CN110951682A - Preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells - Google Patents
Preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells Download PDFInfo
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- CN110951682A CN110951682A CN201911413361.8A CN201911413361A CN110951682A CN 110951682 A CN110951682 A CN 110951682A CN 201911413361 A CN201911413361 A CN 201911413361A CN 110951682 A CN110951682 A CN 110951682A
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- 239000006166 lysate Substances 0.000 title claims abstract description 49
- 210000003074 dental pulp Anatomy 0.000 title claims abstract description 35
- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 210000004369 blood Anatomy 0.000 claims abstract description 20
- 239000008280 blood Substances 0.000 claims abstract description 20
- 239000000706 filtrate Substances 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 17
- 210000004748 cultured cell Anatomy 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 230000004663 cell proliferation Effects 0.000 claims description 5
- 210000004357 third molar Anatomy 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 230000002188 osteogenic effect Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 4
- 239000000126 substance Substances 0.000 abstract description 2
- 210000001772 blood platelet Anatomy 0.000 description 53
- 238000000926 separation method Methods 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 3
- 230000011164 ossification Effects 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000009818 osteogenic differentiation Effects 0.000 description 2
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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Abstract
The invention relates to a preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells, which comprises the following steps: putting the whole blood into a centrifuge to centrifugally separate platelets from other components in the whole blood; then separating and collecting the platelets under aseptic conditions, and then disrupting the platelets; centrifugally separating the cracked platelets, collecting filtrate, and performing aseptic treatment to obtain a platelet lysate; the dental pulp mesenchymal cells of the teeth are cultured by adopting the culture solution, and the optimal platelet lysate concentration is selected by detecting various laboratory indexes of the dental pulp mesenchymal cells. The preparation method of the autologous platelet lysate used for in-vitro culture of the dental pulp mesenchymal cells has no xenogenic substances, and simultaneously, the platelet lysate prepared by mixing a plurality of parts of whole blood can avoid batch-to-batch differences.
Description
Technical Field
The invention relates to a preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells.
Background
The existing technology for amplifying dental pulp mesenchymal cells in vitro needs to add animal serum into a cell culture medium, and the animal serum has great potential risks, such as human and livestock comorbidity, prion infection and the like. The platelet lysate is derived from a healthy donor or a blood bank, has safe sources, contains various growth factors, can increase the safety of cultured cells in later use, and can promote the proliferation and osteogenic differentiation of dental pulp mesenchymal cells. But no research has been developed into healthy donors or blood banks.
Disclosure of Invention
The invention aims to provide a preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells.
The invention relates to a preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells, which comprises the following steps: s101: placing whole blood into a centrifuge to centrifugally separate platelets from other components of the whole blood; s102: then separating and collecting the platelets under aseptic conditions, and then disrupting the platelets; s103: centrifuging the cracked platelets by using a centrifugal machine, collecting filtrate, and performing aseptic treatment to obtain a platelet lysate; s104: the method comprises the steps of culturing dental pulp mesenchymal cells of teeth by adopting a DMEM culture solution with the mass concentration of platelet lysate of 5-20%, and selecting the optimal concentration of platelet lysate by detecting various laboratory indexes of the dental pulp mesenchymal cells.
The preparation method of the autologous platelet lysate used for in-vitro culture of the dental pulp mesenchymal cells has no xenogenic substances, and meanwhile, the platelet lysate prepared by mixing a plurality of parts of whole blood can avoid batch-to-batch differences; the platelet lysate is extracted from autologous whole blood and the quality of the platelet lysate is guaranteed, and the promotion effect of the platelet lysate in the in-vitro amplification and differentiation of dental pulp mesenchymal cells is verified in-vitro research.
In addition, the method for preparing the autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells according to the present invention may further include the following additional technical features:
further, in the step S104, the teeth adopt extracted waste healthy wisdom teeth or corrected extracted healthy orthodontic teeth.
Further, in step S104, the laboratory indexes include the cell proliferation rate, the proportion of living cells, and the osteogenic capacity of the cultured cells compared with the cells cultured in the conventional culture mode.
Further, in the step S101, the whole blood is taken from a healthy human body, and is taken from at least two persons.
Further, in the step S101, the rotation radius during the centrifugal separation is 5 cm-7 cm, the rotation speed is 2500 r/min-3000 r/min, and the centrifugal separation time is 10 min-15 min.
Further, in the step S103, the rotation radius during the centrifugal separation is 8cm to 12cm, the rotation speed is 2700r/min to 3200r/min, and the time of the centrifugal separation is 8min to 12 min.
Further, in the step S102, when the platelets are ruptured, the platelets are first frozen at-82 ℃ to-78 ℃ for 10h to 14h and then thawed at 35 ℃ to 40 ℃ for 1.5h to 2.5h by using a low-temperature and high-temperature freeze-thaw cycle.
Further, in the step S102, the low-temperature/high-temperature freeze-thaw cycle is repeated three times.
Another object of the present invention is to provide autologous platelet lysate prepared by the method as an in vitro culture of dental pulp mesenchymal cells.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and not to be construed as limiting the invention.
Example 1
Example 1 provides a method for preparing autologous platelet lysate for in vitro culture of dental pulp mesenchymal cells, comprising the steps of:
(1) whole blood from two healthy persons was placed in a centrifuge to centrifugally separate platelets from the other components of the whole blood. Wherein the rotation radius during centrifugal separation is 5cm, the rotation speed is 3000r/min, and the centrifugal separation time is 10 min.
(2) The platelets are then separated and collected under sterile conditions, and then disrupted. When the blood platelet is cracked, the blood platelet is firstly frozen for 10 hours at the temperature of minus 78 ℃ and then thawed for 1.5 hours at the temperature of 40 ℃ in a low-high temperature freeze-thaw cycle mode. The low-temperature-high-temperature freeze-thaw cycle is repeated three times.
(3) And (3) centrifugally separating the broken platelets by using a centrifugal machine, collecting filtrate and performing aseptic treatment to obtain a platelet lysate. Wherein the rotation radius during centrifugal separation is 12cm, the rotation speed is 2700r/min, and the centrifugal separation time is 12 min.
(4) The dental pulp mesenchymal cells of the teeth are cultured by DMEM culture solution with the mass concentration of platelet lysate of 5%, and the optimal concentration of platelet lysate is selected by detecting the cell proliferation speed, the living cell proportion and the comparison of the osteogenesis capacity of the cultured cells and the cells under the traditional culture mode. Wherein the teeth are extracted waste healthy wisdom teeth.
Example 2
Embodiment 2 provides a method for preparing autologous platelet lysate used as dental pulp mesenchymal cell culture in vitro, comprising the following steps:
(1) whole blood from three healthy people was placed in a centrifuge to centrifugally separate platelets from the other components of the whole blood. Wherein the rotation radius during centrifugal separation is 7cm, the rotation speed is 2500r/min, and the centrifugal separation time is 15 min.
(2) The platelets are then separated and collected under sterile conditions, and then disrupted. When the blood platelet is cracked, the blood platelet is firstly frozen at the temperature of 82 ℃ below zero for 14 hours and then thawed at the temperature of 35 ℃ for 2.5 hours in a low-high temperature freeze-thaw cycle mode. The low-temperature-high-temperature freeze-thaw cycle is repeated three times.
(3) And (3) centrifugally separating the broken platelets by using a centrifugal machine, collecting filtrate and performing aseptic treatment to obtain a platelet lysate. Wherein the rotation radius during centrifugal separation is 8cm, the rotation speed is 3200r/min, and the centrifugal separation time is 8 min.
(4) The dental pulp mesenchymal cells of the teeth are cultured by DMEM culture solution with the mass concentration of the platelet lysate of 20%, and the optimal concentration of the platelet lysate is selected by detecting the cell proliferation speed, the living cell proportion and the comparison of the osteogenesis capacity of the cultured cells and the cells under the traditional culture mode. Wherein the teeth are healthy orthodontic teeth extracted by correction.
Example 3
Embodiment 3 provides a method for preparing autologous platelet lysate used as dental pulp mesenchymal cell culture in vitro, comprising the following steps:
(1) whole blood from two healthy persons was placed in a centrifuge to centrifugally separate platelets from the other components of the whole blood. Wherein the rotation radius during centrifugal separation is 6cm, the rotation speed is 2800r/min, and the centrifugal separation time is 12 min.
(2) The platelets are then separated and collected under sterile conditions, and then disrupted. When the blood platelet is cracked, the blood platelet is firstly frozen at the temperature of 80 ℃ below zero for 12 hours and then thawed at the temperature of 37 ℃ for 2 hours in a low-high temperature freeze-thaw cycle mode. The low-temperature-high-temperature freeze-thaw cycle is repeated three times.
(3) And (3) centrifugally separating the broken platelets by using a centrifugal machine, collecting filtrate and performing aseptic treatment to obtain a platelet lysate. Wherein the rotation radius during centrifugal separation is 10cm, the rotation speed is 3000r/min, and the centrifugal separation time is 10 min.
(4) The dental pulp mesenchymal cells of the teeth are cultured by DMEM culture solution with 10% of the mass concentration of the platelet lysate, and the optimal concentration of the platelet lysate is selected by detecting the cell proliferation speed, the living cell proportion and the comparison of the osteogenesis capacity of the cultured cells and the cells under the traditional culture mode. Wherein the teeth are extracted waste healthy wisdom teeth.
In conclusion, the invention extracts the platelet lysate by using the whole blood with healthy source, applies the platelet lysate to the in-vitro culture process of the dental pulp mesenchymal cells, does not add animal serum, avoids the potential risk of the animal serum, and ensures that the dental pulp mesenchymal cells cultured in vitro have more stable osteogenic differentiation capacity. The invention has repeatability, the platelet lysate extracted in batches has similar biological characteristics, and can play similar functions in the in vitro culture of the dental pulp mesenchymal cells. The content of the growth factors in the platelet lysate extracted by the method reaches the standard of cell culture, and the optimal concentration of the platelet lysate is explored.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells is characterized by comprising the following steps:
s101: placing whole blood into a centrifuge to centrifugally separate platelets from other components of the whole blood;
s102: then separating and collecting the platelets under aseptic conditions, and then disrupting the platelets;
s103: centrifuging the cracked platelets by using a centrifugal machine, collecting filtrate, and performing aseptic treatment to obtain a platelet lysate;
s104: the method comprises the steps of culturing dental pulp mesenchymal cells of teeth by adopting a DMEM culture solution with the mass concentration of platelet lysate of 5-20%, and selecting the optimal concentration of platelet lysate by detecting various laboratory indexes of the dental pulp mesenchymal cells.
2. The method for preparing an autologous platelet lysate as an in vitro culture of dental pulp mesenchymal cells according to claim 1, wherein, in the step S104, the tooth is an extracted healthy wisdom tooth or a corrected extracted healthy orthodontic tooth.
3. The method for preparing an autologous platelet lysate as claimed in claim 1, wherein in step S104, the laboratory indexes include cell proliferation rate, proportion of living cells and comparison between the cultured cells and the osteogenic capacity of cells in conventional culture mode.
4. The method for preparing an autologous platelet lysate that is used for in vitro culture of dental pulp mesenchymal cells according to claim 1, wherein in step S101, the whole blood is obtained from a healthy human body and at least two persons.
5. The method of preparing an autologous platelet lysate that is used for in vitro culture of dental pulp mesenchymal cells according to claim 1, wherein in step S101, the rotation radius during centrifugation is 5cm to 7cm, the rotation speed is 2500r/min to 3000r/min, and the time for centrifugation is 10min to 15 min.
6. The method of preparing an autologous platelet lysate that is used for in vitro culture of dental pulp mesenchymal cells according to claim 1, wherein in step S103, the rotation radius during centrifugation is 8cm to 12cm, the rotation speed is 2700r/min to 3200r/min, and the time for centrifugation is 8min to 12 min.
7. The method of claim 1, wherein the platelets are first frozen at-82 ℃ to-78 ℃ for 10h to 14h and then thawed at 35 ℃ to 40 ℃ for 1.5h to 2.5h in step S102 by a low-temperature-high-temperature freeze-thaw cycle.
8. The method of preparing an autologous platelet lysate that is obtained by in vitro culturing dental pulp mesenchymal cells according to claim 7, wherein the low-temperature/high-temperature freeze-thaw cycle is repeated three times in step S102.
9. Autologous platelet lysate prepared by the method of any one of claims 1-8 as an in vitro culture of dental pulp mesenchymal cells.
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| CN201911413361.8A CN110951682A (en) | 2019-12-31 | 2019-12-31 | Preparation method of autologous platelet lysate used for in vitro culture of dental pulp mesenchymal cells |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011101834A1 (en) * | 2010-02-22 | 2011-08-25 | Advanced Neuro-Science Allies Private Limited | A method for obtaining mesenchymal stem cells, media, methods and composition thereof |
| CN108753709A (en) * | 2018-06-21 | 2018-11-06 | 深圳至博生物科技有限公司 | A kind of serum free medium and its preparation and cell culture processes |
| CN109402050A (en) * | 2018-11-09 | 2019-03-01 | 沈阳中心血站 | A kind of efficient Mesenchymal stem cell nutrient solution of serum-free ingredient |
| CN109985064A (en) * | 2019-05-16 | 2019-07-09 | 北京京蒙细胞生物科技股份有限公司 | Mescenchymal stem cell secretes the purposes of extract, mescenchymal stem cell secretion extract and preparation method thereof |
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2019
- 2019-12-31 CN CN201911413361.8A patent/CN110951682A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011101834A1 (en) * | 2010-02-22 | 2011-08-25 | Advanced Neuro-Science Allies Private Limited | A method for obtaining mesenchymal stem cells, media, methods and composition thereof |
| CN108753709A (en) * | 2018-06-21 | 2018-11-06 | 深圳至博生物科技有限公司 | A kind of serum free medium and its preparation and cell culture processes |
| CN109402050A (en) * | 2018-11-09 | 2019-03-01 | 沈阳中心血站 | A kind of efficient Mesenchymal stem cell nutrient solution of serum-free ingredient |
| CN109985064A (en) * | 2019-05-16 | 2019-07-09 | 北京京蒙细胞生物科技股份有限公司 | Mescenchymal stem cell secretes the purposes of extract, mescenchymal stem cell secretion extract and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| MATHEW COWPER等: "Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells", 《CELL》 * |
| 谢贤哲: "人血小板裂解液对人牙髓间充质细胞体外增殖与成骨分化的作用研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 谢贤哲等: "人血小板裂解液在人牙髓间充质细胞体外增殖与分化中的应用", 《安徽医科大学学报》 * |
| 陈博: "血小板裂解液（platelet lysate，PL）对牙源性间充质干细胞的生物学影响", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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