POROUS CELLULAR SCAFFOLD COMPRISING SERUM-DERIVED PROTEIN, AND PRODUCTION METHOD THEREFOR

Abstract

Provide are a porous cell scaffold including a serum-induced protein and a method of manufacturing the same. A porous cell scaffold according to an embodiment may stably and continuously incubate cells, show a culture pattern suitable for the characteristics of each cell to simulate actual tissues, and have a stable culture and high in vivo engraftment rate. Accordingly, the porous cell scaffold can be usefully used in the evaluation of drug activity and toxicity using organoids, for use in cell therapy products, or in the production of a target protein.

Claims

1. A porous cell scaffold comprising a serum-induced protein obtained in such a manner that a serum or plasma protein aqueous solution is treated with a cross-linking agent to cross-link proteins therein and then a reducing agent is used to cause a reduction reaction.

2. The porous cell scaffold of claim 1, wherein the serum-induced protein is obtained by homogenizing a reaction product after reduction with the reducing agent.

3. The porous cell scaffold of claim 1, wherein the cross-linking agent comprises one selected from dextran dialdehyde, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, vinylamine, 2-aminoethyl methacrylate, 3-aminopropyl methacrylamide, ethylene diamine, ethylenfor exampleycol dimethacrylate, methyl methacrylate, N,N′-methylene-bisacrylamide, N,N′-methylenebis-methacrylamide, diallyltartardiamide, allyl(meth)acrylate, lower alkylenfor exampleycol di(meth)acrylate, poly lower alkylenfor exampleycol di(meth)acrylate, lower alkylene di(meth)acrylate, divinyl ether, divinyl sulfone, divinylbenzene, trivinylbenzene, trimethylolpropane tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, bisphenol A di(meth)acrylate, methylenebis(meth)acrylamide, triallyl phthalate, diallyl phthalate, allyl glycidyl ether, alkyl choloride, transglutaminase, lysyl oxidase, protein disulfide-isomerase, protein-disulfide reductase, sulfhydryl oxidase, lipoxygenase, polyphenol oxidase, tyrosinase, peroxidase, laccase, horseradish peroxidase, and a combination thereof.

4. (canceled)

5. The porous cell scaffold of claim 1, wherein the reducing agent comprises one selected from beta-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl) phosphine (TCEP), cysteine, glutathione, and tris(3-hydroxypropyl)phosphine (THPP).

6. The porous cell scaffold of claim 1, wherein a pore size of the porous cell scaffold is from 50 μm to 600 μm.

7. The porous cell scaffold of claim 1, wherein the porous cell scaffold has an elastic modulus of 1 kPa to 10 kPa.

8. The porous cell scaffold of claim 1, wherein the porous cell scaffold has a porosity of 10% to 80%.

9-13. (canceled)

14. A composition for tissue repair or tissue regeneration comprising the porous cell scaffold of claim 1 and cells seeded in the porous cell scaffold.

15. The composition of claim 14, wherein the cells comprise any one selected from: chondrocytes; fibrochondral cells; bone cells; osteoblasts; osteoclasts; synovial cells; bone marrow cells; nerve cells; fat cells; mesenchymal cells; epithelial cells, hepatocytes, muscle cells; stromal cells; vascular cells; stem cells; embryonic stem cells; mesenchymal stem cells; progenitor cells derived from adipose tissue; peripheral blood progenitor cells; stem cells isolated from adult tissues; pluripotent stem cells (iPS cells); cancer cells derived therefrom; and a combination thereof.

16. (canceled)

17. (canceled)

18. A method of producing a porous cell scaffold including a serum-induced protein, the method comprising: cross-linking proteins in a serum or plasma protein aqueous solution by treating the serum or plasma protein aqueous solution with a cross-linking agent; reacting the cross-linked proteins in the serum or plasma protein aqueous solution with a reducing agent to obtain a reaction product; and homogenizing the reaction product to obtain a serum-induced protein.

19. The method of claim 18, further comprising aging the serum-induced protein.

20. (canceled)

21. (canceled)

22. The method of claim 18, wherein the cross-linking agent is treated with a specific activity of 2 unit/mg to 100 unit/mg.

23. (canceled)

24. The method of claim 18, wherein the reducing agent has a concentration of 5 mM to 50 mM.

25. The method of claim 18, wherein the homogenizing the reaction product to obtain a serum-induced protein comprises homogenizing the reaction product by using a tissue grinder, followed by centrifuging to recover a suspension of the serum-induced protein.

26. The method of claim 19, wherein the aging of the serum-induced protein comprises washing the serum-induced protein and aging the washed serum-induced protein at a temperature of 1° C. to 15° C.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1 shows results obtained by culturing a SNU017 gastric cancer cell line for 9 days using a porous cell scaffold according to an embodiment.

[0076] FIG. 2 shows results obtained by culturing a SiHA cervical cancer cell line for 14 days using a porous cell scaffold according to an embodiment.

[0077] FIG. 3 shows results obtained by culturing hACs (chondrocytes) for 7 days using a porous cell scaffold according to an embodiment.

[0078] FIG. 4 shows results obtained by culturing enteroendocrine cells for 13 days using a porous cell scaffold according to an embodiment.

[0079] FIG. 5 shows results obtained a week after injecting the three-dimensional cell aggregates of enteroendocrine cells (EEC) into the kidney membrane of a mouse, by using a porous cell scaffold according to an embodiment.

[0080] FIG. 6 shows results obtained by a freeze-thaw test after culturing liver cancer cells for 12 days using a porous cell scaffold according to an embodiment.

[0081] FIG. 7 shows a graph comparing the expression levels of albumin from liver cancer cells cultured using a porous cell scaffold according to an embodiment and liver cancer cells cultured in a positive control group using an ELISA method.

MODE OF DISCLOSURE

[0082] Hereinafter, embodiments are presented to help the understanding of the present disclosure. However, the following examples are provided for easier understanding of the present disclosure, and the contents of the present disclosure are not limited by the following examples.

Example 1. Preparation of Porous Cell Scaffolds

[0083] Porous cell scaffolds using serum-induced proteins are prepared as follows.

[0084] First, the serum protein was cross-linked by using a cross-linking agent and reduced by using a reducing agent. Specifically, in a clean booth of Class 100 or less, the same amount of fetal bovine serum or bovine calf serum was treated with 1 mg/ml microviral transglutaminase (specific activity unit of 20 U/mg, Sigma Aldrich). Thereafter, dithiothreitol (DTT, Sigma Aldrich) as a reducing agent, of which a final concentration was 15 mM, was added thereto, and caused to react at a temperature of 37° C. for 12 hours to prepare a reaction product. Thereafter, washing buffer (6M urea and 0.1M sodium acetate, pH 5.0) was added to the reaction product, and homogenization was performed thereon by using an electric tissue grinder (Homogenizer D1000, Benchmark Scientific), followed by centrifuging to recover a suspension of serum-induced protein. After washing the suspension with lactic acid, the suspension was stirred for 20 minutes at 10,000 rpm using a high-speed stirrer (MaXtir© 500S, DAIHAN-brand), and aged at a temperature of 4° C. for 8 hours. In order to mold the aged serum-induced protein into a target shape, the result was transferred to a casting mold, frozen at a temperature of −80° C. for 1 hour, and then freeze-dried for 24 hours. Thereafter, the dried serum-induced protein was washed 5 times with 100% ethanol and sterilized tertiary distilled water for each at room temperature to wash impurities other than the serum-induced protein to prepare a porous cell scaffold of the serum-induced protein. In subsequent experiments, the porous cell scaffold of the serum-induced protein was immersed in a preservative solution (1×PBS, cell culture media, DW, etc.) and used.

[0085] The characteristics of the prepared cell scaffold are shown in Table 1.

TABLE-US-00001 TABLE 1 Pore size Average diameter of about 200 μm (100 μm to 400 μm) pH about 7.0 to about 7.4 when suspended in PBS or tissue culture medium Storage Room temperature temperature

Experimental Example 1. Three-Dimensional Culture of Cancer Cells Using Porous Cell Scaffold

[0086] Cancer cells were cultured using the porous cell scaffold prepared in Example 1 to identify culture stability and persistence.

[0087] Specifically, in order to remove the preservation solution from the porous cell scaffold prepared in Example 1 as much as possible, 1×PBS was added in such an amount that porous cell scaffold was sufficiently immersed, and rinsed by gently shaking, and then 1×PBS was removed therefrom. This cycle was repeated once more to remove the preservation solution therefrom. Thereafter, a gastric cancer cell line SNU017 (KCLB) and a cervical cancer cell line SiHA (KCLB) were seeded into the porous cell scaffold at a number of 5×10.sup.5 cells. Thereafter, cells seeded on the porous cell scaffold were cultured in a cell culture solution (RPMI, Gibco) in an incubator in conditions including a temperature of 37° C. and 5% CO.sub.2.

[0088] A paraffin block was formed on the 9th day of culture and the 14th day of culture, and was made into a slide, followed by H&E staining to perform histological examination. Results thereof are shown in FIGS. 1 and 2.

[0089] As shown in FIG. 1, as a result of culturing the SNU017 cell line for 9 days using the porous cell scaffold, it was confirmed that the cell line was cultured while forming a spherical cancerous tissue structure, which is similar to that in the actual tissues in the body.

[0090] As shown in FIG. 2, as a result of culturing the SiHA cell line for 14 days using the porous cell scaffold, it was confirmed that the cell line was cultured while forming a spherical cancerous tissue structure, which is similar to that in the actual tissues in the body.

Experimental Example 2. Three-Dimensional Culture of Chondrocytes Using a Porous Cell Scaffold

[0091] It is well known that in vitro culturing of chondrocytes is difficult. In this example, chondrocytes were cultured using the porous cell scaffold prepared in Example 1 to identify culture stability and persistence.

[0092] Specifically, chondrocytes (hACs) were three-dimensionally cultured in the same manner as in Experimental Example 1, except that chondrocytes collected from actual patients were seeded in a number of 5×10.sup.5 cells.

[0093] Thereafter, Live and Dead Cell staining was performed on the three-dimensionally cultured chondrocytes on the 7th day of culture. Thereafter, the stained cells were confirmed with a confocal laser microscope (LSM880 with Airyscan, Zeiss), and the results are shown in FIG. 3. In FIG. 3, green indicates live cells and red indicates dead cells.

[0094] Z1 to Z9 in FIG. 3 show cross-sectional images obtained by fluorescence imaging every 10 μm in height along the vertical axis.

[0095] As shown in FIG. 3, it was confirmed that dead cells indicated in red did no appear for one week. That is, cells were stably cultured. Patient-derived chondrocytes cultured on the porous cell scaffold according to an embodiment showed a high survival rate.

Experimental Example 3. Three-Dimensional Culture of Enteroendocrine Cells Using Porous Cell Scaffold

[0096] Enteroendocrine cells were cultured using the porous cell scaffold prepared in Example 1 to identify culture stability and persistence.

[0097] Specifically, enteroendocrine cells were three-dimensionally cultured in the same manner as in Experimental Example 1, except that enteroendocrine cells collected from mice were seeded in a number of 5×10.sup.5 cells.

[0098] Thereafter, DAPI and Hematoxylin & Eosin (H&E) staining were performed on the enteroendocrine cells 3D cultured on the 11th and 13th days of culture. The stained cells were confirmed with a fluorescence microscope (Axiovert 200, Zeiss), and the results are shown in FIG. 4.

[0099] As shown in FIG. 4, it is usually difficult to culture enteroendocrine cells for more than a week, but it can be seen that the three-dimensional cell aggregate of enteroendocrine cells cultured on a porous cell scaffold according to an embodiment was stably cultured without deteriorating cell status for more than 2 weeks.

[0100] As such, it was found that the porous cell scaffold according to an embodiment can stably and continuously incubate cells, and a culture pattern suitable for the characteristics of the cells for each cell was observed. In addition, it can be seen that even with the conventional 2D culture method of culturing in cell culture dishes and plates, long-term cultivation was able to be achieved.

Experimental Example 4. Engraftment Rate of Three-Dimensional Cell Aggregates Using Porous Cell Scaffolds

[0101] In this example, in order to apply the porous cell scaffold prepared in Example 1 to tissue regeneration treatment, the engraftment rate of the three-dimensional cell aggregate of enteroendocrine cells grafted on the kidney membrane, was measured.

[0102] Specifically, cells were cultured in the same manner as in Example 3, and then grated into the kidney membrane of a mouse by using a Hamilton syringe. One week after grafting, the mouse was euthanized, and the kidneys were separated therefrom and dissected, and the remaining amount of the cell aggregate was directly identified. Later, only the grafted part was removed, the wet weight thereof was measured, and the engraftment rate was confirmed using the number of engrafted cells. Then, a paraffin block was formed and prepared in a slide, followed by H&E staining to perform a tissue examination. The results are shown in FIG. 5.

[0103] As shown in FIG. 5, the cell body obtained by culturing enteroendocrine cells in FBS-free medium for 2 weeks on a porous cell scaffold according to an embodiment was grafted into the kidney membrane of a mouse, and cultured for 1 week, resulting in more than about 80% of the survival rate.

[0104] Considering that the in vivo engraftment rate is usually less than 5% when stem cells are injected, it can be seen that the porous cell scaffold according to an embodiment may be usefully used in the cell therapy for tissue regeneration.

Experimental Example 5. Freeze-Thawing Test of a Three-Dimensional Cell Aggregate Using Porous Cell Scaffold

[0105] In this example, a freeze-thawing experiment was performed to confirm that the porous cell scaffold prepared in Example 1 is easy to commercialize and transport in order to be practically used for commercialization of tissue regeneration treatment.

[0106] Specifically, lung cancer cell lines NCI-H28 and NCI-H2170 (KCLB) were seeded into a porous cell scaffold according to an embodiment in a number of 5×10.sup.5 cells, and then cultured for 28 days. Frozen storage was proceeded in a freezing solution (Media:FBS:DMSO, 5:4:1) in Deepfreezer at a temperature of −80° C. for 30 days, and then thawed and incubated for 12 days. Thereafter, H&E staining was performed on the cells cultured on the scaffold, and the results are shown in FIG. 6.

[0107] As shown in FIG. 6, it was confirmed that even when frozen or thawed, the porous cell scaffold according to an embodiment enabled re-culture of cells.

[0108] These results show that the porous cell scaffold according to an embodiment is advantageous for commercialization and transport as a cell therapy agent.

Experimental Example 6. Protein Production Using Porous Cell Scaffolds

[0109] In order to analyze whether a useful material (target protein) is produced using the porous cell scaffold prepared in Example 1, the protein production ability was evaluated with respect to the three-dimensional cell aggregate of liver cancer cells.

[0110] Specifically, HepG2 cells were seeded in a number of 5×10.sup.5 cells in a 24-well plate, and then cultured in MEM (10% FBS added) medium for 7 days, 14 days and 21 days. Thereafter, the concentration of albumin in the cell culture was measured by the method of Human ALB solid-phase sandwich ELISA. As a control, a conventional two-dimensional cell culture method was performed. Two-dimensional cell culture was performed in a 6-well plate. In addition, Matrigel (Corning, 356231) and Alvetex™ (ReproCell Inc., Glasgow, UK) were used as positive controls. Specifically, cells were cultured in the same manner as described above, except that the commercially available three-dimensional cell culture support was used. The concentration of albumin in the cell culture solutions of the control groups was measured, and the results are shown in FIG. 7.

[0111] As shown in FIG. 7, in the case of the porous cell scaffold according to an embodiment, the expression level of albumin indicating the degree of activation of hepatocellular carcinoma cells was higher than when the positive controls Matrigel and Alvetex™ was used. The above results mean that the porous cell scaffold according to an embodiment may be usefully used in the production of a target material (protein).