CN115521906A - Method for preparing high-quality human-derived dental pulp stem cells - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to the field of cell biology, and discloses a method for preparing high-quality human-derived dental pulp stem cells by in vitro separation, which comprises the following steps: s1, carrying out sample primary screening according to the age and health condition of a tooth supplier, and then collecting teeth and conveying the teeth to a GMP laboratory. S2, crushing teeth by using a tool, taking out exposed dental pulp tissues, further shearing the dental pulp tissues, adding combined digestive enzyme, and vibrating, digesting and decomposing the tissues; after the digestion reaction was terminated, the precipitate was obtained by centrifugation. And S3, adding a complete culture medium to resuspend the tissue cells and filtering to obtain a single cell suspension. S4, inoculating the cells into a culture bottle, changing the culture solution for 2 times every week, adding pancreatin substitute (Tryple-Express) to digest the cells and carrying out passage amplification when the fusion degree of the cells in the bottle reaches 80-90%. The invention ensures the preparation and output of a high-quality clinical-grade dental pulp stem cell by strictly screening a tooth sample and further regulating the separation and culture method of the dental pulp stem cell.
Description
Technical Field
The invention relates to the field of cell biology, in particular to a method for preparing high-quality human-derived dental pulp stem cells through in vitro separation.
Background
Stem cells are the cells of origin of the body and are the progenitor cells that form various tissues and organs of the human body. In recent 20 years, it is a booming period of stem cell therapeutic application; on the basis of this, there is a rapid increase in cell preparation technology. Mesenchymal Stem Cells (MSCs) as a branch of research on stem cells not only have all the commonalities of stem cells, namely self-renewal and multi-directional differentiation capabilities, but also have the advantages not possessed by other stem cells, namely the capability of directionally migrating to damaged tissues and regulating immune response according to specific environments; meanwhile, the stem cells are the most widely used in clinical application because of wide sources and easy separation and preparation. Researchers have discovered and successfully isolated mesenchymal stem cells from the original bone marrow to later human tissue organs such as fat, umbilical cord and placenta.
In 2000, gronthos et al, the american scholars, for the first time, discovered and prepared human-derived Dental Pulp Stem Cells (DPSCs), which are a class of undifferentiated cells or precursor cells with strong self-renewal and multipotential potential in human Dental pulp tissue, and belong to mesenchymal stem cells. Compared with mesenchymal stem cells from other tissues, the dental pulp stem cells are wide in source, and are rich in deciduous teeth naturally shed by children of 6 to 12 years old and wisdom teeth of adults of 19 to 30 years old; and the collection process of the teeth is safe and convenient and does not relate to ethics problems. In addition, dental pulp stem cells are relatively low in immunogenicity and can differentiate into a variety of human cells in a suitable in vivo or in vitro environment; can provide cell sources for tissue and organ repair, autologous and allogeneic transplantation cell therapy and the like, and has wide application prospect.
Although various methods and culture systems are available for preparing stem cells, the proliferation and multipotential differentiation of the prepared dental pulp stem cells are different. On the one hand, there is no clear screening for the quality standard of the tooth sample, so that the success rate of dental pulp stem cell culture is often affected due to the damaged tooth structure, and the worst result may be that no dental pulp tissue is extracted or the extracted dental pulp tissue cannot culture stem cells. On the other hand, the traditional cell culture system has the problems that stem cells are differentiated in advance due to too long culture period, the number of primary cells is small, multiple passages are required, the preparation and culture processes are easily polluted by microorganisms, and antibiotics are abused. The technical difficulties in the preparation and culture processes of the cells can finally influence the application effect of the dental pulp stem cells in clinical research and treatment. The invention aims to provide a preparation method of dental pulp stem cells, which reduces the quality difference of dental pulp stem cells from different donor sources, improves the preparation success rate and the primary culture yield of the cells, and ensures the large-scale production of the clinical grade of the cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the following technical scheme:
a method for preparing high-quality human-derived dental pulp stem cells, comprising the steps of:
s1, screening healthy tooth donors with suitable ages, and carrying out tooth inspection and tooth extraction operations on the donors by professional medical personnel; collected teeth were placed in a preservative solution and transported to a GMP laboratory within 24 hours.
S2, opening a pulp cavity of a tooth by using a bench vice, taking out dental pulp, shearing dental pulp tissue blocks, adding combined digestive enzymes (neutral protease, I-type collagenase and hyaluronidase) with the volume 5 times that of the tissue blocks for constant-temperature oscillation digestion, then adding a proper amount of complete culture medium to stop digestion reaction, and centrifuging to remove supernatant.
And S3, adding a complete culture medium to resuspend the tissue blocks and the cells, and filtering the suspension by adopting a filter screen with the aperture of 80-150 mu m to obtain the single cell suspension.
And S4, inoculating the dental pulp cell suspension obtained in the step S3 into a culture flask, changing the liquid for 2-3 times every week, and adding a pancreatin substitute for digestion and subculture after the fusion rate of the cells in the flask reaches 80-90%.
Preferably, in the step S1, the sources of the teeth include adults aged 19 to 30 years, wisdom teeth that need to be extracted due to orthodontic treatment or arrhythmic, and deciduous teeth aged 6 to 12 years, which are the donors who need to store dental pulp stem cells.
Preferably, the dental donor is screened from a population without the genetic or infectious disease.
Preferably, the timing of deciduous tooth collection is clinically defined secondary loosening; the wisdom tooth needs to select the sample which is complete, has no deep caries, no filling and restoring body, no subfissure and no serious calcification of the dental pulp cavity.
Preferably, the tooth preservation solution is sterile physiological saline containing 1% double antibody.
Preferably, in the step S2, the mass ratio of the neutral protease, the collagenase type I and the hyaluronidase is (2-5): (0.1-0.3).
Preferably, in the step S2, the constant temperature shaking digestion specifically refers to shaking digestion of the tissue mass in a constant temperature oscillator at 37 ℃ at 100-200 rpm for 20-40 min.
More preferably, the tissue mass is digested for 30min with shaking at 150rpm in a 37 ℃ constant temperature shaker.
Preferably, the diameter of the sieve is 100 μm.
Preferably, in the step S3, the complete medium is an alpha-MEM medium containing 5-15% human platelet lysate, 50mg/L L-ascorbic acid, 1mg/L nicotinamide, 100ng/mL biotin, 11mg/L deoxycytidine hydrochloride, 292mg/L L-glutamine and 1% final concentration of the cyan-streptomycin double antibody.
Preferably, in step S4, the cells are seeded at a density of 1 tooth, and the obtained cells are seeded into 1T 25 culture flask, and the volume of the culture medium is 5-10 mL.
Preferably, in step S4, the liquid exchange medium is an α -MEM medium containing 5 to 15% of human platelet lysate.
Preferably, in the step S4, the cells to be transfected are digested by pancreatin substitute, the adding amount is 1-2 mL/T25, and the digestion time is 2-3 min.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a method for preparing high-quality human-derived dental pulp stem cells, which can ensure successful establishment of a human-derived dental pulp stem cell system to the greatest extent through screening of tooth donors.
2. The invention provides a method for screening and preparing high-quality human dental pulp stem cells and fine particles obtained by separationThe cells are excellent in proliferation rate and bioactivity, and dental pulp stem cells obtained by isolated culture of each tooth can be harvested in primary culture at 1-3 × 10 6 The survival rate of each cell is higher than 95%, the purity of the cell is high, the form is good, and each differentiation capability is strong.
3. The invention provides a method for preparing high-quality human-derived dental pulp stem cells, which is characterized in that 1% of green-streptomycin double antibody is added into a sample preservation solution, physiological saline used for preparing and cleaning dental pulp tissues and a culture medium added after cells (tissue blocks) are inoculated for the first time, so that the microbial contamination probability in the primary tissue separation preparation process can be effectively reduced; and no antibiotic is used in the culture medium used in the subsequent liquid change and passage, so that the adverse effects of the antibiotic on the metabolism, proliferation, differentiation, even gene expression and the like of cells can be avoided to the maximum extent.
4. The invention provides a method for preparing high-quality human dental pulp stem cells, which is characterized in that human platelet lysate supplemented in a culture system supports the growth and proliferation of cells, can avoid the heterologous pollution of animal serum and the like, and reduces the immunological rejection reaction of human bodies; the supplemented vitamins and cytidine substances all have the effect of promoting the life activities of stem cells, such as protein metabolism, fat metabolism, carbohydrate metabolism and the like.
5. The invention provides a method for preparing high-quality human dental pulp stem cells, which selects Tryple-Express to replace commonly used pancreatin during cell digestion, belongs to a recombinase without animal sources, can gently process cells, and keeps the reproductive activity of the cells.
6. The high-quality human-derived dental pulp stem cells prepared and cultured by the method form stable cell lines at present; through multiple identification and detection, the requirements of clinical treatment application can be met in both quality and quantity.
Drawings
Fig. 1 is a schematic view of the under-lens morphology of primary dental pulp stem cells at different culture periods.
Fig. 2 is a graph showing the growth curves of the dental pulp stem cells P3 and P5 generations.
Fig. 3 is a schematic view of flow cytometry detection of human dental pulp stem cells prepared according to an embodiment of the present invention.
Fig. 4 is a schematic diagram of human dental pulp stem cell three-line differentiation assay prepared according to an embodiment of the present invention.
Fig. 5 is a schematic flow chart of preparation and detection of dental pulp stem cells according to an embodiment of the present invention.
Detailed Description
The technical solutions related to the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not all described in the following. In the examples, the reagents used were all common commercial products and were commercially available.
The invention provides a method for preparing high-quality human-derived dental pulp stem cells, which improves the success rate of separation and culture of clinical-grade dental pulp stem cells and comprises the following steps:
s1, collecting teeth (including deciduous teeth about to fall off by children and wisdom teeth of adults) of persons who are 6-30 years old and healthy and have no hereditary or infectious diseases, wherein the teeth are required to have complete tooth sample structure and no oral diseases such as periodontal diseases, dental pulp diseases and the like. After the teeth are pulled out, the teeth are immediately put into pre-cooled physiological saline preservation solution containing 1 percent of double antibody, and the cold chain (2-8 ℃) is transported back to a GMP laboratory within 24 hours. In a biosafety cabinet in a clean area, the teeth were taken out with forceps and placed in a sterile petri dish with a diameter of 10cm, and then the remaining tissue on the surface of the teeth was scraped off with a surgical blade. The tooth surface was then spray sterilized with 75% ethanol for 1 minute and the sample was washed 3 times with 1% double antibody in normal saline.
And S2, covering and wrapping the tight tooth with sterile gauze, clamping the whole tooth by using a bench vice, carefully peeling off the tooth fragments by using forceps, and exposing dental pulp tissues. Collecting dental pulp tissue in a 1.5mL sterile EP tube, and cutting the dental pulp tissue into pieces of 0.5-1 mm with medical scissors 3 Size. 1mL of the combined digestive enzymes (mass volume concentration 0.1% neutral protease, 0.15% collagenase type I and 0.025% hyaluronidase) were added to the EP tube. The EP tube was fixed in a constant temperature shaker and digested at 37 ℃ for 30min with shaking at 150 rpm. In a safety cabinet, the mixture in the EP tube was transferred to a new 15mL centrifuge tube by blowing three times, and the tube was filled with 1%The EP tube was washed 3 times with the complete medium containing the diabody, and the wash solution was also added to a 15mL centrifuge tube and supplemented to 14mL with complete medium containing the diabody. 300g, and centrifuging at 4 deg.C for 10min to collect precipitate.
S3, adding 3mL of complete culture medium (containing 10% of human platelet lysate and 1% of double-antibody alpha-MEM culture medium) to carry out centrifugation to obtain precipitates, namely, resuspending the tissue blocks and the cells, filtering the mixed suspension by using a filter screen with the aperture of 100 mu m, and collecting the single cell suspension in a new 50mL centrifuge tube.
S4, inoculating the obtained 3mL of cell suspension into a T25 culture flask, adding 3mL of complete culture medium again to wash the filter screen and the centrifuge tube, adding the washing solution into the culture flask, and shaking up. The culture flask was left standing at 5% CO 2 And culturing in an incubator at 37 ℃. And 2-3 times of liquid change are carried out every week, and the liquid change culture medium is alpha-MEM culture medium containing 10% human platelet lysate. After the cell fusion rate in the bottle reaches 80-90%, adding 1-2 mL pancreatin substitute for digestion and subculture.
According to statistics, in 2 years, 19 teeth from 6-31 year old donors are screened and separated according to the examples in sequence (see table 1 for details), dental pulp stem cells can be successfully separated and prepared, and finally, a stable cell line is established. These cells exhibited typical mesenchymal stem cell morphology, resembling fibroblasts that are adherent (see figure 1 for details).
The results of activity detection and growth curve determination performed on the dental pulp stem cells successfully separated in the examples show that the cells grow adherent to the skin 4 hours after passage, the cells can be observed to be fusiform under a microscope in 1-2 days, the cells enter a logarithmic growth phase in 4-6 days, and the peak is reached in 7-8 days (fig. 2 is a growth curve graph of P3 and P5 generation cells with the number of 20190830004), which accords with the growth rule of general cytology.
Flow assay analysis was performed on the cells after subculture, and as a result, it was determined that the content indexes of specific markers of the surface antigen of the dental pulp stem cells, including CD73+, CD90+, CD105+, and CD34-, CD19-, CD11b-, CD45-, and HLA-DR-, all met the relevant regulations, and high purity and homogeneity of the dental pulp stem cells obtained by preparation were further represented in a data manner (fig. 3 is sample No. 20210202019, a schematic flow result diagram of generation 2 cells).
In order to identify that the cells prepared according to the examples have the characteristics of pluripotent stem cells, the dental pulp stem cells which have been subcultured and expanded are collected and subjected to induction culture in a six-well plate, and induced differentiation media for adipogenesis, osteogenesis and chondrogenesis of the stem cells are added respectively. After several weeks of culture, the cells induced by chondrogenesis were stained with alisnew blue, the cells induced to adipogenesis were stained with oil red O and stained with alizarin red to form the cells induced by osteogenesis and differentiation, and all 3 results were positive. In other words, the culture results of in vitro induction of these dental pulp stem cells showed excellent multipotential differentiation ability (fig. 4, no. 20210202019, schematic diagram of the differentiation results of the 2 nd generation cell three-line).
The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art.
It will be readily apparent to those skilled in the art that various modifications can be made to this embodiment and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.
TABLE 1 DPSCs Primary culture information record Table
Claims (13)
1. A method for preparing high-quality human-derived dental pulp stem cells is characterized by comprising the following key steps:
s1, screening healthy tooth donors with suitable ages, carrying out tooth examination and tooth extraction operations for the tooth donors by professional medical personnel, putting collected teeth into a preservation solution, and conveying the collected teeth to a GMP laboratory within 24 hours.
And S2, opening a pulp cavity of the tooth by using a bench vice, taking out dental pulp, shearing dental pulp tissue blocks, adding combined digestive enzymes (neutral protease, I-type collagenase and hyaluronidase) with the volume 5 times that of the tissue blocks for constant-temperature oscillation digestion, then adding a proper amount of complete culture medium to stop the digestion reaction, and centrifuging to remove the supernatant.
S3, adding a complete culture medium to resuspend the tissue blocks and the cells, and filtering the suspension by adopting a filter screen with the pore diameter of 80-150 mu m to obtain a single cell suspension.
And S4, inoculating the dental pulp cell suspension obtained in the step S2 into a culture flask, changing the liquid for 2-3 times every week, and adding a pancreatin substitute for digestion and subculture after the fusion rate of the cells in the flask reaches 80-90%.
2. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the age of the dental donor is between 6 and 30 years (wisdom tooth: 19 to 30 years, deciduous tooth: 6 to 12 years).
3. The method of claim 1, wherein the health status of the dental donor is selected from the group consisting of non-genetic and non-infectious disease donors based on the physical examination information and health questionnaire of the dental donor in about 3 months.
4. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein clinically defined secondary loose deciduous teeth and wisdom teeth without deep caries, without filling and without prosthesis, without saphenous fissure, without severe calcification of dental pulp cavity, as confirmed by professional qualified doctors, are preferably collected in step S1.
5. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein in step S1, after informed consent is obtained, extracted teeth are collected from a donor, stored in 30mL of a tooth preservation solution, stored and transported at 4 to 8 ℃, delivered to a preparation site as soon as possible, and the best is within 24 hours.
6. The method for preparing high-quality human-derived dental pulp stem cells according to claim 5, wherein the tooth preservation solution is sterile physiological saline containing 1% double antibody (100U/mL penicillin and 100 μ g/mL streptomycin).
7. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the mass ratio of the combined digestive enzyme neutral protease, the collagenase type I and the hyaluronidase is (2-5) to (0.1-0.3).
8. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the constant temperature shaking digestion is performed by shaking digestion of a tissue block in a constant temperature shaker at 37 ℃ at 100-200 rpm for 20-40 min.
9. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the pore size of the mesh is preferably 100 μm.
10. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the complete medium comprises 5-10% human platelet lysate, 50 mg/L-ascorbic acid, 1mg/L nicotinamide, 100ng/mL biotin, 11mg/L deoxycytidine hydrochloride, 292 mg/L-glutamine, and alpha-MEM medium with a final concentration of 1% penicillin-streptomycin diabody.
11. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein in step S4, the seeding density of the cells is 1 tooth and the obtained cells are seeded into 1T 25 cell culture flask, and the volume of the culture medium is 5-10 mL.
12. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein the liquid-changing medium in step S4 is an α -MEM medium containing 5% to 15% of human platelet lysate.
13. The method for preparing high-quality human-derived dental pulp stem cells according to claim 1, wherein in step S4, the cells to be transfected are digested with pancreatin substitute, the amount of T25 added is 1 to 2mL per bottle, and the digestion time is 2 to 3min.
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