CN107267451A - A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse - Google Patents

Abstract

A method for preparing primary dental pulp stem cells and a method for constructing a dental pulp stem cell bank. Using a constant temperature shaker under specific temperature and rotational speed conditions, the dental pulp tissue can be digested within a few minutes, and enough cells with Dental pulp stem cells with better biological activity not only shorten the time for primary extraction of dental pulp stem cells, save time and cost, but also can be produced on a large scale. Compared with the control group with half-digestion of dental pulp tissue for 30 minutes or complete digestion for about 1 hour under conventional conditions, there is no significant statistical difference in the amount of DPSCs, but the cell proliferation activity is significantly higher than control group. Therefore, this method can be quickly carried out in batches during the construction of the DPSCs library, which shortens the time for primary extraction of DPSCs, saves time and cost, and is suitable for large-scale production.

Description

A method for preparing primary dental pulp stem cells and a method for constructing a dental pulp stem cell bank

technical field

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

Background technique

Stem cells are a type of cells with self-renewal, high proliferation and multi-directional differentiation capabilities, which can be induced to differentiate into mature human tissues and organs under specific conditions, and have been widely used in various aspects of regenerative medicine research. The source of stem cells is mainly embryonic stem cells and mesenchymal stem cells, among which embryonic stem cells are limited due to the ethical issues involved in their clinical translation research; the main source of mesenchymal stem cells is bone marrow, that is, bone marrow mesenchymal stem cells, but in the process of clinical translation research There are disadvantages such as invasive damage to the donor. Since Gronthos et al. in 2000 [GRONTHOS S, MANKANI M, BRAHIM J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci USA, 2000, 97(25): 13625-13630.] Since the discovery of dental pulp stem cells (dental pulp stem cells, DPSCs) in the third molars of healthy adults, the exfoliated teeth and wisdom teeth that were previously considered oral medical wastes have "turned waste into treasure" and become the main source of DPSCs research. DPSCs is a kind of mesenchymal stem cells with high proliferative ability and multi-directional differentiation potential. It has the advantages of abundant sources, safe and convenient materials, no ethical issues involved, and low immunogenicity. It has made great progress in the field of regenerative medicine. have a broad vision of application. At the same time, DPSCs can be induced to differentiate into bone tissue, cartilage tissue, adipose tissue, nerve tissue, muscle tissue, cornea and other tissue cells under appropriate conditions, and can be induced into induced pluripotent stem cells with embryonic stem cell-like characteristics ( induced pluripotentstem cells (iPSCs), which can be used as seed cells in the clinical translational research of various diseases in the future. At present, many countries have established dental pulp stem cell banks for clinical treatment or scientific research needs.

The construction process of dental pulp stem cell bank includes three parts: dental pulp stem cell culture, passage of dental pulp stem cell and cryopreservation of dental pulp stem cell. Among them, the culture process of dental pulp stem cells is divided into tooth body collection, tooth body transportation, pulp tissue extraction and stem cell culture, etc., and each step in this process has a greater impact on the biological activity of the final dental pulp stem cells. big impact. At present, for the extraction and culture of dental pulp tissue, the traditional methods are direct culture of tissue blocks and collagenase digestion and culture. Excessive and other shortcomings.

The traditional enzymatic digestion method to prepare dental pulp stem cells takes 20 minutes to 3 hours to digest. As mentioned in the construction method of the patented dental pulp stem cell bank (CN 101717750 A), after cutting the pulp tissue from orthodontic or wisdom teeth Use collagenase and dispase to digest dental pulp stem cells at 37°C and 5% CO ₂ for 50 minutes; patent a dental pulp stem cell for clinical use and its preparation method (CN 104694464 A) use collagenase and dispase II to convert dental pulp tissue Digested for 1-3 hours to obtain dental pulp stem cells; patented a method for preparing dental pulp mesenchymal stem cells and establishing a dental pulp mesenchymal stem cell bank and cell recovery method (CN 104630141 A) using trypsin substitutes to digest dental pulp tissue for 20 minutes to obtain Dental pulp stem cells.

Contents of the invention

In order to overcome the defects in the above-mentioned prior art, a method for preparing primary dental pulp stem cells and a method for constructing a dental pulp stem cell bank are provided, which can digest the dental pulp tissue in a short time (≤10min) and extract enough DPSCs.

The technical solution adopted in the present invention is: a preparation method of primary dental pulp stem cells, comprising the following steps:

(1) Tooth collection;

(2) Pulp extraction: extract the pulp after pre-processing the collected isolated teeth;

(3) Digestion of dental pulp tissue and stem cell culture: Place the extracted dental pulp tissue in sterile PBS, cut the dental pulp tissue to 1mm ² , then collect the dental pulp tissue with a centrifuge tube, and store it at room temperature at 1000rpm, Centrifuge for 10 minutes, discard the supernatant, add dropwise 3mg/mL type I collagenase + 4mg/mL neutral protease mixed enzyme to blow off the pulp tissue, the volume ratio of type I collagenase and neutral protease is 1:1, and place at 37°C , digested in a constant temperature shaker environment at 180rpm, after 5-10min, drop serum medium to stop digestion, under the condition of 1000rpm, centrifuge for 5min to collect tissue pieces and cells, resuspend 1mL complete α-MEM medium, inoculate in 35mm Culture in a sterile culture dish at 37°C in a 5% CO ₂ cell incubator. After 1 day, 2 mL of complete α-MEM medium was replenished, and the α-MEM medium was replaced every 3 days;

(4) Subculture the dental pulp stem cells.

The complete α-MEM medium is 10-20% FBS+100U/mL penicillin+100ug/mL streptomycin.

The pre-treatment in the step (2) of pulp extraction is to use tooth storage solution to rinse the tooth body of the isolated tooth for 2 times, each time for 1 min, then use 1% povidone iodine to disinfect the isolated tooth for 1 min, and finally use Rinse the isolated tooth twice with tooth storage solution, 1 min each time.

The step (4) is to subculture the dental pulp stem cells as follows: when the dental pulp cells reach 80%-90% fusion, digest with trypsin/EDTA mixed enzyme, centrifuge at 1000rpm for 5min, collect the cells, and use The complete α-MEM medium was used to disperse the cells, and the cell suspension was evenly distributed to 2 cell culture dishes, and placed in a 37°C, 5% CO ₂ incubator for culture, and the medium was changed every 3 days.

A method for constructing a dental pulp stem cell bank, comprising the following steps:

(1) Tooth collection;

(2) dental transport;

(3) pulp extraction;

(4) Dental pulp tissue digestion and stem cell culture;

(5) passage of dental pulp stem cells;

(6) Biological function detection of dental pulp stem cells;

(7) Cryopreservation and recovery of dental pulp stem cells.

The biological function detection of dental pulp stem cells includes one or more of CCK-8 detection, immunofluorescence staining detection, adipogenic differentiation detection, osteogenic differentiation detection or chondrogenic differentiation detection.

The cryopreservation of the dental pulp stem cells in the step (7) includes the following steps: take the dental pulp stem cells that meet the standard logarithmic growth phase, and when the cell fusion reaches 80%-90%, digest with trypsin/EDTA mixed enzyme, Centrifuge at 1000rpm for 5min to collect the cells, adjust the cell concentration to 3×106 cells/mL with 10% ^DMSO + 90% FBS cell freezing medium, put 1mL of the cell suspension into cryopreservation tubes, and then place The cryopreservation tubes are numbered sequentially and placed in a special cell freezing box, cooled at a rate of 1°C/min, and stored in a -80°C refrigerator for 12 hours. Finally, the cell cryopreservation tubes are transferred to liquid nitrogen for long-term storage, and corresponding preparations are made. registration.

The recovery of the dental pulp stem cells in the step (7) includes the following steps: after the cell cryopreservation tube is taken out from the liquid nitrogen, it is quickly placed in a 37°C water bath environment for thawing, and then diluted with 5mL of complete α-MEM medium , centrifuged at 1000rpm for 5min, collected cells, blown with complete α-MEM medium, transferred to a 35-mm sterile cell culture dish, and placed in a 37°C, 5% CO ₂ cell culture incubator for 3 days Change medium once.

The beneficial effects of the present invention are: the present invention provides a method for preparing primary dental pulp stem cells and a method for constructing a dental pulp stem cell bank, using a constant temperature shaker under specific temperature and rotational speed conditions, the tooth pulp can be extracted within a few minutes. The pulp tissue is well digested, and enough dental pulp stem cells with good biological activity are extracted, which not only shortens the time for primary extraction of dental pulp stem cells, saves time and cost, can be produced on a large scale, but also obtains a sufficient amount of dental pulp Stem cells can meet the needs of dental pulp stem cells in the construction of stem cell banks, clinical treatment of stem cells in the later stage, and the use of regenerative medicine research. Compared with the control group under normal conditions, the amount of DPSCs is not significant Statistical difference, but the cell proliferation activity was significantly higher than that of the control group. Therefore, this method can be quickly carried out in batches during the construction of the DPSCs library, which shortens the time for primary extraction of DPSCs, saves time and cost, and is suitable for large-scale production.

Instructions attached

Fig. 1 is a flow chart of the extraction of dental pulp tissue in the present invention.

Figure 2 is the light microscope pictures of the first generation of dental pulp stem cells cultured for 8 days and subcultured under different conditions and at different times, where TES represents digestion conditions in a constant temperature shaker, TE represents semi-digestion, and E represents complete digestion.

Figure 3 shows the detection results of dental pulp stem cells CCK-8 under different conditions and at different times, where TES represents digestion conditions in a constant temperature shaker, TE represents half digestion, and E represents complete digestion.

detailed description

The present invention will be described in detail below in conjunction with examples, which are only preferred implementations of the present invention, and are not limitations of the present invention.

dental collection

After the patients made an appointment and registered in advance, the extracted caries-free healthy wisdom teeth (age ≤ 30 years old) were collected in the outpatient room of the designated stomatological hospital. First, rinse the isolated tooth twice with sterile PBS buffer, 1 min each time; secondly, use sterile ophthalmic curved forceps to carefully clean the residual soft tissue on the surface of the isolated tooth, and rinse the cleaned isolated tooth again with sterile PBS buffer. The tooth was wiped twice, each time for 1 min; finally, the isolated tooth was wiped twice with 75% alcohol cotton ball, each time for 1 min, and then placed in a sterile tooth collection tube containing a 4°C tooth storage solution, and stored in an ice box.

Tooth storage solution configuration: sterile PBS buffer + double antibody (100U/mL penicillin + 100ug/mL streptomycin), volume ratio 10:1.

Dental Transport

The collected isolated teeth were transported to the stem cell laboratory at 2-4°C and the pulp tissue was extracted within 24 hours.

pulp extraction

Pre-treatment of the isolated teeth: first, rinse the tooth body of the isolated teeth with tooth storage solution twice, each time for 1 min; then, soak the isolated teeth with 1% povidone-iodine disinfectant (PVP-I) for 1 min; finally , and then rinse the isolated tooth twice with tooth storage solution, 1 min each time.

Pulp extraction: First, use a dental circular split drill to circumscribe the neck of the tooth (without exposing the pulp); then, wrap the tooth with sterile gauze, separate the tooth, expose the pulp, and place it in a tooth storage solution Finally, use medical instruments such as probes and tweezers to separate the dental pulp and place it in sterile PBS, as shown in Figure 1.

Dental pulp tissue digestion and stem cell culture

Place the extracted pulp tissue in sterile PBS, use micro-ophthalmological scissors, cut the pulp tissue (about 1mm ² in size); then, collect the pulp tissue in a centrifuge tube, and centrifuge at 1000rpm for 10min at room temperature . Discard the supernatant, add 3mg/mL type I collagenase + 4mg/mL neutral protease mixed enzyme (1:1 volume ratio) dropwise to blow off the pulp tissue, and place it in a constant temperature shaker environment at 37°C and 180rpm for digestion. After 5, 10, 15 and 20 min respectively, serum medium was added dropwise to stop the digestion, the tissue pieces and cells were collected by centrifugation (1000 rpm, 5 min), and 1 mL of complete α-MEM medium (20% FBS+100U/mL penicillin+100ug/ mL streptomycin), inoculated in a 35-mm sterile petri dish, placed in a 37°C, 5% _CO2 cell incubator for cultivation, 2 mL of liquid was added after 1 day, and the medium was replaced every 3 days. Among them, the pulp tissue was half-digested for 30 minutes and fully digested for 60 minutes in a constant temperature environment at 37°C (the cell suspension was filtered through a 70um cell sieve) as the control group, and other culture conditions were consistent with the experimental group. The experimental results are shown in Figure 2.

Passaging of Dental Pulp Stem Cells

When the cell fusion reaches 80%-90%, digest with trypsin/EDTA mixed enzyme, centrifuge (1000rpm, 5min), collect the cells, disperse the cells with complete α-MEM medium, and distribute the cell suspension evenly to 2 cells Culture dishes were placed in a 37°C, 5% CO ₂ incubator for culture, and the medium was changed every 3 days.

Biological function detection of dental pulp stem cells

CCK- ₈ detection: Select P1 generation dental pulp stem cells with good growth status, adjust the cell concentration to 2.0×10 ³ and inoculate them in 96-well cell culture plates, add recovery medium, and culture at 37°C, 5% CO2 The culture was carried out in the box, and the medium was replaced every 3 days. 1, 3 and 5 days after culture, add 10 μL of CCK-8 solution to each well, put it into the cell culture incubator and continue to culture for 1 hour, then observe the absorbance value at 450 nm (OD value) on a microplate reader, the results are shown in the figure As shown in 3, the proliferative activity of dental pulp stem cells was significantly higher than that of other experimental groups and control groups when TES was 10 min, and there was a significant statistical difference ( ^* P<0.05versus TE30min, ^# P<0.05versus E60min).

Immunofluorescence staining: _P3 generation dental pulp stem cells digested for 10 minutes under the condition of a constant temperature shaker were inoculated on a six-well plate slide, after the culture was completed, fixed with 4% paraformaldehyde for 30 minutes, BSA combined with 0.1TritonX-100 to rupture and seal for 30 minutes; CD146, STRO-1 primary antibody was incubated overnight at 4°C; PBST was washed 3 times, secondary antibody was incubated in the dark for 1 hour, PBST was washed 3 times, DAPI was incubated in the dark for 5 minutes, PBST was washed 3 times; fluorescence microscope observation was taken.

Adipogenic differentiation: _P3 generation dental pulp stem cells in the logarithmic growth phase were selected (digested for 10 min under the condition of a constant temperature shaker), and routinely digested, and the resulting cell suspension was seeded in a six-well plate at a density of 20,000 cells/ ^cm2 . Add complete α-MEM medium dropwise, place in a 37°C, 5% CO ₂ incubator for culture, change the medium every 3 days, and when the cell fusion reaches about 100%, place in complete α-MEM cell culture medium OriCell ^TM adipogenic inducer (Cyagen, USA) was added and cultured in a CO ₂ cell incubator, and the cell culture medium containing adipogenic inducer was replaced every 3 days. After the cells were continuously cultured for 21 days, the induction was terminated, fixed with 4% paraformaldehyde (PFA) for 10 min, stained with Oil Red-O stain, and observed with an optical microscope.

Osteogenic differentiation: _P3 generation dental pulp stem cells in the logarithmic growth phase were selected (digested for 10 min under constant temperature shaker conditions), digested routinely, and the resulting cell suspension was inoculated in 35-mm cells at a density of 20,000 cells/cm ² Add complete α-MEM medium dropwise to the culture dish, and place it in a 37°C, 5% CO ₂ incubator for culture. When the cell confluence reaches about 70%, add OriCell ^TM to the complete α-MEM cell culture medium Osteogenic inducer (Cyagen, USA) was cultured in a CO ₂ cell incubator, and the cell culture medium containing osteogenic inducer was replaced every 3 days. After the cells were continuously cultured for 21 days, the induction was terminated, fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, stained with Alizarin Red-S stain for 3 min, and observed with an optical microscope.

Chondrogenic differentiation: select _P3 generation dental pulp stem cells in the logarithmic growth phase (digested for 10 min under constant temperature shaker conditions), routinely digest, adjust the concentration of the cell suspension to 5×10 ⁵ cells/mL, take 1 mL of the cell suspension and put into a 15mL centrifuge tube, centrifuge at normal temperature (1000rpm, 5min) to collect the cells, discard the supernatant, and directly add the complete α-MEM cell culture medium containing ^OriCellTM chondrogenic inducer (Cyagen, USA) in the centrifuge tube (adding process must be slow, so as not to disperse the cells), the cells are placed in a 37°C, 5% CO ₂ incubator in the form of cell clusters for cultivation, and the medium is changed every 3 days. After 28 days, the induction was terminated, 4% paraformaldehyde (PFA) was fixed overnight, paraffin-embedded sections (4-5 μm), stained with Alcian blue stain, and observed under an optical microscope.

Cryopreservation and recovery of dental pulp stem cells

Cryopreservation of dental pulp stem cells: Take dental pulp stem cells that meet the standard logarithmic growth phase. When the cell fusion reaches 80%-90%, digest with trypsin/EDTA mixed enzymes, centrifuge (1000rpm, 5min), and collect the cells. Use cell freezing solution (10% DMSO + 90% FBS) to adjust the cell concentration to 3×10 ⁶ cells/mL, put 1 mL of the cell suspension into cryopreservation tubes (1.5 mL), and then number the cryopreservation tubes sequentially Put it into a special cell freezing box (temperature drop rate: 1°C/min), put it in a -80°C refrigerator for 12 hours, and finally transfer the cell cryopreservation tube to liquid nitrogen for long-term storage, and make corresponding registration.

Recovery of dental pulp stem cells: The principle of rapid recovery was implemented. After the cell cryopreservation tube was taken out of the liquid nitrogen, it was quickly put into a 37°C water bath environment for thawing, and then diluted with 5mL of complete α-MEM medium, and centrifuged (1000rpm , 5min), collect the cells, blow them with complete α-MEM medium, transfer them to a 35-mm sterile cell culture dish, put them in a 37°C, 5% CO ₂ cell culture incubator, and replace them once every 3 days base.

The above descriptions are only preferred implementations of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principles of the present invention should also be regarded as the protection scope of the present invention.

Claims (8)

1. a kind of preparation method of primary dental pulp stem cell, it is characterised in that comprise the following steps：

(1) tooth body is collected；

(2) dental pulp is extracted：Dental pulp is extracted after the Isolated Tooth of collection is carried out into pre-treatment；

(3) pulp tissue's digestion and stem cell culture：The pulp tissue of extraction is placed in sterile PBS, pulp tissue is shredded extremely 1mm², then, pulp tissue is collected using centrifuge tube, and in 1000rpm under normal temperature condition, centrifuge 10min, abandon supernatant, it is added dropwise 3mg/mL type i collagen enzyme+4mg/mL neutral proteinase mixed enzymes dispel pulp tissue, type i collagen enzyme and neutral proteinase volume Ratio is 1: 1, is placed in 37 DEG C, 180rpm constant-temperature table environment and is digested, after 5-10min, and blood serum medium is added dropwise and terminates Under the conditions of digestion, 1000rpm, centrifugal treating 5min collects tissue block and cell, and the complete α-MEM culture mediums of 1mL are resuspended, are inoculated in In 35mm sterile petri dish, in 37 DEG C, 5%CO₂Cultivated in cell culture incubator, complete α-MEM culture medium fluid infusion after 1d 2mL, a α-MEM culture medium is changed every 3d；

(4) passage processing is carried out to dental pulp stem cell.

2. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described complete α- MEM culture mediums are 10-20%FBS+100U/mL penicillin+100ug/mL streptomysins.

3. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described step (2) pre-treatment is the tooth body 2 times that Isolated Tooth is rinsed using tooth storing liquid during dental pulp is extracted, and then each 1min gathers using 1% Ketone iodic disinfecting liquid bubble Isolated Tooth 1min is tieed up, finally Isolated Tooth is rinsed 2 times with tooth storing liquid again, each 1min.

4. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described step (4) passage process step is carried out to dental pulp stem cell is：It is mixed with pancreatin/EDTA when pulp cells fusion reaches 80%-90% Synthase is digested, and 5min is centrifuged under the conditions of 1000rpm, collects cell, and with complete α-MEM culture medium cell dispersions, cell hangs Liquid is evenly distributed in 2 Tissue Culture Dish, is placed in 37 DEG C, 5%CO₂Cultivated in incubator, liquid is changed once every 3d.

5. a kind of method for building dental pulp stem cell storehouse, it is characterised in that comprise the following steps：

(1) tooth body is collected；

(2) tooth body is transported；

(3) dental pulp is extracted；

(4) pulp tissue's digestion and stem cell culture；

(5) passage of dental pulp stem cell；

(6) the biological function detection of dental pulp stem cell；

(7) dental pulp stem cell freezes and recovered.

6. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described dental pulp is dry thin The biological function detection of born of the same parents includes CCK-8 detections, immunofluorescence dyeing detection, examined into fat to differentiation detection, skeletonization to differentiation Survey or the one or more into cartilage into differentiation detection.

7. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described step (7) Freezing for dental pulp stem cell comprises the following steps：The dental pulp stem cell for meeting standard logarithmic growth period is taken, when cell fusion reaches During 80%-90%, digested with pancreatin/EDTA mixed enzymes, 5min is centrifuged under the conditions of 1000rpm, cell is collected, with 10% DMSO+90%FBS cells frozen storing liquid, adjustment cell concentration is 3 × 10⁶Individual/mL, cell suspension 1mL is respectively charged into and frozen Cryopreservation tube number consecutively, is then put into the special freezing storing box of cell by Guan Zhong, is cooled with 1 DEG C/min speed, is put into -80 DEG C of refrigerators 12h is frozen, last cell cryopreservation tube is transferred in liquid nitrogen to be stored for a long time, and carries out corresponding registration.

8. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described step (7) The recovery of dental pulp stem cell comprises the following steps：After cell cryopreservation tube takes out from liquid nitrogen, quickly it is put into 37 DEG C of water baths Thawed, be then diluted with 5mL complete α-MEM culture mediums, 5min is centrifuged under the conditions of 1000rpm, collect cell, used Complete α-MEM culture mediums are dispelled, and are transferred in 35-mm steril cell culture dishes, are put into 37 DEG C, 5%CO₂Carried out in cell culture incubator Culture, changes a subculture in 3 days.