JP7803507B2 - Cell Treatment Agents - Google Patents

Description

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/suspending, dormancy, maintaining viability, preventing cell death, and activating suspended cells.

With the exception of special cells (non-adherent cells) such as unicellular organisms, blood cells, and cancer cells, the vast majority of cells, called adherent cells (hereafter sometimes simply referred to as "cells"), survive and proliferate by adhering to a scaffolding made up of the extracellular matrix that constitutes the body in the body, or to a scaffolding such as the walls 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 their scaffolding and suspended without weakening them.

The detachment and suspension process typically utilizes the cell detachment properties 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 using a cell detachment agent such as a cell detachment enzyme or 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 for detaching and suspending cultured cells from a specified scaffold is to add a solution containing a cell detachment agent such as trypsin to a culture vessel and allow the cell detachment agent to act on the cultured cells, detaching and suspending the cells (Non-Patent Document 2).

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 continue to act 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, the suspended cells are separated from the surfactant or cell detachment enzyme after suspension, or the separated cells are washed or the surfactant or enzyme is 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 unavoidable during the suspension process. Furthermore, for use in research and medical treatment, it is necessary to ensure that the cells maintain their viability without weakening during the detachment and suspension process.

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 cells are detached and suspend- ed by heating this polymer to a certain temperature (Non-Patent Document 3). However, this method has been criticized for several issues, including the high cost of using a special polymer, the need for a special temperature control device, difficulty in temperature control and other aspects leading to inconsistent results, the inability to use it in complex, three-dimensional devices for culturing large numbers of cells, and the inability to detach and suspend cells within tissues from living organs.

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

Even when refrigerated 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 and mixing 10% fetal bovine serum (FBS) or the patient's own serum into the liquid used to store and transport the cells. It is known that mixing fetal bovine serum (FBS) or the patient's own serum dramatically increases cell viability (cell protective effect) compared to when these ingredients are not added (Non-Patent Documents 4-6).

However, the addition of FBS or autologous serum has been criticized for its potential problems, including the fact that these are biological preparations, as well as issues related to infection, allergies, ease of use, and ethics. However, currently, no substance is known that can adequately replace FBS or autologous serum.

Furthermore, cells, particularly 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 the 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 a means of achieving this has not yet been established, and the establishment of a solution to this problem is desired.

Crapo PM,.et al., An overview of tissue and whole organ decellularization processes. Biomaterials 32,3233-3243 (2011) Hayflick L., et al., Subculturing human diploid fibroblast cultures. Edited by Kruse, P. F. et al., Tissue Culture Methods and Applications.220-223 Academic Press (New York) (1973) 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) 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) Liang C., et al. Serotonin promotes the proliferation of serum3 deprived hepatocellular carcinoma cells via upregulation of FOXO3a. Molecular Cancer 12 (2013) (open access) 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)

As mentioned above, when performing a process to detach and suspend intracellular cells or cultured cells from a living organ or a culture substrate for living cells, it is necessary to (a) prevent the cells from dying, and (b) obtain suspended cells in a state where the cell viability is maintained during the detachment and suspending process, i.e., not to reduce the cell viability.

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

In addition, 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.

Furthermore, when attaching suspended, dormant cells to living organs, culture substrates, etc., it is desirable to be able to do so easily, and it is necessary to activate and proliferate the attached cells.

However, for example, to allow detached and suspended cells to adhere to a scaffold, which is a culture substrate such as the wall of a culture vessel or a specific carrier, i.e., to "implant" such cells, the cells 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 on the scaffold. In particular, to implant cells into a specific localized area of the body, the cells must be kept in contact with the biological scaffold for 12 to 24 hours.

For this purpose, a scaffold such as a nonwoven fabric is typically brought into contact with cells outside the body for 12 to 24 hours to allow the cells to adhere to the nonwoven fabric scaffold, and the scaffold with the cells attached is then transplanted into the desired location in the body. However, this type of transplantation is problematic in that it takes time and effort.

The object of the present invention is to provide a method for safely and easily performing processes such as detachment/floating, dormancy (dormancy not only enhances protective effects but also suppresses unnecessary differentiation of cells, maintaining an undifferentiated/underdifferentiated state), and protective processes 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 easily performing cell activation processes for transplanting dormant cells (e.g., suspended cells, etc.).

The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they discovered 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:

(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 paragraph (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 treatment 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 treatment agent according to any one of the preceding items (1) to (7) and a solid or liquid preparation containing a polyvalent cation.

By "scaffold" is meant what is known in the art as one of the three elements of regenerative medicine.
Adhesive cells, which account for 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 either artificial or natural. An example of an artificial scaffold is the wall of an artificial cell culture substrate such as a petri dish. In addition, when cells are supported and attached to artificial fibers or the like 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.

"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.

"Protection" of cells means maintaining their viability, that is, preventing the loss of cell function or cell death.

Suspension cell activation means initiating proliferation, respiration, metabolism, and maintenance of differentiation of suspension cells in the aforementioned "dormant" or "protected" state, or restoring adhesiveness, respiration, metabolism, and maintenance of differentiation of suspension cells in the aforementioned "dormant" or "protected" state. Cell activation also refers to initiating proliferation, respiration, metabolism, and maintenance of differentiation of dormant cells, including cells not in suspension, initiating proliferation, respiration, metabolism, and maintenance of differentiation of cells in the aforementioned "dormant" or "protected" state, or restoring adhesiveness, respiration, metabolism, and maintenance of differentiation of cells in the aforementioned "dormant" or "protected" state, and is synonymous with "awakening," described below.

Viability does not simply mean that a cell is alive and not dying, but that the cell retains the ability to perform its inherent functions in the body (including proliferation, respiration, metabolism, and maintaining a differentiated state).

The present invention provides a method for safely and easily performing processes such as detachment/floating, 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 easily performing cell activation processes for transplanting dormant cells.

10 shows an image of the surface of a calcium phosphate film when cells detached and suspended with dextran sulfate were cultured in a culture dish with a calcium phosphate film in Experimental Example 5. 10 shows an image of the surface of a calcium phosphate film when cells detached and suspended with heparin sodium were cultured in a culture dish with a calcium phosphate film in Experimental Example 5. 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. 10 shows an image of the bottom surface of a culture dish in Experimental Example 5, in which cells detached and suspended with sodium heparin were cultured in the culture dish without a calcium phosphate film.

A 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 a protease inhibitor (hereinafter sometimes referred to as the "active ingredient of the treatment agent" or "active ingredient").

Alginic acid 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. Furthermore, when alginic acid is dissolved in water, it forms a smooth, viscous aqueous solution (colloidal solution). The viscosity of this solution is proportional to the degree of polymerization of the alginic acid, with the viscosity increasing with increasing degree of polymerization. Various types of alginic acid with different viscosities are commercially available, including those that form standard viscous aqueous solutions and those with a lower viscosity than standard solutions. While various types can be used, depending on the viscosity, more effective methods are available. Here, the following three types of alginic acid viscosity, conveniently classified as (a) to (c), will be described as examples: (a) high-viscosity alginic acid: for example, alginic acid with a relatively high viscosity of 60 mPa·s or more in a 1% by mass aqueous solution at 20°C; (b) low-viscosity alginic acid: for example, alginic acid with a relatively low viscosity of 5 mPa·s or more but less than 60 mPa·s in a 1% by mass aqueous solution at 20°C; and (c) very low-viscosity alginic acid: for example, alginic acid with a very low viscosity of 5 mPa·s or less in a 1% by mass aqueous solution at 20°C or 30 mPa·s or less in a 10% by mass aqueous solution at 20°C. (a) high-viscosity alginate, which has a high viscosity, tends to function more effectively than (b) low-viscosity and (c) very low-viscosity alginate, for example, in a solution prepared to have a concentration of 5 mg/ml or less when stored for a relatively short period of time, such as 24 hours, at room temperature (22°C). (b) low-viscosity alginate, which has a relatively low viscosity, tends to 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, such as 120 hours, at room temperature (22°C). (c) very 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 have a concentration of 10 mg/ml or more when stored for a relatively long period of time, such as 168 hours, at refrigerated (4°C). The various effects of alginic acid as a cell treatment agent generally tend to vary depending on the concentration, but the extent of this varies depending on the type of cell, concentration, etc., so the concentration ranges mentioned above represent general trends.

"Alginic acid" includes pharmacologically acceptable salts of alginic acid. Such pharmacologically acceptable salts of alginic acid are formed when the hydrogen ion of the carboxyl group of alginic acid is released and bound to a cation. Such cations may be any cation capable of forming a pharmacologically acceptable salt, and examples 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 organic polyvalent ions such as polylysine.

Commercially available alginic acid can be used as described above.

Heparins refer to heparin and heparin-like substances. 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. Heparin-like substances are modified and partially degraded heparin and have similar physiological activity to heparin. Heparinoids include, for example, salts of heparin that can form 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. Low molecular weight heparins preferably have a number average molecular weight of 2,000 to 8,000. Commercially available heparins can be used.

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 when the hydrogen ion of the sulfonic acid group of dextran sulfate is released and bound to a cation. Such cations may be any cation capable of forming a pharmacologically acceptable salt, including 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, and more preferably 0.001 to 2, 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.

Protease inhibitors (hereinafter sometimes simply referred to as "inhibitors") are also called protease inhibitors, and are not particularly limited, with a variety of commercially available inhibitors being applicable. 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, and atazanavir sulfate. These may be used alone or in combination.

Alginic acid, heparins, dextran sulfate, and protease inhibitors may be used alone or in combination of two or more.

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

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 infusions alone, and their safety for administration to the body has been established. Due to their similar electrolyte compositions to those surrounding the cells and their safety, extracellular fluid replenishers and maintenance infusions are suitable as excipients for cell treatments used in administering cells to the body in regenerative medicine. Examples of such extracellular fluid replacement solutions include so-called replacement infusion solutions used to replenish extracellular fluid loss, such as Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, Hartmann's solution, physiological saline, plasma substitutes, and plasma preparations. Among these, replacement infusion solutions that are not derived from humans, such as plasma substitutes and plasma preparations, are desirable. 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.

However, when preparing a cell treatment agent by separating and washing cells from a typical cell culture medium, followed by immersion in a so-called extracellular fluid replenisher, as described above, this procedure can potentially reduce the cell viability or expose the cells to infection. However, the inventors have discovered that the active ingredient of the treatment agent possesses cytoprotective properties in the extracellular fluid replenisher, similar to those found 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 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 reduced viability and infection that may occur when cells are transferred from culture medium to a so-called extracellular fluid replenisher or maintenance infusion.

The cell treatment agent described above can be used as a reagent in a predetermined dosage form containing the specific active ingredient described above. By adding or applying the reagent to a culture medium, a culture substrate such as a culture vessel or 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.

The aforementioned cell treatment agent can stop proliferation and adhesion of cells within living tissues and cultured cells on scaffolds of culture substrates or scaffolds of living tissues while maintaining their viability, and can 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, the cells cease proliferation, but are able to awaken, re-attach to the culture substrate or living tissue, and begin proliferation when the conditions are right. 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 aforementioned cell treatment agent can maintain cells in an undifferentiated or underdifferentiated state, it is also suitable for cell preservation, enabling the regeneration and formation of tissues and organs exhibiting desired properties after awakening from a dormant state.

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, this 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, cells can be safely protected by preventing cell death without the use of FBS or human serum. This cell treatment agent with a cytoprotective effect is suitable, for example, for the storage and transportation of cells. In particular, as described above, a cell treatment agent containing an extracellular fluid replacement solution or maintenance infusion as an excipient and the specific active ingredient described above can protect cells even during storage and transportation, and can be administered immediately as is, greatly contributing to the safety and convenience of medical care.

Cell treatment agents 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. Therefore, when the cell treatment agent is in liquid 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, extracellular fluid replenisher, maintenance infusion, or the like.

As mentioned above, cell treatment agents can be used to induce cell dormancy. However, treatment of dormant cells with preparations containing polyvalent cations can awaken these cells. Examples of 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 polyvalent cations used can be determined appropriately depending on the target of application, the composition of the active ingredients in the treatment agent, the form of the preparation, etc. As described above, by combining a cell treatment agent with a preparation containing polyvalent cations, a set reagent can be created that can awaken dormant cells.

When applying a cell treatment agent for various purposes, the amount added can be determined appropriately depending on the application target, the composition of the treatment agent's active ingredients, etc. When applying the cell treatment agent to a culture medium for cell protection, for example, the cell treatment agent can be added so that the alginic acid concentration is 200-0.001 mg/ml, the dextran sulfate concentration is 100-0.0001 mg/ml, and the heparin concentration is 1000-0.001 units/ml. The amount of inhibitor 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 2500-0.01 units/ml, the nafamostat mesilate concentration is 5-0.00001 mg/ml, and the gabexate mesilate concentration is 5-0.00001 mg/ml.

Embodiments of the present invention will be described in detail below based on examples. In the examples shown below, various effects on cells in cell culture vessels or storage vessels 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 formed into spheroids by three-dimensional cell culture, etc.

(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.

(Cell preparation)
The aforementioned cells were cultured in a culture vessel under standard cell culture conditions (37°C, 100% humidity, 5% _CO2 ) in a standard serum-free cell culture medium supplemented with 10% FBS. The cells in the culture vessel were then detached and suspended by 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.

The cell culture medium in the cell culture vessel was aspirated and discarded, and the cell surface was washed with approximately half the amount of PBS(-) (Ca ²⁺ /Mg ²⁺ -free PBS) used for culture. Then, 1 mL of 0.2% trypsin/EDTA solution was spread throughout the vessel for a 25 cm ² cell surface area to allow trypsin to act. Excess trypsin solution was then removed. The culture vessel was incubated in a CO ₂ incubator for 2 minutes under normal conditions (37°C, 5% CO ₂ , 100% humidity), and cell detachment from the inner wall of the culture vessel was confirmed. Then, when using serum-free cell culture medium, trypsin was completely inactivated with trypsin inhibitor. Furthermore, when using FBS-supplemented cell culture medium, FBS-supplemented cell culture medium was added to completely inactivate trypsin.

(Experimental Example 1: Cytoprotective effect)
(1-1) Alginic Acid (1-1-1) Storage at Room Temperature for 24 Hours Cell preservation solutions were prepared by using a standard serum-free cell culture medium or Ringer's solution (Otsuka Pharmaceutical Co., Ltd., Lactec Injection, Japanese Pharmacopoeia L-lactate Sodium Ringer's Solution) containing no FBS as the preparation solution used for cell storage and transportation, and adding sodium alginate (Na alginate) (Fuji Chemical Industry Co., Ltd., Snow Algin SSL, 1% aqueous solution viscosity 30 mPa·s at 20°C) to the concentrations shown in Table 1. Each of the aforementioned cells was added to each cell preservation solution at a concentration of 10 ⁶ cells/ml to achieve the conditions shown in Table 1. After leaving the cells in a room at room temperature (22°C) for 24 hours, the viability of the cells was assessed, and the percentage of viable cells (average viability (%)) was calculated and compared. Cell viability was assessed using a trypan blue exclusion test, a common method for confirming cell viability. This excretion 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.

(1-1-2) Refrigerated storage for 72 hours The average survival rate (%) was calculated in the same manner as in Experimental Example (1-1-1), except that the storage conditions were changed to leaving the cells in a refrigerator at a set temperature of 4°C for 72 hours. The results are shown in Table 2. N=4.

As shown in Tables 1 and 2, cell preservation solutions containing alginate exhibit superior cell viability and cell protection effects when stored at room temperature for 24 hours and refrigerated (4°C) for 72 hours compared to serum-free cell culture medium or Ringer's solution that does not contain alginate. 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 fibroblast cells, which are mesenchymal cells.

(1-2) Dextran sulfate (1-2-1) Storage 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 shown in Table 3 were used. The results are shown in Table 3. N=4.

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

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 that did not contain 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 improvement effect, i.e., the cell protection effect, due to the addition of dextran sulfate was also observed in Chinese hamster fibroblasts, a type of mesenchymal cell.

(1-3) Heparins (1-3-1) Storage 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 sodium heparin (Mochida Pharmaceutical Co., Ltd., Heparin Na Mochida, unfractionated heparin) was used instead of alginic acid and the conditions shown in Table 5 were used. The results are shown in Table 5. N=4.

(1-3-2) Refrigerated storage 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 the conditions shown in Table 6, in which the cells were left in a refrigerator at a set temperature of 4°C for 72 hours. The results are shown in Table 6. N=4.

As shown in Tables 5 and 6, cell preservation solutions containing heparin sodium, regardless of the preparation used, exhibited superior cell viability and cell protection effects compared to cell preservation solutions not containing heparin sodium (i.e., preparation solution only) when stored at room temperature for 24 hours and refrigerated (4°C) for 72 hours. This cell viability improvement effect, i.e., cell protection effect, due to the addition of heparin sodium was also confirmed to be observed in Chinese hamster fibroblasts, a type of mesenchymal cell. Furthermore, when a similar experiment was performed using low-molecular-weight heparin sodium instead of heparin sodium, it was confirmed that the cell viability improvement effect, i.e., cell protection effect, was similarly observed in each cell type.

(1-4) Protease inhibitors (1-4-1) Urinastatin (inhibitor a)
(1-4-1-1) Refrigerated Storage 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 shown in Table 7 were used. The results are shown in Table 7. N=4. 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 of 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.

(1-4-2) Nafamostat mesylate (inhibitor b)
(1-4-2-1) Refrigerated Storage for 72 Hours Cell preservation solutions were prepared by adding the protease inhibitor nafamostat mesilate (Nichi-Iko Pharmaceutical Co., Ltd., Fusan for Injection) to a 5% glucose solution as the adjusting solution at the concentrations shown in Table 8. (Fusan for Injection (Nichi-Iko Pharmaceutical Co., Ltd.) specifies that 5% glucose solution be used as its diluent.) Highly metastatic mouse melanoma cells were added to each cell preservation solution at a concentration of ¹⁰⁶ cells/ml. After leaving the cells in a refrigerator set at 4°C for 48 hours, the average cell viability (%) was calculated in the same manner as in Experiment (1-4-1-1). The results are shown in Table 8. N=4 results.

(1-4-3) Mixture of protease inhibitors (inhibitor c)
(1-4-3-1) Refrigerated Storage 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.2H ₂ O, 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.

As shown in Tables 7 to 9, cell preservation solutions containing protease inhibitors exhibited superior cell viability and had excellent cell protective 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). It was also confirmed that the cell viability-enhancing effect, i.e., cell protective effect, of the addition of these protease inhibitors was also observed in Chinese hamster fibroblasts, a type of mesenchymal cell.

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

(2-1) Induction of Cell Dormancy Cell cycle analysis was performed by flow cytometry according to the method described in a published 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 was compared depending on the active ingredient of the cell treatment agent. Specifically, the procedure is as follows. First, a normal serum-free cell culture medium was used as the conditioning solution. This was used as (a) a control. Furthermore, the following cell preservation solutions were prepared: (b) a normal serum-free cell culture medium to which sodium alginate (Na alginate) was added to a concentration of 2.5 mg/ml; (c) a normal serum-free cell culture medium to which DS was added to a concentration of 0.1 mg/ml; and (d) a normal serum-free cell culture medium to which heparin sodium (Na heparin) was added to a concentration of 50 units/ml. Rat corneal epithelial cells were added to these cell preservation solutions to a cell concentration of ¹⁰ cells/ml and cultured for 48 hours under normal cell culture conditions (37°C, 5% _CO2 , 100% humidity). The number of cells in each cell cycle was determined by flow cytometry cell cycle analysis for each of the resulting cultured cells, and the proportions of cells in the S, G2, and M phases, which are considered to be periods during the cell division and proliferation process, were compared with the proportions 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.

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 serum-free cell culture medium was used alone (the control). In other words, when each component was added to serum-free cell culture medium, the cells' division and proliferation function was reduced, causing them to enter a dormant state. Therefore, it was found that cell preservation solutions containing each component are effective as cell treatments for cell dormancy, putting cultured cells into a dormant state.

(2-2) Maintenance of Differentiation State. Dextran sulfate (DS) was used as the active ingredient in the cell treatment agent. The cell dormancy effect of DS was investigated using its ability to maintain cells in an undifferentiated state and inhibit differentiation as an indicator. Undifferentiated human amnion-derived mesenchymal stem cells were used. A standard serum-free cell culture medium was used as the conditioning solution. Cell preservation solution A was prepared by adding DS to 0.1 mg/ml, and cell preservation solution B was prepared by adding FBS to 10%. The aforementioned stem cells were added to each of cell preservation solutions A and B at a concentration of ¹⁰ cells/ml, and then cultured for 5 days under standard cell culture conditions (37°C, 5% _CO2 , 100% humidity). The degree of differentiation of the stem cells before and after 5 days of culture was evaluated and compared by flow cytometry. The method used was in accordance with the method described in Masoumeh Fakhr Taha and Vahideh Hedayati, "Identification and multipotential differentiation of mouse adipose tissue-derived stem cells," Tissue and Cell 42 (2010) 211-216. Evaluation was performed using CD29 and CD44 as undifferentiated markers, and CD11b, CD31, and CD45 as mesenchymal cell differentiation markers. The results are shown in Table 11.

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. In contrast, 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 cells began to differentiate. Thus, it was revealed that DS suppresses differentiation by keeping cells dormant in an undifferentiated state, whereas FBS, which also has a cytoprotective effect, differentiates cells but does not suppress them.

(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), or 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.

(3-1) Awakening from Cell Dormancy First, a standard serum-free cell culture medium was used as the conditioning solution. This served as (a) a control. Cell preservation solutions were prepared by adding (b) DS to the standard serum-free cell culture medium at 0.1 mg/ml, (c) heparin sodium (heparin Na) to a concentration of 50 units/ml, and (d) sodium alginate (alginate Na) to a concentration of 2.5 mg/ml. Next, rat corneal epithelial cells obtained as described above were added to the cell preservation solutions (a) to (d) to a cell density of 1 x ¹⁰ cells/ml, and these were 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) thus obtained were designated (a') to (d'), respectively.

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 calticol injection solution 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.

The cell-containing cell preservation solutions (a) to (d) obtained as described above after 48 hours of refrigerated storage were centrifuged, and the cells (a') to (d') were separated and collected by centrifugation, and the cells (a') to (d') were added to the cell preservation solutions a-1 to d-2 with the corresponding alphabetical sample numbers, respectively, so that the cell content was 6 x ¹⁰ cells/ml.

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%).

After culturing, the number of viable cells per unit volume contained in cell preservation solutions a-1 to d-2 was quantified using the MTT assay. The MTT assay was performed according to a known publication (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 are shown.

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 when 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 by including the components used as active ingredients in the cell treatment agent, the reduced proliferation and division ability of dormant cells was awakened by the addition of calcium ions, and conversely, the proliferation and division ability of the cells was activated.

(Experimental Example 4: Cell Detachment and Floating Action)
<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 a concentration of 10% and placing it in a culture dish. Rat corneal epithelial cells were added to the medium at a concentration of ¹⁰ cells/ml and cultured for 24 hours under normal cell culture conditions (37°C, 5% _CO2 , 100% humidity) to allow the cultured cells to fully adhere to the wall of the culture dish, which served as a scaffold.

<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 solutions at the concentrations shown in Table 13, which were obtained 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 culture was then continued 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. A detachment of 90% or more of the cells was considered to be 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.

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 conducted 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.

(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.

<Detachment and suspension of adherent cells>
The culture medium in the culture dish containing the cultured cells obtained as described above was prepared with or without each active ingredient added to the concentrations shown in Table 14. Next, each culture dish was placed on the platform of a shaker (manufactured by Biocraft Co., Ltd., product name: Laboshaker Model BC-740) with the shaking intensity set to the lowest setting and the shaking frequency set to 4 times/minute. The culture dish was cultured for 24 hours under normal cell culture conditions (37°C, 5% _CO2 concentration, 100% humidity) while gently shaking. After 24 hours of culture, the cells were observed for detachment and floating. Detachment and floating were determined by observing whether the cells detached and floated from the dish wall while the culture dish was being shaken. Cells that detached and floated were judged as "detached and floated," with "detached and floated" cells marked as "○" and "not detached and floated" cells marked as "×." The results are shown in Table 14. The result is N=4.

<Re-adhesion of detached and floating cells>
We investigated whether the detached and suspended cells re-adhere via calcium ions. First, a slurry of calcium phosphate fine powder mixed with distilled water was applied to a portion of the bottom of a culture dish, which was then heat-dried to prepare a separate culture dish with a calcium phosphate fine powder film attached to the bottom. Next, a cell culture medium containing the detached and suspended cells was added to the calcium phosphate-film-covered culture dish and a control culture dish without the film. The culture dishes were then placed on a shaker (Biocraft, Product Name: Laboshaker Model BC-740) with the shaking intensity set to the lowest setting and the culture dish gently shaken at 4 shakes per minute. The culture dishes were then cultured for 24 hours under normal cell culture conditions (37°C, 5% _CO2 , 100% humidity) with the shaking intensity set to the lowest setting and the shaking frequency set to 4 shakes per minute. Thereafter, the culture medium in each culture dish was removed, and the culture dish was gently washed with 10% FBS cell culture medium. Thereafter, live cells were stained with a fluorescent dye (CytoPainter F-actin Staining Kit, manufactured by Abcam) and the live cells adhering to the inner wall of the culture dish were observed under a microscope (Olympus Corporation, inverted fluorescent and visible microscope with cooled CCD camera, IX83) by fluorescent staining of F-actin. 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.

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

As shown in Figures 1 and 2, numerous viable cells were found attached to the surface of the calcium phosphate film, either singly or in cell clusters. On the other hand, as shown in Figures 3 and 4, very few viable cells were found attached to the bottom of the culture dish without a calcium phosphate film. A similar experiment was performed using Chinese hamster fibroblasts, yielding similar results. Thus, it can be seen that calcium phosphate, a solid formulation, activates cells by re-attaching them after they have been detached and suspended by active ingredients such as DS or heparin sodium. In other words, by combining a cell treatment agent containing an active ingredient with a specific formulation of polyvalent cations, it is possible to awaken dormant cells that have been rendered dormant by the cell treatment agent. Therefore, a combined reagent consisting of these two agents is suitable for awakening dormant cells.

(Experimental Example 6: Relationship between viscosity and concentration of alginic acid and cell protective 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 conditioning solution used, for example, when storing and transporting cells. The three types of alginic acid with different viscosities were: (a) sodium alginate (Snow Algin L, manufactured by 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, manufactured by 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 (low-molecular-weight sodium alginate, manufactured by Kyosei Pharmaceutical Co., Ltd.) with a 10% aqueous solution and a viscosity of 30 mPa·s (very low viscosity (ultra-low viscosity)) at 20°C. The Ringer's solution used was the same as in Experiment 1.

(6-1) Storage at Room Temperature for 24 Hours Cell preservation solutions were prepared by adding the aforementioned alginates of different viscosities to Ringer's solution to the concentrations shown in Table 15. The aforementioned rat corneal epithelial cells were added to this cell preservation solution at a concentration of 10 ⁶ cells/ml. After leaving the solution in a room at room temperature (22°C) for 24 hours, the viability of the cells was assessed, and the percentage of viable cells (average survival rate (%)) was calculated and compared. Cell viability was assessed using a trypan blue exclusion test, a common method for confirming cell viability. The results are shown in Table 15. N=4 results.

(6-2) Refrigerated storage 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.

As shown in Table 15, when the storage time is relatively short, 24 hours, at room temperature (22°C), alginic acid (high viscosity) with a relatively high viscosity (1% aqueous solution, 120 mPa·s (20°C)) exhibits more effective cell protection in a cell preservation solution prepared at a relatively low concentration (approximately 2.5 mg/ml or less) than other viscosities (low viscosity, very low viscosity). As shown in Table 16, when cells are stored for a very long period of time, such as 7 days, alginic acid (very low viscosity) with a very low viscosity (10% aqueous solution, 30 mPa·s (20°C)) exhibits more effective cell protection in a cell preservation solution prepared at a relatively high concentration (approximately 10 mg/ml or more) than other viscosities (high viscosity, low viscosity). Furthermore, Table 16 shows that alginic acid with a very low viscosity is expected to be more effective at higher concentrations.

Claims (8)

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 . A cell- protective cell treatment agent containing alginic acid as an active ingredient and at least one selected from extracellular fluid replacement solutions and maintenance infusion solutions, which maintains the viability of cells in tissues or cultured cells and suppresses their death. A cell treatment agent for cell preservation for maintaining cells in an undifferentiated or less differentiated state , comprising alginic acid as an active ingredient and at least one selected from an extracellular fluid replacement solution and a maintenance infusion solution . 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 scaffold of biological tissue or a scaffold of a culture substrate, or intracellular cells of biological tissue from a scaffold of biological tissue. 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. 3. The cell-protecting cell treating agent according to claim 2, which is a liquid for cell, tissue or organ preservation. 4. The cell treating agent for cell preservation according to claim 3, which is a liquid for cell, tissue or organ preservation. 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.