JP7470893B2 - Cell culture substrate and method for producing same - Google Patents

Description

The present invention relates to a cell culture substrate and a method for producing the same.

In medical and biological research, cell culture is an important method for growing target cells. When culturing cells outside of a living body, many cells need to be attached to a scaffolding substrate. Cells are bound via proteins that act as adhesion molecules, such as integrins, and are firmly attached to the substrate. Therefore, when harvesting cultured cells, they must be detached from the substrate. In many cases, proteolytic enzymes are used for detachment. Proteolytic enzymes break down proteins on the cell surface, so cells can be detached by breaking the bond between the cell and the substrate. However, they also break down proteins on the cell surface, damaging the cells. For these reasons, there has been a demand for technology that can detach cells from substrates without damaging them.

As a substrate that combines cell adhesion and detachability, much research and development has been carried out on cell culture substrates whose surfaces are coated with stimuli-responsive polymers. Known stimuli-responsive polymers include materials that are temperature-responsive, pH-responsive, photoresponsive, etc. Patent Document 1 describes a cell culture substrate that achieves cell adhesion and detachment by cooling treatment by coating a commercially available cell culture substrate with a temperature-responsive polymer. However, general cell culture substrates are not designed to be coated with other substances on the culture surface. In order for the coating polymer to fully exert its function, a certain amount of coating or more is necessary, but if more than a certain amount is coated, there is a problem that cell adhesion decreases, hindering cell proliferation.

JP 2018-174919 A

The object of the present invention is to provide a cell culture substrate coated with a polymer and a method for producing the same.

In view of the above, the inventors conducted extensive research and discovered that a cell culture substrate having a substrate surface with a specific zeta potential coated with a polymer can achieve both cell adhesion and the functionality of the polymer, thus completing the present invention.

That is, the present invention includes the following aspects.
<1> A cell culture substrate, which is formed by coating a surface of a substrate having a zeta potential of -50 mV to 0 mV with a polymer.
<2> The cell culture substrate according to <1>, wherein the contact angle of water with respect to the substrate before being coated with the polymer is 20° to 110°.
<3> The cell culture substrate according to <1> or <2>, wherein the material of the substrate is at least one selected from the group consisting of polyolefin, polystyrene, and polyethylene terephthalate.
<4> The cell culture substrate according to any one of <1> to <3>, wherein the coating amount of the polymer is 0.1 μg/cm ² to 10 μg/cm ² .
<5> The cell culture substrate according to any one of <1> to <4>, wherein the polymer is a temperature-responsive block copolymer.
<6> A method for producing a cell culture substrate, comprising: coating a surface of a substrate having a zeta potential of −50 mV to 0 mV with a polymer.
<7> A method for producing a cell culture substrate according to <6>, comprising the steps of: subjecting the substrate to a plasma treatment; and applying a polymer solution to cover the substrate surface with a polymer.

The polymer-coated cell culture substrate of the present invention can achieve both cell adhesion and the functionality of the polymer.
In particular, it was found that the surface characteristics of the substrate before being coated with a polymer are related to the adhesion of cells to the polymer-coated cell culture substrate, and it became possible to obtain any stimulus response after coating with the polymer without performing a surface treatment on the cell adhesion surface.

The following describes in detail the form for carrying out the present invention, but it is not intended that the present invention be limited to the following content. The present invention can be carried out with appropriate modifications within the scope of its spirit.

The substrate in this invention refers to a material that is not coated with a polymer, and its surface properties can be controlled by performing an appropriate surface treatment.

The cell culture substrate in the present invention is a material that combines the functions of cell adhesion and polymer by coating the substrate with a polymer.

To obtain the effects of the present invention, a substrate with a specific zeta potential is required, specifically -50 mV to 0 mV, preferably -50 mV to -20 mV. When the substrate is plate-shaped, a flat sample cell is used to measure the zeta potential. With the substrate placed in the cell, a monitor particle solution is injected into the cell and the electrical mobility of the monitor particles is measured, thereby determining the zeta potential. The following Smoluchowski formula is generally used to determine the zeta potential from the electrical mobility.

ζ = (4πηU) / ε
(In the above formula, ζ is the zeta potential, η is the viscosity of the solvent, ε is the dielectric constant of the solvent, and U is the electrical mobility.)
A substrate having a specific zeta potential can be obtained by subjecting the substrate to a hydrophilic treatment such as a plasma treatment described below.

The contact angle of water with a substrate (hereafter referred to as contact angle) is the angle between the liquid surface and the substrate surface when a drop of water is dropped onto the surface of the substrate and the drop is stationary. Methods for measuring contact angles include the θ/2 method, the tangent method, and the curve fitting method, with the θ/2 method being the most commonly used. The contact angle using the θ/2 method can be calculated using the following formula:

θ=2arctan(h/r)
(In the above formula, θ is the contact angle, h is the height of the droplet, and r is the radius of the droplet.)
The smaller the contact angle, the more hydrophilic the surface. Cells are less likely to adhere to hydrophobic surfaces, but are more likely to adhere to surfaces with moderate hydrophilicity. To obtain the effects of the present invention, the contact angle is preferably 20° to 110°, and more preferably 40° to 60°.

By using a substrate having the surface properties as described above, the function of the polymer can be imparted to the substrate surface without surface treatment of the cell adhesion surface while maintaining the cell adhesiveness, which is a property of the substrate surface, even after coating with the polymer. In the present invention, examples of a method for coating the substrate with the polymer include coating methods such as dipping, bar coating, slit die coating, gravure coating, casting, spray coating, and spin coating. The polymer may be applied to only one side or to all sides of the substrate. The drying method may be natural drying or heat drying.

In order to apply the polymer to the substrate, it is necessary to dissolve the polymer in a solvent and use it as a polymer solution. There are no particular limitations on the solvent used, as long as it dissolves the polymer and does not dissolve the substrate. Examples of the solvent include methanol, ethanol, isopropyl alcohol, butanol, 2-butanol, tert-butanol, and 2-methoxyethanol. The solvent may be used alone or in combination of two or more types.

The polymer content in the polymer solution is preferably 0.01 to 10% by mass, and more preferably 0.05 to 5% by weight. If the polymer content is low, the substrate surface will not be sufficiently coated. If the polymer content is high, the viscosity of the polymer solution will increase, resulting in poor coatability.

In the present invention, the coating amount of the polymer is preferably 0.1 to 10 μg/cm ² , and more preferably 2 to 8 μg/cm ^2. The coating amount may be measured by a commonly known method, and as an example, a total reflection type Fourier transform infrared spectroscopy (FT-IR/ATR) method can be used.

In the present invention, the substrate material is preferably one whose zeta potential is controllable and which is easy to mold. Examples of such materials include polyolefin, polystyrene, and polyethylene terephthalate. Polyolefin is a general term for polymers that have hydrocarbon double bonds inside, and among polyolefins, it is preferable to use polyethylene or polypropylene, which are versatile. In addition, the substrate may be a mixture or copolymer of these materials, and may contain additives such as plasticizers.

The substrate in the present invention is preferably in the form of a film. A cell culture substrate prepared using a film-like substrate can be used by laying it on a petri dish or plate, or the film can be welded and used in the form of a cell culture bag. The film may be a single layer or a multilayer structure.

There are no particular limitations on the method for controlling the zeta potential of the substrate, but one example is plasma treatment. Plasma treatment is a method for improving the hydrophilicity of the substrate surface by irradiating the substrate with plasma generated by ionization after discharging the gas in an atmosphere containing oxygen or nitrogen atoms. Discharge in plasma treatment includes corona discharge (high pressure, low temperature), arc discharge (high pressure, high temperature), glow discharge (low pressure, low temperature), and atmospheric pressure plasma. Glow discharge or atmospheric pressure plasma is preferably used for thin substrates such as films, as they cause less damage.

In the present invention, there is no particular limitation on the structure of the coating polymer, but one example is a block copolymer. 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 limitations on the characteristics of the polymer, but it is preferable that it has stimuli responsiveness, and one example is that it has temperature responsiveness. A temperature-responsive block copolymer is a block copolymer in which at least one of the repeating units contains a temperature-responsive repeating unit. Each temperature-responsive repeating unit has its own lower critical solution temperature (LCST). The LCST is the temperature below which a polymer dissolves in water and becomes a transparent solution, but above which it becomes insoluble and becomes cloudy or precipitates, resulting in phase separation. The presence of an LCST can be treated as synonymous with the presence of temperature responsiveness. The LCST is preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C. Examples of temperature-responsive repeating units and their LCSTs with respect to water include 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 = 35°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), and N-methyl-N-n-propylacrylamide (LCST = 20°C). The temperature at which the LCST appears varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because it has low concentration dependency for the appearance of the LCST. The temperature-responsive repeating units used in the temperature-responsive block copolymer of the present invention may be one type only, or two or more types may be combined.

When a temperature-responsive block copolymer is coated on a substrate, it is preferable that the block copolymer contains a repeating unit for fixing the block copolymer to the substrate in addition to the temperature-responsive repeating unit. Examples of the repeating units include styrene and its derivatives, 2-methoxyethyl acrylate, n-propyl acrylate, n-propyl methacrylate, n-butyl acrylate, and n-butyl methacrylate. A temperature-responsive block copolymer containing a repeating unit of 2-methoxyethyl acrylate, a repeating unit of n-butyl acrylate, and a repeating unit of N-isopropylacrylamide is particularly preferable. The polymer is not particularly limited, but the synthesis method thereof can be a method using the living radical polymerization technique described in "Radical Polymerization Handbook" published by NTS Co., Ltd., pp. 161-225 (2010).

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.

<Composition of block copolymer>
The values were determined by proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) spectrum analysis using a nuclear magnetic resonance measuring device (manufactured by JEOL Ltd., product 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 HLC-8320GPC, and two Tosoh TSKgel Super AWM-H columns were used. The column temperature was set to 40°C, and the eluent was a 2,2,2-trifluoroethanol solution containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg/mL and measured. For the molecular weight calibration curve, polymethyl methacrylate (Polymer Laboratories) with a known molecular weight was used.

<Synthesis example of block copolymer>
To a 200 mL two-neck flask, 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added, and then 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of tert-butyl alcohol were 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) was added, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butyl alcohol. 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, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol. After replacing 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 separatory funnel. The collected chloroform phase was concentrated with 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 a 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.

<Zeta potential of substrate>
The zeta potential of the substrate was measured using a zeta potential meter (ELS-2, manufactured by Otsuka Electronics). The sample was set in a flat cell unit. The monitor particle solution (manufactured by Otsuka Electronics) was diluted 100 times with a 10 mM aqueous solution of sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare an electrophoretic liquid for monitoring. The zeta potential was calculated using the Smoluchowski method. <Contact angle of water on substrate>
The contact angle of water with respect to the substrate was measured using a contact angle meter (DMs-401, manufactured by Kyowa Interface Science Co., Ltd.). 1 μL of pure water was applied to the sample surface, and the contact angle was measured by analysis using the θ/2 method.

<Preparation of Block Copolymer Solution>
29.970 g of ethanol was added to 30 mg of the block copolymer and completely dissolved by stirring to prepare a 0.10 wt % block copolymer solution 1.

29.985 g of ethanol was added to 15 mg of block copolymer and completely dissolved by stirring to prepare a 0.05 wt % block copolymer solution 2.

<Coating amount of block copolymer>
The coating amount of the block copolymer was measured by total reflection Fourier transform infrared spectroscopy (ATR/FT-IR). The intensity ratio of the peak specific to the substrate and the peak specific to the block copolymer was analyzed. A calibration curve was created using a model sample with a known coating amount, and the coating amount of the block copolymer was measured.

Example 1
A low-density polyethylene film (manufactured by Tosoh) was cut into a circle with a diameter of 5 cm, and was subjected to a plasma treatment in air at 10 Pa and 10 mA for 5 minutes using a soft plasma etching device (manufactured by Meiwa Force Systems, SEDE-P). The zeta potential of this film was measured to be -33 mV. The contact angle of water with this film was also measured to be 38°.

The above-mentioned film was immersed in the block copolymer solution 1 and naturally dried to coat the film with the block copolymer, thereby producing a cell culture substrate. The coating amount of the block copolymer on the surface of this cell culture substrate was measured and found to be 7.3 μg/ ^cm2 .

The cell culture substrate prepared by the above method was placed on a non-treated dish for floating cells (AGC Technoglass, 1010-060), and bone marrow-derived human mesenchymal stem cells (Lonza Japan, Product Code: PT-2501, Lot Number: 0000603525) were seeded at 1.0 x 10 ⁵ cells/dish, and cultured at 37 ° C and 5% CO ₂ concentration. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS/DMEM) containing 10 vol% fetal bovine serum (Colombia). After culturing for 6 days, the culture medium was removed, and a new culture medium cooled to 4 ° C was added, and cooled at room temperature for 20 minutes. After 20 minutes, the culture medium was pipetted using a pipetter so that the culture medium was applied to the entire surface of the culture surface, and the culture medium was collected together with the cells. The number of cells was measured, and the number of cells recovered by the cooling treatment (number of cells recovered after cooling) was 1.11 x 10 ⁵ cells. The cells remaining on the substrate after the cooling treatment were recovered by trypsin treatment, and the total number of cells was calculated by adding this to the number of cells recovered after cooling, and the total number of cells was 1.99 x 10 ⁵ cells. From the above, the cell recovery rate by the cooling treatment was 56%. The cell proliferation rate was calculated from the number of cells after proliferation relative to the initial seeding number, and was 199%.

Example 2
A cell culture substrate was prepared in the same manner as in Example 1, except that block copolymer solution 2 was used. The zeta potential of the film before coating with the block copolymer was −33 mV, and the contact angle was 38°. The coating amount of the block copolymer was 3.0 μg/ ^cm2 . Cell culture and recovery by cooling were performed in the same manner as in Example 1. As a result, the cell recovery rate by cooling treatment was 46%. In addition, the cell proliferation rate was 161%.

(Comparative Example 1)
A cell culture substrate was prepared in the same manner as in Example 1, except that the film was not subjected to plasma treatment. The zeta potential of the film before coating with the block copolymer was −51 mV, and the contact angle was 119°. The coating amount of the block copolymer was 3.8 μg/ ^cm2 . Cell culture was performed in the same manner as in Example 1, but the cells did not adhere to the substrate and did not proliferate.

(Comparative Example 2)
A cell culture substrate was prepared in the same manner as in Example 1, except that the block copolymer solution was not applied. The zeta potential of the film before coating with the block copolymer was −33 mV, and the contact angle was 38°. Cell culture and recovery by cooling were performed in the same manner as in Example 1. As a result, the cell recovery rate by cooling treatment was 10%. In addition, the cell proliferation rate was 192%.

Claims (6)

A cell culture substrate, comprising a substrate having a zeta potential of -50 mV to 0 mV, the substrate surface being coated with a temperature-responsive block copolymer comprising a repeating unit of 2-methoxyethyl acrylate, a repeating unit of n-butyl acrylate, and a repeating unit of N-isopropylacrylamide . 2. The cell culture substrate according to claim 1, wherein the contact angle of water with respect to the substrate before being coated with the temperature-responsive block copolymer is 20° to 110°. The cell culture substrate according to claim 1 or 2, characterized in that the substrate material is at least one selected from the group consisting of polyolefin, polystyrene, and polyethylene terephthalate. 4. The cell culture substrate according to claim 1, wherein the coating amount of the temperature-responsive block copolymer is 0.1 μg/cm ² to 10 μg/cm ² . The method for producing a cell culture substrate according to any one of claims 1 to 4 , wherein the surface of a substrate having a zeta potential of -50 mV to 0 mV is coated with the temperature-responsive block copolymer . 6. The method for producing a cell culture substrate according to claim 5 , comprising the steps of: subjecting a substrate to a plasma treatment; and applying a solution of the temperature-responsive block copolymer to cover the substrate surface with the temperature-responsive block copolymer .