Cell Treatment Agents

Abstract

To provide a method for suppressing extinction of cells in a tissue or cultured cells, holding viability of the cells, for example, easily performing peeling / floating processing and dormancy processing (by dormancy processing, it is possible to suppress unnecessary differentiation of cells to maintain undifferentiation and low-differentiation state, in addition to enhancing a protection effect), and easily performing protection processing for preservation and carrying, in a safe manner, and a method for easily and safely performing cell activation processing for implanting floated cells in a dormant state.SOLUTION: There is provided a cell processing agent including, as an active ingredient, at least one kind selected from a group consisting of: alginic acid; heparin; sulfate dextran; and a protein decomposition enzyme inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell treatment agent, and in particular to a cell treatment agent used to treat cells in tissues of living organs or cultured cells, such as cell detachment / suspension, dormancy, viability maintenance, death prevention, and suspension cell activation. [Background technology]

[0002] With the exception of unicellular organisms and specialized cells (non-adherent cells) such as blood cells and cancer cells, the vast majority of cells, known as adherent cells (hereafter simply referred to as "cells"), survive and proliferate by adhering to a scaffold made up of the extracellular matrix that constitutes the body in the body, or to a scaffold such as the wall of a culture vessel or a specific carrier in a culture substrate, thereby functioning and proliferating. When using such cells in research or medical applications, they need to be detached from the scaffold and suspended without weakening the cells.

[0003] The detachment and suspension process typically utilizes the cell detachment action of surfactants or cell detachment enzymes such as trypsin. For example, when detaching and suspending cells from a living organ and harvesting only the cells, a known processing method involves digesting the components that adhere the cells to the scaffold with a cell detachment agent such as a cell detachment enzyme or a surfactant, detaching and suspending the cells, and then washing and collecting the cells from the living organ (see Non-Patent Document 1 for the surfactant method using a surfactant). Another known processing method involves detaching and suspending cultured cells from a predetermined scaffold by adding a solution containing a cell detachment agent such as trypsin to a culture vessel and allowing the cell detachment agent to act on the cultured cells, resulting in the detachment and suspension of the cells (Non-Patent Document 2).

[0004] However, the surfactants and cell detachment enzymes used in these detachment and suspension processes also digest important components of the cells themselves. Therefore, if surfactants or cell detachment enzymes are left on cells after detachment and suspension, the suspended cells will weaken and eventually die. Thus, surfactants and cell detachment enzymes are cytotoxic. To protect suspended cells from this cytotoxicity, they are separated from the surfactant or cell detachment enzyme after suspension, or the separated cells are washed or inactivated to remove the cytotoxic effects of the surfactant or cell detachment enzyme. However, when using such cytotoxic cell detachment agents, some cell death is inevitable during the suspension process. Furthermore, for use in research and medical treatment, it is necessary to maintain the viability of suspended cells without weakening them during the detachment and suspension process.

[0005] In response to this situation, a method has been developed in which a special polymer is used as a temperature-sensitive cell culture scaffold, and the polymer is heated to a certain temperature to detach and suspend cells (Non-Patent Document 3). However, it has been pointed out that this method is expensive because it uses a special polymer, requires a special temperature control device, produces unstable results due to difficulties in temperature control, cannot be used in complex three-dimensional devices for culturing large amounts of cells, and is unable to detach and suspend cells from tissues in living organs.

[0006] Furthermore, when cells are used for research or medical purposes, they must be stored and transported safely. In this case, cells are generally stored in a liquid (storage and transport liquid) for transport and storage, but because cells weaken over time at room temperature, they are stored and transported in a refrigerated or frozen state, and then returned to room temperature before use.

[0007] Even when stored in a refrigerator or at room temperature, cells lose viability over time and die. To prevent this cell death and maintain cell viability, a conventional method involves adding 10% fetal bovine serum (FBS) or the patient's own serum to the liquid used to store and transport the cells. It is known that the addition of fetal bovine serum (FBS) or the patient's own serum dramatically increases cell viability (cytoprotective effect) compared to when these substances are not added (Non-Patent Documents 4-6).

[0008] However, the addition of FBS or autologous serum has been criticized for its potential problems, including the fact that these are biological preparations, infection, allergies, ease of handling, and ethics. At present, however, no substance is known that can adequately replace FBS or autologous serum.

[0009] Furthermore, cells, especially those currently in an undifferentiated or underdifferentiated state that have the potential to proliferate and regenerate or form tissues or organs in the future, may undergo unnecessary differentiation when preserved, resulting in changes that result in cells exhibiting properties different from those of the tissues or organs that are desired to be regenerated or formed using those cells. Maintaining an undifferentiated or underdifferentiated state by making the cells dormant throughout the period in which they are preserved and suppressing this unnecessary differentiation is an important aspect of regenerative medicine, but the means to achieve this have not yet been established, and the establishment of a solution to this problem is desired. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Crapo PM,.et al., An overview of tissue and whole organ decellularization processes. Biomaterials 32,3233-3243 (2011) [Non-patent document 2] Hayflick L., et al., Subculturing human diploid fibroblast cultures. Edited by Kruse, PFet al., Tissue Culture Methods and Applications.220-223 Academic Press (New York) (1973) [Non-patent document 3] Yamato M., et al., Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature.Tissue Engineering, 7, 473-480(2001) [Non-patent document 4] Juan Jose'Dorantes-Aranda et al. Novel application of a fish gill cell line assay to assess ichthyotoxicity of harmful marine microalgae.Harmful Algae.,10,366-373(2011) [Non-Patent Document 5] Liang C., et al. Serotonin promotes the proliferation of serum3 deprived hepatocellular carcinoma cells via upregulation of FOXO3a. Molecular Cancer 12 (2013) (open access) [Non-patent document 6] Julia C. et al. Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage. J. Immunol. Methods 382, ​​24-31 (2012) Summary of the Invention [Problem to be solved by the invention]

[0011] As mentioned above, when carrying out a process to detach and suspend intracellular cells or cultured cells from a biological organ or a culture substrate for biological cells, it is required that (a) cell death be prevented, and (b) the viability of the detached and suspending cells be maintained during the detachment and suspending process, i.e., the viability of the cells is not reduced.

[0012] Furthermore, when cells are preserved and transported, in order for the cells to function properly again, they must be protected to prevent cell death without reducing their viability.

[0013] Furthermore, cells, particularly those currently in an undifferentiated or underdifferentiated state that have the potential to proliferate and regenerate and form tissues and organs in the future, must be kept in an undifferentiated or underdifferentiated state by making their cellular functions dormant throughout the preservation period and suppressing unnecessary differentiation.

[0014] Furthermore, when cells that have been suspended and are in a dormant state are to be attached to living organs, culture substrates, etc., it is desirable that they can be attached easily, and it is necessary to activate and proliferate the attached cells.

[0015] However, for example, to allow detached and suspended cells to adhere to the wall of a culture vessel or a scaffold, i.e., to "implant" such cells, they must be kept in contact with the scaffold for 12 to 24 hours in a device that maintains normal culture conditions. If the contact is poor, the cells will not be able to implant. In particular, to implant cells in a specific localized area of ​​the body, it is necessary to maintain the cells in contact with the biological scaffold for 12 to 24 hours.

[0016] For this purpose, a scaffold such as a nonwoven fabric is usually contacted with cells ex vivo for 12 to 24 hours to allow the cells to adhere to the nonwoven fabric scaffold, and then the scaffold with the cells attached is transplanted into the desired location in the body. However, this transplantation method has the problem of being time-consuming and labor-intensive.

[0017] Therefore, an object of the present invention is to provide a method that can prevent the death of cells in tissues or cultured cells and maintain their viability, and that can safely and easily perform processes such as detachment / suspension treatment, dormancy treatment (dormancy not only enhances the protective effect but also suppresses unnecessary differentiation of cells, maintaining an undifferentiated / low-differentiated state), and protective treatment for storage and transportation, as well as a method that can safely and easily perform cell activation treatment for transplanting dormant cells (e.g., suspension cells, etc.). [Means for solving the problem]

[0018] The present inventors have conducted extensive research to solve the above-mentioned problems. As a result, they have found that the above-mentioned problems can be solved by using at least one selected from alginic acid, heparins, sulfated dextran, and protease inhibitors. The gist of the present invention is as follows.

[0019] (1) A cell treatment agent containing, as an active ingredient, at least one selected from alginic acid, heparins, dextran sulfate, and protease inhibitors. (2) The cell treatment agent according to the preceding item (1), which contains at least one selected from a cell culture medium, an extracellular fluid replacement solution, and a maintenance infusion solution. (3) The cell treatment agent according to the above (1) or (2), which is used for causing cells in tissues or cultured cells to become dormant. (4) The cell treatment agent according to the above (1) or (2), which is used for cell protection, maintaining the viability of cells in tissues or cultured cells and suppressing their death. (5) The cell treatment agent according to the above (1) or (2), which is used for cell preservation to maintain cells in an undifferentiated or less differentiated state. (6) The cell treatment agent according to the preceding paragraph (1) or (2), which is used for cell detachment and suspension, for detaching and suspending cultured cells from a biological tissue scaffold or a culture substrate scaffold, or intracellular cells of biological tissue from a biological tissue scaffold. (7) The cell treating agent according to any one of the above (1) to (5), which is a liquid for cell preservation, tissue preservation, or organ preservation. (8) A set reagent for awakening dormant cells, which is a combined reagent of the cell treating agent according to any one of the above (1) to (7) and a solid or liquid preparation containing a polyvalent cation.

[0020] By "scaffold" is meant what is known in the art as one of the three elements of regenerative medicine. Adhesive cells, which make up the majority of cells that make up living organisms, need to be attached to a fixed base in order to perform their original functions (including proliferation). In regenerative medicine, this base is called a "scaffold." Adhesive cells cannot perform their original functions when suspended in a liquid. These scaffolds can be artificial or natural. An example of an artificial scaffold is the wall of an artificial cell culture substrate such as a petri dish. Also, when cells are supported and attached to artificial fibers and then implanted into the body, the fibers that support the cells are considered a scaffold. Living tissues and organs are composed of cells and the extracellular matrix (including collagen fibers and proteoglycans) that surrounds them. Adhesive cells exist by adhering to this extracellular matrix and perform their functions in the body. An example of a scaffold in its natural state is the extracellular matrix to which cells adhere in living tissues. The three elements of regenerative medicine are the "cells" that make up tissues and organs, the "biologically active substances" that act as signaling factors for cell function, and the "scaffold" that allows the cells and bioactive substances to move about.

[0021] "Dormancy" means that cells cease proliferation, respiration, metabolism, and maintenance of a differentiated state while retaining viability (cessation of maintenance of a differentiated state also means maintenance of a dedifferentiated state, i.e., a less differentiated or undifferentiated state), or that cells cease adhesion.

[0022] "Protection" of cells means maintaining viability, that is, preventing the loss of the ability of cells to perform their functions and preventing cell death.

[0023] "Activation of suspended cells" refers to initiating proliferation, respiration, metabolism, and maintenance of differentiation of suspended cells in the "dormant" or "protected" state, or restoring adhesion, respiration, metabolism, and maintenance of differentiation of suspended cells in the "dormant" or "protected" state. Cell activation also refers to initiating proliferation, respiration, metabolism, and maintenance of differentiation of dormant cells, including non-suspended cells, in the "dormant" or "protected" state, or restoring adhesion, respiration, metabolism, and maintenance of differentiation of dormant cells in the "dormant" or "protected" state, and is synonymous with "awakening," described below.

[0024] Viability does not simply mean that a cell survives and does not die, but that the cell retains the ability to perform its original functions in the body (including proliferation, respiration, metabolism, and maintenance of a differentiated state). [Effects of the Invention]

[0025] According to the present invention, it is possible to provide a method for safely and simply performing processes such as exfoliation and suspension, dormancy, and protection for storage and transportation on cells in tissues or cultured cells while preventing their death and maintaining their viability, as well as a method for safely and simply performing cell activation processes for transplanting dormant cells. [Brief explanation of the drawings]

[0026] [Figure 1] In Experimental Example 5, the image of the calcium phosphate membrane surface when cells detached and suspended with dextran sulfate were cultured in a culture dish with a calcium phosphate membrane is shown. [Figure 2] In Experimental Example 5, the image of the calcium phosphate film surface when cells detached and suspended with heparin sodium were cultured in a culture dish with a calcium phosphate film was shown. [Figure 3] 10 shows an image of the bottom surface of a culture dish in Experimental Example 5, in which cells detached and suspended with dextran sulfate were cultured in the culture dish without a calcium phosphate membrane. [Figure 4] 10 shows an image of the bottom surface of a culture dish in Experimental Example 5, in which cells detached and suspended with heparin sodium were cultured in the culture dish without a calcium phosphate membrane. DETAILED DESCRIPTION OF THE INVENTION

[0027] The cell treatment agent according to an embodiment of the present invention contains at least one active ingredient selected from alginic acid, heparins, dextran sulfate, and protease inhibitors (hereinafter sometimes referred to as the "active ingredient of the treatment agent" or "active ingredient").

[0028] Alginate is a linear polysaccharide composed of two monosaccharides, β-D-mannuronic acid and α-L-guluronic acid, found in various brown algae worldwide, such as kelp and wakame seaweed. Its structure consists of an M block consisting of 1,4-linked β-(1-4)-D-mannuronic acid, a G block consisting of 1,4-linked α-(1-4)-L-guluronic acid, and an MG block consisting of alternating 1,4-linked mannuronic acid and guluronic acid. When dissolved in water, alginate forms a smooth, viscous aqueous solution (colloidal solution). The viscosity of this solution is proportional to the degree of polymerization of the alginate, with higher degrees of polymerization resulting in higher viscosity. Various types of alginate with different viscosities are commercially available, including those that form standard viscous solutions and those with a lower viscosity than standard solutions. However, different viscosity levels can be used for different applications. Here, we will explain the viscosity of alginic acid by dividing it into three types (a) to (c) for convenience. (a) High-viscosity alginic acid: For example, a 1% by weight aqueous solution has a relatively high viscosity of 60 mPa·s or more at 20°C. (b) Low-viscosity alginic acid: For example, a 1% by weight aqueous solution has a relatively low viscosity of 5 mPa·s or more but less than 60 mPa·s at 20°C. (c) Very low-viscosity alginic acid: For example, a 1% by weight aqueous solution has a very low viscosity of 5 mPa·s or less at 20°C, or a 10% by weight aqueous solution has a very low viscosity of 30 mPa·s or less at 20°C. High-viscosity (a) alginic acid tends to function more effectively than low-viscosity and very low-viscosity (c) alginic acid in a solution prepared to a concentration of 5 mg / ml or less, for example, when stored at room temperature (22°C) for a relatively short period of time, such as 24 hours. (b) Low-viscosity alginate, which has a relatively low viscosity, tends to be able to perform its function more effectively than (a) high-viscosity and (c) very low-viscosity alginate, for example, in a solution prepared to have a concentration of 0.5 to 10 mg / ml, when stored for a relatively long period of time, for example, 120 hours at room temperature (22°C).(c) Ultra-low viscosity alginate, which has a very low viscosity, tends to function more effectively than (a) high viscosity alginate and (b) low viscosity alginate, for example, in a solution prepared to a concentration of 10 mg / ml or more when stored for a relatively long period, for example, 168 hours, in a refrigerator (4°C). The various effects of alginate as a cell treatment agent generally tend to vary depending on the concentration, but the extent of this also varies depending on the type of cell, concentration, etc., so the concentration ranges mentioned above represent general trends.

[0029] "Alginic acid" includes pharmacologically acceptable salts of alginic acid. Such pharmacologically acceptable salts of alginic acid are formed by freeing the hydrogen ion of the carboxyl group of alginic acid and bonding it to a cation. Such cations may be any cation capable of forming a pharmacologically acceptable salt, and examples thereof include monovalent cations such as sodium ions, potassium ions, and ammonium ions, inorganic polyvalent ions such as calcium ions, magnesium ions, iron ions, and ammonium ions, and polyvalent cations such as organic polyvalent ions such as polylysine.

[0030] As the alginic acid described above, commercially available products can be used.

[0031] Heparins refer to heparin and heparinoids. Heparin is a type of glycosaminoglycan produced in the body primarily in the liver. Its number-average molecular weight is said to be approximately 3,000 to 35,000. It is an N-sulfate, N-acetyl, and O-sulfate-substituted linear polysaccharide in which uronic acid (D-glucuronic acid or L-iduronic acid) and D-glucosamine are alternately linked, and is said to have approximately three sulfate groups per disaccharide, making it the most sulfated acidic polysaccharide. Heparin possesses anticoagulant activity, lipid-clearing activity, etc. Heparinoids are modified and partially degraded heparin and have similar physiological activity to heparin. Heparinoids include, for example, salts of heparin capable of forming pharmacologically acceptable salts (e.g., alkali metal salts, alkaline earth metal salts, etc.), unfractionated heparin, low molecular weight heparin, danaparoid, and salts thereof, as well as anticoagulants such as fondaparinux. Examples of heparin salts include sodium salts, potassium salts, and ammonium salts. The low molecular weight heparin preferably has a number average molecular weight of 2000 to 8000. Commercially available heparins can be used.

[0032] Dextran sulfate (hereinafter sometimes referred to as DS) is dextran sulfate or a pharmacologically acceptable salt thereof. A pharmacologically acceptable salt of dextran sulfate is formed by freeing the hydrogen ion of the sulfonic acid group of dextran sulfate and binding it to a cation. Such a cation may be any cation capable of forming a pharmacologically acceptable salt, and examples thereof include monovalent cations such as sodium ion, potassium ion, and ammonium ion. The average molecular weight (Mw) of dextran sulfate is preferably 200 to 1,000,000. The sulfur content is preferably 0.00001 to 2, more preferably 0.001 to 2, as the number of sulfate groups bound per monosaccharide. The average molecular weight (Mw) and sulfur content can be measured according to the method described in the Japanese Pharmacopoeia. Commercially available dextran sulfate can be used.

[0033] The protease inhibitor (hereinafter sometimes simply referred to as "inhibitor") is also called a protease inhibitor, and is not particularly limited, and various commercially available inhibitors can be used. Examples of such protease inhibitors include, but are not limited to, urinastatin, benzamidine, phenylmethylsulfonyl fluoride (PMSF), 4-(2-aminoethyl)benzene fluoride (AEBSF), aprotinin, E-64, ethylenediaminetetraacetic acid (EDTA), glycoletherdiaminetetraacetic acid (EGTA), leupeptin, leupeptin hemisulfate, antipain, chymostatin, pepstatin A, phosphoramidon, bestatin, sivelestat sodium hydrate, fosamprenavir calcium hydrate, darunavir ethanolate, lopinavir, ritonavir, aprotinin and pharmacologically acceptable salts thereof, as well as nafamostat mesylate, alafenamide fumarate, camostat mesylate, atazanavir sulfate, etc. These may be used alone or in combination.

[0034] Alginic acid, heparins, dextran sulfate and protease inhibitors may be used singly or in combination of two or more.

[0035] The dosage form of the cell treatment agent is not particularly limited, and can be powder, liquid, or the like, using an appropriate excipient depending on the intended use. Various additives can also be added as needed.

[0036] For example, if desired cells are cultured in a standard cell culture medium and then administered to the body, the cells can be separated from the cell culture medium, washed to remove various reagents not used in medical applications, and then immersed in an extracellular fluid replenisher or maintenance infusion, which can then be administered to the body. Extracellular fluid replenishers are a group of fluids with electrolyte compositions similar to those of the extracellular fluid surrounding cells in vivo. Maintenance infusions are infusions that contain the daily amount of water and electrolytes required to sustain human life, plus nutrients and micronutrients such as sugars, proteins (amino acids), and fats. These extracellular fluid replenishers and maintenance infusions are medical fluids used as solvents for injections of other active ingredients or for intravenous injections alone, and their safety for administration to the body has been established. Due to their electrolyte compositions similar to those of the pericellular environment and their safety, extracellular fluid replenishers and maintenance infusions are suitable as excipients for cell treatment agents used in the administration of cells to the body in regenerative medicine. Examples of such extracellular fluid replacement solutions include so-called replacement infusion solutions used for the purpose of replenishing extracellular fluid loss, and more specifically, include Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, Hartmann's solution, physiological saline, plasma substitutes, plasma preparations, etc. Among these, replacement infusion solutions that are not derived from humans, such as plasma substitutes and plasma preparations, are preferable. Examples of maintenance infusion solutions include amino acid-free glucose / electrolyte infusion solutions, glucose / electrolyte / amino acid infusion solutions, glucose / electrolyte / amino acid / multivitamin liquid preparations, glucose / electrolyte / amino acid / multivitamin / trace element liquid preparations, and glucose / electrolyte / amino acid / fat emulsions.

[0037] However, when preparing a cell treatment agent by isolating cells from a typical cell culture medium, washing them, and then immersing them in a so-called extracellular fluid replenisher, as described above, this procedure may result in a decrease in cell viability or infection. However, the present inventors have discovered that the active ingredient of the treatment agent exhibits cytoprotective effects in the extracellular fluid replenisher, similar to those in typical cell culture medium. In other words, by including the active ingredient of the treatment agent, the extracellular fluid replenisher or maintenance infusion can be used as a substitute for the cell culture medium, for example, during storage and transportation, and the cells can be administered immediately while still immersed in the extracellular fluid replenisher. This prevents the risk of a decrease in viability or infection that may occur when cells are transferred from the culture medium to a so-called extracellular fluid replenisher or maintenance infusion.

[0038] The cell treatment agent can be used as a reagent in a predetermined dosage form containing the specific active ingredient, and by adding and applying the reagent to a culture medium, a culture substrate such as a culture vessel or a cell carrier, or a biological organ, (a) cultured cells grown on a culture substrate scaffold or a biological tissue scaffold can be detached from the culture substrate scaffold or the biological tissue scaffold and suspend them, and (b) intracellular cells of a biological tissue can be detached from the biological tissue scaffold (such as a biological organ) and suspend them. Therefore, the cell treatment agent can be suitably used for cell detachment and suspending.

[0039] The cell treatment agent described above can stop proliferation and adhesion of cells in living tissues and cultured cells on scaffolds of culture substrates or scaffolds of living tissues, while maintaining their viability, and maintain an undifferentiated or underdifferentiated state without differentiating the cells, i.e., put them into dormancy. Therefore, it can be suitably used as a cell treatment agent for putting such cells into dormancy. Due to this dormancy effect, cells cease proliferation, but are able to awaken, reattach to culture substrates or living tissues, and begin proliferation under the right conditions. Therefore, it is an effective cell treatment agent in that it can temporarily suspend cell activity for preservation and transportation, and resume activity at a desired time, and it can also keep suspended cells obtained by detachment and suspension treatment dormant as they are. Furthermore, because the cell treatment agent described above can maintain cells in an undifferentiated or underdifferentiated state, it is also suitable for cell preservation, enabling the regeneration and formation of tissues or organs with desired properties after awakening from dormancy.

[0040] The cell treatment agent described above has a cytoprotective effect, which prevents the death of cells in cell containers, including culture containers, or in tissues of living tissues or cultured cells while maintaining their viability. Therefore, the cell treatment agent can be used in place of FBS or human serum, which have traditionally been used as agents with a protective effect to prevent cell death. Therefore, cell death can be safely prevented and cells can be protected without the use of FBS or human serum. This cell treatment agent with a cytoprotective effect is suitable, for example, for preserving and transporting cells. In particular, as described above, a cell treatment agent containing an extracellular fluid replenisher or maintenance infusion as an excipient and the specific active ingredient described above can protect cells even during preservation and transport, and can be administered immediately as is, greatly contributing to the safety and convenience of medical care.

[0041] The cell treatment agent can function, for example, as described above, for cell dormancy, cell protection, or cell preservation to maintain cells in an undifferentiated or less differentiated state, and therefore, when the cell treatment agent is in a liquid dosage form, it is suitable as a liquid for preserving cells, tissues, or organs. In this case, the dosage form used is preferably a cell culture medium, an extracellular fluid replenisher, a maintenance infusion, or the like.

[0042] As mentioned above, cell treatment agents can be used to induce cell dormancy, but applying a preparation containing polyvalent cations to dormant cells can also awaken them. Examples of such preparations containing polyvalent cations include aqueous solutions containing polycations such as chitosan, aluminum ions, iron ions, magnesium ions, and calcium ions; chelating preparations of these polyvalent cations; water-insoluble solids that generate polyvalent cations, such as powders of aluminum compounds, iron compounds, magnesium compounds, and calcium compounds; and fabrics impregnated with or bearing these polyvalent cation donors. Examples of calcium ion sources include calcium chloride, calcium gluconate, calcium carbonate, and calcium phosphate; examples of iron ion sources include iron hydroxide; and examples of aluminum ion sources include aluminum hydroxide. Examples of polyvalent cation donors include chelated polyvalent cations. Examples of fabrics include gauze made of biocompatible fibers that can be used in living organisms. The amount of the preparation containing a polyvalent cation to be used can be determined appropriately depending on the target of application, the composition of the active ingredients of the treatment agent, the form of the preparation, etc. As described above, by combining the cell treatment agent and the preparation containing a polyvalent cation, a set reagent can be prepared that can awaken dormant cells.

[0043] The amount of cell treatment agent added for various applications can be determined appropriately depending on the target of application, the composition of the active ingredients of the treatment agent, etc. When used in a culture medium for cell protection, the cell treatment agent can be added so that the alginic acid concentration is 200 to 0.001 mg / ml, the dextran sulfate concentration is 100 to 0.0001 mg / ml, and the heparin concentration is 1000 to 0.001 units / ml. The inhibitor amount can be determined appropriately depending on the type of inhibitor, etc. For example, the cell treatment agent can be added so that the urinastatin concentration is 2500 to 0.01 units / ml, the nafamostat mesilate concentration is 5 to 0.00001 mg / ml, and the gabexate mesilate concentration is 5 to 0.00001 mg / ml. [Example]

[0044] Hereinafter, embodiments of the present invention will be described in detail based on examples. In the examples shown below, various effects on cells in a cell culture vessel or a storage vessel were verified, but it goes without saying that the present invention is not limited to these, and similar effects are also exhibited on cells in biological tissues and cells spheroidized by three-dimensional cell culture or the like.

[0045] (Cells used) The experiments were conducted using Chinese hamster fibroblasts as mesenchymal cells, rat corneal epithelial cells as epithelial cells, human amnion-derived mesenchymal stem cells as undifferentiated cells, and a highly metastatic mouse melanoma strain as a malignant tumor.

[0046] (Cell preparation) The aforementioned cells were cultured in a culture vessel under standard cell culture conditions (37°C, 100% humidity, 5% CO2 concentration) in an FBS-supplemented cell culture medium prepared by adding 10% FBS to a standard serum-free cell culture medium. The cells in the culture vessel were then detached and suspended using a standard trypsin method. The resulting detached and suspended cells were used in the following experimental examples. The standard trypsin method was performed as follows.

[0047] Discard the cell culture medium in the cell culture vessel by suction and add approximately half the amount of PBS(-)(Ca2+ / Mg 2+ The cell surface was washed with PBS (containing no PBS). 2 Trypsin was applied to the cell surface by spreading 1 mL of 0.2% trypsin / EDTA solution throughout the vessel. Excess trypsin solution was then removed. The culture vessel was incubated in a CO2 incubator for 2 minutes under normal conditions (37°C, 5% CO2, 100% humidity), and the cells were confirmed to have detached from the inner wall of the culture vessel. If serum-free cell culture medium was used, trypsin was then completely inactivated with trypsin inhibitor. If FBS-supplemented cell culture medium was used, trypsin was completely inactivated by adding FBS-supplemented cell culture medium.

[0048] (Experimental Example 1: Cell protection effect) (1-1) Alginic acid (1-1-1) Store at room temperature for 24 hours As a preparation solution used for storing and transporting cells, for example, a normal serum-free cell culture medium without FBS or Ringer's solution (Otsuka Pharmaceutical Co., Ltd., Lactec Injection, Japanese Pharmacopoeia L-lactate Ringer's solution) was used, to which sodium alginate (Na alginate) (Fuji Chemical Industry Co., Ltd., Snow Algin SSL, 1% aqueous solution has a viscosity of 30 mPa·s at 20°C) was added to prepare cell preservation solutions at the concentrations shown in Table 1. Each of the above-mentioned cells was added at 10 mL to each cell preservation solution to achieve the conditions shown in Table 1. 6Cells / ml were added to the cells and left to stand at room temperature (22°C) for 24 hours. After that, the viability of the cells was assessed, and the percentage of viable cells (average viability (%)) was calculated and compared. Cell viability was assessed by the trypan blue exclusion test, a standard method for confirming cell viability. This exclusion test was performed according to the method described in Denizot F., et al., "Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability." J. Immunol. Methods, 89, 271-277 (1986)." The results are shown in Table 1. N=4.

[0049] [Table 1]

[0050] (1-1-2) Refrigerate for 72 hours The average survival rate (%) was calculated in the same manner as in Experiment (1-1-1), except that the storage conditions were changed to a refrigerator set at 4°C for 72 hours. The results are shown in Table 2. N=4.

[0051] [Table 2]

[0052] As shown in Tables 1 and 2, cell preservation solutions containing alginate exhibited superior cell viability and cell protection effects compared to serum-free cell culture medium or Ringer's solution that did not contain alginate, after storage at room temperature for 24 hours and refrigerated (4°C) for 72 hours. Furthermore, this cell viability improvement effect, i.e., cell protection effect, due to the addition of alginate was observed in both rat corneal epithelial cells, which are epithelial cells, and Chinese hamster fibroblasts, which are mesenchymal cells.

[0053] (1-2) Dextran sulfate (1-2-1) Store at room temperature for 24 hours The average cell viability (%) was determined in the same manner as in Experimental Example (1-1-1), except that dextran sulfate (Nacalai Tesque, Inc., special grade dextran sulfate reagent) was used instead of sodium alginate and the conditions were as shown in Table 3. The results are shown in Table 3. N=4 results.

[0054] [Table 3]

[0055] (1-2-2) Refrigerate for 72 hours The average cell survival rate (%) was determined in the same manner as in Experimental Example (1-2-1), except that the cells were left in a refrigerator at a set temperature of 4°C for 72 hours, as shown in Table 4. The results are shown in Table 4. When serum-free cell culture medium was used, N=3 results, and when Ringer's solution was used, N=4 results.

[0056] [Table 4]

[0057] As shown in Tables 3 and 4, cell preservation solutions containing dextran sulfate, regardless of the preparation used, exhibited superior cell viability and cell protection effects compared to cell preservation solutions not containing dextran sulfate when stored at room temperature for 24 hours and refrigerated (4°C) for 72 hours. Furthermore, it was confirmed that the cell viability-enhancing effect of adding dextran sulfate, i.e., the cell protection effect, was also observed in Chinese hamster fibroblasts, a type of mesenchymal cell.

[0058] (1-3) Heparins (1-3-1) Store at room temperature for 24 hours The average cell viability (%) was determined in the same manner as in Experimental Example (1-1-1), except that heparin sodium (Mochida Pharmaceutical Co., Ltd., Heparin Na Mochida, unfractionated heparin) was used instead of alginic acid and the conditions were as shown in Table 5. The results are shown in Table 5. N=4.

[0059] [Table 5]

[0060] (1-3-2) Refrigerate for 72 hours The average cell survival rate (%) was calculated in the same manner as in Experimental Example (1-3-1), except that the storage conditions were changed to 72 hours in a refrigerator set at 4°C, as shown in Table 6. The results are shown in Table 6. N=4.

[0061] [Table 6]

[0062] As shown in Tables 5 and 6, cell preservation solutions containing heparin sodium exhibited superior cell viability and cytoprotective effects compared to cell preservation solutions not containing heparin sodium (i.e., the preparation solution alone) when stored at room temperature for 24 hours and refrigerated (4°C) for 72 hours. Furthermore, it was confirmed that the cell viability-enhancing effect of adding heparin sodium, i.e., the cytoprotective effect, was also observed in Chinese hamster fibroblasts, a type of mesenchymal cell. Furthermore, when similar experiments were performed using low-molecular-weight heparin sodium instead of heparin sodium, the cell viability-enhancing effect, i.e., the cytoprotective effect, was similarly observed in each cell type.

[0063] (1-4) Protease inhibitors (1-4-1) Urinastatin (inhibitor a) (1-4-1-1) Refrigerate for 72 hours The average cell viability (%) was determined in the same manner as in Experimental Example (1-3-2), except that urinastatin (Miraclid Injection, manufactured by Mochida Pharmaceutical Co., Ltd., containing 25,000 units) was used as the protease inhibitor instead of heparin sodium, and the conditions were as shown in Table 7. The results are shown in Table 7. N=4 results. Note that the "unit (U)" in the concentration unit (units / mL) of inhibitor a in Table 7 can be measured, for example, by the urinastatin quantification method in the Japanese Pharmacopoeia, 15th Edition. Specifically, the absorbance of the sample solution at 405 nm can be measured using a UV240 spectrophotometer, and the urinastatin concentration can be calculated from a previously prepared calibration curve.

[0064] [Table 7]

[0065] (1-4-2) Nafamostat mesylate (inhibitor b) (1-4-2-1) Refrigerate for 72 hours A 5% glucose solution was used as the adjusting solution, and cell preservation solutions were prepared by adding the protease inhibitor nafamostat mesilate (Nichi-Iko Pharmaceutical Co., Ltd., Fusan for injection) to the concentration shown in Table 8 (Fusan for injection (Nichi-Iko Pharmaceutical Co., Ltd.) is specified to be diluted with 5% glucose solution). 10 6 The cells were added at a concentration of 0.1% cells / ml and left in a refrigerator set at 4°C for 48 hours. The average cell survival rate (%) was calculated in the same manner as in Experimental Example (1-4-1-1). The results are shown in Table 8. N=4.

[0066] [Table 8]

[0067] (1-4-3) Protease inhibitor mixture (inhibitor c) (1-4-3-1) Refrigerate for 72 hours The average cell viability (%) was determined in the same manner as in Experiment (1-4-1-1), except that a protease inhibitor mixture (Fujifilm Wako Pure Chemical Industries, Ltd., Protease Inhibitor Cocktail Set I) was used instead of urinastatin, and the conditions shown in Table 9 were used. The results are shown in Table 9. N=4. When one vial of the protease inhibitor mixture was dissolved in 1 ml of distilled water, it contained 50 mmol / L AEBSF hydrochloride, 15 μmol / L aprotinin (recombinant), 0.1 mmol / L E-64, 50 mmol / L EDTA·2Na·2H2O, and 0.1 mmol / L leupeptin hemisulfate. This was used as the stock solution and added to the adjusted solution at the dilution ratio shown in Table 9.

[0068] [Table 9]

[0069] As shown in Tables 7 to 9, cell preservation solutions containing protease inhibitors exhibited superior cell viability and cytoprotective effects when stored refrigerated (4°C) for 72 or 48 hours compared to cell preservation solutions not containing protease inhibitors (i.e., preparation solution only). Furthermore, it was confirmed that the cell viability-improving effect, i.e., cytoprotective effect, of the addition of these protease inhibitors was also observed in Chinese hamster fibroblasts, a type of mesenchymal cell.

[0070] (Experimental Example 2: Cell dormancy effect) The active ingredients of the cell treatment agents used were sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd., sodium alginate first-class reagent; viscosity of 1% aqueous solution is 120 mPa·s at 20°C), DS (same as in Experimental Example 1), and heparin sodium (same as in Experimental Example 1).We investigated whether or not these agents would cause cells to become dormant by comparing the activity of cell division and proliferation, the most common cellular activity, as described below.

[0071] (2-1) Induction of cell dormancy The cell cycle was analyzed by flow cytometry according to the method described in a known literature (Piotr Pozarowski and Zbigniew Darzynkiewicz, "Analysis of Cell Cycle by Flow Cytometry," Methods in Molecular Biology, vol. 281: Checkpoint Controls and Cancer, Volume 2), and the frequency of cell division and proliferation due to differences in the active ingredients of the cell treatment agents was compared. Specifically, the procedure is as follows. First, a normal serum-free cell culture medium was used as the adjustment solution, which served as (a) a control. Cell preservation solutions were prepared by adding (b) sodium alginate (Na alginate) to a concentration of 2.5 mg / ml, (c) DS to a concentration of 0.1 mg / ml, and (d) heparin sodium (Na heparin) to a concentration of 50 units / ml. Rat corneal epithelial cells were cultured in these cell preservation solutions at a cell concentration of 10 5 The cells were added to a concentration of 1000 cells / ml and cultured for 48 hours under standard cell culture conditions (37°C, 5% CO2, 100% humidity). The resulting cultured cells were subjected to cell cycle analysis by flow cytometry to determine the number of cells in each cell cycle, and the proportion of cells in the S, G2, and M phases, which are considered to be periods during the cell division and proliferation process, was compared with the proportion of cells in the G0 and G1 phases, which are considered to be other periods. The results are shown in Table 10. N=4 results.

[0072] [Table 10]

[0073] As shown in Table 10, when each component was added, the percentage of cells in the S, G2, and M phases, which are considered to be periods during the cell division and proliferation process, was significantly lower than when the serum-free cell culture medium was used alone (the control). In other words, when each component was added to a serum-free cell culture medium, the cell's division and proliferation function was reduced, and the cells entered a dormant state. Therefore, it was found that the cell preservation medium containing each component was effective as a cell treatment agent for causing cell dormancy, which puts cultured cells into dormancy.

[0074] (2-2) Maintaining the differentiated state Dextran sulfate (DS) was used as the active ingredient of the cell treatment agent, and the cell dormancy effect of DS was investigated using as an index the effect of maintaining the cells in an undifferentiated state and suppressing differentiation. Undifferentiated human amniotic membrane-derived mesenchymal stem cells were used as the cells. A normal serum-free cell culture medium was used as the adjusting solution, and cell preservation solution a was prepared by adding DS to it at 0.1 mg / ml, and cell preservation solution b was prepared by adding FBS to it at 10%. 10% of the above-mentioned stem cells were added to each of cell preservation solutions a and b. 5 After adding 1000 mg of HCl to the cells at a concentration of 10 ...

[0075] [Table 11]

[0076] As shown in Table 11, when serum-free cell culture medium containing DS was used as the cell preservation medium, the positivity rates of all three differentiation markers increased slightly after culture compared to before culture, with no significant difference, and the cells maintained an undifferentiated state, whereas when serum-free cell culture medium containing FBS was used as the cell preservation medium, the positivity rates of all three differentiation markers increased significantly after culture compared to before culture, indicating the beginning of a shift toward cell differentiation. Thus, it was revealed that DS suppresses differentiation by keeping cells dormant in an undifferentiated state, whereas FBS, which also has a cytoprotective effect, suppresses differentiation without causing cells to differentiate.

[0077] (Experimental Example 3: Awakening from Cell Dormancy) As shown in the above experimental examples, we investigated whether cells that had been rendered dormant by the action of sodium alginate (same as in Experimental Example 2), DS (same as in Experimental Example 1), and sodium heparin (same as in Experimental Example 1), which were used as active ingredients in cell treatment agents, would be awakened by a preparation containing calcium ions (Calcicol Injection, calcium gluconate hydrate solution, manufactured by Nichi-Iko Pharmaceutical Co., Ltd.), using the resumption of cell division and proliferation, the most common cellular activity, by adding a calcium agent as an indicator.

[0078] (3-1) Awakening from cellular dormancy First, a normal serum-free cell culture medium was used as the adjustment solution, and this was used as (a) a control. The following cell preservation solutions were prepared: (b) a normal serum-free cell culture medium to which DS was added to give a concentration of 0.1 mg / ml, (c) a normal serum-free cell culture medium to which heparin sodium (heparin Na) was added to give a concentration of 50 units / ml, and (d) a normal serum-free cell culture medium to which sodium alginate (alginate Na) was added to give a concentration of 2.5 mg / ml. Next, 1 x 10 rat corneal epithelial cells obtained as described above were added to the cell preservation solutions (a) to (d). 5 The cells were added to give a cell count of 1000 / ml and stored in a refrigerator at 4°C for 48 hours. The sample numbers of the cells contained in the cell preservation solutions (a) to (d) obtained in this way were designated (a') to (d'), respectively.

[0079] Similarly, cell preservation solutions (a) to (d) were prepared separately and divided into two, designated sample numbers a-1 to d-1 and a-2 to d-2, respectively. Nothing was added to the cell preservation solutions of sample numbers a-1 to d-1, while calcicol injection was added to the cell preservation solutions of sample numbers b-2 to d-2 so that the calcium concentration was 5.23 mg / ml.

[0080] The cell preservation solutions (a) to (d) containing cells after 48 hours of refrigeration obtained as described above were centrifuged, and the cells (a') to (d') were separated and collected by centrifugation. The cells (a') to (d') were then placed in the cell preservation solutions a-1 to d-2 with the corresponding alphabetical sample numbers, respectively, at a cell content of 6 × 10 4 The cells were added so that the concentration was 1 / ml.

[0081] The cell preservation solutions a-1 to d-2 containing the specified cells (a') to (d') were cultured for 120 hours under normal cell culture conditions (37°C, CO2 concentration 5%, humidity 100%).

[0082] After culturing, the number of viable cells per unit volume contained in cell preservation solutions a-1 to d-2 was quantified by MTT assay. The MTT assay was performed according to a known literature (Priti Kumar et al. "Analysis of Cell Viability by the MTT Assay" Cold Spring Harbor Protocols. 6; 2018 DOI: 10.1101 / pdb.prot095505). The culturing conditions and measurement results are shown in Table 12. N = 12 results.

[0083] [Table 12]

[0084] As shown in Table 12, when calcium ions were not added, the increase in viable cell count was suppressed when the components used as active ingredients in the cell treatment agent were included (b-1 to d-1) compared to the case where these components were not included (a-1). On the other hand, when calcium ions were added (b-2 to d-2), the viable cell count increased. Similar experiments were conducted using Chinese hamster fibroblasts, and similar results were obtained. These experiments revealed that the addition of calcium ions to the components used as active ingredients in the cell treatment agent awakened the cells, and conversely, activated the cells' ability to divide and proliferate.

[0085] (Experimental Example 4: Cell detachment and floating effect) <Preparation of adherent cells> A culture medium (10% FBS cell culture medium) was prepared by adding FBS to a normal serum-free cell culture medium to make the concentration of FBS 10%. The culture medium was placed in a culture dish. 10% rat corneal epithelial cells were added to the culture medium. 6 The cells were added at a concentration of 1000 cells / ml and cultured for 24 hours under normal cell culture conditions (37°C, CO2 concentration 5%, humidity 100%) to allow the cultured cells to fully adhere to the scaffolding wall of the culture dish.

[0086] <Detachment and suspension of adherent cells> The culture medium in the culture dish containing the cultured cells obtained as described above was replaced with dextran sulfate aqueous solution (shown in Table 13) and dextran aqueous solution (shown in Table 13) prepared by dissolving dextran sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., dextran sodium sulfate MW 36,000-50,000) and dextran (Fujifilm Wako Pure Chemical Industries, Ltd., dextran MW 35,000-50,000) in purified water. The cells were cultured under standard cell culture conditions (37°C, 5% CO2, 100% humidity). The adherent cells were observed over time for detachment and floating from the dish wall. Detachment and floating were assessed by gently shaking the culture dish manually to observe whether the cells detached from the dish wall and became floating. The cells were considered to have become detached and floating when 90% or more of the cells had become detached and floating. The results for the dextran sulfate aqueous solution are shown in Table 13. N=4 results. In Table 13, "○" means that the cells were detached and floated, and "×" means that the cells were not detached and floated. When the medium was replaced with an aqueous dextran solution, the adherent cells were not detached and floated in either case.

[0087] [Table 13]

[0088] As shown in Table 13, when DS was added, the adherent cells attached to the inner wall of the culture dish were detached and floated within a specified time period, depending on the concentration of DS. A similar experiment was performed using Chinese hamster fibroblasts, and similar results were obtained. Therefore, it was found that DS, unlike ordinary dextran, has the effect of detaching and floating adherent cells.

[0089] (Experimental Example 5: Cell detachment / floating action and cell re-adhesion action) <Preparation of adherent cells> In the same manner as in Experimental Example 4, rat corneal epithelial cells were allowed to adhere sufficiently to the wall of the culture dish, which served as a scaffold.

[0090] <Detachment and suspension of adherent cells> The culture medium in the culture dishes containing the cultured cells obtained as described above was prepared by adding or not adding each active ingredient to the concentrations shown in Table 14. Each culture dish was then placed on a shaker (Biocraft, product name: Laboshaker Model BC-740) and gently shaken at the lowest setting, 4 shakes per minute, for 24 hours under standard cell culture conditions (37°C, 5% CO2, 100% humidity). After 24 hours of culture, the cells were observed for detachment and floating. Detachment and floating were assessed by observing whether the cells detached and floated from the dish wall while the dish was being shaken. Cells that detached and floated were assessed as "detached and floated," with "○" indicating that they were detached and floated, and "×" indicating that they were not detached and floated. The results are shown in Table 14. N=4 results.

[0091] [Table 14]

[0092] <Re-adhesion of detached and floating cells> We investigated whether the detached and suspended cells reattached as described above in the presence of calcium ions. First, a slurry of calcium phosphate powder mixed with distilled water was applied to a portion of the bottom of a culture dish, which was then heat-dried to create a layer of calcium phosphate powder attached to the bottom. Next, cell culture medium containing the detached and suspended cells was added to the calcium phosphate-coated culture dish and a control culture dish without the calcium phosphate coating. The culture dishes were then placed on a shaker (Biocraft, Product Name: Laboshaker Model BC-740) and gently shaken at the lowest setting (4 shakes / min) for 24 hours under standard cell culture conditions (37°C, 5% CO2, 100% humidity). After that, the culture medium in each culture dish was removed, and the culture dishes were gently washed with 10% FBS cell culture medium. After that, live cells were stained with a fluorescent dye (CytoPainter F-actin Staining Kit, Abcam) and the live cells adhering to the inner wall of the culture dish were observed under a microscope (Olympus Corporation, inverted fluorescence / visual microscope with cooled CCD camera, IX83) using F-actin fluorescent staining. Fluorescent staining of F-actin can be performed, for example, according to the method described in Vera DM, Robert JE, Ved PS, Orrin S and John SC, “Optimizing leading-edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities” BIOTECHNIQUES, VOL. 66, NO. 3, (2019), or Michael M, Matthias P and Robert G, “Actin visualization at a glance” J. Cell Sci. 130, 525-530. (2017) doi:10.1242 / jcs.20448.

[0093] Microscopic images are shown in Figures 1 to 4. Figure 1 shows an image of the surface of a calcium phosphate film when cells detached and suspended using DS were cultured in a culture dish with a calcium phosphate film. Figure 2 shows an image of the surface of a calcium phosphate film when cells detached and suspended using heparin sodium were cultured in a culture dish with a calcium phosphate film. Figure 3 shows an image of the bottom of a culture dish when cells detached and suspended using DS were cultured in a culture dish without a calcium phosphate film. Figure 4 shows an image of the bottom of a culture dish when cells detached and suspended using heparin sodium were cultured in a culture dish without a calcium phosphate film. In Figures 1 to 4, the white or gray areas represent live cells stained with fluorescent dye.

[0094] As shown in Figures 1 and 2, numerous viable cells were observed adhering to the surface of the calcium phosphate coating, either singly or in clusters. In contrast, as shown in Figures 3 and 4, very few viable cells were found adhering to the bottom of the culture dish without a calcium phosphate coating. Similar experiments were performed using Chinese hamster fibroblasts, and similar results were obtained. Thus, it is clear that solid calcium phosphate preparations can re-attach and activate cells that have been detached and suspended by active ingredients such as DS and heparin sodium. In other words, the combination of a cell treatment agent containing an active ingredient and a specific formulation of polyvalent cations can awaken dormant cells that have been rendered dormant by the cell treatment agent. Therefore, a combined reagent consisting of these two components is suitable for awakening dormant cells.

[0095] (Experimental Example 6: Relationship between viscosity and concentration of alginate and cell protection effect) The cell-protective effects of sodium alginate with different viscosities were investigated using three types of alginic acid with different viscosities as the active ingredient of a cell treatment agent and Ringer's solution as the preparation used for, for example, preserving and transporting cells. The three types of alginic acid with different viscosities were: (a) sodium alginate (Snow Algin L, Fuji Chemical Industry Co., Ltd.) with a 1% aqueous solution and a viscosity of 120 mPa·s (high viscosity) at 20°C; (b) sodium alginate (Snow Algin SSL, Fuji Chemical Industry Co., Ltd.) with a 1% aqueous solution and a viscosity of 30 mPa·s (low viscosity) at 20°C; and (c) sodium alginate (depolymerized sodium alginate, Kyosei Pharmaceutical Co., Ltd.) with a 10% aqueous solution and a viscosity of 30 mPa·s (very low viscosity) at 20°C. The Ringer's solution used was the same as in Experiment 1.

[0096] (6-1) Store at room temperature for 24 hours Cell preservation solutions were prepared by adding the above-mentioned alginic acids with different viscosities to Ringer's solution to the concentrations shown in Table 15. 10% of the above-mentioned rat corneal epithelial cells were added to the cell preservation solutions. 6 Cells / ml were added and left to stand at room temperature (22°C) for 24 hours, after which the viability of the cells was assessed and the percentage of viable cells (average viability (%)) was calculated and compared. The viability of cells was assessed by trypan blue exclusion test, a standard method for confirming cell viability. The results are shown in Table 15. N=4 results.

[0097] [Table 15]

[0098] (6-2) Refrigerate for 168 hours (7 days) The average survival rate (%) was calculated in the same manner as in (6-1), except that the storage conditions were changed to a refrigerator set at 4°C for 7 days and the sodium alginate concentration was set as shown in Table 16. The results are shown in Table 16. N=4 results.

[0099] [Table 16]

[0100] As shown in Table 15, for a relatively short storage time of 24 hours at room temperature (22°C), alginate (high viscosity) with a relatively high viscosity (1% aqueous solution, 120 mPa·s (20°C)) exhibits more effective cell protection in cell preservation solutions prepared at relatively low concentrations (approximately 2.5 mg / ml or less) than other viscosities (low viscosity, very low viscosity). As shown in Table 16, for a very long storage time of 7 days, alginate (very low viscosity) with a very low viscosity (10% aqueous solution, 30 mPa·s (20°C)) exhibits more effective cell protection in cell preservation solutions prepared at relatively high concentrations (approximately 10 mg / ml or more) than other viscosities (high viscosity, low viscosity). Furthermore, Table 16 indicates that alginate with a very low viscosity is expected to be more effective at higher concentrations.

Claims

1. A cell treatment agent for cell dormancy that contains alginic acid as an active ingredient and at least one selected from extracellular fluid replacement solutions and maintenance infusion solutions, and that puts intracellular or cultured cells into dormancy, i.e., stops proliferation, respiration, metabolism, and maintenance of a differentiated state of intracellular or cultured cells while maintaining their viability.

2. A cell treatment agent for cell protection, which contains alginic acid as an active ingredient and at least one selected from extracellular fluid replacement solutions and maintenance infusion solutions, and which maintains the viability of cells in tissues or cultured cells and prevents their death.

3. A cell treatment agent for cell preservation, which contains alginic acid as an active ingredient and at least one selected from extracellular fluid replacement solutions and maintenance infusion solutions, for maintaining cells in an undifferentiated or low-differentiated state.

4. A cell treatment agent for cell detachment and suspending, which contains alginic acid as an active ingredient and at least one selected from water, cell culture medium, extracellular fluid replenisher, and maintenance infusion fluid, and which detaches and suspends cultured cells from a biological tissue scaffold or a culture substrate scaffold, or intracellular cells of biological tissue from a biological tissue scaffold.

5. 2. The cell treatment agent for causing cell dormancy according to claim 1, which is a liquid for cell preservation, tissue preservation, or organ preservation.

6. A cell treatment agent for cell protection according to claim 2, which is a liquid for cell preservation, tissue preservation or organ preservation.

7. A cell treatment agent for cell preservation according to claim 3, which is a liquid for cell preservation, tissue preservation or organ preservation.

8. A set reagent for awakening dormant cells, which is a combined reagent of the cell treatment agent for causing cell dormancy according to claim 1 or 5 and a solid or liquid preparation containing a polyvalent cation.

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