JP2002171967A - Method for in vitro culture of hematopoietic cell group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and effective method for culturing a hematopoietic cell group such as a hematopoietic stem cell, a hematopoietic precursor cell, etc. SOLUTION: A substrate composed of a fabric is arranged in a medium, a stroma cell in a concentration of 1×102-4×105 cells/ml is inoculated into the medium and cultured. Then a hematopoietic cell in a concentration of 1×100-1.8×106 cells/ml is inoculated into the medium and cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for in vitro culture of a group of hematopoietic cells. More specifically, the invention of the present application relates to an in vitro culture method for a hematopoietic cell group capable of efficiently expanding a hematopoietic cell group including at least hematopoietic stem cells and hematopoietic progenitor cells such as bone marrow, peripheral blood, and umbilical cord blood in vitro. .

[0002]

2. Description of the Related Art In 1990, about 5.2 million people died from cancer worldwide, but in 2010,
It is estimated to approach 8 million (Pisani, P., Pa
rkin, DM, Bray, F., Ferlay, J., Int. J. Cancer 8
3, 18-29 (1999)), and the number of cancer deaths in Japan in 1999 exceeded 290,000 (Ministry of Health and Welfare 1999 demographic statistics). Cancer control is regarded as one of the most important issues in disease control worldwide, and research on therapeutic agents and treatments is being conducted from various angles.

[0003] As a method of treating cancer, chemotherapy to kill cancer cells by administration of an anticancer drug, surgical treatment to remove the affected part, radiation treatment to irradiate the affected part, and leukemia have been hitherto. Hematopoietic stem cell transplantation (bone marrow transplantation) for replacing bone marrow with normal bone marrow is generally performed. Antibiotics and anticancer drugs used in chemotherapy are
All are accompanied by strong side effects such as nausea, vomiting, fever, decreased white blood cells and platelets, hair loss, and stomatitis simultaneously with the therapeutic effect. Surgical treatment is effective at an early stage when the cancer is limited to a specific site, but has little effect on the progression of the cancer and metastasis or leukemia. Radiation therapy is likewise a topical therapy and is not suitable for systemic cancer. As described above, all of the conventional cancer treatment methods have problems such that the burden on the patient is about the same as or better than the effect, and the effect on systemic cancer is low.

[0004] On the other hand, the hematopoietic stem cell transplantation method involves administering a large amount of an anticancer agent to a patient or irradiating the whole body with radiation in advance to suppress hematopoiesis throughout the body, and then converting hematopoietic stem cells or hematopoietic progenitor cells to produce normal blood cells. It is a method of supplying and has a high effect on leukemia, severe aplastic anemia, and systemic cancer with metastasis. Because hematopoietic stem cells are abundant in bone marrow, it is generally called a bone marrow transplantation method.In recent years, however, it has been revealed that peripheral blood contains hematopoietic stem cells as well as bone marrow. Since general anesthesia is not required, clinical application is actively performed.
Furthermore, it has recently become clear that umbilical cord blood contains hematopoietic stem cells at a much higher concentration than peripheral blood, and clinical application of this has also begun.

[0005] Such hematopoietic stem cell transplantation methods include autologous transplantation using the patient's own cells and allogeneic transplantation using cells provided by others of the same leukocyte type. In autologous bone marrow transplantation, it is difficult to obtain the required amount of cells. In allogeneic bone marrow transplantation, the concordance rate of leukocyte types is as low as 25% for siblings and less than 1% for parents and relatives. In many cases, we have to rely on provisions from people other than people. In any case, the donor must be hospitalized for a minimum of several days, which imposes a heavy burden on blood collection for blood transfusion, general anesthesia, collection of bone marrow more than 50 times each several ml, blood transfusion, and the like. Furthermore, after the collection, symptoms such as pain and fever often appear in the donor, so that collection may not be possible depending on the health condition of the donor. When using peripheral blood stem cells, general anesthesia and blood transfusion to the donor are not necessary,
Since the content of hematopoietic stem cells in peripheral blood is usually quite low, a method of increasing the content by administering an anticancer drug or a cytokine such as G-CSF (granulocyte colony stimulating factor) that causes proliferation of leukocytes is used. . The administration of such a drug is performed to the donor for 5 days or more, but the long-term side effects and the like are not clear at present. Therefore, in the conventional hematopoietic stem cell transplantation method, in order to collect a sufficient amount of hematopoietic stem cells and transplant them into a patient, not only the leukocyte type must be matched, but also there is a problem that the burden on the donor is large. In fact, transplantation was not always possible. On the other hand, when using cord blood stem cells, there is almost no burden on the donor,
There is almost no risk of infection such as virus, but it is necessary to preserve blood from the placenta at the time of childbirth, and usually it is done through cord blood banks except between siblings, but the amount of cells obtained is limited However, there is a problem that patients who can be transplanted are limited to children.

In order to solve these problems, attempts have recently been made to collect a small amount of hematopoietic stem cells, culture them outside the body, and proliferate them instead of collecting the necessary amount of hematopoietic stem cells from a donor. I have. For example, JP-A-10-13697
8 and Japanese Patent Application Laid-Open No. 10-295369, a method of culturing a liquid from an hematopoietic stem cell having a specific phenotype (CD34 ⁺ and / or c-kit ⁻ ) using an adherent incubator in the presence of a hematopoietic stem cell growth factor. Is disclosed. However, in these methods, in order to start the culture, it is necessary to isolate only the specific hematopoietic stem cells from cord blood, and there is a problem that a complicated operation is required.

[0007] Japanese Patent Application Laid-Open No. 2000-189157 discloses that
Methods for obtaining human hematopoietic-derived progenitor cell expansions ex vivo are disclosed. However, in such a method, it is necessary to control the rate at which the liquid culture medium is replaced or the cell density in the replaced medium according to the density of the cells to be cultured,
The operation was complicated and it was hard to say that it was efficient.

[0008] Therefore, at present, a simple and efficient method for expanding a large number of hematopoietic cells is not known.

Therefore, the invention of this application has been made in view of the circumstances described above, and solves the problems of the prior art, and provides a simple method for culturing a group of hematopoietic cells such as hematopoietic stem cells and hematopoietic progenitor cells. The task is to provide an effective method.

[0010]

Means for Solving the Problems According to the invention of this application, as a solution to the above-mentioned problems, first, a support made of cloth is placed on a medium, and stromal cells are reduced to 1 × 10 ² to 10 × 10 ² .
Provided is an in vitro culture method for a group of hematopoietic cells in which hematopoietic cells are inoculated and cultured at a concentration of 1 × 10 ^{0 to} 1.8 × 10 ⁶ cells / ml after being inoculated and cultured at a concentration of 4 × 10 ⁵ cells / ml. I do.

Secondly, the invention of the present application provides an in vitro culture method for a group of hematopoietic cells in which the cloth is a nonwoven fabric.

[0012] Thirdly, the invention of the present application is to provide a medium in which a support made of cloth is placed on a medium, and stromal cells are 1 × 10 5
Provided is a hematopoietic cell group culture kit for in vitro culture of a hematopoietic cell group inoculated and cultured at a concentration of ² to 4 × 10 ⁵ cells / ml.

Fourth, the invention of the present application is directed to the first aspect.
To provide a method for producing blood cells that differentiates blood cells from any of the hematopoietic cells of the group of hematopoietic cells cultured by any one of the third to third methods.

Fifth, the invention of this application also provides blood cells produced by the method of the fourth invention.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The inventors of the present application have previously shown that mouse bone marrow cells can be cultured in a medium spread with a nonwoven fabric or a woven fabric as a support (Cytotech).
nology, 34, 121-130 (2000)). Subsequently, as a result of intensive research, it was clarified that a group of hematopoietic cells such as hematopoietic stem cells and hematopoietic progenitor cells were proliferated, particularly in the range of specific hematopoietic cell inoculation concentrations and stromal cell inoculation concentrations. It has been reached.

Therefore, in the method for culturing a group of hematopoietic cells according to the invention of the present application, at least a woven or non-woven fabric is placed in a medium to serve as a culture support, and stromal cells are cultured at 1 × 10
After inoculating and culturing at a concentration of ² to 4 × 10 ⁵ cells / ml, it is essential to inoculate and culture hematopoietic cells at a concentration of 1 × 10 ^{0 to} 1.8 × 10 ⁶ cells / ml.

In the method of culturing a group of hematopoietic cells of the present invention, the cloth acts as a support for the adhesive stromal cells to spread on the fibers. In such a cloth, any fiber material may be used. For example, natural fibers such as cellulose, synthetic fibers such as polyester and polypropylene, and blended fibers thereof can be used. Fiber thickness and spacing between fibers,
The thickness of the cloth and the like can be appropriately selected according to the cell density of the group of hematopoietic cells to be cultured. In addition, these cloths may be those in which fibers are coated with a natural extracellular matrix such as collagen, gelatin, fibronectin, or the like, or a synthetic matrix such as ethylene vinyl alcohol copolymer, and are made hydrophilic. Also, these cloths
The fibers may be modified with diethylamine, diethylaminoethyl, or the like, or charged groups may be introduced to the surface by plasma discharge treatment or the like. Further, the cloth as described above may be placed in a culture medium, and its shape is not particularly limited. For example, it may be cut into an arbitrary shape such as a circle or a rectangle, may be formed into a columnar shape, an annular shape, a cubic shape, or the like, or may be a plurality of pieces. The cloth as the support as described above may be a woven cloth or a nonwoven cloth, but is preferably a nonwoven cloth capable of freely controlling the density between fibers and the thickness of the cloth.

In the method for in vitro culture of a group of hematopoietic cells of the invention of the present application, it is not clear why the group of hematopoietic cells can be cultured more efficiently and favorably than before by arranging the cloth on the medium. By providing the support, it is considered that stromal cells extend on the fiber, the hematopoietic cell group can come into contact with a plurality of stromal cells, and the culturing and propagation of the hematopoietic cell group can proceed efficiently. In fact, in the body, it is known that hematopoiesis is performed in a state where the group of hematopoietic cells adheres to the inner surface of bone marrow, or in a state where the group of stromal cells adheres loosely to the inner surface of bone marrow. Therefore, in the invention of this application, it is considered that the cloth in the culture medium functions as the inner surface of the bone marrow, the group of hematopoietic cells is efficiently cultured, and the production of blood cells proceeds.

In the method of in vitro culture of a group of hematopoietic cells of the invention of the present application, any medium may be used for the culture. In general, various liquid media used for culturing animal cells are preferable.
PMI medium, Dulbecco MEM medium and the like are exemplified.
Such a medium is preferably adjusted to conditions suitable for animal cell culture. For example, serum components such as bovine serum, human serum, fetal bovine serum, salts, amino acids and the like may be added. Furthermore, various hematopoietic growth factors considered useful for the production of blood cells may be added. For example, IL-1, IL-
3, IL-6, MGF (SCF), GM-CSF / IL
-3, one of the cytokines selected from the group consisting of
08987), IL-1, 1L-3, IL-6, EPO
(Tokuhyohei 5-50238)
5) Further, various known compositions such as cytokines combining IL-3, IL-2, and SCF (JP-A-7-135969) may be used.

In the invention of this application, first, stromal cells are placed in a culture medium provided with a cloth in an amount of 1 × 10 ² to 4 × 10 ⁵ cells.
Inoculate at a concentration of s / ml and culture. Stromal cells refer to hematopoietic cells in the bone marrow and umbilical cord and adherent cells other than blood cells.Hematopoiesis occurs when both come into contact via a ligand on the surface of hematopoietic cells and a receptor on the surface of stromal cells. It is believed to be regulated. Examples of such stromal cells include various mammal-derived stromal cells capable of growing a hematopoietic cell group. Specifically, fibroblasts, endothelial cells, adipocytes, macrophages and the like derived from bone marrow, umbilical cord, spleen, uterus, placenta, blood vessels and the like of humans, monkeys, cows, pigs, sheep, horses, mice and the like can be mentioned. . Also, these cells, even primary cells,
It may be a cell line. In the body, these stromal cells adhere to the inner surface of bone marrow and the like. However, in the method of in vitro culture of the group of hematopoietic cells of the present invention, the inoculated cells are cultured as described above by culturing. Stretch on the fiber. Conditions for culturing stromal cells are as follows:
Inoculation concentration of stromal cells of 1 × 10 ² -4 × 10 ⁵ cells /
There is no particular limitation as long as it is within the range of ml. According to the study of the inventors, even if the inoculum concentration is less than 1 × 10 ² cells / ml,
At more than 4 × 10 ⁵ cells / ml, the hematopoietic cell population does not grow efficiently. Other conditions may be selected in various media as described above, such as pH and temperature generally applied when culturing animal cells. For example, the culture temperature
The temperature can be 20 to 39 ° C, preferably 33 to 37 ° C.

In the in vitro culture method of the group of hematopoietic cells of the invention of the present application, the group of hematopoietic cells is further added to the medium in which the stromal cells are cultured at a concentration of 1 × 10 ^{0 to} 1.8 × 10 ⁶ cells / ml. By inoculating and culturing, a large number of hematopoietic cell groups can be obtained.

In the invention of the present application, the "hematopoietic cell group"
The term “hematopoietic stem cells capable of self-renewal” and hematopoietic progenitor cells differentiated from hematopoietic stem cells. At this time, hematopoietic progenitor cells refer to cells included in an early stage of differentiation among blood cells differentiated from hematopoietic stem cells. Specific examples include myeloid progenitor cells, lymphoid progenitor cells, erythroid progenitor cells, and the like. It is almost impossible to know exactly whether hematopoietic cells in bone marrow collected from a patient or donor are hematopoietic stem cells or hematopoietic progenitor cells at the stage of inoculation into the culture medium. Therefore, in the invention of this application, these are collectively referred to as “hematopoietic cell group”. That is, in the in vitro culture method of the group of hematopoietic cells of the invention of the present application, the group of hematopoietic cells as a raw material includes bone marrow, peripheral blood, and umbilical cord blood collected from humans, and further, centrifuged from these bloods. Cells separated by a filter, cells from which unnecessary cells have been removed by a filter, cells from which unnecessary cells have been removed by a monoclonal antibody-fixed cell separator such as immunomagnetic beads, C
Examples include cells purified as D34-positive cells.

In the in vitro culture method of hematopoietic cell group of the present invention, conditions for culturing these hematopoietic cell group are as follows:
Like the stromal cell culture conditions, there is no particular limitation. Conditions generally applied as a method for culturing animal cells, such as the temperature range as described above, can be set.

The hematopoietic cell group obtained by the in vitro culture method of the hematopoietic cell group of the invention of the present application may be used as it is, or after removing unnecessary cells by centrifugation or a monoclonal antibody-fixed cell separator, and After performing necessary operations such as purification of necessary cells and gene transfer by various known methods, the patient can be infused.
Preferably, the purified product is stored for transfusion or blood transfusion to prevent infectious diseases and the like. Such a group of hematopoietic cells works normally in a patient's body and can differentiate to produce blood cells.

The invention of this application also provides a kit for culturing a hematopoietic cell group by the above-described method for in vitro culturing a hematopoietic cell group. That is, in such a kit for culturing a group of hematopoietic cells, a support made of cloth is provided in a medium, and stromal cells are inoculated and cultured at a concentration of 1 × 10 ² to 4 × 10 ⁵ cells / ml. In such a kit, cultured stromal cells adhere to a cloth placed on a medium, and a group of hematopoietic cells collected from a patient or a donor is added to the kit at 1 × 10 ^{0 to} 1.8 × 10 ^6. If inoculated and cultured at a concentration of cells / ml, hematopoietic cells can be cultured in vitro. The group of hematopoietic cells thus obtained can be transfused into a patient as described above, and blood cells can be produced by differentiation in the patient's body.

Further, according to the invention of this application, blood cells are differentiated and obtained from a group of hematopoietic cells cultured in vitro by the method described above. Here, “blood cells” means cells at all stages from hematopoietic stem cells to differentiated blood cells. Specifically, hematopoietic stem cells self-renewed from hematopoietic stem cells, differentiated myeloid progenitor cells, lymphoid progenitor cells, erythroblasts, megakaryocytes, pre-B lymphocytes, pre-T lymphocytes, myeloblasts , Promyelocytes, myelocytes, neutrophils, eosinophils, basophils, erythrocytes, platelets, T lymphocytes, B lymphocytes, plasma cells, natural killer cells, monocytes, macrophages, dendritic cells, osteoclasts Examples include cells, stem Kupffer cells, and the like.

The blood cells obtained by the method for producing blood cells of the invention of the present application may be used as they are, or after removal of unnecessary cells by centrifugation or a monoclonal antibody-fixed cell separator, After performing necessary operations such as purification of necessary cells and gene introduction by various known methods, the patient can be infused. Of course, it may be stored for blood transfusion.

Hereinafter, examples will be shown, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.

[0029]

<Example 1> 12-well plate for suspension cells (3.6 cm ² SUMIRON, MS-80120R)
4 non-woven fabric carriers (Fibra-Cel, NBS, polyester, disc shape, diameter 6 mm, thickness 0.7 mm)
And a mouse stromal cell line ST2 (RIKEN Cell Bank R) using RPMI1640 medium containing 10% fetal bovine serum.
CB224), and the solution volume was 3 ml (inoculation concentration of 3.9 × 1).
( ⁴ cells / ml) at 37 ° C. in an atmosphere of 5% CO ₂ for about 1 hour.

12-well plate for adhesion culture (3.6 cm ²
(SUMILON, MS-80120) under the same conditions without laying a non-woven fabric on the bottom of the well.
2 was used as a control.

Thereafter, the supernatant of each well was removed, and a hematopoietic cell suspension obtained from the bone marrow of the femur and tibia of a mouse (Balb / c, 8 to 12 weeks old, male) was hydrocortisone (1 mM), 5 × 10 ⁵ cells using McCoy's 5A powder medium (GIBCO BRL) containing fetal calf serum (12.5%) and horse serum (12.5%)
/ Ml at a concentration of 3 ml, at 33 ° C,
The cells were cultured under a 5% CO ₂ atmosphere for one week.

The suspended cells contained in the culture solution were collected, the density of surviving hematopoietic cells was measured by the trypan blue dye exclusion method, and a methylcellulose medium (Stem Cell Technology, Methocult GF M343) was used.
The proportion of progenitor cells (CFU-Mix) in the hematopoietic cells was measured by the colony forming unit assay using 4).

Further, the density of the precursor cells was calculated by multiplying the density of the hematopoietic cells by the ratio of the precursor cells.

Table 1 shows the density of hematopoietic cells and the density of precursor cells (CFU-Mix) before and after culture.

[0035]

[Table 1] From Table 1, in the control containing no nonwoven fabric, the density of hematopoietic cells and the density of progenitor cells (CFU-Mix) were drastically reduced by culturing for 7 days after the addition of hematopoietic cells, but the density of hematopoietic cells was slightly reduced in the culture containing nonwoven fabric. As a result, the density of the progenitor cells increased about three times, and the proliferation of the progenitor cells was confirmed.

[0036]

As described above, according to the present invention, a simple and effective method for culturing a group of hematopoietic cells and a culture kit are provided. Further, according to the present invention, blood cells can be differentiated from a hematopoietic cell group. Further, the present invention enables a large amount of hematopoietic cell group to be cultured from a small amount of hematopoietic stem cells, so that it can be applied as a method for treating cancer or leukemia.

Claims (5)

[Claims] [Claim 1] established a support consisting of fabric to the medium, inoculated stromal cells at a concentration of ^{1 × 10 2 ~4 × 10 5} cells / ml, were cultured, hematopoietic cell group 1 × 10 ⁰ to 1 .8 × 1
0 ⁶ cells / ml of inoculated at a concentration of hematopoietic cell population method of in vitro culture to culture. 2. The method according to claim 1, wherein the cloth is a non-woven fabric. 3. A group of hematopoietic cells for in vitro culturing of a group of hematopoietic cells in which stromal cells have been inoculated and cultured at a concentration of 1 × 10 ² to 4 × 10 ⁵ cells / ml by placing a cloth support on the medium. Culture kit. 4. A method for producing blood cells, wherein blood cells are differentiated from any one of the group of hematopoietic cells cultured by the method according to claim 1. 5. A blood cell produced by the method of claim 4.