JP7480485B2 - Cell Isolation Methods - Google Patents

Description

The present invention relates to a cell separation method.

In fields such as regenerative medicine and tailored treatments that require cells derived from specimens, there is a need for technology to efficiently separate, detect, and culture target cells. To date, the mainstream methods for separating target cells have been immunoaffinity-related technologies, such as flow cytometry, in which fluorescently labeled antibodies are attached to the surface proteins of target cells and then separated, and magnetic cell separation, in which magnetically labeled antibodies are attached to the surface proteins of target cells and then separated using an external magnetic field. However, there are issues with the high running costs, as the reagents used, such as antibodies, are expensive. In addition, immunoaffinity technology has the issue that it can damage cells by attaching antibodies to the surface proteins of cells. For the future development of fields such as regenerative medicine, there is a need for cell separation technology that can separate target cells inexpensively and minimally invasively.

The object of the present invention is to provide a cell separation method that can separate target cells in a minimally invasive manner.

In light of the above, the inventors conducted extensive research and completed the present invention.

That is, one aspect of the present invention is a cell separation method comprising the steps of seeding two or more types of cells on a cell culture vessel coated with a block copolymer containing a segment exhibiting a lower critical solution temperature (LCST), removing cells that do not adhere to the block copolymer from the cell culture vessel, and cooling the cell culture vessel to a temperature below the LCST to recover the cells that have adhered to the block copolymer.

This invention allows for inexpensive, minimally invasive isolation of target cells.

One aspect of the present invention is described in detail below, but the present invention is not limited to the following content. The present invention can be modified as appropriate within the scope of its intent.

LCST stands for Lower Critical Solution Temperature (LCST), below which the polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and either becomes cloudy or precipitates, resulting in phase separation.

A block copolymer is a polymer consisting of two or more types of repeating units, in which polymer chains consisting of the same type of repeating units are bonded together in a single chain.

There are no particular limitations on the block copolymer containing a segment exhibiting a lower critical solution temperature (LCST), but it is preferable that the block copolymer has a structure containing an acryloyl group or a methacryloyl group, since this facilitates polymerization. The repeating unit of the segment exhibiting a lower critical solution temperature (LCST) (hereinafter sometimes referred to as the "LCST repeating unit") and its LCST with respect to water are, for example, N-ethylacrylamide (LCST = 72°C), N-cyclopropylacrylamide (LCST = 46°C), N-isopropylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfuryl acrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N,N-diethylacrylamide (LCST = 3 2°C), N-cyclopropyl methacrylamide (LCST = 59°C), N-isopropyl methacrylamide (LCST = 44°C), N-n-propyl methacrylamide (LCST = 28°C), N-tetrahydrofurfuryl methacrylamide (LCST = 35°C), N-methyl-N-ethylacrylamide (LCST = 56°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), N-methyl-N-n-propylacrylamide (LCST = 20°C), or N,N-dimethylaminoethyl methacrylate (LCST = 47°C) can be exemplified. The upper limit of the LCST is preferably 42°C, more preferably 40°C, because cell culture temperatures generally cause damage to cells due to protein denaturation at high temperatures. The lower limit of the LCST is preferably 10°C, more preferably 20°C, in order to avoid a decrease in cell activity at low temperatures. As the LCST repeating unit, only one type of repeating unit may be used, or two or more types may be used in combination. Furthermore, if the block copolymer has the property that the degree of hydrophilicity/hydrophobicity changes with temperature (hereinafter, sometimes referred to as "temperature responsiveness"), it may contain a different repeat unit in addition to the LCST repeat unit.

The number of segments constituting the block copolymer may be 2 or more, and may be 3 or more. The composition ratio of the LCST repeating unit in the block copolymer is preferably 5 to 95 mol%, more preferably 30 to 90 mol%, and even more preferably 40 to 80 mol%. In order to improve temperature responsiveness and coverage of cell culture substrates, the segments constituting the block copolymer preferably further contain, in addition to the segment exhibiting LCST, a hydrophilic polymer segment having no LCST in the range of 0°C to 50°C and an HLB value (Griffin method) in the range of 9 to 20, and a hydrophobic polymer segment having no LCST in the range of 0°C to 50°C and an HLB value (Griffin method) in the range of 0 to less than 9. For these segments, the segments described in JP 2018-087316 A can be used.

There is no particular limitation on the method of coating the cell culture equipment with the block copolymer, but for example, the standard method described in JP 2018-087316 A can be used. The amount of coating of the segment exhibiting the LCST of the block copolymer is preferably 0.1 μg/cm ² or more and 20.0 μg/cm ² or less, and more preferably 0.2 μg/cm ² or more and 10.0 μg/cm ² or less. The amount of coating can be measured according to a standard method, for example, by weighing using a balance. Examples of the shape of the cell culture equipment include a dish, a plate, and a flask.

In the cell culture equipment, the surface of the block copolymer may be further coated with an extracellular matrix. There are no particular limitations on the type of extracellular matrix, and for example, Matrigel containing collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, laminin, collagen IV, heparan sulfate proteoglycan, entactin/nidogen 1, 2, etc. as the main component may be used, or one or more of these may be used. Also, it may be a segment of these extracellular matrices.

The types of cells (hereinafter sometimes referred to as "cell types") are not particularly limited as long as they are two or more types, and may be three or more types. There is no particular limit to the combination of cell types used, but fibroblasts and/or epithelial cells are preferably used in the present invention. In addition, the present invention can be used for a state in which different types of cells, such as blood cells and cells that construct the nervous system or visceral system, are mixed, or for a state in which cells with differentiation ability, such as mesenchymal stem cells, are in different differentiation states, but adhesive cells are preferable. In addition, the cells may be non-human cells, such as cells derived from mammals such as mice, hamsters, rabbits, and monkeys, or cells derived from insects. Furthermore, the cells may be allogeneic cells with different passage numbers.

There are no particular limitations on the medium, and a medium in which serum such as fetal bovine serum or antibiotics have been added to a basal medium such as Eagle's minimum essential medium (MEM) may be used, or a serum-free medium to which an extracellular matrix has been added may be used. There are no particular limitations on the serum concentration or extracellular matrix concentration in the medium, and a concentration suitable for cell separation for each cell type may be used. For example, the concentration of fetal bovine serum is preferably 1 to 30 vol%, and more preferably 5 to 20 vol%.

The cell separation method of the present invention includes a step of seeding two or more types of cells onto a cell culture substrate coated with the above-mentioned block copolymer (hereinafter, sometimes referred to as the "seeding step"); a step of removing cells that are not adhered to the block copolymer from the cell culture substrate (hereinafter, sometimes referred to as the "removal step"); and a step of cooling the cell culture substrate to below the LCST and recovering the cells that have adhered to the block copolymer (hereinafter, sometimes referred to as the "recovery step").

In the seeding process, the amount of block copolymer coated, the serum concentration in the medium, and the culture time are adjusted, either alone or in combination, so that some cell types do not adhere or have a low adhesion rate. However, the culture time is preferably 30 minutes to 12 hours, and more preferably 1 hour to 3 hours.

In the removal step, the non-adherent cells may be collected together with the medium using, for example, a pipetter. After removing the non-adherent cells, the cell culture equipment may be washed by adding PBS or medium (which may contain serum) at a temperature between the LCST and 42°C.

In the recovery step, the cells remaining in the block copolymer are recovered. There is no particular limitation on the cooling method, and the cells may be cooled in a cold place or by replacing the medium with a cooled medium. There is no particular limitation on the cooling temperature as long as it is below the LCST, but in order to promote recovery of the remaining cells, a temperature that is at least 2°C lower than the LCST is preferred, and a temperature that is at least 4°C lower than the LCST is more preferred. The cooling time is preferably less than 40 minutes, more preferably less than 20 minutes, and even more preferably less than 10 minutes. After cooling, the cells detached from the cell culture equipment can be recovered, but if detachment from the cell culture equipment is incomplete, physical impact such as pipetting, tapping, or shaking may be applied.

The cells recovered in the recovery step can be further separated and purified by repeating the cell separation method of the present invention. When performing the second cell separation, it is preferable to use a cell culture vessel in which the amount of the segment showing the LCST of the block copolymer coated is equal to or greater than that in the first cell separation. When performing the third cell separation, it is preferable to use the amount of the segment showing the LCST of the block copolymer coated is equal to or greater than that in the second cell separation. For example, when culturing three types of cells a, b, and c (adhesiveness to block copolymer c>b>a), the non-adhesive cell a can be separated in the first operation, and a mixture of b and c can be recovered by cooling treatment. Furthermore, by using a cell culture vessel in which the amount of the segment showing the LCST of the block copolymer coated is increased in the second operation, the non-adhesive cell b can be separated, and a mixture of c can be recovered by cooling treatment.

The following examples of the present invention are provided, but the present invention is not limited to these examples. Unless otherwise noted, commercially available reagents were used.

<Synthesis of block copolymer>
To a 100 mL two-neck flask, 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA, HLB value = 13.5) was added, and 31.8 mg (100 μmol) of cyanomethyl dodecyl carbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added. After replacing with argon gas, the mixture was heated and stirred at 62°C for 24 hours.
After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA, HLB value = 6.9) was added to the above, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of 1,4-dioxane were further added. After replacing with argon gas, the mixture was heated and stirred at 62°C for 48 hours.
After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST = 32°C, HLB value = 7.6) was added to the above, and 1.6 mg (10 µmol) of azobisisobutyronitrile and 35 mL of 1,4-dioxane were further added. After replacing with argon gas, the mixture was heated and stirred at 62°C for 48 hours.
After the third heating and stirring, the reaction liquid was purified by reprecipitation with water and dried under reduced pressure to obtain a yellow solid.
The obtained yellow solid was dissolved in chloroform, and the chloroform phase was collected using a separatory funnel. The collected chloroform phase was concentrated using an evaporator and purified by reprecipitation with hexane. The precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of a block copolymer poly(MEA-BA-IPAAm). The composition ratio of the obtained block copolymer was MEA/BA/IPAAm=5/26/69 (mol%), the number average molecular weight Mn was 85,000, and the molecular weight distribution Mw/Mn was 1.78.

<Composition of block copolymer>
The amount was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectrum analysis using a nuclear magnetic resonance measurement device (manufactured by JEOL Ltd., trade name JNM-ECZ400S/L1).
<Molecular weight and molecular weight distribution of block copolymer>
Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw/Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel SuperAWM-H columns were used. The column temperature was set to 40°C, and the eluent was 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg/mL and measured. For the molecular weight calibration curve, polymethylmethacrylate (Sigma-Aldrich) with a known molecular weight was used.
<Coating amount of segment showing LCST of cell cultureware>
The amount of IPAAm segments (IPAAm) showing LCST on the cell cultureware coated with the block copolymer was analyzed by total reflection Fourier transform infrared spectroscopy (ATR/FT-IR). A calibration curve prepared from samples with known amounts of coating was used for the analysis, and the amount of LCST segments per unit area (μg/ ^cm2 ) was evaluated.

<Counting the number of seeded cells>
The number of cells to be seeded was measured using a hemocytometer. All cells in ¹ mm2 sections at the four corners of the hemocytometer were counted, and the total cell number was calculated by (average number of all live cells in the section) x dilution factor x medium volume (mL) x 10,000.
<Cell type ratio analysis>
The cells were stained in advance using CellTracker ^™ (manufactured by Thermofisher Scientific) with different dyes for each type of cell. The mixed suspension of stained cells was seeded on a cell culture vessel, and after culturing for a predetermined time under conditions of 37°C and 5% _CO2 , non-adherent cells were removed together with the medium, and the remaining cells on the culture vessel were imaged using a confocal quantitative image cytometer CQ1 (manufactured by Yokogawa Electric Corporation) with a 10x objective lens. Image analysis was performed using ImageJ. As an example, the area of each cell was calculated using a hue of 124 to 255 for cells stained with CellTracker ^™ Blue CMAC, a hue of 60 to 100 for cells stained with CellTracker ^™ Green CMFDA, and a hue of 0 to 30 for cells stained with CellTracker ^™ Orange CMTMR, and the proportion of cell types was analyzed by dividing the area by the stained area per cell.

Example 1
A 2.0 wt% solution of the block copolymer in 2-methoxyethanol was prepared. 100 μL of the block copolymer/2-methoxyethanol solution was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin-coated using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a cell culture substrate (1) coated with the block copolymer. The coating amount of the IPAAm segment on the substrate culture surface was 5.8 μg/ ^cm2 .
Using the prepared cell cultureware (1), a cell suspension containing ^1x105 cells each of TIG3-20 cells (stained with CellTracker ^™ blue CMAC), A549 cells (stained with CellTracker ^™ Green CMFDA), and PC9 cells (CellTracker ^™ Orange CMTMR) was seeded. The medium used was D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and 5% _CO2 . The cells that did not adhere to the block copolymer were removed together with the medium. The percentage of remaining cells after removing non-adherent cells is shown in Table 1. The medium cooled to 4°C was added to the cell cultureware containing the remaining cells, and the cells were detached by pipetting after leaving it for 10 minutes, and a cell suspension containing 75.7% PC9 cells was recovered.

Example 2
A 4.0 wt % 2-methoxyethanol solution of the block copolymer was used to prepare a cell culture substrate (2) coated with the block copolymer by the method described in Example 1. The coating amount of the IPAAm segment on the substrate culture surface was 13.3 μg/ ^cm2 .
Culturing was performed in the same manner as in Example 1, except that the cell culture equipment (2) was used. The percentage of remaining cells after removing non-adherent cells is shown in Table 1. The medium cooled to 4°C was added to the cell culture equipment containing the remaining cells, and after leaving it for 10 minutes, the cells were detached by pipetting, and a cell suspension containing 89.4% PC9 cells was recovered.

Comparative Example 1
Except for using an IWAKI tissue culture dish (φ6 cm) as the cell culture equipment, the culture was performed in the same manner as in Example 1. The remaining cell rate after removing non-adherent cells is shown in Table 1. The medium cooled to 4°C was added to the cell culture equipment containing the remaining cells, and pipetting was performed after leaving it for 10 minutes, but the cells did not detach.

Comparative Example 2
Cultivation was performed in the same manner as in Example 1, except that ^UpCell® (φ6 cm) manufactured by CellSeed was used as the cell cultureware. The remaining cell rate after removing non-adherent cells is shown in Table 1. The cells were detached by adding medium cooled to 4°C to the cell cultureware containing the remaining cells and leaving it for 10 minutes and then pipetting, but the collected cell suspension was not cell-separated.

Example 3
Using the cell culture equipment (1), culture was performed in the same manner as in Example 1. The percentage of remaining cells after removing non-adherent cells is shown in Table 2. A medium cooled to 4°C was added to the cell culture equipment containing the remaining cells, and after leaving it for 10 minutes, pipetting was performed to recover a cell suspension containing 76.3% PC9 cells.
The cell suspension containing a high ratio of collected PC9 cells was seeded on cell culture equipment (2). The medium used was D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and 5% _CO2 . Cells that did not adhere to the block copolymer were removed together with the medium.
The remaining cell ratio after removal of non-adherent cells is shown in Table 2. A cell suspension containing 95.0% PC9 cells was recovered by adding medium cooled to 4°C to the cell culture equipment containing the remaining cells and leaving it for 10 minutes, followed by pipetting.

Example 4
A 0.4 wt% solution of the block copolymer in 2-methoxyethanol was prepared. 100 μL of the block copolymer/2-methoxyethanol solution was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin-coated using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a cell culture substrate (3) coated with the block copolymer. The coating amount of the IPAAm segment on the substrate culture surface was 0.5 μg/ ^cm2 .
Using the prepared cell cultureware (3), a cell suspension containing 1 x ¹⁰⁵ human bone marrow-derived mesenchymal stem cells (stained with CellTracker ^™ Orange CMTMR) and RAMOS cells (stained with CellTracker ^™ Green CMFDA) was seeded. The medium used was D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and 5% _CO2 . The cells that did not adhere to the block copolymer were removed together with the medium. The percentage of remaining cells after removing non-adherent cells is shown in Table 3. The medium cooled to 4°C was added to the cell cultureware containing the remaining cells, and the cells were detached by pipetting after leaving it for 10 minutes, and a cell suspension containing 99.0% human bone marrow-derived mesenchymal stem cells was recovered.

Example 5
A 1.5 wt% solution of the block copolymer in 2-methoxyethanol was prepared. 100 μL of the block copolymer/2-methoxyethanol solution was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin-coated using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a cell culture substrate (4) coated with the block copolymer. The coating amount of the IPAAm segment on the substrate culture surface was 4.0 μg/ ^cm2 .
Using the prepared cell cultureware (4), a cell suspension containing 1 x 105 cells of human dental pulp-derived mesenchymal stem cells (stained with CellTracker ^™ Orange CMTMR) and human adipose-derived mesenchymal stem cells (stained with CellTracker ^™ Green CFDA) was seeded. The medium used was a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and ⁵ % _CO2 . The cells that did not adhere to the block copolymer were removed together with the medium. The percentage of remaining cells after removing non-adherent cells is shown in Table 4. The medium cooled to 4°C was added to the cell cultureware containing the remaining cells, and the cells were peeled off by pipetting after leaving it for 10 minutes, and a cell suspension containing 85.2% human adipose-derived mesenchymal stem cells was recovered.

Example 6
A 2.5 wt% solution of the block copolymer in 2-methoxyethanol was prepared. 100 μL of the block copolymer/2-methoxyethanol solution was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin-coated using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a cell culture substrate (5) coated with the block copolymer. The coating amount of the IPAAm segment on the substrate culture surface was 6.9 μg/ ^cm2 .
Using the prepared cell cultureware (5), a cell suspension containing 1 x ¹⁰⁵ cells each of human hepatoma-derived cells HepG2 (stained with CellTracker ^™ ORANGE CMTMR) and human colon adenocarcinoma cells HCT116 (stained with CellTracker ^™ Green CMFDA) was seeded. The medium used was D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and 5% _CO2 . The cells that did not adhere to the block copolymer were removed together with the medium. The percentage of remaining cells after removing non-adherent cells is shown in Table 5. The medium cooled to 4°C was added to the cell cultureware containing the remaining cells, and the cells were detached by pipetting after leaving it for 10 minutes, and a cell suspension containing 98.1% human colon adenocarcinoma cells HCT116 was recovered.

Example 7
A 1.0 wt% solution of the block copolymer in 2-methoxyethanol was prepared. 100 μL of the block copolymer/2-methoxyethanol solution was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin-coated using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a cell culture substrate (6) coated with the block copolymer. The coating amount of the IPAAm segment on the substrate culture surface was 2.6 μg/ ^cm2 .
Using the prepared cell cultureware (6), a cell suspension containing 1 x ¹⁰⁵ TIG3-20 cells (stained with CellTracker ^™ Orange CMTMR) and TIG3-60 cells (stained with CellTracker ^™ Green CMFDA) was seeded. The medium used was a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics, and the cells were co-cultured for 1 hour at 37°C and 5% _CO2 . The cells that did not adhere to the block copolymer were removed together with the medium. The percentage of remaining cells after removing non-adherent cells is shown in Table 6. The medium cooled to 4°C was added to the cell cultureware containing the remaining cells, and the cells were detached by pipetting after leaving it for 10 minutes, and a cell suspension containing 83.3% TIG3-20 cells was recovered.

Claims (3)

A step of seeding two or more types of cells onto a cell culture vessel coated with only a block copolymer containing repeating units of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide ;
removing cells that are not adhered to the block copolymer from the cell culture substrate;
a step of cooling the cell culture substrate to a lower critical solution temperature ( LCST ) or lower to recover the cells adhered to the block copolymer;
A cell separation method comprising:
The cells to be seeded are TIG3-20 cells, TIG3-60 cells, A549 cells, PC9 cells, human bone marrow-derived mesenchymal stem cells, human dental pulp-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human hepatoma-derived cells, and/or human colon adenocarcinoma cells ,
The coating amount of the N-isopropylacrylamide is 0.1 to 20.0 μg/ ^cm2 . 2. The cell separation method according to claim 1 , wherein the shape of the cell culture substrate is a dish, a plate or a flask. A cell separation method, comprising the steps of: recovering cells adhered to the block copolymer; and repeating the method according to claim 1 or 2 , using the cell culture vessel coated with the cells in an amount of N-isopropylacrylamide equal to or greater than that of the previous time.