JP2004254519A - Cell culture method - Google Patents

Abstract

A cell can be grown in a short period of time.
A medium containing a bone marrow fluid is first placed in a culture vessel and allowed to stand. Then, the blood cells are suspended in the bone marrow fluid as they are due to the floating properties, but the mesenchymal stem cells (MSCs) adhere to the bottom surface of the culture vessel due to the adhesion dependence. Thereafter, the medium is replaced to remove the buoyant cells, leaving the MSC adhered to the bottom of the culture vessel. However, since the absolute number of MSCs originally contained in the bone marrow fluid is small, the MSC is sparsely attached to the bottom of the culture vessel. It is just glued. Therefore, the MSC is magnetized by introducing the magnetic fine particles therein and holding the magnetic fine particles on the MSC. Then, the MSC holding the magnetic fine particles is collected near the magnet by the action of the magnetic force by disposing the magnet on the bottom surface of the culture vessel. Then, by culturing the cells after the MSCs have a proper density, the MSCs can be efficiently proliferated.
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cell culture method.
[0002]
[Prior art]
Cells are broadly classified into two types: floating cells such as blood cells and adhesion-dependent cells such as epithelial cells. For example, bone marrow fluid contains hematopoietic stem cells as floating cells and mesenchymal stem cells as adhesion-dependent cells. Mesenchymal stem cells (MSCs, Mesenchymal Stem Cells) have received attention as pluripotent cells, and many studies have been made alongside embryonic stem cells (ESCs, Embryonic Stem Cells). As a method for separating MSC from bone marrow fluid, it is utilized that MSC is an adhesion-dependent cell and that many other cells in bone marrow fluid are floating cells. A method is generally known in which only the MSC adhered to the bottom surface of the container is sorted out by leaving the container still. Further, as in Patent Document 1, an apparatus for sorting cells by magnetic force is also known.
[0003]
[Patent Document 1]
Japanese Patent Publication No. 7-57177
[Problems to be solved by the invention]
By the way, regarding the culture of the adhesion-dependent cells, if the seeding density is too low, the growth becomes extremely slow. Therefore, it is important to adjust the seeding density before culturing. This is thought to be due to the fact that cell proliferation is activated by paracrine or autocrine action of cytokines produced by cells when cultured at an appropriate seeding density.
[0005]
However, since the above-mentioned MSC has a small absolute number that can be collected from the bone marrow fluid, the MSC merely adheres to the bottom of the container in a sparse state, and it takes time for cell proliferation. This is a problem common not only to MSC but also to cells having a small absolute number contained in the extracted tissue / organ.
[0006]
As a method of eliminating the time required for cell proliferation, a method of treating cells adhered to the bottom surface of the container at a low density with an enzyme such as trypsin is used to separate the cells from the bottom surface of the container, collect the separated cells, and properly inoculate the cells. A method is considered in which the cells are seeded in a container so as to have a density and then propagated. In this case, it is possible to efficiently grow the plants. However, there is a problem that it takes time and effort to sow the container so as to have an appropriate sowing density.
[0007]
The present invention has been made in view of the above-described problems, and has as its object to provide a cell growth method capable of growing cells in a short period of time. Another object of the present invention is to provide a cell growth method capable of concentrating cells without trouble.
[0008]
Means for Solving the Problems, Embodiments of the Invention and Effects of the Invention
In order to achieve at least one of the above objects, the present invention employs the following method. That is, the present invention is a cell culture method for culturing cells,
A magnetizing step of magnetizing the cells to be cultured by holding the magnetic microparticles on the cells,
An assembling step of collecting the cells that have been magnetized in the magnetizing step by magnetic force so that the cells have a density that allows the cells to proliferate properly;
Culturing the cells collected in the assembling step.
[0009]
In this cell culture method, even if the cells exist in a sparse state before the aggregation step, the cells will grow in a relatively short period of time after the aggregation step because they will be dense enough to proliferate properly. Can be done. In addition, since the cells are densely packed by the magnetic force in the assembling step, the cells can be densely packed without trouble.
[0010]
Here, "the density at which the cells can grow properly" means that the density suitable for the growth of the cells to be cultured is previously determined experimentally or theoretically, and based on the density. This is the set value. Whether or not the cell is suitable for proliferation may be determined by whether or not a preset standard is cleared.For example, a standard value may be set for a culture time until a predetermined number of cells is reached and the standard value may be cleared. Alternatively, the determination may be made by setting a reference value for the number of cells grown within a predetermined culture time and clearing the reference value.
[0011]
In the cell culture method of the present invention, the cells to be cultured are preferably adhesion-dependent cells. The adhesion-dependent cells, which are also called anchorage-dependent cells, are cells that once attach to the inner surface of the container and then extend and divide. If the adhesion-dependent cells adhere to the inner surface of the container in a sparse state, they can be cultured as they are, or they can be treated with an enzyme, peeled off from the inner surface of the container, collected, and then seeded to obtain an appropriate seeding density. However, the former requires a long time to proliferate, and the latter requires time. Therefore, by applying the present invention to the adhesion-dependent cells, the cells can be densely grown without any trouble and can be efficiently proliferated.
[0012]
Here, the “adhesion-dependent cells” are, for example, collected from warm-blooded animals such as humans, mice, rats, guinea pigs, hamsters, chickens, rabbits, pigs, sheep, cows, horses, dogs, cats, monkeys, etc. Various cells are included. Examples of the cells of this warm-blooded animal include keratinocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, Muscle cells, adipocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, as well as embryonic stem cells (ESC) And stem cells such as mesenchymal stem cells (MSCs) and adhesion-dependent cancer cells. Among them, ESC and MSC are preferable, and MSC is particularly preferable. If the adhesion-dependent cells to be cultured are adhered to the bottom surface, they may be treated with an enzyme such as trypsin and peeled off before being concentrated by magnetic force, or the magnetic force may be superior to the adhesive force. If it is not treated with an enzyme, it may be condensed by magnetic force, or cells may be condensed by magnetic force after treatment using a lower amount of enzyme than usual amount of enzyme so that the adhesive force is weaker than magnetic force Is also good.
[0013]
In the cell culture method of the present invention, when the cells to be cultured are MSCs, the medium is replaced after the adhesion-dependent MSCs have adhered to the bottom surface of the container in which the solution containing the bone marrow fluid has been seeded before the magnetizing step. It is preferable to carry out a pretreatment step of obtaining MSCs to be cultured by removing floating cells by carrying out the above. Specifically, for example, the cells may be cultured according to the procedure shown in FIG. That is, first, a culture medium containing a bone marrow fluid is placed in a culture vessel and allowed to stand (see FIG. 1A). Then, the blood cells are suspended in the bone marrow fluid as they are due to the buoyancy, but the MSCs adhere to the bottom surface of the culture vessel due to adhesion dependence (see FIG. 1 (b)). Thereafter, the medium is replaced to remove the buoyant cells, leaving the MSC adhered to the bottom of the culture vessel. However, since the absolute number of MSCs originally contained in the bone marrow fluid is small, the MSC is sparsely attached to the bottom of the culture vessel. They are merely adhered (see FIG. 1 (c)). After that, if necessary, treatment with an enzyme such as trypsin is performed, and then the magnetic fine particles are charged and the MSCs are magnetized by holding the magnetic fine particles on the MSC (see FIG. 1 (d)). Thereafter, the MSC holding the magnetic fine particles is collected near the magnet by the action of the magnetic force by disposing the magnet on the bottom surface of the culture vessel (see FIG. 1 (e)). At this time, the magnetic force and size (dimensions) of the magnets are selected in advance so that the density of MSCs obtained by experiments is appropriate. Then, by culturing the cells after the MSCs have a proper density, the MSCs can be efficiently proliferated.
[0014]
In the cell culture method of the present invention, the confluence is preferably such that the cell density of a portion where cells are gathered is 1000 cells / cm ² or more, and particularly preferably 4000 to 6000 cells / cm ² . Within this range, the cells tend to proliferate due to the action of cytokines produced by the cells (especially adhesion-dependent cells such as MSC). It should be noted that the expression "cell density of a portion where cells are gathered" instead of simply "cell density" means that a predetermined number of cells occupy an entire area when simply expressed as "cell density". Since it is impossible to numerically distinguish between the case where the cells are distributed and the case where the same number of cells are densely packed in a part of the same area, the two can be distinguished by expressing them as `` the cell density of the area where the cells are gathered '' It was.
[0015]
In the cell culture method of the present invention, the magnetic microparticles are magnetic microparticles in a magnetic microparticle-encapsulated positively charged liposome (MPCL), and the cells to be cultured incorporate the MPCL in the magnetizing step. Preferably, the cells are thereby magnetized. The MPCL has a structure in which magnetic fine particles such as magnetite are encapsulated in liposomes and a positively charged lipid is provided between the liposomes, as shown in an example in FIG. Many cells have a negative charge and thus easily bind to MPCL having a positive charge, and MPCL has liposomes and thus is easily taken into cells. Therefore, if MPCL is employed as the magnetic fine particles in the present invention, it can be applied to culture of various cells. MPCL is, for example, Jpn. J. Cancer Res. 87, pp. 1179-1183 (1996) may be prepared by referring to the method for producing magnetite-encapsulated positive liposomes (MCL, Magnetite catic lipsome) described in Vol. As described above, when MPCL is employed in the magnetizing step, it is preferable to use 1 to 150 pg, particularly 20 to 150 pg, of MPCL as magnetic fine particles per cell. It is preferable to proceed to the next step 0.5 to 8 hours, particularly 3 to 5 hours after the start of the contact. In particular, when the cells to be cultured are MSCs, it is preferable to apply these numerical ranges. When the amount of MPCL used is less than 1 pg as magnetic fine particles per cell, it is not preferable because it is difficult to hold the MPCL in a cell in an amount sufficient to aggregate by magnetic force. Not preferred. The amount used is particularly preferably 20 to 150 pg. In addition, since the amount of the cells taking in the MPCL reaches its peak 0.5 to 8 hours after the start of the contact between the cells and the MPCL, it is preferable to proceed to the next step after 0.5 to 8 hours. The peak range is particularly preferably after 3 to 5 hours.
[0016]
In the cell culture method of the present invention, the magnetic microparticles are magnetic microparticles in a magnetic microparticle-encapsulated liposome (AML, Antibody-immobilized magnetosome) immobilized with an antibody, and the cells to be cultured and the cells are used in the magnetizing step. It is also preferable to magnetize the cells by binding to AML on which an antibody that specifically binds to is immobilized. As shown in FIG. 3, AML has a structure in which magnetic fine particles such as magnetite are encapsulated in liposomes and an antibody is provided between the liposomes. An antibody that specifically binds to a specific cell to be cultured is selected. In this case, a specific cell can be selected from a large number of cells and aggregated. For example, when the cells to be cultured are MSC and the magnetic fine particles are AML, the cells may be cultured according to the procedure shown in FIG. That is, first, a solution containing a bone marrow fluid is placed in a culture vessel, and AML is added thereto (see FIG. 4A). Bone marrow fluid contains a large amount of buoyant cells in addition to a small amount of MSC. However, cells other than MSC do not bind to the AML antibody, so only MSC binds to the AML antibody and is selectively magnetized. (See FIG. 4B). After binding to the AML antibody, MSC takes up AML into cells due to the presence of the liposome. Subsequently, by arranging a magnet on the bottom surface of the culture vessel, MSCs holding the magnetic fine particles are collected near the magnet by the action of magnetic force (see FIG. 4C). At this time, the magnetic force and size (dimensions) of the magnets are selected in advance so that the density of MSCs obtained by experiments is appropriate. Subsequently, the medium is exchanged while keeping the magnetized MSCs dense by magnetic force to remove the floating cells (see FIG. 4D). Thereafter, the MSCs are cultured. At this time, since the MSCs have an appropriate density, they grow efficiently. In addition, examples of the antibody that specifically binds to MSC include CD105, CD73, CD29, and CD44. Further, in this method, even when the cells to be cultured are adhesion-dependent cells, the aggregation step can be performed before the cells adhere, so that treatment using an enzyme such as trypsin can be omitted, and cell damage can be prevented. Can be reduced.
[0017]
The above-described AML is described in, for example, Chem. Eng. Jpn. It may be prepared by referring to the method described in Vol. 34, pp. 66-72 (2001). As described above, when AML is employed in the magnetizing step, it is preferable to use 1 to 150 pg, particularly 20 to 150 pg, of AML as magnetic fine particles per cell. It is preferable to proceed to the next step 0.5 to 8 hours, particularly 3 to 5 hours after the start of the contact. If the amount of AML used is less than 1 pg as magnetic fine particles per cell, it is not preferable because it is difficult to hold a sufficient amount of AML in the cells to aggregate by magnetic force, and if it exceeds 150 pg, the amount of AML that is not held increases and it is not economical. Not preferred. The amount used is particularly preferably 20 to 150 pg. In addition, the amount of MPCL taken up by the cells becomes 0.5 to 8 hours after the start of the contact between the cells and the AML. Therefore, it is preferable to proceed to the next step after 0.5 to 8 hours. The peak range is particularly preferably after 3 to 5 hours.
[0018]
The present invention can be applied when cells are separated and cultured from bone marrow, tissue, blood, and the like. In particular, when AML is used as the magnetic fine particles, it is effective for separating and culturing cells from blood or the like. The magnetic fine particles are not particularly limited to MPCL and AML as long as they adhere (retain) to cells. For example, they are used for MACS (Magnetic Cell Sorting and Separation of Biomolecules) of Daiichi Kagaku Co., Ltd. Magnetic microbeads, magnetic nanoparticles of Veritas (EasySep), or the like may be used. Among these magnetic fine particles, the magnetic fine particles containing liposomes, such as MPCL and AML, are taken into cells by the presence of the liposome, so that one cell can take in many magnetic fine particles and can be densely packed by magnetic force. This is preferable because the magnetism can be easily obtained.
[0019]
【Example】
[Example 1]
(1) Cells Used and Culture Conditions Human mesenchymal stem cells (MSCs) were manufactured by Bio Whittaker. In the experiment, an MSC growth medium MSCGM (Bio Whittaker) was used as a medium, and a 100 mm tissue culture dish (tissue culture dish, Asahi Techno Glass Co., Ltd.) was used as a culture vessel. The medium contained 37 ° C. and 5% CO ₂ . Culture was performed in a humidified air environment.
[0020]
(2) Preparation of Magnetite Encapsulated Positively Charged Liposomes (MCL, Magnetite Cationic Lipsome) MCL was prepared according to Jpn. J. Cancer Res. 87, pages 1179 to 1183 (1996). Specifically, first, three types of phospholipids, namely, N- (α-trimethylammonioacetyl) didodecyl-D-glutamate chloride (manufactured by Mutual Pharmaceutical Co., Ltd.), dilauroylphosphatidyl chloride (manufactured by Sigma Chemical Company), and dioleyl A liposome membrane containing phosphatidylethanolamine (Sigma Chemical Co., Ltd.) at a molar ratio of 1: 2: 2 was prepared, followed by addition of 1 mL of colloidal magnetite (magnetite amount 20 mg, magnetite manufactured by Toda Kogyo Co., Ltd.) MCL was prepared by the vortex method described above. The magnetite concentration was 18 mg / ml as measured by the potassium thiocyanate method.
[0021]
(3) Incorporation of MCL into MSC MCL was taken into MSC by replacing the medium of MSC in the logarithmic growth phase with MSCGM containing MCL in a tissue culture dish. The MCL concentration was added so as to be 100 pg of magnetite per cell with respect to the number of MSC cells.
[0022]
(4) Cell Culture Method Using Magnet Four hours after the addition of MCL, the MSC into which MCL was taken was adhered to the bottom surface of the tissue culture dish, so that it was peeled off from the bottom surface by treatment with trypsin, and the number of cells was 1000. Individually prepared in 10 ml MSCGM and seeded in 100 mm tissue culture dishes. At that time, the cells were shaken in a figure 8 at 60 rpm for 10 minutes using a shaker to inoculate the cells. At this time, a neodymium magnet (outer diameter 2.2 cmφ, surface magnetic flux density 0.45 T) was placed on the bottom of the 100 mm tissue culture dish at the center of the bottom of the tissue culture dish. As a control, shaking was performed in the same manner without installing a neodymium magnet. The MSCs (1000 cells) loaded with MCL were densely packed in a 2.2 cmφ circle at the center of the bottom of the tissue culture dish when the neodymium magnet was installed, whereas no neodymium magnet was installed. In some cases, it was distributed sparsely over the entire bottom surface of the tissue culture dish. When the neodymium magnet was installed, cells gathered toward the edge of the magnet (the part having a high magnetic flux density), and the area where such cells gathered was 1 cm ² . Therefore, the cell density (density) of the portion where the cells are gathered is 1000 cells / cm ² .
[0023]
(5) Measurement of Number of Cells The number of cells after 7 days of culture was measured using a cell counting kit-8 (Cell Counting Kit-8 (manufactured by Dojindo Chemical)) using WST-8 as a coloring substrate. The measuring method followed the instruction manual of the cell counting kit-8.
[0024]
(6) Experimental Results The experimental results are shown in FIG. FIG. 5 shows the number of MSC cells after 7 days of culture. The left side shows a control example (without magnet), and the right side shows this example (with magnet). As is clear from FIG. 5, the number of MSC cells was approximately four times higher than that in the control experiment by densely packing the MCL-loaded MSCs with a magnet.
[0025]
[Example 2]
In order to examine the toxicity of MCL to dog MSC, dog MSCs were cultured under the same conditions except that a medium containing MCL and a medium without MCL were used, and the change in cell number over time was graphed. FIG. 6 shows the result. As is clear from FIG. 6, no remarkable difference was observed between the case where the medium in which MCL was added to give 100 pg of magnetite per cell was used and the case where the medium in which MCL was not added was used. From this, it was found that MCL did not inhibit cell growth of MSC at a concentration of 100 pg as magnetite per cell.
[0026]
[Example 3]
In order to examine the amount of MCL taken up by dog MSCs, the cells were cultured using a medium in which MCL was added at 100 pg as magnetite per cell, and the change in MCL amount per cell over time was graphed. FIG. 7 shows the result. As apparent from FIG. 7, about 4 hours after the addition of MCL, the amount of MCL magnetite per cell showed a peak. At this peak, 30 pg of the added magnetite per cell to 100 pg was incorporated into dog MSCs.
[0027]
It should be noted that the present invention is not limited to the above embodiment at all, and it is needless to say that the present invention can be implemented in various forms without departing from the technical scope of the present invention.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing one embodiment of the cell culture method of the present invention.
FIG. 2 is a schematic diagram of an example of a positively charged liposome (MPCL) encapsulating magnetic fine particles.
FIG. 3 is a schematic diagram of an example of a magnetic microparticle-encapsulated liposome (AML) on which an antibody is immobilized.
FIG. 4 is an explanatory diagram showing one embodiment of the cell culture method of the present invention.
FIG. 5 is a graph showing the number of MSC cells after 7 days of culture.
FIG. 6 is a graph showing the results of a toxicity test of MCL on dog MSC.
FIG. 7 is a graph showing the amount of MCL uptake per cell over time.

Claims (9)

A cell culture method for culturing cells, comprising:
A magnetizing step of magnetizing the cells to be cultured by holding the magnetic microparticles on the cells,
An assembling step of collecting the cells that have been magnetized in the magnetizing step by magnetic force so that the cells have a density that allows the cells to proliferate properly;
Culturing the cells collected in the assembling step. The cell culture method according to claim 1, wherein the cells are adhesion-dependent cells. The cell culture method according to claim 2, wherein the adhesion-dependent cell is a mesenchymal stem cell (MSC). Before the magnetizing step, before the adhesion-dependent MSC adheres to the bottom of the container in which the solution containing the bone marrow fluid has been inoculated, the culture medium is replaced to remove the floating cells before obtaining the MSC to be cultured. The cell culture method according to claim 3, wherein a treatment step is performed. The cell culture method according to any one of claims 1 to 4, wherein the confluence is such that a cell density of a portion where cells are collected is 1000 to 6000 cells / cm ² . The magnetic particles are magnetic particles in a positively charged liposome (MPCL) encapsulating magnetic particles, and in the magnetizing step, the cells to be cultured are magnetized by incorporating the MPCL into the cells. 6. The cell culture method according to any one of 5. The cell culture method according to claim 6, wherein in the magnetizing step, the MPCL is used in an amount of 1 to 150 pg as magnetic fine particles per cell. The cell culture method according to claim 6, wherein in the magnetizing step, the cell proceeds to the next step 0.5 to 8 hours after the contact of the cell to be cultured with the MPCL is started. The magnetic microparticles are magnetic microparticles in a magnetic microparticle-encapsulated liposome (AML) in which an antibody is immobilized. In the magnetizing step, the cells to be cultured and the AML in which an antibody that specifically binds to the cells is immobilized. The cell culture method according to any one of claims 1 to 5, wherein the cells are magnetized by binding to

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