Cell cryopreservation composition and cryopreservation method

The present invention relates to cryopreservation compositions that are used to cryopreserve cells, and cryopreservation methods using the same.

Cell cryopreservation is widely used as an essential technique to prevent cell degeneration due to passaging, prevent contamination with bacteria associated with passaging, transport cells, etc. However, it is known that, in the process of freezing cells, water in and out of the cells turns into ice crystals and the ice crystals damage the cells (Non-Patent Document 1). It I therefore desired to protect cells from damage during freezing and thawing and to cryopreserve cells while maintaining their properties.

In common cryopreservation, cells are typically suspended in a culture solution containing bovine serum etc. with a cryoprotectant for protecting the cells from damage from ice crystals, and the cell suspension thus obtained is placed in a cryotube etc., cooled, and eventually cryopreserved at a cryogenic temperature of −80° C. or −196° C. The cryoprotectant is 5 to 20% of dimethyl sulfoxide (DMSO), glycerin (Gly), ethylene glycol (EG), propylene glycol (PG), etc. (Patent Documents 1, 2). The most effective and most common cryoprotectant among these is DMSO (Non-Patent Document 2). However, a cryopreservation solution containing DMSO as a cryoprotectant does not have satisfactory preservation efficiency, and it cannot necessarily be said that this cryopreservation solution sufficiently inhibits ice crystal formation.

There are a cryopreservation solution further containing polyether to enhance the effect of DMSO (Patent Document 3), a cryopreservation solution containing a fructan to enhance the cytoprotective effect (Patent Document 4), and a cryopreservation solution containing carboxylated polylysine to enhance the stem cell storing effect (Patent Document 5). However, it cannot be said that these cryopreservation solutions sufficiently inhibit ice crystal formation. There is a concern about cytotoxicity of residual polyether, and a less toxic cryopreservation solution is desired. Moreover, since the cryopreservation solution contains as high as 30% of fructan, this is economically disadvantageous, and it is also difficult to remove fructan after preservation. Cell preservation using carboxylated polylysine focuses only on stem cells and is therefore not so versatile. Furthermore, since carboxylated polylysine is a polypeptide, there is also a concern about its impact on cell functionality etc. The above cryopreservation solutions are thus not always satisfactory with respect to cell preservation. Accordingly, a low-toxic cryopreservation solution is desired which can preserve all the cells.

Patent Documents

PATENT DOCUMENT 1: Japanese Patent No. 5940975

PATENT DOCUMENT 2: Japanese Unexamined Patent Publication No. 2001-247401

PATENT DOCUMENT 3: Japanese Unexamined Patent Publication (Japanese Translation of PCT Application) No. H10-511402

PATENT DOCUMENT 4: Japanese Unexamined Patent Publication No. 2012-235728

PATENT DOCUMENT 5: Japanese Patent No. 5630979

PATENT DOCUMENT 6: Japanese Unexamined Patent Publication No. 2016-160244

Non-Patent Documents

NON-PATENT DOCUMENT 1: Mazur, Am. J. Physiol., 247:C125-142, 1984

NON-PATENT DOCUMENT 2: Kovelock J E and Bioshop M W H, Nature 183:1394-1395, 1959

Technical Problem

Since current cryopreservation methods do not sufficiently inhibit ice crystal formation and do not sufficiently protect cells from freezing damage, development of a novel low-toxic cryopreservant is desired.

Solution to the Problem

The inventors intensively studied to solve the above problems and found that a sophorose lipid (SL) can inhibit ice crystal formation and can easily reduce freezing damage to cells. Based on these findings, the inventors found that adding 0.01 wt % to 20 wt % of SL to a cell culture solution before cryopreservation improves cell preservation efficiency and that adding 0.01 wt % to 20 wt % of SL during cryopreservation reduces DMSO toxicity. The inventors also found that mixing 1 wt % to 50 wt % of a polyhydric alcohol with 0.01 wt % to 20 wt % of SL improves cell viability after cryopreservation without adding DMSO. The present invention provides the following:

[1] A composition that contains 0.01 wt % to 20 wt % of a sophorose lipid and improves cell viability after cryopreservation. [2] The composition of [1], containing 5 wt % to 10 wt % of dimethyl sulfoxide (DMSO). [3] The composition of [1] or [2], containing 1 wt % to 50 wt % of a polyhydric alcohol. [4] The composition of [3], containing at least one of glycerin, ethylene glycol, and propylene glycol as the polyhydric alcohol. [5] A cell cryopreservation method in which 1 volume % of the composition of [1] is added to a cell culture medium just before or up to 6 hours before cryopreserving cells. [6] A cell cryopreservation method in which 10 volume % to 99 volume % of the composition of any of [2] to [4] is added to a cell culture medium when cryopreserving cells.

Addition of SL can reduce freezing damage to cells. As a result, a certain level or higher of cell viability can be obtained even without relying on the effects of DMSO and serum.

FIG. 1 (A) -(H) are micrographs showing inhibition of ice crystal formation, where (A) is ultrapure water, (B) is 10% SL, (C) is 0.1% SL, (D) is 10% DMSO, (E) is 10% Gly, (F) is 10% PG, (G) is 10% EG, and (H) is 10% APG.

FIG. 2 is a graph showing expression of cell markers.

FIG. 3 (A) -(K) are micrographs showing cell morphology, where (A) is 0.2% SL+30% Gly, (B) is 0.2% SL+20% Gly, (C) is 0.2% SL+15% Gly, (D) is 0.1% SL+30% Gly, (E) is 0.1% SL+20% Gly, (F) is 0.1% SL+15% Gly, (G) is 0.05% SL+30% Gly, (H) is 0.05% SL+20% Gly, (I) is 0.05% SL+15% Gly, (J) is 30% Gly, and (K) is 20% DMSO.

SL is a low-toxic glycolipid biosurfactant and is a fermentation product produced by fermentation of yeast. SL has the following effects. When added before freezing cells (addition before freezing), SL is taken into the cells and inhibits ice crystal formation within the cells. When added upon freezing, SL inhibits ice crystal formation outside the cells. SL used in the following experimental examples and examples was prepared according to the description in Japanese Unexamined Patent Publication No. 2016-160244. SL adjusted to pH 6 to 8 was used. A pH adjuster is an alkaline agent, an acid, etc.

A polyhydric alcohol is glycerin, ethylene glycol, propylene glycol, etc. and is preferably propylene glycol.

Cells for a cell preservation solution are animal and plant cells such as somatic cells, cancer cells, cell lines, and stem cells.

The cell preservation solution is also similarly applicable to tissues formed by cells, organs, and individuals such as animals and plants. The cell preservation solution can be expected to keep plant- and animal-derived foods fresh.

The freezing and thawing methods using the present invention are not particularly limited. Fine temperature control need not necessarily be performed during freezing, and common slow freezing and rapid thawing may also be used.

EXPERIMENTAL EXAMPLES: ICE CRYSTAL FORMATION INHIBITING COMPOSITION AND ICE CRYSTAL FORMATION INHIBITING EFFECT

Each of the compositions shown in Table 1 was mixed with Dulbecco's modified Eagle's medium (DMEM), which is a medium most commonly used for cell preservation, at a volume ratio of 7:3. Each sample was dispensed into a 15 ml centrifuge tube (Thermo Scientific BioLite) and cooled at 4° C. for 5 minutes, −20° C. for 20 minutes, and −80° C. in this order, and the appearance of each sample was visually observed after cooling at −80° C. for 10 minutes.

The results in Table 1 show that Examples 1 to 4 inhibited ice crystal formation more than Examples 5 and 6 did.

Ultrapure water solutions with compositions as shown in Table 2 were prepared. Each sample was frozen by a cooling stage of a scanning probe microscope (AFM5000/AFM5300, Hitachi High-Tech Science Corporation) and observed with an optical microscope. This test was conducted with support of Nanotechnology Open Facilities in Osaka University. Each obtained image was analyzed using image analysis software (OLYMPUS cellSens Standard) to measure the area per ice crystal.

FIG. 1 , (A)-(H) are micrographs showing inhibition of ice crystal formation, where (A) is ultrapure water, (B) is 10 wt % SL, (C) is 0.1 wt % SL, (D) is 10 wt % DMSO, (E) is 10 wt % Gly, (F) is 10 wt % PG, (G) is 10 wt % EG, and (H) is 10 wt % APG. As shown in FIGS. 1 B and 1 C , the solutions containing SL inhibited ice crystal formation regardless of whether the SL content was 0.1 wt % or 10 wt %. As shown in FIGS. 1 , (A), (D), (E), (F) and (G), ultrapure water and the solutions containing a substance other than SL did not sufficiently inhibit ice crystal formation. As shown in FIG. 1 , (H), however, the solution containing APG that is the same glycolipid surfactant as SL inhibited ice crystal formation.

[1. Effect of SL Addition Upon Culturing Before Cryopreservation (Human Normal Fibroblasts, Serum-Containing Medium)]

Human normal fibroblasts (KURABO) were sown on a 6-well plate at 3.2×10 4 cells/ml and cultured for 48 hours. After culturing, the culture medium was removed, and a 5 wt % SL aqueous solution was diluted with DMEM containing fetal bovine serum to prepare a 0.05 volume % SL solution. The solution thus prepared was added to the cultured cells. After culturing for a predetermined time, the viable cell count was measured by trypan blue staining (viable cell count before freezing). The remaining cells were suspended in 1 ml of a 10% DMSO/fetal bovine serum-containing DMEM solution in CRYOGENIC VIAL (Sansyo Co., Ltd.), and the cell suspension thus obtained was cooled at 4° C. for 5 minutes, −20° C. for 20 minutes, and −80° C. in this order. After overnight storage at −80° C., the cell suspension was rapidly thawed at 37° C., and the viable cell count was measured by trypan blue staining (viable cell count after thawing). The remaining cell suspension was sown on a 6-well plate and the cells were cultured for 72 hours. The viable cell count after culturing was measured by trypan blue staining (viable cell count after culturing).

The viability upon thawing and the proliferation rate were calculated by the following equations.

Viability upon thawing (%)=viable cell count after thawing/total cell count after thawing×100

Proliferation rate (%)=viable cell count after culturing/viable cell count after thawing×100

The results in Table 3 show culturing with SL added 1 to 6 hours before cell cryopreservation resulted in higher cell viability upon thawing and a higher cell proliferation rate than culturing with no SL added.

Not only the viability upon thawing but also the viability before and after freezing were examined Specifically, human normal fibroblasts (KURABO) were sown on a 10 mm dish at 4.0×10 4 cells/ml and cultured for 72 hours. After culturing, a 5 wt % SL aqueous solution was diluted to prepare a 0.05 volume % SL solution, and the solution thus prepared was added to the cultured cells. After culturing for a predetermined time, the resultant cell suspension was sown on a 96-well plate at 2.0×10 4 cells/ml, and the cells were cultured for 6 or 72 hours. Absorbance was then measured with Cell Counting Kit-8 (DOJINDO LABORATORIES) (absorbance before freezing). The remaining cells were suspended at a cell density of 4.0×10 5 cells/ml, and the cell suspension thus prepared and 20% DMSO were mixed at a volume ratio of 1:1 in CRYOGENIC VIAL (Sansyo Co., Ltd.). The resultant cell suspension was placed in a freezing container, BICELL (Nihon Freezer Co., Ltd.), and cooled at −80° C. After overnight storage, the cell suspension was rapidly thawed at 37° C., 100 μl of the cell suspension was sown on a 96-well plate, and the cells were cultured for 6 or 72 hours. Absorbance was then measured with Cell Counting Kit-8 (DOJINDO LABORATORIES) (absorbance after cryopreservation).

The viability before and after freezing and the proliferation rate were calculated by the following equations.

Viability before and after freezing (%)=absorbance after cryopreservation (6-hour culture)/absorbance before freezing (6-hour culture)

Proliferation rate (%)=absorbance after cryopreservation (72-hour culture)/absorbance before freezing (72-hour culture)

The results in Table 4 show that culturing with SL added 1 to 6 hours before cell cryopreservation resulted in higher cell viability before and after freezing and a higher cell proliferation rate than culturing with no SL added.

[2. Effect of SL Addition Upon Culturing Before Cryopreservation (Human Normal Fibroblasts, Serum-Free Medium)]

Human normal fibroblasts (KURABO) were sown on a 6-well plate at 3.2×10 4 cells/ml and cultured for 48 hours. After culturing, the culture medium was removed, and a 5 wt % SL aqueous solution was diluted with a DMEM medium to prepare a 0.05 volume % SL solution. The solution thus prepared was added to the cultured cells. After culturing for 6 hours, the viable cell count was measured by trypan blue staining (viable cell count before freezing). The remaining cells were suspended in 1 ml of a 10% DMSO/DMEM solution in CRYOGENIC VIAL (Sansyo Co., Ltd.), and the cell suspension thus obtained was cooled at 4° C. for 5 minutes, −20° C. for 20 minutes, and −80° C. in this order. After overnight storage at −80° C., the cell suspension was rapidly thawed at 37° C., and the viable cell count was measured by trypan blue staining (viable cell count after thawing).

The viability before and after freezing was calculated by the following equation.

Viability before and after freezing (%)=viable cell count after thawing/viable cell count before freezing×100

The results in Table 5 show that culturing with SL added 6 hours before cell cryopreservation resulted in higher cell viability before and after freezing than culturing with no SL added.

[3. Effect of SL Addition Upon Culturing Before Cryopreservation (Mesenchymal Stem Cells, Serum-Free Medium)]

Mesenchymal stem cells (Lonza) were sown on a 6-well plate at 3.2×10 4 cells/ml and cultured for 48 hours. After culturing, the culture medium was removed, and a 5 wt % SL aqueous solution was diluted with an MSCGM-CD medium to prepare a 0.05 volume % SL solution. The solution thus prepared was added to the cultured cells. After culturing for 6 hours, the viable cell count was measured by trypan blue staining (viable cell count before freezing). The remaining cells were suspended in 1 ml of a 10% DMSO/MSCGM-CD medium in CRYOGENIC VIAL (Sansyo Co., Ltd.), and the cell suspension thus obtained was cooled at 4° C. for 5 minutes, −20° C. for 20 minutes, and −80° C. in this order. After overnight storage at −80° C., the cell suspension was rapidly thawed at 37° C., and the viable cell count was measured by trypan blue staining (viable cell count after thawing).

The viability before and after freezing was calculated by the following equation.

Viability before and after freezing (%)=viable cell count after thawing/viable cell count before freezing×100

The results in Table 6 show that culturing with SL added 6 hours before cell cryopreservation resulted in higher cell viability before and after freezing than culturing with no SL added.

[4. Effect of SL Addition without Culturing Before Cryopreservation (Human Normal Fibroblasts, Serum-Containing Medium)]

The viable cell count of human normal fibroblasts (KURABO) was measured by trypan blue staining (viable cell count before freezing). The cells were suspended in DMEM containing fetal bovine serum. Each of the samples shown in Tables 7 and 8 and the cell suspension were mixed at a volume ratio of 3:7 in CRYOGENIC VIAL (Sansyo Co., Ltd.), and each of the resultant cell suspensions was cooled at 4° C. for 5 minutes, −20° C. for 20 minutes, and −80° C. in this order. After overnight storage at −80° C., each cell suspension was rapidly thawed at 37° C., and the viable cell count was measured by trypan blue staining (viable cell count after thawing). Each of the remaining cell suspensions was sown on a 6-well plate and the cells were cultured for 72 hours. The viable cell count after culturing was measured by trypan blue staining (viable cell count after culturing).

The viability before and after freezing and the proliferation rate was calculated by the following equations.

Viability before and after freezing (%)=viable cell count after thawing/viable cell count before freezing×100

Proliferation rate (%)=viable cell count after culturing/viable cell count after thawing×100

The results in Table 7 show that Examples 12 and 13 had high cell viability and Example 13 had higher viability than Comparative Examples 4 to 6.