KR102024742B1 - Cryopreservation medium for hematopoietic stem cells and cryopreservation method for hematopoietic stem cells using the same - Google Patents

Abstract

The present invention provides a cryopreservation medium of hematopoietic stem cells and a cryopreservation method of hematopoietic stem cells using the same, which can increase the survival rate of cells after thawing by containing a specific ice-binding protein.

Description

Cryopreservation medium for hematopoietic stem cells and cryopreservation method for hematopoietic stem cells using the same}

The present invention relates to a cryopreservation medium of hematopoietic stem cells and a cryopreservation method of mesenchymal stem cells using the same. More specifically, by containing a specific ice-binding protein, the survival rate of the cells after thawing increases. The present invention relates to a cryopreservation medium of hematopoietic stem cells, and a cryopreservation method of hematopoietic stem cells using the same.

Many clinical trials have been made due to the possibility of treating intractable diseases using stem cells. Among stem cells, hematopoietic stem cells refer to stem cells derived from bone marrow, umbilical cord blood, peripheral blood, and the like. Hematopoietic stem cells have been studied as a raw material of cell therapy that can replace the existing bone marrow transplantation.

On the other hand, hematopoietic stem cells have a limited number of high purity cells that can be secured immediately after separation. In particular, it is very important to store pure hematopoietic stem cells isolated from living organisms under appropriate conditions without degrading their function. The alteration of cell specific ability due to improper storage affects the direct therapeutic efficacy or intrinsic differentiation ability, making it difficult to prepare a smooth therapeutic agent or causing side effects due to deterioration of cell function.

Cryopreservation is commonly used for storage of cells, including stem cells. Since cells have different physiological characteristics depending on their origin, tissue, and microenvironment in the body, cryopreservation methods suitable for the target cells should be applied even when cryopreserving each cell. In particular, stem cells require optimized cryopreservation conditions to maintain their specific specific dividing ability and differentiation ability, and it is essential to use a preservative that can ensure high efficiency, for example, high survival rate.

The cryopreservation medium of stem cells contains cryoprotectants such as dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, etc. in the cell culture medium, depending on the characteristics of the stem cells, serum, plasma (such as dextran Plasma), disaccharides (sucrose, trehalose, etc.) or polyvinylpyrrolidone (PVP). Currently, cryopreservation media containing about 10 w / v% of cryopreservatives such as dimethyl sulfoxide and about 10 w / v% of dextran, etc. are used for cryopreservation of hematopoietic stem cells.

The present inventors conducted various studies to develop a method capable of achieving high cryopreservation efficiency in cryopreservation of stem cells, particularly hematopoietic stem cells. The present inventors have noted the applicability of ice-binding proteins (IBPs), which have recently been reported in the academic community, and found that using certain ice-binding proteins can achieve high cryopreservation efficiency.

Therefore, an object of the present invention is to provide a cryopreservation medium of hematopoietic stem cells comprising the specific ice-binding protein.

In addition, an object of the present invention is to provide a cryopreservation method of hematopoietic stem cells using the medium.

According to one aspect of the invention, a protein consisting of the amino acid sequence of SEQ ID NO: 1; Cryopreservatives selected from one or more of the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And it provides a cryopreservation medium of hematopoietic stem cells comprising dextran.

According to another aspect of the invention, a protein consisting of the amino acid sequence of SEQ ID NO: 1; Cryopreservatives selected from one or more of the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And adding hematopoietic stem cells in a medium containing dextran, and freezing the obtained culture solution.

It has been found by the present invention that the use of certain ice-binding proteins, ie proteins consisting of the amino acid sequence of SEQ ID NO: 1, can achieve high cryopreservation efficiency, ie high cell viability after thawing. Therefore, the cryopreservation medium and cryopreservation method using the same according to the present invention can be usefully used to obtain hematopoietic stem cells with high efficiency.

1 is a result of evaluating cryopreservation efficiency during cryopreservation using the protein of SEQ ID NO: 1 (LeIBP) (*, p <0.05).
Figure 2 shows the results of the CFU test after thawing of hematopoietic stem cells using a frozen medium containing the protein of SEQ ID NO: 1 (LeIBP).
Figure 3 is the result of measuring the change in recovery rate according to the freezing medium of hematopoietic stem cells. Although no statistical significance was observed in comparison with the CFU formation ability before freezing of the experimental group to which the protein of SEQ ID NO: 1 (LeIBP) was added, the CFU formation ability was higher than that of the non-addition group.

The present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1; Cryopreservatives selected from one or more of the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And it provides a cryopreservation medium of hematopoietic stem cells comprising dextran.

The hematopoietic stem cells include, without limitation, hematopoietic stem cells derived from bone marrow, hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem cells derived from peripheral blood, and the like. Methods of isolating the hematopoietic stem cells are well known in the art.

The protein consisting of the amino acid sequence of SEQ ID NO: 1 is a polar yeast in the leucosporidium , that is, Leucosporidium species sp . A protein isolated at AY30, which has properties as an antifreeze protein (Lee et al., Cryobiology. 2010. 60: 222-228). The protein consisting of the amino acid sequence of SEQ ID NO: 1 has a structural characteristic that does not have a single cysteine amino acid residue, unlike other antifreeze proteins. The inventors noted the nature of the antifreeze protein (or 'ice-binding protein') to test its applicability in cryopreservation of hematopoietic stem cells. In particular, various anti-freezing proteins were tested and found to be unsuitable for lower viability of cells compared to conventional cryopreservatives and plasma. Surprisingly, however, it has been found that the use of a protein consisting of the amino acid sequence of SEQ ID NO: 1 significantly increases the survival rate of cells. This is presumably due to the structural properties of the protein consisting of the amino acid sequence of SEQ ID NO: 1 and its compatibility with hematopoietic stem cells.

In the cryopreservation medium of the hematopoietic stem cells of the present invention, the concentration of the protein consisting of the amino acid sequence of SEQ ID NO: 1 may range from 0.0003 to 0.3 w / v%, preferably 0.003 to 0.03 w / v% . In addition, the concentration of the cryopreservative and dextran may be used in an amount commonly used for cryopreservation of hematopoietic stem cells, for example, may be used at a concentration of about 10 w / v%, respectively. In one embodiment, the cryopreservation medium of the mesenchymal stem cells of the present invention is 0.0003 to 0.3 w / v% protein consisting of the amino acid sequence of SEQ ID NO: 1; About 10 w / v% of at least one cryopreservative selected from the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And dextran may comprise about 10 w / v%. In another embodiment, the cryopreservation medium of the mesenchymal stem cells of the present invention is 0.003 to 0.03 w / v% protein consisting of the amino acid sequence of SEQ ID NO: 1; About 10 w / v% of at least one cryopreservative selected from the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And dextran may comprise about 10 w / v%.

The cryopreservation medium of mesenchymal stem cells of the present invention is a physiologically compatible buffer solution such as phosphate buffered saline (PBS) or a basal medium commonly used for cell culture. May be). The basal medium includes, for example, without limitation, media such as DPBS, HBSS, DMEM, IMDM, RPMI, MEM, and the like. In one embodiment, the basal medium may be Dulbecco's Modified Eagle Medium (DMEM).

The invention also relates to a protein comprising (a) the amino acid sequence of SEQ ID NO: 1; Cryopreservatives selected from one or more of the group consisting of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; And it provides a method for cryopreservation of hematopoietic stem cells comprising the step of adding hematopoietic stem cells in a medium containing dextran, and (b) freezing the culture solution obtained in step (a).

In the cryopreservation method of hematopoietic stem cells according to the present invention, the medium used in step (a) is as described above. The number of hematopoietic stem cells added to the medium in step (a) is not particularly limited, and may be, for example, in the range of 1 × 10 ⁵ to 2 × 10 ⁷ cells per ml of medium.

The freezing of step (b) may be carried out according to a conventional freezing method used in the cryopreservation field. For example, the freezing may be performed using a manual method, an automatic controlled rate freezing method, or the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example

The protein consisting of the amino acid sequence of SEQ ID NO: 1 used in the following test was supplied from the Korea Polar Research Institute.

1. Test Method

(1) Isolation and Culture of Hematopoietic Stem Cells

Hematopoietic stem cells derived from umbilical cord blood were collected from the umbilical vein prior to delivery of the placenta at delivery of healthy volunteers who indicated their intention to prenatal care. Samples were transferred to a laboratory using a dedicated transfer container containing CPDA (citrate / phosphate / dextrose / adenine) anticoagulant. Hematopoietic stem cells were isolated by Rubinstein et al. (Rubinstein et al., 1995). For counting hematopoietic stem cells, the cell layer was suspended in DPBS, and the cells were divided for hoechst staining, trypanblue staining, and CD34 labeling staining.

(2) Freezing and thawing stem cells

The protein of SEQ ID NO: 1 was added to DMEM at a predetermined concentration, and a cryopreservative (dimethyl sulfoxide) and dextran were added at a predetermined concentration to prepare a cryopreservation medium. 1 ml of the obtained cryopreservation medium was placed in frozen vials and suspended by adding 1 × 10 ⁶ cells of hematopoietic stem cells per frozen vial. The resulting mixture was frozen using an automatic freezer (Ice cube, SY Lab.). After freezing, the frozen vials were stored in a liquefied nitrogen tank until analysis.

Thawing of the frozen vials was performed using a 37 ° C. hot water bath, and the thawed frozen vials were quickly washed with DPBS containing 2% fetal calf serum (FBS). The washing was performed three times, and the cell count and analysis were performed by floating the cell layer in a fresh wash solution after centrifugation at 300 x g for 10 minutes.

(3) survival rate of cells after thawing

The group using 10 w / v% DMSO and 10 w / v% dextran was group 1 (control); Cryopreservation of test groups (Groups 2 and 3, respectively) containing 0.003 w / v% or 0.03 w / v% of the protein of SEQ ID NO: 1 in 10 w / v% DMSO and 10 w / v% dextran Survival was evaluated for comparison of yield. All experimental groups were frozen using automatic freezers and stored in liquefied nitrogen. The thawing test was performed after being stored in liquefied nitrogen for at least 7 days, and cell viability of each experimental group was confirmed through cell counts immediately after thawing.

(4) Examination of differentiation ability of stem cells after thawing

Differentiation capacity of hematopoietic stem cells after thawing was performed using colony-forming unit (CFU) method. After Hoechst staining, certain cells were taken on the basis of the results of mononuclear cell count counts, mixed in CFU medium (Miltenyi Biotec) using methyl cellulose, and then spread evenly by inoculating into non-coated well plates. CO ₂ After incubation for 2 weeks in the incubator, colonies by differentiation type were distinguished under inverted microscope (CFU-erythrocyte, burst forming unit-erythrocyte, CFU-granulocyte, CFU-macrophage, CFU-granulocyte and macrophage, CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte), counted. The criteria for sorting and counting colonies after differentiation were performed according to the manufacturer's instructions from Miltenyi Biotec.

(5) statistical analysis

In order to confirm the proliferative effect of the frozen medium composition, the experimental results were analyzed for significance with the control results for each experiment. The analytical method performed paired t 'test and statistically significant cases where P value was lower than 0.05 were marked with an asterisk (*).

2. Test Results and Discussion

Group 1 (control) using 10 w / v% DMSO and 10 w / v% dextran; Thaw for test groups (groups 2 and 3, respectively) containing 0.003 w / v% or 0.03 w / v% of the protein of SEQ ID NO: 1 in 10 w / v% DMSO and 10 w / v% dextran Post cell viability was measured, and the results are shown in FIG. 1. The result of FIG. 1 is the result converted into 100% survival rate of a control group. As can be seen from the results of Figure 1, when treated with the protein of SEQ ID NO: 1 than when only treated with DMSO and dextran statistically significantly increased the survival rate of hematopoietic stem cells. This shows that stable cell storage is possible by the protein of SEQ ID NO: 1.

CFU assay was performed after thawing to determine whether the hematopoietic stem cells cryopreserved using the protein of SEQ ID NO. 1 showed a difference in hematopoietic differentiation capacity after cryopreservation (FIG. 2). For analysis, BFU-E, CFU-G, CFU-M, and CFU-GM in the colony for hematopoietic differentiation assay were counted after the differentiation culture and the total number of colonies was checked and compared with the control group (FIG. 3). As a result of the rate and count of colony formation, similar hematopoietic differentiation ability was observed in comparison with non-freezing hematopoietic stem cells. When the protein concentration of SEQ ID NO: 1 was increased, the recovery capacity for CFU formation was increased, but there was no statistical significance.

3. Conclusion

When the protein of SEQ ID NO: 1 is used for cryopreservation, it was found in this study that the survival rate of hematopoietic stem cells after thawing can be significantly increased. That is, when using a cryopreservation medium containing the protein of SEQ ID NO: 1, high-efficiency cryopreservation of hematopoietic stem cells is possible without affecting the differentiation capacity of the cells.

<110> HURIMBIOCELL CO. LTD.          Korea Institute of Ocean Science and Technology <120> Cryopreservation medium for hematopoietic stem cells and          cryopreservation method for hematopoietic stem cells using the          same <130> PN0606 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 261 <212> PRT <213> Leucosporidium sp. AY30 <400> 1 Met Ser Leu Leu Ser Ile Ile Thr Ile Gly Leu Ala Gly Leu Gly Gly   1 5 10 15 Leu Val Asn Gly Gln Arg Asp Leu Ser Val Glu Leu Gly Val Ala Ser              20 25 30 Asn Phe Ala Ile Leu Ala Lys Ala Gly Ile Ser Ser Val Pro Asp Ser          35 40 45 Ala Ile Leu Gly Asp Ile Gly Val Ser Pro Ala Ala Ala Thr Tyr Ile      50 55 60 Thr Gly Phe Gly Leu Thr Gln Asp Ser Ser Thr Thr Tyr Ala Thr Ser  65 70 75 80 Pro Gln Val Thr Gly Leu Ile Tyr Ala Ala Asp Tyr Ser Thr Pro Thr                  85 90 95 Pro Asn Tyr Leu Ala Ala Ala Val Ala Asn Ala Glu Thr Ala Tyr Asn             100 105 110 Gln Ala Ala Gly Phe Val Asp Pro Asp Phe Leu Glu Leu Gly Ala Gly         115 120 125 Glu Leu Arg Asp Gln Thr Leu Val Pro Gly Leu Tyr Lys Trp Thr Ser     130 135 140 Ser Val Ser Val Pro Thr Asp Leu Thr Phe Glu Gly Asn Gly Asp Ala 145 150 155 160 Thr Trp Val Phe Gln Ile Ala Gly Gly Leu Ser Leu Ala Asp Gly Val                 165 170 175 Ala Phe Thr Leu Ala Gly Gly Ala Asn Ser Thr Asn Ile Ala Phe Gln             180 185 190 Val Gly Asp Asp Val Thr Val Gly Lys Gly Ala His Phe Glu Gly Val         195 200 205 Leu Leu Ala Lys Arg Phe Val Thr Leu Gln Thr Gly Ser Ser Leu Asn     210 215 220 Gly Arg Val Leu Ser Gln Thr Glu Val Ala Leu Gln Lys Ala Thr Val 225 230 235 240 Asn Ser Pro Phe Val Pro Ala Pro Glu Val Val Gln Lys Arg Ser Asn                 245 250 255 Ala Arg Gln Trp Leu             260

Claims (6)

delete delete delete 0.003 to 0.03 w / v% of a protein consisting of the amino acid sequence of SEQ ID 1; Dimethyl sulfoxide as a cryopreservative; And adding hematopoietic stem cells in a medium containing dextran, and freezing the obtained culture solution. delete delete

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