JP2018157758A - Cell culture apparatus and biological sample production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture apparatus and a biological sample production method in which various types of cells can be adsorbed on a container surface while the cells are cultured, formation of the cells into tissue can be promoted, obtained biological sample can be released without losing the original functions of the cell, and cells being cultured can be prevented from being released.SOLUTION: A cell culture apparatus 1 comprises: a transparent culture container 10 in which culture solution is housed; a dielectric layer 12 which is formed such that an inner bottom surface 10a of the culture container 10 and a first surface 12a come into contact with the culture solution; a first electrode 14 provided at a second surface 12b on the opposite side to the first surface 12a of the dielectric layer 12; a second electrode 16 provided in the culture container 10 separately from the dielectric layer 12; and a voltage application mechanism 18 which applies voltage between the first electrode 14 and the second electrode 16, and the surface potential of the first surface 12a of the dielectric layer 12 is designed to change by applying voltage between the first electrode 14 and the second electrode 16.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture device and a biological sample manufacturing method.

  In recent years, in the field of regenerative medicine, attempts have been made to enhance the therapeutic effect by placing a thick cell sheet obtained by culturing cells at high density in an affected area and engrafting it. In cell culture for producing an organized biological sample such as a cell sheet, a scaffold for organizing the cells is required. Conventionally, as a culture container, a petri dish or the like in which the inner surface is hydrophobic and cells are adsorbed on the inner surface during culture has been used. However, when culturing is performed in a culture vessel having high cell adsorptivity, the formed biological sample is difficult to be detached from the culture vessel, and the biological sample is easily damaged during the separation.

  In addition, a caged peptide in which a photolabile protecting group is introduced to an RGD peptide that is a cell adhesive peptide portion is bound on a substrate, and ultraviolet rays are irradiated at any timing during culture to remove the protecting group, thereby removing cells. A method of bonding is known (Patent Document 1). However, this method cannot control the desorption of cells reversibly, and there is a concern about the adverse effect on cells caused by ultraviolet rays irradiated to desorb the photolabile protecting group or the detached protecting group. Difficult to remove with stimulation and low load.

  In addition, a biological sample is produced using a culture vessel in which the inner surface of the vessel is coated with a temperature-responsive polymer that is hydrophobic at the culture temperature (eg, 37 ° C.) and hydrophilic at room temperature (20-25 ° C.). Methods have been proposed (Patent Documents 2 and 3). In this method, since the temperature-responsive polymer on the surface of the container is hydrophobic and contracted during culture, the cells are adsorbed on the surface of the container and the organization thereof is promoted. In addition, when the temperature of the culture vessel is lowered to room temperature after culturing, the temperature-responsive polymer becomes hydrophilic and swelled, and water enters between the formed biological sample and the surface of the vessel, so that the biological sample becomes the surface of the vessel. Detaches intact. Since the biological sample obtained in this way does not lose the original function of the cell, it can be rapidly engrafted in the biological tissue.

Japanese Patent No. 4682364 Japanese Patent No. 3441530 Japanese Patent No. 3641301

  However, in a culture container using a temperature-responsive polymer, the temperature may drop when the culture container is taken out of the incubator for observation, medium replacement, or the like, and the cells in the middle of the culture may peel off. In addition, in the culture container, the surface of the container cannot be adjusted to conditions suitable for adhesion for each type of cell, and depending on the type of cell, the adhesion to the surface of the container becomes insufficient, and biological samples such as cell sheets Manufacturing becomes difficult.

  In the present invention, various types of cells can be adsorbed on the surface of a container at the time of culturing to promote the organization thereof, and the obtained biological sample can be detached without impairing the original function of the cells, It is an object of the present invention to provide a cell culture device and a biological sample manufacturing method capable of suppressing the detachment of cells.

The present invention has the following configuration.
[1] A transparent culture container in which a culture solution is stored, a dielectric layer formed on the inner surface side of the culture vessel so that a first surface is in contact with the culture solution, and the dielectric layer A first electrode provided on a second surface opposite to the first surface, a second electrode provided in the culture vessel and spaced apart from the dielectric layer, and the first electrode An applied voltage mechanism for applying a voltage between the second electrode and the surface potential of the first surface is applied between the first electrode and the second electrode. A cell culture device that changes depending on the situation.
[2] A biological sample manufacturing method for manufacturing a biological sample by cell culture using the cell culture device according to [1], wherein the first electrode is applied to the second electrode by the applied voltage mechanism. By culturing the cell with a potential of E ₁ (V) and obtaining a biological sample adsorbed on the first surface, and the applied voltage mechanism, the potential of the first electrode with respect to the second electrode is changed to E And a desorption step of desorbing the biological sample from the first surface, wherein E ₂ (V) is lower than ₁ (V).
[3] The biological sample manufacturing method according to [2], wherein the biological sample is a cell sheet.

By using the cell culture apparatus of the present invention, various types of cells can be adsorbed on the surface of the container during culture to promote their organization, and the obtained biological sample can be detached without impairing the original function of the cells. In addition, detachment of cells during the culture can be suppressed.
According to the method for producing a biological sample of the present invention, various types of cells can be adsorbed on the surface of a container at the time of culturing to promote the organization thereof, and the obtained biological sample can be detached without impairing the original function of the cells. And the detachment of cells during the culture can be suppressed.

It is the schematic diagram which showed an example of the cell culture apparatus of this invention. FIGS. 2A and 2B are diagrams showing an example of a biological sample manufacturing method using the cell culture apparatus of FIG. 1, FIG. 2A is a schematic diagram showing a state of a culture process, and FIG. 2B is a desorption process. It is the schematic diagram which showed the mode of. It is the graph which showed the relationship between the surface potential of the inner bottom face of a recessed part, and the protein adsorption rate Q in the protein adhesiveness test of an Example. It is the graph which showed the relationship between the stationary time and the protein adsorption rate Q in the surface potential -3V of the inner bottom face of the recessed part in the protein desorption test of an Example.

The following definitions of terms apply throughout this specification and the claims.
The “fluorinated polymer” means a polymer compound having a fluorine atom in the molecule.
The “unit” means a part derived from a monomer that exists in the polymer and constitutes the polymer. The unit derived from the monomer resulting from addition polymerization of a monomer having a carbon-carbon unsaturated double bond is a divalent unit generated by cleavage of the unsaturated double bond. Moreover, what unitally converted the structure of a unit after polymer formation is also called a unit. Hereinafter, in some cases, a unit derived from an individual monomer is referred to as a name obtained by adding “unit” to the monomer name.
“The fluoropolymer has an aliphatic ring in the main chain” means that at least one of the carbon atoms constituting the ring skeleton of the aliphatic ring is a carbon atom constituting the main chain of the fluoropolymer. Means that.
“Transparent” means that the average transmittance of visible light (360 nm to 830 nm) is 10% or more. When observing the inside of a culture vessel in a state where a conductive fluid such as a medium is accommodated from the bottom side, the visible light transmission haze measured in that state is preferably 30% or less.

[Cell culture equipment]
The cell culture device of the present invention includes a transparent culture container that contains a culture solution, a dielectric layer formed on the inner surface side of the culture vessel so that a first surface is in contact with the culture solution, A first electrode provided on a second surface opposite to the first surface of the dielectric layer; a second electrode provided in the culture container and spaced apart from the dielectric layer; A voltage applying mechanism for applying a voltage between the first electrode and the second electrode is provided. In the culture container of the cell culture device of the present invention, the surface potential of the first surface of the dielectric layer is changed by applying a voltage between the first electrode and the second electrode. ing.
The cell culturing apparatus of the present invention can be suitably used for searching for optimal substrate surface properties according to various biological samples and their intended use, and for cell culturing for producing cell sheets.

FIG. 1 is a cross-sectional view showing an example of the cell culture apparatus of the present invention.
The cell culture device 1 includes a transparent culture vessel 10 in which a culture solution is accommodated, a dielectric layer 12, a first electrode 14, a second electrode 16, and a voltage application mechanism 18.
The dielectric layer 12 is formed on the inner bottom surface 10a side of the culture vessel 10 so that the first surface 12a is in contact with the culture solution. The first electrode 14 is provided on the second surface 12 b opposite to the first surface 12 a of the dielectric layer 12. In this example, the first electrode 14 is formed on the inner bottom surface 10 a of the culture vessel 10, and the dielectric layer 12 is further formed on the surface of the first electrode 14.

The second electrode 16 is provided in the culture vessel 10 so as to be separated from the dielectric layer 12. The applied voltage mechanism 18 is electrically connected to the first electrode 14 and the second electrode 16, respectively, so that a voltage can be applied between the first electrode 14 and the second electrode 16. Yes.
The surface potential of the first surface 12 a of the dielectric layer 12 is changed by applying a voltage between the first electrode 14 and the second electrode 16 by the applied voltage mechanism 18.

(Culture container)
The culture vessel may be any transparent vessel that can accommodate the culture solution, and for example, a known vessel used for culturing a petri dish or the like can be used.
It does not specifically limit as a material which forms a culture container, Resin, such as a polystyrene, Glass etc. are mentioned.
The material forming the culture vessel may be one type or two or more types.

(Dielectric layer)
The first surface of the dielectric layer formed in the culture vessel serves as a cell adsorption surface during culture. The dielectric layer may be provided on a part of the inner surface side of the culture vessel, or may be provided on the entire inner surface side of the culture vessel.
The dielectric layer is preferably provided on the inner bottom surface side of the culture vessel like the dielectric layer 12 in the cell culture device 1 from the viewpoint that the cells are adsorbed on the first surface and the organization is easily promoted. The dielectric layer may be provided on the inner side surface of the culture vessel.

The material for forming the dielectric layer is not particularly limited, and examples thereof include organic polymer materials such as a fluorine-containing polymer, polyethylene terephthalate, polyimide, polycarbonate, acrylic resin, parylene C, and the like. Coalescence is preferred.
The material for forming the dielectric layer may be one type or two or more types.

  The fluorine-containing polymer is not particularly limited, and examples thereof include a fluorine-containing polymer having a fluoroolefin unit, a fluorine-containing polymer having an aromatic ring, a fluorine-containing polymer having a fluoropolyether chain, and an aliphatic ring in the main chain. And fluorine-containing polymers, fluororubbers and the like.

Examples of the fluoropolymer having a fluoroolefin unit include polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoro (alkyl vinyl ether) copolymer (PFA), and the like.
Examples of the fluorine-containing polymer having an aromatic ring include fluorine-containing aromatic compounds (perfluoro (1,3,5-triphenylbenzene) and the like) and phenolic compounds (1,3,5-trihydroxybenzene and the like). And a polymer obtained by reacting a crosslinkable functional group-containing aromatic compound (such as pentafluorostyrene) in the presence of a dehydrohalogenating agent (such as potassium carbonate).
Examples of the fluoropolymer having a fluoropolyether chain include OPTOOL DSX (registered trademark, manufactured by Daikin Industries), KY-100 series (manufactured by Shin-Etsu Chemical Co., Ltd.), and the like.
Examples of the fluorine-containing polymer having an aliphatic ring in the main chain include CYTOP (registered trademark, manufactured by Asahi Glass Co., Ltd.), Teflon (registered trademark) AF (manufactured by DuPont), and the like.
Examples of the fluororubber include vinylidene fluoride-based fluororubbers mainly composed of vinylidene fluoride (VDF), propylene-tetrafluoroethylene-based fluororubber, tetrafluoroethylene-perfluoroalkylvinylether-based fluororubber, and the like.
As a fluoropolymer, 1 type may be used independently and 2 or more types may be used in combination.

  As a material for forming the dielectric layer, one or more metal oxides selected from the group consisting of silica, alumina, ceria, titania and zirconia may be used.

  The thickness of the dielectric layer is preferably 0.1 to 10 μm, and more preferably 0.5 to 1 μm. If the thickness of the dielectric layer is equal to or greater than the lower limit, dielectric breakdown is less likely to occur within a practical working voltage. If the thickness of the dielectric layer is not more than the upper limit value, the dielectric layer can be efficiently polarized by applying a voltage.

(First electrode)
The material for forming the first electrode may be a conductive material, such as indium tin oxide (ITO); metal such as Pt, Au, Ni, Al; SnO ₂ , In ₂ O ₃ , RuO _2, etc. Examples include conductive metal oxides; semiconductors such as Ge, Si, and GaAs; carbon-based materials such as graphite, glassy carbon, and diamond; and conductive polymers such as polyacetylene, polypyrrole, polyaniline, and polythiophene.
The shape of the first electrode is not particularly limited, and examples thereof include a flat plate shape and a comb shape.
The number of first electrodes is not limited to one and may be two or more.

(Second electrode)
The second electrode is provided so as to be separated from the dielectric layer, and comes into contact with the culture solution when the culture solution is stored in the culture vessel.
The material for forming the second electrode may be any conductive material, and examples thereof include the same materials as those described for forming the first electrode.
The shape of the second electrode is not particularly limited, and examples thereof include a flat plate shape and a rod shape.

(Applied voltage mechanism)
As the applied voltage mechanism, any mechanism can be used as long as an arbitrary voltage can be applied between the first electrode and the second electrode, and a known mechanism can be adopted.
As the applied voltage mechanism, a mechanism including a switch for switching ON / OFF of voltage application is preferable. Further, as a voltage application mechanism, a voltage is applied between the first electrode and the second electrode from any one of the positive voltage output unit, the negative voltage output unit, and the positive voltage output unit and the negative voltage output unit. What is provided with the switch which switches whether to do is preferable. Furthermore, as an applied voltage mechanism, what is equipped with the voltage adjustment mechanism which can adjust the voltage applied between a 1st electrode and a 2nd electrode is preferable.

(Production method)
The method for producing the cell culture device of the present invention is not particularly limited, and examples thereof include the following methods.
After the first electrode is formed on the inner surface of the culture vessel by sputtering, vapor deposition or the like, a coating solution in which a fluoropolymer or the like is dissolved in a solvent is applied on the first electrode and dried by baking or the like. A dielectric layer is formed. Next, the second electrode is disposed in the culture container so as to be separated from the dielectric layer, and the first electrode and the second electrode are electrically connected to the applied voltage mechanism, thereby the cell culture device Get.

  The coating method for the coating solution is not particularly limited, and examples thereof include spin coating, dip coating, cast coating, spray coating, die coating, scan coating, brush coating, and potting.

[Biological sample manufacturing method]
The biological sample manufacturing method of the present invention is a method of manufacturing a biological sample by cell culture using the cell culture apparatus of the present invention. The biological sample manufacturing method of the present invention includes the following culture process and desorption process.
Culturing step: Using the cell culturing apparatus of the present invention, the cell is cultured by applying a voltage mechanism to the potential of the first electrode with respect to the second electrode as E ₁ (V), and adsorbed on the first surface of the dielectric layer A biological sample is obtained.
Desorption step: The potential of the first electrode with respect to the second electrode is set to E ₂ (V) lower than E ₁ (V) by the applied voltage mechanism, and the biological sample is desorbed from the first surface of the dielectric layer. Let

(Culture process)
In a state where the culture solution is accommodated in the culture vessel in the cell culture apparatus of the present invention, the culture is performed with the potential of the first electrode with respect to the second electrode as E ₁ (V) by the applied voltage mechanism. Thereby, the cell adsorbability of the first surface of the dielectric layer is improved, so that a biological sample in which cells are grown and organized in a state where the cells are adsorbed on the first surface of the dielectric layer is obtained.

When the cell culture apparatus 1 illustrated in FIG. 1 is used, as shown in FIG. 2 (A), the second voltage 16 is applied to the second electrode 16 by the applied voltage mechanism 18 while the culture solution 100 is accommodated in the culture vessel 10. Incubation is performed with the potential of one electrode 14 being E ₁ (V). Thereby, the biological sample 110 in a state of being adsorbed on the first surface 12a of the dielectric layer 12 in the culture vessel 10 is obtained.

The potential E ₁ (V) of the first electrode with respect to the second electrode in the culturing step can be appropriately determined according to the type of cells to be cultured.
Cells are usually negatively charged. Therefore, when the potential E ₁ (V) is a positive potential, and the surface potential of the first surface of the dielectric layer is a positive potential and the first surface is positively charged, electrostatic interaction with the cells is performed. The action increases the adsorptivity of the cells.
In the case where the first surface of the dielectric layer is hydrophobic and the adsorptivity of cells is high, the potential E ₁ (V) may be set to 0 (V).

In addition, some cells require more time for adsorption than electrostatic adsorption, but there are some cells whose biological adsorption to the surface increases as the surface potential decreases. When culturing such a cell, the potential E is applied as long as the biological adsorption force is more dominant than the electrostatic repulsion force between the first surface of the dielectric layer and the cell during the culturing. ₁ (V) may be a negative potential.
In the culturing step, it is preferable to set the potential E ₁ (V) to a positive potential from the viewpoint that cells are easily adsorbed to the first surface of the dielectric layer and the organization of the cells is easily promoted.

  The cells used for the culture are not particularly limited. For example, gingival fibroblasts, periodontal ligament cells, epidermis cells, fibroblasts, liver parenchymal cells, liver non-parenchymal cells (endothelial cells, Kupffer cells, stellate cells, etc.) Osteoblasts, epithelial cells, chondrocytes, nerve cells, muscle cells, mesenchymal stem cells, islet cells, macrophages, various tumor cells and functional cells, and the like. The cell used for culture may be an artificial cell.

  It does not specifically limit as a liquid medium used for a culture solution, The well-known liquid medium according to the cell to culture can be used.

(Desorption process)
After completion of the culture, the potential of the first electrode with respect to the second electrode is set to E ₂ (V) lower than E ₁ (V) by the applied voltage mechanism. As a result, the biological sample organized in a state of being adsorbed to the first surface of the dielectric layer is detached from the first surface.
When the cell culture apparatus 1 illustrated in FIG. 1 is used, as shown in FIG. 2B, the potential of the first electrode 14 with respect to the second electrode 16 is set to be higher than E ₁ (V) by the applied voltage mechanism 18. The biological sample 110 is desorbed from the first surface 12a of the dielectric layer 12 with a low E ₂ (V).

The potential E ₂ (V) of the first electrode with respect to the second electrode in the desorption process is set lower than the potential E ₁ (V) of the first electrode with respect to the second electrode in the culture process.
When the potential E ₁ (V) is set to a positive potential in the culturing step and the cells are adsorbed on the first surface of the dielectric layer by electrostatic interaction, the potential E ₂ (V) is set to a negative potential in the detachment step. It is preferable that Accordingly, the biological sample can be easily detached from the first surface due to the influence of electrostatic repulsion with the negatively charged first surface of the dielectric layer.

When the first surface of the dielectric layer is hydrophobic and the potential E ₁ (V) is set to 0 (V) in the culture process, the potential E ₂ (V) is set to a negative potential in the desorption process. Thereby, the biological sample can be detached from the first surface of the dielectric layer. In this case, in addition to the effect due to electrostatic repulsion of the negatively charged first surface and the biological sample, the wettability of water to the first surface is improved by electwetting, and the first surface and the biological sample It is thought that the intrusion of water molecules during this period affects the detachment of the biological sample.

When the potential E ₁ (V) is set to a negative potential in the culturing process and the cells are adsorbed on the first surface of the dielectric layer by the biological adsorption force, the detachment process is more static than the biological adsorption force. The potential E ₂ (V) is lowered until the electric repulsive force becomes dominant. Thereby, the biological sample can be detached from the first surface of the dielectric layer.

The use of the biological sample desorbed from the first surface of the dielectric layer in the desorption step is not particularly limited. For example, the desorbed biological sample can be held and transported by a nonwoven fabric or the like, and placed in the affected area to be used for treatment of the affected area.
In the biological sample manufacturing method of the present invention, it is preferable to manufacture a cell sheet.

  As described above, in the present invention, by adjusting the surface potential of the first surface of the dielectric layer and changing the surface properties of the first surface to change the cell adhesion, Can control the detachment of cells to the surface. Therefore, the cells can be adsorbed on the first surface of the dielectric layer during the culture to promote the organization thereof, and the obtained biological sample can be detached without impairing the original function of the cells. In the present invention, since the desorption of the cells is not affected by the temperature, it is possible to suppress unintentional detachment of the cells in the middle of the culture during observation or medium exchange. In the present invention, the surface potential of the first surface of the dielectric layer can be adjusted to adjust the surface properties of the first surface to conditions suitable for adsorption for each cell. A biological sample can be produced by culturing. Furthermore, in the present invention, since the biological sample can be collected with a low load without applying a stimulus such as heat, light, vibration, etc., it is possible to suppress the loss of the original function of the biological sample.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by the following description.
[Example 1]
(Preparation of coating solution)
Using CYTOP (registered trademark, manufactured by Asahi Glass Co., Ltd.), a coating solution was prepared by dissolving in CT-solv100E (manufactured by Asahi Glass Co., Ltd.) so that the concentration thereof was 0.9% by mass.
(Production of evaluation device)
An ITO film having a thickness of 150 nm and a sheet resistance value of 10 Ω / cm ² was formed as an electrode on a glass substrate. Further, the coating solution was applied onto the electrode by spin coating to form a dielectric layer having a thickness of 0.5 μm. In spin coating, the glass substrate was spun at 500 rpm for 10 seconds and then spun at 1000 rpm for 20 seconds.
Next, a polystyrene petri dish having a through hole having a diameter of 35 mm formed on the bottom surface portion is adhered to the dielectric layer using an adhesive, and an evaluation device including a recess having an inner bottom surface formed of the dielectric layer is manufactured. did.

[Protein adhesion test]
In order to simply evaluate the cell adhesiveness of the evaluation device, the adhesiveness of a protein considered to behave in the same manner as a cell with respect to the adhesiveness was measured and evaluated. For the measurement, Advances in Polymer Science 57 Polymers in Medicine. (Western book) and Surface modification of polymers for medical applications. Biomaterials 1994. Vol.15 No.10 725-736 were referred.
(1) Preparation of color developing solution and protein solution As a color developing solution, 50 mL of peroxidase coloring solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), manufactured by KPL) and TMB Peroxidase Substrate (manufactured by KPL) ) Was mixed with 50 mL.
As a protein solution, a protein (POD-goat anti mouse IgG, Biorad) diluted 16,000 times with a phosphate buffer solution (D-PBS, Sigma) was used.
(2) Protein adsorption 3 mL of the protein solution is dispensed into the recess of the evaluation device, the counter electrode is arranged, a voltage of 3 V is applied to the evaluation device, and the electrode potential of the evaluation device is set to 3 V at room temperature. The protein was adsorbed by standing for 1 hour. Separately from this, the same operation was performed except that the electrode potential of the evaluation device was set to -3V. In addition, a voltage was not applied to the evaluation device and the electrode potential of the evaluation device was set to 0 V, and the device was left at room temperature for 1 hour in the same manner as described above. In these, it can be considered that the surface potential of the inner bottom surface of the recess of the evaluation device is equivalent to the electrode potential.
As a blank, 2 μL of the protein solution was dispensed into 3 wells in a 96-well microplate per well.
(3) Well Washing Phosphate buffer solution (D-PBS, manufactured by Sigma) containing 0.05% by mass of a surfactant (Tween 20, manufactured by Wako Pure Chemical Industries, Ltd.) in the recess of the evaluation device on which the protein is adsorbed ) 4 times (using 16 mL).
(4) Coloring solution dispensing 3 mL of the coloring solution was dispensed into the recesses of the evaluation device that had been washed, and a coloring reaction was performed for 7 minutes. The color reaction was stopped by adding 1.5 mL of 2N sulfuric acid.
For the blank, 100 μL of the coloring solution was dispensed into 3 wells of a 96-well microplate for 7 minutes, and then the coloring reaction was performed by adding 50 μL of 2N sulfuric acid to each well. Stopped.
(5) Preparation for absorbance measurement 150 μL of the liquid was taken from the recess of the evaluation device and transferred to a 96-well microplate.
(6) Absorbance measurement and protein adsorption rate Q
Absorbance was measured at 450 nm using MTP-810Lab (Corona Electric Co., Ltd.). Mean absorbance of blank (N = 3) was _{A 0.} The inner bottom surface of the surface potential of the recess (electrode potential of the evaluation device) 3V, -3 V, for each of those and 0V, the absorbance of the liquid is moved from the evaluation device to a 96-well microplate was A _1.
The protein adsorption rate Q was determined by the following equation.
Q = A ₁ / {A ₀ × (100 / amount of dispensed blank protein solution)} × 100
= A ₁ / {A ₀ × (100/2 μL)} × 100 [%]
The results are shown in FIG.

  As shown in FIG. 3, in the evaluation device of Example 1, the protein adsorption rate Q increased in the order of the surface potential of the inner bottom surface of the recess (electrode potential of the evaluation device) of −3V, 0V, and 3V. This result shows that the adsorptivity of the cells in culture to the first surface can be controlled by controlling the surface potential of the first surface of the dielectric layer.

[Protein desorption test]
(1) Protein adsorption 3 mL of the protein solution is dispensed into the concave portion of the evaluation device, a counter electrode is placed, a voltage of 3 V is applied to the evaluation device, and the surface potential of the inner bottom surface of the concave portion (device for evaluation) The electrode potential was set at 3 V and allowed to stand at room temperature for 1 hour to adsorb proteins.
As a blank, 2 μL of the protein solution was dispensed per well into 3 wells in a 96-well microplate.
(2) Protein Desorption A voltage of −3 V was applied to the evaluation device on which the protein was adsorbed, and the surface potential of the inner bottom surface of the recess (the electrode potential of the evaluation device) was set to −3 V, and left at room temperature for 1 minute. Next, the recess was washed four times with 4 mL of a phosphate buffer solution (D-PBS, manufactured by Sigma) containing 0.05% by mass of a surfactant (Tween 20, manufactured by Wako Pure Chemical Industries, Ltd.).
Moreover, it carried out similarly about what set the standing time in the state which set the surface potential of the inner bottom face of a recessed part to -3V as 0 minute, 5 minutes, and 10 minutes. Note that the standing time of 0 minutes means that cleaning was performed with the surface potential of the inner bottom surface of the recess (electrode potential of the evaluation device) set to 0V.
(3) Coloring solution dispensing, absorbance measurement preparation, absorbance measurement, and protein adsorption rate Q
In the same manner as in (4) to (6) of [Protein Adhesion Test], the protein adsorption rate Q was measured for each of the standing times of 0 minutes, 1 minute, 5 minutes, and 10 minutes.
The results are shown in FIG.

  As shown in FIG. 4, in the evaluation device of Example 1, after allowing the protein to be adsorbed, the standing time with the surface potential of the inner bottom surface of the recess (electrode potential of the evaluation device) being −3 V was 10 minutes. As a result, the protein adsorption rate Q was significantly lower than that when the standing time was 0V. As a result, by controlling the surface potential of the first surface of the dielectric layer, the adsorptivity of the cells to the first surface is controlled after culturing, and the biological sample can be detached at an arbitrary timing. Indicates that is possible.

DESCRIPTION OF SYMBOLS 1 Cell culture apparatus 10 Culture container 10a Inner bottom surface 12 Dielectric layer 12a 1st surface 12b 2nd surface 14 1st electrode 16 2nd electrode 18 Applied voltage mechanism

Claims (3)

A transparent culture container in which a culture solution is accommodated; a dielectric layer formed on the inner surface side of the culture vessel so that a first surface is in contact with the culture solution; and the first of the dielectric layers A first electrode provided on a second surface opposite to the surface, a second electrode provided in the culture vessel so as to be separated from the dielectric layer, the first electrode, and the second electrode An applied voltage mechanism for applying a voltage between the electrode and
The cell culture device, wherein the surface potential of the first surface is changed by applying a voltage between the first electrode and the second electrode. A biological sample production method for producing a biological sample by cell culture using the cell culture apparatus according to claim 1,
A culture step of culturing cells by the applied voltage mechanism with the potential of the first electrode with respect to the second electrode as E ₁ (V) to obtain a biological sample adsorbed on the first surface;
Desorption that causes the potential of the first electrode with respect to the second electrode to be E ₂ (V) lower than E ₁ (V) and desorbs the biological sample from the first surface by the applied voltage mechanism. A biological sample manufacturing method comprising: a step.   The biological sample manufacturing method according to claim 2, wherein the biological sample is a cell sheet.

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