JP2023042352A - Method for separating cells having different sizes - Google Patents

Abstract

To provide a technique capable of acquiring cells having different sizes at high recovery rate from a mixture of two or more kinds of cells having different sizes in preparation of cells for regenerative medicine application.SOLUTION: A column, into which a particulate carrier insoluble into water having specific particle size distribution is filled, is used, mixed-cell liquid of cells having different sizes is brought in contact with the carrier, after smaller cells having passed through gaps of the carrier are recovered, larger cells not having passed through the carrier gap and been captured are recovered from the carrier by cleaning operation.SELECTED DRAWING: None

Description

The present invention relates to a method for separating cells of different sizes using water-insoluble particulate carriers, in which a mixture of cells of different sizes is passed through a column filled with particulate carriers having a certain particle size distribution. After recovering the cells that have passed through the gaps of the particulate carriers, the cells captured by the particulate carriers are recovered by a washing operation, thereby converting the former into small-sized cells and the latter into large-sized cells, respectively. Regarding how to get in efficiency.

In terms of cell function, the parameter of cell size can be said to be the most fundamental factor that characterizes cells, along with shape and specific gravity. Cells exist in various sizes. For example, E. coli has a diameter of 1-2 μm, baker's yeast has a diameter of about 5 μm, platelets, which are human blood cells, have a diameter of 2-4 μm, red blood cells have a diameter of 6-8 μm, and white blood cells have a diameter of 2-4 μm. It is 10 to 15 μm, but leukocyte subtypes such as B cells, T cells, and NK cells are 8 to 9 μm, which is small among leukocytes. It is also known that megakaryocytes have a size of 35-160 μm, CTCs (circulating cancer cells) have a size of 10-20 μm, and cultured cell spheroids have a size of 200-300 μm. Furthermore, it is known that the cell size varies greatly depending on the type of organism. eggs are 15 cm in size.

Cell separation methods using differences in cell size and density include the elutriation method (Non-Patent Document 1), methods using microfluidic devices (Non-Patent Documents 2 and 3), and methods using the principle of dielectrophoresis ( Non-Patent Document 4) is known. However, these methods have problems such as a small number of cells that can be treated as separation targets or a low recovery amount of the separated cells because the cells are severely damaged.

As a general means for identifying and separating cells, a method using marker molecules present on the cell surface is known, and is widely used in FACS (Fluorescence-Activated Cell Sorting) and MACS (Magnetic-activated Cell Sorting). used. However, in these methods, there was concern that labeling cells expressing marker molecules with antibodies labeled with fluorescent molecules or magnetic particles would have some effect on the properties and functions of cells. However, at present, there is a demand for the development of a label-free method for separating cells from a large amount of cells with a high yield using parameters such as cell size.

Grosse, J et al., Prep Biochem Biotechnol. 2012, 42(3):217-233. Davis, JA et al., Proc Natl Acad Sci USA. 2006, 103(40): 14779-14784. Takagi, J et al., Lab Chip. 2005, 5(7):778-784. Flanagan, LA et al., Stem Cells. 2008, 26(3):656-665.

An object of the present invention is to provide a technique for separating and purifying target cells of different sizes from a large amount of cells in a short period of time and obtaining them with high efficiency in the preparation of cells for regenerative medicine. Specifically, a column packed with particulate carriers having a specific particle size distribution is used to bring the cell population into contact, collect the cells that have passed through the interstices of the particulate carriers, and then remove the particulate carriers from the column. By detaching and recovering the cells trapped in between by pipetting and other operations, the former are collected as small-sized cells and the latter as large-sized cells, and both are separated and purified at a high recovery rate. to provide.

As a result of intensive studies by the present inventors to solve the above problems, a column filled with particulate carriers having a particle size of 150 μm or more and less than 250 μm and a frequency peak particle size of around 200 μm prepared by classification, A mixture of cells with different sizes is passed through, and the cell groups that have passed through the gaps of the particulate carriers are first recovered, then the particulate carriers are removed from the column, and the cell clusters trapped between the particulate carriers are pipetted. The former can be obtained as a cell sap containing small cells with a size of less than 10 μm, and the latter can be obtained as a cell sap containing large cells with a size of 25 μm or more. rice field. In addition, when a column is filled with a particulate carrier having a particle size of 150 μm or more and less than 250 μm in a wet state and a frequency peak particle size of about 200 μm, the size per 1 mL of the particulate carrier is 10 μm or more and less than 25 μm. 1×10^4 to 1×10^5 cells can be captured between the particulate carriers. It has been found that less than 100% of the cells can be collected as a group of cells passing through the gaps of the particulate carriers or a group of cells trapped between the particulate carriers.

That is, a mixture of cells with different sizes is passed through a column filled with particulate carriers, and after collecting the cells that have passed through, the particulate carriers are taken out of the column and washed to trap them between the particulate carriers. The collected cells are collected, and each cell fraction is passed through the column filled with the particulate carrier again by adjusting the packing amount of the particulate carrier, and the cell collection is repeated by the same operation to obtain a separation target. The inventors have found that cells with a size of less than 10 μm, cells with a size of 10 μm or more and less than 25 μm, and cells with a size of 25 μm or more can be recovered from a cell mixture with high efficiency, and have completed the present invention.

That is, the present invention includes the inventions described in [1] to [2] below.
[1]
A step of contacting a water-insoluble particulate carrier packed in a column with a cell mixture containing two or more types of cells, a step of recovering cells that have passed through the gaps of the particulate carrier packed in the column, and including a step of recovering the captured cells by a washing operation, wherein the water-insoluble particulate carrier has a particle size of 150 to 250 μm, and the cell mixture containing two or more types of cells contains cells with a size of less than 10 μm. A method for isolating cells, characterized by containing at least one type of cell and at least one type of cell having a size of 10 μm or more.
[2]
The cell mixture containing two or more types of cells contains at least one type of cell with a size of less than 10 μm, at least one type of cell with a size of 10 μm or more and less than 25 μm, and at least one type of cell with a size of 25 μm or more. The method for separating cells according to [1], comprising the above.
The present invention will be described in detail below.

The method for separating cells of different sizes of the present invention (hereinafter sometimes abbreviated as the cell separation method of the present invention) comprises filling a water-insoluble particulate carrier having a specific particle size distribution. A mixture of cells with different sizes is brought into contact with the column, and the cell clusters that have passed through the gaps of the particulate carriers are collected first, and then the particulate carriers are washed after being taken out of the column to be captured between the particulate carriers. The former is collected as a cell sap containing small-sized cells, and the latter as a cell sap containing large-sized cells. This is a cell separation method for separating small-sized cells, medium-sized cells, and large-sized cells by repeating the steps.

Next, the particulate carrier in the cell separation method of the present invention will be explained. The raw materials for the water-insoluble particulate carrier in the cell separation method of the present invention are not particularly limited, and inorganic carriers such as silica gel and glass deposited with a thin gold film, and polysaccharides such as agarose, cellulose, chitin and chitosan are used as raw materials. Water-insoluble polysaccharide carriers and crosslinked polysaccharide carriers obtained by cross-linking them with a cross-linking agent, cross-linked polysaccharide carriers obtained by cross-linking water-soluble polysaccharides such as dextran, pullulan, starch, alginate and carrageenan with a cross-linking agent, poly(meth) ) Synthetic polymer carriers such as acrylate, polyvinyl alcohol, polyurethane, polystyrene, etc., and crosslinked synthetic polymer carriers obtained by crosslinking these with a crosslinking agent.

Among these carriers, uncharged polysaccharide carriers such as agarose, cellulose, dextran, and pullulan, which have hydroxyl groups and can be easily modified with a hydrophilic polymer to be described later, and their cross-linking agents. Crosslinked crosslinked polysaccharide carriers, hydrophilic synthetic polymer carriers such as poly(meth)acrylate and polyurethane, and crosslinked hydrophilic synthetic polymer carriers obtained by crosslinking these with a crosslinking agent are preferred. Furthermore, the presence or absence of pores in the water-insoluble carrier is not particularly limited, and may be either porous or non-porous. In addition, the water-insoluble carrier used in the adsorbent of the present invention may be a commercially available product. (manufactured by Asahi Kasei Corp.), Cellophia (manufactured by Asahi Kasei Corp.) using cellulose as a raw material, and the like can be used.

The surface of the water-insoluble carrier used in the cell separation method of the present invention is preferably modified with a hydrophilic polymer in order to suppress non-specific adsorption of cells, and the hydrophilic polymer is shared with the water-insoluble carrier. It is more preferable to be fixed by binding.

Hydrophilic polymers that modify the surface of water-insoluble carriers include neutral polysaccharides such as agarose, cellulose, dextran, pullulan and starch, and hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. can be exemplified by synthetic polymers having Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan and starch are preferred, and dextran and pullulan are more preferred, because they are highly hydrophilic and can be easily fixed to the surface of an insoluble carrier by covalent bonding. . Although the molecular weights of dextran and pullulan are not particularly limited, those having a number average molecular weight of 10,000 to 1,000,000 are preferred in terms of sufficient hydrophilic modification of the surface of the insoluble carrier.
In addition, the shape of the water-insoluble carrier is not particularly limited, and may be particulate, spongy, flat membrane, tabular, hollow, or fibrous. A spherical particulate carrier is preferable, and since cells of a single type generally have a specific size distribution, in order to pass cells of a certain size or less, the particle size is It is more preferable to have as uniform a particle size distribution as possible.

The average particle diameter (median diameter) of the water-insoluble carrier used as the adsorbent in the cell separation method of the present invention in a state of being swollen in water is an appropriate particle size depending on the size of the cells to be separated. What is necessary is just to classify and prepare and use. Specific classification methods include a wet classification method of classifying a particulate carrier swollen in water using a stainless steel JIS standard sieve, and a particulate carrier dried using a stainless steel JIS standard sieve. A dry classification method can be exemplified. Theoretically, when spherical particulate carriers are closely packed, the diameter of the true spheres that can pass through the gaps of the particulate carriers is considered to be 16% of the diameter of the closest packed particulate carriers.

Therefore, assuming that cells are aggregates of true spheres with a specific normal particle size distribution, for example, if cells with a diameter of less than 8 μm are to be passed through, a particulate carrier with a particle size of less than 50 μm can be used. In addition, if it is desired to pass cells with a diameter of 25 μm or less, a particulate carrier with a particle diameter of 156 μm or less may be used. However, in reality, the size of the cells fluctuates depending on the culture conditions, and expansion and contraction occur due to the difference in osmotic pressure between the cell suspension and the cell suspension, so the composition and osmotic pressure of the cell suspension were taken into account. It is desirable to determine the optimum particle size and particle size distribution of the particulate carrier.

The particle size of the water-insoluble carrier in the cell separation method of the present invention can be measured using, for example, a precision particle size distribution analyzer manufactured by Beckman Coulter (product name: "Multisizer 3"). Alternatively, after photographing an image of a slide glass with a scale using an optical microscope, images of a plurality of particles to be measured are photographed at the same magnification, and the particle size of the photographed plurality of carriers is measured using a ruler. , can be obtained by calculating the average value thereof. In addition, the average size and size distribution of each cell type in the present invention can be measured with a particle size measuring device such as a Coulter counter, or can be measured visually using a hemocytometer and a microscope. good.

In the cell separation method of the present invention, a mixture of cells with different sizes is passed through a column filled with particulate carriers having a specific particle size distribution, and the cells that have passed through the gaps between the particulate carriers are separated into and recovering the cells trapped between the particulate carriers by washing the particulate carriers taken out from the column. At that time, in the former step, small-sized cells can be obtained by passing through the gaps of the particulate carriers, while large-sized cells are trapped between the particulate carriers and do not flow out, so they were removed from the column. It can be recovered by a washing operation of the particulate carrier. In addition, by performing this operation in several steps while changing the filling amount of the particulate carrier and the number of liquid passages, it is possible to separate cells of different sizes.

Examples of methods for separately separating cells of three or more different sizes include the following methods. Cells with a size of less than 10 μm and cells with a size of 10 μm or more and less than 25 μm using a column filled with a particulate carrier having a particle size of 150 μm or more and less than 250 μm in a wet state and a frequency peak particle size of about 200 μm. When separating a mixture of cells of 25 μm or larger, the number of captured cells per amount of particulate carrier used will be cells of 25 μm or larger > cells of 10 μm or more and less than 25 μm > cells of less than 10 μm. Most of the cells with a diameter of less than 10 μm pass between the particulate carriers, and the number of cells with a size of 10 μm or more and less than 25 μm is 2 to 7×10^4 per 1 mL of the particulate carrier, and the cells with a size of 25 μm or more are It is believed that 1-2×10̂5 are trapped in the interstices between particulate carriers.

Therefore, for example, 6.7×10̂4 cells with a size of 10 μm or more and less than 25 μm, and 1.7×10̂5 cells with a size of 25 μm or more are captured in the gaps between the particulate carriers per 1 mL of the particulate carriers. Assuming that 1 × 10^6 cells each having a size of 25 µm or more, cells having a size of 10 µm or more and less than 25 µm, and cells having a size of less than 10 µm were passed through the mixture, 1 × 10^6 cells The amount of particulate carrier required to capture all cells with a size of 25 μm or more is 5.4 mL, and the amount of particulate carrier required to capture all 1×10^6 cells with a size of 10 μm or more and less than 25 μm is 5.4 mL. The amount of carrier is calculated to be 10.1 mL.

Therefore, when cells are passed through 10 mL of particulate carriers, only cells with a size of less than 10 μm pass through between the particulate carriers, but both cells with a size of 10 μm or more and less than 25 μm and cells with a size of 25 μm or more pass through. Almost all will be captured between the particulate carriers. In this case, when 10 mL of the particulate carrier is used, both cells with a size of 10 μm or more and less than 25 μm and cells with a size of 25 μm or more can be recovered by washing the particulate carrier removed from the column. On the other hand, when 5 mL of particulate carriers were used, almost all of the cells with a size of less than 10 μm and about half of the cells with a size of 10 μm or more and less than 25 μm flowed out through the gaps between the particulate carriers. The remaining half of the cells with a size of 10 μm or more and less than 25 μm and all the cells with a size of 25 μm or more are captured between the particulate carriers. Therefore, when 5 mL of filler is used, about 50% of the cells with a size of 10 μm or more and less than 25 μm and almost all the cells with a size of 25 μm or more can be recovered by washing the particulate carrier removed from the column.

In other words, the cell mixture collected as the outflow cells from 5 mL of the packing material and the cell mixture collected by the washing operation have a distribution ratio of each cell due to the difference in size. By repeating the same operation multiple times, cells with a size of 10 μm or more and less than 25 μm and cells with a size of 25 μm or more in the outflow cells and washed cells are collected. It is possible to obtain a cell sap with an even higher time distribution ratio.

As a result, cells with a size of 25 μm or more, cells with a size of 10 μm or more and less than 25 μm, and cells with a size of less than 10 μm can be separated. In addition, in this cell separation method, the number of captured cells per volume of the particulate carrier is: cells with a size of 25 μm or more > cells with a size of 10 μm or more and less than 25 μm > cells with a size of less than 10 μm. Therefore, when the particulate carrier is removed from the column in which the cells are trapped and the cells are recovered by washing, the column is cut at a specific site to recover the cells, resulting in a washed cell solution with a higher cell distribution ratio. It is possible to recover.

In other words, for example, even when all cells with a size of 25 μm or more and cells with a size of 10 μm or more and less than 25 μm are all trapped in 10 mL of the particulate carrier as described above, the actual number of cells trapped at the top of the column is 25 μm. The above cells > cells with a size of 10 μm or more and less than 25 μm, and by cutting the upper part of the column and collecting the washed cells from the particulate carrier, a cell solution with a high distribution ratio of cells with a size of 25 μm or more can be obtained. can get.

The cell separation method of the present invention can separate cells of various sizes by setting the maximum frequency particle size and particle size distribution of the particulate carrier according to the particle size distribution of cells to be separated. In addition, the relationship between the size and the number of cells captured by the particulate carrier per unit volume is affected by the column diameter (cross-sectional area) and column length. In general, the cross-sectional area of the column should be small as long as there is no cell clogging, and the longer the column length, the better the ability to separate cells of different sizes.

Since the cell separation method of the present invention allows cell separation using only cell size as a parameter, the composition of the cell suspension only needs to consider expansion and contraction of cells due to osmotic pressure. is not particularly limited as long as it does not interfere with For example, water, MACS buffer (0.5% (w / v) bovine serum albumin (hereinafter abbreviated as BSA) and 2 mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) in PBS, which is a phosphate buffer) )), or an aqueous solution of mannitol, etc., can be used. In addition, it is also possible to add a reagent that enhances cell viability, or to use a buffer solution as the base solution to suppress pH fluctuations. Furthermore, in the step of recovering the cells trapped between the particulate carriers by washing the particulate carriers taken out from the column, in order to further promote the detachment of the cells, EDTA of about 20 mM, a surfactant, and cell detachment were added. A liquid or the like can be added as appropriate to the extent that it does not interfere with the survival of the cells.

In the cell separation method of the present invention, a large amount of cell fluid can be continuously treated by using an adsorbent packed in a column. This is an extremely effective means for directly separating and recovering cells with a desired size at a high recovery rate from a cell solution with a low cell concentration and a high liquid volume, such as a suspension of cultured cells recovered by the method. In addition, since pretreatment of cells with a drug or the like is unnecessary, even cell fluid such as whole blood extracted from humans can be directly subjected to cell separation, and the loss of recovered cells can be reduced.

In the cell separation method of the present invention, a mixture of cells with different sizes is passed through a column filled with particulate carriers having a certain particle size distribution, and after collecting the cells that have passed through the gaps of the particulate carriers, the particles are This is an effective cell separation method because the cells trapped in the carrier are collected by washing, and the former are obtained as small cells and the latter as large cells, respectively, with high efficiency. By the cell separation and collection method of the present invention, cells of various sizes, such as small cells less than 10 μm in size, medium cells 10 to 25 μm in size, and large cells 25 μm or more in size, can be collected at high efficiency. It is possible to separate and purify by the recovery rate, and the standard of cell size to be separated can be controlled by appropriately selecting the particle size of the particulate carrier and the filling amount of the column.

In addition, the cell separation method of the present invention can easily process a large amount of cells in a short time compared to existing methods such as density gradient centrifugation, microfluidic devices, dielectrophoresis, and labeling of cell surface marker molecules. Furthermore, since separation is possible without labels or pretreatment, it is an extremely effective means for large-scale purification of high-quality medical cells, especially for regenerative medicine applications.

1 is a diagram showing the particle size distribution of Toyopearl HW-40EC having a particle size of 150-250 μm, produced in Production Example 1. FIG. Ramos cells, RD cells, human iPS cell 201B7 strain (hereinafter abbreviated as 201B7 cells), and human mesenchymal stem cells used in Example 1, Example 2, Comparative Example 1, and Example 3 were treated with MACS buffer. It is a photograph of cells taken by introducing them into a hemocytometer after washing. The scale bar is 100 μm, and the size is less than 10 μm for Ramos cells, 10-25 μm for RD cells and 201B7 cells, and greater than 25 μm for human mesenchymal stem cells. 1 is a graph showing the recovery rates of Ramos cells, RD cells, 201B7 cells, and human mesenchymal stem cells in each of the first and second passages of cells through the column in Example 1. FIG. 2 is a graph showing recovery rates of Ramos cells, RD cells, and human mesenchymal stem cells in the effluent cell fluid in Example 2 and Comparative Example 1. FIG. 2 is a graph showing recovery rates of Ramos cells, RD cells, and human mesenchymal stem cells in washed cell sap in Example 2 and Comparative Example 1. FIG. In Example 3, the effluent cell fluid Fr. 1 is a graph showing recovery rates of Ramos cells and human mesenchymal stem cells in 1 to 10. FIG. Washed cell suspension UP. 1 is a graph showing recovery rates of Ramos cells and human mesenchymal stem cells in 1 to 5. FIG. The applied cells (App.) and the effluent cell fluid Fr. 1 is a graph showing the cell ratios of Ramos cells and human mesenchymal stem cells in 1-10. The applied cells (App.) and the washed cell suspension UP. 1 is a graph showing cell ratios of Ramos cells and human mesenchymal stem cells in 1 to 5. FIG.

The present invention will be described in more detail below with reference to Production Examples, Examples, and Comparative Examples, but the present invention is not limited to these.

Preparation Example 1 Preparation of particulate carrier having a particle size of 150 to 250 μm Using a glass filter, Toyopearl HW-40EC (manufactured by Tosoh, particle size of 100 to 300 μm), which is a porous synthetic polymer particulate carrier, was washed with water. Then, in a state of being suspended in water, by wet classification using 150 μm and 250 μm stainless steel standard sieves (manufactured by Tokyo Screen), Toyopearl HW-40EC with a particle size of 150-250 μm was produced (hereinafter referred to as HW -40EC/150-250μm may be abbreviated). FIG. 1 shows the particle size distribution of the produced HW-40EC/150-250 μm.

Example 1 Separation of human mesenchymal stem cells from mixture of Ramos cells, RD cells, 201B7 cells and human mesenchymal stem cells using HW-40EC/150-250 μm packed column (1) Packed with particulate carrier Preparation of column Between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G), a nylon mesh filter with an opening of 35 μm (taken out from a 5 mL polystyrene round tube with a cell strainer and cap manufactured by Japan BD) was used. A mounted column was prepared. Next, HW-40EC/150-250 μm prepared in Preparation Example 1 was replaced with MACS buffer, and after standing for 12 hours or more, a 50% suspension of the particulate carrier was adjusted so that the sedimentation volume was 50%. A liquid was prepared, 4.0 mL of the liquid was added to the prepared column, and the column was packed with the particulate carrier (carrier packing volume: 2.0 mL). Two identical columns were prepared.

(2) Culture of Ramos Cells and Preparation of Cell Suspension Ramos cells (human Burkitt's lymphoma cells, JCRB9119), which are free-floating cells, were mixed with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, Using RPMI 1640 medium (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) supplemented with the cells (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), cells were seeded in petri dishes for suspension culture (manufactured by Sumitomo Bakelite) and cultured at 37 ° C. in a 5% CO2 atmosphere. .

The Ramos cells were collected in a 50 mL tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Cells were then washed by suspending the cell pellet in serum-free RPMI 1640 medium (without phenol red), centrifuging again at 1500 rpm for 5 minutes, and discarding the supernatant. The cell pellet was suspended in a solution obtained by dissolving Cell Tracker Green in serum-free RPMI medium (without phenol red) at a final concentration of 0.3 μM, transferred to a culture petri dish, and cultured at 37° C. for 1 hour in a 5% CO atmosphere. bottom.
Next, the cells were collected in a 50 mL tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. was cultured overnight. Next, the cells were collected in a 50 mL tube, centrifuged at 1500 rpm for 5 minutes to sediment, suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells.

After repeating the cell washing operation twice, the cells were suspended in a MACS buffer and filtered using a cell strainer to prepare a cell suspension of Ramos cells. A portion of the obtained Ramos cell suspension was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column. In addition, the size of the Ramos cells measured by a hemocytometer was 8 μm on average. In the present invention, it corresponds to cells of less than 10 μm. FIG. 2 shows microscopic images of Ramos cells.

(3) Cultivation of RD Cells and Preparation of Cell Suspension RD cells (human fetal rhabdomyosarcoma cells, cell Jp. EC85111502-G0), which are adherent cells, were grown in cell growth medium No. 104 (cell Jp. DSGM104), the cells were seeded in an adhesive culture flask (manufactured by Sumitomo Bakelite) and cultured at 37° C. in a 5% CO 2 atmosphere. Next, RD cells were fluorescently stained using Cell Tracker Orange (manufactured by Thermo Fisher Scientific) by the following method.
After discarding the medium in the flask during culturing of the RD cells, serum-free RPMI 1640 medium was added to wash the cells, and then the serum-free RPMI 1640 medium was discarded. Next, Cell Tracker Orange dissolved in serum-free RPMI 1640 medium to a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, cell growth medium No. 104 was added and cultured at 37°C for 1 hour in a 5% CO2 atmosphere. Next, after discarding the medium, new cell growth medium No. 1 was added again. 104 was added and cultured overnight at 37°C in a 5% CO2 atmosphere. Next, cell collection and cell suspension preparation were performed by the following methods.

After discarding the medium in the flask during cell culture and adding D-PBS(-), the cells were washed and D-PBS(-) was discarded. Next, an appropriate amount of Accutase (manufactured by Innovative Cell Technology) was added and left for several minutes to detach the RD cells and collect them in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in MACS buffer and filtered using a cell strainer to prepare a cell suspension of RD cells stained with Cell Tracker Orange. A portion of the obtained RD cell suspension was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column. In addition, as a result of measuring the size of the RD cells with a hemocytometer, the average size was 15 μm. In the present invention, this corresponds to cells of 10-25 μm. FIG. 2 shows microscopic images of RD cells.

(4) Culture of 201B7 Cells and Preparation of Cell Suspension Human iPS cell 201B7 strain (purchased under license from iPS Academia Japan), which is an adhesive cell, was cultured in an adhesive culture flask (manufactured by Sumitomo Bakelite). was used in the following manner. iMatrix-511 (manufactured by Nippi) prepared in advance was diluted to 3 μg/mL in D-PBS (−), added to the petri dish, and left at 4° C. overnight or longer to spread iMatrix-511 onto the petri dish culture surface. was coated.
After discarding the iMatrix-511 solution in the coated petri dish, StemFit AK02N medium (manufactured by Ajinomoto Co., Ltd.), which is an iPS cell culture medium, was added and washed. 27632: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was suspended in the same medium to which 10 μM was added and seeded. After culturing overnight at 37°C in a 5% CO2 atmosphere, the StemFit AK02N medium containing Y-27632 was discarded, and the medium was replaced with StemFit AK02N medium not containing Y-27632. , cell harvesting and passaging were performed.

Next, cell collection and cell suspension preparation were performed by the following methods. After washing the cells by adding D-PBS (-) to the petri dish and rinsing the cells, discarding the D-PBS (-) was repeated twice, and then the cells were washed with CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific). and Versene Solution (manufactured by Thermo Fisher Scientific) were added at a ratio of 1:1 and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere. After confirming that the cells were detaching in a round shape, the cells were detached by repeating pipetting in the detachment solution and collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells.

After repeating the cell washing operation twice, the cells were suspended in a MACS buffer and filtered using a cell strainer to prepare a cell suspension of 201B7 cells. A portion of the obtained 201B7 cell suspension was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column. In addition, as a result of measuring the size of the 201B7 cells with a hemocytometer, the average size was 15 μm. In the present invention, this corresponds to cells of 10-25 μm. FIG. 2 shows microscopic images of 201B7 cells.

(5) Culture of Human Mesenchymal Stem Cells and Preparation of Cell Suspension Adhesive human mesenchymal stem cells UE6E7T-2 (hereinafter referred to as human mesenchymal stem cells) were obtained from the JCRB cell bank. . In addition, culture was performed according to the following procedure. Human mesenchymal stem cells, which are adherent cells, were prepared with 10% FBS (manufactured by Biological Industries), 4 mM L-glutamine (manufactured by Fujifilm Wako Pure Chemical Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries). was added to the D-MEM medium (Low Glucose, Sigma-Aldrich), cells were seeded in an adherent culture flask (Sumitomo Bakelite), and cultured at 37° C. in a 5% CO 2 atmosphere.

Next, fluorescent staining of human mesenchymal stem cells using Cell Tracker Red (manufactured by Thermo Fisher Scientific) was performed by the following method. After discarding the medium in the petri dish in which the human mesenchymal stem cells were being cultured, serum-free RPMI 1640 medium was added to wash the cells, and then the serum-free RPMI 1640 medium was discarded. Next, Cell Tracker Red dissolved in serum-free RPMI 1640 medium to a final concentration of 10 μM was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, D-MEM medium supplemented with 10% FBS, 4 mM L-glutamine and an antibiotic solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the medium, fresh D-MEM medium supplemented with 10% FBS, 4 mM L-glutamine and an antibiotic solution was added, and cultured overnight at 37° C. in a 5% CO 2 atmosphere. Next, cell collection and cell suspension preparation were performed by the following methods. After the medium in the petri dish during cell culture was discarded and D-PBS(-) was added, the cells were washed and D-PBS(-) was discarded. Next, an appropriate amount of Accutase (manufactured by Innovative Cell Technology) was added and left for several minutes to detach the human mesenchymal stem cells, which were collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in MACS buffer and filtered using a cell strainer to prepare a cell suspension of human mesenchymal stem cells stained with Cell Tracker Red. A portion of the obtained human mesenchymal stem cell suspension was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Hereinafter, based on this cell density, the amount of cells to be added to the column was calculated from cell density×amount of cell solution applied to the column.

In addition, as a result of measuring the size of human mesenchymal stem cells with a hemocytometer, the average size was 35 μm or more. In the present invention, it corresponds to cells of 25 μm or more. FIG. 2 shows microscopic images of human mesenchymal stem cells.

(6) Separation of Human Mesenchymal Stem Cells Using a Particulate Carrier-filled Column The number of Ramos cells is 6.7×10^5, the number of RD cells is 8.9×10^5, the number of 201B7 cells is 8.9×10^5, and the number of human mesenchymal stem cells is 8.9×10^. A column filled with 2 mL of HW-40EC/150-250 μm was vertically placed and added from the top of the column. 2 mL of the effluent cell sap was collected from the 22G syringe needle at the bottom of the column (hereinafter referred to as effluent cell sap-1). After collecting a small amount of the effluent cell fluid-1 for measuring the cell recovery rate, another column filled with 2 mL of HW-40EC/150-250 μm was placed vertically, and from the top of the column, By adding 2 mL of the effluent cell sap-1, 2 mL of the effluent cell sap was collected from the 22G syringe needle at the bottom of the column (hereinafter referred to as effluent cell sap-2).

(7) Measurement of cell recovery rate in effluent cell effluent After adding MACS buffer to effluent cell effluent-1 and 2 obtained by the above operation and diluting, 2 mL of each 5 mL polystyrene round with cell strainer cap It was separated into a tube (manufactured by BD Japan). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a cell viability determination reagent, the number of cells was measured using a cell sorter BD FACSAria (manufactured by BD Japan). gone.
Ramos cells, RD cells, 201B7 cells, and human mesenchymal stem cells contained in each cell solution were identified using dot plots that were double developed using the fluorescence intensity of FITC, PE, APC, etc. as parameters, The number of cells was calculated by proportional calculation based on the number of particles of the internal standard beads (this number of cells is defined as the number of outflow cells -1 and 2, respectively). The recovery rate of each cell in the effluent cell sap-1, 2 was calculated by dividing the effluent cell number-1, 2 by the number of each cell applied to the column.

Table 1 and FIG. 3 show the results of calculating the recovery rate of each cell. From this result, when a mixed cell solution of Ramos cells, RD cells, 201B7 cells, and human mesenchymal stem cells was passed through a column filled with 2 mL of a particulate carrier, the cells passed through once. It was found that the recovery rate of human mesenchymal stem cells decreased by 29.3% in the cell effluent cell sap-1. In addition, it was found that the recovery rate of human mesenchymal stem cells was further reduced by 38.1% in the effluent cell sap-2, in which the cells were passed through separate columns twice. This result was considered to be due to the fact that human mesenchymal stem cells are larger than other cells in the mixed cells, averaging 35 μm, and thus easily captured in the gaps between the granular carriers.

In addition, 1.310×^5 human mesenchymal stem cells per 1 mL of the particulate carrier were caught in the gaps between the particulate carriers in the first passage, and 1.7×10^5 in the second passage. , It is clear that 2.7×10^4 RD cells per 1 mL of the particulate carrier were caught in the gaps between the particulate carriers in the first passage, and 6.7×10^4 were caught in the gaps between the particulate carriers in the second passage. became. The particulate carriers are removed from the two columns after the passage of the liquid, and washed on a cell strainer with a MACS buffer solution containing 20 mM EDTA by pipetting, resulting in a high abundance ratio of human mesenchymal stem cells. A cell suspension could be obtained. From these results, it was clarified that the larger the size of the cells, the larger the number of cells sandwiched between the particulate carriers per volume, and that by utilizing this difference, cells of different sizes can be separated from each other. Theoretically, when spherical particulate carriers are closely packed, the maximum diameter of the true spheres that can pass through the gap is estimated to be about 16% of the diameter of the particulate carriers. Since the wet state diameter of the particulate carrier is >190 μm, the diameter of the cells that can pass through the interstices was estimated to be <30 μm, which could prove to support the results of this example.

Example 2 Separation of Ramos cells from mixture of Ramos cells, RD cells and human mesenchymal stem cells using HW-40EC/150-250 μm packed column (1) Preparation of column filled with particulate carrier 20.0 mL A column was prepared by attaching a nylon mesh filter with an opening of 35 μm (taken out from a 5 mL polystyrene round tube with a cell strainer cap and used) between a holding syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G).

Next, HW-40EC/150-250 μm prepared in Preparation Example 1 was replaced with MACS buffer solution, and after leaving for 12 hours or longer, the particulate carriers were adjusted so that the sedimentation volume of the particulate carriers was 50%. A 50% suspension was prepared, 20.0 mL of the suspension was added to the prepared column, and the column was filled with the particulate carrier (packed volume of the particulate carrier: 10.0 mL).

(2) Culture of Ramos cells, RD cells, and human mesenchymal stem cells and preparation of cell suspension ), (3) and (5).

(3) Separation of Ramos cells using a particulate carrier-filled column The cell suspension prepared by the above method was mixed and diluted with MACS buffer to obtain a cell suspension volume of 0.5 mL and the number of added cells. was prepared to have 1.0 × 10^7 Ramos cells, 6.8 × 10^5 RD cells, and 9.1 × 10^5 human mesenchymal stem cells, and 10 mL of HW- A column filled with 40EC/150-212 μm was placed vertically and added from the top of the column. Subsequently, 4.0 mL of MACS buffer was added from the top of the column, and a total of 4.5 mL of effluent cell fluid was recovered from the 22G syringe needle at the bottom of the column (hereinafter referred to as effluent cell fluid). Next, the particulate carriers are removed from the column and repeatedly washed with 4 mL of MACS buffer containing 20 mM EDTA on a cell strainer by pipetting to remove the cells trapped between the particulate carriers. Then, 4 mL of cell fluid was collected (hereinafter referred to as washed cell fluid).

(4) Measurement of cell recovery rate in effluent cell sap and washed cell sap, after dilution by adding MACS buffer to the effluent cell sap and washed cell sap obtained by the above operation, 2 mL of each cell strainer cap It was fractionated into a 5 mL polystyrene round tube (manufactured by BD Japan). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a cell viability determination reagent, the number of cells was measured using a cell sorter BD FACSAria (manufactured by BD Japan). gone. Ramos cells, RD cells, and human mesenchymal stem cells contained in each cell solution were discriminated using dot plots that were double-developed using the fluorescence intensity of FITC, PE, APC, etc. as parameters. It was calculated by proportional calculation based on the number of particles of the internal standard beads (this number of cells is defined as the number of outflow cells and the number of washed cells, respectively). The recovery rate of each cell in the effluent cell sap and the washed cell sap was calculated by dividing the number of effluent cells and the number of washed cells by the number of each cell applied to the column.

Table 2, FIG. 4 and FIG. 5 show the results of calculating the recovery rate of each cell in the effluent cell sap and the washed cell sap. The cell recovery rate in the effluent cell liquid was 80.3% for Ramos cells, 9.0% for RD cells, and 2.3% for human mesenchymal stem cells, and the cell recovery rate in the washed cell liquid was 13.1% for Ramos cells. , RD cells 81.5%, and human mesenchymal stem cells 89.9%.

From this result, when a mixed cell sap of Ramos cells, RD cells, and human mesenchymal stem cells was passed through a column filled with 10 mL of a particulate carrier, the effluent cell sap, which is the cells that passed through, was RD cells and human cells. It was found that the recovery rate of mesenchymal stem cells was low and the recovery rate of Ramos cells was high. Conversely, in the washed cell solution, which is a cell solution obtained by collecting the cells trapped between the particulate carriers by pipetting, the recovery rate of RD cells and human mesenchymal stem cells is high, and the recovery rate of Ramos cells is low. I found out. That is, when a large amount of 10 mL of particulate carriers is used, both human mesenchymal stem cells with an average size of 35 μm and RD cells with an average size of 15 μm are sandwiched between the particulate carriers, resulting in only Ramos cells with an average size of 8 μm. It was also shown that a mixture of RD cells and human mesenchymal stem cells can be separated by washing and recovering the cells from the particulate carrier.

Furthermore, in Example 1, a maximum of 1.7×10̂5 human mesenchymal stem cells and 6.7×10̂4 RD cells per 1 mL of particulate carriers were captured between the particulate carriers. Based on this result, 9.1 × 10^5 human mesenchymal stem cells and 6.8 × 10^5 RD cells, the number of cells applied in Example 2 It was estimated that the amount of particulate carrier required to capture 100% of each was 5.4 mL for human mesenchymal stem cells and 10.1 mL for RD cells. That is, in Example 2, compared to the amount of the particulate carrier used in Example 1, which was 2 mL, 10 mL of the particulate carrier was used, so that both the human mesenchymal stem cells and the RD cells were sufficiently captured between the particulate carriers. , it was thought that only Ramos cells could be separated.

Therefore, by appropriately adjusting the amount of the particulate carrier used according to the number of cells, from a mixture of Ramos cells with an average size of 8 μm, RD cells with an average size of 15 μm, and human mesenchymal stem cells with an average size of 35 μm, in Example 1, between humans It was demonstrated that leaf stem cells alone and a mixture of Ramos cells and RD cells, and in Example 2, Ramos cells alone and a mixture of RD cells and mesenchymal stem cells can be separated. In addition, from the results of both, it was found that a mixture of Ramos cells, RD cells, and human mesenchymal stem cells was first passed through a column filled with a small amount of particulate carriers capable of sufficiently capturing human mesenchymal stem cells. A mixture of Ramos cells and RD cells can be obtained as a cell sap that has passed through the gaps between the particulate carriers, and then the cell sap containing human mesenchymal stem cells can be recovered by washing the particulate carriers, and the effluent cell sap can be obtained. Then, the mixture of Ramos cells and RD cells collected as RD cells is passed through a column filled with a larger amount of particulate carriers that can sufficiently trap RD cells, thereby removing Ramos cells from the gaps between the particulate carriers. Then, by washing and collecting the cells trapped between the particulate carriers, it is possible to collect the cell liquid containing the RD cells. , it was thought possible to separate three types of human mesenchymal stem cells with different sizes.

Comparative Example 1 Separation of Cells of Different Sizes from a Mixture of Ramos Cells, RD Cells, and Human Mesenchymal Stem Cells Using a Commercially Available Membrane Filter Cells were prepared in the same manner as in Example 1. As in Example 1, a mixed cell suspension containing 1.0 × 10^7 Ramos cells, 6.8 × 10^5 RD cells, and 9.1 × 10^5 human mesenchymal stem cells 500 μL of the liquid was prepared, and a commercially available mesh size of 3 μm (3 μm membrane strainer manufactured by Pullreselect Life Science), 8 μm (8 μm membrane strainer manufactured by Pullreselect Life Science), and 20 μm (20 μm pull restrainer manufactured by Pullreselect Life Science). A total of 2.5 mL of the filtered cell sap was collected by passing the liquid through each filter and then washing the filter with 2.0 mL of MACS buffer solution from the top of the filter (hereinafter referred to as the effluent cell sap). bottom. Next, the filter was placed upside down in another collection tube, and then reverse washed with 4.0 mL of MACS buffer containing 20 mM EDTA to collect the cell fluid trapped in the filter (hereinafter referred to as washed cell fluid ).

Table 2, FIG. 4 and FIG. 5 show the results of calculating the recovery rate of each cell in the effluent cell sap and the washed cell sap. From this result, it was found that in the case of the 3 μm filter, the recovery rate of each cell sap in the effluent cell sap and the washed cell sap was low. In addition, the effluent cell sap from the 8 μm filter, and the effluent cell sap and the washed cell sap from the 20 μm filter all had the same recovery rate of each cell, indicating that the cells could not be separated by size.

Example 3 Separation of both cells from mixture of Ramos cells and human mesenchymal stem cells using HW-40EC/150-250 μm packed column (1) Preparation of pipette column filled with particulate carrier 10.0 mL disposable A pipette column was prepared by attaching a nylon mesh filter with an opening of 35 μm (taken out from a 5 mL polystyrene round tube with a cell strainer cap and used) between a pipette (357530 manufactured by Falcon) and an injection needle (22G manufactured by Terumo).

Next, HW-40EC/150-250 μm prepared in Preparation Example 1 was replaced with MACS buffer solution, and after leaving for 12 hours or longer, the particulate carriers were adjusted so that the sedimentation volume of the particulate carriers was 50%. A 50% suspension was prepared, 20.0 mL was added to the prepared column, and the particulate carrier was packed in the pipette column (packed volume of particulate carrier: 10.0 mL).

(2) Culturing of Ramos cells and human mesenchymal stem cells and preparation of cell suspension I did it in a similar way.
(3) Separation of Ramos cells and human mesenchymal stem cells using a particulate carrier-filled column The cell suspension prepared by the above method is mixed and diluted with MACS buffer to obtain a cell suspension volume of 1 Column filled with HW-40EC/150-250 μm, prepared so that the number of added cells was 2.110×^7 for Ramos cells and 1.8×10^6 for human mesenchymal stem cells. was added from the top of the column while standing vertically. Subsequently, 1.0 mL of MACS buffer was added from the top of the column, and a total of 2.0 mL of effluent cell fluid was recovered from the 22G syringe needle at the bottom of the column (hereinafter referred to as effluent cell fluid Fr.1). Next, 2.0 mL of MACS buffer was added from the top of the column, and 2.0 mL of effluent cell fluid was collected from the 22G syringe needle at the bottom of the column (hereinafter referred to as effluent cell fluid Fr.2). This operation was repeated eight more times, and each 2.0 mL of effluent cell effluent Fr. 3-10 were obtained similarly. Next, the column was cut from the upper part into 5 parts with a particulate carrier capacity of 2 mL according to the scale, and 2 mL of the particulate carrier was taken out from each part and washed with 8 mL of MACS buffer containing 20 mM EDTA on a cell strainer. By doing so, the cells trapped between the particulate carriers were discharged, and 8 mL of the washed cell sap was recovered (hereinafter, in order from the top of the column to the bottom of the column, each 2 mL of the washed cell sap collected from each 2 mL of the particulate carrier was collected. listed as UP.1 to UP.5).

(4) Measurement of cell recovery rate in effluent cell sap and washed cell sap The effluent cell sap Fr. 1 to 10 and the washed cell suspension UP. After adding MACS buffer to 1 to 5 to dilute, 2 mL of each was dispensed into a 5 mL polystyrene round tube with a cell strainer cap (manufactured by BD Japan). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a cell viability determination reagent, the number of cells was measured using a cell sorter BD FACSAria (manufactured by BD Japan). gone. Ramos cells and human mesenchymal stem cells contained in each cell solution were discriminated using dot plots that were double-developed using the fluorescence intensity of FITC, APC, etc. as parameters. Based on the number, it was calculated by proportional calculation (this cell number is defined as the outflow cell number and the washed cell number, respectively). The recovery rate of each cell in the effluent cell sap and the washed cell sap was calculated by dividing the number of effluent cells and the number of washed cells by the number of each cell applied to the column.

Effluent cell fluid Fr. 1 to 10 and the washed cell suspension UP. Table 3, FIG. 6 and FIG. 7 show the results of calculating the recovery rate of each cell in 1 to 5. In addition, the applied cells and the effluent cell fluid Fr. 1 to 10 and the washed cell suspension UP. Table 4, FIG. 8 and FIG. 9 show the results of calculating the cell ratios in 1 to 5. The total cell recovery rate in the effluent cell solution was 76.5% for Ramos cells and 5.0% for human mesenchymal stem cells, and the total cell recovery rate for the washed cell solution was 11.4% for Ramos cells and between humans. Leaf stem cells were 80.5%, and it was found that the recovery rate of Ramos cells was high in the effluent cell sap, and the recovery rate of human mesenchymal stem cells was high in the washed cell sap.

In addition, the efflux rate of Ramos cells among the efflux cells was determined by Fr. 2 at 62.1%, Fr. 3, and the cell number ratio of Ramos cells:human mesenchymal stem cells was 92.0%:8.0% in the applied cells, whereas in Fr. 99.8% at 2:0.2%, Fr. 3 is 98.2%:1.8%, these Fr. It was found that a cell sap with a high proportion of Ramos cells could be recovered. On the other hand, the recovery rate of Ramos cells in washed cells is as low as 1.8-3.2%, and the recovery rate of human mesenchymal stem cells is UP. 1 at 29.2%, UP. 2 at 23.7%, UP. 3 at 11.6%, UP. 4 at 12.2%, UP. 5 is 3.7%, and the UP. 1, UP. In 2, the recovery rate was particularly high, and it was found that the number of captured human mesenchymal stem cells decreased toward the bottom of the column. In addition, the cell number ratio of Ramos cells:human mesenchymal stem cells was 92.0%:8.0% in the applied cells, whereas UP. 1: 46.0%: 54.0%, UP. 2: 53.4%: 46.6%, UP. 3: 63.7%: 36.3%, UP. 75.2% at 4: 24.8%, UP. 5, 84.7%: 15.3%, the cell ratio of human mesenchymal stem cells is higher in the upper part of the column, and the cell ratio of Ramos cells is higher in the lower part of the column. UP. In all of 1 to 5, it was revealed that the cells were recovered at a higher cell ratio of human mesenchymal stem cells than the applied cells.

Based on this result, a mixed cell solution of Ramos cells having an average size of 8 μm and human mesenchymal stem cells having an average size of 35 μm was passed through a column filled with particulate carriers, and the cells that passed through the gaps between the particulate carriers were treated with Fr. As a result, the cell fluid Fr. is obtained. In addition, by dividing and collecting the particulate carrier from the upper part of the column and obtaining the cells trapped between the particulate carriers from each fraction, the cell sap collected from the particulate carrier at the upper part of the column is more likely to be interhuman. It was proved that a cell sap with a high cell ratio of leaf stem cells can be obtained. Furthermore, from the results of Example 2, it is estimated that the necessary amount of particulate carrier required to capture 1.8×10^6 human mesenchymal stem cells applied in Example 3 is 10.8 mL. However, in this example, when 10.0 mL of the particulate carrier was used to capture and wash the human mesenchymal stem cells between the particulate carriers, the recovery rate of the human mesenchymal stem cells in the washing and recovery solution was The total was 80.5%, indicating that most of the applied human mesenchymal cells were captured, supporting this result.

Claims (2)

A step of contacting a water-insoluble particulate carrier packed in a column with a cell mixture containing two or more types of cells, a step of recovering cells that have passed through the gaps of the particulate carrier packed in the column, and including a step of recovering the captured cells by a washing operation, wherein the water-insoluble particulate carrier has a particle size of 150 to 250 μm, and the cell mixture containing two or more types of cells contains cells with a size of less than 10 μm. A method for isolating cells, characterized by containing at least one type of cell and at least one type of cell having a size of 10 μm or more. The cell mixture containing two or more types of cells contains at least one type of cell with a size of less than 10 μm, at least one type of cell with a size of 10 μm or more and less than 25 μm, and at least one type of cell with a size of 25 μm or more. 2. The method for separating cells according to claim 1, comprising the above.

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