JPH06217764A - Composition for cryogenic preservation of cell - Google Patents

Abstract

PURPOSE:To obtain a composition for suspending an animal cell and protecting a cell from freezing at the time of freezing in crgogenically preseving the animal cell for a long period. CONSTITUTION:The composition for cyrogenical preservation contains 5-60% milk or milk-derived protein fraction, 5-20% dimethylsulfoxide(DMSO) and 20-90vol% basal medium for cell culture. Thereby, survival rate of frozen cell is improved and stable supply is made possible and the composition is free from problem such as contamination by virus an can be supplied at a low cost, compared with a composition for cryogenic preservation containing fetal calf serum(FCS).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition for cryopreservation having a protective function for cryopreserved cells.

[0002]

2. Description of the Related Art Animal cells and tissues can only survive for a short period in vitro. Therefore, in order to survive in vitro for a long period of time, it is necessary to prepare a cell line transformed from a state of primary culture so that it can withstand long-term culture. This cell line can be subcultured for a long period of time by exchanging the culture medium. By subculturing in this way, it became possible to freely utilize the target cells when necessary. However, such subculture often has problems such as bacterial contamination and transformation of cultured cells due to mutation, resulting in loss of required traits. Therefore, a method for cryopreserving cells and tissues has been developed. With the development of this method, cells and tissues were frozen under specific conditions,
It became possible to store the cells in the following ultra-low temperature freezer or liquid nitrogen, thaw frozen cells when necessary, and use the target cells or tissues.

When cryopreserving animal cells, about 10% of dimethyl sulfoxide (DMS) is used as a protective component.
O) and 10 to 50% of animal serum such as fetal calf serum (FCS) are generally used as a composition for cryopreservation. DMS for cryopreservation of these cells
A method using O and FCS is disclosed in US Pat. No. 3,852,155. In particular, DMSO and FCS are considered to be essential components in the medium used for this cryopreservation. Animal cells are suspended in such a composition for cryopreservation, and the cells are slowly frozen using a program freezer or an ultra-low temperature freezer.

However, animal serum is extremely expensive,
This is a cause of increasing storage costs when cryopreserving a large number of various cell lines. Further, it has been pointed out that the serum may contain viruses and mycoplasma other than those guaranteed by the manufacturer, and that the quality of the serum varies greatly from lot to lot. Furthermore, when cryopreserving cells in liquid nitrogen, liquid nitrogen may enter the ampoule from a slight gap in the lid of the frozen ampoule, which may contaminate other stored cell ampoules with serum and other viruses. Can be a problem. In addition, the use of components obtained from animal tissues is an excellent target from animal welfare groups, and there is a high possibility that the use of serum components will be restricted in the future.

For this reason, investigations have been made on additional components to replace serum. WO 91/11101 describes that nonionic surfactants, particularly Triton-100 and octylphenoxypolyethoxyethanol, are effective. Also, for the purpose of cryoprotection, a cryopreservation medium supplemented with carboxymethyl cellulose has come to be marketed. When such a composition that does not use serum is used, there is an advantage that cells thawed from frozen can be immediately transferred to serum-free culture. Also, because it does not use fetal bovine serum,
It can be stably supplied without contamination by viruses and the like. However, cryoprotective agents containing substances that replace these sera were not always satisfactory in terms of cryoprotective effect, and their prices were high. In recent years, when a composition for cryopreservation culture to which carboxymethylcellulose is added is used as a protective component other than serum, the protective effect on frozen cells is often unsatisfactory. That is, the viability of cells when frozen and thawed was low, and there remained a problem in cell recovery.

[0006]

DISCLOSURE OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION The present inventors have studied a composition for cryopreservation of cells, and as a result, DMS
It was discovered for the first time that a composition containing O and milk or a milk-derived protein fraction was effective for cryoprotection of cells. The present invention is
An object of the present invention is to provide a composition for cryopreserving cells, which comprises dimethyl sulfoxide (DMSO) and milk or a protein derived from milk as active ingredients.

[0007]

The present invention is characterized in that milk is added as a protective component in place of animal serum which has been added as a protective component for cryopreservation of animal cells until now. Examples of the composition which has been used for cryopreservation of animal cells so far include the composition disclosed in US Pat. No. 3,852,155. This is 8-10% DMSO
It is a solution containing 80% or more sheep serum or FCS. According to this U.S. patent, it is confirmed that when the serum content is 60% or less, the recovery of cells from freezing is reduced.

The present invention contains DMSO in an amount of 5 to 20%, particularly preferably 10 to 15%, and further contains milk, skim milk, casein,
A composition containing 5 to 60% whey protein, particularly preferably 10 to 50% is used as a cryopreservation solution. Components other than the above include serum-free medium generally used for cell culture. The milk for obtaining skim milk, casein, and whey protein may be milk of any mammal origin, but milk is an easily available milk. Milk is cheaper than animal serum. Milk is homogenized raw milk, whose fat concentration is adjusted by centrifugation (low-fat milk or skim milk),
A product obtained by dissolving powdered milk in water and reducing it (reduced milk) is easily available and can be used in the present invention.

Casein, which is a major protein isolated from milk, whey protein which is a milk protein other than casein, whey protein concentrate (WPC) containing whey protein concentrated, isolated whey protein (WPI) and the like can also be used. When milk is used, it is preferable to homogenize it with a high-pressure homogenizer or the like. If it is not homogenized, the fat will separate, which may adversely affect the freezing results of the cells by contacting them. Even skim milk is preferably homogenized in order to stabilize components such as proteins.

Sterilization of milk and milk components can be carried out under various conditions. From pasteurization method of 63 ℃ for 30 minutes, 120 ℃
Alternatively, a high temperature sterilization method such as heating at 140 ° C for several seconds can be adopted. Sterilization under normal autoclave conditions at 121 ° C for 15 minutes is not preferable because it causes severe browning and coagulation of milk protein. The concentration of milk, skim milk, casein, and whey protein is preferably 10 to 50% (volume). Milk at concentrations above 50% can significantly reduce the viable cell rate. On the contrary, at a concentration lower than 10%, it is not possible to prevent the pH of the basal medium during freezing from increasing, which is not suitable for long-term storage.

As the serum-free medium to be added to the mixed solution of DMSO and milk, skim milk, casein or whey protein, any kind may be used as described above. Typical examples of such a medium include the minimum essential medium, Dulbecco's modified minimum essential medium, and RP.
MI1640 medium, Ham's F12 medium, ERDF medium and the like can be mentioned. The ERDF medium used in the examples of the present invention has excellent properties as a basal medium for cell culture and is particularly preferable. This ERDF
The medium used as an index the proliferation of mouse myeloma NS1
It is a known basal synthetic medium for serum-free culture developed by Murakami et al. (See Japanese Patent Laid-Open No. 3-180175). Dimethyl sulfoxide (DMSO) added as a cryoprotective ingredient
Regarding the concentration of the substance, its content is determined in consideration of toxicity to cells and cryoprotective effect, but it can be used within the concentration range of 5 to 20%. Particularly preferably, 10 to 15% is optimum in the present invention. If the concentration of DMSO is low, the protective effect is not sufficient, and if it is too high, cytotoxicity is exhibited. Tables 1 and 2 show typical compositions of the cryopreservation composition.

[0012]

[Table 1] [Composition 1] ────────────────────── ERDF medium 80% DMSO 10% Skim milk ^* 10% ──────── ────────────── ^* Skim milk powder is reduced to 10% (v / v) in water and sterilized by heating at 140 ° C for 2 seconds.

[0013]

[Table 2] [Composition 2] ────────────────────── ERDF medium 40% DMSO 10% Skim milk ^* 50% ───────── ────────────── ^* Skim milk as composition 1.

When cryopreserving animal cells using the above composition, it can be carried out according to a method generally used for cryopreserving cells. Freezing is performed as follows. Animal cells are harvested from the culture vessel, placed in a centrifuge tube and spun down. After removing the supernatant, an appropriate amount of the composition for cryopreservation is added to the cell precipitate, and the cells are well suspended and then placed in a cell freezing container such as a freezing tube (cryotube) to freeze the cells. As a container used for freezing, a freezing tube made of resin, a glass ampoule, a cell freezing bag (such as a blood bag), or the like can be used. As a cooling device used for freezing cells, there are a program freezing device capable of controlling the temperature with liquid nitrogen, a deep freezer capable of cooling to -80 ° C, and the like. In most cases, there is no problem in putting the cell ampoules in a suitable box and freezing directly in a deep freezer at -80 ° C. Use liquid nitrogen containers for long-term (more than half a year) frozen storage.

The present invention will be described in detail below with reference to examples.

Example 1 This example shows an example of cryopreservation of cells by a composition containing DMSO and skim milk. [Cells] Mouse hybridoma HB8852 (ATCC-HB8
852) (H. Kawakami et al., J. Dairy Sci., Vol. 70, 752-75
9, 1986) was used. The cells were subcultured in ITES-ERDF medium containing insulin, ethanolamine, transferrin, and sodium selenite as growth factors (see JP-A-3-180175). [Cryopreservation Medium] As a composition containing DMSO and skim milk, a cryopreservation medium having the composition shown in Table 3 was prepared.
The skim milk was prepared by dissolving skim milk powder in water, adjusting the fat content to 1% and the milk solid content to 10%, homogenizing, and sterilizing at ultrahigh temperature for 2 seconds at 140 ° C. Fetal bovine serum was purchased from Flow Laboratory and ERDF medium was purchased from Kyokuto Pharmaceutical.

[0016]

[Table 3] [Composition of medium used for freezing] ────────────────────────────────── Number composition (% ) DMSO non-fat milk fetal bovine serum ERDF medium ────────────────────────────────── 1 1 10 10 0 80 2 10 50 0 40 3 10 90 0 0 0 4 10 0 10 80 5 5 10 0 50 40 40 6 10 0 90 90 7 (control) 10 0 0 90 90 ─────────────────── ───────────────

[Cryopreservation of cells and measurement of viable cell rate] HB
8852 cells 4 × 10 ⁶ cells were suspended in freezing medium 4 ml, it was dispensed 2ml increments content in polypropylene cryotubes of 2ml volume (Corning). Freezing tubes were placed in a cryopreservation rack paper box (Intermet) and stored at -80
It was frozen and stored in a deep freezer at ℃. One week and one month after freezing, the frozen tube was immersed in a large amount of water (25 ° C.) to thaw the cells, and immediately suspended in 8 ml of ERDF medium. After the cells were precipitated by centrifugation at 1,000 rpm for 10 minutes, the cells were well suspended in 3 ml of ERDF medium. The thawed cells were mixed with an equal amount of trypan blue solution (Gibco), and the ratio of unstained cells was calculated using a hemocytometer. HB8852 before cryopreservation
The cell viability was 82.9%.

[Results] Table 4 shows the viable cell rate of HB8852 after 1 week and 1 month of freezing at -80 ° C. One week later, a free medium containing all fetal bovine serum and a free medium containing 10% skim milk gave a high viable cell rate of over 80%. After 1 month, the freezing medium containing 10% skim milk gave the highest viable cell rate.

[0019]

[Table 4] [Viable cell rate after cryopreservation of hybridoma] ──────────────────────────────── Viable cell rate ( %) Freezing medium 1 week later 1 month later ─────────────────────────────── 1 81.6 (98.4) 84.4 (101.8) 2 76.9 (92.8) 70.7 (85.3) 3 35.8 (43.2) 12.8 (15.4) 4 82.1 (99.0) ) 77.6 (93.6) 5 81.3 (98.1) 75.3 (90.8) 6 83.7 (101.0) 80.0 (96.5) 7 (control) 75.0 (90.5) 65.8 (79.4) ─────────────────────────────── () is before freezing Ratio of viable cells

[0020]

Example 2 In this example, the effect on the cryoprotection of a composition containing casein, whey protein or lactose, which are the main components in skim milk, and DMSO is shown. [Cells] As in Example 1, HB8852 was used. [Freezing Medium] As a composition containing DMSO and each component, a medium for cryopreservation having the composition shown in Table 5 was prepared. All added milk components are in saline and 10% casein
The solution (Sigma Co.), WPC 10% solution (Taiyo Kagaku), and lactose 2% solution (1 water product, Wako Pure Chemical Industries) were dissolved and heat-sterilized in a medium bottle at 63 ° C. for 30 minutes.

[0021]

[Table 5] [Composition of medium used for freezing] ───────────────────────────────────── Number composition (%) DMSO Casein solution WPC solution Lactose solution ERDF medium ───────────────────────────────────── 1 10 10 0 0 80 2 10 50 0 0 0 40 3 10 0 10 10 0 80 4 4 0 0 50 0 40 40 5 5 10 0 0 10 10 80 6 10 0 0 0 50 40 40 ───────────────── ────────────────────

[Cryopreservation of cells and measurement of viable cell rate] The same procedure as in Example 1 was performed. The viable cell rate of HB8852 cells before cryopreservation was 84.0%. [Results] HB8 frozen at -80 ° C for 1 week and 1 month
The viable cell rate of 852 is shown in Table 6. In the WPC-containing freezing medium, a high viable cell rate exceeding 80% was maintained even after 1 month of freezing. Further, 75% or more of the cells survived in the casein-containing medium. The cryoprotective action of lactose, which is often added as a protective substance for proteins and microorganisms, was not high.

[0023]

[Table 5] [Ratio of live cells after cryopreservation of hybridomas] ──────────────────────────────── %) Freezing medium 1 week later 1 month later ──────────────────────────────── 181.5 (97.0) 76.6 (91.2) 2 79.0 (94.0) 76.4 (91.0) 3 82.9 (98.7) 81.7 (97.3) 4 84.9 (101.1) ) 80.2 (95.5) 5 74.1 (88.2) 65.2 (77.6) 6 69.1 (82.3) 59.3 (70.6) ─────── ──────────────────────── () indicates the ratio to the viable cell ratio before freezing

EFFECT OF THE INVENTION The present invention provides a novel composition for cryopreserving cells. Since the composition of the present invention has a large cell protection effect upon freezing, it is possible to cryopreserve animal cells while maintaining a high viable cell rate. Further, skim milk, casein, and whey protein are used in place of animal serum, and a safe and inexpensive cryopreservation composition is provided, which is free from contamination of cells by viruses and bacteria.

Claims (2)

[Claims] 1. A milk or protein fraction derived from milk, and
A composition for cryopreservation of cells, which comprises dimethyl sulfoxide as an active ingredient. 2. The composition for cryopreservation of cells according to claim 1, wherein the protein fraction is a casein fraction or a whey protein fraction.