JP2005052071A - Culturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a desired number of cells efficiently in a short period of time by improving the adhesion efficiency of to-be-cultured adhesive cells to the bottom surface of a culture vessel to increase the number of cells in their early culture stages. <P>SOLUTION: A culturing method is provided, comprising the steps S1, S2 and S3 of charging an erythrocyte C-coagulative substance into a body fluid A sampled from a biological tissue and removing the resultant coagulated erythrocyte C from the body fluid and the step S4 of charging a culture vessel 2 with the remaining body fluid B freed from the erythrocyte C and conducting a culture under specified culture conditions. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a culture method, and more particularly to a method for culturing adherent cells such as mesenchymal stem cells.

In order to culture adherent cells, a suspension obtained by mixing cells to be cultured in a predetermined medium is stored in a culture container and allowed to stand. It is known that the cells to be cultured settle in the culture container while being placed in a stationary state, and contact with the bottom surface of the culture container to adhere to the bottom surface and start growing (for example, non-culture). (See Patent Document 1).
Yoshikawa, “Cultivated dermis and bone marrow with bone marrow mesenchymal cells—Regenerative medicine with bone marrow mesenchymal cells”, Bioindustry, CMC Publishing Co., Ltd., 2001, Vol. 18, No. 7, p. 46-53

However, if erythrocytes are present in the medium, the erythrocytes have a relatively large specific gravity, so that the erythrocytes first settle in the culture container and may cover cell adhesion by covering the bottom surface of the culture container.
It may be possible to separate and remove red blood cells with a centrifuge using the difference in specific gravity, but it is difficult to finely adjust the centrifugal rotation speed, and the inconvenience that tissues other than red blood cells are also separated together. is there.

  The present invention has been made in view of the above-described circumstances, and improves the adhesion efficiency of adherent cells to be cultured to the bottom surface of the culture container, increases the number of cells in the initial stage of culture, and efficiently and in a short period of time. It is an object of the present invention to provide a culture method that makes it possible to obtain a desired number of cells.

In order to achieve the above object, the present invention provides the following means.
In the invention according to claim 1, a substance for aggregating red blood cells is introduced into a body fluid collected from a biological tissue, a removal step for removing the aggregated red blood cells from the body fluid, and a remaining body fluid from which the red blood cells have been removed are introduced into a culture container. And a culture step of culturing under predetermined culture conditions.

The invention according to claim 2 provides the culture method according to claim 1, wherein the substance that agglutinates red blood cells is dextran.
The invention according to claim 3 provides the culture method according to claim 1, wherein the substance that aggregates red blood cells is an anti-A antibody, an anti-B antibody, or an anti-H antibody.

The invention according to claim 4 is the culture method according to claim 1, wherein the substance that agglutinates red blood cells physically adsorbs anti-A antibody, anti-B antibody or anti-H antibody to microparticles such as polystyrene particles. A culture method is provided.
The invention according to claim 5 is the culture method according to claim 1, wherein the substance that aggregates red blood cells is a substance obtained by physically adsorbing anti-A antibody, anti-B antibody or anti-H antibody to magnetic particles, A culture method for aggregating red blood cells by applying a magnetic force is provided.

  According to the present invention, since the red blood cells are selectively aggregated and removed from the body fluid by the substance that aggregates red blood cells, which is input in the removal step, it is possible to improve the efficiency of cell attachment to the bottom surface of the culture container. . As a result, since a sufficient number of cells can be attached to the bottom surface of the culture vessel at the initial stage of culture and growth can be started, it is possible to grow to the desired number of cells within a short period of time. Play.

The culture method according to the first embodiment of the present invention will be described below with reference to FIG.
In the culture method according to the present embodiment, as shown in FIG. 1, a body fluid such as bone marrow fluid A collected from a patient is accommodated in a predetermined container 1 and a 6% dextran solution is injected into the container 1. (Step S1). Next, the container 1 is allowed to stand for about 1 hour while maintaining the temperature at about 4 ° C. (step S2). Then, the supernatant B is sucked from the container 1 and put into the culture container 2 together with a predetermined medium (step S3). Thereafter, the cells are placed in a CO ₂ incubator maintained at a temperature of 37 ° C. ± 0.5 ° C., a humidity of 100%, and a CO ₂ concentration of 5% (step S4).

According to the culturing method according to the present embodiment, red blood cells C in the bone marrow fluid A are aggregated by the action of dextran added together with the bone marrow fluid A, and are precipitated by specific gravity during the standing in step S2. Therefore, the red blood cells C are removed from the supernatant B sucked in step S3. Then, when the supernatant B from which the red blood cells C have been removed in this way is put into the culture vessel 2 together with a predetermined medium, the adherent mesenchymal stem cells contained in the bone marrow fluid A floated in the medium. Later, it settles and attaches to the bottom surface of the culture vessel 2 to start growing.
The medium is, for example, a mixture of MEM (Minimal Essential Medium), FBS (Fetal Bovine Serum: fetal bovine serum), and an antibiotic at a ratio of 84: 15: 1, for example.

In this case, since the red blood cells C are not contained in the bone marrow fluid mixed with the culture medium, the bottom surface is not covered with the red blood cells C in the culture vessel 2. Therefore, mesenchymal stem cells can easily reach the bottom surface of the culture vessel 2 without being inhibited by the red blood cells C.
As a result, in step S4, from the initial stage of the culture in the CO ₂ incubator, many cells can be attached to the bottom surface of the culture vessel 2 and growth can be started. That is, since the starting culture number can be increased, the culture time required to reach the desired number of cells can be greatly shortened.

Next, a culture method according to the second embodiment of the present invention will be described.
In the culture method according to this embodiment, erythrocyte antibodies are added in place of the dextran solution introduced into the bone marrow fluid A in the first embodiment (step S11).
According to the culturing method according to the present embodiment, erythrocytes C are mediated through antibodies by erythrocyte antibodies injected into bone marrow fluid A, for example, anti-A antibody, anti-B antibody or anti-H antibody (or anti-H lectin). Will be agglomerated.

  As a result, similar to the culture method according to the first embodiment, since the red blood cells C can be removed before mixing with the medium, the mesenchymal stem cells are efficiently attached to the bottom surface of the culture vessel 2 and cultured. There is an effect that the period can be greatly shortened.

Next, a culture method according to the third embodiment of the present invention will be described below with reference to FIG.
In the culturing method according to the present embodiment, instead of the single red blood cell antibody introduced into the bone marrow fluid A in the second embodiment, a microparticle that is physically adsorbed with the above red blood cell antibody is introduced (step S12). As the microparticles, for example, arbitrary particles such as polystyrene particles having a diameter of about 0.5 to 5 μm may be adopted, and the microparticles are made of a material having a specific gravity sufficiently higher than other components contained in the bone marrow fluid A. It is desirable.

  According to the culture method according to the present embodiment configured as described above, the red blood cells C are aggregated by the action of the red blood cell antibody. In this case, since the microparticles are physically adsorbed to the erythrocyte antibody, the aggregate of the erythrocytes C is formed larger than in the second embodiment. Therefore, the agglomerates easily settle in the container 1 and can be easily separated. Furthermore, by setting the specific gravity of the microparticles to be sufficiently larger than that of other components, separation by specific gravity can be further facilitated, and the standing time can be shortened.

Next, a culture method according to the fourth embodiment of the present invention will be described.
The culture method according to the present embodiment uses magnetic particles as the microparticles according to the third embodiment. That is, a magnetic particle obtained by physically adsorbing erythrocyte antibodies is introduced into the bone marrow fluid A (step S13) and aggregated for a predetermined time (step S14). Further, after waiting for a time necessary for the aggregation to occur, the magnet 3 is moved closer to the outside of the container 1 (step S15).
Examples of the magnetic particles include magnetic gelatin particles (manufactured by Olympus Optical Co., Ltd.), magnetic particles for diagnostic agents alone “Ferisphere” (manufactured by Nippon Paint), and the like.

  According to the culture method according to this embodiment configured as described above, when the erythrocytes C are aggregated by the action of the erythrocyte antibody, the magnetic particles physically adsorbed on the erythrocyte antibody are also aggregated together. The As a result, the point that a large aggregate is formed is the same as in the third embodiment. Furthermore, according to the present embodiment, the magnetic particles in the container 1 can be adsorbed to the inner wall surface of the container 1 by applying a magnetic force from the outside of the container 1 in an aggregated state.

  That is, unlike in the above-described embodiments, unlike the method of separation based on the action of gravity and the difference in specific gravity, the separation is performed by the magnetic force applied from the outside, so it is more reliable and quick in a time sufficiently shorter than the standing time. There is an effect that it can be separated. Further, if the magnet 1 is attracted to the inner wall surface of the container 1 by tilting the container 1, the remaining supernatant B can be flowed out of the container 1 while leaving only the red blood cells C.

  The body fluid is not limited to the above-mentioned bone marrow fluid, but may be peripheral blood or umbilical cord blood as long as it contains somatic cells such as ES cells, somatic stem cells, bone cells, chondrocytes, and nerve cells.

It is a flowchart which shows the culture method which concerns on 1st Embodiment of this invention. It is a flowchart which shows the culture method which concerns on 2nd Embodiment of this invention. It is a flowchart which shows the culture method which concerns on 3rd Embodiment of this invention. It is a flowchart which shows the culture method which concerns on 4th Embodiment of this invention.

Explanation of symbols

S1, S11, S12, S13 A step of introducing a substance that causes red blood cells to aggregate S2, S3, S14, S15 A removal step of removing aggregated red blood cells from the body fluid S4 Incubation step

Claims (5)

A removal step of introducing a substance that aggregates red blood cells into a body fluid collected from a biological tissue and removing the aggregated red blood cells from the body fluid;
A culture method comprising a culture step in which the remaining body fluid from which red blood cells have been removed is put into a culture container and cultured under predetermined culture conditions.   The culture method according to claim 1, wherein the substance that agglutinates erythrocytes is dextran.   The culture method according to claim 1, wherein the substance that causes red blood cells to aggregate is an anti-A antibody, an anti-B antibody, or an anti-H antibody.   The culture method according to claim 1, wherein the substance that aggregates red blood cells is a substance obtained by physically adsorbing anti-A antibody, anti-B antibody or anti-H antibody to microparticles such as polystyrene particles.   The culture method according to claim 1, wherein the substance that agglutinates erythrocytes is a substance obtained by physically adsorbing anti-A antibody, anti-B antibody or anti-H antibody to magnetic particles, and agglutinates erythrocytes by applying a magnetic force from the outside. .

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