CN111662867A - Human tooth marrow cell separation culture method - Google Patents

Abstract

The invention provides a human dental pulp cell isolated culture method, which is characterized in that glucomannan and sodium alginate are added into a primary culture medium, so that adherent growth and replication of cells in the primary culture process can be effectively promoted, an adherent effect equivalent to that of a traditional coated culture dish or by adding fibronectin can be achieved, pre-coating operation can be omitted, and the method is more economic compared with laminin, so that the time cost and the economic cost are effectively reduced while the adherent effect is improved, and a large number of dental pulp stem cells with high activity are obtained; meanwhile, the culture medium provided by the invention does not need serum, can effectively promote the growth and replication of dental pulp cells and improve the differentiation capacity of the cells by matching various components, and can overcome the defects of the serum on the premise of ensuring the performance, thereby effectively improving the culture effect of the dental pulp cells.

Description

Human tooth marrow cell separation culture method

Technical Field

The invention belongs to the technical field of human cell culture, and particularly relates to a human dental pulp cell separation culture method.

Background

The dental pulp is a loose connective tissue in the pulp chamber, and has the ability of repair and regeneration, including nerve, blood vessel, lymph and connective tissue, and also the artificial dentin cells arranged at the periphery of the pulp and the pulp stem cells inside the pulp. The dental pulp stem cell is an adult stem cell which is kept in an undifferentiated state, has multiple differentiation abilities, can be used for tissue repair and regeneration of dentin, tooth bodies and dental crowns, and is an important basis in tooth development research and tooth regeneration research. However, the existing dental pulp stem cell culture has certain difficulty, when the dental pulp tissue is cultured, fibroblasts are often obtained instead of the dental pulp stem cells, and the dental pulp stem cells are not easy to adhere to the wall in the culture process, so that the culture effect is seriously influenced; in the existing method, the adhesion of cells is promoted by pre-coating a culture dish or adding fibronectin into a culture medium, but the former method consumes a lot of time, and the latter method costs a lot, which results in higher cost of the existing method.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for separating and culturing human dental pulp cells.

The specific technical scheme of the invention is as follows:

the invention provides a method for separating and culturing human dental pulp cells on one hand, which comprises the following steps:

s1: collecting the completely extracted teeth, extracting dental pulp tissues, and digesting to obtain standby dental pulp cells;

s2: inoculating the dental pulp cells into a serum-free primary culture medium added with glucomannan and sodium alginate for primary culture;

s3: and transferring the cells subjected to primary culture into a serum-free subculture medium for subculture for later use.

The addition of glucomannan and sodium alginate in the primary culture medium can significantly improve the anchorage rate of cells in the primary culture process, thereby promoting cell amplification, avoiding pre-coating the culture dish, effectively saving time and cost and improving experiment efficiency.

Further, the specific method of step S1 is as follows:

has been collected completelyCleaning the surface of the extracted tooth with 75% ethanol, drilling grooves with the depth of about 1mm on two symmetrical surfaces along the long axis of the tooth with a split drill, cutting the tooth into two halves with a large-sized steel wire, and taking out the marrow tissue with a dental forceps; washing with 1% double antibody-containing PBS buffer solution, and cutting into pieces smaller than 1mm³The fragments are digested and stored for later use.

Further, in step S1, a specific method of digesting the pulp tissue is as follows:

adding 0.3% type I collagenase and 0.4% neutral protease into the cut pieces, mixing, sterilizing at 37 deg.C for 1 hr, slightly blowing to obtain discrete single cell mass, and storing.

The method can fully digest and disperse the cells, thereby facilitating the subsequent operation.

Further, the specific method of step S2 is as follows:

washing dental pulp cells with PBS buffer solution, centrifuging for 5min under the condition of 1000r/min, collecting cells, and repeating washing and centrifuging operations; resuspending the pelleted cells in primary medium supplemented with glucomannan and sodium alginate at 1 x 10⁴～1*10⁵Inoculating at a density of one/ml, placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the culture solution once every 3d, stopping primary culture when the cell density reaches 80-90%, and preparing for subculture.

Further, in step S2, the primary culture medium is a DMEM medium supplemented with the following components:

8-12% of plant source recombinant human serum albumin, 0.1-0.2% of glucomannan, 0.1-0.2% of sodium alginate, 5-10% of sodium lactate and 0.05-0.1% of naringenin.

At present, the conventional cell preservation solution mainly uses fetal bovine serum or human serum albumin, the higher the cell value is, the higher the requirement on nutrition is, and the higher the dosage of the components is; the serum components are complex, and besides components for promoting cell growth, the serum components also contain other harmful components such as endotoxin, hemoglobin, complement, antibodies and the like, which can influence the cell growth; the plant source recombinant human serum albumin is manufactured based on plant production, can provide nutrient components equivalent to serum, does not contain harmful components in the serum, and can replace fetal bovine serum, so that the cost can be reduced, and the adverse effect on cells can be reduced.

Sodium lactate Green is sterilized water solution of sodium lactate, sodium chloride, potassium chloride and calcium chloride, and can be used for providing environment with moderate osmotic pressure similar to human body to facilitate cell survival

The naringenin is a natural mixture extracted from orange peel, has strong antibacterial effect, can inhibit growth and proliferation of bacteria, has no toxic or side effect, does not influence cell growth, and does not cause drug resistance of pathogenic microorganisms.

Further, in the primary culture medium, the ratio of glucomannan to sodium alginate is 1:1 to 1.5.

Further, the specific method of step S3 is as follows:

and when the cell growth density reaches 80-90%, washing the cells with PBS, digesting, adding the washed cells into the subculture medium in a ratio of 1:3 to stop digestion after the cells are completely shed under a microscope, culturing in an incubator at 37 ℃ and 5% CO2, and changing the medium once every 3 days until the cell fusion degree reaches 80-90%.

Further, the subculture medium is a DMEM medium added with the following components:

12-15% of plant source recombinant human serum albumin, 2-4% of mannatide, 0.05-0.1% of quercetin and 0.05-0.1% of allantoin.

The mannatide is a mixture with stable chemical properties and certain uniformity, does not contain micromolecule saccharides such as monosaccharide, disaccharide and the like and free amino acid, and is completely composed of mannatide molecules with different chain lengths. Mannan peptide has direct activation effect on human immune cells, can promote immune cell proliferation and immune factor generation, and improve hematopoietic function.

Quercetin is a flavonol compound widely distributed in plant world, has strong biological activity in the aspects of resisting free radicals, inflammation, bacteria and oxidation, and also has certain treatment effect on cardiovascular diseases and tumors.

Allantoin promotes cell growth, softens keratin, and accelerates tissue turnover and wound healing.

Further, in step S3, the specific method for digesting the primary cells is as follows:

and (3) uniformly adding 0.01% EDTA (ethylene diamine tetraacetic acid) added with 0.1% trypsin into the washed primary cells, and digesting for 1-3 min at 37 ℃.

The invention also provides the application of the method in the separation and culture of human dental pulp cells.

The invention has the following beneficial effects: the invention provides a human dental pulp cell isolated culture method, which is characterized in that glucomannan and sodium alginate are added into a primary culture medium, so that adherent growth and replication of cells in the primary culture process can be effectively promoted, an adherent effect equivalent to that of a traditional coated culture dish or by adding fibronectin can be achieved, pre-coating operation can be omitted, and the method is more economic compared with laminin, so that the time cost and the economic cost are effectively reduced while the adherent effect is improved, and a large number of dental pulp stem cells with high activity are obtained; meanwhile, the culture medium provided by the invention does not need serum, can effectively promote the growth and replication of dental pulp cells and improve the differentiation capacity of the cells by matching various components, and can overcome the defects of the serum on the premise of ensuring the performance, thereby effectively improving the culture effect of the dental pulp cells.

Detailed Description

Preparation of main reagent and culture medium

Dmem medium: the concrete components are as follows:

serial number	Name of Compound	Content (mg/L)	Serial number	Name of Compound	Content (mg/L)
						1	Anhydrous calcium chloride	265.00	18	L-serine	42.00
2	Ferric nitrate	0.10	19	L-threonine	95.00
						3	Potassium chloride	400.00	20	L-tryptophan	16.00
4	Anhydrous magnesium sulfate	97.67	21	L-tyrosine	72.00
						5	Sodium chloride	6400.00	22	L-valine	94.00
6	Anhydrous sodium dihydrogen phosphate	109.00	23	D-calcium pantothenate	4.00
						7	Succinic acid	75.00	24	Tartaric acid choline	7.20
8	Succinic acid sodium salt	100.00	25	Folic acid	4.00
						9	L-arginine hydrochloride	84.00	26	Inositol	7.20
10	L-cystine hydrochloride	63.00	27	Nicotinamide	4.00
						11	Glycine	30.00	28	Riboflavin	0.40
12	L-histidine hydrochloride	42.00	29	Thiamine hydrochloride	4.00
						13	L-isoleucine	105.00	30	Pyridoxine hydrochloride	4.00
14	L-leucine	105.00	31	Glucose	1000.00
						15	L-lysine hydrochloride	146.00	32	Pyruvic acid sodium salt	110.00
16	L-methionine	30.00	33	Phenol Red	9.30.00
						17	L-phenylalanine	66.00

PBS buffer:

weighing 8g NaCl, 0.2g KCl and 1.44g Na₂HPO₄And 0.24g KH₂PO₄Dissolving in 800mL of distilled water, adjusting pH to 7.4 with HCl, adding distilled water to a constant volume of 1L, steam sterilizing at 15lbf/in2(1034 × 105Pa) under high pressure for at least 20 min, and storing in refrigerator at room temperature or 4 deg.C.

The present invention will be described in further detail with reference to the following examples. Note that the DMEM medium described in the following examples was added in the formulation described in a, and "further addition" means addition based on the DMEM medium described in a. The DEME medium and PBS buffer described in the following examples were formulated as described above.

Examples

A method for separating and culturing human dental pulp cells comprises the following steps:

s1: collecting completely extracted (12-25 years old) orthodontic teeth of healthy people, cleaning the surfaces of the orthodontic teeth by using 75% ethanol, drilling grooves with the depth of about 1mm on two symmetrical surfaces along the long axis of the teeth by using a split drill, cutting the teeth into two halves by using a large-sized steel wire, and taking out tooth marrow tissues by using dental forceps; cleaning with 1% double-antibody-added PBS buffer solution, cutting into pieces smaller than 1mm3, adding 0.3% type I collagenase and 0.4% neutral protease into the cut pieces, mixing, sterilizing at 37 deg.C for 1 hr, gently blowing to obtain discrete single-cell mass, and storing;

s2: washing dental pulp cells with PBS buffer solution, centrifuging for 5min under the condition of 1000r/min, collecting cells, and repeating washing and centrifuging operations; resuspending the pelleted cells in primary medium supplemented with glucomannan and sodium alginate at 1 x 10⁴～1*10⁵Inoculating at a density of one/ml, placing in 5% CO₂Performing primary culture in an incubator at 37 ℃, changing the culture solution once every 3d, stopping the primary culture when the cell density reaches 80-90%, and preparing for subculture; wherein the primary culture medium is a DMEM culture medium added with the following components: 8-12% of plant source recombinant human serum albumin, 0.1-0.2% of glucomannan, 0.1-0.2% of sodium alginate, 5-10% of sodium lactate and 0.05-0.1% of naringenin;

s3: when the cell growth density reaches 80-90%, washing the cells with PBS, digesting, uniformly adding 0.01% EDTA (ethylene diamine tetraacetic acid) added with 0.1% trypsin into the washed primary cells, digesting at 37 ℃ for 1-3 min, adding the subculture medium at a ratio of 1:3 to stop digestion after the cells are completely detached under a microscope, and placing the cells in 5% CO₂Subculturing in an incubator at 37 ℃, changing the culture solution once every 3d, culturing until the cell fusion degree reaches 80-90%, and storing the cultured cells for later use; wherein the subculture medium is a DMEM medium added with the following components: 12-15% of plant source recombinant human serum albumin, 2-4% of mannatide, 0.05-0.1% of quercetin and 0.05-0.1% of allantoin.

Experimental example 1

Identification experiment

(1) Immunofluorescence assay (experiment carried out after passage of more than 12 generations)

The dental pulp cells are cultured by adopting the method, and the obtained passage cells are subjected to immunofluorescence detection, and the result shows that the vimentin, the I-type collagenase, the III-type collagenase, the alkaline phosphatase, the BMP and the dentin specific non-collagen phosphoprotein are all positive, which indicates that the dental pulp cells are indeed obtained by culturing.

(2) Cell morphology observation experiment

During the culture process, the cells are continuously observed under an inverted phase contrast microscope every day, and the growth condition and the basic morphology of the cells can be seen: the cells can grow on the iron wall after 2 days of culture, part of the cells grow in a colony shape, the cells growing in the colony shape are round or oval, the cells are small and are closely arranged, the cells at the edge of the colony are fusiform, the cells are large and long, and the cells among the colonies are more dispersed; the cell shape is fusiform, is in a fibroblast-like shape, and is larger; day 14, unilaterally fused cells.

(3) Flow assay test (passage over 12 generations)

The surface antigen analysis of the cells by a flow analyzer shows that after 12 generations of subculture, the cells with positive stem cell surface marker STRO expression are more than 8%, about 8.85%, and CD34, CD45 and CD146 are all negative, which meets the requirements of stem cells, and the obtained cells are proved to be dental pulp stem cells.

Experimental example 2

Comparison experiment of adherence situation

The formula shown in table 1 is adopted to prepare primary culture media with different proportions, primary culture is carried out on dental pulp tissues from the same source by the method provided by the embodiment, and the growth conditions of all groups of cells are compared; the plates were further coated with gelatin and hyaluronic acid and control experiments were performed in the same manner, and the cell growth of each group was as shown in table 2 (wherein the adhesion was observed every 15min and the fusion was observed every 12 h).

TABLE 1 Primary Medium formulation (ingredients not listed in the Table are the same as in the examples)

TABLE 2 cell growth

As can be seen from table 2, the adherence time and the time for the fusion degree of 1-5 groups to reach 80-90% are significantly lower than those of 6-11 groups and are equivalent to those of 12-13 groups, which indicates that addition of glucomannan and sodium alginate to the primary culture medium according to the method provided by the present application can effectively promote cell adherence growth, can achieve an effect equivalent to coating a culture dish or adding fibronectin, can omit the operation of pre-coating (13 groups), and can save cost compared with laminin (12 groups); wherein the time of 3-5 groups is the shortest, indicating that the ratio (1: 1-1.5) is the optimal ratio. In addition, the cell adherence time and fusion time of 6-8 groups are longer than those of 1-5 groups, which indicates that the adherence growth effect of the cells can be influenced by adjusting the dosage of glucomannan and sodium alginate.

Experimental example 3

Comparative experiment of cell primary culture conditions

Primary culture media with different ratios are prepared according to the formula shown in table 3, primary culture is performed on dental pulp tissues from the same source by the method provided in the example, the same culture operation is performed by taking serum-free DMEM medium as 14 groups, and after 15 days, the growth conditions of cells of each group are compared and counted, and the results are shown in table 4.

TABLE 3 Primary Medium formulation (ingredients not listed in the table are the same as in the examples)

TABLE 4 Primary cell growth

As can be seen from Table 4, the time that the cell fusion degree reaches 80-90% in groups 1-3 and the number of living cells after the culture is finished are both significantly lower than groups 4-9 and 12-14, and are equivalent to groups 10-11, which indicates that the plant-derived recombinant human serum albumin in the primary culture medium provided by the application can effectively promote the growth and replication of dental pulp cells, and can completely replace fetal bovine serum or bovine serum albumin, so that the defects of serum can be overcome, and the cost can be effectively reduced; the time of the 4-7 groups is prolonged compared with that of the 1-3 groups, and the number of living cells is also obviously reduced, which shows that the excessive or insufficient content of the sodium lactate and the naringenin can influence the growth of the cells; except 14 groups, all the other groups have no mixed bacteria pollution, which shows that the naringenin can play an antibacterial role equivalent to that of double antibiotics in a culture medium; the time of the 12-13 groups is prolonged and the number of living cells is reduced compared with that of the 1-3 groups, which shows that under the condition that other components are the same or have equivalent effects, the naringenin can play a certain role in promoting cell growth besides sterilization; since sodium lactate greens were not added to both groups 9 and 14, the above criteria were significantly different from the other groups, indicating that the addition of sodium lactate greens was effective in promoting the growth and replication of dental pulp cells.

Experimental example 4

Comparative experiment of cell subculture

The primary culture media with different proportions are respectively prepared by the formula shown in table 5, primary culture is carried out on dental pulp tissues from the same source by the method provided by the embodiment, DMEM culture medium added with fetal calf serum is used as 12 groups for carrying out the same culture operation, the culture is continued for 28 days by using osteogenic induction culture medium after 15 days, and the results are shown in table 6 by comparing osteogenic indexes.

TABLE 5 subculture medium formulation (ingredients not listed in the tables are the same as in the examples)

TABLE 6 osteogenic differentiation of cells (mean)

As can be seen from Table 6, the contents of alkaline phosphatase and osteocalcin in groups 1-3 are significantly higher than those in the other groups, which indicates that the subculture medium provided by the application can effectively promote the growth and replication of dental pulp subculture cells and can improve the differentiation performance of the cells; the indexes of the 4-9 groups are obviously reduced compared with those of the 1-3 groups, which shows that the excessive or insufficient content of mannatide, quercetin and allantoin in the culture medium can influence the growth and differentiation performance of cells; after quercetin and allantoin are replaced by common IGF and dexamethasone in 10 groups, the indexes reach a higher level but are obviously lower than those of 1-3 groups, meanwhile, mannatide is not used in 11 groups, so that the indexes are obviously reduced, and all indexes obtained after the conventional culture medium is adopted in 12 groups are the lowest, which shows that the subculture medium provided by the application can effectively improve the growth, replication and differentiation performances of dental pulp cells compared with the conventional culture medium.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A method for separating and culturing human dental pulp cells is characterized by comprising the following steps:

s1: collecting the completely extracted teeth, extracting dental pulp tissues, and digesting to obtain standby dental pulp cells;

s2: inoculating the dental pulp cells into a serum-free primary culture medium added with glucomannan and sodium alginate for primary culture;

s3: and transferring the cells subjected to primary culture into a serum-free subculture medium for subculture for later use.

2. The method for isolated culture of human dental pulp cells according to claim 1, wherein the specific method of step S1 is as follows:

collecting completely extracted teeth, cleaning the surface with 75% ethanol, drilling grooves with a depth of about 1mm on two symmetrical surfaces along the long axis of the teeth with a split drill, cutting the teeth into two halves with a large-sized steel wire, and taking out tooth marrow tissues with dental forceps; washing with 1% double antibody-containing PBS buffer solution, and cutting into pieces smaller than 1mm³The fragments are digested and stored for later use.

3. The method for isolated culture of human dental pulp cells according to claim 2, wherein the specific method for digesting the dental pulp tissue in step S1 is as follows:

adding 0.3% type I collagenase and 0.4% neutral protease into the cut pieces, mixing, sterilizing at 37 deg.C for 1 hr, slightly blowing to obtain discrete single cell mass, and storing.

4. The method for isolation and culture of human dental pulp cells according to claim 1, wherein the specific method of step S2 is as follows:

washing dental pulp cells with PBS buffer solution, centrifuging for 5min under the condition of 1000r/min, collecting cells, and repeating washing and centrifuging operations; resuspending the pelleted cells in primary medium supplemented with glucomannan and sodium alginate at 1 x 10⁴～1*10⁵Inoculating at a density of one/ml, placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution every 3d, stopping primary culture when the cell density reaches 80-90 percent, and preparing to transferAnd (5) performing generation culture.

5. The method for isolated culture of human dental pulp cells according to claim 4, wherein in step S2, the primary culture medium is DMEM medium supplemented with:

8-12% of plant source recombinant human serum albumin, 0.1-0.2% of glucomannan, 0.1-0.2% of sodium alginate, 5-10% of sodium lactate and 0.05-0.1% of naringenin.

6. The method for isolated culture of human dental pulp cells as claimed in claim 5, wherein the ratio of glucomannan to sodium alginate in the primary culture medium is 1: 1-1.5.

7. The method for isolation and culture of human dental pulp cells according to claim 1, wherein the specific method of step S3 is as follows:

when the cell growth density reaches 80-90%, washing the cells with PBS, digesting, adding the subculture medium at a ratio of 1:3 to stop digestion after the cells are observed under a microscope to completely shed, and placing the cells in 5% CO₂And culturing in an incubator at 37 ℃ until the cell fusion degree reaches 80-90% after replacing the culture solution every 3 days.

8. The method for isolated culture of human dental pulp cells according to claim 7, wherein the subculture medium is a DMEM medium supplemented with:

12-15% of plant source recombinant human serum albumin, 2-4% of mannatide, 0.05-0.1% of quercetin and 0.05-0.1% of allantoin.

9. The method for isolation and culture of human dental pulp cells according to claim 7, wherein the primary cells are digested in step S3 as follows:

and (3) uniformly adding 0.01% EDTA (ethylene diamine tetraacetic acid) added with 0.1% trypsin into the washed primary cells, and digesting for 1-3 min at 37 ℃.

10. Use of the method of any one of claims 1 to 9 in isolated culture of human dental pulp cells.