CN110951683A - Preparation method of 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
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a preparation method of dental pulp stem cells, which comprises the following steps: removing pulp tissue from the tooth and cutting the pulp tissue; placing the cut pulp tissue in a pulp stem cell culture medium, centrifuging, and removing supernatant to obtain pulp tissue liquid; adding collagenase type I into pulp tissue fluid, and digesting at a specific temperature; continuously adding a dental pulp stem cell culture medium into the digested dental pulp tissue fluid, and purifying to obtain a dental pulp stem cell single cell suspension; the dental pulp stem cells are prepared based on the single cell suspension of the dental pulp stem cells. The dental pulp stem cells are prepared by using an enzyme digestion method, the enzyme digestion method has the advantage of higher efficiency than a tissue adherence method for culturing the cells, and the cells can adhere to the walls in a short time, so that the subsequent stem cell culture is simpler and faster; the prepared cells have low immunogenicity, wide applicable population range, convenient subsequent amplification and high cell activity, and meet the requirements of clinical application.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a preparation method of dental pulp stem cells.
Background
The dental pulp stem cell is derived from the mesenchymal stem cell of the tooth, and has strong self-renewal capacity and multidirectional differentiation potential. The dental pulp stem cells can be induced and differentiated into various histiocytes such as odontoblasts, chondrocytes, myocytes, hepatocytes and pancreatic cell-like cells by artificially adding different inducers in vitro or transplanting the dental pulp stem cells into a specific in vivo environment, and the dental pulp stem cells have lower immunogenicity than general autologous cells, so that the dental pulp stem cells have great research value in the future cell therapy progress.
In the cell culture preparation, the dental pulp stem cells are difficult to separate by only using dental pulp tissues in the traditional technology, and the dental pulp stem cells can normally grow and proliferate only by processing the dental pulp tissues.
Because of the residual gum and periodontal tissue on the tooth surface, contamination is very likely to occur during the process of taking out the dental pulp and culturing the cells. Meanwhile, the success rate of the tissue adherence method is low, the tissue is easy to inactivate, dental pulp stem cells are probably not obtained when the tissue adheres to the wall for 14 days, and the problems of low efficiency, time waste and the like are caused.
Disclosure of Invention
The embodiment of the invention mainly provides a preparation method for preparing dental pulp stem cells by a tissue digestion method, and aims to solve the technical problems that the traditional dental pulp stem cells are difficult to culture and low in preparation efficiency.
In order to solve the above technical problem, one technical solution adopted by the embodiment of the present invention is: provided is a method for preparing dental pulp stem cells, the method including: removing pulp tissue from a tooth and shearing the pulp tissue; placing the cut dental pulp tissue in a dental pulp stem cell culture medium, centrifuging, and then removing supernatant to obtain dental pulp tissue fluid; adding collagenase type I into the dental pulp tissue fluid, and digesting the pulp tissue fluid at a specific temperature; continuously adding the dental pulp stem cell culture medium into the digested dental pulp tissue fluid, and purifying to obtain a dental pulp stem cell single cell suspension; and preparing the dental pulp stem cells based on the single cell suspension of the dental pulp stem cells.
Optionally, the collagenase type I is present at a concentration of 10 mg/ml.
Optionally, the dental pulp tissue in the dental pulp tissue fluid is digested in collagenase type i at a digestion temperature of 37 ℃ for 30-45 min.
Optionally, the pulp tissue fluid is oscillated every 10min while the pulp tissue in the pulp tissue fluid is digested in collagenase i.
Alternatively, when the minced dental pulp tissue is placed in a dental pulp stem cell culture medium and then centrifuged, the centrifugation conditions are as follows: the centrifugal temperature is 20 ℃, the centrifugal force is 1200 rpm/min-1500 rpm/min, and the centrifugal time is 10 minutes.
Optionally, the dental pulp stem cells are prepared based on the single cell suspension of dental pulp stem cells, specifically as follows: taking out the 6-hole plate; inoculating the dental pulp stem cells in the dental pulp stem cell single cell suspension into a 6-well plate for culture; and replacing the cell culture solution at regular time, and finishing the culture to obtain the dental pulp stem cells.
Optionally, the teeth are cleaned prior to removing the pulp tissue from the teeth.
Optionally, the cleaning mode when cleaning the teeth is as follows: primarily cleaning the surface of teeth by using alcohol or iodophor; and (3) immersing the primarily cleaned teeth into a sterile PBS solution containing penicillin and streptomycin, and sterilizing to obtain the cleaned teeth.
Optionally, the teeth are wisdom teeth.
Optionally, the method further comprises: performing flow cytometric identification on the dental pulp stem cells.
The preparation method of the dental pulp stem cells provided by the embodiment of the invention has the following beneficial effects: the immune cells obtained by the invention have low immunogenicity and wide applicable population range, and the prepared immune cells have high purity, convenient subsequent amplification and high cell activity; the dental pulp stem cells are prepared by a tissue digestion method, the collagenase type I is used, the collagenase type I is mainly used for hydrolyzing collagen components in connective tissues and is used for digesting the connecting parts in the tissues to enable the connecting parts to become single cells, the enzyme digestion method has the advantage of higher efficiency than the general tissue adherence method for culturing the cells, the cells can adhere to the walls in a short time, and the subsequent stem cell culture is simpler and faster; the preparation method of the invention comprises the steps of cleaning the surface of teeth by using alcohol or iodophor, and then soaking the teeth in sterile PBS solution containing penicillin and streptomycin to simply sterilize tissues and avoid the pollution of subsequent culture; in the preparation process, the sample is not subjected to cryopreservation, and the addition of a cryopreservation protective agent is avoided, so that the final stem cells do not contain any newly added components, and the application risk of the population is reduced.
Drawings
Fig. 1 is a schematic diagram of a method for preparing dental pulp stem cells according to an embodiment of the present invention.
Detailed Description
In order to make the objects, aspects and advantages of the present invention more apparent, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
An embodiment of the present invention first provides a method for preparing dental pulp stem cells, as shown in fig. 1, the method includes the following steps:
The "pulp tissue" in embodiments of the present invention may be from any tooth in any oral cavity, e.g., deciduous teeth, wisdom teeth, etc. Pulp tissue can be removed from either a dental extracted tooth or a naturally extracted or exfoliated tooth at a dental care facility.
The "removal of pulp tissue from a tooth" is not limited to collection of pulp tissue from a living body, and includes thawing pulp tissue after cryopreservation. Also, the source of the dental pulp tissue is not limited to humans, and may be other mammals.
This step requires that the clinically extracted normal tooth be cleaned first before the pulp tissue is removed from the tooth. The cleaning method comprises the following steps: the method comprises the steps of removing residual gum and periodontal tissues on the surface of teeth, cleaning the surface of the teeth with essence or iodophor, and sterilizing the teeth in a sterile Phosphate Buffered Saline (PBS) solution containing penicillin and streptomycin to obtain cleaned teeth.
After cleaning the teeth, the teeth are cut transversely with a high-speed split drill, and are broken with bone forceps, and then the pulp tissue is taken out with sterile forceps or a broach and cut into pieces with surgical scissors, to obtain the cut pulp tissue.
This step consists in performing a preliminary treatment of the pulp tissue to obtain a pulp tissue fluid that can be subsequently cultured. Specifically, the cut pulp tissue is first placed in a pulp stem cell culture medium, and after centrifugation, the supernatant is removed (cells are collected by centrifugation to help exchange the solution), and finally pulp tissue solution is obtained, which contains pulp tissue.
Different cell tissues are suitable for different centrifugal forces, and in one embodiment, the centrifugal conditions are room temperature of 20 ℃, the centrifugal force is 1200rpm/min to 1500rpm/min, and the centrifugal time is 10 minutes. Small centrifugal force causes the cells not to be easily separated because of small centrifugal force, while larger centrifugal force causes the cells to be easily collided and cracked in the centrifugal process.
The pulp stem cells are inhibited from contacting each other in the pulp tissue fluid, and thus cannot proliferate in a large amount on a large scale, and cannot divide each other. When the dental pulp stem cells need to be proliferated, the adhesive proteins in the dental pulp stem cells need to be decomposed by digestion, so that the dental pulp stem cells are in a dispersed state, and the dental pulp stem cells are beneficial to nutrient exchange.
This step is carried out by digesting it with collagenase, an enzyme extracted from the bacteria, which has a strong digestion effect on collagen. It is suitable for digesting fibrous tissue, epithelial tissue and cancer tissue, has good digestion effect on intercellular substance, has little influence on cell itself, and can separate cell from collagen component without damage.
Specifically, the collagenase type I is selected for digesting the cells of epithelial, lung, fat and adrenal gland tissues, and is used for digesting the connecting parts in the tissues to form single cells. The preparation of collagenase type I is well known to those skilled in the art and will not be described further herein.
During digestion, the temperature is increased, the activity of the collagenase type I is enhanced before the optimal temperature is reached, the activity of the collagenase type I is increased until the optimal temperature is reached, then the activity of the collagenase is gradually reduced when the temperature is increased, and the collagenase is denatured and inactivated when the temperature is increased to a certain temperature, which is the optimal temperature for the activity of the collagenase type I and is defined by the invention at 37 ℃. The digestion time is 30-45min, and in some embodiments, the pulp tissue fluid may be oscillated every 10min in order to prevent incomplete digestion due to pulp tissue deposition.
And 240, continuously adding the dental pulp stem cell culture medium into the digested dental pulp tissue fluid, centrifuging and filtering to obtain a dental pulp stem cell single cell suspension.
After digestion, the dental pulp stem cell culture medium needs to be added to the digested dental pulp tissue fluid.
In this embodiment, a 5-fold volume of dental pulp stem cell culture medium may be added to the digested dental pulp tissue fluid, followed by centrifugation, so that the cells in the dental pulp tissue fluid are placed in the complete culture medium, and then filtered through a 70 μm cell sieve, to obtain a single cell suspension of dental pulp stem cells.
And 250, preparing the dental pulp stem cells based on the marrow stem cell single cell suspension.
After obtaining the single cell suspension of dental pulp stem cells, the dental pulp stem cells can be inoculated and cultured, and in one embodiment, a 6-well plate (the bottom area of the 6-well plate is 9.6 cm) can be taken out2The amount of culture medium was 2.5ml, and the amount of cell inoculum was 1.2 x 106) The dental pulp stem cells were seeded in 6-well plates and cultured. Then, the cells were changed every 2 days, passaging was performed according to the growth of the cells, and after the culture was completed, dental pulp stem cells were obtained. In actual operation, of course, the experimenter can select a suitable orifice plate and exchange the liquid for the cells in time according to actual conditions to finally obtain the dental pulp stem cells, which is not limited by the invention.
The preparation method of the dental pulp stem cells provided by the embodiment of the invention has the following beneficial effects:
1. the immune cells obtained by the invention have low immunogenicity and wide applicable population range, and the prepared immune cells have high purity, convenient subsequent amplification and high cell activity;
2. the preparation method of the invention uses I-type collagenase, which mainly hydrolyzes collagen components in connective tissues and is used for digesting the connective parts in the tissues to make the connective parts become single cells, compared with the common tissue adherence method, the enzyme digestion method has the advantage of higher efficiency, the cells can adhere to the walls in a short time, so that the subsequent stem cell culture is simpler and faster;
3. the preparation method of the invention comprises the steps of cleaning the surface of teeth by using alcohol or iodophor, and then soaking the teeth in sterile PBS solution containing penicillin and streptomycin to simply sterilize tissues and avoid the pollution of subsequent culture;
4. in the preparation process, the sample is not subjected to cryopreservation, and the addition of a cryopreservation protective agent is avoided, so that the final stem cells do not contain any newly added components, and the application risk of the population is reduced.
To illustrate the preparation process of the present invention in detail, the preparation of multiple dental pulp stem cells is further illustrated by the following examples. The specific preparation process is as follows:
(1) collecting normal teeth which are clinically extracted;
(2) cleaning teeth;
removing residual gum and periodontal tissue on tooth surface, cleaning tooth surface with alcohol or iodophor, and sterilizing in sterile PBS solution containing penicillin and streptomycin to obtain cleaned tooth.
(3) Taking out dental pulp tissue;
the specific taking-out mode is as follows: firstly, the cleaned teeth are transversely cut by a high-speed split drill, then the cleaned teeth are broken by bone tongs, the dental pulp tissue is taken out by using sterile forceps or a broach, and the dental pulp tissue is cut into pieces by using surgical scissors.
(4) Centrifuging and separating;
placing the cut pulp tissue in a test tube filled with pulp stem cell culture medium, centrifuging, and removing supernatant, wherein the centrifugation condition is 20 ℃ at room temperature, the centrifugal force is 1500rpm/min, and the centrifugation time is 10 minutes.
(5) Adding 10mg/ml collagenase type I into dental pulp tissue at the bottom of the test tube, digesting at 37 deg.C for 30-45min, and shaking every 10 min.
(6) Culturing in culture medium;
after digestion, adding a dental pulp stem cell culture medium with 5 times of volume into the test tube, centrifuging, placing the dental pulp stem cells into the complete culture medium, and filtering by using a 70-micrometer cell sieve to obtain a dental pulp stem cell single cell suspension;
(7) subculturing;
the dental pulp stem cells were inoculated into 6-well plates and cultured, the cells were changed every 2 days on average, and passaged according to the growth of the dental pulp stem cells to P4 generation, and after the culture was completed, flow cytometry was performed (corresponding to example 1).
(8) The preparation method according to the above steps (1) to (7) was repeated 19 times to prepare 20 parts of dental pulp stem cells (corresponding to example 2 to example 20 in table 1).
(9) Cell counting and cell activity detection;
the total of 20 dental pulp stem cells obtained by the above preparation method were counted, digested with P4 generation cells, and then counted and cell activity was measured, respectively, and the statistical results are shown in table 1 (the number of stem cells in table 1 was measured by Countstar cell counter IM1200, and cell activity was measured by trypan blue staining method).
TABLE 1
The results of dental pulp stem cells prepared by the same method from example 1 to example 20 can be concluded as follows:
the invention provides a method for preparing dental pulp stem cells, which can increase the total number of the dental pulp stem cells to 1.0 to 1.6 x 10 to P4 generations7A conventional tissue patchThe total number of dental pulp stem cells prepared by wall method is less than 1 x 107And the number of the dental pulp stem cells prepared by the tissue digestion method is obviously increased compared with the dental pulp stem cells prepared by the traditional method.
The activity of the dental pulp stem cells induced by the preparation method is more than 96 percent, and the activity of the dental pulp stem cells prepared by the traditional tissue wall pasting method is less than 96 percent, wherein the activity of the dental pulp stem cells is nearly 50 percent; in examples 2 to 5, the activity of the dental pulp stem cells obtained by the tissue adherence method was less than 95%, and the activity of the dental pulp stem cells obtained by the tissue digestion method was more than 97%, and it was found that the dental pulp stem cells obtained by the tissue digestion method were greatly improved in cell activity and were good in growth of dental pulp stem cells, and thus the dental pulp stem cells were in accordance with the clinical application requirements.
The type I collagenase has slow digestion effect, is simple and convenient to operate, can improve the survival rate of cells, but has relatively high price, and the concentration of the type I collagenase is limited to 10mg/ml in the embodiment of the invention because: if the concentration of the collagenase type I is too low, the digestion time is too long, the digestion effect is not obvious, while if the concentration of the collagenase type I is too high, the experiment cost is greatly increased, and the type I glue with the concentration of 10mg/ml is obtained through creative work of the inventor.
The embodiment of the invention limits the digestion temperature of dental pulp tissues in collagenase type I to be 37 ℃ and the digestion time to be 30-45 min. During digestion, the temperature is increased, the activity of the collagenase type I is enhanced before the optimal temperature is reached, the activity of the collagenase type I is increased until the optimal temperature is reached, then the activity of the collagenase is gradually reduced when the temperature is increased, and the collagenase is denatured and inactivated when the temperature is increased to a certain temperature, which is the optimal temperature for the activity of the collagenase type I and is defined by the invention at 37 ℃.
When the minced dental pulp tissue is placed in a dental pulp stem cell culture medium and then centrifuged, the centrifugation conditions defined in the embodiments of the present invention are as follows: the centrifugal temperature is 20 ℃, the centrifugal force is 1200 rpm/min-1500 rpm/min, and the centrifugal time is 10 minutes.
Different cell tissues are suitable for different centrifugal forces, the centrifugal force is limited to be 1200 rpm/min-1500 rpm/min in the embodiment of the invention, so that cells are not easy to separate due to small centrifugal force, and the cells are easy to collide and crack in the centrifugal process due to large centrifugal force; the present invention also limits the centrifugation temperature to 20 ℃, because higher centrifugation temperatures tend to make the pulp stem cells potentially inactive due to high temperatures.
By the preparation method of the dental pulp stem cells provided by the embodiment of the invention, the dental pulp stem cells digested by dental pulp can be amplified, and meanwhile, due to the low immunogenicity of the dental pulp stem cells, the immunogenicity of the obtained cells is also low, so that the preparation method can be suitable for a wide range of people in the future and establishes a good direction for future research.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for preparing dental pulp stem cells, comprising:
removing pulp tissue from a tooth and shearing the pulp tissue;
placing the cut dental pulp tissue in a dental pulp stem cell culture medium, centrifuging, and then removing supernatant to obtain dental pulp tissue fluid;
adding collagenase type I into the dental pulp tissue fluid, and digesting the pulp tissue fluid at a specific temperature;
continuously adding the dental pulp stem cell culture medium into the digested dental pulp tissue fluid, and purifying to obtain a dental pulp stem cell single cell suspension;
and preparing the dental pulp stem cells based on the single cell suspension of the dental pulp stem cells.
2. The method according to claim 1, wherein the collagenase type I is present at a concentration of 10 mg/ml.
3. The method according to claim 1, wherein the dental pulp tissue in the dental pulp tissue fluid is digested with collagenase type I at a temperature of 37 ℃ for a period of 30 to 45 min.
4. The method according to claim 3, wherein the pulp tissue fluid is oscillated every 10min while the pulp tissue in the pulp tissue fluid is digested with collagenase type I.
5. The method according to claim 1, wherein when the minced dental pulp tissue is placed in a dental pulp stem cell culture medium and then centrifuged, the centrifugation conditions are as follows:
the centrifugal temperature is 20 ℃, the centrifugal force is 1200 rpm/min-1500 rpm/min, and the centrifugal time is 10 minutes.
6. The method according to claim 1, wherein the dental pulp stem cells are prepared based on the single cell suspension of dental pulp stem cells as follows:
taking out the 6-hole plate;
inoculating the dental pulp stem cells in the dental pulp stem cell single cell suspension into a 6-well plate for culture;
replacing the cell culture solution at regular time;
after the culture, dental pulp stem cells were obtained.
7. The method of claim 1, wherein the tooth is cleaned prior to removing the pulp tissue from the tooth.
8. The method for preparing a tooth cleaner according to claim 7, wherein the teeth are cleaned in the following manner:
primarily cleaning the surface of teeth by using alcohol or iodophor;
and (3) immersing the primarily cleaned teeth into a sterile PBS solution containing penicillin and streptomycin, and sterilizing to obtain the cleaned teeth.
9. The method of claim 1, wherein the teeth are wisdom teeth.
10. The method of any one of claims 1-9, further comprising:
performing flow cytometric identification on the dental pulp stem cells.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010011360.7A CN110951683A (en) | 2020-01-06 | 2020-01-06 | Preparation method of dental pulp stem cells |
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| CN113151163A (en) * | 2021-04-23 | 2021-07-23 | 优牙生物科技(上海)有限公司 | Method for repairing endometrial injury by using dental pulp stem cell preparation |
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