-concentration cell preservation solutions (cells / mL), high cell viability was observed even after 72 hours of storage, regardless of the cell type. Specifically, when cells were stored at 4°C in a high-concentration cell preservation solution, the cell viability after 72 hours was found to be 80% or higher, more specifically 85% or higher, more specifically 90% or higher, and even more specifically 95% or higher (Example 1: 98.3%, Example 2: 97.8%, Example 3: 95.9%). The above tests were conducted multiple times, and similar test results were confirmed.

[0048] A decrease in cell viability was observed in the high-concentration cell suspensions of Comparative Examples 1 and 2. Specifically, when cells were stored at 4°C in a ready-made cell preservation solution mainly containing Ringer's solution, the cell viability after 72 hours was found to be less than 60% (Comparative Example 1: 55.4%, Comparative Example 2: 51.5%). Note that when the cell concentration is low (e.g., 1 × 10 ７ It has been confirmed that when the cell concentration is high (for example, 1 × 10 cells / mL), even cell preservation solutions based on Ringer's solution, such as those in Comparative Examples 1 and 2, can preserve the cells. ８ It was shown that it is difficult to achieve a high concentration of erythrocytes (cells / mL) using a composition such as Ringer's solution.

[0049] Test Example 2: Evaluation of cell viability by measuring intracellular dehydrogenase activity Test Example 1 examined only cell morphology. To confirm whether the cells were properly preserved while maintaining their functionality, cells were seeded into culture vessels after preservation, and cell viability was evaluated by measuring metabolic activity after 20 hours. For the high-concentration cell suspensions of Examples 1, 2, and 3 and Comparative Examples 1 and 2, cell suspensions were sampled from each preservation vessel 0, 48, and 72 hours after the start of preservation at 4°C, and serially diluted with each cell culture medium to 4 x 10 ４ The cell suspension was adjusted to 100 μL / mL and seeded in a 96-well plate. Also prepared were wells to which only the medium containing no cells was added. These were incubated at 37°C, 5% CO ２ After culturing for 20 hours in an incubator, the test was performed according to the protocol attached to Cell Counting Kit-8 (Dojindo Laboratories), and optical density (OD) values ​​were obtained by measuring absorbance at a wavelength of 450 nm. The OD values ​​of each test sample were subtracted from the OD value of the medium alone to calculate the relative value (optical density ratio) when the value at 0 hours of storage was set to 1.

[0050] (Results and Discussion of Test Example 2) Figure 2 shows a summary of the changes over time in the OD value ratio (optical density ratio) in each Example and Comparative Example. The results of Test Example 2 indicate that, unlike the high-concentration cell suspensions of Comparative Examples 1 and 2, the high-concentration cell suspensions of Examples 1, 2, and 3 retained high metabolic activity even 72 hours after the start of storage (as indicated by high OD value ratios), demonstrating that cell viability was maintained. More specifically, when cells were stored at 4°C in the high-concentration cell preservation solution of Examples 1, 2, and 3, the OD value ratios after 72 hours relative to the value stored at time 0 were 0.5 or higher, more specifically 0.6 or higher, and even more specifically 0.7 or higher (Example 1: 0.931, Example 2: 0.876, Example 3: 0.708).

[0051] Test Example 3: Evaluation of cytotoxicity by detecting lactate dehydrogenase (LDH) in culture medium To evaluate damage to cells preserved in a high-concentration cell preservation solution, the amount of free LDH induced by cell membrane damage was measured. Cells were extracted from the high-concentration cell suspensions of Examples 1, 2, and 3 at 0, 48, and 72 hours after the start of storage at 4°C, respectively, and serially diluted with each cell culture medium to 4 × 10 ４ The solution was adjusted to 1000 μL / mL and seeded in two wells of a 12-well plate. Two wells were also prepared to contain only the medium without cells. These were incubated at 37°C in 5% CO ２ After culturing for 20 hours in an incubator, the lysis buffer included in the LDH cytotoxicity assay kit (manufactured by Nacalai Tesque) was added to one of the two wells of each test sample, and the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 30 minutes. After 30 minutes, the culture medium from each test sample was collected and centrifuged at 5,000 rpm at 4°C for 5 minutes. The supernatant after centrifugation was collected and frozen at -80°C until analysis. The test was performed according to the protocol attached to the LDH cytotoxicity assay kit, and the OD value was obtained by measuring absorbance at a wavelength of 492 nm. The OD value of each test sample was calculated by subtracting the OD value of the medium alone, and the relative value to the sample to which lysis buffer had been added was calculated.

[0052] (Results and Discussion of Test Example 3) Figure 3 shows a summary of the changes in relative OD values ​​over time in each Example. The results of Test Example 3 indicate that the high-concentration cell suspensions of Examples 1, 2, and 3 showed low relative OD values ​​even 72 hours after the start of storage, indicating that no cell damage was observed. More specifically, when cells were stored at 4°C in the high-concentration cell preservation solutions of Examples 1, 2, and 3, the relative OD values ​​after 72 hours compared to samples containing lysis buffer were 10% or less, more specifically, 8% or less (Example 1: 2.33%, Example 2: 3.75%, Example 3: 7.80%).

[0053] Example 4 A high-concentration cell suspension of Example 4 was obtained by suspending "human preadipocytes" in a high-concentration cell preservation solution prepared as in Example 2. The "cell concentration" of the high-concentration cell suspension was adjusted to 1 x 10 ８ The "basal medium" was RPMI1640 (containing L-glutamine) medium, and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentrations were 0.1 (w / v)%, 0.2 (w / v)%, and 2.0 (w / v)%, and the ascorbic acid concentrations were 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL.

[0054] Test Example 4: Calculation of cell viability based on cell morphology (additives at different concentrations) In the same manner as Test Example 1, the cell viability was evaluated for the high-concentration cell suspensions of Example 4, which had different albumin and ascorbic acid concentrations.

[0055] (Results and Discussion of Test Example 4) Figures 4 and 5 show a summary of the time-dependent changes in cell viability (cell survival rate) in Example 4, where the albumin concentration and ascorbic acid concentration were different. Recombinant human serum albumin is abbreviated as "rHA" and ascorbic acid as "AA." From the results of Test Example 4-1, it was found that the high-concentration cell suspension (1 x 10 ８In the case of the high-concentration cell suspension of Example 4 (rHA concentration 0.1 (w / v)%: 94.8%, rHA concentration 0.2 (w / v)%: 94.1%, rHA concentration 2.0 (w / v)%: 97.1%), the cell viability was high even after 72 hours from the start of storage. Specifically, when the cells were stored at 4°C, the cell viability was found to be 90% or higher after 72 hours (rHA concentration 0.1 (w / v)%: 94.8%, rHA concentration 0.2 (w / v)%: 94.1%, rHA concentration 2.0 (w / v)%: 97.1%). Furthermore, from the results of Test Example 4-2, the high-concentration cell suspension of Example 4, in which the ascorbic acid concentration (AA concentration) was 0.1 mg / mL to 10 mg / mL, showed a high viability even after 72 hours from the start of storage. Specifically, when cells were stored at 4°C, the cell viability after 72 hours was found to be 90% or higher (AA concentration 0.1 mg / mL: 94.4%, AA concentration 0.5 mg / mL: 94.1%, AA concentration 1 mg / mL: 95.0%, AA concentration 2 mg / mL: 96.7%, AA concentration 5 mg / mL: 97.5%, AA concentration 10 mg / mL: 97.4%). The above test was conducted multiple times, and similar test results were confirmed. The high-concentration cell suspension of Example 4 was subjected to a cell viability evaluation similar to that in Test Example 2, confirming that the cells remained viable. Furthermore, a cytotoxicity evaluation similar to that in Test Example 3 was conducted, confirming that no cytotoxicity was observed.

[0056] <Examples 5 and 6> As in Example 1, "LCAT gene-introduced human preadipocytes" were suspended in a high-concentration cell preservation solution to obtain high-concentration cell suspensions of Examples 5 and 6. For Example 5, the "cell concentration" was 1 x 10 ８ The "basal medium" was RPMI 1640 (containing L-glutamine), and the "additives" were recombinant human serum albumin (rHA), ascorbic acid (AA), and sodium pyruvate (SP). The albumin concentration was 0.5 (w / v)%, the ascorbic acid concentration was 5 mg / mL, and the sodium pyruvate concentration was 1 mM. For Example 6, the "cell concentration" was 1 x 10 ８The "basal medium" was DMEM (low glucose) (manufactured by Nacalai Tesque), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was 0.5 (w / v)%, and the ascorbic acid concentration was 5 mg / mL. Note that, since the RPMI 1640 in Example 5 does not contain sodium pyruvate, sodium pyruvate was added separately. Since the DMEM in Example 6 contains sodium pyruvate, the same composition conditions were used.

[0057] Test Example 5: Calculation of cell viability based on cell morphology (different basal media) In the same manner as in Test Example 1, the viability was evaluated for the high-concentration cell suspensions of Examples 5 and 6, which were prepared using different basal media.

[0058] (Results and Discussion of Test Example 5) Figure 6 shows a summary of the changes over time in the viability (cell survival rate) in Examples 5 and 6. Recombinant human serum albumin is abbreviated as "rHA," ascorbic acid as "AA," and sodium pyruvate as "SP." From the results of Test Example 5, it was found that the high-concentration cell suspensions (1 x 10 ８ The high-concentration cell suspensions (Examples 5 and 6) showed high cell viability even after 72 hours from the start of storage. Specifically, when the cells were stored at 4°C, the cell viability was shown to be 80% or higher after 72 hours (Example 5: 90.9%, Example 6: 84.2%). Furthermore, it was shown that the cell viability was higher when the basal medium was "RPMI 1640" than when it was "DMEM". The above test was performed multiple times, and similar test results were confirmed. The high-concentration cell suspensions of Examples 5 and 6 were subjected to cell viability evaluation in the same manner as in Test Example 2, and the maintenance of cell viability was confirmed.

[0059] Example 7 A high-concentration cell preservation solution was prepared according to the following steps (7-1) to (7-3), and a high-concentration cell suspension was prepared by suspending "FVIII gene-transduced human preadipocytes" in the cell preservation solution, and the cell suspension was then stored. Step (7-1) Preparation of FVIII gene-transduced human preadipocytes Human preadipocytes were collected from adipose tissue obtained from a healthy human according to the method in step (1-1) of Example 1. The cells were suspended in a cell freezing preservation solution (CELLBANKER1) and stored frozen at -80°C until the start of the test.

[0060] Step (7-2) Preparation of FVIII Gene-Transduced Human Preadipocytes For gene transfer, a lentiviral vector was prepared as follows. First, a "FVIII variant" was prepared in which the number of Asn residues to be N-glycosylated in the B domain of the human FVIII gene was set to 5, counting from the N-terminus, and the function of the furin recognition site was deleted, with the 758th Pro residue being replaced with a Thr residue. A base sequence in which a signal peptide was added to this "FVIII variant" was inserted into a pLVSIN vector plasmid (manufactured by Takara Bio Inc.) to prepare a lentiviral vector plasmid carrying the human FVIII gene. From this plasmid, a lentiviral vector solution carrying the human FVIII gene that can be introduced into human cells was obtained using a packaging system (manufactured by Takara Bio Inc.). The preadipocytes frozen in the above step (7-1) were thawed, and 150 cm ２ The flasks were incubated at 37°C and 5% CO ２ The cells were cultured overnight in an incubator. The next day, the culture medium was removed, the culture surface was washed with D-PBS, and then the cells were treated with trypsin to recover the preadipocytes. The human FVIII gene-carrying lentiviral vector solution obtained in this step was added to the cells at a concentration of 1 × 10 ９ RNA copies / mL, containing 100 μg / mL protamine sulfate, cell concentration 2.5 × 10 ４ The concentration was adjusted with MSF-BM / MSF-Supplement A to 25 cm ２Five mL of the medium was seeded into a flask to initiate gene transfer, and the medium was replaced the next day. The same gene transfer procedure was repeated on days 2 and 7 after the first gene transfer (a total of three times) to obtain "FVIII gene-transfected human preadipocytes." Prior to the start of the test, the cells were precipitated by centrifugation at 300 x g and 4°C for 5 minutes, suspended in a cell cryopreservation medium (CELLBANKER1), and cryopreserved at -80°C.

[0061] Step (7-3) Recovery, washing, concentration, and storage of cells A cell preservation solution was prepared using "RPMI1640 (L-glutamine-containing) medium" as the basal medium according to the method of step (1-3) in Example 1, and cells were recovered, washed, concentrated, and stored according to the method of step (1-4). Through the above, "FVIII gene-transfected human preadipocytes" were suspended in a high-concentration cell preservation solution using "RPMI1640 (L-glutamine-containing) medium" as the basal medium, thereby obtaining the high-concentration cell suspension of Example 7.

[0062] Example 8 A high-concentration cell preservation solution was prepared as described below, and a high-concentration cell suspension was prepared by suspending "human bone marrow-derived mesenchymal stem cells" in the cell preservation solution and storing it. Human bone marrow-derived mesenchymal stem cells (PromoCell) were cultured and proliferated. The cells were suspended in a cell cryopreservation solution (CELLBANKER1) and stored frozen at -80°C until the start of the test. A cell preservation solution was prepared according to the method in step (1-3) of Example 1, and cells were recovered, washed, concentrated, and stored according to the method in step (1-4). Through the above process, the high-concentration cell suspension of Example 8 was obtained by suspending "human bone marrow-derived mesenchymal stem cells" in a high-concentration cell preservation solution using "RPMI 1640 medium" as the basal medium.

[0063] Test Example 6: Calculation of cell viability based on cell morphology (different cell types) In the same manner as Test Example 1, cell viability was evaluated for the high-concentration cell suspensions of Examples 1, 2, 7, and 8 and Comparative Examples 1 and 2, which contained different cell types. Note that tests were conducted on multiple samples of Examples 1, 2, and 7.

[0064] (Results and Discussion of Test Example 6) Figure 7 shows a summary of the changes over time in the cell viability (cell survival rate) in Examples and Comparative Examples using different cell types. Recombinant human serum albumin is abbreviated as "rHA" and ascorbic acid as "AA." From the results of Test Example 6, the high-concentration cell suspensions (1 x 10 ８ In the case of cells stored in a high-concentration cell preservation solution (cells / mL), high cell viability was observed even after 72 hours of storage, regardless of the cell type. Specifically, when cells were stored at 4°C in a high-concentration cell preservation solution, the cell viability after 72 hours was 80% or higher, more specifically 85% or higher, more specifically 90% or higher, and even more specifically 95% or higher (Example 1: 98.4% (n=3), Example 2: 96.7% (n=4), Example 7: 96.7% (n=3), Example 8: 95.3% (n=1)). Cell viability and cytotoxicity evaluations similar to those in Test Examples 2 and 3 were also performed on Examples 7 and 8, confirming that the cells retained high metabolic activity and no cytotoxicity were observed even after 72 hours of storage.

[0065] <Examples 9 and 10> As in Example 2, non-transfected "human preadipocytes" were suspended in a high-concentration cell preservation solution to obtain high-concentration cell suspensions of Examples 9 and 10. For Example 9, the "cell concentration" was 1 x 10 ８ The "basal medium" was RPMI 1640, and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was 0.2 (w / v)%, and the ascorbic acid concentration was 0.5 mg / mL. For Example 10, the "cell concentration" was 1 x 10 ８ The "basal medium" was DMEM (low glucose), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was 0.2 (w / v)%, and the ascorbic acid concentration was 0.5 mg / mL.

[0066] <Examples 11 and 12> As in Example 1, "LCAT gene-introduced human preadipocytes" were suspended in a high-concentration cell preservation solution to obtain high-concentration cell suspensions of Examples 11 and 12. For Example 11, the "cell concentration" was 1 x 10 ８ The "basal medium" was RPMI 1640 (containing L-glutamine), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was 0.2 (w / v)%, and the ascorbic acid concentration was 0.5 mg / mL. For Example 12, the "cell concentration" was 1 x 10 ８ The "basal medium" was DMEM (low glucose), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was 0.2 (w / v)%, and the ascorbic acid concentration was 0.5 mg / mL.

[0067] Although the RPMI 1640 of Examples 9 and 11 does not contain sodium pyruvate, it was confirmed that the presence or absence of sodium pyruvate has almost no effect on cell viability, cell survival, or cell damage, and therefore sodium pyruvate was not added.

[0068] Test Example 7: Calculation of cell viability based on cell morphology (different basal media) In the same manner as in Test Example 5, the viability of the high-concentration cell suspensions of Examples 9, 10, 11, and 12, which were prepared using different basal media, was evaluated.

[0069] (Results and Discussion of Test Example 7) Figure 8 shows a summary of the changes over time in the cell viability (cell survival rate) in Examples 9, 10, 11, and 12. Recombinant human serum albumin is abbreviated as "rHA," and ascorbic acid is abbreviated as "AA." From the results of Test Example 7, it was found that the high-concentration cell preservation solutions (1 x 10) of Examples 9, 10, 11, and 12, in which the basal media were "RPMI1640" and "DMEM," and the preserved cells were "LCAT gene-introduced human preadipocytes" and "human preadipocytes," respectively, were used. ８In the case of the cells stored at 4°C, the cell viability was 96% or more after 72 hours (Example 9: 97.6%, Example 10: 96.8%, Example 11: 98.8%, Example 12: 97.4%).

[0070] Furthermore, it was shown that using "RPMI 1640" as the basal medium rather than "DMEM" resulted in higher cell viability (the cell viability in Examples 9 and 11 was higher than that in Examples 10 and 12, respectively). The above test was performed multiple times, and it was confirmed that similar test results were obtained. Furthermore, it was confirmed that the cell viability was higher in Examples 9 and 11 when sodium pyruvate was not intentionally added. Note that for Examples 9 and 10, the same cell viability evaluation and cell damage evaluation as in Test Examples 2 and 3 were performed, and it was confirmed that the cells remained viable and that no cell damage was observed.

[0071] Test Example 8: Calculation of cell viability based on cell morphology (long-term viability) In the same manner as in Test Example 1, the long-term viability was evaluated for the high-concentration cell suspensions of Examples 9 and 10, which were prepared using different basal media.

[0072] (Results and Discussion of Test Example 8) The time course changes in the cell viability (cell survival rate) in Examples 9 and 10 are summarized in Figure 9. From the results of Test Example 8, the high concentration cell suspensions (1 x 10 ８ In Example 9, where the basal medium was "RPMI 1640 medium," the cell viability was found to be 90% or higher after 170 hours (Example 9: 94.0%, Example 10: 81.4%). The above test was repeated multiple times, and similar test results were confirmed.

[0073] <Additional Notes> Examples 1 to 12 can be summarized as follows. Example 1: LCAT gene-transfected human preadipocytes, RPMI1640 Example 2: Human preadipocytes, RPMI1640 Example 3: Human adipose-derived stem cells, RPMI1640 Example 4: Human preadipocytes, RPMI1640, different additive concentrations Example 5: LCAT gene-transfected human preadipocytes, RPMI1640, sodium pyruvate Example 6: LCAT gene-transfected human preadipocytes, DMEM Example 7: FVIII gene-transfected human preadipocytes, RPMI1640 Example 8: Human bone marrow-derived mesenchymal stem cells, RPMI1640 Example 9: Human preadipocytes, RPMI1640 Example 10: Human preadipocytes, DMEM Example 11: LCAT gene-transfected human preadipocytes, RPMI1640 Example 12: LCAT gene-transfected human preadipocytes, DMEM

Claims

1. A cell preservation solution for preserving cells in an unfrozen state, A basal medium; An additive added to the basal medium, The basal medium is a medium used for cell culture and for preserving the cells, The additive contains at least recombinant albumin and ascorbic acid or an ascorbic acid derivative, The cells are at least 1 x 10 ７ cells / mL, A high-concentration cell preservation solution characterized in that when the cells are stored at 4°C at a cell concentration of 1 x 107 cells / mL, the cell viability after 72 hours is 80% or more, the cells maintain their functionality, and the cells are preserved in a state where no cell damage is observed.

2. The cell is a cell derived from a mammal, The recombinant albumin is recombinant human serum albumin, The highly concentrated cell preservation solution according to claim 1 , wherein the cell preservation solution is used for cell transplantation.

3. The cells were 1×10 ８ ~1 x 10 ９ 2. The high-concentration cell preservation solution according to claim 1, wherein the cell is preserved at a cell concentration of 1000 cells / mL.

4. The cell is a somatic cell of human origin or a stem cell of human origin, 2. The highly concentrated cell preservation solution according to claim 1, wherein the basal medium is at least one medium selected from the group consisting of RPMI1640 and DMEM.

5. The concentration of the recombinant albumin is 0.1 to 2.0 (w / v)%, 2. The high-concentration cell preservation solution according to claim 1, wherein the concentration of the ascorbic acid or the ascorbic acid derivative is 0.1 to 10 mg / mL.

6. When the cells are stored at a cell concentration of 1 x 107 cells / mL at 4°C, The OD value ratio (optical density ratio) after 72 hours using Cell Counting Kit-8 relative to the value stored at 0 hours shows a value of 0.7 or more, and the cells are stored in a state where they maintain their function, and The high-concentration cell preservation solution described in claim 1, characterized in that the cells are preserved in a state where the relative OD value (optical density) after 72 hours using an LDH cytotoxicity assay kit shows a value of 8% or less, indicating no cell damage.

7. The cells were 1×10 ８ 7. The high-concentration cell preservation solution according to claim 1, wherein when the high-concentration cell preservation solution is stored at 4°C at a cell concentration of 1000 cells / mL, the cell viability after 72 hours is 80% or more, the cells maintain their functionality, and the cells are preserved without any cell damage.

8. The cells were 1×10 ８ 7. The high-concentration cell preservation solution according to claim 1, wherein when the high-concentration cell preservation solution is stored at 4°C at a cell concentration of 170 cells / mL, the cell viability after 170 hours is 80% or more, the cells maintain their functionality, and no cell damage is observed during preservation.

9. Suspend the cells in a cell preservation solution and store the cells in a medium containing at least 1 x 10 cells in an unfrozen state. ７ A method for preserving cells at a cell concentration of 1000 cells / mL, comprising: The cell preservation solution contains a basal medium and an additive added to the basal medium, The basal medium is a medium used for cell culture and for preserving the cells, The additive contains at least recombinant albumin and ascorbic acid or an ascorbic acid derivative, This high-concentration cell preservation method is characterized in that when the cells are stored at 4°C at a cell concentration of 1 x 107 cells / mL, the cell viability after 72 hours is 80% or more, the cells maintain their functionality, and the cells are preserved in a state where no cell damage is observed.