JP2002233356A - Cell storage liquid and method for storing cell using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a cell storage liquid having excellent performances without containing blood serum and a culture solution during the cell storage, and a method for storing the cell using the same. SOLUTION: This cell storage liquid without containing the blood serum and the culture solution is prepared by using a substance as the main ingredient prepared by mixing albumins included in a phosphate buffer solution conventionally used for storing a germ cell instead of dimethylsulfoxide conventionally used as an additive to a blood serum-containing storage liquid and adding an auxiliary agent such as phosphate ion, a divalent metal ion and a monosaccharide thereto if necessary. Therefor, the storage liquid having clear ingredients thereof and an extremely stable quality and having excellent storage performances of the cell without requiring including both the blood serum and the culture solution and thereby having dangers of changing properties of the stored cell and contaminating with unknown viruses and the method for storing the cell using the storage liquid are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a preservation solution for cells containing purified albumin and dimethyl sulfoxide and a method for preserving cells using the preservation solution. The purpose is to measure.

[0002]

2. Description of the Related Art The preservation solution and preservation method of cells are not uniform depending on the type of cells from which they are derived. For example, in the case of cells such as microorganisms, the microorganisms are generally suspended in a microorganism culture medium containing about 10% glycerol and stored at a low temperature, or the microorganisms themselves are lyophilized. It is done to save in. In the case of mammalian cells, they are generally suspended in a culture solution containing fetal bovine serum to which about 10% of dimethyl sulfoxide has been added, and after pre-cooling, they are stored in a liquid nitrogen storage. And save it.

In the case of germ cells such as sperm, a phosphate buffer containing albumin alone is used as a preservative solution. In the case of the above-mentioned mammalian cell preservation, there are a type containing serum in a preservation solution and a type not containing serum. Examples of the type containing serum include cells containing serum and dimethyl sulfoxide in a culture solution. Preservatives are known. In the type without serum,
A cell preservation solution containing carboxymethylcellulose instead of serum is known (JP-A-6-4684).
No. 0).

[0004] However, those containing no serum have low cell preservation performance, and the usual culture solution contains serum. Therefore, a cell preservation solution containing serum is often used. In addition, since mammalian cells are generally more difficult to preserve than microbial cells and germ cells, a preservation solution for mammalian cells can be used to preserve microbial cells and germ cells, which are easier to preserve than mammalian cells. Is also being done.

[0005]

However, in a preservation solution containing serum, not only a different culture solution is used for each cell to be cultured, but also a different lot for serum is used for each cell. Since it is used, it is difficult to use a cell preservation solution prepared in-house as a preservation solution for other cells. Not only that,
Serum-containing cell preservation solutions not only contain various cytokines, growth factors, hormones, etc. as serum-containing components, but also many components that are not necessary for cell preservation, including amino acids and various ions. As a result of using a culture solution containing, there is a possibility that the properties of the preserved cells may be changed, and further, there is an inherent problem of an unknown virus that may be contained in serum.

[0006]

As described above, in the serum-containing cell preservation solution, there is a possibility that the properties of the stored cells may be changed, the risk of contamination with unknown viruses, and the components of the preservation solution. However, the present invention has various problems that it is difficult to stabilize the quality because it is unclear, so that in the present invention neither serum nor culture medium is contained, and the components are clear so that these problems do not occur at all. A cell preservation solution having excellent cell preservation performance and a cell preservation method using the preservation solution were developed, and various experiments were repeated.

As a result, surprisingly, dimethyl sulfoxide, which has been used as an additive to a serum-containing preservation solution, is contained in a phosphate buffer solution, which has been used for preserving germ cells. A serum-free and culture medium-free cell preservation solution containing a mixture of albumin as the main agent and supplements such as phosphate ions, divalent metal ions, and monosaccharides as necessary It has been found that it has excellent cell preservation performance for preserving cells, microbial cells, germ cells, and the like.

Specifically, the invention of claim 1 relates to a cell preservation solution containing purified albumin and dimethyl sulfoxide. The invention of claim 2 relates to the cell preservation solution according to claim 1, wherein the purified albumin is derived from bovine. Furthermore, the invention of claim 3 relates to the cell preservation solution according to claims 1 or 2, which contains a saccharide in addition to purified albumin and dimethyl sulfoxide. Furthermore, the invention of claim 4 relates to the cell preservation solution according to claims 1 to 3, wherein the sugar is a monosaccharide.

Further, the invention of claim 5 relates to the cell preservation solution according to claims 1 to 3, wherein the sugar is glucose. Furthermore, the invention of claim 6 relates to the cell preservation solution according to claims 1 to 5, which contains phosphate ion in addition to purified albumin and dimethyl sulfoxide.
Furthermore, the invention of claim 7 relates to the cell preservation solution according to claims 1 to 5, which contains a metal ion in addition to purified albumin and dimethyl sulfoxide.

Further, in the invention according to claim 8, the metal ion is 2
The cell preservation solution according to claim 7, which is a valent metal ion. Further, the invention of claim 9 relates to the cell preservation solution according to claims 1 to 8, wherein the concentration of the purified albumin is in the range of 0.01 to 25% (w / w).
Further, the invention according to claim 10 is characterized in that the concentration of purified albumin is in the range of 0.1 to 5% (w / w).
8. A preservation solution for cells according to item 8. Claim 11
The invention of claim 1 is characterized in that the concentration of dimethyl sulfoxide is 0.1 to 5
11. The preservation solution for cells according to claims 1 to 10, which is in the range of 0% (w / w). Further, in the invention of claim 12, the concentration of dimethyl sulfoxide is 1 to 20% (w / w).
11. The cell preservation solution according to claim 1, wherein the cell preservation solution falls within the range.

Further, the invention of claim 13 relates to the cell preservation solution according to claims 1 to 12, wherein the concentration of the metal ion is in the range of 0.0000001 to 5% (w / w). Further, the invention according to claim 14 is characterized in that the concentration of metal ions is in the range of 0.1 to 5% (w / w).
12. A cell preservation solution according to item 12. Claim 1
The invention of 5 relates to the cell preservation solution according to any one of claims 1 to 14, wherein the sugar concentration is in the range of 0.1 to 10% (w / w). Further, in the invention according to claim 16, the sugar concentration is 1%.
15. The composition of claim 1, wherein the concentration is in the range of -5% (w / w).
A preservation solution for the cells described in 1.

The invention of claim 17 is the invention of claims 1 to 16
Cell preservation characterized by suspending the cells in the cell preservation solution described in the above, dispensing the cells into a plurality of cell preservation containers, and cryopreserving the cells without preliminary freezing. About the method. Further, the invention of claim 18 is
Cells that are dispensed and stored in a cell storage container are 1 × 1
0 ³ suspended in a cell preservation solution such that 1 × 10 ⁸ cells / ml, a method for the storage cell of claim 17 which is adapted to cryopreservation.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The specific contents of the present invention will be further described. The present invention is characterized by containing purified albumin and dimethyl sulfoxide as a cell preservation solution. As the dimethyl sulfoxide to be used, generally known commercially available ones can be used. With respect to the amount of dimethyl sulfoxide used, if the concentration is less than 0.1% (w / w), the storage capacity of the cells is reduced, and conversely, 50% (w / w) based on the total amount of the storage solution.
Exceeds 0.1% (w / w), more preferably 1% to 20% (w / w) in the range of 0.1% (w / w) to 50% (w / w) in view of the effect and economy. ).

As the albumin to be used in addition to dimethyl sulfoxide, any type of albumin can be selected depending on the type of cells to be preserved. Generally, human albumin is used for preserving human cells for human treatment. The use of is best. However, the present invention is not necessarily limited to this, and those derived from cattle or other species can also be used. Albumin may be purified by any method. Purified albumin can be used with a purity of about fraction V.

The concentration of albumin used is 0.0
If it is less than 01% (w / w), there is no effect, and conversely, 25% (w / w)
/ w) does not change the effect. Therefore 0.0
It can be used within a range of from 01% to 25% (w / w), and more preferably within a range of from 0.1% to 5% (w / w) in terms of economy and handling.

The addition of metal ions improves the viable cell rate (effective cell rate) when cells are restored and the subsequent cultivation efficiency. The metal ions used in this case are various divalent or trivalent metal ions, and the use of divalent metal ions is particularly preferred.Other than this, for example, calcium ions, magnesium ions, iron ions, Use of zinc ions, copper ions, aluminum ions, or the like is conceivable. In addition, the use concentration may be very small, but 0.0000001%
(W / w) is less effective and 1% (w / w)
The effect does not change even if it exceeds. Therefore 0.00
Use in the concentration range of 00001 to 5% (w / w), more preferably in the concentration range of 0.1 to 5% (w / w) is economical.

Further, the sugar is added by adding
Again, the viable cell rate (effective cell rate) when cells are restored and the subsequent culturing efficiency are improved. Various sugars, such as glucose, galactose, fructose, ribose, trehalose, glucose, etc., can be used as the sugars, and various sugars such as glucose, glucose, etc. are considered. Among them, glucose is economical and easy to handle. Can be recommended. About addition amount, if less than 0.1% (w / w), there is no effect,
Further, since the effect does not change even if it exceeds 10% (w / w), a concentration range of 0.1 to 10% (w / w), more preferably a concentration range of 1 to 5% (w / w) is preferred. Good.

Further, phosphate ions can be used as a phosphate buffer, and Dulbecco's phosphate buffer, phosphate buffer and the like can be used. In addition, various culture solutions can be used in place of the divalent or trivalent metal ions or the phosphate buffer described above. Further, as an example of preservation of the cells, the cells are suspended in a cell preservation solution at a concentration of 1 × 10 ³ to 1 × 10 ⁸ cells / ml in a tube for low-temperature preservation, and this is suspended at −80 ° C. Alternatively, store in liquid nitrogen. In this case, if the storage is for a short period, the temperature may be about -4C to -20C. In addition, the cells can be suspended in the above-mentioned cell preservation solution, dispensed into a plurality of cell preservation containers, and cryopreserved without preliminary freezing.

In preservation, the temperature of the cells suspended in the cell preservation solution can be gradually lowered by a program freezer or the like, but it is also possible to directly store the cells at a rapidly lowered temperature. Further, regarding the restoration of the cryopreserved cells, the cryopreserved cells were stored at room temperature or for 3 hours.
The cells can be melted in a constant temperature bath at 7 ° C., and the cells can be separated from the cell storage solution and restored by an operation such as centrifugation using a centrifuge. After the separated cells are washed with an appropriate culture solution or the like, they can be cultured by a usual method.

The method for evaluating the storage performance of cells is as follows. Cells are suspended in a cell preservation solution, frozen, and then stored frozen for several hours to several days, and in some cases for several years. At the same time, the present cryopreserved cells are returned to room temperature, cultured, and the number of living cells can be counted at this time.

[0021]

The present invention will be described in more detail with reference to the following examples. Preparation of Cell Preservation Solution] Using an electronic balance (Tokyo Glass Instruments Co., Ltd., 416-65-81-01), sodium hydrogencarbonate (Wako Pure Chemical Industries, Ltd., 191-01305 reagent special grade) was 1.8.
g, D (+) glucose (Wako Pure Chemical Industries, Ltd., 041-00
595 reagent special grade) 300 g, HEPES (Dojindo Laboratories, 324-01375 reagent special grade) 3.6 g, PBS
(-) 96 g of the powder (Nissui Pharmaceutical Co., Ltd., 05913) was weighed and placed in a 10-liter beaker (Asahi Techno Glass Co., Ltd., 1000BK10000).

Water for injection (Hikari Pharmaceutical Co., Ltd.) with a 10-liter measuring cylinder (1079).
8 liters were weighed, added to the above 10 liter beaker, and stirred and dissolved with a magnetic stirrer (Asahi Techno Glass Co., Ltd., BS-38). 2 liter measuring cylinder (Asahi Techno Glass Co., Ltd., 3022CYL2000
In S), 1 liter of DMSO (Nacalai Tesque, Inc., 134-07) was weighed, added to the above 10 liter beaker, and stirred with a magnetic stirrer.

After dissolution, 1 g of calcium chloride (Wako Pure Chemical Industries, Ltd., 039-00475) and magnesium chloride hexahydrate (Wako Pure Chemical Industries, 135-00165) are taken on an electronic balance.
Were weighed, added to the above 10 liter beaker, and stirred and dissolved with a magnetic stirrer (Asahi Techno Glass Co., Ltd., BS-38). BSA
(Sigma-Aldrich Japan Co., Ltd., A-2934)
00g was weighed and placed in the 10 liter beaker. After dissolution, the volume was increased to 10 liters with water for injection to sufficiently dissolve, followed by filtration with a spiral cap PF (Nihon Genetics Co., Ltd., 12981) to prepare a cell preservation solution.

[2. Confirmation of cell preservation performance of cell preservation solution using P3U1 cells] In advance, P3U1 cells derived from mouse myeloma were cultured in a carbon dioxide incubator for culture (Hirasawa Co., CPD-300A) in RPMI1640 + 7S
(Nikken Biomedical Research Institute, Inc., GM1104) 450ml and fetal bovine serum (Lifetech Oriental Co., Ltd., 2614)
0-079) was mixed as a culture medium and cultured in a culture flask (Sumitomo Bakelite Co., Ltd., MS-2080R).

From the P3U1 cell suspension, 10 μl was taken out with a pipette (Nichiyo Corporation, NPX-20), and a 500 μl tube (Asahi Techno Co., Ltd.) in which 20 μl of trypan blue solution (Sigma-Aldrich Japan Co., Ltd., T-8154) was previously placed. Glass Co., Ltd., 12301MTB0.5), lightly agitate, put a part of the solution on a hemocytometer (Elma Sales Co., Ltd., 03-202-2), and use an inverted microscope (Olympus Optical Co., Ltd., CK- 2) Count cells underneath.

The number of viable cells is 7.0 × 10 ⁵ cells / ml
Met. Each 50 ml of the cell suspension was transferred to two centrifuge tubes (Nippon Becton Dickinson Co., Ltd., 2070) and centrifuged at 1500 rpm for 10 minutes at 20 ° C. (Kokusan Co., H700FR) to precipitate P3U1 cells. I let it. The supernatant is discarded by decantation, and the precipitated cells are transferred to a test tube mixer (Asahi Techno Glass Co., Ltd.
The cells were uniformly loosened using MS-1) and the cell concentration was 4.4.
× 10 ⁶ cells / ml [1. In the cell preservation solution prepared in [1], cells are suspended so as to be uniform, and the total number of cells is set to 4.4 × 10 ⁶ cells / tube in a serum tube (Asahi Techno Glass Co., Ltd., 2722-002). Dispensed. Thereafter, the tube was placed in a tube rack (Asahi Techno Glass Co., Ltd., 9791-081) and stored in a -80 ° C. freezer (Sanyo Electric Co., Ltd., MDF-382).

[3. Removal and restoration of cryopreserved P3U1 cells] [2. One year after storing the P3U1 cells stored in [1], remove the cells from the freezer at -80 ° C.
The solution was placed in a heat block (TAL-TEC, TAL-1G) that had been warmed to ℃ for 4 minutes to dissolve it. In a clean bench (Sanyo Electric Co., MCV-131BNF), aseptically put 10 ml of a washing medium (Niken Biomedical Research Institute, CM1101) into a 15 ml centrifuge tube (Nippon Becton Dickinson Co., Ltd., 2095). The P3U1 cells that had been heated and lysed with a heat block were dropped into the dropper (Asahi Techno Glass Co., Ltd.
SM251-1S), ambient temperature 20 ° C, rotation speed 1200
Centrifugation was performed for 5 minutes under the condition of rpm.

After centrifugation, the supernatant was discarded by decantation, and the precipitated cells were loosened uniformly using a test tube mixer, suspended in 2 ml of a culture medium, and then cultured in a 24-well culture dish (Asahi Techno Glass Co., Ltd. 1820-024N). Pipette out 10 ml of the P3U1 cell suspension, place it in a 500 ml tube previously containing 20 ml of trypan blue solution, and gently agitate.
A part of the solution was placed on a hemocytometer, and the number of cells was counted under an inverted microscope.

As a result, the viable cell number (effective cell number) of the stored P3U1 cells was 4.2 × 10 ⁶ cells, and the cell number immediately before cell preservation was 4.4 × 10 ⁶ cells / tube. From the results, it was confirmed that the viable cell rate was 96%. When observed under a microscope three days after the culture, it was confirmed that the P3U1 cells were growing. From these results, it was found that the cell preservation solution of the present invention had an excellent cell preservation effect.

[4. Confirmation of Cell Preservation Performance of Cell Preservation Solution Using K562 Cells] K562 cells, which are cells derived from human leukemia, were previously used in [2. ] By the same operation as in
When the number of cells was counted, the number of viable cells was 7.0 × 1
0 was ⁵ / ml. Transfer 50 ml of the cell suspension to a centrifuge tube (Nippon Becton Dickinson Co., Ltd., 2070).
Transfer to a book, ambient temperature 20 ° C, rotation speed 1500rpm,
, And centrifuged for 10 minutes using a centrifugal separator (Kokusan Co., Ltd., H700FR) to precipitate K562 cells.

The supernatant is discarded by decantation and settled.
Test cell mixer (Asahi Techno Glass Co., Ltd., MS
-1), the cells are loosened uniformly, and the cell concentration is 3.0 ×
10 ⁶Cells in a cell preservation solution to make cells / ml
Suspended in a serum tube (Asahi Techno Glass Co., Ltd.)
Formula Company, 2722-002) has a total cell count of 3.0 × 10⁶Ko / chi
It became a tube. Then the tube rack
(Asahi Techno Glass Co., Ltd., 9791-081)
Stored in a freezer (Sanyo Electric Co., Ltd., MDF-382)
Was.

[5. Removal and revival of cryopreserved K562 cells] One year after storing the K562 cells preserved in [4.], [3. ] And removal and resurrection. As a result, the number of viable K562 cells was 2.9 × 10 ⁶
And the number of cells immediately before cell storage was 3.0 × 10 ⁶ cells /
Since it was a tube, the viable cell rate was confirmed to be 95%. Next, K5 was added to a 24-well culture dish.
62 cells were transferred and cultured. When observed under a microscope three days after the culture, it was confirmed that the K562 cells were growing.
From these research results, it was found that the cell preservation solution of the present invention had an excellent cell preservation effect.

[0033]

As described in detail above, the present invention contains purified albumin and dimethyl sulfoxide as cell preservatives, and optionally contains phosphate ions, divalent metal ions, monosaccharides and the like. To add an auxiliary agent,
It is not necessary to include both serum and culture solution, and as a result, it is possible to use a cell preservation solution prepared in-house as a preservation solution for other cells, and as a serum-containing component, like a cell preservation solution containing serum. Various cytokines and growth factors,
Since it is not necessary to use a culture solution containing many components that are not necessary for cell preservation, such as hormones, amino acids, various ions, etc., use a different culture solution for each cell to be cultured. There is no danger of altering cell properties or allowing the contamination of unknown viruses, and it is not necessary to use different lots of serum for each cell.

In addition, it is possible to provide a preservation solution and a cell preservation method in which the preservation solution components are clear, the quality is extremely stable, and the cell preservation performance is excellent. The cell preservation solution of the present invention is particularly suitable for preserving mammalian cells, but can also be used for preserving plant cells such as protoplasts and germ cells. Furthermore, various cell banks can be efficiently constructed using the cell preservation solution of the present invention.

Claims (18)

[Claims] 1. A cell preservation solution containing purified albumin and dimethyl sulfoxide. 2. The cell preservation solution according to claim 1, wherein the purified albumin is derived from bovine. 3. The cell preservation solution according to claim 1, which contains a saccharide in addition to purified albumin and dimethyl sulfoxide. 4. The cell preservation solution according to claim 1, wherein the sugar is a monosaccharide. 5. The method of claim 1, wherein the sugar is glucose.
4. A preservative solution for cells according to 3. 6. The method according to claim 1, which contains phosphate ions in addition to purified albumin and dimethyl sulfoxide.
2. A preservation solution for cells according to item 1. 7. The cell preservation solution according to claim 1, which contains a metal ion in addition to purified albumin and dimethyl sulfoxide. 8. The cell preservation solution according to claim 7, wherein the metal ion is a divalent metal ion. 9. The concentration of purified albumin is 0.01 to 25%.
The cell preservation solution according to any one of claims 1 to 8, which is in the range of (w / w). 10. The concentration of purified albumin is 0.1 to 5% (w
The cell preservation solution according to any one of claims 1 to 8, which is within the range of (w). 11. A dimethyl sulfoxide having a concentration of 0.1
Claims 1 to 10 in the range of ~ 50% (w / w).
2. A preservation solution for cells according to item 1. 12. A dimethyl sulfoxide having a concentration of 1-2.
The cell preservation solution according to any one of claims 1 to 10, which is in a range of 0% (w / w). 13. The method according to claim 1, wherein the concentration of the metal ion is 0.0000001.
The cell preservation solution according to any one of claims 1 to 12, wherein the cell preservation solution is within a range of ５5% (w / w). 14. The metal ion concentration of 0.1 to 5% (w / w).
The cell preservation solution according to any one of claims 1 to 12, which is within the range of (1). 15. The cell preservation solution according to claim 1, wherein the sugar concentration is in the range of 0.1 to 10% (w / w). 16. The cell preservation solution according to claim 1, wherein the sugar concentration is in the range of 1 to 5% (w / w). 17. The cell preservation solution according to claim 1, wherein the cells are suspended in the cell preservation solution, dispensed into a plurality of cell preservation containers, and cryopreserved without preliminary freezing. A method for preserving cells, characterized in that: 18. The cells to be aliquoted and stored in a cell storage container are suspended in a cell preservation solution at a concentration of 1 × 10 ³ to 1 × 10 ⁸ cells / ml, and the cells are stored in a frozen state. The method for preserving cells according to claim 17.