KR102041223B1 - 3 Dimensional Shape-Specific Cell Sheet and Method for Producing the Same - Google Patents

Abstract

The present invention relates to a cell sheet prepared using a shape memory polyisopropylacrylamide for the production of a patient-specific cell sheet. To prepare a cell sheet. The patient-specific cell sheet produced through this can promote the healing rate for various wounds of the patients.

Description

3D shape-specific cell sheet and method for producing the same}

The present invention relates to a three-dimensional shape customized cell sheet and a method of manufacturing the same. More specifically, the present invention utilizes the shape memory phenomenon of the water-swellable resin support to disperse seed the cells on the support to incubate the cell layer on the support to produce a cell layer sheet conforming to the three-dimensional shape of the patient wound. It's about how to do it.

The shape-customized cell layer sheet prepared by the method of the present invention may be covered with wound areas of various shapes of the patient to promote the speed and effect of wound healing.

Korean Patent Application No. 10-2008-0110395 (published May 17, 2010)
Korean Patent Application No. 10-2014-0120024 (2015.08.03)
Korean Patent Application No. 10-2012-0130729 (2014.05.28 publication)
Korean Patent Application No. 10-2014-0117892 (published Mar. 14, 2016)
Korean Patent Application No. 10-2015-0065479 (published Nov. 22, 2016)
Korean Patent Application No. 10-2017-0121344 (Registration No. 10-1963781)
Tissue engineering, which emerged after the 1980s, is a method for regenerating and treating damaged living tissue. In the biological tissue regeneration method, a cell tissue cultured using a support is implanted or injected into a living body. In this case, various problems occur due to various three-dimensional shapes of the tissue that requires regeneration.

Biodegradable scaffolds for processing or supporting cells are used in transplanted or implanted regenerated tissues produced through such tissue engineering techniques, and these are usually implanted or injected into the living body together with the cell tissues.

In this case, the number of tissue cells injected into the body by the volume occupied by the support in the regenerated tissue to be transplanted is reduced, thereby reducing the therapeutic effect.

In addition, when the support is transplanted with the cells, an immune response to the support occurs after transplantation, and furthermore, the biodegradable support may be induced even when the biodegradable support is degraded in the body.

In order to solve these problems recently, in cell sheet engineering, a new technique of tissue engineering, only cell tissues, except scaffolds, are transplanted into a wound of a patient during cell tissue transplantation.

This cell sheet transplantation technology has the advantage of being able to transplant relatively high density of cells into patient tissues, compared to conventional cell tissue transplantation methods in which scaffolds are transplanted together.

In order to effectively regenerate or treat living tissue, it is necessary to transplant tissue consisting of cells of high density. For this reason, attention has been paid to cell sheet engineering in which cell tissues are cultured in the form of implantable sheets to remove scaffolds and then only cell sheets are used for the treatment and regeneration of tissues.

Since the cell sheet technology developed to date is mainly a method of manufacturing a planar cell sheet, there are physical limitations in transplanting a two-dimensional planar cell sheet tissue covering a curved lesion of a three-dimensional shape.

Therefore, in the art, the development of a technique for culturing cell tissue in the form of a three-dimensional sheet that conforms to the shape of the living body to be implanted in order to accurately integrate the three-dimensional curved living tissue and improve the therapeutic effect It has long been required.

Treatment by culturing extracellular matrix (ECM) and cell-laminated sheets consisting of cells only on the support, then removing the cultured cell sheet from the support and allowing the isolated cell sheet to be implanted into the patient wound tissue site There is a need for a new biological tissue manufacturing technology required for regenerative medicine technology to increase the effect.

However, a technique for manufacturing a three-dimensional curved cell layer sheet that can be accurately incorporated into various types of biological tissues of three-dimensional shape has not been developed until now.

Therefore, the present inventors fabricate a resin support, which is a polymer having a temperature-sensitive shape recovery ability, to store a three-dimensional shape of a patient wound in order to cultivate a curved cell sheet corresponding to the three-dimensional shape of a patient's wound. Next, a technique for culturing cell tissues in sheet form on such a shape memory resin supporter is intended.

Accordingly, an object of the present invention is to provide a method for producing a shape-specific cell sheet for each patient site by culturing the cell sheet on a water-swellable polymer support sheet that stores a three-dimensional shape corresponding to a complex three-dimensional living tissue.

An object of the present invention is to provide a cell sheet having a shape to be stored in a shape memory water swellable resin sheet support,

1) adding a crosslinking agent to the mixture of the water-soluble monomer and water and administering to the mold in which the shape to be remembered is engraved;

2) preparing a cell support sheet, comprising a water-swellable resin having a shape recovery ability at the time of the polymerization reaction by polymerizing and crosslinking the monomer filled in the mold;

3) applying a pressure to the cell support sheet made of the water-swellable polymer resin to transform the sheet into a flat sheet;

4) inoculating by spreading the cells on the support sheet deformed into a flat shape;

5) culturing the cells inoculated on the cell support sheet to form a cell sheet layer on the support sheet;

6) restoring the shape of the cell support on which the cell sheet layer is formed to the shape at the time of polymerization of the step 2); And

7) Support The present invention provides a method for producing a three-dimensional cell sheet, comprising the step of separating the three-dimensional cell sheet layer required from the cell support restored to the shape at the time of the polymerization reaction.

It is still another object of the present invention to provide a cell sheet having a three-dimensional shape in which a tissue is tailored, which is cultured in a water-swellable resin sheet support having a property of being restored to the shape at the time of a polymerization reaction and manufactured in a three-dimensional shape.

The present invention is to produce a shape-customized cell sheet of a patient wound site or a tissue site requiring regeneration using a shape memory water swellable polymer support sheet, wherein the patient-customized cell sheet produced through the present invention is applied to various wounds of patients. Can speed up the healing process. Hereinafter, the present invention will be described in more detail.

As an example of the present invention, a polyacrylamide support sheet was prepared by irradiating ultraviolet rays to a composition comprising an aqueous acrylamide-based monomer solution, a crosslinking agent and a photoinitiator, and the support sheet exhibits a property of restoring to the shape at the time of the polymerization reaction. .

More specifically, in the present invention, an aqueous solution of N-isopropylacrylamide (NIPAAm) monomer was prepared and polymerized to prepare a polyisopropylacrylamide support sheet having a restoring force restored to its shape at the time of polymerization.

The molar ratio of N-isopropylacrylamide and water in the aqueous monomer solution used to prepare the polyisopropylacrylamide support sheet of the present invention is 0.045: 1 or less, and water is foamed through a solution polymerization process using water as a solvent. Serves to form polymeric foams of porous structure.

By the way, the porous structure is a factor that damages the mechanical properties of the polyisopropyl acrylamide. The material with low mechanical properties may change the shape of the material during the deformation process when the external pressure is applied, and even if it is not changed, the deformation shape may not be maintained.

This is the reason why the polyisopropylacrylamide support produced in the prior art cannot maintain shape memory. The fact that the support sheet has a shape restoring force means that even if it deforms when a pressure is applied, the pressure is removed and restored to the shape at the time of polymerization when exposed to a specific temperature.

In order to solve the problem of poor mechanical properties of the support sheet made of polyisopropylacrylamide foam, in the present invention, polyisopropylacryl having a shape recovery ability using an aqueous solution having a relatively high N -isopropylacrylamide monomer concentration An amide support sheet was prepared.

The polyisopropylacrylamide support sheet having the desired shape restoring force required to prepare the three-dimensionally shaped cell layer sheet of the present invention has an improved elasticity than the dried state when it is swollen with water or another solvent. It is possible to deform into a flat shape by applying external pressure in the swollen state.

When the solvent or water molecules that swell the polyisopropylacrylamide support sheet at room temperature are removed while maintaining the state in a flat shape, the polyisopropylacrylamide support sheet can maintain such a flat shape.

When the flat sheet of dried polyisopropylacrylamide is swollen by a solvent such as an external magnetic pole and water and reaches a certain condition, the polyisopropylacrylamide polymer is restored to the shape at the time of the polymerization reaction.

N -isopropyl acrylamide aqueous solution of the present invention is characterized by containing a relatively high concentration of N -isopropyl acrylamide compared to the prior art. For example, the weight ratio of N -isopropylacrylamide to water in the aqueous monomer solution of the present invention may be 87:13 to 95: 5, preferably 87:13 to 88:12.

The crosslinking agent used in the present invention can be used without limitation as long as it is a crosslinking agent that can be used in the conventional polymerization method used for producing polyisopropylacrylamide. Preferably, in the support of the present invention, N, N'-methylenebisacrylamide or tetramethylethylenediamine (TEMED) may be used as a crosslinking agent, but is not limited thereto.

In particular, the time for restoring the shape memory polyisopropylacrylamide support sheet shape may vary according to the amount of crosslinking agent added to the N -isopropylacrylamide monomer aqueous solution, so that shape recovery time may be controlled by controlling the content of the crosslinking agent.

For example, when the content of the crosslinking agent is 0.02% by weight based on 100% by weight of N -isopropylacrylamide, the time required for shape restoration is 115 to 125 minutes, and the shape restoration time is 185 to 195 minutes when the content is 0.1% by weight. Can be.

In addition, the optical crosslinking agent may be used, but is not limited thereto, 2-hydroxy-1-1 [4- (hydroxyethoxy) phenyl] -2-methyl-1propanone.

For example, the photoinitiator may be included in an amount of 0.01 to 0.05% by weight based on 100% by weight of N -isopropylacrylamide.

In one embodiment of the present invention, the polyisopropylacrylamide support is characterized in that it does not exhibit a porous structure or only shows a very low level of porosity.

As a special effect of minimizing the porous structure of the polyisopropylacrylamide support of the present invention, the mechanical strength of the support sheet is relatively excellent.

For example, the polyisopropylacrylamide support of the present invention may have a Young's modulus in the range of 14 to 15 MPa.

In another embodiment of the present invention, a polymerization reaction is performed by irradiating ultraviolet rays to a composition consisting of a monomer solution having a weight ratio of N -isopropylacrylamide and water of 87:13 to 95: 5, and a crosslinking agent and a photoinitiator. There is provided a shape memory polyisopropylacrylamide support having the property of restoring to a shape that was stored when swollen.

Various known techniques related to the production of shape memory polyisopropylacrylamide known to the art to date can be applied to the production process of the support for producing a cell sheet of the present invention.

Another embodiment of the invention N - mixing to prepare an aqueous monomer solution of isopropyl acrylamide (N-isopropylacrylamide) and water; Supplying the aqueous monomer solution to a mold having a three-dimensional shape; And a method of manufacturing a shape memory polyisopropylacrylamide support sheet comprising irradiating ultraviolet light to the aqueous monomer solution in the mold to polymerize the polyisopropylacrylamide.

In the method for producing a support sheet used in the present invention, the mold used is a shape in which the three-dimensional shape of the living tissue of the patient to be stored is engraved or embossed. One embodiment of the present invention used a L-shaped mold.

In the manufacturing method of the present invention, after the step of irradiating ultraviolet rays, further comprising the step of strengthening the mechanical properties of the polyisopropyl acrylamide support sheet which is polymerized through a crosslinking reaction, modified to a shape different from the previously memorized shape You can.

For example, when manufacturing a shape memory polyisopropyl acrylamide support for use in cell sheet production, in order to facilitate cell culture, polyisopropyl acrylamide is applied by applying physical pressure to the shape memory support sheet in which a specific shape is stored. Deformation can be made to flatten the support.

In one embodiment of the present invention, the step of immersing the prepared polyisopropyl acrylamide support sheet in water after the step of ultraviolet irradiation may be added to enhance the elasticity of the polyisopropyl acrylamide.

The N -isopropylacrylamide solution used in the production method of the present invention is characterized by containing a high concentration of monomer, for example, the weight ratio of N -isopropylacrylamide and water in the aqueous monomer solution is 87:13 to 95: 5 , Preferably from 87:13 to 88:12.

In addition, a crosslinking agent and / or a photoinitiator may be further mixed in the preparation of the aqueous monomer solution for the preparation of the shape memory polyisopropylacrylamide sheet.

The shape memory polyisopropylacrylamide support prepared by the manufacturing method of the present invention is characterized by recovering the shape memorized by swelling with a solvent, and the solvent can be used without limitation as long as the solvent can absorb the hydrogel. For example, water may be used.

The shape restoration time may vary depending on the temperature of the solvent used for shape restoration of the support sheet of the present invention, so that the shape restoration time may be adjusted by adjusting the temperature of the solvent.

For example, when the temperature of the solvent is 37 ℃ shape recovery time is 860 to 880 minutes, when the temperature of the solvent is 80 ℃ shape recovery time may be 100 to 110 minutes.

As described above, the shape memory polyisopropylacrylamide support of the present invention is very useful because the shape recovery time can be controlled by controlling the content of the crosslinking agent added to the monomer solution at the time of manufacture or the temperature of the solution used at the time of shape recovery.

In particular, when the shape memory polyisopropyl acrylamide is used as a support for producing a cell sheet, the time of attachment depending on the cells is different, so that the shape memory polyisopropyl acrylamide is particularly suitable for the production of various kinds of patient biometric customized cell sheets.

The shape memory polyisopropylacrylamide support of the present invention can be used to prepare cell sheets, for example, a shape can be stored in a desired shape to produce a cell sheet of the shape.

The cell sheet produced by the production method of the present invention may exhibit the same shape as that stored in the shape memory polyisopropylacrylamide, and the shape may have various shapes as necessary.

In particular, when the inoculation of the cells to the support having a shape including the reverse direction of the gravity direction is caused by the action of gravity causes the side cells to rush to the lower surface, the physical pressure in the shape memory polyisopropyl acrylamide of the present invention After the addition of a flat strain to the cell seeding (seed) is spread evenly spread on the front of the support (seed) to be able to be cultured into a cell layer of uniform thickness.

By restoring the shape of the support sheet in the state in which the cell layer sheets uniformly cultured are bound to the originally stored shape, the cell layer sheet having the uniform thickness is also restored to the three-dimensional shape originally stored.

Through such a process, after the original stored shape is restored, the cell layer sheet can be separated from the support sheet, thereby preparing the cell layer sheet of the present invention having the original stored shape.

In addition, the shape-memory water-swellable support sheet used in the present invention may further include a step of flatly deforming the O ₂ (oxygen) plasma, water-swellable support sheet by O ₂ (oxygen) plasma treatment Can enhance cell adhesion to cells.

In the manufacturing method of the present invention, the step of restoring to the shape originally stored is preferably performed after the cells are adhered to the surface of the shape memory support sheet. This is because when the water-swellable support sheet recovers the three-dimensional curved shape before being attached, the cells move again to the lower side under the influence of gravity (FIG. 3).

In one example of the present invention, the shape recovery time can be controlled by adjusting the content of the crosslinking agent added in the aqueous monomer solution or the temperature of the solution used to recover the shape when the shape memory polyisopropylacrylamide support is prepared. Shape recovery time can be controlled by adjusting various factors such as (adhesion time).

Another embodiment of the present invention includes a cell sheet manufactured by the method of the present invention, wherein the cell sheet may be characterized in that it is patient-specific.

The cell sheet of the invention is characterized in that it consists of cell layers of relatively uniform distribution. In general, when the cell sheet has various shapes, a cell distribution in the direction of gravity is concentrated to form a layer having a non-uniform cell sheet thickness. However, the cell sheet according to the present invention is obtained by flatly deforming a polyisopropylacrylamide support sheet. After forming the cell layer and restoring it to its original shape, the cell sheet shows a three-dimensional shape with uniform thickness through recovery through shape memory.

Cell sheet of the present invention is for use in the regeneration of human tissues or organs, for example, may be characterized by regenerating cells, tissues and / or organs, specifically can be used for skin regeneration or wound treatment. .

As with the present invention, using a cell sheet prepared using a shape memory polyisopropylacrylamide cell support can produce a cell sheet having a shape consistent with a patient wound site, which is a time required to heal the wound. You can save.

In addition, the time required for the polyisopropylacrylamide support to restore the original shape through the shape memory phenomenon can be adjusted as needed, which is useful for various types of patient-specific cell sheets using cells having different attachment times. Enable production.

1 is a process diagram showing a process of manufacturing a polyisopropyl acrylamide support of a desired shape, in accordance with one embodiment of the present invention.
Fig. 2 is a schematic diagram illustrating the manufacturing process of the shape memory polyisopropylacrylamide support of the present invention.
FIG. 3 illustrates a cell sheet manufacturing process using polyisopropylacrylamide (FIG. 3A) and a shape memory polyisopropylacrylamide (FIG. 3B) support according to the present invention.
Figure 4 shows the results of preparing a monomer solution containing N-isopropyl acrylamide in a low or high ratio.
FIG. 5 is a scanning electron microscope (SEM) showing an internal structure when a polyisopropylacrylamide is prepared using an aqueous monomer solution having a high ratio of N-isopropylacrylamide according to one embodiment of the present invention. It is a photograph.
FIG. 6 is a scanning electron microscope (SEM) photograph showing the internal structure when polyisopropyl acrylamide is prepared using a monomer solution having a low ratio of N-isopropyl acrylamide according to a comparative example.
7 is a beaker containing polyisopropyl acrylamide support made of L shape according to one embodiment of the present invention by applying pressure to a flat sheet shape to fix one side with a PDMS holder and distilled water (80 ° C.). The pictures were taken in 20-minute intervals.
Figure 8a is a result of measuring the mechanical strength of the polyisopropyl acrylamide prepared by containing a low proportion of N-isopropyl acrylamide according to the comparative example, Figure 8b is a high proportion of N- isopropyl acrylamide of the present invention The result of measuring the mechanical strength of polyisopropyl acrylamide is shown.
Figure 9 is made of a flat shape by adding an external pressure to the polyisopropyl acrylamide produced in the L shape after seeding the cells and incubated in the incubator (32 ℃) and stored at room temperature for a period of time desired cells Figure shows how the sheet is retrieved.
10 is a photograph showing a cell sheet cultured in an L shape according to one embodiment of the present invention.
Figure 11 is a photograph showing the cell death on the top of the cell sheet cultured in L shape in accordance with one embodiment of the present invention.
12 is a photograph showing cell death on the underside of a cell sheet cultured in an L shape according to one embodiment of the present invention.
FIG. 13 is a graph showing that the time required for the polyisopropylacrylamide support to restore its original shape through a shape memory phenomenon can be adjusted according to the temperature of distilled water according to one embodiment of the present invention.
14 is a graph showing that the time required for polyisopropylacrylamide to restore its original shape through shape memory phenomenon can be controlled according to the composition of N-isopropylacrylamide according to one embodiment of the present invention. .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Preparation of Shape Memory Polyisopropylacrylamide Support Sheet

1.1. N-isopropylacrylamide solution preparation

Phase separation in high concentration N -isopropylacrylamide solution was used to prepare a high content ratio (> 87%) N -isopropylacrylamide solution and a low content ratio (<13%) solution of N -isopropylacrylamide. Prepared. The low content ratio of polyisopropylacrylamide was prepared as a comparative group for comparison with the high ratio of polyisopropylacrylamide of the present invention.

Specifically, N -isopropyl acrylamide and distilled water were mixed in a 1: 1 mass ratio, and stirred for 5 minutes so that N -isopropyl acrylamide was sufficiently dissolved in distilled water. Over time, the low concentration of N -isopropylacrylamide solution and the high concentration of N -isopropylacrylamide solution were divided stably, and finally, when the N -isopropylacrylamide solution was stably separated, transferred to each of the solutions using a pipette to the vial N - isopropyl acrylamide: water weight ratio of 87: 100 in a high content ratio of N - isopropyl acrylamide solution, 5 ml of age farm: water weight ratio of 13: 100 in a low 5 ml of napalm solution in content proportions were obtained.

Next, 0.05 mg (0.1%) of N, N'-methylenebisacrylamide (MBAAm), a crosslinking agent, to react with ultraviolet rays when subjected to UV irradiation, and 2-hydroxy-1, a photoinitiator, were used. 0.005 mg (0.01%) of −1 [4- (hydroxyethoxy) phenyl] -2-methyl-1-propanone were added to each of the obtained napalm solutions.

1.2. Preparation of Polyisopropylacrylamide Using High Ratio of Monomer Solution

Example 1.1. To prepare polyisopropylacrylamide using a high proportion of N -isopropylacrylamide solution prepared in the above, a polymethylmethacrylate (PMMA) mold was manufactured by using a laser cutter, as shown in FIG. 1. Polydimethylsiloxane (PDMS) was filled in the prepared PMMA mold and placed in an oven for about one day to prepare a PDMS mold.

Then, the N -isopropyl acrylamide solution was filled in the prepared PDMS mold, and a polyisopropyl acrylamide support having a desired shape was prepared by irradiating a UV light source.

Specifically, a desired shape was completed using 3D modeling software, and a mold made of poly (methyl methacrylate) (PMMA) material having the shape was manufactured using a laser cutting machine. Next, a polydimethylsiloxane (PDMS) mold was manufactured using the prepared PMMA mold.

Thereafter, the prepared PDMS mold was filled with the high proportion of the nifam solution obtained in Example 1.1. To produce a poly-isopropyl acrylamide support of desired shape, N-isopropyl acrylamide solution is cold PDMS covering the upper surface with a cover glass N of the mold filled-acrylamide solution isopropyl surface that occur when it is filled in the PDMS mold The tension is minimized.

Finally, an N -isopropylacrylamide solution filled in a desired shape was irradiated with a UV light source for 30 minutes to prepare a polyisopropylacrylamide support using a high proportion of N -isopropylacrylamide solution. 4 shows a scanning electron microscope (SEM) photograph showing the internal structure of the manufactured polynipal support.

1.3. Preparation of Polyisopropylacrylamide Support Using Low Ratio N -isopropylacrylamide Solution (Comparative Example)

Polyisopropylacrylamide was prepared under the same conditions and methods as in Example 1.2, except that the low ratio of the naphage solution obtained in Example 1.1 was used instead of the high ratio of the naphage solution. A scanning electron microscope (SEM) photograph showing the internal structure of the prepared polyisopropyl acrylamide is shown in FIG. 5.

Example 2 Confirmation of Shape Memory Capability of Shape Memory Polyisopropyl Crylamide Support

2.1. Confirmation of shape resilience of shape memory polyisopropylacrylamide

In order to check whether the polyisopropylacrylamide support prepared in the aqueous solution having high concentration of N -isopropylacrylamide monomer prepared in Example 1 has shape memory characteristics, first, the method of Example 1 has an L-shape. A high proportion of polyisopropylacrylamide support was produced.

Then, in order to increase its elastic force, the produced L-shaped polyisopropylacrylamide support was immersed in distilled water at 80 ° C. for 15 minutes and flattened by applying pressure thereto. The flattened polyisopropylacrylamide support removes water molecules contained within the polyisopropylacrylamide support while drying at room temperature (25 ° C.) to lower its elasticity and increase the hardness.

Then, one side of the flattened polyisopropylacrylamide support was fixed with a PDMS holder made in the same manner as in Example 1 to produce a PDMS mold of the desired shape, which was placed in a beaker containing 80 ° C. distilled water. It was confirmed whether the shape is restored over time by 20 minutes. The results are shown in FIG.

As can be seen in Figure 7, it was confirmed that the shape is maintained when the pressure is applied to the polyisopropyl acrylamide polymerized in the aqueous solution having a high monomer ratio, when it is put in distilled water, polyisopolyacryl with time It was confirmed that the amide support was returned to its original shape before the pressure was applied while absorbing the water molecules in the distilled water.

The polyisopropyl acrylamide polymerized with such a high concentration of monomer was memorized in an L-shape, and when the pressure was applied, the polyisopropyl acrylamide could withstand the corresponding pressure. .

2.2. Property comparison

The internal structure and physical properties of the two kinds of polyisopropylacrylamide supports prepared in Example 2.1 were compared.

The results are shown in FIGS. 5, 6 and 8A, 8B, and the porosity of the polyisopropylacrylamide support polymerized with the low concentration aqueous monomer solution and the polyisopropylacrylamide support polymerized with the high monomer concentration solution. The low concentration of polyisopropylacrylamide has a young's modulus of 0.0607 MPa, whereas the high concentration of polyisopropylacrylamide supports has a young's modulus of 14.814 MPa, resulting in a difference of 250 times the Young's modulus (elastic modulus). We believe this is due to a difference.

Example 3. Preparation of Cell Sheet of Desired Shape

3.1. Cell sheet manufacturers

In order to confirm whether a cell sheet having a desired shape is recovered from the polyisopropyl acrylamide support polymerized with a high concentration aqueous monomer solution having the shape memory phenomenon prepared in Example 1, the L-shaped polyisopropyl according to Example 1 In order to increase the elastic force of the acrylamide support, it was immersed in distilled water at 80 ℃ for 15 minutes. After the elastic force of the polyisopropyl acrylamide support was secured, it was flattened by applying pressure, and plasma treatment was performed to make cells adhere to the flat polyisopropylacrylamide support better.

Then, to increase biocompatibility, the polyisopropyl acrylamide support was sterilized by irradiating the UV light source for 4 hours. Epithelial tumor cells (epidermoid carcinoma (A431)) were seeded on a polyisopropylacrylamide scaffold after sterilization, and cultured for 7 days in a cell incubator at 37 ° C. After 7 days of cell culture, the polyisopropylacrylamide support was stored at room temperature for 40 minutes in a culture medium. At this time, the culture medium was absorbed by the polyisopropylacrylamide support to obtain a cell sheet having a desired shape. Recovered.

The cell sheet recovery process diagram of the desired shape is shown in FIG. 9.

3.2. Confirm shape of recovered cells

In order to confirm whether the state of the cell sheet of the desired shape recovered in Example 3.1 can be used for the purpose of wound healing, it was confirmed whether the overall shape of the manufactured cell sheet has an L shape.

Specifically, the cell sheet cultured in L shape was stained using calcein AM (Sigma-Aldrich, USA), which is a dye capable of confirming that the cells of the fluorescent dyes are alive, and the result of confirming the fluorescence microscope in FIG. 10. Indicated.

As can be seen in Figure 10, it was confirmed that the cells maintain the L shape. In the process of remembering the shape of polyisopropyl acrylamide in a cell incubator at 37 ° C, the migration / proliferation of the cells proceeded while the cells adhered well to each side of the L shape without falling down by gravity. It means.

3.3. Confirm the degree of death of recovered cells

Further staining using ethidium homodimer-1, a dye capable of confirming cell death in fluorescent dyes, to more accurately determine the degree of cell death on each side of the cell sheet of the desired shape (L) prepared in Example 3.1 Proceeded. After cell staining was completed, the results of confirming the degree of cell death on each side using a fluorescence microscope at high magnification 200 are shown in FIGS. 11 (upper surface of the cell sheet) and FIG. 12 (lower surface of the cell sheet).

In cell experiments, when cells are stained with fluorescent dyes (calcein AM and ethidium homodimer-1), they have different colors depending on the state of the cells. When the cells become green, this means that the cells are in the live state. The red light indicates that the cell is dead. 11 and 12 it can be seen that there are significantly more cells in the live state compared to the cells in the dead state, which means that it can be used for the purpose of wound healing further determined to be a positive result.

Example 4. Shape Recovery Experiment with Temperature

In order to confirm how the high ratio of the polyisopropylacrylamide support shape memory properties produced in Example 1 had a change according to the temperature of the distilled water, a high proportion of the polyisopropylacrylamide having an L shape according to Example 1 Two supports were produced. Then, in order to increase the elastic force of the produced L-shaped polyisopropyl acrylamide support, it was immersed in distilled water at 80 ° C. for 15 minutes and applied to pressure to make it flat.

Then, one side of the flattened polyisopropylacrylamide support was fixed with a PDMS holder, and each high proportion of polyisopropylacrylamide was placed in a beaker containing distilled water at 37 ° C. and distilled water at 80 ° C. The effect on the shape memory phenomenon of the polyisopropyl acrylamide support was confirmed, and the results are shown in FIG. 13 and Table 1. FIG.

At this time, empty circle (○) shows the degree of restoration by time when the temperature of distilled water is 80 ℃ and filled circle (●) shows the degree of restoration by time when the temperature of distilled water is 37 ℃.

In order to quantitatively analyze the degree of shape recovery of polyisopropylacrylamide over time, the shape recovery ratio (R _r ) was defined as follows.

[Calculation 1]

Time (Min)
Recovery ratio (80)
Recovery ratio (37)
0
0
0
7
0.91

14
1.54

21
2.64

28
5.43
0.061
35
10.20

42
15.10

49
16.84

56
26.11
0.095
63
35.53

70
43.77

77
52.71

84
73.30
0.17
91
85.29

98
93.89

105
100

133

0.90
161

0.96
189

1.15
217

1.48
245

2.16
273

3.03
301

4.39
329

5.44
357

6.14
385

9.28
413

10.77
441

12.06
469

13.36
497

14.39
525

17.17
553

19.97
581

25.85
609

34.13
637

40.54
665

47.04
693

56.24
721

67.50
749

74.47
777

83.73
805

92.08
833

97.40
861

100
889

99.18

As can be seen in Figure 13 and Table 1, the shape recovery rate of the polyisopropyl acrylamide support is the rate of water molecules entering the polyisopropyl acrylamide as the molecular weight of water in the distilled water increases as the temperature of the distilled water increases It can be seen that since the increase in the temperature of the distilled water is increased, so that the shape recovery of the shape memory polyisopropylacrylamide support of the present invention can be adjusted according to the temperature of the solution to be treated for shape recovery. .

Example 5. Shape recovery experiment according to the amount of crosslinking agent used

In order to confirm what shape memory properties of the high proportion of the polyisopropylacrylamide support fabricated in Example 1 vary depending on the amount of cross-linker used, first of all, Two polyisopropylacrylamide supports were prepared by varying the content of the crosslinking agent N, N'-methylenebisacrylamide (MBAAm).

Specifically, the amount of crosslinking agent used was adjusted to 0.02% mass and 0.1% mass based on the total weight of polyisopropylacrylamide, respectively. The L-shaped polyisopropylacrylamide support prepared with different crosslinking agent content was immersed in distilled water at 80 ° C. for 15 minutes to increase elasticity, and was flattened by applying pressure thereto.

Next, one side of the flattened polyisopropylacrylamide was fixed with a PDMS holder and placed in a beaker containing distilled water at 80 ° C to check the effect of crosslinking agent content on the polyisopropylacrylamide shape memory phenomenon. The results are shown in FIG. 14 and Table 2.

At this time, the empty circle (○) indicates that the cross-linker concentration of polyisopropylacrylamide is 0.02% by weight? The degree of restoration was shown over time, and filled circle (●) is the degree of restoration over time when the cross-linker concentration of polyisopropylacrylamide is 0.1% by weight.

In order to quantitatively analyze the degree of shape recovery of different polyisopropylacrylamide supports at time, shape recovery ratio (R _r ) was defined as follows.

[Calculation 2]

Time (Min)
Recovery ratio (CS-0.02%)
Recovery ratio (CS-0.1%)
0
0
0
7
0.11
0.14
14
1.54
0.39
21
0.91
1.24
28
2.65
2.02
35
5.43
0.47
42
5.49
1.12
49
10.21
1.49
56
15.10
1.07
63
16.84
0.91
70
26.11
2.47
77
35.54
2.69
84
43.78
8.00
91
52.72
16.71
98
73.30
19.97
105
85.30
23.80
112
93.90
33.01
119
100
42.43
126
99.83
49.92
133
99.83
65.88
140

71.00
147

85.25
153

89.44
160

92.00
167

97.99
173

98.10
180

96.34
187

100

As can be seen in Figure 14 and Table 2, the shape recovery rate of the polyisopropyl acrylamide was found to decrease as the amount of crosslinking agent in the polynipalm increases. This means that as the crosslinking density in the polynipalm increases, crosslinking between the main chains forming the polyisopropylacrylamide support becomes dense and water molecules in distilled water have difficulty in penetrating into the polyisopropylacrylamide. Therefore, it was confirmed that the shape recovery ability of the shape memory polyisopropylacrylamide support of the present invention can be adjusted according to the crosslinking density of the polyisopropylacrylamide support.

Claims (21)

In the method of manufacturing the cell sheet of the shape memorized using the shape memory water-swellable resin support body,
1) administering N-isopropylacrylamide monomer aqueous solution and a crosslinking agent to a mold of a shape to be memorized;
2) polymerizing and crosslinking the N-isopropylacrylamide monomer filled in the mold to prepare a water swellable polyN-isopropylacrylamide resin support sheet;
3) depressurizing the water-swellable polyN-isopropylacrylamide resin support sheet prepared in step 2) into a flat shape;
4) dispersing and inoculating cells in the water-swellable polyN-isopropylacrylamide resin support sheet deformed into a flat shape;
5) culturing the inoculated cells dispersed in the water-swellable polyN-isopropylacrylamide resin support sheet prepared in step 4) to form a cell sheet on the water-swellable polyN-isopropylacrylamide resin support sheet ;
6) The cell sheet formed on the water-swellable polyN-isopropylacrylamide resin support sheet in step 5) and the water-swellable polyN-isopropylacrylamide resin support sheet are the polyN-isopropylacrylates of step 2). Maintaining the cell sheet and the water-swellable polyN-isopropylacrylamide resin support sheet shape to the shape of the mold by maintaining at an amide resin support sheet polymerization and crosslinking reaction temperature; And
6) separating the formed cell sheet from the water-swellable polyN-isopropylacrylamide resin support sheet restored to the mold shape
Including, the cell sheet manufacturing method restored to the mold shape.
delete delete delete The water-swellable polyN-isopropylacrylamide resin support sheet in which the polyN-isopropylacrylamide resin support sheet is crosslinked with N, N'-methylenebisacrylamide (MBAAm) in the cell sheet preparation method of claim 1. The cell sheet manufacturing method characterized by the above-mentioned. The method for producing a cell sheet according to claim 1, wherein the water-swellable polyN-isopropylacrylamide resin support sheet is a polyN-isopropylacrylamide resin support sheet crosslinked with tetramethylethylenediamine (TEMED). Cell Sheet Fabrication Method. The cell sheet manufacturing method of claim 1, wherein the elastic modulus of elasticity of the crosslinked polyN-isopropylacrylamide resin support sheet is 14 to 15 megapascals (Mpa). The method for producing a cell sheet according to claim 1, wherein a photoinitiator is used for the polymerization reaction of the polyN-isopropylacrylamide resin support sheet. The method for producing a cell sheet according to claim 8, wherein the photoinitiator is 2-hydroxy-1-1 [4- (hydroxyethoxy) phenyl] -2-methyl-1propanone. . The cell sheet manufacturing method of claim 1, wherein the weight ratio of the N-isopropylacrylamide monomer and water used to prepare the water-swellable polyN-isopropylacrylamide resin support is 87:13 to 95: 5. Cell sheet production method. The cell sheet manufacturing method of claim 10, wherein the weight ratio of the N-isopropylacrylamide monomer and water used to prepare the polyN-isopropylacrylamide resin support sheet is 87:13 to 88:12. , Cell sheet making method. Cells are dispersed and seeded in the shape memory water-swellable polyN-isopropylacrylamide resin support sheet, wherein the cells are restored to a three-dimensional shape at the time of the polymerization reaction, and dispersed and inoculated onto the water-swellable polyN-isopropylacrylamide resin support. A three-dimensional cell sheet produced by culturing the prepared cells. The cell sheet according to claim 12, wherein the cells are derived from living tissue cells. The cell sheet according to claim 13, wherein the cells are derived from living epithelial tissue cells. The cell sheet according to claim 13, wherein the cells are derived from organ tissue cells of a living body.
delete delete The cell sheet according to claim 12, wherein the polyN-isopropylacrylamide is a crosslinked resin support sheet. The cell sheet of claim 18, wherein the weight ratio of the N-isopropylacrylamide monomer and water used to prepare the polyN-isopropylacrylamide support sheet is 87:13 to 95: 5. Shaped cell sheet. The cell sheet of claim 19, wherein the weight ratio of the N-isopropylacrylamide monomer and water used to prepare the poly N-isopropylacrylamide support sheet is 87:13 to 88:12, 3 Cell sheet in dimensional shape.
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