KR20220122430A - Method for extracting and culturing dental stem cell for tissue regeneration - Google Patents

Abstract

The present invention relates to a method for extracting and culturing dental pulp stem cells, and dental pulp stem cells produced therefrom, wherein the method comprises the following steps: (a) exposing dental pulp by splitting a tooth by applying a hammer or a handpiece; (b) segmenting the dental pulp; (c) culturing the result of step (b) in a culture medium after enzymatic digestion; and (d) subculturing the product of step (c). The dental pulp stem cells produced by using the method are excellent in colony formation, proliferative ability, and differentiation into bones, fat, cartilages, nerves, and the like, and thus can be usefully used as transplant materials for treatment.

Description

Extraction of pulp stem cells for tissue regeneration and culturing method thereof {Method for extracting and culturing dental stem cell for tissue regeneration}

The present invention relates to a method for extracting and culturing pulp stem cells from teeth.

Stem cells are cells capable of differentiating into various cells constituting biological tissues, and collectively refer to undifferentiated cells in the pre-differentiation stage obtained from each tissue of the embryo, fetus, and adult. Stem cells are differentiated into specific cells by differentiation stimuli, have the characteristics of producing cells identical to themselves by cell division, and have the flexibility to differentiate into different cells according to differentiation stimuli. is characterized.

Stem cells can be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation capacity. Pluripotent stem cells are pluripotent cells with the potential to be differentiated into all cells. Embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells, iPS) and the like. Examples of multipotent and/or unipotent stem cells include adult stem cells, and include hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like. Mesenchymal stem cells have the ability to differentiate into various cells such as adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, cardiomyocytes, hepatocytes, pancreatic islet beta cells, and vascular cells. It is a stem cell that is expected to be applied in

As one of the stem cells classified as mesenchymal stem cells, pulp stem cells derived from pulp tissue are known. Among pulp stem cells, dental pulp stem cells derived from dental pulp have been reported to have therapeutic effects in various disease models (Seo et al., Oral Dis. (2008) 14:428-434; Yamaza et al., Stem Cell Res). Ther. (2010) 1:5), pulp stem cells are attracting attention as a source of stem cells used in regenerative medicine due to these therapeutic effects.

Patent Literature: Domestic Patent Publication No. 10-2021-0041578

As a result of the study on the extraction and culture method of pulp stem cells from teeth, the present inventors confirmed that pulp stem cells with excellent cell proliferation ability and stem cell characteristics can be obtained according to the manufacturing method according to the present invention. has been completed

Accordingly, the problem to be solved by the present invention is to expose the dimension by dividing the tooth (a); (b) dividing the dimension; (c) decomposing the product of step (b) with a protease and then culturing initially; (d) removing the supernatant by centrifugation after adding a culture medium to the resultant of step (c); (e) culturing by adding a culture medium to the supernatant after filtering the resultant of step (d) using a cell strainer; (f) washing after confirming that the resultant cells of step (e) are attached to the culture dish; (g) culturing after adding a culture medium to the resultant of (f); (h) relates to a pulp stem cell culture method comprising the step of sub-culturing the result of step (g).

Another object to be solved by the present invention is to provide pulp stem cells prepared by the above method.

Another object to be solved by the present invention is to provide a method for manufacturing a graft material for treatment, including the step of manufacturing a material for treatment using the pulp stem cells prepared by the above method.

Another object to be solved by the present invention is to provide a therapeutic graft including pulp stem cells prepared by the above method.

The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention comprises the steps of (a) dividing the tooth to expose the pulp; (b) dividing the dimension; (c) decomposing the product of step (b) with a protease and then culturing initially; (d) removing the supernatant by centrifugation after adding a culture medium to the resultant of step (c); (e) culturing by adding a culture medium to the supernatant after filtering the resultant of step (d) using a cell strainer; (f) washing after confirming that the resultant cells of step (e) are attached to the culture dish; (g) culturing after adding a culture medium to the resultant of (f); (h) provides a pulp stem cell culture method comprising the step of sub-culturing the resultant of step (g).

In the present invention, the 'puss' refers to soft tissue intrinsic tissue in the center of the inside of the tooth, and the pulp is surrounded by dentin and includes nerve fibers, blood vessels, and lymphatic vessels inside.

The 'pull stem cells' refer to stem cells present in the pulp, and the pulp is easily accessible and has a biological status with a high ratio between stem cells and collected tissue volume, so pulp stem cells are cells for regenerative medicine. It is useful as a cell source for the development of therapeutic agents.

The 'tooth' is not particularly limited, and includes teeth having all dimensions derived from humans, such as primary teeth, permanent teeth, wisdom teeth, and excess teeth.

In the present invention, the step of exposing the pulp by dividing the tooth was applied with a hammer or handpiece. A hammer or handpiece is a dental bur that has been used to expose pulp in conventional dental treatment. Alternatively, as compared to a barbed brochae, the crown can be easily broken and the pulp is not directly in contact with the pulp, so that the pulp can be exposed as much as possible without loss, which is preferable for extraction of pulp stem cells.

In the present invention, the protease of step (c) includes collagenase, dispase, or a mixture thereof. In the example of the present invention, a mixed solution of collagenase II and dispase II was used.

In the present invention, the initial incubation of step (c) is performed at 35 °C to 40 °C for 30 minutes to 2 hours. Optionally, vortexing may be performed for 2 to 5 seconds at a minimum speed (600 rpm) at 15-minute intervals during the incubation. When the initial incubation is less than 30 minutes, it is difficult for the protease to sufficiently digest the protein component of the pulp tissue, so that it is difficult to effectively separate the pulp cells from the tissue.

In the present invention, the culture medium of step (d) includes alpha MEM, FBS, penicillin-streptomycin, L-glutamine and L-ascorbic acid phosphate (L-ascorbic acid phosphate). The addition of the culture medium is added after confirming the cotton-like structure of the pulp cells, and the centrifugation in step (d) is performed at 1000 rpm to 1500 rpm for 1 to 5 minutes. In the above condition range, it is possible to obtain pulp stem cells having high cell proliferation ability and stem cell characteristics. After confirming the cotton-like structure, the medium is added after the protease performs the tissue decomposition function properly. When the centrifugation rpm is lower than 1000 rpm, it is difficult to obtain a large amount of cells, and when it is higher than 1500 rpm, it damages the cells affect activity. In order to maintain the integrity of stem cells, the centrifugation speed should not exceed “1,500 rpm” and the time should not exceed “5 minutes”.

In the present invention, the cell strainer of step (e) has a pore size of 60 μm to 80 μm. Contaminants are efficiently removed from the pore-range strainer, and thereafter, adherent cells can be selected, enabling efficient separation of pulp stem cells.

In the present invention, the subculture of step (d) is performed once every 2-3 days at a cell density of less than 90%. Compared to the case of not performing subculture, it is possible to obtain a large amount of pulp stem cells that maintain stem cell proliferation and stem cell characteristics when subculture is performed. Specifically, the subculture is performed by dispensing the cells into a new culture vessel so that 1/20 to 1/3 of the lower area of the culture vessel is covered with the cells. If more than 70%, 80% or more of the bottom area of the culture vessel is covered with cells due to cell division, the passage is repeated and the cell density less than 90% must be maintained to prevent the proliferation of stem cells and stem cells. characteristics can be maintained.

In the present invention, the 'culture dish' is not particularly limited as long as it is an instrument that can be used for culturing cells, and a commercially available cell culture well or the like can be used. As used herein, "resultant cells adhere to the culture dish" refers to adhesion of pulp cells to the culture dish by seeding and culturing pulp cells recovered from pulp tissue on a culture dish containing a culture solution. In order for the pulp cells to become adherent on the culture dish, it can be achieved, for example, by culturing in a culture medium for 3 to 48 hours. Preferably, the culture is 24 to 48 hours. If the culture time is shorter than this, the number of adherent cells decreases, and if the culture time is longer, the possibility of cell adhesion other than stem cells occurs, which is not preferable.

Another aspect of the present invention provides pulp stem cells produced by the method described above. The pulp stem cells prepared by the present invention provide superior stem cell characteristics by confirming a higher level of oct4 expression compared to bone marrow-derived mesenchymal stem cells (hMSCs) classified as mesenchymal stem cells.

Another aspect of the present invention provides a method for manufacturing a graft material for treatment, including the step of preparing a material for treatment using the pulp stem cells prepared by the method described above. The pulp stem cells included in the transplant material for treatment may include pulp stem cells prepared by the method for producing pulp stem cells of the present invention as they are, and pulp stem cells are cultured under specific conditions, differentiation induction, etc. for a state suitable for a desired therapeutic use. cells may be included. The graft material for treatment can be injected into the target site by a syringe or the like, can be directly placed by incising the target site, and can be used in a desirable form according to the disease or site of the target. In addition, therapeutic grafts may be used in combination with immunosuppressants or other drugs.

Another aspect of the present invention provides a therapeutic graft, including pulp stem cells produced by the method described above.

In another aspect of the present invention, in addition to the steps (a) to (h), (i) attaching the product of step (h) to a culture dish at a density of 70 to 80% and then culturing to adhere the cells at a density of 80 to 90% to confirm; (j) exchanging the (i) result with α-MEM medium or commercial medium without FBS and antibiotics; and (k) passing the supernatant through a 0.1 to 0.3 um pore filter after culturing the (j) product.

Another aspect of the present invention provides an anti-inflammatory composition comprising a stem cell culture medium prepared by the method described above.

The pulp stem cell extraction and culture method according to the present invention can supply a sufficient amount of pluripotent stem cells, and provides a method for efficiently producing pulp-derived pluripotent stem cells, wherein the pulp stem cells produced thereby It has excellent colony-forming ability and stem cell characteristics, so it can be usefully used as a therapeutic graft material.

1 is a result of (a) the number of pulp stem cells prepared by the present invention, (b) stem cell ability-related oct4 protein expression, (c) colony formation ability, and (d) differentiation ability into bone, cartilage, fat, and nerve. to be.
2 shows the tissue engineering efficacy of a stem cell culture medium containing pulp stem cell-derived material. It is the result of confirming the effect of LPS-derived inflammation reduction on (a) cell migration and (b) growth promotion, and (c) inflammatory cells (Raw cells and THP1) on angiogenic cells (HUVEC).

Hereinafter, the present invention will be described in detail with reference to Preparation Examples, Examples and Experimental Examples.

However, the following Preparation Examples, Examples and Experimental Examples are merely illustrative of the present invention in detail, and the content of the present invention is not limited by the Preparation Examples, Examples and Experimental Examples.

Example 1: Isolation of pulp tissue

Teeth were extracted from patients who agreed to donate teeth. Extracted teeth were immersed in HBSS (Hanks' Balanced Salt Solution) containing 2% Antibiotic-Antimycotic (Thermo fhisher scientific) or similar antibiotics 2% and stored at 4 °C. Thereafter, the tooth was immersed in 70% ethanol for about 1 minute to disinfect the entire tooth, washed twice with HBSS to remove residual ethanol, and then the pulp tissue was separated within 1 hour by the following method.

Separation of pulp tissue using a hammer

A glove was placed on the sterilized gauze, the sterilized tooth was placed on the glove, and the crown was broken using a hammer weighing 0.5 to 2 kg. After removing all the broken dentin plates, separating only the pulp and putting it on a Petri dish, 1 to 2 drops of HBSS were dropped on the pulp using a 200 μl tip to prevent the pulp from drying out. Using small scissors or a knife, the dimensions were finely cut to 0.1 mm or less.

Separation of pulp tissue using a handpiece

The entire sterilized tooth was removed from the crown using a high-speed handpiece (300,000 to 450,000 rpm) and a dental handpiece bur (diamond, tungsten carbide, or any type of bur that can be applied to a high-speed handpiece) to expose the pulp. . At this time, sterilized handpieces and handpiece burs were used, and this was carried out in a sterile clean bench. The high-speed handpiece is applied until the dimension is slightly visible between 0.5 mm and 1 mm, then the high-speed handpiece and burr are applied to expose the dimension as large as possible when the dimension is visible between 1 mm and 2 mm. When the dimensions of 2 mm to 20 mm became visible, all of the dentin plates were broken and removed using a sterilized dental hand instrument. After exposing the pulp as wide as possible, apply a sterilized root canal file to obtain the pulp, put it on a sterilized Petri dish, and use a 200 μl tip to drop 1 to 2 drops of PBS or HBSS on the pulp and use small scissors or a knife. was used to cut the dimensions to a size of 0.1 mm or less.

Example 2: Protease Enzyme Treatment and Pulp Cell Culture

The cut pulp samples were treated with a protease enzyme solution. Dispase II and collagenase II were dissolved in PBS or HBSS to make concentrations of 4 mg/ml and 2 mg/ml, respectively, and then filtered using a 0.22 μm pore size syringe filter. 1 ml of dispase II solution was prepared in a 50 ml tube, and the dispase II solution was dropped on the pulp using a 200 μl tip, and then transferred to a 50 ml tube by pipetting. Using the collagenase II solution, it was carried out in the same manner as above and then transferred to the same tube. A 50 ml tube containing the cut pulp, 1 ml dispase II solution, and 1 ml collagenase II solution was placed in a constant temperature water bath at 37° C. and incubated for 1 hour. Optionally, during incubation, vortexing may be performed for about 3 seconds at a minimum rpm (600 rpm) at 15 minute intervals.

After incubation for about 1 hour, after confirming the cotton-like structure of the cells, 4 ml of growth medium (GM, alpha MEM, 15% FBS, 1% Penicillin-Streptomycin, 2mM L-Glutamine, 0.1mM L-ascorbic acid phosphate) ) was dropped in a dropwise manner and centrifuged at 1,500 rpm for 5 minutes. After removing the supernatant, 1 ml growth medium was added and pipetted. Thereafter, a 70 um pore size cell strainer was placed in a new 50 ml tube, and the cell supernatant was dropped in the center of the strainer in a dropwise manner. After 3 ml of growth medium was added in the same way, the filtered cell supernatant was placed in a 60 mm culture dish, and then cultured in an incubator under 5% CO 2 , 37° C. conditions. After 24 to 48 hours, it was confirmed that the cells were attached to the culture dish, washed twice with PBS, and 4 ml of growth medium was added. The medium was replaced once a week, and after a total of 2 weeks of culture, subculture was performed in a 100 mm culture dish every 2-3 days so that the cell density did not exceed 90%. After washing once with PBS during subculture, the cells were centrifuged at 1,500 rpm for 3 minutes.

Example 3: Confirmation of pulp stem cell characteristics

Oct4 expression analysis

In order to measure the stem cell ability of pulp stem cells prepared by the present invention, the amount of Oct4 protein was measured by Western blot.

As a result, it was confirmed that the pulp stem cells of the present invention had a higher expression level of Oct4 compared to bone marrow-derived mesenchymal stem cells (hMSCs). Regardless of the pulp stem cell extraction method of Hammer or Handpiece, the expression level of Oct4 at a similar level was confirmed (FIG. 1b). Oct4 is a transcription factor expressed in embryonic stem cells, plays a role in preventing cell differentiation, and disappears when natural cell differentiation begins, and is known as a marker of pluripotent stem cells (Donovan, P. J., Nature Genet., 29:246, 2001; Pesce, M. and Scholer, H. R., Stem Cells, 19:271, 2001). Therefore, high Oct4 expression of pulp stem cells means that stem cellularity is high, which means that differentiation into various mesodermal lineage cells such as osteoblasts, myoblasts, adipocytes, chondrocytes, and fibroblasts is increased. Through this, it can be confirmed that the pulp stem cells of the present invention and a therapeutic transplant material utilizing the same can be effectively applied to regenerative medicine.

Colony forming ability analysis

Colony formation analysis (CFU-F) was performed to confirm whether pulp stem cells prepared by the present invention have colony forming ability, which is a basic characteristic of stem cells. 1000 stem cells obtained through subculture were cultured on a plastic culture dish to confirm the colony-forming ability according to the pulp separation method (Hammer or Handpiece). As a result, the pulp stem cell extraction method of Hammer or Handpiece Regardless, a similar level of colony-forming ability was confirmed (FIG. 1c).

Differentiation ability analysis

An experiment was performed to confirm whether the pulp stem cells prepared by the present invention have differentiation ability. As a result of differentiating the stem cells obtained through three passages into the differentiation medium for 14 to 21 days, it was confirmed that they can be differentiated into four types of cells: bone, fat, cartilage, and nerve cells. Differentiation medium was each bone cell differentiation medium (MEM supplemented with 10% FBS, 1% P/S, 50ug/ml Ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone), adipogenic differentiation medium (StemXVivo Adipogenic Base Media supplemented adipogenic). Supplement), Chondrogenic Base Media supplemented chondrogenic Supplement, Neurobasal media (Neurobasal media, 2% B27, 1%P/S, 1% glutamax, and 20 ng/ml EGF and FGF) were used. did. In addition, it was confirmed that differentiation into 4 cells is possible regardless of the pulp stem cell extraction method of a hammer or a handpiece (FIG. 1d).

Example 5: Confirmation of characteristics of pulp stem cell culture medium

Confirmation of cell migration and proliferative capacity

After attaching the pulp stem cells prepared by the present invention to a plastic culture dish at a density of 70% to 80%, and checking the cell density of 80% to 90% the next day (provided that the cell density should not be 100%) ), alpha-MEM without FBS and antibiotics, or normal commercial medium was replaced. A stem cell culture solution cultured for 48 hours was obtained, centrifuged at 3000 rpm for 5 minutes, and then the supernatant was passed through a 0.22 um pore filter and stored at -80°C. As a result of treating the cell culture medium prepared by the present invention on HUVEC cells, it was confirmed that the number of Edu positive cells was higher than that of the commercial medium as a control, thereby confirming high proliferation and cell migration ability. In addition, excellent cell migration and proliferation ability was confirmed regardless of the pulp stem cell extraction method of a hammer or a handpiece (FIGS. 2a, b).

Check anti-inflammatory effect

In order to confirm the anti-inflammatory effect of the cell culture medium of pulp stem cells prepared by the present invention, raw cells, which are mouse macrophages, and THP-1 cells, which are human monocytes, were treated with LPS and pulp stem cell culture medium. was compared with the commercial medium as a control. The cell culture medium of the present invention confirmed the anti-inflammatory effect by confirming that the expression of TNF-alpha and IL-1beat, which are genes related to inflammation, was lower than that of the control. In addition, an excellent anti-inflammatory effect was confirmed regardless of the pulp stem cell extraction method of a hammer or a handpiece (FIG. 2c).

Claims (13)

(a) segmenting the tooth to expose the pulp;
(b) dividing the dimension;
(c) decomposing the product of step (b) with a protease and then culturing initially;
(d) removing the supernatant by centrifugation after adding a culture medium to the resultant of step (c);
(e) culturing by adding a culture medium to the supernatant after filtering the resultant of step (d) using a cell strainer;
(f) washing after confirming that the resultant cells of step (e) are attached to the culture dish;
(g) culturing after adding a culture medium to the resultant of (f); and
(h) pulp stem cell culture method comprising the step of sub-culturing the resultant of step (g). The method of claim 1, wherein the step of segmenting the tooth to expose the pulp is to apply a hammer or handpiece. The method of claim 1, wherein the culture medium in step (c) is α-MEM, FBS, penicillin-streptomycin (Penicillin-Streptomycin), L- glutamine (Glutamine) and L- ascorbic acid phosphate (L-ascorbic acid phosphate) ). The method of claim 1, wherein the culturing in step (c) is performed for 30 minutes to 2 hours. The method of claim 1, wherein the protease in step (c) comprises collagenase, dispase, or a mixture thereof. The method according to claim 1, wherein the centrifugation in step (d) is performed at 1000 rpm to 1500 rpm for 1 minute to 5 minutes. The method according to claim 1, wherein the cell strainer in step (e) has a pore size of 50 μm to 90 μm. The method of claim 1, wherein the subculture of step (h) is performed at a cell density of less than 90%. The pulp stem cells produced by the method according to any one of claims 1 to 8. A method for manufacturing a therapeutic graft, comprising the step of manufacturing a therapeutic graft using the pulp stem cells produced by the method according to any one of claims 1 to 8. A therapeutic transplant comprising pulp stem cells produced by the method according to any one of claims 1 to 8. In addition to the method according to any one of claims 1 to 8,
(i) confirming cell adhesion of 80 to 90% density by culturing after attaching the resultant of step (h) to a culture dish at a density of 70 to 80%;
(j) exchanging the (i) result with α-MEM medium or commercial medium without FBS and antibiotics; and
(k) a method of obtaining a stem cell culture medium comprising the step of passing the supernatant through a 0.1 to 0.3 um pore filter after culturing the resultant (j). An anti-inflammatory composition comprising a stem cell culture medium prepared by the method according to any one of claims 1 to 8.

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