KR20120125702A - Patterned substrate for culturing cells and preparation method of cell sheet using the same - Google Patents

Abstract

PURPOSE: A patterned cell cultivation plate and a producing method of a cell sheet using thereof are provided to rapidly obtain the cell sheet without damaging by culturing cells by using thermo-sensitive hydrogel and cell adhesion peptide as a culture plate. CONSTITUTION: A patterned cell cultivation plate includes thermo-sensitive hydrogel and cell adhesion peptide. The patterned cell cultivation plate has patterns on one surface. The thermo-sensitive hydrogel is obtained by adding carrot peroxidase and hydrogen peroxide to a polymer mixture containing more than one kind of polymer, and dehydrogenation bonding phenol analogs.

Description

Patterned cell culture substrate and cell sheet manufacturing method having a pattern using the same {PATTERNED SUBSTRATE FOR CULTURING CELLS AND PREPARATION METHOD OF CELL SHEET USING THE SAME}

The present invention relates to a patterned cell culture substrate capable of easily separating and recovering a cell sheet according to temperature change, and a method of manufacturing a cell sheet having a pattern using the same.

Transplantation methods for reconstructing damaged tissues of the human body include xenografts, allografts, and autografts. Xenografts have problems with transfer of common pathogens, including immune incompatibility and retroviruses. In addition, allografts have problems of donor immunity and availability, and autografts have difficulty obtaining adequate amounts of adequate tissue and problems with increased trauma to the patient.

In order to solve such a problem, the technique which tries to transplant artificial substitute or the thing which cultured and cultured as it is attracting attention is attracting attention. In 2004, a study on retinal regeneration using cell sheets made from pure cells itself was published in the New England Journal of Medicine. It is becoming.

Recently, Professor Okano of Tokyo Women's University in Japan has suggested that cells can be used as a "sheet-shaped" tissue by simply lowering the temperature of the cultured cells through cell sheet engineering.

Specifically, poly (N-isopropylacrylamide) (PIAAm), a temperature-sensitive polymer, was fixed to the surface of a commercially available polystyrene culture dish by an electron beam by a radical polymerization method. As described above, when the temperature-sensitive polymer is formed on the surface, the hydrophilic property is exhibited at 32 ° C as a critical temperature at a higher temperature and at a lower temperature than this. Therefore, in the culture environment at 37 ° C, the bottom of the culture dish is cell adhesive, and the cells adhere to the culture dish. Adhesion cells adhered to the dish soon increase in number due to cell division, leading to a confluency state in which the entire surface of the culture dish is filled with cells. When the confluence is reached, the low temperature treatment below the critical temperature of 32 ° C. causes the surface of the culture dish to become non-adhesive, resulting in a continuous sheet-like structure coming out of the culture dish. At this time, the polymer strongly adhered to the culture dish is separated from the cell sheet and remains in the culture dish.

However, such a technique is slow in recovering the cells and the cell sheet because water infiltrates from the edges of the cells and the cell sheet during the low temperature treatment, thereby causing the hydrophilization of the temperature-sensitive polymer grafted on the surface to proceed slowly.

On the other hand, in order to develop a culture matrix having a pattern having a temperature sensitive characteristic, a method of forming a matrix having a pattern by strongly pressing the surface of the matrix by an embossing process or the like and then grafting a temperature sensitive material using an electron beam has been proposed. The disadvantage is that the process is complicated.

In addition, the culture matrix material that is being studied a lot, such as polyethylene glycol-diacrylate (PEG-DA), alginate, PDMS, poly (N-isopropylacrylamide) (PIPAAm), etc. Since there are few ligands such as biomolecules required when attached, there is a significant limitation in the interaction between cell-matrix.

The present invention is to solve the above problems, an object of the present invention by culturing the cells using a temperature-sensitive hydrogel and cell-attached peptides as a culture substrate that reversibly shows swelling and contraction according to temperature changes, It is to provide a patterned culture substrate that can easily and quickly recover the cell sheet without damaging the cells.

It is also an object of the present invention to provide a method for producing a cell sheet having a pattern that can be used as an artificial tissue for regenerating directional tissue using the patterned culture substrate.

In order to solve the problems of the present invention as described above, the present invention

It provides a patterned cell culture substrate comprising a temperature-sensitive hydrogel and cell adhesion peptides, the pattern is formed on one side.

In addition,

Preparing a cell culture substrate comprising a temperature-sensitive hydrogel and the cell adhesion peptide, the pattern is formed on one surface;

Culturing the cells on the culture substrate; And

The temperature of the culture substrate is lowered below the low critical solution temperature and the temperature is raised above the low critical solution temperature to recover the cell sheet from the culture substrate.

It provides a cell sheet manufacturing method comprising a.

According to the present invention, the cell sheet can be easily recovered from the culture substrate quickly without damaging the cells while maintaining the pattern, and the cell sheet having the pattern can be very useful as an artificial tissue for regenerating oriented tissues among human tissues. Can be.

Figure 1 is a schematic diagram showing the process of the cell sheet manufacturing method according to the present invention.
Figure 2 is a schematic diagram showing the cell sheet separation recovery step according to the present invention.
3 is a scanning electron micrograph of the surface of the PDMS mold used in one embodiment of the present invention, and an optical microscope photograph of a patterned cell culture substrate prepared using the same.
Figure 4 is a photograph of the size change at room temperature, 37 ℃ and 4 ℃ of the Tetronic-tyramine conjugated hydrogel culture substrate prepared in one embodiment of the present invention.
Figure 5 is a graph showing the swelling ratio and size change of the Tetronic-Tyramine conjugate hydrogel culture substrate prepared in one embodiment of the present invention.
Figure 6 shows the separation pattern of the cell sheet according to temperature change using Tetronic TA-gel hydrogel (Tet-TA hydrogel) and alginate hydrogel (Alginate hydrogel) having a non-temperature sensitive characteristics as a substrate for cell culture The results observed with an optical microscope are shown.
Figure 7 shows the cell morphology and adhesion of the cell sheet prepared using a patterned Tetronic-tyramine conjugate hydrogel culture substrate and a patternless Tetronic-Tyramine conjugate hydrogel culture substrate in one embodiment of the invention. The results confirmed through immunofluorescence staining are shown.
FIG. 8 is a photograph illustrating a process of separating and recovering a cell sheet through temperature change of a patterned tetronic-tyramine conjugate hydrogel culture substrate in one embodiment of the present invention and moving it to a cover glass.
FIG. 9 is a cell sheet having a recovered pattern and a cell sheet having no pattern in the embodiment of the present invention are transferred to a fibronectin-coated glass surface, and then cultured for 24 hours and 72 hours, respectively, and then The cytoskeleton of, the extracellular matrix protein fibronectin assembly (fibronectin assembly), the adhesion protein paxillin is shown through the results of immunostaining.
FIG. 10 is a graph showing the results of measuring an average degree of orientation of nuclei of myoblast nuclei in the cell sheets separated and recovered in one embodiment of the present invention.
Figure 11 is a graph showing the results of measuring the ratio of the cell sheet myoblast cell nuclei separated and recovered in one embodiment of the present invention.
FIG. 12 is a cell sheet having a separated and recovered pattern in one embodiment of the present invention moved to a fibronectin-coated glass surface, and then cultured again. The observed results are shown.
Figure 13 is a graph showing the results of measuring the average orientation of the root canal by immunostaining myotubes of myoblasts after culturing by moving the cell sheet having a pattern and the cell sheet without a pattern to the cover glass.

Hereinafter, the present invention will be described in more detail.

The present invention provides a patterned cell culture substrate, a method for producing a cell sheet having a pattern of a certain direction using the same.

Patterned cell culture substrate according to the present invention comprises a temperature-sensitive hydrogel and cell adhesion peptide, the pattern is formed on one side.

The temperature sensitive hydrogel is a polymer having a crosslink, and reversibly shows shrinkage and swelling instead of sol-gel behavior with temperature change. As a result, the water content increases below the lower critical solution temperature (LCST) and the swelling phenomenon decreases above the low critical solution temperature. By using the temperature sensitive property of the hydrogel, the cell sheet can be separated and recovered while maintaining the pattern from the culture substrate by only controlling the temperature.

The cell adhesion peptide may be used as long as it has an amino acid sequence that is responsible for cell adhesion activity. In one example, a peptide comprising the amino acid sequence RGD, RGDS, REDV, YKNR, QPQGLAK or YIGSR is possible.

The culture substrate of the present invention can be manufactured using a polymer replication mold having a plurality of negative patterns as a template, and in particular, it is preferable to use a PDMS (polydimethylsiloxane) mold. In this case, the PDMS mold may be manufactured using a silicone mold having a plurality of embossed patterns as a basic mold, and may be prepared by pouring and curing a mixed solution of a polymer prepolymer and an initiator forming the PDMS, and then separating it from the silicone mold. have. The PDMS mold thus prepared can be used repeatedly several times.

The prepared PDMS mold is injected with a starting material capable of producing a temperature sensitive hydrogel and a cell-attached peptide together and gelled to obtain a culture substrate on which the pattern of the PDMS mold is replicated.

At this time, the temperature-sensitive hydrogel is a branched polymer having a phenol analog represented by the following formula (II) a, and a phenol analog represented by the following formula (II) b as shown in Scheme 1 below. Prepared by dehydrogenation between phenol analogs by adding carrot peroxidase (HRP) and hydrogen peroxide to one or more polymer mixtures selected from linear polymers linked to a type polymer and a phenol analog represented by formula (II) c:

[Reaction Scheme 1]

In this way, a patterned cell culture substrate can be obtained through a simple process by adding carrot peroxidase and hydrogen peroxide to a polymer in which a phenol analog is bound to a PDMS mold and gelling them at once.

Preferably, a phenol analog is added to the copolymer / tyramine conjugate of polyethylene oxide-polypropylene oxide-polyethylene oxide to form a crosslink via dehydrogen bonds between the conjugates by adding carrot peroxidase and hydrogen peroxide to form polyethylene oxide-polypropylene. A copolymer of oxide-polyethylene oxide / tyramine hydrogel is obtained. As the copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a copolymer having a trade name of Pluronic, Poloxamer, or Tetronic may be used. Tetronic-tyramine conjugates used in the examples of the present invention exhibit low critical solution temperatures at room temperature (15-25 ° C.).

The patterned cell culture substrate has convex portions having a width of 1 to 100 μm, and the spacing between the convex portions is preferably 1 to 100 μm.

Since the culture substrate of the present invention exhibits reversible swelling and contraction according to temperature change, the cell sheet can be easily and quickly recovered without damaging the cells, and thus can be used for producing cell sheets of various cells.

Hereinafter, a method for producing a cell sheet having a pattern using the patterned cell culture substrate of the present invention will be described in detail.

Figure 1 is a schematic diagram showing the process of the cell sheet manufacturing method according to the present invention. Referring to FIG. 3, the above steps will be described in detail.

First, a cell culture substrate including a temperature sensitive hydrogel and a cell attachment peptide and a pattern formed on one surface thereof is prepared.

Cell culture substrate preparation is as described above.

Next, the cells are cultured on the culture substrate.

Cells to be cultured are not particularly limited in the present invention, for example, liver, kidney, spleen, bone, bone marrow, thymus, heart, muscle, lungs, brain, testes, ovaries, islets, intestines, ears, skin Cells that can be isolated or activated from, gallbladder tissue, prostate, bladder, embryo, immune system and hematopoietic system can be used. Preferably, fibroblasts, osteoblasts, myoblasts, kidney cells, vascular endothelial cells, chondrocytes, hepatocytes, adipocytes, corneal epithelial cells, various stem cells, neurons, keratinocytes, muscle cells, cardiomyocytes or esophagus Incubate epithelial cells.

At this time, the culture conditions may vary depending on the type of cells, and the details thereof are omitted since they are well known in the art.

The cell adhesion peptide on the pattern surface formed on the culture substrate during the culturing process effectively induces cell adhesion and proliferation by acting as a ligand to the cell, and the cells proliferate along the pattern formed on the culture substrate. As a result, the cells grow while maintaining a constant specificity like the pattern of the culture substrate.

Subsequently, the temperature of the culture substrate is lowered to less than the low critical solution temperature, and the temperature is raised above the low critical solution temperature to recover the cell sheet from the culture substrate.

In the temperature sensitive hydrogel of the present invention, the water content is increased below the low critical solution temperature, the swelling phenomenon is reduced, and the water content is decreased above the low critical solution temperature. Using this property, the cell sheet is separated and recovered from the culture substrate.

Figure 2 is a schematic diagram showing the cell sheet separation recovery step according to the present invention.

Referring to FIG. 2, since the cell culture is performed at a low critical solution temperature or higher, the cell-side receptor (eg, integrin) and the cell adhesion peptide of the culture substrate are connected through interaction. However, if the culture substrate is lowered below the low critical solution temperature, the water content of the temperature sensitive hydrogel increases and swells and becomes large. .

At this time, it is preferable to lower the temperature of the culture substrate to 2 to 8 ℃ which is less than the low critical solution temperature and maintain for 5 to 15 minutes, and then to raise the temperature to 35 to 38 ℃ which is above the low critical solution temperature.

As such, the method of separating and recovering the cultured cell sheet from the substrate by changing only the temperature of the substrate can recover the cells without damaging the cells, unlike the method using a protease such as trypsin. Accordingly, the cell sheet obtained through the production method of the present invention is not only recover the adhesion protein molecules, extracellular matrix, membrane proteins and adhesion proteins of the cells secreted themselves during the cultivation, but also maintain a new pattern while maintaining the formed pattern. It is possible to move to the surface and have good viability even on the moved surface. In addition, the recovery time is shortened because the cell sheet is recovered through the size change rather than the change of the properties of hydrophilicity and hydrophobicity, such as poly (N-isopropylacrylamide) (PIPAAm), which is a temperature-sensitive polymer used in the related art. There is this.

In this manner, various cells are sheeted through the production method of the present invention, and the obtained sheets are stacked in multiple layers, so that tissues can be constructed in vitro for various organs. In addition, the cell sheet having a pattern can be very useful as an artificial tissue for regeneration of tissues such as oriented tissues, for example, muscle tissues, among human tissues.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

< Manufacturing example  1>

Tetronic-tyramine conjugates were prepared by carrying out as disclosed in Published Patent Publication No. 2010-0076173. Solution A was prepared by dissolving 5.2 wt% tetronic-tyramine conjugate (Tet-TA) and RGD peptide (2.0 mg / ml) in 0.025 mg / ml carrot peroxidase (HRP). Solution B was prepared by dissolving 5.2 wt% tetronic-tyramine conjugate (Tet-TA) in 0.0125 wt% hydrogen peroxide. Each solution A and B was placed in a dual syringe, and the two solutions were evenly spaced in a space with a 0.5 mm thick Teflon sheet between the PDMS mold and a glass plate having a 10 μm wide engraved pattern at 25 μm intervals. After gelation by injection, a hydrogel culture substrate having a pattern was obtained.

< Experimental Example  1>

The surface of the PDMS mold used in Preparation Example 1 was performed by scanning electron microscopic analysis, and the surface morphology of the hydrogel culture substrate having a pattern formed using the same was observed through optical microscopic analysis, which is shown in FIG. 3. .

As shown in Figure 3, it was confirmed that the hydrogel was produced in the same pattern form as the PDMS mold.

< Experimental Example  2>

In order to determine the physical properties of the hydrogel culture substrate prepared in Preparation Example 1, the swelling rate and size of the hydrogel according to the temperature difference was observed by changing the temperature to 4 ℃ and 37 ℃ at 30 minutes intervals, this is Figure 4 And FIG. 5.

4 and 5, the temperature sensitive hydrogel swells at 4 ° C. below the low critical solution temperature (LCST) and increases by 1.5 to 1.7 times, and at 37 ° C. above the low critical solution temperature (LCST). It was confirmed that the degree and size were reduced.

< Experimental Example  3>

In order to examine the adhesion of the cells on the surface of the patterned hydrogel culture substrate prepared in Preparation Example 1, the myoblasts were formed on the surface of the patterned hydrogel culture substrate and the glass substrate at 1 x 10 ⁵ cell / cm ² , respectively. Incubated for 24 hours at 37 ℃. In order to separate and recover the cell sheet, the temperature of the substrate was lowered to 4 ° C. and maintained for 10 minutes, and then raised to 37 ° C.

Figure 6 shows the separation pattern of the cell sheet according to the temperature change using Tetronic TA-gel hydrogel (Tet-TA hydrogel) and alginate hydrogel (Alginate hydrogel) having a non-temperature sensitive characteristics as a substrate for cell culture The results observed with an optical microscope are shown.

As shown in FIG. 6, when the temperature sensitive hydrogel was used as the culture substrate, as the hydrogel swelled and contracted according to the temperature change based on the low critical solution temperature (LCST), the cell sheet was brought to the surface of the culture substrate. On the other hand, when the alginate hydrogel having non-temperature sensitive characteristics was used as the culture substrate, the cell sheet was still attached to the substrate even if the temperature was changed.

< Experimental Example  4>

Hydrogel without pattern and hydrogel with pattern formed at 1 x 10 ⁵ cell / cm ² in order to examine the morphology and adhesion of cells on the surface of the hydrogel culture substrate having the pattern prepared in Preparation Example 1 Incubate on the surface for 37 hours at 37 ℃. In order to separate and recover the cell sheet, the temperature of the substrate was lowered to 4 ° C., and then raised to 37 ° C. The morphology and adhesion of the cells were confirmed by immunofluorescence staining, which is shown in FIG. 7.

Referring to FIG. 7, it was confirmed that the expression-related protein expression and the actin filament were formed according to the orientation of the pattern on the surface of the hydrogel having paxilin, which is an adhesion-related protein, and actin filament, which is a cytoskeletal structure. In the case of cells cultured on the surface of the hydrogel culture substrate without a pattern, the cytoskeleton also showed no directivity.

< Experimental Example  5>

In Experimental Example 3, a cell sheet having a pattern recovered and separated into a hydrogel culture substrate through temperature change was fixed to a cover glass coated with fibronectin, which is one of the extracellular matrix proteins, and this process is illustrated in FIG. 8. .

As shown in FIG. 8, it was confirmed that the cell sheet cultured on the surface of the temperature sensitive hydrogel culture substrate was completely moved away from the hydrogel according to the temperature change and moved to the cover glass through the method proposed in the present invention.

< Experimental Example  6>

The cell sheet having the recovered and recovered pattern in Experimental Example 3 and the cell sheet without the pattern were moved to the fibronectin-coated glass surface, and then cultured for 24 hours and 72 hours, respectively.

After culturing, the cytoskeleton of cells, fibronectin assembly, an extracellular matrix protein (fibronectin assembly), and paxillin, an adhesion protein, were observed through immunostaining and are shown in FIG. 9. The observed degree of quantitative analysis of the orientation degree of nuclei and the aspect ratio of the nuclei of the cell sheet myoblast nuclei, and the results are shown in FIGS. 10 and 11. At this time, the average orientation was measured based on the orientation of the pattern (0 °), or the average orientation was measured based on the orientation indicated by the attached cells.

Referring to FIGS. 10 and 11, even when a cell sheet cultured in a patterned hydrogel culture substrate moves on a glass having a flat surface, the orientation of the cells initially attached to the hydrogel culture substrate and the aspect ratio of the nucleus are maintained. It was confirmed that the expression of actin filament, fibronectin, and paxillin also maintained the orientation of the pattern.

< Experimental Example  7>

The cell sheet having the separated and recovered pattern in Experimental Example 3 and the cell sheet without the pattern were transferred to a fibronectin-coated glass surface, and then cultured for another 24 hours to obtain 48 cells under differentiation medium (5% horse serum) conditions. After further incubation, myotubes, which are a measure of differentiation of myoblasts, were observed through immunostaining, which is shown in FIG. 12.

Referring to FIG. 12, even when a cell sheet cultured in a patterned hydrogel culture substrate is moved to a glass having a flat surface, the cell sheet is differentiated to form a myotube. It was found that the root canal (myotube) having a pattern orientation was formed while maintaining the orientation of the cells attached to the culture substrate.

Figure 13 is a graph showing the results of measuring the average orientation of the root canal by immunostaining myotubes of myoblasts after culturing by moving the cell sheet having a pattern and the cell sheet without a pattern to the cover glass.

As shown in FIG. 13, the cell sheets cultured and recovered in the patterned cell sheet were confirmed to maintain the orientation of the pattern even after moving to another culture substrate.

Claims (10)

A patterned cell culture substrate comprising a temperature sensitive hydrogel and cell adhesion peptide, the pattern being formed on one surface. The method of claim 1, wherein the temperature-sensitive hydrogel is a branched polymer (branched) polymer is bound to the phenol analog represented by the following formula (II) a, as shown in Scheme 1 below, represented by the formula (II) b Prepared by dehydrogenation between phenol analogs by adding carrot peroxidase and hydrogen peroxide to one or more polymer mixtures selected from star-type polymers bound with phenol analogs and linear polymers bound with phenol analogs represented by formula (II) c Incubation substrate:
[Reaction Scheme 1]

The culture substrate of claim 1, wherein the temperature-sensitive hydrogel is a copolymer / tyramine conjugate hydrogel of polyethylene oxide-polypropylene oxide-polyethylene oxide. The culture substrate of claim 1, wherein the cell adhesion peptide is a peptide comprising an amino acid sequence RGD, RGDS, REDV, YKNR, QPQGLAK or YIGSR. Preparing a culture substrate comprising a temperature-sensitive hydrogel and the cell adhesion peptide, the pattern is formed on one surface;
Culturing the cells on the culture substrate; And
The temperature of the culture substrate is lowered below the low critical solution temperature and the temperature is raised above the low critical solution temperature to recover the cell sheet from the culture substrate.
Cell sheet manufacturing method having a pattern comprising a. The method according to claim 5, wherein the temperature-sensitive hydrogel is a branched polymer having a phenol analog represented by the following formula (II) a, represented by the following formula (II) a, represented by the formula (II) b Prepared by dehydrogenation between phenol analogs by adding carrot peroxidase and hydrogen peroxide to at least one polymer mixture selected from star polymers bound with phenol analogs and linear polymers bound with phenol analogs represented by formula (II) c Manufacturing method
[Reaction Scheme 1]

The method of claim 5, wherein the temperature sensitive hydrogel is polyethylene oxide-polypropylene oxide-polyethylene oxide / tyramine conjugate hydrogel. The method of claim 5, wherein the cell adhesion peptide is a peptide comprising the amino acid sequence RGD, RGDS, REDV, YKNR, QPQGLAK or YIGSR. The method according to claim 5, wherein the temperature of the culture substrate is lowered to 2 to 8 ℃ which is less than the low critical solution temperature and maintained for 5 to 15 minutes, the temperature is raised to 35 to 38 ℃ or more above the low critical solution temperature to separate the cell sheet from the culture substrate The manufacturing method which collect | recovers. According to claim 5, wherein the cells are myoblasts, fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, osteoblasts, kidney cells, adipocytes, corneal epithelial cells, stem cells, neurons, keratinocytes, muscle It is one method selected from the group consisting of cells, cardiomyocytes and esophageal epithelial cells.

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