JP2005021082A - System and method for controlling culture of cell or tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple system and a method for artificially controlling the adhesion position and extending direction of cells or tissue without the need of pattering cell-adhesive and cell-nonadhesive regions prior to cell culture. <P>SOLUTION: The system for culturing cells or tissue and controlling the adhesion position and extending direction of the cells or tissue at that time comprises a substrate immobilized with a cell-nonadhesive substance, electrodes placed closely to the substrate, and a computer for controlling both the function and action of the electrodes. This system works as follows: By applying oxidation potential onto the electrodes, active oxidative species are formed electrochemically, modifying the cell-nonadhesive substance immobilized on the substrate into a cell-adhesive substance. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a cell or tissue culture control apparatus and method. More specifically, the invention of this application relates to a culture control apparatus and method for artificially controlling the adhesion position and extension direction of cells or tissues without requiring a large-scale apparatus.
[0002]
[Prior art]
The cells have many functions such as adhesion, growth, proliferation, secretion, gene expression, etc., and form a complex network to exhibit various physiological functions. In order to elucidate such complicated life phenomena, various approaches are conceivable, and one of the approaches is pattern culture in which the adhesion position and extension direction of cultured cells are controlled and cultured in an arbitrary pattern. It has been. Many researches and studies have been made on methods for controlling the patterning of the cultured cells. For example, a pattern culturing method using photolithography has been proposed (Non-Patent Document 1). This method of Non-Patent Document 1 uses a so-called mask (or negative) in which a pattern such as an adhesion position of cultured cells is copied on a microchip in advance, and transfers the pattern onto a substrate. Lithography is used. By this photolithography, cell adhesion and non-cell adhesion regions are patterned on a substrate and used for pattern culture of cultured cells. However, various reagents (organic solvents) used for photolithography have an adverse effect such as dissolving many polymer materials such as polystyrene used as a scaffold for cell adhesion in culturing cells and tissues. In addition, the scaffold for cultured cells is lost, and it is difficult to maintain good culture conditions.
[0003]
In recent years, a pattern culture method using polydimethylsiloxane (PDMS) has been developed (Non-patent Document 2). This pattern culturing method is called soft lithography in which a cell adhesion substance is transferred to a substrate using a surface having a fine structure made of polydimethylsiloxane (PDMS), which is a rubbery polymer, to produce a pattern. Based on technology. By adopting such a technique using PDMS, photolithography on the culture substrate becomes unnecessary, and the adverse effects on the culture conditions can be reduced without causing dissolution of the scaffold by the organic solvent as described above. it can.
[0004]
However, since the pattern culture shown in Non-Patent Document 2 is based on photolithography or electron beam lithography for surface processing of PDMS, there is a problem that a large-scale apparatus is required and costs are increased. . In addition, cell adhesion and cell non-adhesion areas need to be patterned prior to cell culture, limited to the location where cells adhere, and the subsequent control of cell elongation and proliferation In addition, it was difficult to pattern a plurality of types of cells step by step. Such a problem can be expected to be solved by making the surface of the substrate non-cell-adhesive and modifying and changing any part to cell-adhesiveness, but such technology has not yet been reported or published. Absent.
[0005]
[Non-Patent Document 1]
Yoshihiro. Ito, Biomaterials 20; 2333-2342, 1999
[Non-Patent Document 2]
Ravi S. Kane, Shuichi Takayama, et al. , Biomaterials 20; 2363-2376, 1999
[0006]
[Problems to be solved by the invention]
Accordingly, the invention of this application has been made in view of the circumstances as described above, solves the problems of the prior art, does not require a large-scale device, and is an area of cell adhesion and cell non-adhesion. It is an object of the present invention to provide a culture control apparatus and method capable of artificially controlling the adhesion position and extension direction of cells or tissues without the need for patterning prior to cell culture.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the invention of this application is first a culture control apparatus for culturing a cell or tissue and controlling the adhesion position and extension direction of the cell or tissue at that time. A substrate on which a non-adhesive substance is fixed, an electrode disposed in the vicinity of the substrate, and a computer for controlling the function and operation of the electrode; by applying an oxidation potential to the electrode, Provided is a culture control apparatus characterized in that a chemically active oxidized species is generated and a cell non-adhesive substance immobilized on a substrate is modified into a cell adhesive substance.
[0008]
In addition, the invention of this application is, secondly, a culture control apparatus characterized in that the cell non-adhesive substance is albumin, and thirdly, albumin is bovine serum albumin (BSA). A culture control device characterized in that it is derived from a fourth, and fourthly, a culture control device characterized in that the active oxidation species is an active halogen species generated by oxidizing halide ions. Provides a culture control device characterized in that the active halogen species is either hypobromite (HOBr) or hypochlorous acid (HOCl).
[0009]
Furthermore, according to the sixth aspect of the present invention, sixthly, a cell or tissue is cultured using the culture control device according to any one of the first to fifth aspects, and the adhesion position of the cell or tissue at that time and A method for controlling the extension direction, wherein an electrode is disposed in the vicinity of a substrate on which a cell non-adhesive substance is immobilized, and a computer for controlling the function and operation of the electrode is connected to the electrode, and this By applying an oxidation potential to the electrode under the control of the computer, an active oxidizing species is generated electrochemically, and the non-cell-adhesive substance immobilized on the substrate is modified into a cell-adhesive substance, A control method characterized by controlling the adhesion position and extension direction of a cell or tissue is provided. And, seventhly, by performing the control method of the sixth invention step by step, a further new detail is provided. Or seeded with tissue, and co-cultured to provide a control method and controlling the adhesion position and extension direction Saishi this culture.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The invention of this application has the features as described above, and the embodiments thereof will be described in detail below.
[0011]
The first invention of this application is a culture control apparatus that can artificially control the adhesion position and extension direction of cells and tissues without requiring a large-scale apparatus. In this culture control device, a cell non-adhesive substance having an activity that inhibits adherence of cultured cells and tissues to a scaffold or the like is immobilized on a substrate using a hydrophobic interaction, and an electrode such as a microelectrode is attached. It is arranged in the vicinity of this substrate. As the substrate, a semiconductor, a thin glass plate, or the like can be used, and is not particularly limited. Further, the electrodes can be arbitrarily changed in arrangement, and the movement and operation thereof can be controlled by a computer connected to the electrodes.
[0012]
Then, by applying an oxidation potential to the electrode under the control of the computer, the active oxidized species can be generated locally electrochemically. By this activated species, the non-cell-adhesive substance can be modified into a cell-adhesive substance. That is, by applying an oxidation potential (or sometimes an oxidation pulse if the application time is short) to an electrode at an arbitrary location on the substrate, active oxidized species are generated, and only that location is cell adhesive. It can be modified into a substance, allowing cells or tissues to adhere to the scaffold. Since this electrode is small in size, these series of reactions are performed locally as described above.
[0013]
The cell adhesion area depends on the distance between the electrode and the substrate and the time of the electrode reaction, and is governed by the diffusion supply of the active chemical species generated at the electrode tip.
[0014]
The “cell” in this application may be a cultured cell of any origin, for example, a plant cell, an insect cell, or an animal cell, and cells of different origins or non-cells such as a collagen gel membrane, silk thread, nylon mesh, etc. Or a fused cell. Of course, primary cells or established cells may be used. Particularly preferred are animal cells. Further, primary cells in animal cells include chicken embryo-derived cells (PSG), rat primary cardiomyocytes, rat primary hepatocytes, mouse primary bone marrow cells, porcine primary hepatocytes, bovine vascular endothelial cells, human primary umbilical cord blood cells, human primary Examples include human primary nerve cells such as bone marrow hematopoietic cells and dorsal root ganglion cells (DRG). Examples of the established cell line include CHO cells derived from Chinese hamster ovary cells, HeLa cells derived from human uterine cancer, Huh7 cells and HepG2 cells derived from human liver cancer. In addition, cells obtained by gene manipulation such as plasmid introduction or virus infection into these cells can also be used in the invention of this application.
[0015]
The cell may be an adherent cell or a floating cell, but is preferably an adherent cell because the effects of the invention of this application can be obtained more remarkably.
[0016]
On the other hand, examples of the “tissue” include liver, heart, kidney, skin, bone, cartilage, bone marrow, and the like, and a tissue derived from these exemplified tissues.
[0017]
In this application, the non-cell-adhesive substance means a substance having a non-adhesive activity that inhibits adhesion because many cells and tissues often adhere to the scaffold in order to grow and elongate themselves. Point to. Therefore, the second invention of this application is that, when albumin is used as a non-cell-adhesive substance in the above-described culture control apparatus, the rate of the albumin modification reaction itself is extremely high. It is possible to induce adhesion of many cultured cells such as endothelial cells, which is a preferred embodiment. Furthermore, as a third invention, there is provided a culture control apparatus characterized in that the albumin is derived from bovine serum albumin (BSA). Of course, the cell non-adhesive substance is not limited to albumin, and any substance may be used as long as it has an activity to prevent cells from adhering to a substrate, that is, a scaffold. Examples of albumin include fetal bovine serum (FBS), ovalbumin contained in egg white, lactalbumin in milk, and the like.
[0018]
The invention of this application eliminates the non-adhesive activity of cell non-adhesive substances such as albumin by using a denaturing agent, active oxidation species, etc., and allows cells and tissues to adhere to the scaffold. That is, it can be modified into a cell adhesive substance. Examples of the denaturing agent include guanidine hydrochloride.
[0019]
The invention of this application can use either a modifier or an active oxidized species as described above, but it is preferable to use an active oxidized species. That is, as a fourth aspect of the present invention, there is provided a culture control apparatus characterized by being an active halogen species generated by oxidizing halide ions as an active oxidizing species. The “halide ion” is an ion of a halogen element belonging to Group 17 (7B) of the periodic table, and any halide ion can be used. Specifically, it is fluorine (F), chlorine (Cl), bromine (Br), iodine (I), or asterathion (At). These active halogen species are preferably either hypobromite (HOBr) or hypochlorous acid (HOCl), and can be provided as the culture control device of the fifth invention.
[0020]
Furthermore, as a sixth invention, there is provided a method for culturing a cell or tissue using the culture control device of the above invention, and controlling the adhesion position and extension direction of the cell or tissue at that time. In this control method, an electrode is disposed in the vicinity of a substrate on which a cell non-adhesive substance such as albumin is immobilized, and a computer for controlling the operation of the electrode is connected to the electrode. Then, by applying an oxidation potential to the electrodes under the control of this computer, the electrochemically active oxidized species can be locally generated, and the non-cell-adhesive substance can be modified into a cell-adhesive substance. it can. Thereby, the adhesion position and extension direction of the cell or tissue can be controlled.
[0021]
And as 7th invention, the control method characterized by performing the control method of 6th invention in steps can be provided. In the case of this control method, new cells or tissues can be further seeded and co-cultured with previously cultured cells or tissues, and the adhesion position and extension direction during this culture can be controlled. That is, with respect to a substrate on which cells and tissues are already cultured, an electrode is two-dimensionally scanned at an arbitrary position where cells and tissues are not cultured, and an oxidation potential (oxidation pulse or the like) is applied to the electrodes, Generate active oxidizing species. This active oxidized species again transforms the non-cell-adhesive substance at any point (local) of the substrate into a cell-adhesive substance, and seeds new cultured cells or tissues there, thereby performing pattern culture. It can be carried out. The kind and origin of cells and tissues to be newly seeded at this time are not particularly limited, and may be different from the previously cultured cell types and tissues. According to the purpose of research, experiment, etc., the type and origin of cells and tissues can be appropriately selected.
[0022]
As described above, the invention of this application does not require a large-scale apparatus, eliminates the need for patterning cell adhesive and non-cell-adherent areas prior to cell culture, and artificially regulates cell or tissue. The culture control device and method capable of controlling the adhesion position and extension direction will be realized, and it can be provided as an innovative tool in a wide range of fields such as medical engineering and sensor engineering. It can be expected to bring about great economic effects.
[0023]
Examples will be described below, and the invention of this application will be described in more detail. Of course, the invention is not limited by the following examples.
[0024]
【Example】
<Example 1> A pretreated glass plate of a substrate was used as a substrate. The substrate was washed and immersed in a 10 mM n-octadecyltriethoxysilane / benzene solution for 2 hours to make the substrate surface hydrophobic. Next, the BSA was immersed in a phosphate buffer (PBS) solution (0.5 mg to 10 mg mL ⁻¹ , pH 7.4) containing bovine serum albumin (BSA) for 30 minutes, thereby hydrophobizing the BSA. To be fixed by adsorption.
<Example 2> Effect of sodium hypobromite on BSA (1) Examination using cultured cells The effect of sodium hypobromite (NaBrO) on BSA immobilized on a substrate was evaluated. NaBrO is easily decomposed by a weak acid and is characterized by liberating hypobromous acid (HBrO) having a strong oxidizing power. An arbitrary portion of the substrate was immersed in a NaBrO solution having the above-described characteristics (a solution obtained by diluting NaBrO with 9% effective bromine 10 times with PBS) on a substrate having BSA immobilized on the entire surface of the substrate. Next, HeLa cells were seeded on the substrate as cultured cells and cultured.
[0025]
The result is as shown in FIG. This was confirmed by fluorescent staining by a known method. The left side of the broken line indicates that NaBrO treatment is not performed, and the right side of the broken line indicates that NaBrO treatment is performed. By performing NaBrO treatment, HeLa cells adhere to the substrate surface and are cultured. This is considered to mean that the cell non-adhesive activity of BSA was lost and modified to cell adhesiveness.
(2) Surface Plasmon (SPR) Measurement The effect of sodium hypobromite on BSA was evaluated using a surface plasmon biosensor (SPR 670M, Nippon Laser Electronics) for surface plasmon (SPR) measurement. When a culture medium containing serum was flowed after BSA was immobilized on the entire surface of the substrate, as shown in FIG. 1b, the resonance angle, which is a reference for the signal after BSA immobilization, increased. On the other hand, when the NaBrO solution was brought into contact with the substrate on which BSA was immobilized, a decrease in the resonance angle was observed as shown in FIG. 1c, and further, when the culture medium containing serum was brought into contact, the resonance angle was increased. It was.
[0026]
From these results, it can be considered that BSA immobilized on the substrate is oxidized with a NaBrO solution (that is, HBrO), so that serum proteins are easily adsorbed on the substrate. That is, cell adhesion substances (in this case, cell adhesion proteins) such as fibronectin (FN) and laminin (LN) contained in serum adhere to the substrate, thereby promoting adhesion of the cells to the substrate. it is conceivable that.
[0027]
Although not shown in the figure, the cell non-adhesive activity of BSA is lost by exposing a substrate on which a cell non-adhesive substance such as BSA is immobilized to an aqueous solution of a denaturing agent such as guanidine hydrochloride. The cultured cells can adhere to and grow on the substrate surface.
<Example 3> Use of electrochemical method (1) Production of electrode The electrode used for generation of the active halogen species was produced as a microelectrode. The platinum (Pt) wire thinned by electrolytic etching was inserted into a glass capillary and heated to melt the glass, and the Pt wire was enclosed in the capillary. Next, by polishing the tip of the capillary, the Pt portion was exposed to produce a Pt microdisk electrode. Obtained in a 4 mM potassium ferrocyanide solution and from a voltammograph, the electrode radius was 15 μm. The chip diameter including the insulating glass portion was about 30 μm.
(2) Electrode operation and control The prepared electrodes were placed near the substrate and under the control of a computer. That is, the electrode position was used by fixing the microelectrode to a stepping motor drive type xyz stage under the control of a computer. An Ag / AgCl electrode was used as a reference electrode, and the oxidation current of halide ions was controlled by a computer.
[0028]
FIG. 2a is a diagram schematically illustrating how a BSA is modified locally in a substrate using HOBr generated by a microelectrode, and FIG. 2b is a graph in which HeLa cells are cultured after applying a Br ^- oxidation potential. It is the figure which showed a mode.
[0029]
As illustrated in FIG. 2a, the BSA-immobilized substrate prepared in Example 1 was immersed in an aqueous solution containing 25 mM KBr, 0.1 M KCl, 0.1 M phosphate buffer (pH 7.5). The distance between the microelectrode and the substrate was kept at d to 5 μm. Then, by applying a 1.7V Br ^- oxidation potential to the electrode, HOBr was generated locally on the substrate, and the cell non-adhesive activity of BSA on the substrate was modified to cell adhesiveness.
[0030]
The result is as shown in FIG. 2b. A Br ^- oxidation potential was applied to the electrode for 60 seconds, and the electrode was locally placed and scanned on the substrate on which BSA was immobilized. Then, HeLa cells were seeded and cultured as cultured cells. It was possible to grow HeLa cells only at the locations where the electrodes to which the Br ^- oxidation potential was applied were arranged and scanned.
[0031]
When using HOCl to modify a cell non-adhesive substance (BSA in the examples) fixed to the substrate to a cell adhesive substance, 0.1 M KCl, 0.1 M phosphate buffer ( Immerse in an aqueous solution containing pH 7.5) and apply a 1.9 V Cl ^- oxidation potential to the electrodes.
<Example 4> Effect on culture of cultured cells by two-dimensional movement of electrode As exemplified in Example 3 (2), a microelectrode and a BSA-immobilized substrate were installed. Then, Br ^- and while the electrode holding the state of applying an oxidation potential to the electrode at a speed of 1250 micrometers / s, by scanning two-dimensionally, and the cell non-adhesive activity of BSA was modified to cell adhesion. At this time, the pattern width changes depending on the moving speed of the electrode, and an array of single cells can be formed by moving the electrode at a high speed. This movement of the electrode is performed under the control of a computer, and arbitrary pattern culture can be performed. By utilizing this feature, in the case of nerve cells, the extension direction of axons can also be controlled.
[0032]
The results were as shown in FIG. 3 and FIG. FIG. 3 is a view in which electrodes are two-dimensionally scanned in a spiral shape to modify BSA, and HeLa cells are seeded and cultured as cultured cells. As shown in FIG. 3, HeLa cells adhered in a spiral shape and proliferated.
[0033]
FIG. 4a is a diagram showing how dorsal root ganglion cells (DRG) are cultured by a normal method, and FIG. 4b is a diagram showing the result of seeding and culturing DRG on a substrate modified with BSA. is there. When the modification is not performed, the cells proliferate randomly as shown in FIG. 4a. On the other hand, as shown in FIG. 4b, when the modification is performed, the axon along the portion where the modification is performed. Was confirmed to elongate.
[0034]
Thus, it was confirmed that cells can be cultured along a pattern (shape) by two-dimensionally scanning the electrode with an arbitrary pattern (shape) while maintaining the electrode potential.
<Example 5> Re-seeding of cultured cells In FIG. 5a, the operation of Example 3 (2) is performed on a substrate on which cells are already cultured to locally modify the cell non-adhesive activity. The process of seeding and culturing cultured cells at the locations is schematically illustrated.
[0035]
As illustrated in FIG. 5a, by repeating the electrode operation of Example 3 (2), another cultured cell is seeded and cultured later at an arbitrary position even on a substrate on which cultured cells have already been cultured. can do. Of course, by repeating such electrode operation step by step, it is possible to inoculate and culture cultured cells (also different types of cells) in multiple steps.
[0036]
The results are as shown in FIGS. 5b, c and d. FIG. 5b shows HeLa cells divided into two groups and subjected to pattern culture. Further, as shown in FIG. 5c, since the calcein treatment was performed before pattern culture of the right cell population, it was confirmed that only the left cell population was fluorescently stained. FIG. 5d is a diagram showing a state after 24 hours of culture. As shown in FIG. 5d, it was confirmed that the cell population was cultured in a circular shape only at the location where the local modification by the electrode operation was performed.
<Example 6> Use of albumin-fixed substrate and substrate electrode As illustrated in Fig. 6a, a substrate provided with an electrode part is placed on a substrate on which BSA is fixed via a spacer, and Example 3 (2) starts from the electrode part. In this way, active halogen species were generated, and only the portion corresponding to this electrode part was modified by removing BSA from cell non-adhesive activity, and cultured cells were cultured.
[0037]
The results were as shown in FIGS. 6b and 6c. And the electrode portion is arranged on the thick line position shown in 6b, the using the substrate having the electrode portion, Br ^- and the oxidation potential is applied for 10 seconds. Next, HeLa cells were seeded on this modified BSA-fixed substrate and cultured. As shown in FIG. 6c, it was confirmed that cultured cells (HeLa cells in this example) were adhered, grown, and induced along the shape of the electrode part.
[0038]
【The invention's effect】
As described above in detail, according to the invention of this application, a large-scale apparatus is not required, and it is not necessary to pattern cell adhesion and cell non-adhesion regions prior to cell culture. A culture control apparatus and method capable of controlling the adhesion position and elongation direction of a tissue are provided.
[Brief description of the drawings]
FIG. 1 is a diagram showing the effect of sodium hypobromite on BSA, a is a diagram confirmed by fluorescent staining of cell culture state, and b and c are the results of surface plasmon measurement, respectively. .
FIG. 2a is a diagram schematically illustrating how BSA is locally modified in a substrate using HOBr generated by a microelectrode, and FIG. 2b is a diagram in which a Br ^- oxidation potential is applied to the electrode; It is the figure which showed a mode that the HeLa cell was culture | cultivated, after modifying the albumin of a board | substrate.
FIG. 3 is a diagram showing a state in which cells are cultured on a substrate by two-dimensionally scanning an electrode in a spiral shape and applying an oxidation potential to the electrode.
FIG. 4 is a diagram showing the culture state of dorsal root ganglion cells before (a) and after (b) modification of albumin on a substrate by an oxidation potential.
FIG. 5a shows that a substrate on which cells are already cultured is applied with an oxidation potential to an electrode to locally modify cell non-adhesion activity, and cultured cells are seeded and cultured at this location. It is the figure which illustrated schematically the process to perform, As a result, the figure (b) which showed the mode of HeLa cell culture for 2 hours, the fluorescence photograph figure at the time of performing a calcein process before the pattern culture of the right cell group ( c) A diagram (d) showing a state after 24 hours of HeLa cell culture.
FIG. 6a shows a substrate on which albumin is immobilized and a substrate having an electrode part installed through a spacer, an oxidation potential is applied to the electrode part, and the cell non-adhesive activity of albumin is locally lost. FIG. 6B is a diagram schematically illustrating a state in which cultured cells are cultured after being modified, FIG. 6B is a diagram illustrating the shape of the used electrode part, and FIG. 6C is the electrode illustrated in FIG. 6B. It is the figure which showed the result of the cell culture at the time of using a board | substrate with a part.

Claims (7)

A culture control apparatus for culturing cells or tissues and controlling the adhesion position and extension direction of the cells or tissues at that time, a substrate on which a cell non-adhesive substance is immobilized, an electrode disposed in the vicinity of the substrate, And a computer for controlling the function and operation of the electrode, and by applying an oxidation potential to the electrode, an electrochemically active oxidized species is generated and the non-cell-adhesive substance immobilized on the substrate A culture control device characterized by reforming the material into a cell adhesive substance. The culture control apparatus according to claim 1, wherein the non-cell-adhesive substance is albumin. The culture control apparatus according to claim 2, wherein the albumin is derived from bovine serum albumin (BSA). 4. The culture control apparatus according to claim 1, wherein the active oxidation species is an active halogen species generated by oxidizing halide ions. The culture control apparatus according to claim 4, wherein the active halogen species is either hypobromite (HOBr) or hypochlorous acid (HOCl). A method for culturing a cell or tissue using the culture control device according to any one of claims 1 to 5 and controlling an adhesion position and an extension direction of the cell or tissue at that time, wherein the cell non-adhesive substance comprises: An electrode is arranged in the vicinity of the fixed substrate, and a computer for controlling the function and operation of the electrode is connected to the electrode, and by applying an oxidation potential to the electrode under the control of the computer, It is characterized by controlling the adhesion position and extension direction of cells or tissues by electrochemically generating active oxidized species and modifying the cell non-adhesive substance immobilized on the substrate into a cell adhesive substance. Control method to do. A control method comprising: stepwise performing the control method according to claim 6 and further seeding and co-culturing new cells or tissues, and controlling an adhesion position and an extension direction during the culture.

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