JP2008104407A - Cell preservation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the refrigerating preservation of an adherent cell, more particularly, to provide a method for the refrigerating preservation of an adherent cell to enable effective preservation without removing a hitherto indispensable cryopreservant in the case of transferring from a frozen cell storage state to the normal cell culture condition. <P>SOLUTION: Adherent cells proliferated on a culture substrate are peeled off from the culture substrate by using a cell peeling liquid, suspended in a cell preservation liquid containing a reducing sugar and preserved under a refrigeration condition. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for preserving adherent cells, which is preserved under refrigerated conditions.

  Living cells have physiological functions and biological activities, and are useful in many fields. However, if the cells are left under high temperature conditions, the cells are denatured by metabolism, so that the physiological functions and biological activities of the cells are reduced or lost. Therefore, it is necessary to freeze and store the cells. For cell cryopreservation, DMSO (dimethyl sulfoxide), ethylene glycol, glycerol and the like have been conventionally used as cell membrane permeation cryopreservation agents, and polyvinylpyrrolidone and the like have been used as cell membrane non-permeation cryopreservation agents. Further, it is disclosed that if a cell preservation solution containing purified albumin and DMSO is used, the cells can be preserved at −80 ° C. and for a short period of time at about −4 ° C. (Patent Document 1).

  However, generally, these cryopreservatives have a problem that they are toxic to cells at high concentrations. In addition, when using cryopreserved cells for culture or the like, it is necessary to remove the cryopreservative from the cells in advance, which is complicated. Moreover, although cryopreservation is performed at very low temperature conditions, such as -80 degreeC, for example, this has the problem that a specialized expensive freezing apparatus is required. In addition, the process of freezing / thawing may cause significant damage to the cells, thus reducing the survival rate. Furthermore, some cell types cannot be cryopreserved.

  As a preservation solution for cells / tissues and the like that does not contain a toxic substance such as DMSO for cryopreservation, a preservation solution containing polysaccharide levan or levan oligosaccharide (Patent Document 2), fructooligosaccharide A preservation solution containing 1-kestose as an active ingredient (Patent Document 3) is disclosed.

  On the other hand, a composition containing trehalose, a matrix protein filler, and a monosaccharide in a buffer (Patent Document 4), a preservation solution containing an antioxidant polyphenol (Patent Document 5), and water A solution in which a chelating agent or the like is added to a miscible alcohol is disclosed (Patent Document 6).

In addition to the above-mentioned solutions, conventionally used cell / tissue preservation solutions include Euro-Collins solution (Non-patent document 1), UW solution (University of Wisconsin solution) (Non-patent document 2). ), ET-Kyoto solution (Patent Document 7), and a solution obtained by adding epigallocatechin galade of green tea catechin as an active ingredient to Euro-Collins solution or UW solution (Patent Document 8).
JP 2002-233356 A JP-A-9-255501 Japanese Patent Laid-Open No. 5-38284 Special Table 2003-505024 International Publication WO02 / 017952 Japanese Patent Laid-Open No. 2002-168858 Japanese Patent Laid-Open No. 6-40801 JP 2003-267801 A Squifflet, JP, et al., Transplant Proc., 13, 693, 1981 Wahlberg, JA, et al., Transplantation, 43, pp. 5-8, 1987

  An object of the present invention is to provide a method for refrigerated storage of adherent cells. More specifically, when transferring from frozen cell storage to cell culture conditions using a cell growth medium, it can be effectively stored without using a cryopreservative so that the necessary cryopreservative need not be removed. It is an object to provide a method for refrigerated storage of adherent cells.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research, and as a result, reduced adherent cells grown on the culture substrate from the culture substrate using a cell detachment solution. The present invention has been completed by finding that adherent cells can be stored under refrigerated conditions by suspending in a cell preservation solution containing sugar.

That is, this invention consists of the following.
1. Adherent cells grown on the culture substrate are detached from the culture substrate using a cell detachment solution, and the detached adherent cells are suspended in a cell preservation solution containing a reducing sugar and subjected to refrigeration conditions. A method for preserving adherent cells, comprising:
2. 2. The method for preserving adherent cells according to item 1 above, wherein the reducing sugar contained in the cell preservation solution is any one or more selected from the group consisting of glucose and maltose.
3. 3. The method for preserving adherent cells according to item 1 or 2, wherein the cell preservation solution further contains hydrogen carbonate ions and / or carbonate ions.
4). 4. The method for preserving adherent cells according to item 3 above, wherein the content of hydrogen carbonate ions and / or carbonate ions contained in the cell preservation solution is 1 to 75 mM.
5. The cell preservation solution further contains any one or more kinds of ions selected from the group consisting of calcium ions, magnesium ions, chloride ions, phosphate ions, sulfate ions and lactate ions, 5. The method for preserving adherent cells according to any one of 4 above.
6. The method for preserving adherent cells according to item 1 above, wherein adherent cells are preserved under refrigerated conditions using a cell preservation solution containing the following composition in a 1,000 ml aqueous solution.
Reducing sugar 0-150 mM
Sodium ion 20-260 mM
Potassium ion 3 to 125 mM
Bicarbonate ion and / or carbonate ion 1-75 mM
Calcium ion 1-4 mM
Magnesium ion 0 to 1 mM
Chloride ion 75-245 mM
Phosphate ion 0.5 to 65 mM
Sulfate ion 0 to 1 mM
Lactate ion 0-20 mM
7). 7. The method for preserving adherent cells according to any one of items 1 to 6, wherein the refrigeration condition is 0 to 25 ° C.
8). By seeding the adherent cells after being stored by the method for preserving adherent cells according to any one of items 1 to 7 in a cell growth medium without passing through a step of washing and removing the cell preservation solution. A method for culturing adherent cells.
9. A cell preservation solution containing a reducing sugar for preservation of adherent cells and a kit for preservation of adherent cells comprising a cell detachment solution.
10. 10. The kit for preserving adherent cells according to item 9, wherein the cell preservative solution is a cell preservation solution used for the method for preserving adherent cells according to any one of items 1 to 8.

  According to the method for preserving adherent cells of the present invention, when the adherent cells are transferred from a cryopreserved state to a cultured state, a washing step for removing a cryopreservative by centrifugation or the like that is normally required is provided. Without passing, the cells can be seeded in a medium while being suspended in a cell preservation solution to culture the cells. The cell preservation solution used for refrigerated preservation of the adherent cells of the present invention contains at least a reducing sugar. When cells are preserved using such a cell preservation solution, cell viability is increased and cell physiology is maintained. And cell preservation performance can be maintained at a high level.

  In the method for preserving adherent sperm cells of the present invention, adherent cells cultured on a culture substrate using a normal cell growth medium are used, for example, a cell detachment solution known per se containing trypsin and / or EDTA or the like. It is related with the preservation | save method which carries out refrigeration preservation | save by detaching, suspending to the said density | concentration in a cell preservation solution, and making it float. Here, the normal cell growth medium differs depending on the cell type and purpose of use, but a necessary amount of fetal bovine serum (hereinafter referred to as “FBS”) or the like is appropriately added to a commercially available medium. Examples of the medium for cell growth known per se can be mentioned.

  In the present invention, the cell culture substrate refers to a substrate used by a person skilled in the art for cell culture, such as a cell culture petri dish or a cell culture flask, and is not particularly limited. The material for the cell culture substrate is not particularly limited as long as cell culture can be performed. Examples thereof include fibers, woven fabrics, non-woven fabrics, sponges, cottons, and films in addition to plastic plates.

  Examples of adherent cells that can be preserved by the preservation method of the present invention include various cells isolated from mammals including humans, such as periosteum cells, mesenchymal stem cells, nerve cells, hepatocytes, kidney cells, Examples include epithelial cells, fibroblasts, hematopoietic stem cells, mesenchymal stem cells and ES cells. The adherent cells may be established adherent cells. Examples of established adherent cells include 3T3 cells (derived from mouse fibroblasts), normal human fibroblasts, A549 cells (derived from human lung epithelial cells), HepG2 cells (derived from human liver cancer), Vero cells (African green monkey kidney). Cell-derived).

  According to the method for storing adherent cells of the present invention, cells are stored under refrigerated conditions. Refrigerated conditions are conditions in which the solution does not freeze and are not particularly limited as long as the temperature is low, but is preferably 0 to 25 ° C, more preferably 0 to 10 ° C, and particularly preferably 4 to 6 ° C. Range. The cell storage device is not particularly limited as long as it can achieve the refrigeration conditions as described above, and examples thereof include an incubator and a refrigerator.

  The cell storage container used in the method for storing adherent cells of the present invention is appropriately selected from known containers in consideration of the cell storage device, cell type, storage temperature, storage period, use of cells after storage, etc. Can be used. For example, when storing cells in a refrigerator, it is preferable to use a sealed container.

  Further, the cell storage period according to the cell storage method of the present invention depends on conditions such as the cell type and storage temperature, and thus cannot be uniformly defined, but as a guideline, it is about 4 to 12 days, preferably 4 to 4 days. Up to about 6 days is possible. As a specific use mode, for example, it can be used as a preservation method when transporting adherent cells from a contract culture facility to a patient. In the case of such short-term storage, the storage method of the present invention is advantageous in that it can be stored under refrigerated conditions.

Storage by the cell storage method of the present invention can be performed by suspending the cells in a storage solution even if they are adherent cells. The cell concentration suspended in the cell preservation solution during storage depends on conditions such as cell type, cell size, storage period, etc., and is appropriately determined by those skilled in the art, and is not particularly limited. However, it is about 1 × 10 ⁴ to 1 × 10 ⁸ cells / ml, for example.

  The method for preserving adherent cells according to the present invention uses adherent cells cultured on a culture substrate using a normal cell growth medium, for example, a cell detachment solution known per se containing trypsin and / or EDTA or the like. This is achieved by detaching, suspending in the cell preservation solution shown below at the above concentration, and suspending. In order to make it float in a cell preservation solution so that it may become the said density | concentration, after removing the cell detachment liquid by centrifuging the detached cell in a cell preservation solution or other solution and removing the cell detachment liquid by centrifugation, the cell preservation solution is made so that the said density | concentration Cells may be suspended.

  The cell preservation solution used in the method for preserving adherent cells of the present invention is characterized by containing at least a reducing sugar. As used herein, reducing sugar refers to monosaccharides and oligosaccharides in which aldose 1-position or ketose 2-position, that is, the anomeric carbon atom is not substituted, such as glucose or maltose. These sugars may be used alone or in combination. Of these, glucose is preferred. Commercially available saccharides can be used. The saccharide content in the cell preservation solution is 0.5 to 150 mM, preferably 0.5 to 100 mM, and more preferably 0.5 to 50 mM. If the saccharide content exceeds 150 mM, the cells may aggregate and form a lump during cell storage, and a complicated operation is required to loosen the lump.

The cell preservation solution used in the present invention preferably further contains hydrogen carbonate ions and / or carbonate ions. Here, the bicarbonate ion indicates HCO ₃ ⁻ , and the carbonate ion indicates CO ₃ ²⁻ . These ions are in an equilibrium state in the solution and can change depending on conditions such as pH. Hydrogen carbonate ions and / or carbonate ions can be included in the preservation solution by adding various carbonates and bicarbonates to the cell preservation solution. Examples of such compounds include sodium hydrogen carbonate (sodium bicarbonate), sodium carbonate, calcium carbonate, calcium bicarbonate, magnesium carbonate and the like. Baking soda is particularly suitable in terms of economy and ease of handling, but the present invention is not limited to this. A commercially available baking soda can be used (in the following examples, Wako Pure Chemical Industries, Ltd. is used). The hydrogen carbonate ion and / or carbonate ion content in the cell preservation solution can be selected from 1 to 75 mM, preferably 10 to 50 mM, more preferably 20 to 30 mM, but is not particularly limited. Absent.

In the cell preservation solution used in the present invention, it is preferable to improve cell viability by preventing cells and tissues from expanding or contracting during preservation. For this reason, the osmotic pressure of the cell preservation solution is adjusted to 200 to 500 mOsm / kg, preferably 200 to 400 mOsm / kg, more preferably 250 to 350 mOsm / kg. The osmotic pressure of the preservation solution is determined by the concentration of all substances including ions contained in the preservation solution. The osmotic pressure of the preservation solution can be appropriately adjusted by changing the content of saccharides, hydrogen carbonate ions and / or carbonate ions, and other ions contained in the preservation solution, or harmless such as hydroxyethyl starch. An osmotic pressure adjusting agent may be added.
Further, the cell preservation solution used in the present invention is adjusted to a pH of 5.0 to 8.5, preferably 6.0 to 8.5, more preferably 7.0 to 8.0. Although pH can be adjusted by adding electrolytes, such as a sodium salt or potassium salt of the organic acid mentioned above, it is not limited to this.

  The cell preservation solution used in the present invention may further contain metal ions such as calcium ions and magnesium ions, chloride ions, phosphate ions, nitrate ions, sulfate ions, and lactate ions. These ions may be used alone or in combination, and are not particularly limited. The concentration of these ions depends on the osmotic pressure, pH, and content of other substances in the preservation solution, and can be appropriately set by those skilled in the art, and is not particularly limited. For example, magnesium ion is 0.0 to 1 mM, preferably 0.2 to 0.8 mM, calcium ion is 1 to 4 mM, preferably 1.5 to 3.5, chloride ion is 75 to 245 mM, preferably 100 to 150 mM, phosphate ion is It can be selected from 0.5 to 65 mM, preferably 0.5 to 2 mM, sulfate ion 0 to 1 mM, preferably 0.5 to 1 mM, and lactate ion 0 to 20 mM, preferably 0 to 15 mM.

  The cell preservation solution used in the present invention appropriately contains other components in the conventional preservation solution, such as antibiotics, antibacterial agents, antioxidants, serum, vitamins, proteins, amino acids, pH indicators, chelating agents, etc. You can also.

In addition, the cell preservation solution used in the present invention is aseptic so that a necessary concentration of a dehydration replenisher that is a commercially available infusion preparation is included in the Japanese Pharmacopoeia Ringer's solution and the Japanese Pharmacopoeia sodium bicarbonate solution. It can be prepared by a method of mixing under conditions. As another method, for example, a necessary amount of each necessary component may be weighed, mixed with pure water, and the mixed solution may be sterilized and filtered.
As a dehydration replenisher which is a commercially available infusion preparation, for example, a drug sold as a general electrolyte infusion or an electrolyte for infusion can be used. More specifically, an injection solution having the following composition 1) or 2) can be mentioned.

1) Injection solution containing the following ingredients in 1,000 ml of aqueous solution 1.92 g of sodium chloride
Potassium chloride 1.00 g
Sodium lactate 2.80 g
Magnesium chloride 0.10 g
Monosodium phosphate 0.14 g
Dipotassium phosphate 1.00 g
Glucose 23.5 g

2) Injection solution consisting of the following composition Sodium chloride 0.270 w / v%
Potassium chloride 0.149 w / v%
Sodium lactate 0.224 w / v%
Sodium dihydrogen phosphate 0.031 w / v%
Sodium monohydrogen phosphate 0.287 w / v%
Glucose 3.2 w / v%
L-lactic acid 8 mEq / L

  The cell preservation solution of the present invention can be prepared by mixing the Japanese Pharmacopoeia Ringer's solution, the Japanese Pharmacopoeia sodium bicarbonate solution and the commercially available infusion preparation at a volume ratio of 80: 3: 20-50. Specifically, for example, it can be prepared by mixing 250 to 300 ml of the injection solution of 1) above with 800 ml of Japanese Pharmacopoeia Ringer's solution and 30 ml of Japanese Pharmacopoeia sodium bicarbonate solution. As another method, for example, 800 ml of Japanese Pharmacopoeia Ringer's solution and 30 ml of Japanese Pharmacopoeia hydrogen carbonate can be prepared by mixing 400 to 450 ml of the injection solution of the above 2).

  Cells stored by refrigerated storage for a certain period of time as described above may be cultured after removing the cell storage solution by centrifugation or the like and then seeded and cultured in a normal cell growth medium. Can be cultivated as it is in a normal cell growth medium to perform normal culture. The cell concentration for resuming the culture varies depending on the cell type, but can be, for example, the cell concentration at the time of cell passage. The present invention also extends to a method of performing cell culture using a normal cell growth medium using cells that have been stored in a cell preservation solution.

  The present invention also extends to an adherent cell storage kit containing a cell storage solution and a cell detachment solution.

  In the following, reference examples, examples, experimental examples, etc. are shown to specifically describe the preservation solution, the cell preservation method, and the cell viability resulting therefrom, which can be used in the method for preserving adherent cells of the present invention. The present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention.

(Reference Example 1) Cell preservation solution containing glucose and sodium bicarbonate A cell preservation solution was prepared by dissolving the following components in pure water. The osmotic pressure was about 283.5 mOsm / kg. The osmotic pressure was measured using OSMOMETER OM 802-D (manufactured by VOGEL).
Glucose (Wako Pure Chemical Industries, Ltd.) 1,000 mg
Sodium chloride 6,976.8 mg
Potassium chloride 400 mg
Calcium chloride 200 mg
Sodium bicarbonate 2,000 mg
Sodium hydrogen phosphate hydrate 140 mg
Magnesium sulfate 98 mg
1N hydrochloric acid 2.8 ml
Total water 1,000 ml

Therefore, the ionic composition is as follows.
Glucose 5.5 mM
Sodium ion 143.8 mM
Potassium ion 5.4 mM
Bicarbonate ion and / or carbonate ion 23.7 mM
Calcium ion 1.8 mM
Magnesium ion 0.8 mM
Chloride ion 130.8 mM
Phosphate ion 1.0 mM
Sulfate ion 0.8 mM
Total water 1,000 ml

(Reference Example 2) Cell preservation solution comprising a commercially available MEM medium A commercially available MEM medium (GIBCO) was used as a cell preservation solution. It consists of the following compositions.
Glucose 5.6 mM
Sodium ion 145.3 mM
Potassium ion 5.4 mM
Bicarbonate ion and / or carbonate ion 23.7 mM
Calcium ion 1.8 mM
Magnesium ion 0.8 mM
Chloride ion 132.4 mM
Phosphate ion 1.0 mM
Sulfate ion 0.8 mM
Total water 1,000 ml

Therefore, the ionic composition is as follows.
Sodium ion 143.8 mM
Potassium ion 5.4 mM
Bicarbonate ion and / or carbonate ion 23.7 mM
Calcium ion 1.8 mM
Magnesium ion 0.8 mM
Chloride ion 130.8 mM
Phosphate ion 1.0 mM
Sulfate ion 0.8 mM
Total water 1,000 ml

(Reference Example 3) Cell preservation solution obtained by adding FBS to commercially available MEM medium A cell preservation solution obtained by adding 10% FBS to the MEM medium in Reference Example 2 was used.

(Reference Example 4) Cell preservation solution containing maltose and sodium bicarbonate A cell preservation solution similar to Reference Example 1 was prepared, except that glucose in Reference Example 1 was replaced with maltose (manufactured by Wako Pure Chemical Industries, Ltd.). Incidentally, the maltose content is the same as the glucose content of Reference Example 1.

(Reference Example 5) Cell preservation solution containing mannitol and sodium bicarbonate A cell preservation solution similar to Reference Example 1 was prepared except that glucose in Reference Example 1 was replaced with mannitol (manufactured by Wako Pure Chemical Industries, Ltd.). Incidentally, the content of mannitol is the same as the content of glucose in Reference Example 1.

(Reference Example 6) Cell preservation solution containing sucrose and sodium bicarbonate A cell preservation solution similar to Reference Example 1 was prepared except that glucose in Reference Example 1 was replaced with sucrose (manufactured by Sigma). Incidentally, the sucrose content is the same as the glucose content of Reference Example 1.

(Reference Example 7) Cell preservation solution containing trehalose and sodium bicarbonate A cell preservation solution similar to Reference Example 1 was prepared except that glucose in Reference Example 1 was replaced with Trehalose (manufactured by Wako Pure Chemical Industries, Ltd.). Incidentally, the trehalose content is the same as the glucose content of Reference Example 1.

(Reference Example 8) Cell preservation solution containing no saccharide A cell preservation solution similar to Reference Example 1 was prepared except that glucose was not contained. Therefore, it consists of the following compositions.
Glucose (Wako Pure Chemical Industries, Ltd.) 0 mg
Sodium chloride 6,976.8 mg
Potassium chloride 400 mg
Calcium chloride 200 mg
Sodium bicarbonate 2,000 mg
Sodium hydrogen phosphate hydrate 140 mg
Magnesium sulfate 98 mg
1N hydrochloric acid 2.8 ml
Total water 1,000 ml

Therefore, the ionic composition is as follows.
Sodium ion 143.8 mM
Potassium ion 5.4 mM
Bicarbonate ion and / or carbonate ion 23.7 mM
Calcium ion 1.8 mM
Magnesium ion 0.8 mM
Chloride ion 130.8 mM
Phosphate ion 1.0 mM
Sulfate ion 0.8 mM
Total water 1,000 ml

Reference Example 9 Phosphate buffer solution Phosphate buffer solution was used as a cell preservation solution.

(Example 1) Refrigerated storage of 3T3 cells using the cell preservation solution of Reference Example 1 First, a cell suspension of 3T3 cells (provided by RIKEN) was prepared. As a dispersion medium, a 10% FBS-containing MEM medium (GIBCO) was used, and the concentration was about 1 × 10 ⁴ cells / ml. Next, 10 ml of this cell suspension was developed and seeded on a 10 cm polystyrene dish. Then, the cells were cultured in an incubator set to 5% CO ₂ and 37 ° C.

With the cultured cells becoming confluent, the medium was removed by suction. Next, 3 ml of a phosphate buffer containing 0.001% trypsin and 0.02% EDTA was added to the polystyrene dish. While observing under a microscope, trypsin and EDTA-containing phosphate buffer were removed by suction before the cells were detached. 5T FBS-containing MEM medium was added to detach 3T3 cells from the petri dish, and then transferred to a centrifuge tube (manufactured by Greiner). After centrifugation at 300 × g, the supernatant was removed. And in order to wash | clean FBS, the operation | work which centrifuges and removes a supernatant liquid with a phosphate buffer solution was repeated twice. With the above operation, a cell pellet was formed at the bottom of the centrifuge tube. A cell suspension of about 220 × 10 ⁴ cells / ml was prepared by adding 1 ml of the cell preservation solution of Reference Example 1 to the cell pellet. By sealing the centrifuge tube, the cell suspension suspended in the cell preservation solution was stored in an environment of 6 ° C.

(Example 2) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 2 Example except that the cell preservation solution of Reference Example 2 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Example 3) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 3 Example except that the cell preservation solution of Reference Example 3 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Example 4) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 4 Example except that the cell preservation solution of Reference Example 4 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Example 5) Refrigerated storage of normal human fibroblasts using the cell preservation solution of Reference Example 1 Normal human fibroblasts (provided by RIKEN) were used in place of the 3T3 cells used in Example 1. Except for this, the same procedure as in Example 1 was performed.

(Example 6) Refrigerated storage of normal human fibroblasts with the cell preservation solution of Reference Example 2 As in Example 2, except that normal human fibroblasts were used instead of the 3T3 cells used in Example 2. went.

(Example 7) Refrigerated storage of normal human fibroblasts using the cell preservation solution of Reference Example 3 As in Example 3, except that normal human fibroblasts were used instead of the 3T3 cells used in Example 3. went.

(Example 8) Refrigerated storage of A549 cells with the cell preservation solution of Reference Example 1 Example 1 except that A549 cells (provided by RIKEN) were used instead of the 3T3 cells used in Example 1. As well as.

(Example 9) Refrigerated storage of A549 cells with the cell preservation solution of Reference Example 2 The same procedure as in Example 2 was conducted except that A549 cells were used instead of the 3T3 cells used in Example 2.

(Example 10) Refrigerated storage of A549 cells with the cell preservation solution of Reference Example 3 The same procedure as in Example 3 was conducted except that A549 cells were used instead of the 3T3 cells used in Example 3.

(Example 11) Refrigerated storage of HepG2 cells with the cell preservation solution of Reference Example 1 Example 1 except that HepG2 cells (provided by RIKEN) were used instead of the 3T3 cells used in Example 1. As well as.

(Example 12) Refrigerated storage of HepG2 cells with the cell preservation solution of Reference Example 2 The same procedure as in Example 2 was conducted except that HepG2 cells were used instead of the 3T3 cells used in Example 2.

(Example 13) Refrigerated storage of HepG2 cells with the cell preservation solution of Reference Example 3 The same procedure as in Example 3 was conducted except that HepG2 cells were used instead of the 3T3 cells used in Example 3.

(Example 14) Refrigerated storage of Vero cells with the cell preservation solution of Reference Example 1 Example 1 except that Vero cells (provided by RIKEN) were used instead of the 3T3 cells used in Example 1. As well as.

(Example 15) Refrigerated storage of Vero cells with the cell preservation solution of Reference Example 2 The same procedure as in Example 2 was performed except that Vero cells were used instead of the 3T3 cells used in Example 2.

(Example 16) Refrigerated storage of Vero cells using the cell preservation solution of Reference Example 3 The same procedure as in Example 3 was performed except that Vero cells were used instead of the 3T3 cells used in Example 3.

(Comparative Example 1) Cryopreservation of 3T3 cells First, a cell suspension of 3T3 cells (provided by RIKEN) was prepared. As a dispersion medium, a 10% FBS-containing MEM medium (GIBCO) was used, and the concentration was 1 × 10 ⁴ cells / ml. Next, 10 ml of this cell solution was developed and seeded on a 10 cm polystyrene dish. Then, the cells were cultured in an incubator set to 5% CO ₂ and 37 ° C.

With the cultured cells becoming confluent, the medium was removed by suction. Then, the cells were detached by trypsin and EDTA treatment in the same manner as in Example 1, and 3T3 cells were recovered with 10% FBS-containing MEM. Next, the FBS was washed by centrifuging twice using a phosphate buffer. Thereafter, MEM containing 10% FBS and 10% DMSO was added to the cell pellet formed at the bottom of the centrifuge tube to prepare a cell suspension having a concentration of 830 × 10 ⁴ cells / ml. Then, the cell suspension added to the cryopreservation tube was slowly frozen in a −80 ° C. freezer. The temperature was lowered to −80 ° C. and then transferred into liquid nitrogen.

(Comparative Example 2) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 5 Example except that the cell preservation solution of Reference Example 5 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Comparative Example 3) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 6 Example except that the cell preservation solution of Reference Example 6 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Comparative Example 4) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 7 Example except that the cell preservation solution of Reference Example 7 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 1 was performed.

(Comparative Example 5) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 8 Example except that the cell preservation solution of Reference Example 8 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 3T3 cells were preserved as in 1.

(Comparative Example 6) Refrigerated storage of 3T3 cells with the cell preservation solution of Reference Example 9 Example except that the cell preservation solution of Reference Example 9 was used instead of the cell preservation solution of Reference Example 1 used in Example 1 3T3 cells were preserved as in 1.

(Comparative Example 7) Refrigerated storage of normal human fibroblasts with the cell preservation solution of Reference Example 9 As in Comparative Example 3, except that normal human fibroblasts were used instead of the 3T3 cells used in Comparative Example 3. went.

(Comparative Example 8) Refrigerated storage of A549 cells with the cell preservation solution of Reference Example 9 The same procedure as in Comparative Example 6 was conducted except that A549 cells were used instead of the 3T3 cells used in Comparative Example 6.

(Comparative Example 9) Refrigerated storage of HepG2 cells with the cell preservation solution of Reference Example 9 The procedure was the same as Comparative Example 6 except that HepG2 cells were used instead of the 3T3 cells used in Comparative Example 6.

(Comparative Example 10) Refrigerated storage of Vero cells with the cell preservation solution of Reference Example 9 The same procedure as in Comparative Example 6 was conducted except that Vero cells were used instead of the 3T3 cells used in Comparative Example 6.

(Comparative Example 11) Refrigerated storage of 3T3 cells without detachment First, a cell suspension of 3T3 cells (provided by RIKEN) was prepared. As a dispersion medium, a 10% FBS-containing MEM medium (GIBCO) was used, and the concentration was 1 × 10 ⁴ cells / ml. Next, 2 ml of this cell solution was developed and seeded on a 10 cm polystyrene dish. Then, the cells were cultured in an incubator set to 5% CO ₂ and 37 ° C.

  With the cultured cells becoming confluent, the medium was removed by suction. Then, 2 ml of the cell preservation solution of Reference Example 1 was added and stored in an environment of 6 ° C.

(Comparative Example 12) Refrigerated storage of HepG2 cells without detachment The same procedure as in Comparative Example 11 was performed except that HepG2 cells were used instead of the 3T3 cells used in Comparative Example 11.

(Experimental example 1)
In the storage conditions of Examples 1 to 3 (cell preservation solution: Reference Examples 1 to 3) and Comparative Example 1 (freeze storage), the cell viability with respect to the storage days when 3T3 cells were stored was examined. The result is shown in FIG. The cell viability was measured by viability determination by trypan blue staining. From these results, it was shown that the cell storage method of the present invention can store cells with a survival rate equivalent to that of cryopreservation if stored within 10 days.

(Experimental example 2)
In the storage conditions of Example 1 (cell preservation solution: Reference Example 1), Example 4 (Cell preservation solution: Reference Example 4) and Comparative Examples 2 to 6 (Cell preservation solution: Reference Examples 5 to 9), 3T3 cells were used. The cell viability with respect to the preservation days when preserved was examined. The result is shown in FIG. The cell viability was measured in the same manner as in Experimental Example 1. In the cell preservation solution containing no reducing sugar (Comparative Examples 2 to 6), the cells were almost killed in 3 days, but in the cell preservation solution containing reducing sugar, the cells were alive even after 6 days. In particular, the results of storage using the cell preservation solution containing glucose of Example 1 and the cell preservation solution containing maltose of Example 4 were more excellent.

(Experimental example 3)
Cell survival rate relative to the number of storage days when normal human fibroblasts were stored under the storage conditions of Examples 5 to 7 (cell storage solution: Reference Examples 1 to 3) and Comparative Example 7 (cell storage solution: Reference Example 9) It was investigated. The result is shown in FIG. The cell viability was measured in the same manner as in Experimental Example 1.

(Experimental example 4)
In the storage conditions of Examples 8 to 10 (Cell preservation solution: Reference Examples 1 to 3) and Comparative Example 8 (Cell preservation solution: Reference Example 9), the cell viability with respect to the storage days when A549 cells were stored was examined. . The result is shown in FIG. The cell viability was measured in the same manner as in Experimental Example 1.

(Experimental example 5)
In the storage conditions of Examples 11 to 13 (cell preservation solution: Reference Examples 1 to 3) and Comparative Example 9 (cell preservation solution: Reference Example 9), the cell viability with respect to the storage days when HepG2 cells were stored was examined. . The result is shown in FIG. The cell viability was measured in the same manner as in Experimental Example 1.

(Experimental example 6)
In the storage conditions of Examples 14 to 16 (cell preservation solution: Reference Examples 1 to 3) and Comparative Example 10 (cell preservation solution: Reference Example 9), the cell viability with respect to the storage days when Vero cells were stored was examined. . The result is shown in FIG. The cell viability was measured in the same manner as in Experimental Example 1.

  From the results of the above Experimental Examples 1 and 3-6, the cell storage method of the present invention is not limited to the storage of adherent cells such as normal human fibroblasts, A549 cells, HepG2 cells, Vero cells as well as 3T3 cells. It was clear that there was a special effect.

(Comparative Experimental Example 1)
With respect to 3T3 cells and HepG2 cells, the cell viability with respect to the storage days was examined in the storage without comparison (Comparative Examples 11 and 12). Cell viability was measured by trypan blue staining. Specifically, after removing the cell preservation solution of Reference Example 1, the cells were detached by adding an appropriate amount of 0.001% trypsin and 0.02% EDTA-containing phosphate buffer, and the remaining cells were collected. did. And the life and death judgment was performed by trypan blue dyeing | staining. As a result, all the cells were dead on the third day of storage.

As described above, the method for preserving adherent cells of the present invention can improve cell preservation performance even under refrigerated conditions, and increase cell viability. In addition, according to the present invention, since a cell storage method under refrigerated conditions is provided, cell types that have been difficult to store can be stored. In addition, even when an expensive device or facility such as a programming freezer required for cryopreservation cannot be prepared, cells can be preserved. Furthermore, if the cell preservation solution of the present invention is used, the cryoprotectant or the like normally used during cryopreservation is not subjected to a washing step or the like, and the cells are seeded in a medium while being suspended in the preservation solution. Since it becomes possible to culture, cell culture can be performed simply. For example, the cell preservation solution of the present invention can be used when transporting adherent cells from a contract culture facility to a patient.
Thereby, it is considered that the decrease in cell viability is suppressed and the quality of the cells is maintained, and finally, it is useful for therapeutic use.

  Since culturing of cells must always observe the state of culturing, if culturing cannot be observed for a certain period of time due to emergency use, etc., cells must be frozen and stored in a complicated operation. Even in such a case, provision of a cell preservation solution that does not require complicated operations is required. The method for preserving adherent cells according to the present invention is highly useful in a test research institution or the like where a cell preservation manager may need a sudden business trip of about one week, for example.

It is a figure which shows the survival rate by floating storage and freezing storage of 3T3. (Experimental example 1) It is a figure which shows the survival rate when the saccharide added to a cell preservation solution is changed. (Experimental example 2) It is a figure which shows the survival rate by the floating preservation | save of a normal human fibroblast. (Experimental example 3) It is a figure which shows the survival rate by floating storage of A549. (Experimental example 4) It is a figure which shows the survival rate by the floating storage of HepG2. (Experimental example 5) It is a figure which shows the survival rate by the floating preservation | save of Vero. (Experimental example 6)

Claims (10)

Adherent cells grown on the culture substrate are detached from the culture substrate using a cell detachment solution, and the detached adherent cells are suspended in a cell preservation solution containing a reducing sugar and subjected to refrigeration conditions. A method for preserving adherent cells, comprising: The method for preserving adherent cells according to claim 1, wherein the reducing sugar contained in the cell preservation solution is any one or plural kinds selected from the group consisting of glucose and maltose. The method for preserving adherent cells according to claim 1 or 2, wherein the cell preservation solution further contains hydrogen carbonate ions and / or carbonate ions. The method for preserving adherent cells according to claim 3, wherein the content of hydrogen carbonate ions and / or carbonate ions contained in the cell preservation solution is 1 to 75 mM. The cell preservation solution further contains any one or more kinds of ions selected from the group consisting of calcium ions, magnesium ions, chloride ions, phosphate ions, sulfate ions and lactate ions. The preservation | save method of the adherent cell of any one of -4. The method for preserving adherent cells according to claim 1, wherein the adherent cells are preserved under refrigerated conditions using a cell preservation solution containing the following composition in a 1,000 ml aqueous solution.
Reducing sugar 0-150 mM
Sodium ion 20-260 mM
Potassium ion 3 to 125 mM
Bicarbonate ion and / or carbonate ion 1-75 mM
Calcium ion 1-4 mM
Magnesium ion 0 to 1 mM
Chloride ion 75-245 mM
Phosphate ion 0.5 to 65 mM
Sulfate ion 0 to 1 mM
Lactate ion 0-20 mM Refrigeration conditions are 0-25 degreeC, The preservation | save method of the adherent cell of any one of Claims 1-6. Seeding the adherent cells after being stored by the method for preserving adherent cells according to any one of claims 1 to 7 in a cell growth medium without undergoing a step of washing and removing the cell preservation solution. Method for culturing adherent cells by A cell preservation solution containing a reducing sugar for preservation of adherent cells and a kit for preservation of adherent cells comprising a cell detachment solution. The cell preservation solution according to claim 9, wherein the cell preservation solution is a cell preservation solution used in the method for preserving adherent cells according to any one of claims 1 to 8.

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