JP4260139B2 - Cell culture membrane support device - Google Patents

Description

  The present invention relates to a support device for a cell culture membrane, and more particularly, to culture a desired cell on both the upper and lower sides of the membrane without using separate fastening means for supporting the cell culture membrane. The present invention also relates to a support device for a membrane for cell culture, which is capable of performing tilted vibration culture during gas-liquid interface culture of cells attached to the membrane.

  Due to the rapid development of medicine along with the development of science, it has now reached a level where almost all organs can be transplanted. However, the development of such organ transplantation technology plays a very important role in promoting human health, but there are limitations such as technical difficulties in organ transplantation surgery, high costs, and side effects caused by the use of immunosuppressive agents. is there. Under these circumstances, some scientists have conducted research on methods of artificially creating and transplanting living tissues or organs, resulting in the creation of a new field called tissue engineering. .

  Tissue engineering is a discipline related to the field of producing cells-matrix complexes by three-dimensionally culturing cells on a biocompatibility substrate, and using this to regenerate living tissues and organs. Technology and biomaterial technology are combined and applied in the field of artificial tissue and organ development, gene and cell therapy. More specifically, development of artificial organs such as bone and cartilage, muscle, skin, liver, kidney, heart, and valve membrane, and replacement through tissue engineering regeneration, artificial blood vessels made using vascular constituent cells and biodegradable polymers The tissue engineering technique can be applied to the development of the above and the production of an artificial cornea made by transplanting corneal epithelial cells into a transparent biomaterial.

  In order to regenerate biological tissues and organs using such tissue engineering techniques, it is first necessary to culture cells on a biocompatible membrane or a substrate membrane, and thus culture cells on the membrane. This requires an instrument or device that supports the membrane.

  As a specific example of the membrane, an amniotic membrane can be used. The amniotic membrane is the innermost membrane of the fetal membrane and serves as an important barrier to protect the fetus from various infections and immune reactions from the maternal side, consisting of a thick basement membrane and an avascular stroma (Jerzy et al., Ann. Of Trans., 4: 85-90 (1999); Nancy et al., Aorn Jour., 39: 894-899 (1984)). Such amniotic membranes are currently used not only as a dressing and post-transplant cover for burn patients, but also as a support for the reconstruction of neural crests, airways, and mucosal tissues. In some cases, a model or patient having an abnormal surface of the eyeball is transplanted using amniotic membrane as a substrate for autologous transplantation of corneal epithelial cells (Koizumi et al., Cornea, 19: 65-71 (2000); Tsai et al., The New Eng. Jour. Of Med., 343: 86-93 (2000)).

  On the other hand, in connection with the instrument for supporting the amniotic membrane, Noguchi et al., Biochem. And Biophy. Res. Com., 210: 302-309 (1995)) described two amniotic membranes. Tissue support device for fixing between upper and lower rings consisting of concentric polycarbonate rings, inoculating airway epithelial cells on the amnion epithelial surface, monolayer culture, and differentiation US Pat. No. 4,446,234 discloses an amniotic membrane derived from a mammalian placenta in an in vitro assay method for assessing cell interaction with a natural barrier tissue. As an example, the amnion is fixed between the upper ring and the lower ring by using a fastening means such as a bolt.

In addition, in experiments and research methods for culturing corneal epithelial cells on existing amniotic membrane, a device such as a culture plate insert (Corning Inc., Corning, NY) or Trentwell insert (Transwell ^TM insert) was used. However, such a commercial instrument has the inconvenience that the amniotic membrane must be sutured before cell culture and the suture must be removed before transplantation, and can only be cultured on the upper surface of the amniotic membrane, Among the three types of cells of the cornea, there was a problem that only one type of cell could be cultured.

U.S. Pat. No. 4,446,234 Jerzy et al., Ann. Of Trans., 4: 85-90 (1999); Nancy et al., Aorn Jour., 39: 894-899 (1984) Koizumi et al., Cornea, 19: 65-71 (2000); Tsai et al., The New Eng. Jour. Of Med., 343: 86-93 (2000) Noguchi et al., Biochem. And Biophy.res.com., 210: 302-309 (1995)

  The present invention has been devised to solve the above-mentioned problems, and the object of the present invention is to eliminate the inconvenience in the amnion stitching process and the process of removing the suture when transplanted, It is an object of the present invention to provide a support device for a cell culture membrane that allows not only the desired cell culture on both the upper and lower sides of the cell but also the tilted vibration culture during the gas-liquid interface culture of the cells attached to the membrane.

  Another object of the present invention is to provide a method for culturing target cells using the cell culture membrane support apparatus and membrane.

  In order to achieve the above object, the present invention includes a through hole at the center, a protrusion formed on one surface along the side surface of the through hole, and three or more legs on the other surface. 1 member; and a through hole at the center, a receiving part for taking in the protrusion of the first member along one side surface of the through hole, and three or more legs on the other side Provided is a support device for a cell culture membrane that includes a second member, and the protruding portion of the first member and the receiving portion of the second member mesh with each other so that the membrane for cell culture can be fixed therebetween.

  The expression “reconstruction” used in the present invention means making an artificial tissue in a form similar to an actual tissue outside the body.

  The cell culture membrane support apparatus of the present invention can support a membrane without using a separate fastening means, and can culture a target cell on both the upper and lower surfaces of the membrane. Since tilted vibration culture can be performed during liquid interface culture, the tissue reconstruction period can be shortened. Furthermore, the tissue reconstituted in this way can be used not only for toxicity models but also for transplantation.

Hereinafter, the present invention will be described in detail.
The present invention is characterized by providing an apparatus that plays a role of supporting a membrane when cells constituting actual tissue are cultured on a membrane in the production of a prosthesis using tissue engineering.

  Specifically, the present invention is a first member having a through hole at the center, a protrusion formed on one surface along the side surface of the through hole, and three or more legs on the other surface; A hole, a receiving portion for taking in the protruding portion of the first member on one surface along the side surface of the through-hole, and a second member having three or more legs on the other surface; The protrusion part and the receiving part of the second member provide a support device for the cell culture membrane that is meshed so that the cell culture membrane can be fixed therebetween.

  FIGS. 1a to 1e are a perspective view (FIG. 1a), a side separation degree (FIG. 1b), a plan view (FIG. 1c), and a side coupling diagram (FIG. 1d) for a cell culture membrane support device according to the first embodiment of the present invention. ) And a design drawing (FIG. 1e), and FIG. 2 shows a photograph of the support device for the cell culture membrane manufactured in a circular shape according to the first embodiment of the present invention. Hereinafter, description will be given with reference to the accompanying drawings.

  The leg (13) formed on one surface of the first member (15) and the second member (16) is formed by the protruding portion (12) of the first member (15) and the second member (16). When a cell culture membrane is placed between the cell culture medium and the cell culture medium is placed in the cell culture medium and the cell culture medium is cultivated by placing a cell culture membrane between the cell culture medium and the receiving portion (14) of the cell culture medium, the bottom surface of the cell culture medium By providing a space between the membrane, air liquid interface culture can be performed.

  The gas-liquid interface culture is also called raft culture, and means that the medium is cultured on the lower surface of the membrane to which the cells to be cultured adhere and the air is in contact with the upper surface. When performing gas-liquid interface culture in this way, there is an advantage that differentiation can be induced in a state similar to the environmental state of the actual tissue (e.g., epithelial cells of the skin) by contact with the nutrient supply from the lower part and the air from the upper part. is there.

  The legs (13) may be manufactured to have an appropriate number in consideration of the fluidity of the culture medium, and it is preferable that three or more legs (13) are provided for maintaining the balance of the support device on the culture medium. The legs (13) may be manufactured to have various heights in consideration of the height of the cell culture container, the amount of the medium in the cell culture container, the medium replacement time, and the like. There is an advantage that the medium does not need to be changed frequently as the legs are high, but there is a disadvantage that the medium is excessively required if it is too high, and the flow of the medium inside and outside the support device is not appropriate if the legs are too low. There is a disadvantage that culture is not performed sufficiently. Specifically, the leg is preferably manufactured to have a height of 1 mm to 5 mm, more preferably 2 mm to 4 mm.

  The protruding length of the protruding portion (12) of the first member (15) is the same as the receiving depth of the receiving portion (14) of the second member (16), and the protruding portion (12) of the first member (15). ) And the receiving part (14) of the second member (16) with a membrane for cell culture applied on both sides to push the protruding part (12) into the receiving part (14) and assemble it. The membrane is fixed and supported while meshing (12) and the receiving portion (14). The receiving part 14 is preferably in the form of a concave groove, and can be manufactured in various shapes according to the shape of the protrusion 12.

  On the other hand, the first member (15) and the second member (16) of the device have a through hole (11) in the center in consideration of the form of the membrane in which the cells are cultured and the form of the site where the cultured cells are transplanted. Although it can be manufactured in a circular shape or a polygonal shape such as a triangle or a quadrangle, it is preferably manufactured in a ring type member in which the force for fixing and pulling the amniotic membrane is constant in all directions.

  In addition, the material of the first member (15) and the second member (16) is preferably a polymer material that is excellent in biocompatibility and has a specific gravity that does not float on the medium during fluid culture. Specifically, it is preferable to use polytetrafluoroethylene (PTFE; trade name: Teflon (registered trademark)), polyethylene, polymethyl methacrylate (PMMA), polyacrylonitrile (PAN), polyethylene terephthalate (PET), silicone, and the like. Most preferably, Teflon (registered trademark) is used.

  The apparatus for supporting a membrane for cell culture according to the present invention can be manufactured by adjusting the size arbitrarily according to the size of the tissue to be cultured and the culture medium. However, considering that the size of the tissue and the membrane can be supported in a stretched state, etc. When manufacturing in a circular shape, the diameter is preferably 10 mm to 150 mm.

  The membrane that can be fixed to the supporting device according to the present invention can be used without particular limitation as long as it can be used for membranes for cell culture in the technical field of tissue engineering, but it is most preferable to use amniotic membrane. In addition to amniotic membranes, natural materials such as collagen membranes, gelatin membranes, chitin membranes, chitosan membranes, alginate membranes, hyaluronic acid membranes and their derivatives can be used as synthetic substrates. A PLGA (poly (D, L-lactic-co-glycolic acid)) film, a PGA (polyglycolic acid) film, a PLA (poly (lactic acid)) film, or the like may be used. The membrane is preferably a bioabsorbable material, taking into account the inconvenience that must be removed after transplanting tissue consisting of cultured cells for treatment, and in particular, the degradation product does not cause an inflammatory reaction. Those having high biocompatibility are preferred.

  In addition, the cell culture membrane support device according to the first aspect of the present invention includes, as shown in FIG. 1f, between the protruding portion of the first member (15) and the receiving portion of the second member (16). An intermediate ring (17) can be inserted. The cell culture membrane support device according to the second aspect of the present invention in which the intermediate ring is inserted in this way is particularly in a dry state (when the eyes are open) and a wet state (when the eyes are open). It is useful to reconstitute the cornea by culturing the corneal cells in an environment where the crushing occurs repeatedly.

  FIG. 1g is a side-separated view of the device according to the second aspect of the present invention, in which an intermediate ring (17) is inserted between the protruding part of the first member (15) and the receiving part of the second member (16). FIG. 1h shows a design drawing for manufacturing the cell culture membrane support device according to the second embodiment of the present invention together with the size of each part (gas-liquid interface: mm). It is shown.

  A specific corneal cell culture method using the support device according to the second aspect of the present invention is as follows. First, corneal epithelial cells are inoculated on the epithelial surface of the amniotic membrane and subjected to liquid culture, and then the second member (16) is removed. Even if the second member (16) is removed, the amniotic membrane is still fixed by the intermediate ring (17) and the first member (15), and the leg (13) of the first member (15) When placed toward the bottom, there will be no barrier around the surface of the amniotic membrane. This corresponds to the case where the first member (15) coupled to the intermediate wheel (17) is reversely covered with the second member (16) removed in FIG. 1g. In this way, in the apparatus according to the first aspect of the present invention, unlike the presence of a wall due to the first member or the second member around the amniotic membrane, there is no wall around the amniotic membrane, and the medium becomes amniotic membrane. The surface can be more easily contacted. Thereafter, the first member coupled to the intermediate ring is shifted to one of the wall surfaces of the culture vessel, and then fluid culture is performed. When fluid culture is performed, the corneal epithelial layer on the amniotic membrane is repeatedly immersed in the medium and exposed to air, but such a culture environment is similar to the actual environment of the corneal epithelial cells. Can be easily reconfigured.

  On the other hand, the present invention provides a method for culturing a target cell on a membrane (reconstructing a tissue) using the cell culture membrane support apparatus. By using such a method, for example, a corneal epithelial layer, a monolayer of fibroblasts and a corneal epithelial layer can be reconstructed, and a partial layer or all layers of the cornea having a structure similar to the actual cornea can be reconstructed. can do.

  When transplanting cultured tissue after reconstituting tissues such as skin, cornea, mucous membrane, etc. by fixing the membrane to the support device according to the present invention, the reconstructed tissue is separated by separating the first member and the second member. Just transplant.

  The cell culture method using the apparatus according to the present invention not only allows stationary culture, but also enables dynamic culture such as tilting dynamic culture or fluid culture to shorten the differentiation induction period, In fact, it has an advantage that a basement layer similar to that of a tissue can be obtained, and is particularly advantageous for a tissue structure that requires a strong basement membrane like the cornea. That is, in the dynamic culture method, a stable basal layer and a desired cell layer can be obtained in a short time by smoothly supplying nutrients in fresh medium to cells on the amniotic membrane, compared to stationary culture that supplies medium nutrients by diffusion. There is an advantage.

  FIG. 7 shows a schematic diagram of a fluid culture method using the apparatus according to the present invention. In FIG. 7, if the stirring device is moved at such a slow speed that the supporting device to which the amniotic membrane is fixed does not move, the nutrient medium can be smoothly supplied to the cells on the amniotic membrane while the medium under the amniotic membrane moves. Therefore, the medium can be supplied more smoothly than the existing stationary culture in which the medium is supplied to the amniotic membrane by diffusion.

  Although the specific fluid culture conditions vary depending on the type of cells to be cultured, it is preferable to perform culture for 3 to 12 days at a speed of 6 to 10 rpm that does not move the support device.

  Furthermore, by using the cell culture method of the present invention, it is possible not only to create an epithelial cell toxicity model that is difficult to culture by culturing and differentiating epithelial cells in support of an amnion or a collagen membrane that can be used as a basement membrane. A full-thickness cornea similar to the actual cornea can be reconstructed.

  In one embodiment of the present invention, a single layer of corneal fibroblasts was cultured on the stroma side of the amniotic membrane using the above device, and the corneal epithelial layer was reconstructed on the epithelial side of the amniotic membrane. . As a result, as shown in FIG. 12B, it can be seen that the corneal epithelial layer is well formed on the amniotic membrane, and it can be confirmed that a transparent corneal epithelial layer is obtained. Further, it can be confirmed that the supporting device of the present invention is useful not only for the amniotic membrane but also for supporting (fixing) the collagen membrane (see FIG. 14).

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

<Example 1> Manufacture of a support device for a corneal cell culture membrane For manufacturing a corneal cell cornea in a format similar to an actual tissue, the corneal cell culture can be used to support an amniotic membrane used as a basement membrane for corneal cell culture. An amnion support device was manufactured. Teflon (registered trademark) (trade name) was used as a material, and was manufactured in a circular shape in consideration of the size of the cornea and the ease of culture. The inner diameter of the first member and the second member of the support device is set to 14 mm in consideration of the diameter of the cornea (average: 10 mm, in the case of a full-layer cornea transplant, punching an intermediate cornea having a diameter of about 7 mm) In order to fix the amniotic membrane between the first member and the second member, the outer diameter was 22 mm (26 mm when the intermediate ring was inserted between the first member and the second member). Design drawings for manufacturing the support device are shown in FIGS. 1e and 1h together with the size of each part (gas-liquid interface: mm). Legs were provided for culturing the gas-liquid interface, three legs were provided for the flow of the medium, and the leg height was 2 mm. The support device in which the first member and the second member are assembled has a height of 9 mm in order to use a commercially available culture vessel.

<Test example> Cell culture using corneal cell culture membrane support device <Test example 1> Preparation of amniotic membrane Amniotic membrane was used as a membrane for culturing corneal cells. The amniotic membrane is negative in serum tests for hepatitis B and C, human immunodeficiency virus (HIV) antibody, laboratory for sex diseases (VDRL), and placenta obtained by caesarean section from a healthy mother without disease Was washed with physiological saline and then separated by a method performed in an aseptic environment (Jerzy et al., Ann. Of Trans., 4: 85-90 (1999)). The separated amniotic membrane was washed 3 to 4 times with a phosphate buffer solution (PBS) containing 0.3% ofloxacin and then attached to a nitrocellulose membrane (NC, Osmonics, USA).

<1-1> Preparation of cryopreserved amniotic membrane The amniotic membrane attached to the nitrocellulose membrane (NC, Osmonics, USA) was cut into 2 cm × 2 cm and DMEM (Dulbecco's modified essential medium) and glycerol (Sigma, USA) were 1: It was stored at −80 ° C. in an amniotic membrane preservation solution mixed at a ratio of 1. For culturing, the cryopreserved amniotic membrane was first quickly thawed. The thawed amnion was washed 3 times with PBS and then 0.05% trypsin (Sigma, T-4799, USA) /0.02% ethylenediaminetetraacetic acid (Gibco BRL, Cat. No. 11266-012, USA) After treatment at 37 ° C. for 30 minutes, amniotic epithelial cells were removed with a cell scraper (Cell Scraper; Nunc, 179693, USA).

  Amniotic epithelial cells randomly cover the surface of the amniotic membrane without any gaps, and the surface of such an irregular amniotic membrane makes epithelial cells of other tissues on the amniotic membrane by making the corneal epithelial cell attachment surface irregular. It is preferable to remove amniotic epithelial cells in order to provide a flat culture surface because it interferes with the culture.

  The amniotic membrane from which the amniotic epithelial cells were removed was further washed 3 to 4 times with PBS, and then fixed to the supporting device produced in Example 1 and used for culture.

<1-2> Preparation of freeze-dried amniotic membrane A previously washed amniotic membrane was treated with 0.05% trypsin (Sigma, T-4799, USA) /0.02% ethylenediaminetetraacetic acid (Gibco BRL, Cat. No. 11266-012, USA) for 30 minutes at 37 ° C., and amniotic epithelial cells were removed with a cell scraper (Nunc, 179693, USA). The amniotic membrane from which the amniotic epithelial cells were removed was further washed 3 to 4 times with PBS. The washed amniotic membrane was frozen at 80 ° C. for 24 hours and then lyophilized at −80 ° C. for 48 hours (FREEZE DRY SYSTEM, SAMWON, SFDSM06, KOREA). The lyophilized amniotic membrane was fixed on the support device manufactured in Example 1 and then sterilized (25 kGy, Green Pier, Korea) and used for culture.

<Test Example 2> Preparation of collagen membrane A collagen membrane was produced with a 0.3% collagen solution (BioLand Ltd., Korea). First, taking into consideration the shape and size of the target collagen membrane, the collagen solution is divided into a circular or polygonal container so as to have a height of 2 mm, frozen at 80 ° C. for 24 hours, and then at −80 ° C. A collagen sponge was produced by freeze-drying for 48 hours, and the freeze-dried collagen sponge was slightly wetted with a 0.3% collagen solution, placed in a flat container, and dried at 4 ° C. to produce a collagen membrane. The degradation rate of the collagen membrane can be adjusted by the degree of crosslinking. The collagen membrane thus produced was fixed on the support device produced in Example 1, and then sterilized (25 kGy, Green Pier, Korea) and used for culture.

<Test Example 3> Primary culture of corneal fibroblasts, corneal epithelial cells, corneal endothelial cells (Soverin Karmiol, Methods of Tissue Engineering, Chapeter 2 (Cell isolation and selection), page 20, Academic Press, 2002) Corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells were primarily cultured by the method of explant culture.

The specific primary culture method is as follows. First, a corneal scleral button was obtained from a rabbit eyeball using a surgical instrument. After complete removal of the conjunctiva from the keratoconus, the cornea and limbus were each obtained with a limbus containing 1 mm, and the remaining central cornea was centered on the stromal site using a scalpel. Separated into a layer with epithelial cells and a layer with endothelial cells. Three types of cells of the cornea were obtained through the tissue attachment method from the three parts of the tissue thus separated. Corneal epithelial cells, corneal fibroblasts, and corneal endothelial cells were obtained from a limbal region, a tissue partially including an epithelial cell and a corneal stromal layer, and a tissue partially including an endothelial layer and a corneal stromal layer, respectively. Each tissue was cut to a size of 22 mm. Corneal epithelial cells are obtained by attaching the limbal part in contact with air in the direction of the culture container, and corneal fibroblasts are tissues that partially contain epithelial cells and stromal layers, while stromal layers are placed in the container. It was obtained by attaching in the direction. Corneal endothelial cells were obtained by attaching endothelial cells in the direction of the container. The attached tissue was cultured overnight with a small amount of culture medium so that the tissue was not completely dried. The medium was added the next day, and cultured for about 7 days to 10 days while changing the medium once every three days. From the attached tissue, cells were spread around the tissue and cultured to obtain primary cells. As a culture medium for corneal epithelial cells, HCGS (Human corneal growth supplement; Cascade Biologics, USA) and EpiLife (Cascade Biologics, USA) supplemented with 0.06 mM CaCl ₂ were used. HCGS includes hydrocortisone 0.18 μg / ml, transferrin 5 μg / ml, bovine pituitary extract (BPE) 0.2% v / v and mouse epidermal growth factor (mEGF) 1 μg / ml was contained.

The culture medium of corneal fibroblasts is DMEM medium (Dulbecco's modified Eagle's medium; GIBCO, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, USA). The corneal endothelial cell culture medium is Opti-MEM medium ( Optimal essential medium (GIBCO), FBS 8%, EGF (Sigma, USA) 10 ng / ml, nerve growth factor (NGF; R & D, USA) 20 μg / ml, basic fibroblast growth factor (bFGF; Sigma, USA) 10 ng / Ml, RPMI vitamin mixer (Sigma, USA) 1% v / v, ascorbic acid (Sigma, USA) 5 μg / ml, insect lipid (Sigma, USA) 0.2 mg / ml, CaCl ₂ A medium supplemented with 0.2 mg / ml of (GIBCO), 0.2 mg / ml of CaCl ₂ (GIBCO) and 0.08% chondrotin sulfate (Sigma, USA) was used.

<Test Example 4> Reconstitution of corneal epithelial layer using third generation cells Primary cells were obtained by a tissue adhesion method in a culture vessel. Depending on the amount of primary cells, the cells were subcultured 1: 2 or 1: 3 to obtain 2 generation cells after 5 to 7 days, and subcultured at about 1: 4 to obtain 3 generation cells after 4 to 6 days. The 3rd generation cells thus obtained were used for reconstitution of the corneal epithelial layer.
5 × 10 ⁵ 3 generation corneal epithelial cells were inoculated on the amniotic membrane. After liquid culture for 1-2 days, the corneal epithelial layer was reconstituted by gas-liquid interface culture for 6 days. EpiLife (Cascade Biologics, USA) supplemented with HCGS (Human corneal growth supplement) was used as the medium.

The reconstructed corneal epithelial layer was subjected to the following <Test Example 9> histological examination, and the results are shown in FIGS.
FIG. 3 is an H / E-stained photograph of the reconstructed corneal epithelial layer. In the epithelial layer (A) that has been subjected to the gas-liquid interface culture for 3 days, the inoculated cells are stabilized and two to three layers of epithelial cells are formed. It was confirmed that the cells were well attached, and the epithelial layers (B and C) subjected to the gas-liquid interface culture for 6 days were confirmed to have a form similar to the actual epithelial layer of the cornea.

On the other hand, FIG. 4 is a photograph obtained by immunohistochemical staining of the reconstructed corneal epithelial layer with AE5. The surface cells (superficial cells) of the epithelial layer (C) cultured on the air-liquid interface for 3 days and 6 days on freeze-dried amniotic membrane (A and B) and the cryo-preserved amniotic membrane on the air-liquid interface for 6 days are the corneal epithelial layer. Differentiation was often performed, and a morphology similar to that of corneal surface cells was confirmed.
FIG. 5 is a photograph of the reconstructed corneal epithelial layer immunohistochemically stained with PCNA (proliferating cell nuclear antigen), in which active proliferating cells are observed near the basal layer from the actual cornea (D). As described above, it can be confirmed that the corneal epithelial cells (A to C) cultured in the amniotic membrane have active proliferation ability like the actual corneal epithelial cells.

  Further, FIG. 6 is a transmission electron microscope (TEM) photograph of the reconstructed corneal epithelial layer, which shows that the corneal epithelial layer is well attached on the cryopreserved amniotic membrane (A) and the freeze-dried amniotic membrane (D). It can be confirmed that the formation of hemidesmosome not only shows that the corneal epithelial cells are well attached to the amniotic membrane (B and E), but also that the exchange between the cells is facilitated by the formation of desmosomes. Can be confirmed (C and F).

<Test Example 5> Reconstitution of corneal epithelial layer using flow culture method 5 × 10 ⁵ corneal epithelial cells of 3 generations and 4 th generation of thawing were inoculated on the amniotic membrane. Thawed 4th generation cells were mixed with EpiLife, serum, and frozen cytoprotective agent (Dimethyl sulfoxide (DMSO)) at 8: 1: 1 respectively at a concentration of 1 × 10 ⁶ cells / ml. Cryopreserved cells were rapidly thawed and then expanded. After liquid culture for 1-2 days, the corneal epithelial layer was reconstituted by gas-liquid interface culture using 9-day fluid culture, for example, tilting dynamic culture (RP: 6). As the medium, EpiLife (Cascade Biologics, USA) supplemented with HCGS was used.

  The reconstructed corneal epithelial layer was subjected to the following <Test Example 9> histological examination, and the results are shown in FIGS.

  FIG. 8 is an H / E-stained photograph of the corneal epithelial layer reconstructed on the amniotic membrane for 9 days (A, B) and 6 days (C, D), respectively, using the flow culture method. When comparing A and B in FIG. 8, it can be seen that the flow culture method is cultured in more corneal epithelial layers during the same period than the stationary culture. This is because fluid culture smoothly supplies nutrients and oxygen of the medium to corneal epithelial cells on the amniotic membrane than stationary culture to promote the growth and differentiation of epithelial cells. Such a tendency can be seen by comparing C and D in FIG. FIGS. 9C and 9D show thawing four-generation corneal epithelial cells obtained by reconstituting the corneal epithelial layer on the amniotic membrane by stationary culture and fluid culture, respectively. Usually, when thawing after storing cells in liquid nitrogen, depending on the storage and thawing method, cells become damaged, and fluid culture that can sufficiently supply nutrients to such damaged cells If the method is used, an epithelial layer similar to the corneal epithelial layer can be reconstructed as shown in FIG. 9D. Although the corneal epithelial layer could hardly be reconstructed by static culture (see FIG. 9C), a corneal epithelial layer similar to the basal layer of the actual cornea was obtained through fluid culture.

  FIG. 10 is a PCNA immunohistochemically stained photograph of the corneal epithelial layer reconstituted on the amniotic membrane using the flow culture method. Compared with FIG. 5B, the cells have excellent proliferation ability. Is present in the basal layer of the epithelial layer obtained by fluid culture.

<Test Example 6> Reconstitution of monolayer corneal fibroblasts and corneal epithelial layer on the stromal and epithelial surfaces of the amniotic membrane When culturing cells on the stromal side of the lyophilized amnion, an amnion sponge It is necessary to culture after removing the layer (Spongy layer) and the mother cell layer (Fibroblast layer). The amniotic membrane layer is composed of an epithelial side to an epithelial layer, a basement membrane, a dense layer, a mother cell layer, and a sponge layer. In particular, since the sponge layer on the interstitial surface is a site that adheres to the chorion and is mucous, cells cannot adhere to it and are necrotized halfway in a spherical state. Therefore, when culturing on the interstitial surface of the amniotic membrane, the treatment of the amniotic membrane is important. Therefore, 2 × 10 ⁵ corneal fibroblasts were inoculated on the stromal surface of the lyophilized amniotic membrane from which the sponge layer and mother cell layer had been removed, and then cultured for 5 days. After culturing for 5 days, the amnion was covered and 5 × 10 ⁵ epithelial cells were inoculated on the epithelial surface, cultured for 2 days, and then cultured for 6 days.

After culturing, the histological examination of <Test Example 9> was performed, and the results are shown in FIGS.
FIG. 11 is a H / E-stained photograph in which corneal fibroblasts are monolayer cultured on the interstitial surface of the amniotic membrane and the corneal epithelial layer is reconstructed on the epithelial surface, A is stationary culture, and B is fluid culture. It is a photograph. It can be seen that two corneal cells can be easily cultured on both sides of the amniotic membrane by the support device according to the present invention.

  On the other hand, FIG. 13 is a TEM photograph in which corneal fibroblasts are monolayer cultured on the interstitial surface of the amniotic membrane and the corneal epithelial layer is reconstructed on the epithelial surface. Since hemidesmosomes (B and D) between epithelial cells can be observed, the reconstituted corneal epithelial layer is very well connected between cells and between amnion and corneal epithelial cells. I understand.

<Test Example 7> Reconstruction of skin epidermis layer using collagen membrane Enzymatic digestion was used for primary culture of keratinocytes. A collagenase solution 3 mg (1000 U) / mL in which the skin tissue (foreskin) is dissolved in a calcium acetate buffer solution (130 mM NaCl, 10 mM calcium acetate, 20 mM HEPES, pH 7.2) and dispase in PBS A solution of 1 mg (1.5 U) / mL and 0.05% trypsin enzyme solution was used. In order to prevent keratinocyte differentiation and fibroblast contamination, keratinocyte specific growth hormone such as hydrocortisone, insulin, and triiodothyronine was added to the keratinocyte culture. EGF (epidermal growth factor, 5.0 g / L) and BPE (bovine pituitary extract, 50 mg / L) to K-SFM (serum-free keratinocyte medium, Gibco. Cat. # 320-7005PJ) with low Ca ²⁺ concentration The added medium was used. The 3rd generation cells were obtained from the keratinocytes obtained in the primary culture through two subcultures of 1: 4. 5 × 10 ⁵ 3 generation keratinocytes were inoculated on the collagen membrane. After liquid culture for 1-2 days, the skin epidermis layer was reconstituted by gas-liquid interface culture for 7 days. Of the histological examination of <Test Example 9> below, H / E staining was performed on the reconstructed skin epidermal layer, and the results are shown in FIG. As can be seen from FIG. 14, a reconstructed skin epidermis layer was obtained on the collagen membrane.

<Test Example 8> Reconstruction of the corneal epithelial layer in the amniotic membrane fixed to the support device with the intermediate ring inserted Four generations of corneal epithelial cells thawed on the amniotic epithelial surface with the intermediate ring inserted and fixed to the support device 5 × 10 ⁵ cells were inoculated and liquid cultured for 1-2 days. EpiLife (Cascade Biologics, USA) supplemented with HCGS (Human corneal growth supplement) was used as the medium. Thereafter, the second member is removed from the support device in which the intermediate ring is inserted, and the first member coupled to the intermediate ring is positioned so as to be offset to one of the wall surfaces of the culture vessel with the legs facing the bottom surface, Fluid culture was carried out at a speed for 6 days.

  Of the histological examination of <Test Example 9> below, H / E staining was performed on the reconstructed corneal epithelial layer, and the results are shown in FIG. It was confirmed that the corneal epithelial layer in FIG. 15 was reconstructed in a form similar to the actual cornea even when compared with the photographs in C and D in FIG.

<Test Example 9> Histological Examination Histological examination is performed with reference to histopathology (National Council of Clinical Pathology, Korean Medicine, 1992) and pathological tissue special staining picture book (Kim Ju-ho, Singhwan Publishing Co., 1993). It was.
(1) Hematoxylin / eosin (H / E) staining
After the specimen was dripped with a 4% formaldehyde solution, a section obtained by cutting the paraffin-fixed one was stained with hematoxylin-eosin.

(2) AE5 staining A specimen obtained by treating a specimen with a 4% formaldehyde solution and then cutting a sample fixed to paraffin was stained with AE5, which is a differentiation-specific protein of corneal epithelial cells. It was observed whether the reconstructed corneal epithelial layer was correctly differentiated through this staining.
(3) PCNA staining After the specimen is treated with a 4% formaldehyde solution, a section obtained by cutting a paraffin-fixed piece is subjected to PCNA staining. Proliferating cells were observed through this staining.

(4) Electron Microscopy The surface and cross section of the amniotic membrane from which the amniotic epithelial cells were removed and the non-removed amniotic membrane, and the cross section of the reconstructed corneal epithelial layer were observed with an electron microscope. The tissue was fixed with 4% glutaraldehyde and 2% formaldehyde and then re-fixed with 2% osmium tetroxide. After dehydrating the re-fixed tissue, coating and scanning electron microscope (SEM), JSM-35CF, Jeol, Japan and transmission electron microscope (TEM), JEM-200CX, Jeol, Observed in Japan).

The perspective view (FIG. 1a) with respect to the support apparatus of the film | membrane for cell cultures by the 1st aspect of this invention is shown. FIG. 2 shows a side separation view (FIG. 1b) of the apparatus for supporting a membrane for cell culture according to the first embodiment of the present invention. The top view (FIG. 1c) with respect to the support apparatus of the film | membrane for cell cultures by the 1st aspect of this invention is shown. FIG. 2 shows a side view (FIG. 1d) of the cell culture membrane support device according to the first embodiment of the present invention. 1 shows a design diagram (FIG. 1e) for a cell culture membrane support device according to a first embodiment of the present invention. The perspective view (FIG. 1f) with respect to the support apparatus of the film | membrane for cell cultures by the 2nd aspect of this invention is shown. FIG. 3 shows a side separation view (FIG. 1g) of the cell culture membrane support device according to the second embodiment of the present invention. FIG. 1h shows a design diagram (FIG. 1h) for a cell culture membrane support apparatus according to a second embodiment of the present invention. FIG. 4 is a photograph (A) of a support device manufactured in a ring type according to the first embodiment of the present invention, where the membrane fixed by the support device is a freeze-dried amniotic membrane (B) and a frozen storage amniotic membrane. Case (C) is shown as an example. The photograph which culture | cultivated the corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention, and dye | stained this with hematoxylin / eosin (H / E) (hematoxylin / eosin) is shown. A: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 3 days B: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 6 days C: Gas-liquid interface culture was performed on frozen storage amniotic membrane for 6 days D: The epithelial layer of the actual rabbit cornea The photograph which culture | cultivated the corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention, and immunostained this with AE5 which is a differentiation specific protein of a corneal epithelial cell is shown. A: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 3 days B: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 6 days C: Gas-liquid interface culture was performed on frozen storage amniotic membrane for 6 days D: The epithelial layer of the actual rabbit cornea The photograph which culture | cultivated the corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention, and immunostained this with PCNA (proliferating cell nuclear antigen) is shown. A: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 3 days B: Gas-liquid interface culture was performed on freeze-dried amniotic membrane for 6 days C: Gas-liquid interface culture was performed on frozen storage amniotic membrane for 6 days D: The epithelial layer of the actual rabbit cornea It is the photograph observed after cultivating the corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention, and using the transmission electron microscope (TEM). A: TEM photograph of corneal epithelial layer cultured on cryopreserved amniotic membrane (basal layer) B: TEM photograph of corneal epithelial layer cultured on cryopreserved amniotic membrane (hemidesmosome) C: Corneal epithelial layer cultured on cryopreserved amniotic membrane TEM photograph (desmosome) D: TEM photograph of corneal epithelial layer cultured on freeze-dried amniotic membrane (basal layer) E: TEM photograph of corneal epithelial layer cultured on freeze-dried amniotic membrane (hemidesmosome) F: On freeze-dried amniotic membrane Photograph of corneal epithelial layer cultured in potato (desmosome) It is a schematic diagram of the fluid culture method using the apparatus by this invention. The photograph which dye | stained by H / E after culture | cultivating a corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention using a fluid culture method is shown. A: Gas-liquid interface culture by static culture for 9 days B: Gas-liquid interface culture by flow culture for 9 days C: Gas-liquid interface culture by stationary culture for 6 days using thawed cells D: Using thawed cells Gas-liquid interface culture in fluid culture for 6 days The photograph which carried out the immunohistochemical dyeing | staining by AE5 after culture | cultivating a corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention using a fluid culture method is shown. A: Gas-liquid interface culture by fluid culture for 6 days B: Gas-liquid interface culture by fluid culture for 9 days The photograph which carried out immunohistochemical dyeing | staining by PCNA after culture | cultivating a corneal epithelial cell on the amniotic membrane fixed to the apparatus by the 1st aspect of this invention using a fluid culture method is shown. A: Gas-liquid interface culture by fluid culture for 6 days B: Gas-liquid interface culture by fluid culture for 9 days A photograph of corneal fibroblasts monolayer cultured on the stromal surface of the amniotic membrane using the apparatus according to the first aspect of the present invention and corneal epithelial cells cultured on the epithelial surface and then stained with H / E is shown. A: cultured by static culture B: cultured by fluid culture Using the apparatus according to the first aspect of the present invention, corneal fibroblasts are monolayer-cultured on the stromal surface of the amniotic membrane, and the epithelial surface shows a photograph after culturing the corneal epithelial cells. ) And a photograph (B) after removing the first member and the second member. It is the photograph observed by TEM, after culturing corneal fibroblasts on the stromal surface of the amniotic membrane and culturing corneal epithelial cells on the epithelial surface using the apparatus according to the first aspect of the present invention. A: cultured by static culture B: cultured by fluid culture The photograph which cultivated the skin epidermal cell on the collagen membrane fixed to the apparatus by the 1st aspect of this invention, and dye | stained this with H / E is shown. The photograph which culture | cultivated the corneal epithelial layer on the amniotic membrane fixed to the apparatus by the 2nd aspect of this invention, and dye | stained this with H / E is shown.

Claims (11)

A through-hole in the center, a protrusion formed on one side along the side of the through-hole, and a first member having three or more legs on the other side; and a through-hole in the center, on the side of the through-hole A receiving portion for taking in the protruding portion of the first member on one surface along the second member, and a second member having three or more legs on the other surface,
A device for supporting a cell culture membrane, wherein the protruding portion of the first member and the receiving portion of the second member mesh with each other so that the membrane for cell culture can be fixed therebetween.   The apparatus according to claim 1, wherein an intermediate ring is inserted between the protruding portion of the first member and the receiving portion of the second member.   The apparatus according to claim 1, wherein the first member and the second member are circular members.   The material of the first member and the second member is polytetrafluoroethylene (PTFE; trade name: Teflon (registered trademark)), polyethylene, polymethyl methacrylate (PMMA), polyacrylonitrile (PAN), polyethylene terephthalate (PET) and silicone. The device of claim 1, wherein the device is a biocompatible polymeric substance selected from the group consisting of:   The apparatus of claim 1, wherein the receiving portion is in the form of a concave groove.   The membrane is an amniotic membrane, collagen membrane, gelatin membrane, chitin membrane, chitosan membrane, alginate membrane, hyaluronic acid membrane, PLGA (poly (D, L-lactic-co-glycolic acid)) membrane, PGA (polyglycolic acid) membrane, PLA The device of claim 1, wherein the device is selected from the group consisting of (poly (lactic acid)) membranes.   A method for culturing target cells using the support device for a membrane for cell culture according to any one of claims 1 to 6 and the membrane.   8. The method according to claim 7, wherein the target cell is selected from the group consisting of corneal epithelial cells, skin epidermal cells, mucosal epithelial cells and airway epithelial cells.   The method according to claim 7, wherein the target cell is cultured by gas-liquid interface culture.   The method according to claim 9, wherein the gas-liquid interface culture is performed by inclined vibration culture.   The method according to claim 10, wherein the tilted vibration culture is cultured at a speed of 6 to 10 rpm for 3 to 12 days.

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