JP7432808B2 - Cell differentiation method - Google Patents

Description

The present invention relates to a method for cell differentiation.

Stem cells are expected to be applied in the field of regenerative medicine because they have self-renewal ability that allows them to multiply themselves almost without limit, and multipotency that allows them to differentiate into various cells. In order to apply stem cells to regenerative medicine such as transplantation, it is generally necessary to expand and culture stem cells existing in the body or stem cells produced outside the body in a cell culture vessel, etc., and differentiate into the target cells on the culture vessel. be. Differentiation of stem cells on a culture vessel has conventionally been carried out by culturing in a differentiation-inducing medium containing a differentiation-inducing factor suitable for differentiation into target cells (see, for example, Patent Document 1). However, the number of days required for differentiation is long when differentiation is induced using a differentiation-inducing medium alone, making it difficult to obtain a sufficient amount of targeted differentiated cells necessary for regenerative medicine.

Japanese Patent Application Publication No. 2000-287680

An object of the present invention is to provide a cell differentiation method with excellent cell differentiation efficiency.

In view of the above points, the present inventors have completed the present invention as a result of extensive research.
That is, one aspect of the present invention is
A step of culturing the stem cells on a culture device exhibiting a lower critical melting temperature (LCST), a step of cooling the culture device below the LCST to detach and collect the stem cells, and a step of culturing the stem cells in a differentiation-inducing medium. A method for differentiating stem cells, which comprises the step of culturing using a .

The present invention can promote differentiation compared to conventional stem cell differentiation methods.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail, but the present invention is not intended to be limited to the following contents. The present invention can be implemented with appropriate modifications within the scope of its spirit.

Stem cells are not particularly limited as long as they have the self-renewal ability to proliferate in an undifferentiated state and the ability to differentiate into other types of cells, and include induced pluripotent (iPS) cells and embryonic stem (ES) cells. , embryonic germ (EG) cells, embryonic cancer (EC) cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germ stem cells, or progenitor cells of each tissue, etc. Mesenchymal stem cells are particularly preferably used.

Mesenchymal stem cells refer to a population of stem cells and their precursor cells that can differentiate into all or some mesenchymal cells such as chondrocytes, osteoblasts, and adipocytes. There are no particular limitations on the origin of mesenchymal stem cells, but examples include those derived from tissues such as bone marrow, adipose, dental pulp, umbilical cord blood, placenta, and synovium, and pluripotent stem cells such as ES cells and iPS cells, especially those derived from bone marrow. Mesenchymal stem cells are preferred. Furthermore, there is no particular limitation on the species from which mesenchymal stem cells are derived, and they may be human, animal, or plant.

Induction of differentiation of stem cells refers to starting differentiation of stem cells from an undifferentiated state, and includes changing the undifferentiated state to a state in which differentiated cell-specific differentiation markers that are not detected in the undifferentiated state can be detected. The above-mentioned differentiation markers and their detection methods may be selected as appropriate, but for example, cell surface markers such as sugars and proteins present in the cell membrane, intracellular markers such as mRNA, proteins, and enzymes present within the cell, and markers outside the cell. Extracellular markers such as secreted peptides, proteins, enzymes, and compounds are listed as differentiation markers, and methods using labeled antibodies (staining method, flow cytometry, ELISA, etc.), staining methods using enzyme activity, RT PCR method etc. are mentioned as a detection method. For example, differentiation of mesenchymal stem cells into chondrocytes can be confirmed by staining the extracellular matrix secreted by the chondrocytes formed by differentiation induction using Alcian blue.

Lower Critical Solution Temperature (LCST) is a polymer that dissolves in water and becomes a clear solution at a temperature lower than this temperature, but becomes insolubilized and becomes cloudy at a temperature higher than this temperature. This is the temperature at which precipitation occurs and phase separation occurs. In this specification, exhibiting LCST is sometimes referred to as having temperature responsiveness. The LCST is preferably in the range of 20°C to 40°C, more preferably in the range of 30°C to 40°C. Repeating units showing LCST and their LCST for water are, for example, 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 = 32°C), N-n-propylmethacrylamide (LCST = 28°C), N-tetrahydrofurfurylmethacrylamide (LCST Examples include N-methyl-N-isopropylacrylamide (LCST=23°C), and N-methyl-Nn-propylacrylamide (LCST=20°C). Although the temperature at which LCST is expressed varies depending on the concentration of the aqueous solution, N-isopropylacrylamide is preferable because the concentration dependence of LCST expression is low. The number of repeating units used in the culture equipment exhibiting LCST in the present invention may be one type or a combination of two or more types. Further, as long as it has temperature responsiveness, it may contain a different repeating unit in addition to the repeating unit showing LCST.

There are no particular limitations on the manufacturing method for the culture equipment; for example, repeating units exhibiting LCST may be polymerized by electron beam irradiation to coat the equipment surface, or a polymer containing repeating units exhibiting LCST may be dissolved in a solvent. An example of this method is to coat a membrane by applying a surface treatment agent to the device. The above-mentioned polymer is not particularly limited, but its synthesis method is described in "Radical Polymerization Handbook" published by NTS Co., Ltd., p. A method of copolymerization using the living radical polymerization technique described in 161-225 (2010) can be used. Cells cultured and proliferated using culture equipment generally need to be detached and recovered from the container surface by treatment with proteolytic enzymes such as trypsin, but by using culture equipment that exhibits LCST, they can be removed from the surrounding environment. The sol transition of the temperature-responsive polymer caused by a drop in temperature weakens the adhesive force on the surface of the device, making it possible to detach and collect cells without using proteolytic enzymes.

When using a polymer membrane containing repeating units exhibiting LCST, it is preferable that the component of the polymer constituting the membrane includes a repeating unit for fixing the membrane to equipment in addition to the repeating units exhibiting LCST. . Examples include styrene and its derivatives, 2-methoxyethyl acrylate, n-propyl acrylate, n-propyl methacrylate, n-butyl acrylate, n-butyl methacrylate, etc. Particularly preferred are block copolymers comprising repeating units and repeating units of N-isopropylacrylamide.

The shape of the culture equipment is not particularly limited, and examples thereof include dishes, flasks, microwell plates, culture bags, tubes, trays, culture vessels, and the like. The materials of these instruments are not particularly limited, and examples include glass, polypropylene, polystyrene, stainless steel, and combinations thereof.

The surface of the culture equipment may be coated with an extracellular matrix. The type of extracellular matrix is not particularly limited, and examples include collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, or laminin, collagen IV, heparan sulfate proteoglycan, entactin/nidogen. Matrigel containing 1, 2, etc. as main components may be used, and one type of these may be used alone, or two or more types may be used in combination. It may also be a segment of these extracellular matrices.

The differentiation method of the present invention is characterized by including the following steps.
A step of culturing stem cells on culture equipment exhibiting a lower critical melting temperature (LCST) (hereinafter referred to as (A) step).
A step of cooling the culture equipment below the LCST to detach and collect the stem cells (hereinafter referred to as (B) step).
A step of culturing the recovered stem cells using a differentiation-inducing medium (hereinafter referred to as step (C)).

1. (A) Regarding the process There are no particular limitations on the composition of the medium used, as long as the stem cells adhere and proliferate while maintaining their ability to differentiate into target cells. Also good. The type of basal medium is not particularly limited, and for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM/Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, RPMI1640, etc. can be used. can. The type of serum is not particularly limited, and examples include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum. However, FBS is commonly used due to its easy availability. The serum concentration in the medium is not particularly limited. Generally, it is often used at a concentration of 20 vol% or less from cost-effectiveness, but it may be used at a concentration of more than 20 vol%. Alternatively, it may be a serum-free medium that does not contain untreated or unpurified serum but contains purified blood-derived components or animal tissue-derived components (growth factors, etc.).
Cell culture is performed at a temperature higher than the LCST of the temperature-responsive polymer coated on the surface of the culture equipment, but when using human-derived cells, it should be performed near body temperature in order to obtain high culture efficiency. is preferred, more preferably carried out at a temperature range of 35 to 39°C, and even more preferably carried out at a temperature range of 36 to 38°C.
The culture density of stem cells is not particularly limited as long as the stem cells adhere and proliferate while maintaining the ability to differentiate into target cells, but in the case of mesenchymal stem cells, for example, 1.0 × 10 ¹ to 1.0 × 10 ⁵ cells/cm ² is preferable, and 1.0×10 ² to 1.0×10 ⁴ cells/cm ² is more preferable. Other culture conditions are not particularly limited, and culture may be performed under conditions commonly used in the art.

2. (B) About the process There are several methods for detaching and collecting stem cells from culture equipment: changing the ambient temperature to a temperature lower than the LCST of the culture equipment and then waiting for the cells to detach naturally; methods using tapping or shaking; A method of locally applying an impact to the cell, a method using a cell scraper, pipetting, etc. can be used. The temperature when cooling the culture equipment is preferably 1° C. or more lower than the LCST. Examples of methods for lowering the temperature include storing in a cold place and pouring cooled liquid after removing the warm medium. The cooled liquid is not particularly limited, and can be selected depending on the purpose, such as a culture solution, other medium solution, or isotonic solution. Moreover, the cooling time is preferably 5 minutes or more and 60 minutes or less.

3. (C) Regarding the process The culture equipment used is not particularly limited, and the surface of the culture equipment may or may not be coated with a temperature-responsive polymer.
The composition of the differentiation-inducing medium is not particularly limited, and in addition to the basal medium and serum, it consists of a differentiation-inducing factor that differentiates stem cells into target cells, and may also contain an antibiotic. The type of basal medium is not particularly limited, and a medium such as that used in step (A) described above can be used. Differentiation-inducing factors that differentiate cultured cells into target cells include fibroblast growth factor (FGF), transforming growth factor (TGF), transforming growth factor β (TGFβ), platelet-derived growth factor (PDFG), and epidermal growth factor. (EGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), activin, bone morphogenetic factor ( BMP), etc., and may be appropriately selected depending on the method of differentiation of stem cells into target cells. In the case of differentiation of mesenchymal stem cells into chondrocytes, examples include TGF-β, IGF, IL, dexamethasone, and BMP. Alternatively, a commercially available differentiation-inducing medium for differentiation into target cells may be used. In the case of differentiation of mesenchymal stem cells into chondrocytes, commercially available differentiation induction media include, for example, Mesenchymal Stem Cell Chondrogenic Differentiation Medium Ready-to-use (manufactured by Promocell Co., Ltd.).
The differentiation method is not particularly limited as long as the stem cells detached and collected in step (B) are used, and differentiation may be induced under conditions commonly used in the art. In addition, adhesive culture, suspension culture, three-dimensional culture using a scaffold such as atelocollagen, etc. can be selected depending on the purpose.
The method may include a step of culturing and proliferating stem cells before inducing cell differentiation with a differentiation-inducing medium. The medium used in this step is not particularly limited as long as the stem cells adhere and proliferate while maintaining the ability to differentiate into target cells, as in the medium used in step (A), and it consists of a basal medium and serum, and does not contain antibiotics. You can leave it there.
The culture density of stem cells may be as long as it does not inhibit the differentiation of stem cells into target cells, and in the case of differentiation of mesenchymal stem cells into chondrocytes, for example, 1.0×10 ¹ to 1.0×10 ⁵ cells. /cm ² is preferable, and 1.0×10 ⁴ to 1.0×10 ⁶ cells/cm ² is more preferable.
There are no particular limitations on the method for evaluating the differentiation state of differentiated cells, but for example, in the differentiation of mesenchymal stem cells into chondrocytes, the extracellular matrix secreted by the chondrocytes formed by differentiation induction is treated with Alcian blue. This can be confirmed by staining with.

Examples of the present invention will be described below, but the present invention is not limited to these Examples in any way. In addition, unless otherwise specified, commercially available reagents were used.

<Composition of block copolymer>
It was determined by proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) spectrum analysis using a nuclear magnetic resonance measuring 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 device used was HLC-8320GPC manufactured by Tosoh Corporation, the columns used were two TSKgelSuperAWM-H manufactured by Tosoh Corporation, the column temperature was set at 40°C, and the eluent was 10mM sodium trifluoroacetate containing 10mM sodium trifluoroacetate. Measured using The measurement sample was prepared and measured at 1.0 mg/mL. For the molecular weight calibration curve, polymethyl methacrylate (manufactured by Polymer Laboratories) with a known molecular weight was used.

<Synthesis example of block copolymer>
Add 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) to a 200 mL two-necked flask, and add 31.8 mg (100 μmol) of cyanomethyldodecyl trithiocarbonate and 1.6 mg (10 μmol) of azobisisobutyronitrile. 10 mL of tert-butyl alcohol was added, and after purging 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) was added, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butyl alcohol were added, and after replacing with argon gas, The mixture was heated and stirred at 62°C for 24 hours.
After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32 ° C.) was added to the above, and further 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62° C. for 24 hours.
After the third heating and stirring, the reaction solution 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 separating funnel. The collected chloroform phase was concentrated using an evaporator and purified by reprecipitation with heptane. The precipitate was collected by filtration and dried under reduced pressure to obtain 8.295 g of block copolymer poly(MEA-BA-IPAAm). The composition of the obtained block copolymer was MEA:BA:IPAAm=5:30:65 (mol%), Mn was 11.8×10 ⁴ , and Mw/Mn was 1.45.

<Preparation of surface treatment agent>
19.910 g of 2-methoxyethanol was added to 90 mg of the block copolymer, and the whole was dissolved by stirring to prepare a 0.45 wt % surface treatment agent for the block copolymer.

<Preparation of culture equipment>
Add 100 μL of surface treatment agent to the center of an IWAKI tissue culture dish (φ6 cm) and spin coat it using a spin coater (manufactured by Mikasa, product name MS-B200) at a rotation speed of 3,000 rpm and a rotation time of 60 seconds. In this way, culture equipment coated with the block copolymer was prepared. The coating amount of the block copolymer on the surface of this device was measured by total reflection Fourier transform infrared spectroscopy (ATR/FT-IR), and it was found to be 0.55 μg/cm ² as a temperature-responsive block.

Example 1
Bone marrow-derived human mesenchymal stem cells (manufactured by Lonza Japan Co., Ltd., Product Code: PT-2501, Lot Number: 0000603525) were seeded at 1.0 × 10 ⁵ cells/dish in culture equipment, and the cells were incubated at 37°C and at a CO ₂ concentration. Cultured at 5%. Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS/DMEM) containing 10 vol% fetal bovine serum (from Colombia) was used as the culture solution.
After culturing for 7 days, the culture solution was drained, a new culture solution cooled to 4°C was added, and the mixture was cooled at room temperature for 10 minutes. After 10 minutes, the culture solution was applied to the entire culture surface of the culture device using a pipetter, and the culture solution was collected together with the cells. The collected culture solution was centrifuged at 160 rcf, 25° C., and for 5 minutes, the supernatant was removed, and 500 μL of the culture solution was added and suspended. 10 μL of the obtained cell suspension was added to a cell counting slide (manufactured by Thermo Fisher Scientific Co., Ltd., trade name: Countess (registered trademark) Cell Counting Chamber Slide), and an automatic cell counter (Thermo Fisher Scientific) was added. ( The number of cells was measured using Countess (registered trademark) II) manufactured by Co., Ltd., trade name: Countess (registered trademark) II).
The mesenchymal stem cells detached and collected by cooling as described above were placed in a 96-well plate for suspension culture (manufactured by Corning, trade name: Corning (registered trademark) 96-well Black Round Bottom Ultra-) to which 200 μL of 10 vol% FBS/DMEM was added. The cells were seeded at 1.0×10 ⁵ cells/well in a Low Attachment Spheroid Microplate and cultured for 2 days at 37° C. and a CO ₂ concentration of 5%. Thereafter, the medium was replaced with Mesenchymal Stem Cell Chondrogenic Differentiation Medium Ready-to-use (manufactured by Promo Cell Co., Ltd.) to induce differentiation into chondrocytes.

After culturing for 25 days while replacing the medium every 3 days, spherical cell aggregates were observed. Cell aggregates were stained with Alcian blue, and the diameter of the positively stained cartilage tissue was measured. After the measurement, the Alcian blue dye was eluted with 150 μL of 6M guanidine hydrochloride, and the amount of extracellular matrix was measured at 630 nm using a plate reader (manufactured by Corona Electric Co., Ltd., trade name: Absorption Grating Microplate Reader SH-1300 Lab). It was measured by the absorbance of As a result, the diameter of the cartilage tissue was 1.75 mm, and the absorbance at 630 nm was 0.912.

Comparative example 1
When detaching and collecting mesenchymal stem cells from culture equipment, instead of adding a culture medium cooled to 4°C, after washing with PBS, a proteolytic enzyme (manufactured by Thermo Fisher Scientific Co., Ltd., product name: TrypLE Express) is added. Differentiation of bone marrow-derived human mesenchymal stem cells into chondrocytes was induced in the same manner as in Example 1, except that 1 mL was added and allowed to stand at 37°C for 10 minutes.

Cell aggregates were stained with Alcian blue, and the diameter of the positively stained cartilage tissue was measured. After the measurement, the Alcian blue dye was eluted with 150 μL of 6M guanidine hydrochloride, and the amount of extracellular matrix was measured by absorbance at 630 nm. As a result, the diameter of the cartilage tissue was 1.22 mm, and the absorbance at 630 nm was 0.314.

Claims (4)

Pre-culturing the stem cells while maintaining an undifferentiated state on a culture device exhibiting a lower critical lysis temperature (LCST);
a step of cooling the culture equipment below the LCST and detaching and collecting the pre-cultured stem cells;
a step of culturing the recovered pre-cultured stem cells using a differentiation-inducing medium;
A method for differentiating stem cells, the method comprising:
The surface of the culture device is coated with a temperature-responsive block copolymer exhibiting LCST,
A method characterized in that the temperature-responsive block copolymer comprises repeating units of 2-methoxyethyl acrylate, repeating units of n-butyl acrylate, and repeating units of N-isopropylacrylamide. 2. The method according to claim 1, wherein the stem cells are mesenchymal stem cells. 3. The method according to claim 2, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. 4. The method according to claim 1, wherein the stem cells differentiate into chondrocytes.