Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• the present invention relates to a multilayer cell structure and a method for manufacturing the same.
• Temperature-responsive culture dishes are widely used in current regenerative medicine research and clinical practice. By culturing cells in this temperature-responsive culture dish and lowering the temperature from 37°C to 20°C in a monolayer state, the cells can be collected in a sheet form.
• the collected cell sheets are single-layered and fragile, and when used for transplantation, it is necessary to stack them one by one using cell sheet supports, which takes time and effort. there were.
• MSCs Mesenchymal stem cells
• stem cells derived from bone marrow, umbilical cord, teeth, etc. are known.
• FBS fetal bovine serum
• FBS-free serum-free culture fluid
• serum-free culture fluid examples include serum-free culture fluid (XFM, manufactured by Irvine Scientific) manufactured under current good manufacturing practices (cGMP).
• XFM cells dental pulp stem cells cultured in XFM
• SCM cells dental pulp stem cells
• Non-Patent Document 1 SCM cells maintain a monolayer at confluency (a state in which cells fill a Petri dish) after the 8th day of culture, whereas XFM cells autonomously become multilayered (hereinafter referred to as Because the number of XFM cells decreased on the 14th day of culture, TUNEL (terminal deoxynucleotidyl transfer-mediated deoxyuridine triphosphate) nick- It has also been reported that when cell death was analyzed by end labeling (end labeling) staining, an extremely large number of TUNEL-positive cells were observed in XFM cells. Therefore, while culturing with XFM can increase the number of cells approximately twice as much as the conventional method, there is a problem in that cells that have become self-multilayered undergo cell death and the number of cells decreases.
• TUNEL terminal deoxynucleotidyl transfer-mediated deoxyuridine triphosphate
• Non-Patent Document 1 As a follow-up to Non-Patent Document 1, in order to solve the problem of cell death caused by self-multilayering, we focused on coating culture equipment (Petri dishes and plates) and reported that type I collagen is the optimal coating substrate. (See Non-Patent Document 2).
• Non-Patent Document 2 dental pulp stem cells (hereinafter sometimes referred to as "COL-XFM cells”) cultured in a collagen-coated culture dish continued to proliferate more sustainably than XFM cells, and on the 20th day of culture. It has been reported that a self-multilayered cell sheet was successfully formed that ultimately reached approximately twice the number of cells without decreasing the number of cells.
• the present invention aims to solve the above-mentioned conventional problems and achieve the following objects. That is, the present invention can shorten the culture period required for manufacturing a multilayer cell structure, suppress cell death in the multilayer cell structure, and further provide multilayer cell structures of various shapes.
• An object of the present invention is to provide a method for manufacturing a multilayer cell structure and a multilayer cell structure.
• the present inventors found that when culturing mesenchymal stem cells derived from dental pulp using culture equipment coated with type I collagen or its fragments using a serum-free medium.
• the seeding number of mesenchymal stem cells derived from the dental pulp is set to more than 0.5 ⁇ 10 4 cells/cm 2 and less than 2 ⁇ 10 4 cells/cm 2 .
• the culture period required for manufacturing the multilayered cell structure can be reduced.
• the present inventors have discovered that it is possible to shorten the structure, suppress cell death in a multilayered cell structure, and form multilayered cell structures in various shapes, and have completed the present invention.
• the present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is, ⁇ 1> Using a serum-free medium, culturing mesenchymal stem cells derived from dental pulp in culture equipment coated with type I collagen or fragments thereof, A method for producing a multilayered cell structure, characterized in that the number of seeded mesenchymal stem cells derived from the dental pulp is more than 0.5 ⁇ 10 4 cells/cm 2 and less than 2 ⁇ 10 4 cells/cm 2 . . ⁇ 2> The method for producing a multilayer cell structure according to ⁇ 1> above, wherein the serum-free medium does not contain a xenobiotic component.
• ⁇ 3> The method for producing a multilayered cell structure according to any one of ⁇ 1> to ⁇ 2>, wherein the shape of the multilayered cell structure is one of a sheet, a rod, and a disk.
• ⁇ 4> The method for producing a multilayer cell structure according to ⁇ 3> above, wherein the multilayer cell structure has a sheet-like shape and a thickness of 5 ⁇ m to 40 ⁇ m.
• ⁇ 5> The method for producing a multilayer cell structure according to any one of ⁇ 1> to ⁇ 4>, wherein the proportion of dead cells in the multilayer cell structure is 5% or less.
• ⁇ 6> The method for producing a multilayered cell structure according to any one of ⁇ 1> to ⁇ 5>, wherein the multilayered cell structure expresses type I collagen, type IV collagen, and type VII collagen. .
• ⁇ 7> A multilayer cell structure of mesenchymal stem cells derived from dental pulp, The multilayer cell structure is characterized in that the percentage of dead cells in the multilayer cell structure is 5% or less.
• ⁇ 8> The multilayer cell structure according to ⁇ 7>, wherein the multilayer cell structure expresses type I collagen, type IV collagen, and type VII collagen.
• ⁇ 9> The multilayer cell structure according to any one of ⁇ 7> to ⁇ 8>, wherein the multilayer cell structure has a sheet-like, rod-like, or disk-like shape.
• ⁇ 10> The multilayer cell structure according to ⁇ 9>, wherein the multilayer cell structure has a sheet-like shape and a thickness of 5 ⁇ m to 40 ⁇ m.
• the present invention it is possible to solve the above-mentioned conventional problems and achieve the above-mentioned objectives, to shorten the culture period required for manufacturing a multilayered cell structure, and to prevent cell death in the multilayered cell structure. It is possible to provide a method for manufacturing a multilayer cell structure and a multilayer cell structure that can be suppressed and can be formed into multilayer cell structures of various shapes.
• FIG. 1 is a graph showing the results of growth curve evaluation in Test Example 1.
• FIG. 2 is a phase contrast micrograph on the 10th day of culture in the growth curve evaluation of Test Example 1.
• FIG. 3 is a graph showing the results of growth curve evaluation in Test Example 2.
• FIG. 4 is a phase contrast micrograph on the 10th day of culture in the growth curve evaluation of Test Example 2.
• FIG. 5 is a stereoscopic microscope image of the cell sheets of Test Examples 2-1 and 2-2 on the 10th day of culture.
• FIG. 6 is a stereomicroscopic image of the cell sheet of Test Example 2-3 on the 10th day of culture.
• FIG. 7 is a diagram showing the results of hematoxylin and eosin (HE) staining and Masson's trichrome (MT) staining of cells on day 10 of culture in Test Example 2.
• FIG. 8 is an immunofluorescence staining diagram showing the results of TUNEL staining and DAPI staining of cells on day 10 of culture in Test Example 2.
• FIG. 9 is a diagram showing the results of immunofluorescence staining of various collagens in the self-multilayered cell sheet of dental pulp stem cells on day 10 of culture in Test Example 2-1.
• FIG. 10 is a diagram showing the appearance of a self-multilayered cell sheet of dental pulp stem cells rolled up into a roll on the 10th day of culture in Test Example 3.
• FIG. 11 is a diagram showing the appearance of the state on the fourth day of culture after rolling in Test Example 3.
• FIG. 12 is a diagram showing the results of hematoxylin and eosin (HE) staining of a cross section of Test Example 3 on the 4th day of culture after rolling.
• FIG. 13 is a diagram showing the results of immunofluorescence staining of various types of collagen in the cross section of the cell tube on the fourth day of culture after rolling in Test Example 3.
• FIG. 14 is a phase-contrast microscopic image of cells in Test Example 4 after one month of culture.
• FIG. 15 is a stereoscopic microscope image of cells of Test Example 4 after 1 month of culture (before being supported with tweezers).
• FIG. 16 is a stereoscopic microscope image of cells of Test Example 4 after 1 month of culture (when supported with tweezers).
• FIG. 17 is a diagram showing the results of hematoxylin and eosin (HE) staining of cells in Test Example 4 after one month of culture.
• FIG. 18 is a diagram showing the results of Masson's trichrome (MT) staining of cells in Test Example 4 after one month of culture.
• FIG. 19 is an immunofluorescence staining diagram showing the results of TUNEL staining and DAPI staining of cells in Test Example 4 after one month of culture.
• FIG. 20 is a diagram showing the results of immunofluorescence staining of various collagens of dental pulp stem cells after 1 month of culture in Test Example 4-1.
• FIG. 21 is a graph showing the results of growth curve evaluation in Test Example 5.
• FIG. 22 is a phase contrast micrograph on the 10th day of culture in the growth curve evaluation of Test Example 5.
• FIG. 23 is a stereoscopic microscope image (when supported with tweezers) of self-multilayered cell sheets of dental pulp stem cells on day 10 of culture in Test Examples 5-1 and 5-2.
• FIG. 24 is a diagram showing the results of hematoxylin and eosin (HE) staining and Masson's trichrome (MT) staining of cells on day 10 of culture in Test Example 5.
• FIG. 25 is a diagram showing the results of immunofluorescence staining of various collagens in the cell sheet of dental pulp stem cells on day 10 of culture in Test Example 5-1.
• FIG. 26 is a diagram showing the results of immunofluorescent staining of various collagens in the cell sheet of dental pulp stem cells on day 10 of culture in Test Example 5-2.
• the method for producing a multilayer cell structure of the present invention includes at least a culturing step, and further includes other steps as necessary.
• the multilayer cell structure of the present invention can be suitably produced by the method for producing a multilayer cell structure of the present invention.
• the multilayer cell structure of the present invention will also be explained in conjunction with the explanation of the method for manufacturing the multilayer cell structure of the present invention.
• the culturing step is a step of culturing dental pulp-derived mesenchymal stem cells (hereinafter sometimes referred to as "dental pulp stem cells”) using a serum-free medium in culture equipment coated with type I collagen or its fragments. be.
• the serum-free medium is not particularly limited as long as it does not contain FBS, and any known mesenchymal stem cell growth medium can be appropriately selected depending on the purpose.
• a commercially available product may be used as the serum-free medium. There are no particular restrictions on the commercially available serum-free medium, and it can be selected as appropriate depending on the purpose.For example, PRIME-XV (registered trademark) MSC Expansion ), etc.
• the serum-free medium may or may not contain xenobiotic components, but is preferably one that does not contain xenobiotic components.
• the culture equipment is not particularly limited as long as it is coated with type I collagen or its fragments, and any known culture equipment can be appropriately selected depending on the purpose.
• the form of the culture equipment is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include culture dishes (culture dishes), culture plates, and the like.
• the type I collagen fragment refers to a fragment having a part of the amino acid sequence of type I collagen.
• the type I collagen fragment may be obtained by linking a plurality of overlapping parts of the type I collagen amino acid sequence.
• the type I collagen fragments may be recombinant proteins (hereinafter sometimes referred to as "recombinant peptides"), or may be fragments obtained by degrading type I collagen.
• the type I collagen or fragment thereof is not particularly limited, and commercially available products can be used as appropriate.
• a commercially available type I collagen fragment includes cellnest human type I collagen-like recombinant peptide (manufactured by Fujifilm).
• the method and amount of application of the type I collagen or its fragments to the culture equipment are not particularly limited as long as they do not impair the effects of the present invention, and known methods and amount of application may be appropriately selected depending on the purpose. For example, the application method and amount recommended by the manufacturer may be used.
• the culture equipment may be coated with type I collagen or its fragments at the time of use, or may be coated culture equipment. Commercially available products may be used as the culture equipment.
• the dental pulp stem cells may be those prepared from teeth at the time of use, or prepared dental pulp stem cells may be used.
• the species of the dental pulp stem cells is not particularly limited and can be appropriately selected depending on the purpose; examples include humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, cows, pigs, monkeys, etc. It will be done.
• the method for preparing the dental pulp stem cells is not particularly limited, and any known method can be selected as appropriate depending on the purpose. For example, in humans, extracted teeth of adults, baby teeth of mixed dentition stage (replacement stage) Examples include a method of preparing from.
• the dental pulp stem cells used in the culture step are not particularly limited and can be appropriately selected depending on the purpose.
• dental pulp stem cells with a passage number of 3 to 5 can be used.
• the number of seeded dental pulp stem cells in the culture step is not particularly limited as long as it is more than 0.5 x 104 cells/ cm2 and less than 2 x 104 cells/ cm2 , and is appropriately selected depending on the purpose. However, it is preferably 0.75 ⁇ 10 4 cells/cm 2 or more and less than 2 ⁇ 10 4 cells/cm 2 , and preferably 0.75 ⁇ 10 4 cells/cm 2 or more and 1.5 ⁇ 10 4 cells/cm 2 or less. is more preferable, and particularly preferably more than 0.75 ⁇ 10 4 cells/cm 2 and less than 1.5 ⁇ 10 4 cells/cm 2 .
• the temperature and carbon dioxide (CO 2 ) concentration in the culture are not particularly limited as long as they do not impair the effects of the present invention, and known conditions for culturing mesenchymal stem cells can be appropriately selected. Examples include 37°C and a CO 2 concentration of 4.7%.
• the period of the culture is not particularly limited and can be appropriately selected depending on the shape of the desired multilayered cell structure. According to the method of the present invention, a self-multilayered cell sheet can be produced in a shorter period of time than conventional methods.
• the multilayer cell structure of the present invention is a multilayer cell structure of mesenchymal stem cells derived from dental pulp.
• the multilayered cell structure is preferably a self-multilayered structure (hereinafter sometimes referred to as a "self-multilayered tissue").
• self-multilayering refers to cells autonomously becoming multilayered, and preferably cells producing ECM.
• the number of layers in the multilayer cell structure is not particularly limited as long as it is two or more layers, and can be appropriately selected depending on the purpose.
• the method for confirming whether the multilayered cell structure is multilayered is not particularly limited and can be appropriately selected depending on the purpose. For example, hematoxylin and eosin (HE) staining, Masson's trichrome (MT), etc. ) A method of histopathological confirmation by staining, a method of fluorescent staining of specific proteins inside and outside the cells, and confirmation using a confocal laser microscope, etc.
• HE hematoxylin and eosin
• MT Masson's trichrome
• a method of histopathological confirmation by staining a method of fluorescent staining of specific proteins inside and outside the cells, and confirmation using a confocal laser microscope, etc.
• a monolayer structure on a culture device that has reached confluence is transparent, a multilayered cell structure will have a thick and cloudy surface
• the shape of the multilayered cell structure is not particularly limited and can be appropriately selected depending on the purpose, for example, sheet-like (hereinafter sometimes referred to as “cell sheet”), rod-like (hereinafter “ ), disk-shaped (hereinafter sometimes referred to as “cell disk”), and the like.
• the thickness of the cell sheet is not particularly limited and can be appropriately selected depending on the purpose, for example, from 5 ⁇ m to 40 ⁇ m, preferably from 10 ⁇ m to 35 ⁇ m. Note that the thickness of the cell sheet refers to the average value of thicknesses measured at 10 randomly selected locations on the cell sheet.
• the cell sheet can be formed by culturing in the above-described culturing process for about 10 days.
• the area of the cell sheet when viewed in plan is not particularly limited and can be appropriately selected depending on the purpose.
• the cell tube is rod-shaped.
• the cell tube can also be referred to as a solid tube.
• the cell tube can be formed, for example, by winding a cell sheet obtained by culturing in the above-mentioned culturing process for about 10 days into a cylindrical shape, and culturing it for about 4 more days.
• the thickness and length of the cell tube are not particularly limited and can be appropriately selected depending on the purpose.
• the method for confirming whether or not the cell tube is solid is not particularly limited and can be appropriately selected depending on the purpose, such as hematoxylin and eosin (HE) staining.
• HE hematoxylin and eosin
• the thickness of the cell disk is not particularly limited and can be appropriately selected depending on the purpose, for example, more than 40 ⁇ m and less than 60 ⁇ m.
• the cell disk can be formed by culturing in the above-described culturing process for about one month.
• the area of the cell disk when viewed in plan is not particularly limited and can be appropriately selected depending on the purpose.
• the mechanical strength of the cell sheet, cell tube, and cell disk is not particularly limited and can be appropriately selected depending on the purpose.
• the cell sheet may have a compressive strength of about 0.02N to 0.04N
• the cell tube may have a tensile strength of about 0.2N to 0.5N
• the cell disk may have a compressive strength of about 0.2N to 0.5N.
• the tensile strength can be about 0.1N to 0.5N.
• the percentage of dead cells in the multilayered cell structure is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 5% or less, more preferably 3% or less, and particularly preferably 1% or less.
• the method for analyzing the percentage of dead cells in the multilayered cell structure is not particularly limited and can be selected as appropriate depending on the purpose, for example, using the Live or Dead Cell Viability Assay Kit (manufactured by AAT Bioquest). Examples include methods.
• the method for confirming whether dead cells are contained in the multilayered cell structure is not particularly limited and can be appropriately selected depending on the purpose.
• TUNEL Terminal deoxynucleotidyl transfer-mediated Examples include a method of confirmation using deoxyuridine triphosphate nick-end labeling) staining.
• the TUNEL staining method is not particularly limited, and can be performed using, for example, TUNEL staining manufactured by Roche Diagnostics.
• the multilayered cell structure is one in which no TUNEL-positive cells are detected when TUNEL staining is performed.
• the multilayered cell structure expresses type I collagen, type IV collagen, and type VII collagen.
• the method for confirming whether the multilayered cell structure expresses type I collagen, type IV collagen, and type VII collagen is not particularly limited, and can be appropriately selected depending on the purpose. For example, , a method of confirming by immunofluorescence staining using antibodies against each collagen, etc.
• the use of the multilayered cell structure is not particularly limited and can be appropriately selected depending on the purpose.
• the cell sheet includes, for example, bone, periodontal tissue, teeth (dental pulp, dentin, etc.), skin (epidermis, dermis, etc.), corneal epithelium, cartilage, brain, heart, liver, pancreas, kidney
• the cell tubes can be used as regenerative medical materials for the uterus, middle ear, thyroid, esophagus, lungs, etc., and for example, as regenerative medical materials for nerves, muscles, blood vessels, teeth (dental pulp, dentin, etc.), tendons, ligaments, etc.
• the cell disk can be suitably used as a regenerative medical material for, for example, teeth (dental pulp, dentin, etc.), skin (epidermis, dermis, etc.), corneal epithelium, temporomandibular joint disc, knee cartilage, bone, etc. can.
• the pulp tissue was incubated at 37°C for 1 hour with a mixture of 3 mg/mL collagenase type I (Merck KGaA) and 4 mg/mL dispase (Fujifilm Wako Pure Chemical Industries, Ltd.), and then incubated with a cell strainer ( ⁇ 70 ⁇ m; (manufactured by Greiner Bio-One) to prepare a suspension of dental pulp stem cells.
• a cell strainer ⁇ 70 ⁇ m; (manufactured by Greiner Bio-One) to prepare a suspension of dental pulp stem cells.
• dental pulp stem cells were seeded in a 60 mm culture dish (manufactured by Thermo Fisher Scientific, subsequent culture dishes were coated with type I collagen) coated with type I collagen (Cellmatrix I; manufactured by Nitta Gelatin Co., Ltd.), and cGMP xeno -/serum-free culture medium (XFM) (PRIME-XV (registered trademark) MSC Expansion .
• XFM xeno -/serum-free culture medium
• the dental pulp stem cells were collected with a mixture of 0.25% trypsin (Becton Dickinson) and 0.02% ethylenediaminetetraacetic acid (EDTA; Dojindo) and cultured at 60 mm.
• the dental pulp stem cells were subcultured in a dish at a cell density of 5 ⁇ 10 3 cells/cm 2 (0.5 ⁇ 10 4 cells/cm 2 ), and the dental pulp stem cells at passage number 3 to 5 were used in the following experiments.
• ⁇ Test Example 1-1 0.5 ⁇ 10 4 cells/cm 2 (“ ⁇ ” in Figure 1) - Test example 1-2: 1 ⁇ 10 4 cells/cm 2 (“ ⁇ ” in Figure 1) - Test example 1-3: 2 ⁇ 10 4 cells/cm 2 (“ ⁇ ” in Figure 1) ⁇ Test Example 1-4: 4 ⁇ 10 4 cells/cm 2 (“ ⁇ ” in Figure 1)
• Test Examples 1-2 to 1-4 cell proliferation was faster than in Test Example 1-1.
• Test Example 1-2 self-multilayering was observed on the 10th day of culture.
• Test Examples 1-3 and 1-4 there was variation in cell proliferation as seen from the large standard deviation ( ⁇ SD), and self-multilayering was not observed even on the 10th day of culture.
• Test Examples 1-2 to 1-4 phase contrast micrographs on the 10th day of culture are shown in FIG.
• the culture dish was completely covered with the cell sheet, which was stable, and no peeling or agglomeration was observed.
• the cell sheet did not peel off from the culture dish or the cells formed an aggregate and did not form into a sheet.
• these Test Examples 1-3 and 1-4 even on the 8th day of culture, there were some areas where there were no cells or where the cell density was low, and cell sheets were not observed, and some of them are not shown in the photos. Similarly, peeling and agglomerates were formed.
• Test Example 1-2 was the optimal seeding condition for forming a self-multilayered tissue. was gotten.
• Test example 2 ⁇ Test Example 2-1> Growth curve evaluation was performed in the same manner as Test Example 1-2. The results are shown in Figure 3.
• Test Examples 2-1 to 2-3 phase contrast micrographs on the 10th day of culture are shown in FIG. Test Example 2-1 formed a stable self-multilayered cell sheet. On the other hand, in Test Example 2-2, although confluence was reached, self-multilayering was not observed. In Test Example 2-3, cells were detached from the culture dish and formed into aggregates.
• FIG. 5 shows the stereomicroscopic images of the cell sheets of Test Examples 2-1 and 2-2 on the 10th day of culture
• FIG. 6 shows the stereoscopic microscopic images of the cell sheets of Test Example 2-3 on the 10th day of culture.
• the self-multilayered cell sheet of dental pulp stem cells of Test Example 2-1 was a durable cell sheet that could not be torn even when grasped with sharp precision tweezers, and had amazing operability and shapeability.
• the bone marrow stem cells of Test Example 2-2 it was impossible to grasp them mechanically because they had only reached confluence and had not been formed into a sheet.
• the umbilical cord stem cells in Test Example 2-3 they were detached from the culture dish and aggregates (arrows in FIG. 6) were observed.
• Test Examples 2-1 to 2-3 on the 10th day of culture was fixed with 4% paraformaldehyde (manufactured by Kanto Kagaku Co., Ltd.) for 3 days, and frozen in an optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.). Buried. Frozen sections were prepared according to conventional methods, and stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The results are shown in FIG. 7 (upper row: HE staining, lower row: MT staining). In Test Example 2-1, a multilayered cell structure due to the production of abundant collagen fibers that was aniline blue positive was observed by MT staining. On the other hand, Test Example 2-2 was negative for aniline blue and had a monolayer structure with no collagen production, and Test Example 2-3 had aggregated cells.
• HE hematoxylin and eosin
• MT staining Masson's trichrome
• Test Examples 2-1 to 2-3 on the 10th day of culture was fixed with 4% paraformaldehyde (manufactured by Kanto Kagaku Co., Ltd.) and frozen embedded in an optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.). .
• Frozen sections were prepared according to a conventional method, and TUNEL staining (manufactured by Roche Diagnostics) and DAPI staining (manufactured by Nacalai Tesque) were performed according to the method recommended by the manufacturer. The results are shown in FIG. 8 (upper row: TUNEL staining, middle row: DAPI staining, lower row: merged).
• Test Example 2-1 and Test Example 2-2 were both TUNEL negative. On the other hand, many TUNEL-positive cells were observed in the aggregate of Test Example 2-3, confirming cell death.
• each cell sheet of Test Example 2-1 and Test Example 2-2 was stained with Live or Dead Cell Viability Assay Kit (manufactured by AAT Bioquest) to remove dead cells. Quantitative evaluation was performed. Each cell was seeded in a 12-well plate under the conditions of Test Example 2-1 or 2-2, and on the 10th day of culture, 10 fields of view (400x magnification) per well were randomly selected to evaluate dead cells. As a result, the percentage of dead cells in Test Example 2-1 was 1.24 ⁇ 0.006%, and the percentage of dead cells in Test Example 2-2 was 1.16 ⁇ 0.007%. Only dead cells were observed.
• FIG. 9 shows the results of immunofluorescence staining of various collagens in the self-multilayered cell sheet of dental pulp stem cells on day 10 of culture in Test Example 2-1.
• the bone marrow stem cells of Test Example 2-2 did not form into a sheet and no collagen production was observed, and the umbilical cord stem cells of Test Example 2-3 formed an aggregate and did not form into a sheet, so they were excluded from this analysis.
• the left column shows the results of staining various collagens (upper row: type I collagen, middle row: type IV collagen, lower row: type VII collagen), the center row shows the results of DAPI staining, and the right column shows the merged results. Show the results.
• the primary antibodies used are as follows.
• Anti-type I collagen antibody collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody collagen type IV (manufactured by Abcam)
• Anti-type VII collagen antibody collagen type VII (manufactured by Abcam)
• type I, type IV, and type VII collagen were positive throughout the entire layer of the cell sheet. These collagens are major constituent proteins of the extracellular matrix (ECM) in vivo, and are thought to provide strength and flexibility (malleability) to the self-multilayered cell sheet.
• ECM extracellular matrix
• cell sheet The mechanical strength of the self-multilayered cell sheet of dental pulp stem cells (hereinafter referred to as "cell sheet”) on day 10 of culture in Test Example 2-1 was measured as follows. -Compressive strength- The cell sheet was placed in a hole with a diameter of 5 mm on the stage of a load-displacement measuring unit (FSA-0.5KE-5N, manufactured by Imada), and a load was applied at a speed of 1 mm/min with a measuring rod of 3 mm in diameter. Quantitative evaluation was performed by monitoring the change in load until the cell sheet broke. As a result, the compressive strength of the cell sheet was 0.028 ⁇ 0.008N.
• FSA-0.5KE-5N load-displacement measuring unit
• Test example 3 The self-multilayered cell sheet of dental pulp stem cells on the 10th day of culture in Test Example 2-1 was rolled up into a roll (see FIG. 10).
• CGMP XENO- / Serum -Free Culture Medium (XFM) (Prime -XV (registered trademark) MSC EXPANSION XSFM 37 ° C, 4.7 by pharmaceutical company) % CO2 environment.
• FIG. 11 shows the state on the fourth day of culture after rolling.
• FIG. 12 shows the results of hematoxylin and eosin (HE) staining on a cross section of a cell tube on the fourth day of culture after rolling. As shown in FIG. 12, solid cell tubes were obtained instead of hollow ones.
• HE hematoxylin and eosin
• FIG. 13 shows the results of immunofluorescence staining of various collagens on the cross section of the cell tube on the fourth day of culture after rolling.
• the left column shows the results of staining various collagens (upper row: type I collagen, middle row: type IV collagen, lower row: type VII collagen), the center row shows the results of DAPI staining, and the right column shows the merged results. Show the results.
• the primary antibodies used are as follows. - Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology) - Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam) - Anti-type VII collagen antibody: collagen type VII (manufactured by Abcam)
• type I, type IV, and type VII collagen were positive throughout the cell tube.
• the mechanical strength of the cell tube on the fourth day of culture after rolling was measured as follows. -Tensile strength- The cell tube was grasped using the force gauge (measuring device) and special jig (clip) of the load-displacement measurement unit (FSA-0.5KE-5N, manufactured by Imada), and pulled upward at a speed of 1 mm/min. Quantitative evaluation was performed by monitoring the change in load until the cell tube broke. As a result, the tensile strength of the cell tube was 0.38 ⁇ 0.130N.
• Test example 4 [Cell disc] ⁇ Test Example 4-1> Dental pulp stem cells were cultured in the same manner as Test Example 2-1 except that the culture period was one month.
• Test Examples 4-1 and 4-2 phase contrast micrographs of cells after 1 month of culture are shown in FIG. Test Example 4-1 formed a stable self-multilayered cell sheet. On the other hand, in Test Example 4-2, the thickness was increased compared to the confluent state of Test Example 2-2, and a multilayered appearance was observed.
• FIG. 15 Stereoscopic microscopic images of cells in Test Examples 4-1 and 4-2 after one month of culture are shown in FIG. 15 (before being supported with tweezers) and FIG. 16 (when being supported with tweezers).
• the self-multilayered cell sheet of dental pulp stem cells of Test Example 4-1 is a durable cell sheet that does not tear even when grasped with precision tweezers with sharp tips; The cell sheet was thicker and stronger than the previous cell sheet, and had a disk-like appearance.
• the bone marrow stem cell sheet of Test Example 4-2 broke when touched with tweezers, making it impossible to grasp it mechanically.
• Test Examples 4-1 and 4-2 after 1 month of culture was fixed with 4% paraformaldehyde (manufactured by Kanto Kagaku Co., Ltd.) for 3 days, and frozen embedded in optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.). did. Frozen sections were prepared according to conventional methods, and stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The results are shown in FIG. 17 (HE staining) and FIG. 18 (MT staining). In Test Example 4-1, the cells were in good condition with no dead cells, and a multilayered cell structure due to the production of abundant collagen fibers that were positive for aniline blue was observed. On the other hand, in Test Example 4-2, although multilayering was observed due to the production of collagen fibers that were positive for aniline blue, many cells were dead and the presence of cells was extremely low.
• Test Examples 4-1 and 4-2 after 1 month of culture was subjected to TUNEL staining (manufactured by Roche Diagnostics) and DAPI staining (manufactured by Nacalai Tesque) according to the method recommended by the manufacturer. The results are shown in FIG. 19 (upper row: TUNEL staining, middle row: DAPI staining, lower row: merged).
• Test Example 4-1 was TUNEL negative and no cell death was observed. On the other hand, in Test Example 4-2, many cells had already died, and TUNEL-positive cell death was confirmed among the observed cells.
• FIG. 20 shows the results of immunofluorescence staining of various collagens of dental pulp stem cells after 1 month of culture in Test Example 4-1.
• the left column shows the results of staining various collagens (upper row: type I collagen, middle row: type IV collagen, lower row: type VII collagen), the center row shows the results of DAPI staining, and the right column shows the merged results. Show the results.
• the primary antibodies used are as follows. - Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology) - Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam) - Anti-type VII collagen antibody: collagen type VII (manufactured by Abcam)
• cell disc The mechanical strength of the disc-shaped self-multilayered cell sheet of dental pulp stem cells (hereinafter referred to as "cell disc") after one month of culture in Test Example 4-1 was measured as follows.
• -Compressive strength The cell disk was placed in a hole with a diameter of 5 mm on the stage of a load-displacement measuring unit (FSA-0.5KE-5N, manufactured by Imada), and a load was applied at a speed of 1 mm/min with a measuring rod of 3 mm in diameter. Quantitative evaluation was performed by monitoring the change in load until the cell disc ruptured. As a result, the compressive strength of the cell disk was 0.75 ⁇ 0.175N.
• the cell disk was grasped using a force gauge (measuring device) and a special jig (clip) of a load-displacement measuring unit (FSA-0.5KE-5N, manufactured by Imada Corporation), and pulled upward at a speed of 1 mm/min. Quantitative evaluation was performed by monitoring the change in load until the cell disc ruptured. As a result, the tensile strength of the cell disk was 0.32 ⁇ 0.155N.
• Test Example 5 ⁇ Test Example 5-1> Growth curve evaluation was performed in the same manner as in Test Example 1, except that the number of dental pulp stem cells seeded was 0.75 ⁇ 10 4 cells/cm 2 . The results are shown in FIG.
• Test Example 5-2 (“ ⁇ " in Figure 21) showed proliferation similar to that in Test Example 1-2, and Test Example 5-1 (" ⁇ " in Figure 21) Cell proliferation equivalent to Test Example 1-1 was observed.
• Test Examples 5-1 and 5-2 on the 10th day of culture was fixed with 4% paraformaldehyde (manufactured by Kanto Kagaku Co., Ltd.) and frozen embedded in an optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.). Frozen sections were prepared according to conventional methods, and stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The results are shown in FIG. 24 (upper row: HE staining, lower row: MT staining). Test Example 5-1 formed a structure densely composed of cells and collagen, but no significant multilayering was observed histologically. On the other hand, in Test Example 5-2, a structure in which cells and collagen were significantly multilayered was formed.
• each cell sheet of Test Example 5-1 and Test Example 5-2 was stained with Live or Dead Cell Viability Assay Kit (manufactured by AAT Bioquest) to remove dead cells. Quantitative evaluation was performed. Each cell was seeded in a 12-well plate under the conditions of Test Example 5-1 or 5-2, and on the 10th day of culture, 10 fields of view (400x magnification) per well were randomly selected to evaluate dead cells. As a result, the percentage of dead cells in Test Example 5-1 was 0.51 ⁇ 0.003%, and the percentage of dead cells in Test Example 5-2 was 1.33 ⁇ 0.010%. Only dead cells were observed.
• Figure 25 shows the results of immunofluorescence staining of various collagens in the cell sheet of dental pulp stem cells on the 10th day of culture in Test Example 5-1.
• the results of immunofluorescence staining are shown in FIG. 26.
• the left column shows the results of staining various collagens (upper row: type I collagen, middle row: type IV collagen, lower row: type VII collagen), the middle row shows the results of DAPI staining, and the right column shows the results of staining with DAPI. Shows the merged result.
• the primary antibodies used are as follows.
• Anti-type I collagen antibody collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody collagen type IV (manufactured by Abcam)
• Anti-type VII collagen antibody collagen type VII (manufactured by Abcam)
• type I, type IV, and type VII collagen were positive throughout the entire layer of the cell sheet. These collagens are major constituent proteins of the extracellular matrix (ECM) in vivo, and are thought to provide strength and flexibility (malleability) to the self-multilayered cell sheet.
• ECM extracellular matrix
• Test Example 6 On the 10th day of culture, under the seeding conditions that formed a self-multilayered cell sheet that could be grasped with tweezers, Test Example 2-1 had the maximum thickness of the cell sheet, and Test Example 5-1 had the minimum thickness. Quantitative evaluation was performed by measuring the thickness of the cell sheet on the 10th day of culture. To measure the thickness, HE-stained tissue photographs were used. Ten locations on the cell sheet were randomly selected and their thicknesses were measured. As a result, the thickness of the cell sheet of Test Example 2-1 was 33.0 ⁇ 6.16 ⁇ m, and the thickness of the cell sheet of Test Example 5-1 was 8.0 ⁇ 2.20 ⁇ m. The cell sheet of Test Example 2-1 was three times or more thicker than the cell sheet of Test Example 5-1.
• the present invention it is possible to shorten the culture period required for manufacturing a cell sheet, which is a multilayered cell structure, and to suppress cell death in the multilayered cell structure. It is possible to manufacture multilayer cell structures with various shapes.

Landscapes

• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Materials For Medical Uses (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=88243015

Family Applications (1)

Country Status (2)

Citations (3)

• 2023
  • 2023-04-07 JP JP2024514321A patent/JP7844055B2/ja active Active
  • 2023-04-07 WO PCT/JP2023/014332 patent/WO2023195531A1/ja not_active Ceased

Patent Citations (3)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events