Production Method For And Use Of Polymer Thin-Film Culture Plat For Production Method For And Application Of Cell Sheet

Abstract

The present invention provides a culture plate comprising a copolymer formed from a first monomer for forming a thin film having high surface free energy and a second monomer for forming a thin film having low surface free energy, a method for producing the culture plate, and a method for producing and transferring a cell aggregate in the form of a cell sheet by using the culture plate. The present invention has the effect of producing a cell aggregate in the form of a cell sheet through an easy and simple process in comparison with prior art.

Claims

1. A culture plate comprising a copolymer, the copolymer being formed of: a first monomer for allowing a thin film having low surface free energy to be formed; and a second monomer for allowing a thin film having a higher surface free energy than the thin film formed of the first monomer to be formed.

2. The culture plate of claim 1, wherein the first monomer has a surface free energy of 60 mJ/m.sup.2 or lower.

3. The culture plate of claim 1, wherein the first monomer is selected from the group consisting of divinylbenzene, vinyl benzoate, styrene, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, isopropyl methacrylate, acryl amide, allyl methacrylate, 2-isocyanatoethyl methacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) methyl ester methacrylate, 2-hydroxyethyle methacrylate, 1,2,4-trivinylcyclohexane, furfuryl methacrylate, tetrahydrofurfuryl methacrylate, hexyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, propargyl methacrylate, 1,4-butanediol divinyl ether, isobornyl acrylate, ethylene glycol diacrylate, propargyl acrylate, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, hexavinyldisiloxane, Hexavinyldisiloxane, 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, 2,4,6-trimethyl-2,4,6-trivinylcyclotrisilazane, dimethylphenylvinylsilane, heptadecafluorodecyl methacrylate, perfluorodecyl acrylate, heptafluorobutyl methacrylate, 1,1,1,3,3,3-Hexafluoroisopropyl methacrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentyl dicrylate, 2-perfluorohexylethyl methacrylate, 2,2,2-trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecafluoroheptyl acrylate, 1H,1H,2H,2H-heptadecaflurodecyl acrylate, di(ethyleneglycol)di(vinyl) ether, 1,9-decadiene, methacrylic anhydride, 1,2,4-trivinylcyclohexane, 2-(methacryloyloxyl)ethyl acetoacetate, allyl acetoacetate, and maleic anhydride.

4. (canceled)

5. The culture plate of claim 1, wherein the second monomer has a surface free energy of 60 mJ/m.sup.2 or higher.

6. The culture plate of claim 1, wherein the second monomer is selected from the group consisting of 2-vinylpyridine, 4-vinylpyridine, vinylimidazole, vinylpyrrolidone, 4-aminostyrene, 9-vinylcabazole, 2-(diethylamino)ethyl acrylate, diethylaminoethylacrylate, dimethylaminoethylacrylate, diethylaminoethylacrylate, 2-chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, 2-(dimethylamino)ethyl methacrylate, t-butylaminoethyl methacrylate, dimethylaminomethyl styrene, methacrylic acid, acrylamide, methacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, vinyl-N-methylpyridinium chloride, N-vinylcaprolactam, allylamine, N-(4-vinylbenzyl)-N-dimethylamine, and acrylonitrile.

7. (canceled)

8. The culture plate of claim 1, wherein the copolymer has a surface free energy of 30-90 mJ/m.sup.2.

9. The culture plate of claim 1, wherein the culture plate is for manufacturing a cell sheet type of cell aggregate.

10. The culture plate of claim 1, wherein a material for the culture plate is selected from the group consisting of glass, a metal, a metal oxide, a fiber, paper, and plastic.

11. The culture plate of claim 10, wherein the plastic is selected from the group consisting of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethanes (PU), polycarbonate (PC), polyvinylidene chloride (PVDC), polytetrafluorethylene (PTFE), polyetheretherrketone (PEEK), and polyetherimide (PEI).

12. (canceled)

13. A method for surface modification of a culture plate, the method comprising: degrading an initiator to form free radicals; subjecting a first monomer and a second monomer to a change polymerization reaction by the free radicals to form a copolymer; and depositing the copolymer on a culture plate to form a thin film, wherein the method employs initiated chemical vapor deposition (iCVD).

14. The method of claim 13, wherein the first monomer has a surface free energy of 60 mJ/m.sup.2 or lower.

15. The method of claim 13, wherein the first monomer is selected from the group consisting of divinylbenzene, vinyl benzoate, styrene, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, isopropyl methacrylate, acryl amide, allyl methacrylate, 2-isocyanatoethyl methacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) methyl ester methacrylate, 2-hydroxyethyle methacrylate, 1,2,4-trivinylcyclohexane, furfuryl methacrylate, tetrahydrofurfuryl methacrylate, hexyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, propargyl methacrylate, 1,4-butanediol divinyl ether, isobornyl acrylate, ethylene glycol diacrylate, propargyl acrylate, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, hexavinyldisiloxane, Hexavinyldisiloxane, 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, 2,4,6-trimethyl-2,4,6-trivinylcyclotrisilazane, dimethylphenylvinylsilane, heptadecafluorodecyl methacrylate, perfluorodecyl acrylate, heptafluorobutyl methacrylate, 1,1,1,3,3,3-Hexafluoroisopropyl methacrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentyl dicrylate, 2-perfluorohexylethyl methacrylate, 2,2,2-trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecafluoroheptyl acrylate, 1H,1H,2H,2H-heptadecaflurodecyl acrylate, di(ethyleneglycol)di(vinyl) ether, 1,9-decadiene, methacrylic anhydride, 1,2,4-trivinylcyclohexane, 2-(methacryloyloxyl)ethyl acetoacetate, allyl acetoacetate, and maleic anhydride.

16. (canceled)

17. The method of claim 13, wherein the second monomer has a surface free energy of 60 mJ/m.sup.2 or higher.

18. The method of claim 13, wherein the second monomer is selected from the group consisting of 2-vinylpyridine, 4-vinylpyridine, vinylimidazole, vinylpyrrolidone, 4-aminostyrene, 9-vinylcabazole, 2-(diethylamino)ethyl acrylate, diethylaminoethylacrylate, dimethylaminoethylacrylate, diethylaminoethylacrylate, 2-chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, 2-(dimethylamino)ethyl methacrylate, t-butylaminoethyl methacrylate, dimethylaminomethyl styrene, methacrylic acid, acrylamide, methacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, vinyl-N-methylpyridinium chloride, N-vinylcaprolactam, allylamine, N-(4-vinylbenzyl)-N-dimethylamine, and acrylonitrile.

19. (canceled)

20. The method of claim 13, wherein the copolymer has a surface free energy of 30-90 mJ/m.sup.2.

21. The method of claim 13, wherein the culture plate is for manufacturing a cell sheet type of cell aggregate.

22. A method for transfer of a cell aggregate, the method comprising: (a) allowing at least one layer of cell sheet type of cell aggregate to adhere onto a surface of a holed structure; and (b) disposing the holed structure such that a cell aggregate-adhering surface of the holed structure faces a site in need of application of the cell aggregate, and then peeling only the holed structure.

23. The method of claim 22, wherein the holed structure is selected from the group consisting of a nitrocellulose membrane, a nylon membrane, a polyvinylidene fluoride (PVDF) membrane, a polytetrafluoroethylene (PTFE) membrane, a polycarbonate membrane, a mixed cellulose ester (MCE) membrane, a polyamide membrane, and a polyethersulfone (PES) membrane, and wherein the holed structure has one or more holes.

24. The method of claim 22, wherein in the peeling of the holed structure in step (b), a phosphate buffer solution or a cell culture medium is dropped on the opposite surface of the cell aggregate-adhering surface of the holed structure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows a structure of a culture plate according to an embodiment of the present invention.

[0051] FIG. 2 is a schematic diagram depicting the type of cell aggregate depending on the injection ratio of monomers in the present invention.

[0052] FIG. 3 shows the results of analyzing, using Fourier transform infrared spectroscopy (FT-IR), chemical structures of the surfaces coated with a first monomer polymer (pDVB), a second monomer polymer (p4VP), and pD4V copolymers prepared in an example of the present invention.

[0053] FIG. 4 shows the results of measuring the contact angle and surface free energy of the surfaces coated with a first monomer polymer (pDVB), a second monomer polymer (p4VP), and pD4V copolymers prepared in an example of the present invention.

[0054] FIG. 5 shows microscopic image results of cells (NIH3T3) cultured on the surfaces coated with a first monomer polymer (pDVB), a second monomer polymer (p4VP), and pD4V copolymers prepared in an example of the present invention.

[0055] FIG. 6 shows images depicting that a cell sheet form cultured on a culture plate of the present invention was spontaneously separated from the culture plate by a buffer.

[0056] FIG. 7 shows optical and fluorescence microscopic images depicting a procedure in which an adult stem cell sheet (hMSC cell sheet) was formed on the surface coated with pD4V prepared in an example of the present invention, spontaneously separated from the surface, and then collected.

[0057] FIG. 8 is a schematic diagram depicting a procedure in which a cell sheet of the present invention was stacked on a holed structure and the resultant structure was applied to a place in need of application of the cell sheet, such as a substrate or a disease model.

[0058] FIG. 9 shows images depicting that two cell sheets formed on a culture plate of the present invention were separated, collected, and then stacked.

DETAILED DESCRIPTION

[0059] The objects, features, and advantages of the present invention would become apparent through the detailed description that is illustrated later with reference to the accompanying drawings. Therefore, a person having ordinary skill in the technical field to which the present invention pertains would easily implement the technical idea of the present invention. Furthermore, in the description of the present invention, detailed descriptions of known techniques associated with the present invention will be omitted when it is determined that the detailed descriptions unnecessarily obscure the gist of the present invention. Hereinafter, preferred embodiments according to the present invention will be described in detail the with reference to the accompanying drawings.

EXAMPLE 1

Preparation of Culture Plate

[0060] In the deposition of PD4V, the deposition was carried out for 1 hour and 30 minutes by allowing a divinylbenzene (DVB) monomer (Sigma-Aldrich), a 4-vinylpyridine (4VP) monomer (Sigma-Aldrich), and tert-butyl peroxide (TBPO, Sigma-Aldrich) as an initiator at a ratio of 60:240:60 to flow into an initiated chemical vapor deposition reactor (iCVD, Daeki Hi-Tech Co., Ltd) while a filament temperature of 140 C., a substrate temperature of 23 C., and a chamber pressure of 300 mTorr were maintained in the reactor, and as a result, a culture plate with a 400-nm thick DVB-4VP copolymer (nPD4V) was obtained.

EXAMPLE 2

Surface Analysis of Cell Sheet Culture Plate

[0061] After the deposition of the polymer thin film, the molecular skeleton and fraction of the polymer were measured using Fourier transform infrared spectroscopy (FT-IR, ALPHA FT-IR absorption mode, Bruker Optics). As a result, as shown in FIG. 3, the presence of the 4VP molecule was confirmed through peaks at 1596 cm-1 and 1415 cm-1 (two dot lines on the left side), and the presence of the DVB molecule and the polymer was confirmed through peaks at 710 cm-1 and 903 cm-1 (two dot lines on the right side).

[0062] After the deposition of the polymer thin film, the surface contact angles on the substrate relative to 5 l of distilled water and diiodomethane (DIM) were measured using a contact angle analyzer (Phoenix 150, SEO, Inc.). As a result, it could be verified as shown in FIG. 4 that the surfaces were modified with the polymers formed of different mixing ratio of monomers, leading to different contact angles. On the basis of the results, the surface free energy of the substrate was calculated using Van Oss-Chaudhury-Good (OCG) equation. The results confirmed that the surface free energy value varies depending on the proportion of a polymer.

EXAMPLE 3

Observation of Cell Morphology According to Proportion of Copolymer

[0063] After NIH3T3 cells were cultured on a cell culture dish coated with DVB-4VP copolymer (PD4V), the formation of a cell sheet was investigated. When the cells were sufficiently grown, the cells were fixed with 4% formaldehyde, the nucleus and actin were stained using DAPI and phalloidin, and then the cells were observed by a fluorescence microscope. It was observed as shown in FIG. 5 that the cells grew well without toxicity on all of the culture plates and that spheroids were formed on the DVB culture surface and the cells adhered to and grew on the 4VP culture surface. It was observed that the cells adhered to and grew on the copolymer culture surfaces (pD4V1 and pD4V2), but the cells were spontaneously separated as a cell sheet type after washing with Dulbecco's Phosphate Buffered Saline (DPBS).

EXAMPLE 4

Formation and Separation of Cell Sheets

[0064] After NIH3T3 and hMSC cells were cultured on 35pi dish on which Pd4V prepared in Example 1 above was deposited, the formation of a cell sheet was investigated. For cell culture, Dulbecco's Modified Eagle Medium (DMEM)/10% FBS/1% antibiotic (penicillin streptomycin, Gibco) were used for NIH3T3 cells, and Minimum Essential Medium (MEM )/17% FBS/1% antibiotic (penicillin streptomycin, Gibco) were used for hMSC cells. Cell sheets were formed when the cells were cultured for 3-5 days. Then, the culture liquid was removed, followed by washing with Dulbecco's Phosphate Buffered Saline (DPBS), and here, it could be confirmed that the formed cell sheets were spontaneously separated from the surface of the culture plate (FIGS. 6 and 7).

EXAMPLE 5

Separation and Collection of Cell Sheets Formed on Culture Plate and then Stacking of Cell Sheets

[0065] In order to manufacture cell sheets, cells were cultured on a cell culture plate coated with PD4V polymer until the intercellular junction could sufficiently occur. Thereafter, the cultured cells were separated as a sheet type by using Dulbecco's Phosphate Buffered Saline (DPBS) solution. After one cell sheet peeled from the culture dish was adsorbed and then transferred to a new cell culture dish, the cell culture dish was left in an incubator containing saturated steam at 37 C. for a proper time (for example, 15-30 minutes). During the time, the cell sheet adhered onto the culture dish. Next, a second cell sheet immediately after peeling was adsorbed together with a culture liquid into a pipette, and dropped on the first cell sheet fixed on the culture dish. A new culture liquid was again slowly dropped on the two dropped sheets, so that the two sheets could join in a state in which the second sheet overlapped the first sheet. Cell sheets could be sequentially stacked by repeating the same procedure.

EXAMPLE 6

Use of Holed Structure when Cell Sheets were Stacked and then Transferred

[0066] In order to stack the cell sheets manufactured according to the present invention and then more easily transfer the staked sheets to another cell culture dish or a subject in need of application of cell sheets, the following method was used.

[0067] First, as shown in FIG. 8, one cell sheet peeled from the culture dish was dropped to adhere onto a surface of a holed structure (for example, a nitrocellulose membrane with one or more holes). Then, another peeled cell sheet was additionally dropped to adhere to a surface of the cell sheet, which had already adhered to the nitrocellulose membrane, and thus two cell sheets were stacked. A desired number of cell sheets can be stacked by repeating the same procedure.

[0068] After the two stacked cell sheets, together with the membrane, were transferred to a new cell culture dish, the cells were incubated in an incubator containing saturated steam at 37 C. for a proper time (for example, 5-30 minutes). As a result, the stacked cell sheets adhered onto the culture dish, and are separated from the membrane as the holed structure. As such, cell sheets, while being stacked on a holed structure, can be transferred to a desired site.

[0069] The present invention described above can be made into various substitutions, transformations, and modifications to a person having ordinary knowledge in the technical field to which the present invention pertains without departing from the spirit and scope of the present invention, and therefore, the present invention is not limited to the above-described examples and accompanying drawings.