CN107267452B - Dental pulp stem cell recovery liquid and recovery method of dental pulp stem cells - Google Patents
Dental pulp stem cell recovery liquid and recovery method of dental pulp stem cells Download PDFInfo
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Abstract
A recovery liquid for dental pulp stem cells and a recovery method for the dental pulp stem cells are disclosed, wherein a certain amount of basic fibroblast growth factor is added into a conventional stem cell culture medium to construct a new stem cell recovery liquid, and the recovery liquid is applied to the recovery process of DPSCs, so that the activity of the DPSCs can be quickly recovered in a short time, the dryness and the multidirectional differentiation potential of the original stem cells are kept, the recovery time of the stem cells can be shortened, the clinical use survival rate of the stem cells is improved, the proliferation speed exceeds that of the conventional culture medium group, the dryness and the multidirectional differentiation potential of the cells are simultaneously maintained, seed cells with better performance and state can be effectively provided for clinical stem cell treatment, and a large amount of time, labor and material cost are saved.
Description
Technical Field
The invention particularly relates to the technical field of stem cell culture, and particularly relates to a dental pulp stem cell recovery liquid and a recovery method of dental pulp stem cells.
Background
Stem cells are cells having self-renewal, high proliferation and multi-directional differentiation ability, can induce differentiation into human mature tissues and organs under specific conditions, and have been widely used in research in various aspects of regenerative medicine. The source of stem cells is mainly embryonic stem cells and mesenchymal stem cells, wherein the embryonic stem cells limit the clinical transformation research of the embryonic stem cells due to the ethical problem; mesenchymal stem cells are mainly derived from bone marrow, namely the mesenchymal stem cells of the bone marrow, but have the defects of invasive damage to donors and the like in the clinical transformation research process. Since 2000 Gronthos et al [ GRONTHOS S, MANKANI M, BRAHIM J, et al. 13625 & 13630.] since Dental Pulp Stem Cells (DPSCs) were found in the third molar of healthy adults, exfoliated teeth and extracted wisdom teeth, which were previously used as oral medical waste, have been "turned into wealth" as a major source of DPSCs research. The dental pulp stem cell is a mesenchymal stem cell with high proliferation capacity and multidirectional differentiation potential, has the advantages of rich source, safe and convenient material taking, no ethical problem, low immunogenicity and the like, is greatly developed in the field of regenerative medicine, and has wide application prospect. Meanwhile, DPSCs can be induced to differentiate into various tissue cells such as bone tissue, cartilage tissue, adipose tissue, nerve tissue, muscle tissue, cornea, and the like under appropriate conditions, and Induced Pluripotent Stem Cells (iPSCs) having embryonic stem cell-like characteristics can be used as seed cells for clinical transformation studies of various diseases in the future.
The DPSCs are used as seed cells in the clinical research process, and are not taken immediately in most of the time, but a stem cell bank is established in advance, the DPSCs are stored, and the cells are recovered and amplified in time when clinical needs are met, so that the requirement of clinical treatment is met. Therefore, it is important to establish an effective method for recovering DPSCs, so that the vitality of the cells is quickly recovered after the cells are recovered and the related dryness function is maintained.
Disclosure of Invention
In order to overcome the defects in the prior art, the dental pulp stem cell recovery liquid and the recovery method of the dental pulp stem cells are provided, so that the activity of DPSCs can be quickly recovered in a short time, the dryness and the multidirectional differentiation potential of the original stem cells are kept, the recovery time of the stem cells can be shortened, and the clinical use survival rate of the stem cells can be improved.
The technical solution adopted by the invention is as follows: the dental pulp stem cell recovery liquid is characterized by consisting of a conventional mesenchymal stem cell culture medium and basic fibroblast growth factor (bFGF), wherein the conventional mesenchymal stem cell culture medium consists of an alpha-MEM culture medium, 10-20% fetal bovine serum, 100U/mL penicillin and 100 mug/mL streptomycin, and the final concentration of the action of the basic fibroblast growth factor (bFGF) is 10-20 ng/mL.
The final concentration of the action of the basic fibroblast growth factor (bFGF) is 20ng/mL
A method for resuscitating dental pulp stem cells, comprising the steps of:
(1) separation, culture and identification of dental pulp stem cells: collecting teeth of healthy patient, wiping tooth surface with 75% alcohol, soaking and washing with sterile PBS containing penicillin and streptomycin for 2 times, cutting teeth with split drill under aseptic condition, taking out dental pulp, cutting into tissue blocks, digesting with type I collagenase and neutral protease mixed enzyme at 37 deg.C for 30min, blowing off after digestion, centrifuging to collect cells, inoculating into sterile culture dish, adding dropwise complete alpha-MEM culture medium, adding 5% CO at 37 deg.C2Culturing in a cell culture box, replacing the first culture medium after 5 days, replacing the first culture medium after 3 days, digesting the cells with pancreatin/EDTA, passaging, selecting the second generation DPSCs with good growth state, adjusting the cell concentration to 1 × 106Loading the cells/mL into 1.5mL EP tubes, respectively adding a proper amount of mouse anti-human CD90, CD73 and CD14, incubating the mixed solution of the cells and the antibodies at room temperature in a dark place for 1h, and detecting by a flow cytometer;
(2) freezing and storing dental pulp stem cells: after the degree of fusion of the second generation dental pulp stem cells reaches 80-90%, digesting by conventional method, centrifuging at 1000rpm for 5min to collect cells, adding cell freezing solution to blow off, and then blowing off the cells at a concentration of 3 × 106Subpackaging each/mL into cell cryopreservation tube, transferring into special cryopreservation box for cells, placing at-80 deg.C overnight, and transferring into liquid nitrogen environment for long termFreezing and storing;
(3) resuscitation of DPSCs: selecting dental pulp stem cells frozen in liquid nitrogen for 3 months, quickly thawing in a constant-temperature water bath environment at 37 ℃, culturing and diluting by using conventional alpha-MEM, centrifugally collecting cells, adding a recovery solution of the dental pulp stem cells containing bFGF, putting the recovery solution into a medium containing 5% CO at 37 DEG C2Culturing in an incubator, and replacing the culture medium every other day.
The complete alpha-MEM medium in step (1) consists of 20% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin.
The cell freezing medium in the step (2) consists of 10% DMSO + 90% FBS.
The conventional alpha-MEM culture in step (3) consists of 10% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin.
The invention has the beneficial effects that: the invention provides a dental pulp stem cell resuscitation liquid and a method for resuscitating dental pulp stem cells, wherein a certain amount of basic fibroblast growth factor is added into a conventional stem cell culture medium to construct a new stem cell resuscitation liquid, and the resuscitation liquid is applied to the resuscitation process of DPSCs, so that the activity of the DPSCs can be quickly recovered in a short time, the dryness and multidirectional differentiation potential of the original stem cells are kept, the stem cell resuscitation time can be shortened, the clinical use survival rate of the stem cells is improved, the proliferation speed exceeds that of the conventional culture medium group, the dryness and multidirectional differentiation potential of the cells are simultaneously maintained, seed cells with better performance and state can be effectively provided for clinical stem cell treatment, and a large amount of time, labor and material cost are saved.
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FIG. 1 shows the optical lens images of DPSCs primary culture at 7d and 14 d.
Fig. 2 shows the flow cytometry detection results of DPSCs surface markers CD90, CD73, and CD 14.
FIG. 3 shows the results of CCK-8 cultures of first generation DPSCs (P1-ac) with different concentrations of bFGF resuscitation fluid.
FIG. 4 shows the results of the recovery of second-generation DPSCs (P2-ac) to CCK-8 in conventional medium after the first-generation DPSCs were subjected to bFGF at different concentrations.
FIG. 5 shows the immunofluorescence results of first generation DPSCs (P2-ac) reverted to conventional media after 20ng/mL bFGF.
FIG. 6 shows the results of western-blotting of first-generation DPSCs (P2-ac) restored to conventional medium after the action of 20ng/mL bFGF.
FIG. 7 shows the immunofluorescence results of the surface marker Nestin/GFAP after the first-generation DPSCs are acted by 20ng/mLbFGF and the second-generation DPSCs (P2-ac) are restored to the conventional culture medium.
FIG. 8 shows the result of lipid droplet oil red-0 staining of first-generation DPSCs after 20ng/mL bFGF and the recovery of second-generation DPSCs (P2-ac) to conventional medium after lipogenic induction.
FIG. 9 shows the result of red-S staining of calcified nodule alizarin after osteogenic induction in conventional medium, which resulted from the recovery of second-generation DPSCs (P2-ac) by 20ng/mL bFGF.
Detailed Description
The present invention will be described in detail with reference to examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Preparing a dental pulp stem cell recovery liquid:
mainly comprises a conventional culture medium of mesenchymal stem cells and basic fibroblast growth factor (bFGF), wherein the conventional culture medium comprises: a-MEM medium (Gibco, USA) + 10% fetal bovine serum (FBS, Gibco, USA) +100U/mL penicillin + 100. mu.g/mL streptomycin (Gibco, USA). Final concentrations of bFGF were 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL and 100ng/mL, respectively.
The recovery process of the dental pulp stem cells comprises the following steps:
separation, culture and identification of DPSCs: clinically, teeth of a healthy patient of 19-29 years old were collected, and the surface of the teeth was first wiped with 75% alcohol, and then rinsed 2 times with sterile PBS containing penicillin and streptomycin for later use. Under aseptic condition, cutting teeth with split drill, taking out dental pulp, and cutting into size of about 1mm2Digesting the tissue block with collagenase I and neutral protease mixed enzyme at 37 deg.C for 30min, blowing off, centrifuging to collect cells, inoculating into sterile culture dish, and adding alpha-MEM (2)0% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin), 5% CO at 37 ℃2Culturing in a cell culture box, replacing the first culture medium after 5d, and replacing the first culture medium after 3d conventionally. After the cell fusion reaches 80-90%, the cells are normally digested by pancreatin/EDTA, passaged, and second generation DPSCs with good growth state are selected, the cell concentration is adjusted to 1 × 106And (4) packaging each cell/mL into 1.5mL EP tubes, adding a proper amount of mouse anti-human CD90, CD73 and CD14 respectively, incubating the mixed solution of the cells and the antibodies at room temperature for 1h in a dark place, and detecting by a flow cytometer.
Freezing and storing DPSCs: after the second generation DPSCs reach 80-90% confluence, the cells are collected by conventional digestion, centrifugation (1000rpm, 5min), cell freezing medium (10% DMSO + 90% FBS) is added for blowing, and then the cells are added at a concentration of 3X 106And (3) subpackaging each cell/mL into a cell cryopreservation tube, transferring into a cell special cryopreservation box, putting into a refrigerator at the temperature of minus 80 ℃ for overnight, and finally transferring into a liquid nitrogen environment for long-term cryopreservation.
Resuscitation of DPSCs: selecting DPSCs frozen in liquid nitrogen for 3 months, quickly thawing in a constant-temperature water bath environment at 37 ℃, then diluting with conventional alpha-MEM (10% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin), centrifuging to collect cells, adding recovery medium containing bFGF (alpha-MEM + 10% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin), placing at 37 ℃, and adding 5% CO2Culturing in an incubator, and replacing the culture medium every other day. The final concentration of the bFGF action is 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL and 100ng/mL respectively, and the bFGF only acts on the first generation of resuscitation DPSCs, and the second generation of resuscitation DPSCs culture medium is replaced by the conventional stem cell culture medium. The DPSCs from which the first and second generations were resuscitated were labeled P1-ac and P2-ac, respectively.
Detecting related biological properties of the dental pulp stem cells after bFGF (basic fibroblast growth factor) action:
cell proliferation activity assay (CCK-8 assay): selecting DPSCs with better growth state(P1-ac)Adjusting the cell concentration to 2.0X 103Inoculating to 96-well cell culture plate, adding resuscitation culture solution, placing at 37 deg.C and 5% CO2Culturing in an incubator, and replacing the culture medium every other day. At the same time, after the cells have been passaged(DPSCs(P2-ac)) The medium was replaced with a conventional medium (α -MEM + 10% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin), 10. mu.L of CCK-8 solution was added to each well at 1, 3, 5 and 7d after the culture, the mixture was placed in a cell incubator and cultured for 1 hour, and then the absorbance value (OD value) at 450nm was observed on a microplate reader. Under the same conditions, the conventional medium was set as a control group.
And (3) immunofluorescence staining: the method mainly detects the dry function of the DPSCs by fluorescence staining of mesenchymal stem cell surface markers CD146 and STRO-1, and comprises the following specific steps: inoculating cells on a six-hole plate climbing sheet, after the culture is finished, washing the cells for 3 times by PBS, fixing the cells by 4% paraformaldehyde for 30min, washing the cells for 3 times by PBS, and sealing the cells for 30min by BSA combined with 0.1TritonX-100 membrane rupture; incubation of CD146, STRO-1 primary antibody at 4 ℃ for 16 h; PBST is washed for 3 times, the second antibody is incubated for 1h in a dark place, PBST is washed for 3 times, DAPI is incubated for 5min in a dark place, and PBST is washed for 3 times; cells were observed by fluorescence microscopy and recorded by photography.
Western-blotting detection: inoculating the cells into a six-hole plate, after the culture is finished, washing the cells for 3 times by PBS, cracking the cells on RIPA and PMSF ice, centrifuging at 4 ℃ to obtain supernatant, and detecting the expression of CD146 and Nanog by western blotting. The protein amount of each hole is consistent, electrophoresis is carried out on 10% SDS-PAGE gel, after the electrophoresis is finished, the protein is transferred to a PVDF membrane, milk is sealed for 2h, TBST is washed for 3 times, primary antibody is incubated for 16h at 4 ℃, TBST is washed for 3 times, secondary antibody is incubated for 1h, and TBST is washed for 3 times. Finally, the amount of relevant target proteins was observed and analyzed by a Bio-rad protein gel imaging system.
And (3) adult nerve induction: a certain density of cells (4X 10)3cells/cm2) After 1 day, the medium was changed every 3 days by changing the neural induction medium (DMEM high-sugar medium containing 10-7M dexamethasone, 50. mu.g/mL vitamin C, 50. mu.M indomethacin, 10. mu.g/mL insulin, 45mM IBMX). After inducing for 6 days, washing with PBS 3 times, fixing with 4% paraformaldehyde for 30min, washing with PBS 3 times, and blocking with BSA combined with 0.1TritonX-100 for 30 min; incubating Nestin and GFAP primary antibody for 16h at 4 ℃; PBST is washed for 3 times, the second antibody is incubated for 1h in a dark place, PBST is washed for 3 times, DAPI is incubated for 5min in a dark place, and PBST is washed for 3 times; cells were observed by fluorescence microscopy and recorded by photography.
Fat forming induction: will have a certain densityNodular cell (2X 10)4 cells/cm2) Inoculating to six-well plate, adding 2mL of conventional culture medium per well, and changing the culture medium to 2mLORICell when the cell fusion degree reaches 100%TMAdipogenic induction differentiation medium (Cyagen, USA) A liquid, after 3d of induction, 2mLORICell was replacedTMAnd (3) replacing the solution A after 1d in the adipogenic differentiation medium B, and after the solution A and the solution B are alternately acted for 4 times (16d), continuing to maintain and culture for 6d by the solution B and replacing the inducing solution every 3 d. After the fatting induction and differentiation is finished, fixing, dyeing by using oil red O dye working solution, and observing the fatting dyeing effect under a microscope.
Osteogenic induction: the cells are arranged according to a certain density (2X 10)4cells/cm2) Inoculating to six-well plate, adding 2mL conventional culture medium into the well, and changing to 2mLORICell when the cell fusion degree reaches 60% -70%TMStem cells were osteogenically induced to differentiate in complete medium (Cyagen, USA). Changing fresh inducing liquid every 3d, fixing after 2w, staining alizarin red, and observing osteogenic staining effect under a microscope.
Results of the experiment
As shown in FIG. 1, it was revealed that dental pulp stem cells had crawled out around the tissue mass at 7d of the primary culture, and the cells were almost confluent at 14d, and had a typical fibroblast-like shape.
As shown in fig. 2, the results showed that DPSCs positively expressed the mesenchymal stem cell surface marker CD90 (98.82%), CD73 (99.72%) and negatively expressed CD14 (0.85%).
As shown in FIG. 3, it was revealed that DPSCs showed a tendency of increasing continuously under the action of bFGF at various concentrations, and the cell proliferation rate was significantly higher than that of the other groups at a final bFGF concentration of 20ng/mL, and the OD values at 5d and 7d were 0.9970 and 2.0912, respectively, which were 1.37 times and 1.24 times that of the control group.
As shown in FIG. 4, the results show that the proliferation rate of DPSCs pretreated by 20ng/mL bFGF is significantly higher than that of other groups, and a high peak value (OD: 2.0482) is reached at 7d, which indicates that only the resuscitation culture solution containing 20ng/mL bFGF is needed to act on the first-generation DPSCs after resuscitation, and after passage, the second-generation DPSCs also have good growth and proliferation effects, so that the concentration of bFGF in the subsequent experimental groups is only 20 ng/mL.
As shown in FIG. 5, the results show that DPSCs (P2-ac) positively express the mesenchymal stem cell surface markers CD146 and STRO-1, and DPSCs (P2-ac) still have the desiccation function of mesenchymal stem cells.
As shown in FIG. 6, the results showed that DPSCs (P2-ac) expressed CD146 and NANOG, indicating that the dry function of DPSCs (P2-ac) was unaffected.
As shown in FIG. 7, the results indicate that DPSCs (P2-ac) positively express the neural stem cell surface markers Nestin and GFAP, and can successfully induce differentiation to neural-like cells.
As shown in FIG. 8, the results showed that DPSCs (P2-ac) exhibited significant lipid droplets, indicating successful differentiation induction into adipogenic cells.
As shown in FIG. 9, the results showed that DPSCs (P2-ac) had distinct calcified nodules, indicating that induced differentiation in the osteoblast direction was successful.
TABLE 1 Effect of bFGF at various concentrations on OD 450nm values of resuscitation first-generation DPSCs (P1-ac)
TABLE 2 OD 450nm values of second-generation DPSCs (P2-ac) pretreated with different bFGF concentrations
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (6)
1. A method for resuscitating dental pulp stem cells by a dental pulp stem cell resuscitating solution, which is characterized by comprising the following steps:
(1) separation, culture and identification of dental pulp stem cells: collecting teeth of healthy patient, wiping tooth surface with 75% alcohol, soaking and washing with sterile PBS containing penicillin and streptomycin for 2 times, performing ring-cutting on teeth with a split drill under aseptic condition, taking out dental pulp, cutting into tissue blocks, digesting with type I collagenase and neutral protease mixed enzyme at 37 deg.C for 30min, blowing off after digestion, centrifuging to collect cells, inoculating into sterile culture dish, and drippingα-MEM Medium, 5% CO at 37 ℃2Culturing in a cell culture box, replacing the first culture medium after 5 days, replacing the first culture medium after 3 days, digesting cells by pancreatin/EDTA (ethylene diamine tetraacetic acid) in a conventional way after the cells are fused to 80-90%, passaging, selecting second generation DPSCs with good growth state, and adjusting the cell concentration to 1 × 106Loading the cells/mL into 1.5mL EP tubes, respectively adding a proper amount of mouse anti-human CD90, CD73 and CD14, incubating the mixed solution of the cells and the antibodies at room temperature in a dark place for 1h, and detecting by a flow cytometer;
(2) freezing and storing dental pulp stem cells: after the degree of fusion of the second generation dental pulp stem cells reaches 80-90%, digesting by conventional method, centrifuging at 1000rpm for 5min to collect cells, adding cell freezing solution to blow off, and then blowing off the cells at a concentration of 3 × 106Subpackaging each/mL into a cell cryopreservation tube, transferring into a cell special cryopreservation box, putting at-80 ℃ for overnight, and finally transferring into a liquid nitrogen environment for long-term cryopreservation;
(3) resuscitation of DPSCs: selecting dental pulp stem cells frozen in liquid nitrogen for 3 months, quickly thawing in a constant-temperature water bath environment at 37 ℃, and then utilizing the conventional methodαCulturing in MEM, diluting, centrifuging to collect cells, adding recovery solution containing bFGF, and adding 5% CO at 37 deg.C2Culturing in an incubator, replacing the culture medium every other day, wherein the final concentration of the bFGF action is 10-20ng/mL, the bFGF only acts on the first-generation recovered DPSCs, and the culture medium of the second-generation recovered DPSCs is replaced by the conventional stem cell culture medium.
2. According to claim 1The method for resuscitating dental pulp stem cells, wherein the step (1) is performed completelyαMEM medium consists of 20% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin.
3. The method for resuscitating dental pulp stem cells of claim 1, wherein the cell cryopreservation solution of step (2) is composed of 10% DMSO + 90% FBS.
4. The method for resuscitating dental pulp stem cells of claim 1, wherein said step (3) is performed by routineαMEM cultures consisted of 10% FBS +100U/mL penicillin + 100. mu.g/mL streptomycin.
5. The method for resuscitating dental pulp stem cells of claim 1, wherein the resuscitating fluid comprises a conventional mesenchymal stem cell culture medium comprising α -MEM culture medium + 10-20% fetal bovine serum +100U/mL penicillin +100 μ g/mL streptomycin, and basic fibroblast growth factor (bFGF) at a final concentration of 10-20 ng/mL.
6. The method for resuscitating dental pulp stem cells of claim 5, wherein the final concentration of basic fibroblast growth factor (bFGF) is 20 ng/mL.
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