JP2000139454A - Separation and recovery of cell and recovery required cell-containing liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating and recovering cells capable of recovering a nuclear cell such as hematopoietic stem cell in high yield by an inexpensive, simple and short time operation. SOLUTION: A cell-containing liquid stored at a cell-containing temperature A and containing a cell required for recovery and a cell required for removal is introduced into a cell capturing means substantially capturing the cell required for recovery and substantially passing the cell required for removal at a cell- separating temperature B, then the liquid containing the cell required for removal is released from the cell capturing means. Next, a liquid is introduced into the cell capturing means and the cell required for recovery capturing in the cell capturing means is recovered from the cell capturing means. The cell-storing temperature A is related to the cell-separating temperature B as follows: 0 deg.C<A<30 deg.C, 0 deg.C<B<30 deg.C and (absolute value of A-B) <15 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating and recovering only necessary cells from a mixed solution of various cells. 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]

2. Description of the Related Art Cord blood stem cells have attracted attention as a source of hematopoietic stem cell transplantation without donor invasion, and clinical applications thereof have been attempted mainly in Europe and the United States. Umbilical cord blood stem cells are rarely transplanted to patients immediately after being collected from a donor like other hematopoietic stem cell transplants, i.e., bone marrow transplants or peripheral blood stem cell transplants. (Especially for unrelated transplants). It is said that umbilical cord blood should be separated from nucleated cells (removal of red blood cells) in order to prevent side effects due to destroyed red blood cells after thawing and to reduce the volume during frozen storage during cryopreservation. In some cases, isolation and preservation are performed (Nankodo, “Peripheral Blood Stem Cell Transplantation”, page 173). Japanese Patent Publication No. 8-69 discloses details of a protocol for separating umbilical cord blood by Ficoll-Hypaque (a centrifugation method using a specific gravity solution, which is hereinafter abbreviated as Ficoll method). However, the Ficoll method has a problem that it is an extremely complicated and time-consuming operation at the laboratory level. WO96 / 17514
The publication discloses a bag system and a method for agglutinating and separating erythrocytes in cord blood using hydroxyethyl starch to obtain a nucleated cell concentrate, and a cell solution obtained by the method. This method is a slight improvement over the conventional Ficoll method in terms of reducing complicated operations, but it also requires a long operation because centrifugation is required twice. On the other hand, hematopoietic stem cell separation methods, which are alternatives to the Ficoll method and the hemagglutination removal method, have also been occasionally found. JP-A-8-104463 discloses a method of collecting hematopoietic stem cells by causing a filter through which red blood cells pass to capture hematopoietic stem cells and then inducing a liquid flow in a direction opposite to the first flow direction. By the way, umbilical cord blood is collected from the placenta and / or umbilical cord at the time of delivery and is usually stored for 10 to 72 hours until it is subjected to cell separation. As the temperature at this time, refrigeration (about 4 ° C.) to room temperature (about 25 ° C.) is employed. From the viewpoint of maintaining cell functions of stem cells, it was reported that storage at room temperature was more preferable (C
ampos et al .: Cryobiology, vol. 3
2, 1995), reports that there is no difference between them (Saito, et al .:
Japanese Society of Transfusion, Vol. 44, No. 5, 1998). However, from the viewpoint of cell separation performance, it is examined what temperature is optimal, more specifically, cell storage temperature (hereinafter simply referred to as “cell storage temperature”) and cell separation temperature before cell separation is performed. Temperature (hereinafter simply referred to as “cell separation temperature”) to improve the cell recovery rate during cell separation and shorten the time required for cell separation processing (hereinafter simply referred to as “cell separation time”). No studies were aimed at. Japanese Patent Application Laid-Open No. 8-104463 described above.
No publication describes the cell storage temperature or cell separation temperature.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for separating and recovering nucleated cells such as hematopoietic stem cells at a high rate at a low cost, with simple and easy operation.

[0004]

Means for Solving the Problems In order to solve these problems, the present inventors have conducted intensive studies focusing on the cell storage temperature and the cell separation temperature, and as a result, the difference between the cell storage temperature and the cell separation temperature has been found. The inventors have found that the present invention has a great effect on cell separation performance, and have completed the present invention. That is, the present invention provides a cell capturing means for substantially capturing a cell containing a cell containing a cell to be recovered and a cell to be removed stored at a cell storage temperature A, and substantially passing the cell to be removed. At a cell separation temperature B, and a liquid containing the cells to be removed is led out from the cell capturing means. Then, a liquid is introduced into the cell capturing means to collect the cells to be collected which have been captured by the cell capturing means. In the cell separation method of recovering from the cell capturing means, the cell storage temperature A and the cell separation temperature B are 0 ° C. <A <30 ° C. and 0 ° C.
C. <B <30.degree. C. and | AB | <15.degree. C. In addition, the present invention provides a cell-containing means containing a cell-containing liquid containing the cells to be recovered and the cells to be removed stored at the cell storage temperature A, which substantially captures the cells to be recovered and substantially passes the cells to be removed. The cell-retaining temperature is introduced at the cell separation temperature B, and the liquid containing the cells to be removed is led out from the cell-capturing means. A cell-containing liquid substantially free of cells to be removed obtained by the cell separation method recovered from the capturing means, wherein the cell storage temperature A and the cell separation temperature B are 0 ° C <A <30 ° C and 0 ° C < B <30 ° C. and | A−B | <15 ° C. The cell-containing solution requires recovery.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The cells requiring recovery referred to in the present invention refer to cells that are separated and recovered and used for some purpose, and the cells to be removed are unnecessary for the above-mentioned purposes or are cells that need to be recovered because they are any pathogenic cells. It is a cell that needs to be actively removed because it causes a problem of contamination. Cell-containing solutions containing these include 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 fluids, and those subjected to some treatment such as centrifugation. Or cells obtained by resuspending cells extracted from various organs or tissues in some liquid. The cell-capturing means for substantially capturing the cells to be recovered and substantially passing the cells to be removed as referred to in the present invention is, for example, a material that substantially captures the cells to be recovered and substantially passes the cells to be removed. And a molded container having a liquid inlet and a liquid outlet filled with, and having a cell trapping surface on the inner surface of the container. Any material can be used as long as the material that substantially captures the cells requiring recovery and the cells to be removed substantially pass is used as long as the material can be selectively captured. Examples of preferable moldability, sterility and low cytotoxicity are polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane and other synthetic polymers, agarose, cellulose,
Examples include natural polymers such as cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metals such as stainless steel, titanium, and aluminum. These trapping materials can be used as they are, but may be surface-modified as necessary, such as for selective passage or trapping of cells. For example, to increase platelet permeability W
O87 / 05812 discloses a method of coating with a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group, and the like.
Ligands having affinity for specific cells, such as amino acids, peptides, saccharides, glycoproteins (including bioligands such as antibodies and adhesion molecules) are disclosed in, for example, JP-A-2-261833.
And a method of fixing by the haloacetamide method proposed in Japanese Patent Application Publication No. Examples of the shape of the trapping material include granular, fibrous mass, woven fabric, nonwoven fabric, sponge-like porous body, and flat plate, but in terms of a large surface area per volume, granular, fibrous mass, woven fabric, nonwoven fabric, A sponge-like porous body is preferable, and a porous structure such as a fiber mass, a woven fabric, a nonwoven fabric, and a sponge-like structure is preferable from the viewpoint of handleability. It is more preferable from the viewpoint of properties. For non-woven fabric,
When so-called bioligands that specifically bind to specific cells such as an anti-CD34 monoclonal antibody are not immobilized on the surface, the fiber diameter is usually 1.0 μm or more and 30 μm or less, preferably 1.0 μm or more and 20 μm or less. ,
Still more preferably, it is 1.5 μm or more and 10 μm or less. If the thickness is less than 1.0 μm, cells that need to be collected may be firmly captured and may be difficult to collect, which is not preferable. 3
If it exceeds 0 μm, the cells that need to be collected are more likely to pass through without being captured by the fibers. In either case, the recovery rate may be reduced, which is not preferable. Also,
In the case of a sponge-like structure, the pore size is usually 2.0 μm or more and 3
0 μm or less, preferably 2.5 μm or more and 25 μm
Or less, still more preferably 3.0 μm or more and 20 μm or more.
m or less. If it is less than 2.0 μm, the flowability is extremely poor, and it may be difficult to pass the liquid itself.
Exceeding the number is not preferred because it leads to a decrease in the capture rate of the cells requiring recovery. Cells that need to be collected are captured, and the cells to be removed are filled with a material that substantially passes through the container. Synthetic polymers such as acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, and vinyl chloride; inorganic materials such as hydroxyapatite, glass, alumina, and titania; stainless steel;
Examples include metals such as titanium and aluminum. In the present invention, "substantially capture the cells required for recovery" means capturing 60% or more of the cells required for recovery, and "substantial passage of the cells to be removed" means removal in the cell-containing liquid. It means that the target cells pass by 60% or more. In the present invention,
Any liquid may be used as a liquid for recovering cells to be recovered which has been captured by being introduced into the cell capturing means, as long as it is a physiological solution.
-Buffers such as PBS and HBSS, and culture media such as RPMI1640. These physiological solutions may be added as necessary for the purpose of protecting cells, supplementing nutrition, preventing frost damage during cryopreservation, improving viscosity (may be effective for improving recovery rate), etc.
Dextran, hydroxyethyl starch, dimethyl sulfoxide, albumin, globulin, gelatin, glucose, saccharose, trehalose and the like may be added.

In the method for separating and recovering cells according to the present invention, the cell-containing solution is stored at a cell storage temperature A. A is usually higher than 0 ° C. and lower than 30 ° C., preferably 2 ° C. or higher and 2 ° C. or lower.
The temperature is 5 ° C or less, and still more preferably 3 ° C or more and 25 ° C or less. If A is 0 ° C or lower or 30 ° C or higher, cell functions may be damaged, which is not preferable. In the method for separating and collecting cells according to the present invention, the cell-containing liquid is introduced into the cell capturing means at the cell separation temperature B. B is usually higher than 0 ° C and lower than 30 ° C, preferably 2 ° C or higher and 2 ° C or higher.
The temperature is 5 ° C or less, and still more preferably 3 ° C or more and 25 ° C or less. If B is 0 ° C. or lower or 30 ° C. or higher, cell functions may be damaged, which is not preferable. In the present invention, the absolute value of the difference between the cell storage temperature A and the cell separation temperature B |
AB | is less than 15 ° C, preferably less than 10 ° C,
Even more preferably, it is below 5 ° C. At 15 ° C. or higher, an increase in cell treatment time and a decrease in cell recovery rate are observed, which is not preferable. The reason why the cell treatment time increases and / or the cell recovery rate decreases when | AB | is 15 ° C or higher is not clear, but the present inventors consider as follows. It is considered that the cell is trapped by the cell capturing means to a considerable extent, causing stress. At that time, the large difference between the cell storage temperature and the cell separation temperature means that stress due to environmental changes around the cells will also be applied,
In the worst case, cells are destroyed and sticky substances such as DNA are released, or even if it is not reached, activation of the coagulation system due to stress stimulation (precipitation of fibrin),
It is considered that an increase in the adhesion ability of the cells themselves is caused. It is presumed that these results lead to an increase in cell treatment time and a decrease in cell recovery rate. The cell separation and collection method according to the present invention is suitably used when the cells requiring collection are nucleated cells. A nucleated cell refers to a cell having a nucleus in the cell, for example, leukocyte, granulocyte, neutrophil, eosinophil, basophil, myelocyte, erythroblast, lymphocyte, T lymphocyte, helper T lymphocytes, suppressor T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, NK cells, NKT cells, monocytes, macrophages, dendritic cells, hematopoietic stem cells, osteoclasts, osteoblasts, bone cells, fibers Blasts, chondroblasts and the like. Further, the nucleated cell-containing liquid in the present invention is a liquid containing the nucleated cells, for example, peripheral blood,
Examples include lymphocytes, bone marrow fluid, umbilical cord blood, and liquids obtained by subjecting these to some treatment.

[0007]

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

Example 1 Cell Separator The outer dimensions of the container (length × width × thickness) were 41 × 41 × 18 mm, and the average fiber diameter was 2.3 μm on the inlet side of a polycarbonate container having a liquid outlet and a liquid inlet on a diagonal line. Eighteen polyester non-woven fabrics were filled on the outlet side with 16 polyester non-woven fabrics having an average fiber diameter of 12 μm to prepare a cell separator. In addition,
The packing density is 0.2 g / cm ³ and the effective filtration area is 12.25.
cm ² and an effective filtration length of 12.4 mm. Further, in order to impart platelet permeability to the cell separator, coating with a hydrophilic polymer was performed. That is, after passing a 1% ethanol solution of a hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer (molar ratio of hydroxyethyl methacrylate to dimethylaminoethyl methacrylate = 97: 3) from the inlet side of the filter, nitrogen gas was passed. Let dry. Cell Separation Procedure Umbilical cord blood was previously collected from the placenta and umbilical cord after delivery using a 50 mL disposable syringe (with an 18 gauge needle).
The blood was collected in 10 10 mL blood collection tubes containing 1.5 mL of PD. The collected cord blood was transferred to a 200 mL blood bag. This blood bag was stored in a thermostat at 22 ° C. for 24 hours. Twenty-four hours later, the blood bag 2 was taken out of the incubator, and the cell separation operation was immediately performed. That is, this blood bag was connected to the inlet side of the cell separator 1 prepared by using a tube having the three-way cock 4 and the mesh chamber 3 to which the cell collection bag 5 was connected in the middle. Cell separator 1
Was connected to a drain bag 7 with a tube having a three-way cock 6 for connecting a syringe for recovery. The nucleated cell-containing liquid in the source blood bag 2 was passed through the cell separator with a head of about 60 cm, and the erythrocyte-containing liquid flowing out of the cell separator 1 was drained into the drain bag 7. Next, three-way cock 6
A 30 mL disposable syringe containing 25 mL of a commercially available dextran 40 physiological saline solution (containing 4% of human serum albumin) is connected thereto, and the three-way cock 6 is turned in a direction in which only the syringe and the cell separator communicate with each other.
Was turned in a direction in which only the cell separator 1 and the cell collection bag 5 communicated with each other, and then the syringe was pushed to collect the cells captured in the cell separator into the cell collection bag 5. Analysis The number of nucleated cells, the number of erythrocytes and the number of platelets were measured by an automatic hemocytometer, and the CD34 positive rate in nucleated cells was determined by FITC-labeled anti-C.
The measurement was performed using the D34 antibody by a flow cytometry method developed on SSC-FITC (Miyazaki, et al .: Daily Practice and Blood, Vol. 5, No. 2, 21-24, 1995). The method of calculating the recovery rate and the removal rate is as follows. Recovery rate (%) = 100 × (number of cells after collection / number of cells in source blood) Removal rate (%) = 100−100 × (number of cells after collection / number of cells in source blood) The time until the end is the cell separation time,
It was measured with a stopwatch. Results Table 1 summarizes the results. It can be seen that nucleated cells and CD34-positive cells were recovered at a high rate in the recovered cell solution, and that the cell separation time was short.

[0008]

Comparative Example 1 Cell Separator The same cell separator as in Example 1 was used. Cell separation operation Preservation of umbilical cord blood using a 4 ° C refrigerator instead of a 22 ° C incubator, and cell separation after leaving at room temperature (24 ° C) for 1 hour instead of promptly performing cell separation Except for the above, the same operation as in Example 1 was performed. Analysis was analyzed in the same manner as in Example 1. Results Table 2 summarizes the results. The recovery rate of nucleated cells and CD34-positive cells was lower than that of Example 1, and the cell separation time was longer, and required about three times that of Example 1.

[0009]

Example 2 Cell Separator The same cell separator as in Example 1 was used. Cell Separation Operation The same operation as in Example 1 was performed except that cord blood was stored using a refrigerator at 4 ° C. instead of a thermostat at 22 ° C. Analysis The same analysis as in Example 1 was performed. Results Table 3 summarizes the results. It can be seen that nucleated cells and CD34-positive cells were recovered at a high rate in the recovered cell solution, and that the cell separation time was short.

[0010]

Comparative Example 2 Cell Separator The same cell separator as in Example 1 was used. Cell Separation Operation Same as Example 1 except that the cord blood stored at 22 ° C. is refrigerated in a refrigerator at 4 ° C. for 1 hour and then subjected to cell separation instead of taking out the cord blood from the incubator and immediately performing cell separation. The operation was performed. Analysis was analyzed in the same manner as in Example 1. Results Table 4 summarizes the results. The recovery rate of nucleated cells and CD34-positive cells was lower than that of Example 1, and the cell separation time was longer, and required about three times that of Example 1.

[0011]

Example 3 Cell Separator The same cell separator as in Example 1 was used. Cell separation operation A 15 ° C incubator was used instead of a 22 ° C incubator, and cord blood stored at 15 ° C was left at room temperature (24 ° C) for 1 hour instead of immediately performing cell separation. The same operation as in Example 1 was performed except that the above operation was performed. Analysis The same analysis as in Example 1 was performed. Results Table 5 summarizes the results. It can be seen that nucleated cells and CD34-positive cells were recovered at a high rate in the recovered cell solution, and that the cell separation time was short.

[0012]

Example 4 Cell Separator The same cell separator as in Example 1 was used. Cell Separation Operation The same operation as in Example 2 except that the cord blood stored at 4 ° C. is left for 1 hour in a 15 ° C. incubator for 1 hour instead of removing the cord blood from the refrigerator and immediately performing cell separation, and then performing cell separation. Was done. Analysis The same analysis as in Example 1 was performed. Results Table 6 summarizes the results. It can be seen that nucleated cells and CD34-positive cells were recovered at a high rate in the recovered cell solution, and that the cell separation time was short.

[0013]

Comparative Example 3 Cell Separator The same cell separator as in Example 1 was used. Cell Separation Operation The same operation as in Example 4 was performed except that a thermostat at 20 ° C. was used instead of a thermostat at 15 ° C. Analysis was analyzed in the same manner as in Example 1. Results Table 7 summarizes the results. The recovery rate of nucleated cells and CD34-positive cells was lower than in Example 4, and the cell separation time was about three times as long as in Example 4. Table 8 summarizes the above Examples and Comparative Examples.

[0014]

As described above, according to the present invention, it is possible to provide a method for separating and recovering nucleated cells, which can recover nucleated cells such as hematopoietic stem cells at a high rate at a low cost, with simple and easy operation. It is a great way to contribute to labor savings in clinical settings.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a cell separation circuit system used in Example 1.

[Explanation of symbols]

 Reference Signs List 1 cell separator 2 blood bag 3 mesh chamber 4 three-way cock 5 cell collection bag 6 three-way cock 7 drain bag

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B029 AA09 BB11 CC01 HA02 HA06 HA09 4B065 AA93X AA94X BD18 4C087 AA04 AA10 BB34 BB43 BB44 BB58 BB59 CA21 DA02 DA03 DA14 DA17 DA21 ZA51

Claims (2)

[Claims] 1. A cell capturing means for substantially capturing a cell containing a cell containing a cell to be recovered and a cell to be removed stored at a cell storage temperature A, and substantially passing the cell to be removed. At a cell separation temperature B, and a liquid containing the cells to be removed is led out from the cell capturing means. Then, a liquid is introduced into the cell capturing means to collect the cells to be collected which have been captured by the cell capturing means. In the cell separation method of recovering from the cell capturing means, the cell storage temperature A and the cell separation temperature B are 0
<A <30 ° C. and 0 ° C. <B <30 ° C. and | AB | <
A method for separating and collecting cells, wherein the temperature is 15 ° C. 2. A cell capturing means for substantially capturing a cell containing a cell containing a cell to be recovered and a cell to be removed stored at a cell storage temperature A, and substantially passing the cell to be removed. At a cell separation temperature B, and a liquid containing the cells to be removed is led out from the cell capturing means. Then, a liquid is introduced into the cell capturing means to collect the cells to be collected which have been captured by the cell capturing means. A cell-containing liquid substantially free of cells to be removed, obtained by the cell separation method recovered from the cell capturing means, comprising a cell storage temperature A and a cell separation temperature B
Is 0 ° C <A <30 ° C and 0 ° C <B <30 ° C and | A−
B | A solution containing cells requiring recovery that is <15 ° C.