JP2001078756A - Washing of eucaryotic cells and washing solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a washing method and a washing solution for eucaryotic cells that permits the centrifugal washing after cell thawing without occurrence of cell aggregate with decreased cell recovery reduction regardless of the eucaryotic cell separation process that is applied before the cryopreservation. SOLUTION: When the eucaryotic cells in a solution is thawed and washed, the cryoprotective agent is removed, as the cell coagulation is inhibited. The objective solution is for washing eucaryotic cells in the solution including post- thawed eucaryotic cells and is a physiological solution including at least polysaccharides and/or chelating agents.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a washing method and a washing solution for removing a cryoprotectant from a nucleated cell-containing solution after thawing. The obtained cells can be used in the treatment of various diseases using cells, such as hematopoietic stem cell transplantation therapy, and in basic science fields such as immunology and cell biology.

[0002]

BACKGROUND OF THE INVENTION Cord blood stem cells have attracted attention as a source of hematopoietic stem cell transplantation without donor invasion, and clinical application has been actively attempted mainly in Western countries. Umbilical cord blood stem cells are rarely harvested from donors and transplanted to patients immediately, as in other hematopoietic stem cell transplants, i.e., bone marrow transplants or peripheral blood stem cell transplants. (Especially for unrelated transplants). Usually, during cryopreservation of cells, a cryoprotectant such as dimethyl sulfoxide (hereinafter abbreviated as DMSO) is added to the cell suspension for the purpose of preventing damage (freezing damage) due to freezing. Since DMSO has cytotoxicity, it is desirable to wash the cells after thawing and to inject the washed cells to a patient. In fact, there are data that compare the presence or absence of lavage and show that clinical results are better with lavage (Ku
rtzberg, et al: New England
J. Med. , Vol. 35, p157-166, 1
996). Conventionally, as a washing solution, physiological saline, D-PB
A buffer such as S (Dulbecoline buffer) or HBSS (Hank's solution) and a medium such as RPMI have been used. American Type Culture C, which describes various common cell treatment methods in the laboratory.
ATCC PRESERVA issued by collection
TION METHODS: Freezing and
In Freeze-drying, 1991, as a method for washing cells after thawing on page 23, a washing solution of a growth medium was used for 100 hours.
A G10 minute centrifugal wash is shown. Also, Consc
ence, et al: Cryobiology, v
ol. No. 2,1985 discloses an IMDM medium supplemented with sodium carbonate, mercaptoethanol, penicillin, streptomycin, and fetal bovine serum as a washing solution for cells after thawing. Further, Japanese Patent Application Laid-Open No. H10-179149 discloses washing with a medium or physiological saline after thawing cells such as blood cells. Further, Japanese Unexamined Patent Publication No.
No. 253195 describes that an isotonic solution is preferred for washing after thawing of cryopreserved umbilical cord blood, but the composition is not described.

On the other hand, it is said that nucleated cells should be separated (removed from red blood cells) for the purpose of preventing side effects due to destructed red blood cells after thawing and reducing the volume during frozen storage during cryopreservation. In most cases, isolation and preservation is performed (Nankodo, “Peripheral blood stem cell transplantation”, p. 173). Initially, as a method for separating nucleated cells, a specific gravity centrifugation method using a specific gravity liquid such as Ficoll-Hypaque which has been widely used at the laboratory level has been used (for example, Harris, et al: Bone Marrow).
Transplantaton, vol. 13,19
In 94, cord blood was separated into mononuclear cells by Ficoll-Hypaque and then cryopreserved.
However, with the increase in the number of specimens to be treated, the problem of this method, which is extremely complicated and time-consuming, has become apparent, and other separation methods have been actively studied. As another separation method, a centrifugal separation-based HES method using hydroxyethyl starch (hereinafter abbreviated as HES) which is an erythrocyte sedimentation agent (for example, Ru)
binstein, et al: Proc. Natl.
Acad. Sci. USA, vol. 92,199
5) Top-and-bottom method using a special bag provided with liquid outlets at the top and bottom (for example, Armitag
e, et al: Bone Marrow Trans
plantaton, vol. 23, 1999). As a method using a principle other than centrifugation, there is a filter method (for example, JP-A-8-104463) in which nucleated cells are collected by a filter and then collected. By the way, the nucleated cells separated using a specific gravity centrifugation method did not cause any problem by washing with a conventionally used physiological saline, a buffer such as D-PBS or HBSS, or a medium such as RPMI, We have found that washing with a conventional washing solution using the above-mentioned three newly developed methods causes cell agglomeration, which often results in a large decrease in cell recovery.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to carry out centrifugal washing after thawing without generating aggregates, regardless of the nucleated cell separation method used before cryopreservation, and to reduce the cell recovery rate. It is an object of the present invention to provide a method for washing cells and a washing solution with a small amount of water.

[0005]

Means for Solving the Problems The present inventors have solved the above problems by using a conventional separation method using specific gravity centrifugation, a HES method, a top and bottom method, which have recently become popular.
The intense study was conducted based on the assumption that the difference in the composition of the cell population obtained by the filter method and the composition of the washing solution might affect the occurrence of aggregation. As a result, in the conventional specific gravity centrifugation method, almost no granulocytes are present after the separation, so that no agglomerated aggregates are generated with a washing solution of any composition. Because of the large number of cells, it has been discovered that conventional washing liquids that have been used in the past tend to cause aggregation due to granulocytes, and the surprising effect that cell aggregation is dramatically improved by suppressing aggregation. The invention has been completed. That is, the present invention is a washing method characterized by removing a cryoprotectant while suppressing cell aggregation when washing nucleated cells in a thawed nucleated cell-containing solution. Further, the present invention is a liquid for washing nucleated cells in a nucleated cell-containing liquid after thawing, which is a physiological solution containing at least a polysaccharide and / or a chelating agent. It is.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The nucleated cells referred to in the present invention refer to cells having a nucleus in the cells, for example, leukocytes, granulocytes, neutrophils, basophils,
Eosinophils, myelocytes, erythroblasts, lymphocytes, T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, suppressor T lymphocytes, B lymphocytes, NK cells, NKT cells, monocytes, macrophages, Dendritic cells, osteoclasts, osteoblasts,
Hematopoietic stem / progenitor cells (hereinafter abbreviated as hematopoietic stem cells), fibroblasts, chondroblasts, mesenchymal stem / progenitor cells (stroma)
stem cell). The nucleated cell-containing solution containing these includes peripheral blood, bone marrow, umbilical cord blood (including not only those collected from umbilical cord blood vessels, but also those collected from placental blood vessels), lymph, and those subjected to some treatment such as centrifugation. Or cells obtained by resuspending cells extracted from various organs or tissues in some liquid. In the present invention, the nucleated cell-containing liquid is thawed after freezing, but upon freezing, a cryoprotectant is added for the purpose of preventing cell frost damage. A typical frost protection agent is DMSO, which is widely used because it has the highest frost protection effect. Other frost protection agents include glycerin. In the present invention, these nucleated cell-containing liquids that have been cryopreserved are thawed and then washed. As a thawing method, rapid thawing in a warm bath at 35 to 40 ° C is generally used. Cell aggregation referred to in the present invention refers to a state in which cells that are originally uniformly dispersed and floating are aggregated and cannot maintain a dispersed state, and are destroyed by, for example, a process of freezing to thawing. Cell fragments and / or released contents (often DNA) may be formed by engulfing cells (often these substances are very sticky).

As means for suppressing cell aggregation according to the present invention, use of a washing solution to which an aggregation inhibitor has been added or performing a washing operation at 0 to 5 ° C. can be mentioned. Polysaccharides and / or chelating agents are suitable as aggregation inhibitors, and polysaccharides are particularly preferred. Polysaccharides include hydroxyethyl starch, dextran, pullulan, alginate, gum arabic and its main components arabate, chitin derivatives, etc., while hydroxyethyl starch and dextran are sterile physiological saline solutions as pharmaceuticals. It is commercially available as an aqueous solution and is most preferred. The concentration of the aqueous solution of the polysaccharide is 0.5% or more and 4/5 of the saturation concentration.
It is preferably less than 1%, more preferably 1% or more and 1/2 or less of the saturation concentration. If it is less than 0.5%, the aggregation suppressing effect may be reduced, and a polysaccharide solution having a saturation concentration of 4/5 or more takes time to prepare (at the time of dissolution), and the viscosity increases so that centrifugation becomes impossible. Could be. The concentrations of commercially available physiological saline solutions of hydroxyethyl starch and dextran are 6% and 10%, respectively, and these are preferably used, but are not limited to these concentrations. EDTA (including its salt) as a chelating agent,
Citric acid (including “CPD” and “ACD” which are anticoagulants to which nutrients such as salts and glucose are added). The concentration of the chelating agent is preferably 0.5% or more and less than 4/5 of the saturation concentration, more preferably 1%.
This is not more than half of the saturated concentration. If it is less than 0.5%, the effect of suppressing aggregation may decrease, and if it is 4/5 or more of the saturation concentration, it takes time to prepare (during dissolution). It can also damage cells. The composition of the chelating agent in the above-mentioned commercially available CPD and ACD is CPD: sodium citrate 2.6%, citric acid 0.3%, AC
D: 2.2% of sodium citrate and 0.8% of citric acid, which are preferably used, but are not limited to these concentrations. When the means for suppressing cell aggregation is to perform washing by lowering the temperature, if the temperature is lower than 0 ° C., the cells may be damaged. If the temperature exceeds 5 ° C., the effect of suppressing cell aggregation is reduced. Further, glucose, saccharose, trehalose, etc. may be added to the washing solution as required for nutritional supplementation, cell membrane protection, and the like. A specific method of washing is performed by adding a washing solution, performing centrifugation, and then removing the supernatant, and is repeated a plurality of times as necessary. In the washing method according to the present invention, the nucleated cell-containing liquid is concentrated before freezing by a method based on the principle of centrifugation such as HES method or top and bottom method (however, except for specific gravity centrifugation) or by a filter method (usually granules) The ball is 100
0 to 10000 / μl). However, it does not matter at all if it is used for a substance concentrated using a specific gravity centrifugation method in which the concentration of granulocytes is much lower than this. The washing solution according to the present invention is a physiological solution containing at least the above-mentioned polysaccharide and / or chelating agent. The term physiological solution as used herein means a liquid that does not harm the living body, such as physiological saline, D-PB
Buffers such as S and HBSS, and culture media such as RPMI1640.

[0008]

EXAMPLES The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Embodiment 1 Cell Separation Five samples were each processed by the following two methods. (1) Filter method: Dextran 40 injection (Dextran 4) disclosed in JP-A-10-313855
0 10% saline solution. The same applies hereinafter) to the recovered solution, and the cells were separated by a known filter method. The obtained cell solution was stored in a cryotube (Nunc, 1.8 m capacity)
l). (2) HES method: Japanese Society of Transfusion, Vol. 44, No. 1, 12-
Cell separation was performed by the known HES method disclosed on page 19. The obtained cell solution was fractionated into a freezing tube (manufactured by Nunc, capacity: 1.8 ml). 2. Cryopreservation (1) Filter method: DMSO was placed in an ice-cooled cryotube.
Was added with a syringe pump over 15 minutes to a final concentration of 5%. Then, it was frozen and stored in a freezer at -80 ° C for one week. (2) HES method: Japanese Society of Transfusion, Vol. 44, No. 1, 12-
Cryopreservation was performed according to the freezing method of the HES method disclosed on page 19. That is, DMSO and dextran 4
0 injection was added to the ice-cooled cryotube to a final concentration of 10% and 1%, respectively, with a syringe pump over 15 minutes. Then, it was frozen and stored in a freezer at -80 ° C for one week. 3. Thawing method The frozen tube was taken out of the freezer, immersed in a 37 ° C. water bath, and rapidly thawed. 4. Washing method After thawing, transfer the cells to a centrifuge tube, and add Dextran 40 injection or 6-HES (hydroxyethyl starch in 6% saline solution) to a final concentration of 2.5% dextran 40 or 2.5% hydroxyethyl starch. The mixture was centrifuged at 2,000 rpm and 10 ° C. for 10 minutes. 5. Evaluation The occurrence of aggregates was visually observed, and the leukocyte recovery rate before and after thawing was determined using a multi-item automatic blood cell analyzer (SF-3 manufactured by Sysmex Corporation).
000). 6. Results A summary of the results is shown in Table 1 (frequency of occurrence of aggregates) and Table 2 (white blood cell recovery rate). Dextran 40 injection or 6-HE
It can be seen that the one washed with the addition of S has no generation of aggregates and a high leukocyte recovery rate.

[0009]

COMPARATIVE EXAMPLE 1 The composition of the washing solution was dextran 40 injection or 6-HES instead of physiological saline, Dulbecco's salt buffer (without Ca and Mg), and RPMI16 with 10% FCS.
The same experiment as in Example 1 was performed except that 40 was used. A summary of the results is shown in Table 1 (frequency of occurrence of aggregates) and Table 2 (white blood cell recovery rate). It can be seen that aggregates are generated with high frequency and the leukocyte recovery rate is also low.

[Table 1] (Aggregate frequency)

[Table 2] (Average of leukocyte recovery rate n = 5)

[0010]

Example 2 The same operation as in Example 1 was performed except that a commercially available ACD-A solution was used as the cleaning solution. However, cell separation was performed only by the filter method. Table 3 summarizes the results.

[Table 3]

[0011]

Example 3 The same operation as in Example 1 was carried out except that the washing solution was cooled to 4 ° C., and a centrifugal separation was performed using a 4 ° C. cooled centrifuge. However, cell separation was performed only by the filter method. Table 4 summarizes the results.

[Table 4]

[0012]

As described above, according to the present invention, cells can be centrifugally washed after thawing without generating clumps, thereby improving cell recovery before and after freezing during cell washing. Therefore, it is expected to contribute to the improvement of clinical results in stem cell transplantation.

Claims (7)

[Claims] 1. A washing method comprising washing a nucleated cell in a thawed nucleated cell-containing solution and removing a cryoprotectant while suppressing cell aggregation. 2. The washing method according to claim 1, wherein the nucleated cell-containing solution contains at least granulocytes. 3. The washing method according to claim 1, wherein the nucleated cell-containing liquid is concentrated by a centrifugation method (excluding a specific gravity centrifugation method) or a filter method and then frozen. 4. The washing method according to claim 1, wherein the means for suppressing cell aggregation uses a washing solution to which an aggregation inhibitor has been added. 5. The cleaning method according to claim 4, wherein the aggregation inhibitor is a polysaccharide and / or a chelating agent. 6. A method for suppressing cell aggregation, wherein the washing operation is performed at zero.
The cleaning method according to claim 1, wherein the cleaning is performed at a temperature of up to 5 ° C. 7. A liquid for washing nucleated cells in a nucleated cell-containing liquid after thawing, comprising at least a polysaccharide and / or
Alternatively, the washing solution is a physiological solution containing a chelating agent.