CN107502588B

CN107502588B - Method for separating and preparing dental pulp stem cells

Info

Publication number: CN107502588B
Application number: CN201710959618.4A
Authority: CN
Inventors: 杨晓光; 鲍永利; 王雪; 李玉新; 石晓川; 李首一
Original assignee: Jilin Sunbird Regeneration Medical Engineering Co ltd
Current assignee: Jilin Sunbird Regeneration Medical Engineering Co ltd
Priority date: 2017-10-16
Filing date: 2017-10-16
Publication date: 2020-04-10
Anticipated expiration: 2037-10-16
Also published as: CN107502588A

Abstract

The invention relates to the technical field of cell culture, in particular to a method for separating and preparing dental pulp stem cells. The natural compound nitidine chloride is used as the bacteriostatic agent in the preparation process of the dental pulp stem cells, so that the dental pulp stem cells have high safety, good stability and obvious bacteriostatic effect, can effectively reduce the infection of bacteria, fungi or mycoplasma, and are superior to the traditional method of simply using antibiotics. In the culture process, the culture dish is coated with hyaluronic acid and gelatin, so that the number of adherent dental pulp stem cells is increased, the growth of the cells is promoted, and the aim of effective amplification is fulfilled. Experiments show that the method provided by the invention has the advantages that the cell quantity obtained by amplifying the dental pulp mesenchymal stem cells is large, the pollution rate is low, the detection of the surface markers of the obtained dental pulp stem cells meets the standard, the positively expressed surface markers are more than or equal to 95 percent, and the negatively expressed surface markers are less than or equal to 2 percent.

Description

Method for separating and preparing dental pulp stem cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a method for separating and preparing dental pulp stem cells.

Background

Stem cells are a type of pluripotent cells with self-replicating ability, which can differentiate into various functional cells under certain conditions, and have the potential function of regenerating various tissues and organs and intact individuals. The stem cells are derived from many sources, including bone marrow, umbilical cord, cord blood, teeth, fat, skin, hair follicles, and the like.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that can form a variety of cell types. Has the characteristics of multidirectional differentiation potential, supporting hematopoiesis, promoting hematopoietic stem cell implantation, regulating immunity, simple and convenient separation culture operation and the like, and is receiving increasing attention. With the increasing maturity of mesenchymal stem cells and related technologies, a plurality of diseases which are difficult to cure have new treatment methods.

Dental Pulp Stem Cells (DPSCs) are multifunctional adult Stem Cells, and are mesenchymal Stem Cells isolated from Dental Pulp tissue. Dental pulp stem cells have now been demonstrated to have a multipotent differentiation capacity, capable of differentiating into tissue cells such as bone, fat and vascular endothelium. The existing research shows that the dental pulp stem cells can be used for tissue repair and regeneration of dentin, dental pulp, tooth body and dental crown, and can also be used for damage repair of skull, jaw bone and facial bone. There are also many applications of dental pulp stem cells in research of other diseases, such as: the dental pulp stem cells can be used for treating diseases such as cerebrovascular accident injury, spinal cord injury, Parkinson's disease, myocardial infarction, diabetes, immunodeficiency and the like. The dental pulp stem cells have strong proliferation capacity, high histocompatibility and convenient storage, and provide the modern people with the opportunity of storing stem cells for the 2 nd time.

At present, methods such as an enzyme-linked digestion method, a tissue block culture method, a tissue block enzyme digestion method and the like are commonly used in a preparation method of dental pulp stem cells, although dental pulp stem cells can be obtained by various methods, the dental pulp stem cells are easy to pollute in the preparation and culture processes due to various bacteria in oral cavities, the number of the separated and extracted dental pulp stem cells is small, the cells grow slowly, and the preparation of the dental pulp stem cells is difficult, which is the biggest problem encountered by storing the dental pulp stem cells at present. Therefore, antibiotics are usually selected to inhibit bacterial proliferation when preparing dental pulp stem cells, and the types of antibiotics commonly used in stem cell culture are: penicillin and streptomycin are commonly used for inhibiting bacteria, amphotericin and nystatin are commonly used for inhibiting fungi, and gentamicin, tetracycline, erythromycin and the like are commonly used for inhibiting mycoplasma pollution. However, the use of the above antibiotics does not ensure that the dental pulp stem cells are not contaminated.

Nitidine chloride (Nitidine chloride) is an effective alkaloid extracted from the dried root of zanthoxylum nitidum of zanthoxylum in rue family, the content of Nitidine chloride in the root of zanthoxylum nitidum is as follows: it is pungent, bitter, slightly warm in nature, and has little toxicity, and can be used for treating stomachache, toothache, neuralgia, rheumatalgia, and venomous snake bite. The nitidine chloride is a main effective component of the nitidine and is a main quality control index component of the nitidine and the preparation thereof, and researches in recent years find that the nitidine chloride has high efficiency and low toxicity and has pharmacological effects of resisting tumors, strengthening heart, reducing blood pressure, resisting fungi and the like.

Hyaluronic acid is an acidic mucopolysaccharide, and its salts are the main components of connective tissues such as human intercellular substance, vitreous body of eye, articular cartilage, synovial fluid of joint, etc. Hyaluronic acid plays a vital role in the physiological activities of the human body, and if the content of hyaluronic acid in tissues and organs such as joint cavities, blood vessels, heart, eyes, brain and the like is reduced, diseases such as arthritis, arteriosclerosis, pulse disorder, brain atrophy and the like may be caused. Hyaluronic acid is a proteoglycan distributed in extracellular matrices of various tissues, has good biocompatibility, plays an important role in the processes of biological development and body injury repair, becomes a promising medical biomaterial, and is widely applied in the fields of three-dimensional cell culture, tissue engineering scaffolds and regenerative medicine.

Gelatin (Gelatin) is a high molecular weight water-soluble protein mixture, mainly present in collagen. It is obtained by boiling skin, tendon, ligament, bone, etc. Type a gelatin is obtained by acid hydrolysis of tissue, whereas type B gelatin is obtained by lime hydrolysis of tissue. Gelatin can be used as a stabilizer and thickener in foods, can inhibit crystallization in bacteriology and cell culture, and can also be used for hybridization reaction, etc. The gelatin with good quality has high protein content, white color, complete water solubility and no peculiar smell after water solubility, is a nontoxic and nonirritating raw material, is collected in the FDA inactive component guide, is suitable for dental preparations, inhalation preparations, injections, oral capsules, pastilles, solutions, syrups, tablets, external preparations and vaginal preparations, and is one of the raw materials approved to be used in medicaments in the United kingdom.

The dental pulp stem cells are easy to pollute in the preparation process, so that the number of the cells is small, and the growth is slow. Furthermore, the dental pulp mesenchymal stem cells are not easy to adhere to the wall, which causes cell loss. However, there is no report on how to solve these problems, and therefore, further improvement of the efficiency of isolation and culture of dental pulp stem cells is still a problem to be solved in the art.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for separating and preparing dental pulp stem cells, which has low contamination rate, short adhesion time and large number of obtained cells.

The invention provides application of nitidine chloride in separation and preparation of dental pulp mesenchymal stem cells.

The research of the prior art shows that the nitidine chloride has the pharmacological actions of resisting tumor, strengthening heart, reducing blood pressure, resisting fungi and the like. The research of the invention shows that the sterilization agent can sterilize teeth or dental pulp tissues and avoid the infection of bacteria, fungi or mycoplasma, thereby improving the yield of cultured cells.

The invention provides a dental pulp mesenchymal stem cell culture solution which consists of fetal bovine serum, transferrin, double antibody, nitidine chloride and a DMEM/F12 culture medium.

In the embodiment of the invention, the dental pulp mesenchymal stem cell culture solution is prepared from 15% V/V fetal calf serum, 1-150 mu g/mL transferrin, 1% V/V double antibody and 0.5-10 mu g/mL nitidine chloride.

In some embodiments, the dental pulp mesenchymal stem cell culture fluid is prepared from 15% V/V fetal bovine serum, 50 μ g/mL transferrin, 1% V/V double antibody, and 0.5 μ g/mL nitidine chloride.

The invention also provides a dental pulp tissue disinfectant, which is a normal saline containing 0.5-10 mug/mL nitidine chloride.

In some embodiments, the endodontic tissue disinfectant is a physiological saline solution containing 0.5 μ g/mL nitidine chloride.

In some embodiments, the endodontic tissue disinfectant is a physiological saline solution containing 5.0 μ g/mL nitidine chloride.

In some embodiments, the endodontic tissue disinfectant is a physiological saline solution containing 10 μ g/mL nitidine chloride.

The invention also provides a tooth transfusion liquid which is a normal saline containing double-antibody and 0.5-10 mu g/mL nitidine chloride.

The invention also provides a tooth transfusion liquid which is normal saline containing double-antibody and 0.5 mu g/mL nitidine chloride.

The invention also provides a tooth transfusion liquid which is normal saline containing double-antibody and 5.0 mu g/mL nitidine chloride.

The invention also provides a tooth transfusion liquid which is normal saline containing double-antibody and 10 mu g/mL nitidine chloride.

In the invention, the double-antibody is penicillin and streptomycin, and the final concentration of the penicillin in a culture solution or a transport solution is 1000-2000U/mL; the final concentration of the streptomycin in the culture solution or the transfusion solution is 1000-2000 mug/mL.

In some embodiments, the final concentration of the penicillin in the culture solution or the transport solution is 1000U/mL; the final concentration of streptomycin in the culture solution or the transfusion solution is 1000 mug/mL.

In the invention, the DMEM/F12 medium is a mixture of a DMEM liquid medium and an F12 liquid medium, wherein the volume ratio of the DMEM medium to the F12 medium is 1: 1.

the conveying fluid provided by the invention is adopted to convey and disinfect teeth, the dental pulp tissue disinfectant provided by the invention is adopted to clean and disinfect dental pulp tissues, and then the dental pulp mesenchymal stem cell culture solution provided by the invention is adopted to separate and culture dental pulp mesenchymal stem cells, so that the pollution risk of the cells can be reduced, the cells are prevented from being infected by bacteria, fungi or mycoplasma, and the yield of the cells is improved.

The invention also provides a separation preparation method of the dental pulp mesenchymal stem cells, which comprises the steps of cleaning and disinfecting dental pulp tissues, digesting the dental pulp tissues, resuspending the obtained cells in the dental pulp mesenchymal stem cell culture solution provided by the invention, inoculating the cells in a culture vessel coated by hyaluronic acid and gelatin, and carrying out primary culture;

the cleaning and disinfection comprises 5 steps, wherein normal saline containing 0.5-10 mug/mL nitidine chloride, ethanol with the volume fraction of 70-75%, normal saline, PBS buffer containing double antibodies and PBS buffer are adopted in sequence.

In the invention, the dental pulp tissue is derived from wisdom teeth, and the sterilization and transportation of the wisdom teeth adopt physiological saline containing double-resistant and 0.5-10 mu g/mL nitidine chloride as a transportation and transfusion liquid.

The transfusion solution contains 1000-2000U/mL penicillin, 1000-2000 mu g/mL streptomycin and 0.5-10 mu g/mL nitidine chloride.

In some embodiments, the delivery fluid comprises 1000U/mL penicillin, 1000. mu.g/mL streptomycin and 0.5. mu.g/mL nitidine chloride.

In some embodiments, the delivery fluid comprises 1000U/mL penicillin, 1000. mu.g/mL streptomycin and 5.0. mu.g/mL nitidine chloride.

In some embodiments, the delivery fluid comprises 1000U/mL penicillin, 1000. mu.g/mL streptomycin and 10. mu.g/mL nitidine chloride.

In the present invention, type I collagen protease and type II collagen protease are used for digestion.

In the digestive juice, the mass fraction of the type I collagenase is 0.05-0.3%, and the mass fraction of the type II collagenase is 0.05-0.3%. The volume of the digestive juice is 5-10 times of that of dental pulp tissues.

The digestion condition is 37 ℃ and the oscillation of 200r/min is 30-40 min.

Digestion was terminated in DMEM/F12 medium containing 15% FBS.

After digestion, centrifuging for 10min at 1200r/min, collecting the precipitate to be the cells to be cultured, and resuspending the dental pulp mesenchymal stem cell culture solution provided by the invention.

The mass ratio of the coated hyaluronic acid to the gelatin is (4-5): (2-3).

In the coating liquid, the mass fraction of hyaluronic acid is 2.5-10%; the mass fraction of the gelatin is 1 to 7.5 percent.

In some embodiments, the mass fraction of hyaluronic acid in the coating solution is 2.5%; the mass fraction of gelatin is 1%.

In some embodiments, the mass fraction of hyaluronic acid in the coating solution is 5%; the mass fraction of gelatin is 3%.

In some embodiments, the mass fraction of hyaluronic acid in the coating solution is 10%; the mass fraction of gelatin is 7.5%.

In the invention, the coating amount is 20-30 mu L/cm²。

In the present invention, the primary culture conditions were 37 ℃ and 5% CO₂Culturing in a constant temperature incubator.

In the present invention, the primary culture further comprises a step of subculture.

The natural compound nitidine chloride is used as the bacteriostatic agent in the preparation process of the dental pulp stem cells, so that the dental pulp stem cells have high safety, good stability and obvious bacteriostatic effect, can effectively reduce the infection of bacteria, fungi or mycoplasma, and are superior to the traditional method of simply using antibiotics. In the culture process, the culture dish is coated with hyaluronic acid and gelatin, so that the adherent number of dental pulp stem cells is increased, the cell growth is promoted, and the aim of effective amplification is fulfilled. Experiments show that the method provided by the invention has large cell quantity when used for amplifying dental pulp mesenchymal stem cells. The pollution rate is low, the detection of the obtained dental pulp stem cell surface markers meets the standard, the positively expressed surface markers are more than or equal to 95 percent, and the negatively expressed surface markers are less than or equal to 2 percent.

Detailed Description

The invention provides a method for separating and preparing dental pulp stem cells, and a person skilled in the art can use the contents to appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 isolation preparation of dental pulp Stem cells Using 0.5. mu.g/mL nitidine chloride treatment

1) Collecting teeth: donation or preservation and publicity are carried out aiming at teenagers, children or adults with teeth needing plastic correction and wisdom tooth extraction of deciduous teeth, peripheral blood samples of volunteers are collected for virology detection, teeth of healthy volunteers are collected after qualification is confirmed, and the teeth are stored in transfusion liquid containing 1000-plus 2000U/mL penicillin, 1000-plus 2000 mu g/mL streptomycin and 0.5 mu g/mL nitidine chloride after conventional treatment, refrigerated at 4 ℃, transported to a laboratory and separated within 24 hours.

2) Cleaning and disinfecting dental pulp: the collected teeth were transferred to a clean sterile plate, grasped with sterile ophthalmic forceps, and washed and sterilized with 0.9% physiological saline, 75% medical alcohol, 0.9% physiological saline, PBS buffer containing 1000U/mL penicillin and 1000. mu.g/mL streptomycin, and PBS buffer, in this order, containing 0.5. mu.g/mL nitidine chloride.

3) Preparing a special culture medium for dental pulp stem cells: DMEM and F12 medium were mixed at equal ratio, and 15% V/VFBS, 50. mu.g/mL transferrin, 1% V/V double antibody, 0.5. mu.g/mL nitidine chloride were added.

4) Coating a culture vessel: mixing 10% W/V hyaluronic acid and 6% W/V gelatin at equal volume, and mixing at 20 μ L/cm²Coating the pulp stem cells in a cell culture vessel to form a gel coating, thereby forming the special culture vessel for the pulp stem cells.

5) Dental pulp stem cell separation: a) separating dental pulp tissues from the cleaned teeth in a biological safety cabinet under the aseptic condition, and fully washing the dental pulp tissues by using PBS (phosphate buffer solution) containing 0.5 mu g/mL nitidine chloride; b) cutting selected dental pulp tissue with medical ophthalmology scissors, adding 5-10 times volume of collagenase mixed solution (0.5% type I collagenase and 0.5% type II collagenase mixed in equal proportion) as digestive juice, shaking up lightly, and placing in a constant temperature shaking incubator at 37 deg.C and 200r/min for shaking culture for 30 min; c) after digestion is finished, adding DMEM containing 15% fetal calf serum and F12 mixed culture medium into the suspension to stop digestion, centrifuging at 1200r/min for 10min, and removing supernatant; d) the cells were resuspended in 10-fold volume of the dedicated culture medium for dental pulp stem cell culture, and transferred to a cell culture vessel previously coated with 10% W/V hyaluronic acid and 6% W/V gelatin for primary culture.

Example 2 isolation preparation of dental pulp Stem cells Using 5. mu.g/mL nitidine chloride treatment

1) Collecting teeth: donation or preservation and publicity are carried out aiming at teenagers, children or adults with teeth needing plastic correction and wisdom tooth extraction of deciduous teeth, peripheral blood samples of volunteers are collected for virology detection, teeth of healthy volunteers are collected after qualification is confirmed, and the teeth are stored in transfusion liquid containing 1000-plus 2000U/mL penicillin, 1000-plus 2000 mu g/mL streptomycin and 5.0 mu g/mL nitidine chloride after conventional treatment, refrigerated at 4 ℃, transported to a laboratory and separated within 24 hours.

2) Cleaning and disinfecting dental pulp: the collected teeth were transferred to a clean sterile plate, grasped with sterile ophthalmic forceps, and washed and sterilized with 0.9% physiological saline, 75% medical alcohol, 0.9% physiological saline, PBS buffer containing 1000U/mL penicillin and 1000. mu.g/mL streptomycin, and PBS buffer, in this order, containing 5.0. mu.g/mL nitidine chloride.

5) Dental pulp stem cell separation: a) separating dental pulp tissue from the cleaned tooth in a biological safety cabinet under aseptic condition and fully washing the dental pulp tissue with PBS buffer solution containing 0.5 mu g/mL nitidine chloride; b) cutting selected dental pulp tissue with medical ophthalmology scissors, adding 5-10 times volume of collagenase mixed solution (0.5% type I collagenase and 0.5% type II collagenase mixed in equal proportion) as digestive juice, shaking up lightly, and placing in a constant temperature shaking incubator at 37 deg.C and 200r/min for shaking culture for 30 min; c) after digestion is finished, adding DMEM containing 15% fetal calf serum and F12 mixed culture medium into the suspension to stop digestion, centrifuging at 1200r/min for 10min, and removing supernatant; d) the cells were resuspended in 10-fold volume of the dedicated culture medium for dental pulp stem cell culture, and transferred to a cell culture vessel previously coated with 10% W/V hyaluronic acid and 6% W/V gelatin for primary culture.

Example 3 isolation preparation of dental pulp Stem cells Using 10. mu.g/mL nitidine chloride treatment

1) Collecting teeth: donation or preservation and publicity are carried out aiming at teenagers, children or adults with teeth needing plastic correction and wisdom tooth extraction of deciduous teeth, peripheral blood samples of volunteers are collected for virology detection, teeth of healthy volunteers are collected after qualification is confirmed, and the teeth are stored in transfusion liquid containing 1000-plus 2000U/mL penicillin, 1000-plus 2000 mu g/mL streptomycin and 10 mu g/mL nitidine chloride after conventional treatment, refrigerated at 4 ℃, transported to a laboratory and separated within 24 hours.

2) Cleaning and disinfecting dental pulp: the collected teeth were transferred to a clean sterile plate, grasped with sterile ophthalmic forceps, and washed and sterilized with 0.9% physiological saline, 75% medical alcohol, 0.9% physiological saline, PBS buffer containing 1000U/mL penicillin and 1000. mu.g/mL streptomycin, and PBS buffer, in this order, containing 10. mu.g/mL nitidine chloride.

Comparative example 1 isolation preparation of dental pulp stem cells without nitidine chloride treatment

1) Collecting teeth: donation or preservation and publicity are carried out aiming at teenagers, children or adults with teeth needing plastic correction and wisdom tooth extraction of deciduous teeth, peripheral blood samples of volunteers are collected for virology detection, teeth of healthy volunteers are collected after qualification is confirmed, the teeth are stored in transfusion liquid containing 1000-plus 2000U/mL penicillin, 1000-plus 2000 mu g/mL streptomycin and transfusion liquid without nitidine chloride after conventional treatment, refrigeration is carried out at 4 ℃, the teeth are transported to a laboratory, and separation treatment is carried out within 24 hours.

2) Cleaning and disinfecting dental pulp: the collected teeth were transferred to a clean sterile plate, grasped with sterile ophthalmic forceps, and washed and sterilized with 0.9% physiological saline, 75% medical alcohol, 0.9% physiological saline, PBS buffer containing 1000U/mL penicillin and 1000. mu.g/mL streptomycin, and PBS buffer in this order.

3) Preparing a special culture medium for dental pulp stem cells: DMEM was mixed with F12 medium at equal ratio and 15% V/VFBS, 50. mu.g/mL transferrin, 1% V/V double antibody were added.

5) Dental pulp stem cell separation: a) separating pulp tissues from the cleaned teeth in a biological safety cabinet under an aseptic condition and fully washing the pulp tissues by using a PBS buffer solution; b) cutting selected dental pulp tissue with medical ophthalmology scissors, adding 5-10 times volume of collagenase mixed solution (0.5% type I collagenase and 0.5% type II collagenase mixed in equal proportion) as digestive juice, shaking up lightly, and placing in a constant temperature shaking incubator at 37 deg.C and 200r/min for shaking culture for 30 min; c) after digestion is finished, adding DMEM containing 15% fetal calf serum and F12 mixed culture medium into the suspension to stop digestion, centrifuging at 1200r/min for 10min, and removing supernatant; d) the cells were resuspended in 10-fold volume of the dedicated culture medium for dental pulp stem cell culture, and transferred to a cell culture vessel previously coated with 10% W/V hyaluronic acid and 6% W/V gelatin for primary culture.

Example 4 comparison of contamination rates of dental pulp stem cells with and without nitidine chloride treatment

The dental pulp stem cells cultured in examples 1 to 3, in which dental pulp stem cells were treated with nitidine chloride at different concentrations, and comparative example 1, in which nitidine chloride was not used, were changed once every three days, and the changed cell waste liquid was poured into a bacterial culture flask, and cultured and observed to see whether or not the cells were contaminated. The results are shown in Table 1.

Table 1: statistical table for pollution condition of dental pulp stem cells

The results in Table 1 show that 6 cells were treated with three different concentrations of nitidine chloride solutions and buffer, for a total of 18 cells, of which 1 cell contaminated with 0.5. mu.g/mL nitidine chloride solution and buffer was mycoplasma contamination, while cells treated with 5. mu.g/mL and 10. mu.g/mL nitidine chloride were not contaminated, and the total contamination probability was 5.6% for the three different concentrations of nitidine chloride solutions and buffer; and the cells treated by the transport solution and the buffer solution which do not contain the nitidine chloride are used for 6 cases, 5 cases are polluted after the cells are cultured, the pollution rate is 83%, wherein 2 cases are polluted by bacteria, 1 case is polluted by fungi and 2 cases are polluted by mycoplasma, and the results show that the nitidine chloride with different concentrations has the pollution resistance effect in the preparation process of the dental pulp stem cells, and the effects are obvious and have the dose-dependent effect.

Table 2: growth state statistical table of dental pulp stem cells

The results in table 2 show that the anchorage time of three cells treated with nitidine chloride containing different concentrations is 1.5h, 1.75h and 2.5h respectively; while the anchorage time of the cells which are not treated by nitidine chloride is 2.5 h. The fusion degree of the three cells containing the nitidine chloride with different concentrations reaches 80% -90% in 5 days, 5 days and 6.5 days respectively; the time for the cells which are not treated by nitidine chloride to reach 80% -90% is 7 days. The results show that the treatment of nitidine chloride does not inhibit the adherence time and proliferation speed of the cells, but rather has a certain promotion effect on proliferation.

EXAMPLE 5 method for preparing dental pulp Stem cells by coating Petri dishes with 5% W/V hyaluronic acid and 2% W/V gelatin

3) Preparing a special culture medium for dental pulp stem cells: DMEM and F12 medium were mixed at equal ratio, and 15% V/VFBS, 1% V/V diabody, 50. mu.g/mL transferrin, 0.5. mu.g/mL nitidine chloride were added.

4) Coating a culture vessel: 5% W/V hyaluronic acid and 2% W/V gelatin were mixed in equal volume, and then 20. mu.L/cm²The amount of the compound is coated in a cell culture vessel to form a gel coating, so that the special culture vessel for the dental pulp stem cells is formed.

5) Dental pulp stem cell separation: a) separating dental pulp tissue from the cleaned tooth in a biological safety cabinet under aseptic condition and fully washing the dental pulp tissue with PBS buffer solution containing 0.5 mu g/mL nitidine chloride; b) cutting selected dental pulp tissue with medical ophthalmology scissors, adding 5-10 times volume of collagenase mixed solution (0.5% type I collagenase and 0.5% type II collagenase mixed in equal proportion) as digestive juice, shaking up lightly, and placing in a constant temperature shaking incubator at 37 deg.C and 200r/min for shaking culture for 30 min; c) after digestion is finished, adding DMEM containing 15% fetal calf serum and F12 mixed culture medium into the suspension to stop digestion, centrifuging at 1200r/min for 10min, and removing supernatant; d) resuspending cells by using a special culture medium for dental pulp stem cell culture with 10 times volume, and transferring the cells into a coated cell culture vessel for primary culture.

EXAMPLE 6 method for preparing dental pulp Stem cells by coating Petri dishes with 10% W/V hyaluronic acid and 6% W/V gelatin

EXAMPLE 7 method for preparing dental pulp Stem cells by coating Petri dishes with 20% W/V hyaluronic acid and 15% W/V gelatin

1) Collecting teeth: the method is characterized in that donation propaganda is carried out for teenagers, children or adults with teeth needing plastic correction and wisdom tooth extraction of deciduous teeth, peripheral blood samples of volunteers are collected for virology detection, teeth of healthy volunteers are collected after qualification determination, the teeth are stored in transfusion liquid containing 1000-plus 2000U/mL penicillin, 1000-plus 2000 mu g/mL streptomycin and 5.0 mu g/mL nitidine chloride after conventional treatment, the liquid is refrigerated at 4 ℃, transported to a laboratory and separated within 24 hours.

4) Coating a culture vessel: mixing 20% W/V hyaluronic acid and 15% W/V gelatin at equal volume, and mixing at 20 μ L/cm²The amount of the compound is coated in a cell culture vessel to form a gel coating, so that the special culture vessel for the dental pulp stem cells is formed.

Comparative example 2 preparation method of dental pulp stem cell without coating Petri dish

4) Dental pulp stem cell separation: a) separating dental pulp tissue from the cleaned tooth in a biological safety cabinet under aseptic condition and fully washing the dental pulp tissue with PBS buffer solution containing 0.5 mu g/mL nitidine chloride; b) cutting selected dental pulp tissue with medical ophthalmology scissors, adding 5-10 times volume of collagenase mixed solution (0.5% type I collagenase and 0.5% type II collagenase mixed in equal proportion) as digestive juice, shaking up lightly, and placing in a constant temperature shaking incubator at 37 deg.C and 200r/min for shaking culture for 30 min; c) after digestion is finished, adding DMEM containing 15% fetal calf serum and F12 mixed culture medium into the suspension to stop digestion, centrifuging at 1200r/min for 10min, and removing supernatant; d) suspending the cells by using a special culture medium for culturing dental pulp stem cells with 10 times of volume, and transferring the cells into an uncoated cell culture vessel for primary culture.

Example 8 comparison of growth of dental pulp Stem cells with plates coated and without coating

Subculturing the dental pulp stem cells cultured in examples 5-7 and comparative example 2, replacing the cell culture medium every three days, and observing the time for the dental pulp stem cells to adhere to the wall and the cell morphology, wherein the cell fusion degree reaches 80-90%. The results are shown in Table 3.

Table 3: growth state statistical table of dental pulp stem cells

The results show that when the dental pulp stem cells cultured in the cell culture dish containing hyaluronic acid and Gelatin coating are cultured until the P2 generation, the attachment time of the cells is faster, the time for the cells to reach 80-90% fusion degree is also faster, and the growth vigor of the cells is good.

Example 9 identification of MSCs

The identification of MSCs was performed when the dental pulp stem cells of examples 1-3, 5-7 and comparative examples 1, 2 were cultured to logarithmic phase (P2 generation).

1) Cell surface marker detection: a) preparing cell suspension from cells with good growth state and cell density of 80-90%, counting with cell counter, centrifuging at 4 deg.C and 400 Xg for 5min, and carefully sucking off supernatant; b) according to the results of cell counting, adding PBS buffer solution to resuspend and prepare into cell suspension, so that the cell density is 0.5X 10⁷Per mL; c) subpackaging the cell suspension into an EP tube added with the antibody in a volume of 100 mu L/tube, and blowing and uniformly mixing; d) placing the sample added with the antibody in a dark place, and incubating for 20-30min at normal temperature; e) after incubation, centrifugation at 400 Xg for 5min at 4 ℃ was performed, the antibody-containing supernatant carefully aspirated, resuspended in 500. mu.L of PBS, and examined by flow cytometry.

2) And (3) cell differentiation identification: a) taking cells with good growth state, digesting and collecting the cells, and culturing the cells in a 6-well plate; b) making two wells for each project of each experimental sample, and a blank control well; c) when the cells are fused by 80-100%, replacing osteogenic culture holes with 1mL of hMSC osteogenic induction culture medium, replacing adipose culture holes with 1mL of adipocyte induction culture medium, replacing cartilage culture holes with cartilage induction culture medium, respectively adding 1mL of complete culture medium into a control group, continuously culturing, and replacing the same culture medium every 3-4 days; d) maintaining the inducing condition, and continuously culturing for 2-3 weeks; e) after the culture is finished, washing cells in the osteogenic culture hole by PBS, fixing for 10 minutes by 95% ethanol, washing for 2 times by pure water, dyeing alizarin red for 30min, washing for 2 times by water, and observing and taking a picture under a microscope; f) washing cells in a fat culture hole by PBS, fixing by proper amount of paraformaldehyde for 10min, washing for 2 times by pure water, dyeing for 1 hour by oil red O, washing for 2 times by pure water, and observing and taking a picture under a microscope; g) cartilage culture wells were washed with PBS, fixed with appropriate amount of paraformaldehyde for 10 minutes, washed 2 times with pure water, stained with toluidine blue for 5 minutes, washed 2 times with pure water, and photographed under microscope.

MSC identification results

Comparing the surface marking and differentiation potential of the dental pulp stem cells of examples 1-3 in which the stem cells were treated with nitidine chloride at different concentrations with comparative example 1 in which the stem cells were not treated with nitidine chloride; the surface marking and differentiation potential of dental pulp stem cells were compared between examples 5-7, in which the plates were coated with different concentrations of gelatin and hyaluronic acid, and comparative example 2, in which gelatin and hyaluronic acid were not used.

And (3) preparing the cells with good growth state and cell density of 80-90% into cell suspension, detecting the cell surface markers by using flow cytometry, and counting the detection results shown in table 4.

Table 4: statistical table of cell surface marker detection results

Name of marker

Example 1

Example 2

Example 3

Example 5

Example 6

Example 7

Comparative example 1

Comparative example 2

CD34+

0.28％

0.32％

0.39％

0.51％

0.32％

0.03％

1.45％

0.83％

CD45+

0.28％

0.32％

0.39％

0.51％

0.32％

0.03％

1.45％

0.83％

CD14+

0.28％

0.32％

0.39％

0.51％

0.32％

0.03％

1.45％

0.83％

CD19+

0.28％

0.32％

0.39％

0.51％

0.32％

0.03％

1.45％

0.83％

HLA-DR+

0.28％

0.32％

0.39％

0.51％

0.32％

0.03％

1.45％

0.83％

CD90+

99.93％

99.94％

99.96％

99.87％

99.94％

99.99％

97.12％

99.88％

CD73+

99.98％

99.99％

99.97％

99.99％

100.00％

98.34％

99.90％

CD105+

99.97％

98.54％

99.50％

97.47％

99.54％

99.65％

95.23％

99.21％

For each cell with good growth state, cell differentiation was identified, and it was observed whether the cells could be differentiated into different cells under different induction media. The differentiation results are shown in Table 5.

Table 5: statistical table of cell differentiation identification results

The results show that the detection of the dental pulp stem cell surface marker by using the transfusion liquid containing the chlorinated nitidine and the buffer solution and the dental pulp stem cell surface marker by using the common transfusion liquid and the buffer solution meets the standard, the positive expression surface marker is more than or equal to 95 percent, the negative expression surface marker is less than or equal to 2 percent, but the cell which is not added with the chlorinated nitidine is polluted and has certain influence on the cell purity, so the content of the negative marker which is not required to be expressed is more. From table 4, the quality of the cells obtained by the embodiments 6 to 7 is significantly better than that of other examples or comparative examples, the expression level of the negatively expressed surface marker is significantly lower than that of other examples or comparative examples, and p is less than 0.05. The above results indicate that the chlorinated nitidine treatment did not affect the dental pulp stem cell characteristics of the cells nor their differentiation potential.

The dental pulp stem cells are inoculated into a cell culture dish coated by gelatin and hyaluronic acid and a cell culture dish not coated by gelatin and hyaluronic acid for culture, the cell surface markers cultured by the two modes are qualified, the results are ideal, only the content of the cell surface markers which are not required to be expressed has slight difference, and the result shows that the dental pulp stem cells cultured in the cell culture dish added with gelatin and hyaluronic acid have higher purity, the content of the non-expressed surface markers is less, and the dental pulp stem cells are more favorable for growth.

When the growth state of the cells is good, the dental pulp stem cells can be differentiated into other cells under the culture of the corresponding induction medium, but the cells of comparative example 1 are contaminated, and the growth state of the cells is influenced, so that the differentiation capability is influenced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A dental pulp mesenchymal stem cell culture solution is characterized by consisting of fetal bovine serum, transferrin, double antibody, nitidine chloride and a DMEM/F12 culture medium; the double-resistant is penicillin and streptomycin.

2. The culture solution of claim 1, comprising 15% V/V fetal calf serum, 1-150 μ g/mL transferrin, 1000-2000U/mL penicillin, 1000-2000 μ g/mL streptomycin, and 0.5-10 μ g/mL nitidine chloride.

3. A method for separating and preparing dental pulp mesenchymal stem cells, which is characterized in that dental pulp tissues are digested after being cleaned and disinfected, the obtained cells are resuspended by the culture solution of claim 1 or 2, and are inoculated into a culture vessel coated by hyaluronic acid and gelatin for primary culture;

the cleaning and disinfection comprises 5 steps, wherein physiological saline containing 0.5-10 mug/mL nitidine chloride, ethanol with the volume fraction of 70% -75%, physiological saline, PBS buffer containing double antibodies and PBS buffer are adopted in sequence; the double-resistant is penicillin and streptomycin.

4. The separation and preparation method according to claim 3, wherein the mass ratio of the hyaluronic acid to the gelatin is (4-5): (2-3).

5. The separation and preparation method of claim 3, wherein the amount of the coating is 20 to 30 μ L/cm²。

6. The isolated preparation method according to claim 3, wherein the dental pulp tissue is derived from wisdom tooth, and sterilization and transportation of the wisdom tooth are performed using physiological saline containing diaschist and 0.5-10 μ g/mL nitidine chloride.

7. The isolation and preparation method according to any one of claims 3 to 6, further comprising a step of passaging after the primary culture.