High-concentration cell preservation solution 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

[Technical Field]

[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 without freezing them. [Background technology]

[0002] In recent years, regenerative medicine using cells collected from patients has been performed. When administering cell-based therapies (cell preparations) directly to patients, it is preferable to use cell preparations prepared with high cell concentrations to reduce the burden on the patient. However, there is a problem in that storing cells at high concentrations reduces cell viability. Therefore, current cell therapy requires the rapid administration of high-concentration cell preparations to patients, necessitating treatment with cell preparations at university hospitals or other facilities equipped with advanced cell processing capabilities.

[0003] Patent Document 1 discloses an invention for a liquid composition for preserving cells in a non-frozen state, but this liquid composition is not intended for preserving cells at high cell concentrations. Furthermore, Patent Document 2 discloses an invention of a cryopreservation solution for preserving stem cells in a frozen state, for example, by storing stem cells in a 1 × 10⁻¹⁶ state. 4 ~1 × 10 8 It is disclosed that cells are stored at a cell concentration of cells / mL. However, since this is a cell preservation solution that stores cells in a frozen state, a separate thawing procedure is required, and the survival rate of viable cells decreases during the freeze-thaw process. Therefore, it is preferable to be able to store cells without freezing them. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2019 / 49985 [Patent Document 2] Japanese Patent Application Laid-Open No. 2019-154329

Summary of the Invention

Problems to be Solved by the Invention

[0005] For example, if there is a cell preservation solution that can preserve cells at a high cell concentration and maintain a high survival rate, not only in university hospitals with cell processing facilities, but also in medical institutions away from cell processing facilities, it will be possible to perform cell therapy. When introducing a non-frozen cell drug (cell preparation) into the market, it is preferable that the cell preservation solution can preserve cells for 72 hours or more in consideration of transportation conditions and patient conditions. In addition, not limited to cell therapy applications, such a cell preservation solution can be widely used for research reagents and other applications, which is beneficial.

[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 survival rate.

Means for Solving the Problems

[0007] As a result of intensive research, the present inventors have clarified that it is possible to produce a cell preservation solution that can preserve cells at a high cell concentration without freezing and maintain a high survival rate, and have thus completed the present invention. Specifically, as a result of intensive research on a suitable combination of mainly a basal medium and additives added to the basal medium, it has been clarified that it is possible to produce a cell preservation solution that can preserve cells at a cell concentration of at least 1×10 7 cells / mL even without freezing the cells. For example, when cells are suspended in the cell preservation solution, the cells are 1×10 8It was revealed that when stored at 4°C at a cell concentration of cells / mL, the cell viability after 72 hours was 80% or more. It was also revealed that it can be produced from pharmaceutically transplantable components.

[0008] Therefore, according to the high-concentration cell preservation solution of the present invention, the above problem is solved by a cell preservation solution for preserving cells without freezing, which contains a basal medium and an additive added to the basal medium. The basal medium is a medium used for cell culture, and the additive contains at least recombinant albumin and ascorbic acid or an ascorbic acid derivative. The cells are stored at a cell concentration of at least 1×10 7 cells / mL, and when the cells are stored at a cell concentration of 1×10 7 cells / mL at 4°C, the cell viability after 72 hours is 80% or more, and the cells are stored in a state where they maintain their functions and no cell damage is observed. The cells are human-derived somatic cells or human-derived stem cells, the basal medium is at least one medium selected from the group consisting of RPMI1640 and DMEM, the concentration of the recombinant albumin is 0.1-2.0 (w / v)%, and the concentration of the ascorbic acid or the ascorbic acid derivative is 0.1-10 mg / mL. This solves the problem. With the above configuration, it is possible to realize 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 can maintain a high survival rate.

[0009] At this time ,before The recombinant albumin is recombinant human serum albumin, and the cell preservation solution is preferably used for cell transplantation. Also, it is preferable to store the cells at a cell concentration of 1×10 8 ~1×10 9 cells / mL. 。 Ma For example, when the cells are stored at a cell concentration of 1×10 8 cells / mL at 4°C, it is preferable that the cell viability after 72 hours is 80% or more. More preferably, it is preferable that the cell viability after 170 hours is 80% or more. The above configuration makes it possible to store cells at a higher cell concentration without freezing, and to realize a high-concentration cell preservation solution that can maintain a higher survival rate. In particular, when directly transplanting cell preparations into patients, it is possible to provide a cell preservation solution that reduces the burden on patients and allows for suitable cell therapy.

[0010] Furthermore, the aforementioned problem is solved by the high-concentration cell preservation method of the present invention, which suspends cells in a cell preservation solution and keeps the cells in a state where they are not frozen, at least 1 × 10⁻¹⁶ 7 A method for preserving cells at a cell concentration of cells / mL, wherein the cell preservation solution contains a basal medium and an additive added to the basal medium, the basal medium being a culture medium used for cell culture, and the additive containing at least recombinant albumin and ascorbic acid or an ascorbic acid derivative, and the cells being 1 × 10 7 When stored at 4°C with a cell concentration of cells / mL, the cell viability after 72 hours is 80% or higher, the cells maintain their function, and no cytotoxicity is observed. The cells are human-derived somatic cells or human-derived stem cells, the basal medium is at least one medium selected from the group consisting of RPMI1640 and DMEM, the concentration of the recombinant albumin is 0.1-2.0 (w / v)%, and the concentration of the ascorbic acid or the ascorbic acid derivative is 0.1-10 mg / mL. This can also be resolved by [method]. [Effects of the Invention]

[0011] According to the high-concentration cell preservation solution and high-concentration cell preservation method of the present invention, it is possible to preserve cells at a high cell concentration without freezing them and to maintain them with a high viability rate. [Brief explanation of the drawing]

[0012] [Figure 1] This graph shows the change over time in the viable cell rate (cell survival rate) in Test Example 1. [Figure 2] This graph shows the results of the cell viability evaluation in Test Example 2. [Figure 3] This graph shows the results of the cell damage evaluation in Test Example 3. [Figure 4] This graph shows the change in the viable cell rate over time in Test Example 4-1. [Figure 5] This graph shows the change in the viable cell rate over time in Test Example 4-2. [Figure 6] This graph shows the change in the viable cell rate over time in Test Example 5. [Figure 7] This graph shows the change in the viable cell rate over time in Test Example 6. [Figure 8] This graph shows the change in the viable cell rate over time in Test Example 7. [Figure 9] This graph shows the change in the viable cell rate over time in Test Example 8. [Modes for carrying out the invention]

[0013] Hereinafter, embodiments of the present invention will be described with reference to Figures 1 to 9. This embodiment relates to a "high-concentration cell preservation solution" that enables the preservation of cells at a high cell concentration without freezing, and the maintenance of a high viability rate. It also relates to a "high-concentration cell preservation method".

[0014] <Overview of cell preservation solution> A "cell preservation solution" is a liquid composition for preserving cells, specifically for storing cells for cell therapy (gene therapy). In this embodiment, it is a liquid composition for preserving cells in a non-frozen state. "Non-frozen state" means a temperature of 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. Furthermore, the cell preservation solution of this embodiment is not limited to use as a cell preparation, but can be widely used as a research reagent and for other purposes.

[0015] A "high-concentration cell preservation solution" is a cell preservation solution that preserves cells at a high cell concentration. For example, cells at least 1 × 10 7 cells / mL (1 × 10) 7 Stored at a cell concentration of cells / mL or higher, preferably 5 × 10 7 cells / mL or more, more preferably 1 × 10 8 The cells are stored at a cell concentration of cells / mL or higher. Alternatively, the cells are stored at a concentration of 1 × 10⁻⁶.7 ~1 × 10 9 cells / mL, more preferably 5×10 7 ~1 × 10 9 cells / mL, more comfortable 1×10 8 ~1 × 10 9 cells / mL, more comfortable 1×10 8 It is stored at a cell concentration of cells / mL. Cells 1 × 10 7 cells / mL or more (preferably 1 × 10) 8 By storing cells at a cell concentration of cells / mL or higher (non-freezing storage), the transplant volume can be reduced when directly transplanting cell preparations (non-frozen cell preparations) into patients, thereby lowering the burden on patients. Also, cells 1 × 10 9 By storing cells at a cell concentration of cells / mL or less, a high cell viability rate can be maintained.

[0016] "High cell viability" means that cells are 1 × 10 7 ~1 × 10 9 cells / mL (preferably 1 × 10) 8 When stored at 4°C with a cell concentration of (cells / mL), it is desirable that the cell viability after 72 hours be 70% or higher, preferably 75% or higher, more preferably 80% or higher, more preferably 85% or higher, and more preferably 90% or higher. Alternatively, when stored at 4°C with the above cell concentration, it is desirable that the cell viability after 96 hours be 70% or higher, preferably 75% or higher, more preferably 80% or higher, more preferably 85% or higher, and more preferably 90% or higher. Alternatively, when stored at 4°C at the above cell concentration, it is desirable that the cell viability after 120 hours be 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, it is desirable that the cell viability after 170 hours be 70% or higher, preferably 75% or higher, more preferably 80% or higher, more preferably 85% or higher, and more preferably 90% or higher. Alternatively, the cell viability after 144 hours and 168 hours should also be similar to that described above. The "cell viability rate" should be calculated using the method used in the example test 1 described below. Furthermore, when introducing a cell preparation to the market in an unfrozen state, it is desirable that the cell preservation solution be capable of storing 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 by 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), cardiomyocytes, muscle cells, pancreatic beta cells, melanocytes, and hematopoietic progenitor cells. Preferably, they are adipocytes, more preferably preadipocytes (adipocytes that have acquired proliferative capacity and can be collected by primary culture), more preferably mammalian-derived preadipocytes, and more preferably human-derived preadipocytes. Adipocytes (preadipocytes) are suitable for cell therapy because there are no restrictions on administration or age restrictions when administering cell preparations, the risk of tumor formation is low, and they are suitable for cell therapy. These somatic cells may also have foreign genes intended for gene therapy incorporated into their genomic DNA. "Stem cells" include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Mesenchymal stem cells are preferable, and human-derived mesenchymal stem cells are even preferable. These stem cells may have foreign genes incorporated into their genomic DNA for the purpose of gene therapy.

[0018] <Composition of cell preservation solution> High-concentration cell preservation solutions contain a basal culture medium and additives added to the basal culture medium. By primarily using a combination of basal culture medium and additives, it is possible to produce a cell preservation solution that allows for the storage of cells at high concentrations while maintaining a high viability rate, even without freezing.

[0019] (Basal culture medium) A "basal culture medium" is a culture medium used for cell culture, and it is preferable that it be a liquid medium. In other words, conventional buffer salt solutions such as Ringer's solution (lactated Ringer's solution) or physiological saline are not used as basal culture media. The "basic culture medium" should preferably be 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 should be RPMI1640 or DMEM, and more preferably RPMI1640. By using these media as basal culture media, it has been confirmed that cells can be preserved at a high cell concentration for a predetermined period of time because they contain amino acids and vitamins necessary for cell survival. In particular, using RPMI1640 and DMEM maintains an appropriate metabolic state and improves cell preservation. Especially with RPMI1640, the presence of glutathione, which has antioxidant properties, further improves cell preservation.

[0020] When RPMI1640 is used as the basal culture medium, it is preferable that it be RPMI1640 (containing L-glutamine). When DMEM is used as the basal culture medium, it is preferable that it be a low-glucose-containing DMEM.

[0021] Furthermore, the culture medium used for the above cell culture may be a "buffer salt solution" containing the essential components of the culture medium. Essential components of the culture medium include, for example, "amino acids," "vitamins," and "other components." Examples of "buffer salt solutions" include Ringer's solution, Ringer's lactate solution, Ringer's acetate solution, physiological saline, Hanks equilibrium salt solution (HBSS), and Dulbecco's phosphate buffer. "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 and L-valine, L-phenylalanine, and L-leucine. "Vitamins" include ascorbic acid (ascorbic acid derivatives), as well as inositol, biotin, pantothenic acid, choline, folic acid, niacin, pyridoxine, riboflavin, thiamine, and vitamin B12. "Other ingredients" include sugars, reduced glutathione, sodium salts, potassium salts, inorganic salts other than sodium and potassium salts, antibiotics, serum, and fatty acids. The above-mentioned "amino acids," "vitamins," and "other additives" may be added as additives as appropriate.

[0022] The basal culture medium is preferably prepared to contain 50(v / v) or more of the total volume of the high-concentration cell preservation solution, more preferably 70(v / v) or more, and more preferably 80(v / v) or more.

[0023] "Additives" have the function of maintaining cell viability when added to the basal culture medium. Genetically modified albumin and ascorbic acid should be added to the basal culture medium as the main "additives."

[0024] (Genetically modified albumin) Albumin has antioxidant properties, inhibits cell aggregation, and plays a role in the transport of nutrients from the basal culture medium to cells. Furthermore, it can inhibit cell adsorption to storage containers, thus preventing difficulties in cell utilization. Albumin can be extracted from blood, but it often contains many unwanted impurities. Genetically modified albumin, on the other hand, is produced using genetic engineering technology and contains extremely low levels of impurities and foreign factors. The albumin to be added is preferably recombinant albumin, and more preferably recombinant human serum albumin (rHA). Genetically modified albumin is albumin produced using genetic engineering technology, from which components of a different species have been removed. Alternatively, genetically modified proteins may be added to the basal culture medium instead of genetically modified albumin. In this case, it is preferable to use proteins with a molecular weight of 20,000 to 100,000 (low molecular weight proteins), similar to albumin.

[0025] The recombinant albumin is preferably prepared to be contained in a concentration of 0.01 to 20% (w / v) of the total volume of the high-concentration cell preservation solution, more preferably 0.01 to 10% (w / v), more preferably 0.01 to 5.0% (w / v), and more preferably 0.1 to 2.0% (w / v). A decrease in cell survival rate has been observed when the concentration is below 0.01(w / v)%. Furthermore, when it exceeds 20(w / v)%, the viscosity of the high-concentration cell preservation solution increases, significantly reducing its operability.

[0026] (Ascorbic acid, ascorbic acid derivatives) Ascorbic acid has antioxidant properties and functions in maintaining cell survival. Ascorbic acid derivatives similarly possess 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-ascorbic acid palmitate, ascorbyl tetrahexyldecanoate, and diphosphate of ascorbic acid. It has been confirmed that adding these additives to the basal culture medium allows for the preservation of cells at high cell concentrations for a predetermined period of time. In particular, adding both recombinant albumin and ascorbic acid allows for even higher cell concentrations to be preserved for a predetermined period of time.

[0027] The ascorbic acid (ascorbic acid derivative) is preferably prepared to be contained in an amount of 0.1 to 10 mg / mL relative to the total volume of the high-concentration cell preservation solution, more preferably 0.1 to 5 mg / mL, more preferably 0.1 to 2 mg / mL, and more preferably 0.1 to 1 mg / mL. It has been confirmed that cell viability decreases when ascorbic acid levels are below 0.1 mg / mL or above 10 mg / mL.

[0028] (Other additives) Other "additives" that may be added to the basal culture medium as appropriate include sodium salts, potassium salts, inorganic salts other than sodium salts and potassium salts, amino acids, vitamins, antibiotics, serum, fatty acids, and sugars. 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. In addition to ascorbic acid mentioned above, other examples of vitamins include biotin, pantothenic acid, choline, folic acid, niacin, pyridoxine, riboflavin, thiamine, and vitamin B12.

[0029] A high-concentration cell suspension, obtained by suspending cells in a cell preservation solution (high-concentration cell preservation solution), is preferably stored in any volume of 0.05 mL or more. Preferably, it is stored in a volume of 0.05 to 500 mL, more preferably 0.05 to 100 mL, and more preferably 0.05 mL to 50 mL. While there are no particular limitations on the storage containers used when storing without freezing, it is preferable to use general-purpose syringes, bags, tubes, vials, or ampoules that can provide the necessary capacity. Furthermore, it is advisable to store the cell preparations aseptically in these storage containers. This allows them to be used directly for cell transplantation even in medical institutions and 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 additives, and keeping the cells in a state where they are not frozen, at least 1 × 10⁶ 7 This method involves preserving cells at a cell concentration of cells / mL.

[0031] <Method for producing a high-concentration cell suspension using 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-transformed human pre-adipocytes," the following steps are mainly performed. This manufacturing method involves a first step of preparing human pre-adipocytes into which the LCAT gene is introduced, a second step of producing human pre-adipocytes into which the LCAT gene is introduced, a third step of preparing a cell preservation solution, and a fourth step of collecting, washing, and concentrating the cells, and storing the concentrated cells in a preservation container, thereby enabling the production of a "high-concentration cell suspension." Further details are provided in the examples described below. [Examples]

[0032] The embodiments of the present invention will be described in detail below. However, the present invention is not limited to these embodiments. Using the high-concentration cell preservation solution of the present invention, five types of cells (LCAT gene-modified human preadipocytes, human preadipocytes, human adipose tissue-derived mesenchymal stem cells, FVIII gene-modified human preadipocytes, and human bone marrow-derived mesenchymal stem cells) were preserved at high concentration at 4°C, and the cell viability after a certain period of time was verified. For the cell preservation solution described above, the "basic medium" was RPMI1640 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 consisting of Ringer's solution was used.

[0033] <Example 1> A high-concentration cell preservation solution was prepared as described in steps (1-1) to (1-4) below, and a high-concentration cell suspension was prepared by suspending "LCAT gene-transformed human pre-adipocytes" in the cell preservation solution, and then stored. Step (1-1) Preparation of human pre-adipocytes into which the LCAT gene will be introduced. Adipose tissue (10g) obtained from healthy individuals was dispensed into 50mL centrifuge tubes in 1g portions. 3mL each of HBSS (Sigma-Aldrich) containing 2mg / mL collagenase (Nordmark), 40μg / mL gentamicin (Takada Pharmaceutical Co., Ltd.), and 4μg / mL vancomycin (Pfizer) was added to each tube, and the mixture was shaken 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 (manufactured by Nichirei Biosciences), 40 μg / mL gentamicin, and 4 μg / mL vancomycin was added and stirred. The mixture was then centrifuged at 400 × g for 1 minute to remove the solution containing the precipitate fraction. Furthermore, 10 mL of DMEM-HAM / 20%FBS(GM-VCM) was added and mixed, then centrifuged at 400 × g for 1 minute. This series of operations, including removing the precipitated fraction, was repeated twice to obtain the supernatant fraction containing human preadipocytes. The supernatant fraction containing human preadipocytes obtained in this manner was filtered through a 440 μm mesh. Then, 10 mL of DMEM-HAM / 20%FBS(GM-VCM) was added to the original 50 mL centrifuge tube for washing, and the washing solution was also filtered. The filtrate obtained in this way is then placed in a 150 cm³ container filled with DMEM-HAM / 20%FBS(GM-VCM) that has been preheated to 37°C. 2 In addition to the flask, DMEM-HAM / 20%FBS(GM-VCM) was added to minimize the amount of air bubbles remaining in the flask, and then the flask was sealed. The flask was placed in a 37°C, 5% CO2 incubator with the bottom (culture surface) facing the ceiling, and cultured on the ceiling for 7 days. After 7 days, the culture medium in the flask was removed, the culture surface was washed with D-PBS, and then treated with trypsin to collect human preadipocytes (4.5 × 10⁶). 7 cells).

[0034] Step (1-2) Creation of human pre-adipocytes with LCAT gene transfer A retroviral vector was prepared for gene transfer as follows. The LCAT gene, cloned from a cDNA library of human hepatocellular carcinoma cell lines, was inserted into a pDON-AI vector plasmid (Takara Bio). A Kozak sequence was added upstream of the 5' start codon (ATG) of the inserted LCAT gene, and the 3' stop codon was modified to remove the polyA signal, thereby creating a retroviral vector plasmid carrying the human LCAT gene. Using this plasmid, a retroviral vector solution containing the human LCAT gene, suitable for introduction into human cells, was obtained using a packaging system (manufactured by Takara Bio).

[0035] Six days after the start of ceiling culture in the above step (1-1) (the day before harvesting human pre-adipocytes), a 20 μg / mL RetroNectin (Takara Bio Inc.) solution diluted in D-PBS is placed in a 225 cm² container for suspension cells. 2 45 mL was added to the flask and left to stand overnight at 4°C. Next, after removing the RetroNectin solution, the flask was washed once with D-PBS and prepared as a RetroNectin-coated flask. The human LCAT gene-containing retroviral vector solution was coated with 20% FBS and DMEM-HAM / 20% FBS(GM) containing 40 μg / mL gentamicin in 5 × 10⁻¹⁴ solutions. 8 After diluting to RNAcopies / mL, 45 mL was added to a RetroNectin-coated flask and allowed to stand at 32°C for 5 hours. After 5 hours, the vector solution was removed, and the flask was converted into a viral vector-coated flask.

[0036] The human preadipocytes collected in the above step (1-1) were 225 cm². 2 2.25 × 10 per flask 6 Cells were seeded in a viral vector-coated flask, and gene transfer was initiated. Two days after gene transfer, the culture medium was changed to "MSF-BM / MSF-supplement A (Shimadzu Diagnostics)" containing 40 μg / mL gentamicin. Two days later, the cells in the flask were harvested and used as LCAT gene-transfected human pre-adipocytes. Before the start of the experiment, the cells were precipitated by centrifugation at 300×g, 4°C, for 5 minutes, suspended in cell cryopreservation solution (CELLBANKER1: manufactured by Nippon Zenyaku Kogyo Co., Ltd.), and cryopreserved at -80°C.

[0037] Steps (1-3) Preparation of cell preservation solution To 500 mL of RPMI1640 (L-glutamine-containing) medium (manufactured by Nacalai Tesque), which serves as the "basic medium," 10 mL of Recombumin Elite (Albumedix), which is "recombinant human serum albumin," was added and mixed. Furthermore, 1 mL of Vitamin C Injection "Fuso" - 500 mg (manufactured by Fuso Pharmaceutical Industries Co., Ltd.), which is "ascorbic acid," was added and mixed. The mixture was then filtered and sterilized to obtain the cell preservation solution (the preservation solution before suspending the cells). The preparation was carried out as described above, resulting in an albumin concentration of 0.2 (w / v)% and an ascorbic acid concentration of 0.5 mg / mL. The concentrations for Examples 2 and 3 were the same.

[0038] Steps (1-4): Cell harvesting, washing, concentration, and storage. In the above steps (1-2), LCAT gene-transformed human pre-adipocytes were thawed and grown in a 37°C, 5% CO2 incubator using a Nunc EasyFill Cell Factory (Thermo Fisher Scientific). After removing the culture medium and washing the culture surface with D-PBS, the cells were treated with trypsin. After collecting the cells using the cell preservation solution prepared in the above steps (1-3), the cells were centrifuged at 3,000 × g at 4°C for 3 minutes, and the supernatant was removed. The cells were then resuspended in the cell preservation solution, centrifuged at 3,000 × g at 4°C for 3 minutes, and the supernatant was removed three times to wash the cells. After measuring the cell concentration, the cells were centrifuged at 3,000 × g at 4°C for 5 minutes, and the concentration was reduced to 1 × 10⁻⁶. 8 The cells were suspended in a cell preservation solution to a concentration of cells / mL. The cell suspension was dispensed into storage containers and stored at 4°C. After following steps (1-1) to (1-4) above, the high-concentration cell suspension of Example 1 was obtained by suspending "LCAT gene-modified human pre-adipocytes" in a high-concentration cell preservation solution.

[0039] <Example 2> As described below, a high-concentration cell preservation solution was prepared, and a high-concentration cell suspension was prepared by suspending "human pre-adipocytes" (without gene introduction) in the cell preservation solution, and then stored. Human preadipocytes were collected from adipose tissue obtained from healthy individuals according to the method of step (1-1) in Example 1. Until the start of the experiment, the cells were suspended in cell cryopreservation solution (CELLBANKER1) and cryopreserved at -80°C. A cell preservation solution was prepared according to the method of steps (1-3) of Example 1, and cells were collected, washed, concentrated, and preserved according to the method of step (1-4). Following the above steps, a high-concentration cell suspension of Example 2 was obtained by suspending "human pre-adipocytes" in a 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 then stored. Human adipose tissue-derived mesenchymal stem cells (manufactured by Thermo Fisher Scientific) were cultured and the cells were proliferated. Until the start of the experiment, the cells were suspended in cell cryopreservation solution (CELLBANKER1) and cryopreserved at -80°C. A cell preservation solution was prepared according to the method of steps (1-3) of Example 1, and cells were collected, washed, concentrated, and preserved according to the method of step (1-4). Following the above steps, a high-concentration cell suspension of Example 3 was obtained by suspending "human adipose tissue-derived mesenchymal stem cells (human adipose-derived stem cells)" in a high-concentration cell preservation solution.

[0041] <Comparative Examples 1 and 2> Commercially available cell suspension preservation solutions, such as dextran 40 and trehalose-containing lactated Ringer's solution, were used as the cell preservation solution in Comparative Example 1. Furthermore, trehalose-containing lactated Ringer's solution, which is commercially available as a cell washing and preservation solution, was used as the cell preservation solution in Comparative Example 2. Specifically, the preparation was carried out as described in the following steps (2-1) to (2-4).

[0042] Step (2-1) Preparation of human pre-adipocytes Following the method of step (1-1) of Example 1, an HBSS containing 2 mg / mL collagenase and 400 μg / mL vancomycin was used instead of an HBSS containing 2 mg / mL collagenase, 40 μg / mL gentamicin, and 4 μg / mL vancomycin. Furthermore, human pre-adipocytes were obtained using DMEM-HAM / 20%FBS(GM) instead of DMEM-HAM / 20%FBS(GM-VCM).

[0043] Step (2-2) Preparation of human pre-adipocytes with LCAT gene transfer Following the procedure in steps (1-2) of Example 1, LCAT gene-transformed human pre-adipocytes were prepared using MesenPRO RS Medium (Thermo Fisher Scientific) instead of MSF-BM / MSF-supplement A. Until the start of the experiment, the cells were suspended in a cell cryopreservation solution (CELLBANKER1) and stored at -80°C.

[0044] Step (2-3) Preparation of cell washing solution for comparison To 500 mL of Ringer's solution (manufactured by Otsuka Pharmaceutical Co., Ltd.), which served as the "cell washing solution" instead of the "basic culture medium," 10 mL of 25% donated blood albumin (manufactured by Japan Blood Products Organization), which served as the "additive," was added and mixed. After filtration, a comparative cell washing solution was prepared.

[0045] Steps (2-4) Cell collection, washing, concentration, and storage Following the method of steps (1-4) in Example 1, the cells were collected and washed using the cell washing solution prepared in step (2-3) above, resulting in 0.5 × 10⁻⁶ cells. 8 The cells were suspended in Comparative Example 1 and Comparative Example 2 respectively to achieve a concentration of 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-modified human pre-adipocytes" in a commercially available cell preservation solution mainly composed of Ringer's solution.

[0046] <Example Test 1: Calculation of Viability Based on Cell Morphology (Different Cell Types)> For the high-concentration cell suspensions of Examples 1, 2, and 3, and Comparative Examples 1 and 2, the viability of cells was evaluated based on the morphology of the cells immediately after storage. High-concentration cell suspensions in each storage container were measured at 0 hours, 48 ​​hours, and 72 hours after the start of storage at 4°C (Example: 1 × 10 8 cells / mL, comparative example: 0.5×10 8 After removing cells / mL and serially diluting them with each cell culture medium, 5 × 10 5 The cells were prepared as a cell suspension at a concentration of cells / mL. The "total cell concentration" and "dead cell concentration" of these cell suspensions were measured using a NucleoCounter NC-100 (Chemometec). The "dead cell concentration" was calculated based on the number of cells stained with propidium iodide. The "live cell percentage (%)" was calculated based on the measured values ​​of both. Note that "live cell rate (%)" is equivalent to "cell viability rate (%)".

[0047] (Results and discussion of Test Example 1) Figure 1 summarizes the changes in the viable cell rate (cell survival rate) over time in each example and comparative example. Recombinant human serum albumin is denoted as "rHA" and ascorbic acid as "AA". From the results of Test Example 1, the high-concentration cell suspensions (1 × 10) of Examples 1, 2, and 3 were found to be 8 At high concentrations (cells / mL), the cell viability remained high even 72 hours after the start of storage, regardless of the cell type. Specifically, when cells were stored at 4°C using a high-concentration cell preservation solution, the cell viability after 72 hours was found to be over 80%, more specifically over 85%, more specifically over 90%, and more specifically over 95% (Example 1: 98.3%, Example 2: 97.8%, Example 3: 95.9%). Furthermore, multiple tests were conducted as described above, and it was confirmed that they yielded similar results.

[0048] In the high-concentration cell suspensions of Comparative Examples 1 and 2, a decrease in cell viability was observed. Specifically, when cells were stored at 4°C using a commercially available cell preservation solution mainly composed of 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 in cases where the cell concentration is low (for example, 1 × 10⁻⁶ 7 When the cell concentration is lower than cells / mL, it has been confirmed that cells can be preserved even with a cell preservation solution mainly composed of Ringer's solution, as in Comparative Examples 1 and 2. On the other hand, when the cell concentration is high (for example, 1 × 10⁻⁶), it has been confirmed that cell preservation is possible. 8 For cells / mL, it has been shown that compositions using Ringer's solution, etc., are not suitable.

[0049] <Example Test 2: Evaluation of Cell Survival by Measuring Intracellular Dehydrogenase Activity> Test Example 1 only examined the morphology of the cells. To confirm whether the cells were properly preserved while maintaining their function, the cells were seeded in a culture vessel after the end of preservation, and cell survival 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, the cell suspensions were removed from each storage container at 0 hours, 48 ​​hours, and 72 hours after the start of storage at 4°C, and after serial dilution with each cell culture medium, 4 × 10⁻⁶ cells were obtained. 4 Cells were prepared as a cell / mL suspension and seeded in 100 μL in each of the 96-well plates. Additionally, wells containing only culture medium without cells were prepared. These samples were incubated at 37°C in a 5% CO2 incubator for 20 hours. Following this, they were tested according to the protocol provided with the Cell Counting Kit-8 (manufactured by Dojin Chemical Laboratories Co., Ltd.), and the optical density (OD value) was obtained by measuring the absorbance at a wavelength of 450 nm. The OD value of each test sample was calculated by subtracting the OD value of the culture medium alone, and the relative value (optical density ratio) was calculated with the value for 0 hours of storage set to 1.

[0050] (Results and discussion of Test Example 2) Figure 2 summarizes the changes over time in the OD value ratio (optical density ratio) for each example and comparative example. The results of Test Example 2 showed that, unlike the high-concentration cell suspensions of Comparative Examples 1 and 2, the high-concentration cell suspensions of Examples 1, 2, and 3 maintained high metabolic activity (high OD ratio) even after 72 hours from the start of storage, indicating that cell survival was maintained. More specifically, in Examples 1, 2, and 3, when cells were stored at 4°C using a high-concentration cell preservation solution, the OD value ratio to the 0-hour storage time after 72 hours was found to be 0.5 or higher, more specifically 0.6 or higher, and 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 detection of lactate dehydrogenase (LDH) in culture medium> To evaluate the damage to cells stored in high-concentration cell preservation solutions, we measured the amount of free LDH induced by cell membrane damage. For the high-concentration cell suspensions of Examples 1, 2, and 3, cells were extracted at 0 hours, 48 ​​hours, and 72 hours after the start of storage at 4°C, respectively, and after serial dilution with each cell culture medium, resulting in 4 × 10⁶ cells. 4 The culture medium was adjusted to cells / mL and seeded in 1000 μL in two wells of a 12-well plate. Two wells containing only culture medium without cells were also prepared. These samples were incubated at 37°C in a 5% CO2 incubator for 20 hours. Then, Lysis Buffer, included with the LDH cytotoxicity assay kit (Nacalai Tesque), was added to one of the two wells of each test sample, and incubated at 37°C in a 5% CO2 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 provided with the LDH cytotoxicity assay kit, and the OD value was obtained by measuring the absorbance at a wavelength of 492 nm. The OD value of each test sample was calculated by subtracting the OD value of the culture medium alone, and the relative value to the sample with Lysis Buffer added was calculated.

[0052] (Results and discussion of Test Example 3) Figure 3 summarizes the changes over time in the relative OD values ​​for each example. The results of Test Example 3 showed that the high-concentration cell suspensions of Examples 1, 2, and 3 exhibited low relative OD values ​​even 72 hours after the start of storage, indicating no cell damage. More specifically, in Examples 1, 2, and 3, when cells were stored at 4°C with a high-concentration cell preservation solution, the relative values ​​after 72 hours compared to the sample with added Lysis Buffer were found to be 10% or less, more specifically 8% or less (Example 1: 2.33%, Example 2: 3.75%, Example 3: 7.80%).

[0053] <Example 4> As described in Example 2 above, a high-concentration cell suspension of Example 4 was obtained by suspending "human pre-adipocytes" in a high-concentration cell preservation solution. For the above high-concentration cell suspension, the "cell concentration" is 1 × 10⁻⁶ 8 The cells / mL ratio was set to RPMI1640 (containing L-glutamine) medium as the "basic medium," and recombinant human serum albumin (rHA) and ascorbic acid (AA) as the "additives." Furthermore, albumin concentrations were set to 0.1(w / v)%, 0.2(w / v)%, and 2.0(w / v)%, and ascorbic acid concentrations were set to 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 viable cell rate based on cell morphology (with different concentrations of additives)> In the same manner as in Example 1, the viability of cells was evaluated for the high-concentration cell suspensions of Example 4, which had different concentrations of albumin and ascorbic acid.

[0055] (Results and discussion of Test Example 4) Figures 4 and 5 summarize the changes in the viability rate (cell survival rate) over time in Example 4, which had different albumin and ascorbic acid concentrations. Recombinant human serum albumin is denoted as "rHA" and ascorbic acid as "AA". From the results of Test Example 4-1, the high-concentration cell suspension of Example 4 (1 × 10) when the albumin concentration (rHA concentration) was set to 0.1-2.0 (w / v)% 8 In the case of cells / mL, a high cell viability rate was observed even after 72 hours from the start of storage. Specifically, it was found that when cells were stored at 4°C, the cell viability rate after 72 hours was 90% or higher (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, the results from Test Example 4-2 showed that the high-concentration cell suspensions of Example 4, with ascorbic acid (AA) concentrations ranging from 0.1 mg / mL to 10 mg / mL, exhibited high survival rates even 72 hours after the start of storage. Specifically, it was found that when cells were stored at 4°C, the cell survival rate after 72 hours was over 90% (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%). Multiple tests were conducted as described above, and it was confirmed that they yielded similar results. Furthermore, the high-concentration cell suspension of Example 4 was evaluated for cell viability in the same manner as in Test Example 2, and the survival of cells was confirmed. In addition, the same cytotoxicity evaluation as in Test Example 3 was performed, and it was confirmed that no cytotoxicity was observed.

[0056] <Examples 5 and 6> As in Example 1 above, high-concentration cell suspensions for Examples 5 and 6 were obtained by suspending "LCAT gene-transformed human pre-adipocytes" in a high-concentration cell preservation solution. For Example 5, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cells / mL ratio was set to RPMI1640 (containing L-glutamine) as the "basic medium," and recombinant human serum albumin (rHA), ascorbic acid (AA), and sodium pyruvate (SP) as "additives." The albumin concentration was set to 0.5 (w / v)%, the ascorbic acid concentration to 5 mg / mL, and the sodium pyruvate concentration to 1 mM. For Example 6, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cells / mL ratio was set to DMEM (low glucose) (Nacalai Tesque Corporation) as the "basic medium," and recombinant human serum albumin (rHA) and ascorbic acid (AA) were used as "additives." The albumin concentration was set to 0.5 (w / v)%, and the ascorbic acid concentration to 5 mg / mL. Since the RPMI1640 in Example 5 does not contain sodium pyruvate, sodium pyruvate was added separately. Since the DMEM in Example 6 contains sodium pyruvate, the same compositional conditions were used.

[0057] <Test Example 5: Calculation of viable cell rate based on cell morphology (different basal media)> In the same manner as in Test Example 1, the viability of high-concentration cell suspensions from Examples 5 and 6, which used different basal media, was evaluated.

[0058] (Results and discussion of Test Example 5) Figure 6 summarizes the changes in the viability rate (cell survival rate) over time in Examples 5 and 6. Recombinant human serum albumin is denoted as "rHA," ascorbic acid as "AA," and sodium pyruvate as "SP." Based on the results of Test Example 5, the high-concentration cell suspensions (1 × 10) of Examples 5 and 6, in which the basal culture medium was "RPMI1640" and "DMEM", were obtained. 8In the case of cells / mL, a high cell viability rate was observed even after 72 hours from the start of storage. Specifically, when cells were stored at 4°C, the cell viability rate after 72 hours was shown to be 80% or higher (Example 5: 90.9%, Example 6: 84.2%). Furthermore, it was shown that using "RPMI1640" as the basal culture medium resulted in higher cell viability than using "DMEM". Multiple such tests were conducted, and similar results were confirmed. Furthermore, the high-concentration cell suspensions of Examples 5 and 6 were evaluated for cell viability in the same manner as in Test Example 2, and the survival of the cells was confirmed.

[0059] <Example 7> A high-concentration cell preservation solution was prepared as described in steps (7-1) to (7-3) below, and a high-concentration cell suspension was prepared by suspending "FVIII gene-transformed human pre-adipocytes" in the cell preservation solution and then stored. Step (7-1) Preparation of human pre-adipocytes into which the FVIII gene is introduced. Human preadipocytes were collected from adipose tissue obtained from healthy individuals according to the method of step (1-1) in Example 1. Until the start of the experiment, the cells were suspended in cell cryopreservation solution (CELLBANKER1) and cryopreserved at -80°C.

[0060] Step (7-2) Preparation of human pre-adipocytes with FVIII gene transfer A lentiviral vector was prepared for gene transfer as follows. First, a "modified FVIII" was created in which the number of N-glycosylated Asn residues in the B domain of the human FVIII gene was reduced to 5 from the N-terminus, the function of the furin recognition site was lost, and the Pro residue at position 758 was replaced with a Thr residue. A nucleotide sequence with a signal peptide added to this "modified FVIII" was inserted into a pLVSIN vector plasmid (manufactured by Takara Bio Inc.) to create a lentiviral vector plasmid carrying the human FVIII gene. Using this plasmid, a lentiviral vector solution containing the human FVIII gene, suitable for introduction into human cells, was obtained using a packaging system (manufactured by Takara Bio Inc.). Thaw the pre-adipocytes that were frozen in the above step (7-1), and 150 cm 2 The samples were incubated overnight in a flask in a 37°C, 5% CO2 incubator. The culture medium was removed the following day, the culture surface was washed with D-PBS, and then pre-adipocytes were recovered by trypsin treatment. The human FVIII gene-containing lentiviral vector solution obtained in this process was 1 × 10⁻⁶ 9 RNAcopies / mL, containing 100 μg / mL protamine sulfate, cell concentration 2.5 × 10⁶ 4 The solution was adjusted with MSF-BM / MSF-Supplement A to achieve a cell / mL concentration. Then, it was 25cm long. 2 Gene transfer was initiated by seeding 5 mL into a flask, and the culture medium was changed the following day. The gene transfer procedure was performed again on the 2nd and 7th days following the first gene transfer (a total of 3 times), resulting in "FVIII gene-transferred human pre-adipocytes." Before the start of the experiment, the cells were precipitated by centrifugation at 300×g, 4°C, for 5 minutes, suspended in cell cryopreservation solution (CELLBANKER1), and cryopreserved at -80°C.

[0061] Step (7-3) Cell harvesting, washing, concentration and storage A cell preservation solution was prepared using "RPMI1640 (L-glutamine-containing) medium" as the basal medium according to the method of steps (1-3) of Example 1, and cells were collected, washed, concentrated, and preserved according to the method of step (1-4). Following the above steps, a high-concentration cell suspension of Example 7 was obtained by suspending "FVIII gene-transformed human pre-adipocytes" in a high-concentration cell preservation solution using "RPMI1640 (L-glutamine-containing) medium" as the base medium.

[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 then stored. Human bone marrow-derived mesenchymal stem cells (PromoCell) were cultured and the cells were proliferated. Until the start of the experiment, the cells were suspended in cell cryopreservation solution (CELLBANKER1) and cryopreserved at -80°C. A cell preservation solution was prepared according to the method of steps (1-3) of Example 1, and cells were collected, washed, concentrated, and preserved according to the method of step (1-4). Following the above steps, a 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 "RPMI1640 medium" as the base medium.

[0063] <Test Example 6: Calculation of viable cell rate based on cell morphology (different cell types)> In the same manner as in Test Example 1, the cell viability of high-concentration cell suspensions from Examples 1, 2, 7, and 8, and Comparative Examples 1 and 2, which use different cell types, was evaluated. Furthermore, multiple samples were used in the tests for Examples 1, 2, and 7.

[0064] (Results and discussion of Test Example 6) Figure 7 summarizes the changes in the viability rate (cell survival rate) over time in the examples and comparative examples using different cell types. Recombinant human serum albumin is denoted as "rHA" and ascorbic acid as "AA". From the results of Test Example 6, the high-concentration cell suspensions (1 × 10) of Examples 1, 2, 7, and 8 were found to be 8 At high cell concentrations (cells / mL), the cell viability remained high even 72 hours after the start of storage, regardless of the cell type. Specifically, when cells were stored at 4°C using a high-concentration cell preservation solution, the cell viability after 72 hours was found to be over 80%, more specifically over 85%, more specifically over 90%, and more specifically over 95% (Example 1: 98.4% (n=3), Example 2: 96.7% (n=4), Example 7: 96.7% (n=3), Example 8: 95.3% (n=1)). Furthermore, for Examples 7 and 8, cell viability and cytotoxicity evaluations similar to those for Test Examples 2 and 3 were also performed, and it was confirmed that high metabolic activity was maintained even 72 hours after the start of storage, and that no cytotoxicity was observed.

[0065] <Examples 9 and 10> As in Example 2 above, high-concentration cell suspensions for Examples 9 and 10 were obtained by suspending "human pre-adipocytes" without gene introduction in a high-concentration cell preservation solution. For Example 9, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cell density was set to cells / mL, the "basic medium" was RPMI1640, and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was set to 0.2 (w / v)%, and the ascorbic acid concentration to 0.5 mg / mL. For Example 10, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cell density was set to cells / mL, the "basic medium" was DMEM (low glucose), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was set to 0.2 (w / v)%, and the ascorbic acid concentration to 0.5 mg / mL.

[0066] <Examples 11, 12> As in Example 1 above, high-concentration cell suspensions for Examples 11 and 12 were obtained by suspending "LCAT gene-transformed human pre-adipocytes" in a high-concentration cell preservation solution. For Example 11, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cell density was set to cells / mL, the "basic medium" was RPMI1640 (containing L-glutamine), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was set to 0.2 (w / v)%, and the ascorbic acid concentration to 0.5 mg / mL. For Example 12, the "cell concentration" was set to 1 × 10⁻⁶. 8 The cell density was set to cells / mL, the "basic medium" was DMEM (low glucose), and the "additives" were recombinant human serum albumin (rHA) and ascorbic acid (AA). The albumin concentration was set to 0.2 (w / v)%, and the ascorbic acid concentration to 0.5 mg / mL.

[0067] Although the RPMI1640 used in Examples 9 and 11 does not contain sodium pyruvate, it was confirmed that the presence or absence of sodium pyruvate has virtually no effect on cell viability, cell survival maintenance, or cytotoxicity, therefore, sodium pyruvate was not added.

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

[0069] (Results and discussion of Test Example 7) Figure 8 summarizes the changes in the viability rate (cell survival rate) over time in Examples 9, 10, 11, and 12. Recombinant human serum albumin is denoted as "rHA" and ascorbic acid as "AA". Based on the results of Test Example 7, the high-concentration cell preservation solutions (1 × 10) for Examples 9, 10, 11, and 12 were prepared using "RPMI1640" and "DMEM" as the basal culture media, and "LCAT gene-transformed human pre-adipocytes" and "human pre-adipocytes" as the preserved cells. 8 In the case of cells / mL, a high cell viability rate was observed even after 72 hours from the start of storage. Specifically, when cells were stored at 4°C, the cell viability rate after 72 hours was shown to be 96% or higher (Example 9: 97.6%, Example 10: 96.8%, Example 11: 98.8%, Example 12: 97.4%).

[0070] Furthermore, it was shown that using "RPMI1640" as the basal culture medium resulted in higher cell viability than using "DMEM" (the cell viability in Examples 9 and 11 was higher than that in Examples 10 and 12, respectively). Multiple such tests were conducted, and similar test results were confirmed. Furthermore, it was confirmed that in Examples 9 and 11, the cell viability was higher when sodium pyruvate was not added. Furthermore, for Examples 9 and 10, cell viability and cytotoxicity evaluations were performed in the same manner as in Test Examples 2 and 3, confirming that cells remained viable and that no cytotoxicity was observed.

[0071] <Example Test 8: Calculation of Viability Rate Based on Cell Morphology (Long-Term Survival Rate)> In the same manner as in Test Example 1, the long-term survival rates of high-concentration cell suspensions from Examples 9 and 10, which used different basal media, were evaluated.

[0072] (Results and discussion of Test Example 8) Figure 9 shows a summary of the changes in the viable cell rate (cell survival rate) over time in Examples 9 and 10. From the results of Test Example 8, the high-concentration cell suspensions (1 × 10) of Examples 9 and 10 were found to be 8 In the case of cells / mL, a high cell viability rate was observed even after 170 hours from the start of storage. Specifically, it was found that when cells were stored at 4°C in a high-concentration cell preservation solution, the cell viability rate after 170 hours was 80% or higher. In particular, in Example 9, where the basal medium was "RPMI1640 medium," the cell viability rate after 170 hours was found to be 90% or higher (Example 9: 94.0%, Example 10: 81.4%). Furthermore, multiple tests were conducted as described above, and it was confirmed that they yielded similar results.

[0073] <Note> Examples 1 to 12 can be summarized as follows: Example 1: LCAT gene-transformed human pre-adipocytes, 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-transformed human pre-adipocytes, RPMI1640, sodium pyruvate Example 6: LCAT gene-transformed human pre-adipocytes, DMEM Example 7: Human pre-adipocytes with FVIII gene transfer, 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-transformed human pre-adipocytes, RPMI1640 Example 12: LCAT gene-transformed human pre-adipocytes, DMEM

Claims

1. A cell preservation solution that preserves cells without freezing them, Basic culture medium and The additive added to the aforementioned basal culture medium contains, The aforementioned basal culture medium is a medium used for cell culture and for preserving the cells, The aforementioned additive comprises at least genetically modified albumin and ascorbic acid or an ascorbic acid derivative. The cells mentioned above are at least 1 × 10 ７ Store at a cell concentration of cells / mL. The aforementioned cells 1 × 10 ７ When stored at 4°C with a cell concentration of cells / mL, the cell viability after 72 hours is 80% or higher, the cells maintain their function, and no cytotoxicity is observed during storage. The aforementioned cells are somatic cells or stem cells of human origin, The basal culture medium is at least one culture medium selected from the group consisting of RPMI1640 and DMEM. The concentration of the recombinant albumin is 0.1 to 2.0 (w / v)%, A high-concentration cell preservation solution characterized in that the concentration of the ascorbic acid or the ascorbic acid derivative is 0.1 to 10 mg / mL.

2. The genetically modified albumin is genetically modified human serum albumin, The high-concentration cell preservation solution according to claim 1, characterized in that the cell preservation solution is used for cell transplantation.

3. The aforementioned cells 1 × 10 ８ ~1 x 10 ９ The high-concentration cell preservation solution according to claim 1, characterized in that it is stored at a cell concentration of cells / mL.

4. The aforementioned cells 1 × 10 ７ When stored at 4°C with a cell concentration of cells / mL, Using Cell Counting Kit-8, the OD value ratio (optical density ratio) to the 0-hour storage value after 72 hours is 0.7 or higher, indicating that the cells are preserved while maintaining their function, and The high-concentration cell preservation solution according to claim 1, characterized in that the cells are preserved in a state in which no cytotoxicity is observed, with a relative OD value (optical density) of 8% or less after 72 hours using an LDH cytotoxicity assay kit.

5. The aforementioned cells 1 × 10 ８ The high-concentration cell preservation solution according to any one of claims 1 to 4, characterized in that, when stored at 4°C with a cell concentration of cells / mL, the cell viability after 72 hours is 80% or more, the cells maintain their function, and the cells are preserved without any cell damage.

6. The aforementioned cells 1 × 10 ８ The high-concentration cell preservation solution according to any one of claims 1 to 4, characterized in that, when stored at 4°C with a cell concentration of cells / mL, the cell viability after 170 hours is 80% or more, the cells maintain their function, and no cytotoxicity is observed during storage.

7. Suspend the cells in a cell preservation solution, and without freezing the cells, store at least 1 × 10⁻¹⁶ cells. ７ A method for preserving cells at a cell concentration of cells / mL, The cell preservation solution comprises a basal culture medium and an additive added to the basal culture medium. The aforementioned basal medium is a medium used for cell culture and for preserving the cells, The aforementioned additive comprises at least genetically modified albumin and ascorbic acid or an ascorbic acid derivative. When the cells are stored at 4°C at a cell concentration of 1×10 ７ cells / mL, the cell viability after 72 hours is 80% or more, the cells are in a state of maintaining their function, and the cells are stored in a state where no cell damage is observed. The aforementioned cells are somatic cells or stem cells of human origin, The basal culture medium is at least one culture medium selected from the group consisting of RPMI1640 and DMEM. The concentration of the recombinant albumin is 0.1 to 2.0 (w / v)%, A method for high-concentration cell preservation, characterized in that the concentration of the ascorbic acid or the ascorbic acid derivative is 0.1 to 10 mg / mL.

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