CN110468096A - A kind of preparation method of periodontal ligament cell microballoon - Google Patents
A kind of preparation method of periodontal ligament cell microballoon Download PDFInfo
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- CN110468096A CN110468096A CN201910698665.7A CN201910698665A CN110468096A CN 110468096 A CN110468096 A CN 110468096A CN 201910698665 A CN201910698665 A CN 201910698665A CN 110468096 A CN110468096 A CN 110468096A
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Abstract
The present invention relates to a kind of preparation method of periodontal ligament cell microballoon, periodontal ligament cell is inoculated on the culture plate of coating chitosan coating and is trained periodontal ligament cell microsphere.The acid solution of chitosan is inoculated on culture plate, aqueous slkali is added after culture plate is dried, in the hole of culture plate and is neutralized, after distilled water flushing is clean, culture plate is soaked in ethyl alcohol, PBS is rinsed, last ultraviolet light irradiation, obtains the culture plate of coating chitosan coating.Compared with prior art, periodontal ligament cell culture is formed microsphere by the present invention on chitosan film, and pelletizing method is simple, is easy production and has repeatability well, compared with traditional two-dimentional cultural method, periodontal ligament cell microballoon culture is able to maintain that the stemness of periodontal ligament cell.
Description
Technical field
The invention belongs to field of biotechnology, more particularly, to a kind of preparation method of periodontal ligament cell microballoon.
Background technique
Periodontitis is the main reason for leading to China's adult's loss of tooth, and traditional periodontal treatment means are only capable of preventing tooth
The further development of all inflammation lesions, and promoting the regeneration of the periodontium destroyed is still clinically one urgently to be resolved
Problem.Development of Periodontal Tissue Engineering makes it possible the reconstruction and regeneration of impaired periodontium, and seed cell is in periodontal group
There is main function, wherein periodontal ligament cell is the seed cell that potentiality are had more in periodontal tissue engineering in weaver's journey.Parodontium
Cell can be obtained very easily from the wisdom tooth or correction tooth pulled out, but during cultivating in vitro, easily generation aging
Application value is lost with from differentiation, shows as the decline of periodontal ligament cell proliferative capacity, downward and alkali at ligament related gene
The active reduction of acid phosphatase.This may be since traditional cultural method is by cell culture in two-dimensional surface, and cell exists
It is to be grown in three-dimensional environment in vivo.
Current dimensional culture system include cell is embedded into Three-dimensional biomaterials or cell itself formed it is three-dimensional thin
Born of the same parents' sphere, and three-dimensional sphere environment and vivo environment that cell itself is formed are even more like, cell three-dimensional sphere can guarantee carefully
It preferably interacts, can be saved when implanting complete extracellular between born of the same parents and cell and between cell and matrix
Matrix, facilitates the expression and performance of cell function, while can also enhance the ability of cell resistance external world undesirable element interference.
The method of cell balling-up at present includes Suspension Culture Flask, sessile drop method and ultralow adherency culture dish etc., but these methods have
Balling-up low efficiency, it is inconvenient for operation the disadvantages of.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of teeth being simple and efficient
The preparation method of all theca cell microballoons, for maintaining periodontal ligament cell stemness.
Periodontal ligament cell culture is formed microsphere, the pelletizing method letter of periodontal ligament cell microballoon by the present invention on chitosan film
It is single, it is easy production, there is repeatability well, and be able to maintain that the stemness of periodontal ligament cell.
The purpose of the present invention can be achieved through the following technical solutions:
Periodontal ligament cell is inoculated in the culture plate of coating chitosan coating by a kind of preparation method of periodontal ligament cell microballoon
On be trained periodontal ligament cell microsphere.
In an embodiment of the invention, the acid solution of chitosan is inoculated on culture plate, culture plate is dried
Afterwards, aqueous slkali is added in the hole of culture plate to be neutralized, after distilled water flushing is clean, culture plate is soaked in ethyl alcohol, PBS punching
It washes, last ultraviolet light irradiation obtains the culture plate of coating chitosan coating.
In an embodiment of the invention, the acid solution of chitosan refers to: dissolving the chitosan in 1-2mol/L's
In acetic acid, the chitosan solution of 0.5-2% (M/M) is prepared.
In an embodiment of the invention, according to 0.25ml/cm2Inoculum concentration the acid solution of chitosan is inoculated in
Culture plate.
In an embodiment of the invention, the condition dried to culture plate is: drying and processing at 50-70 DEG C
24-48h, drying and processing is for 24 hours at preferably 60 DEG C.
In an embodiment of the invention, the aqueous slkali being added in the hole of the culture plate after drying and processing is that NaOH is molten
Liquid.
In an embodiment of the invention, the concentration of NaOH solution is 0.25-1mol/L, preferably 0.5mol/L.
In an embodiment of the invention, the time for carrying out neutralization reaction is 2-6h.
In an embodiment of the invention, the number that distilled water flushing and PBS are rinsed is preferably 3 times.
In an embodiment of the invention, ethyl alcohol is preferably the ethyl alcohol of volume fraction 75%.
In an embodiment of the invention, the time of ultraviolet light irradiation is 6-24h.
In an embodiment of the invention, the periodontal ligament cell is derived from the periodontal membrane tissue in root of the tooth, with group
It knits block method and carries out originally culture.
In an embodiment of the invention, the periodontal ligament cell is according to 0.5-6 × 104/cm2It is inoculated in coating shell
On the culture plate of glycan film.
Compared with prior art, periodontal ligament cell culture is formed microsphere, pelletizing method by the present invention on chitosan film
Simply, it is easy production and there is repeatability well, compared with traditional two-dimentional cultural method, periodontal ligament cell microballoon culture energy
Enough maintain the stemness of periodontal ligament cell.
Detailed description of the invention
Fig. 1: the periodontal ligament cell of various concentration passes through inversion in continuous 5 days after being inoculated in the culture plate of coating chitosan coating
The form of micro- sem observation cell and the formation of microsphere；
Fig. 2: doing Activity determination to microsphere at the 1st, 3,6 day, and as a result the intracorporal cell majority of periodontal ligament cell microballoon is
Living cells (figure A, 200 μm of scale), and have than the cell in conventional two-dimensional culture source from the cell in microsphere source higher
Clonality (figure B) and higher stemness gene expression (figure C)；
In figure, Spheroid: microsphere culture；Monolayer: two dimension culture.
Specific embodiment
Periodontal ligament cell is inoculated in the culture plate of coating chitosan coating by a kind of preparation method of periodontal ligament cell microballoon
On be trained periodontal ligament cell microsphere.
The acid solution of chitosan is inoculated on culture plate, after culture plate is dried, aqueous slkali is added in the hole of culture plate
It is neutralized, after distilled water flushing is clean, culture plate is soaked in ethyl alcohol, PBS is rinsed, and last ultraviolet light irradiation is wrapped
By the culture plate of chitosan coating.
The acid solution of chitosan refers to: dissolving the chitosan in the acetic acid of 1-2mol/L, prepares 0.5-2%'s (M/M)
Chitosan solution.
According to 0.25ml/cm2Inoculum concentration the acid solution of chitosan is inoculated in culture plate.
The condition dried to culture plate is: drying and processing 24-48h at 50-70 DEG C.
The aqueous slkali being added in the hole of culture plate after drying and processing is NaOH solution.
The concentration of NaOH solution is 0.25-1mol/L.
The time for carrying out neutralization reaction is 2-6h.
The number that distilled water flushing and PBS are rinsed is preferably 3 times.
Ethyl alcohol is preferably the ethyl alcohol of volume fraction 75%.
The time of ultraviolet light irradiation is 6-24h.
The periodontal ligament cell is derived from the periodontal membrane tissue in root of the tooth, carries out originally culture with tissue block method.
The periodontal ligament cell is according to 0.5-6 × 104/cm2It is inoculated on the culture plate of coating chitosan coating.
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
It dissolves the chitosan in the acetic acid of 1mol/L, prepares 1% chitosan solution.Then by chitosan solution
0.25ml/cm2It is inoculated on culture plate, culture plate is placed in 60 DEG C of baking ovens for 24 hours.0.5mol/L is added in every hole after drying
In NaOH solution and 2 hours, distilled water flushing 3 times, culture plate being soaked in 75% ethyl alcohol overnight later, PBS is rinsed 3 times,
Last ultraviolet lamp irradiation is overnight.
The preparation of periodontal ligament cell microsphere: taking in root of the tooth 1/3 periodontal membrane tissue, carries out primary training with tissue block method
It supports.It according to various concentration (is 0.5 × 10 respectively by periodontal ligament cell4/cm2、1×104/cm2、3×104/cm2、6×104/cm2)
It is inoculated on the culture plate of coating chitosan coating, separately takes not coated culture plate as a control group, be inoculated with the thin of comparable sodium
Born of the same parents.The form of cell and the formation of microsphere were observed by inverted microscope by continuous 5 days, as a result as shown in Figure 1.
The Activity determination of periodontal ligament cell microsphere: will after cell culture 1,3 and 6 day using LIVE/DEAD kit into
Row dyeing, in the survival condition of fluorescence microscopy microscopic observation cell.As a result as shown in A in Fig. 2, periodontal ligament cell is micro- as the result is shown
The intracorporal cell majority of ball is living cells (A, 200 μm of scale in Fig. 2).
Periodontal ligament cell two-dimension single layer cell (Monolayer) is compared with microsphere (Spheroid): will derive from tooth
The cell of all theca cell two-dimension single layer cells and three-dimensional microsphere makees the dilution of gradient multiple, and every group of cell is respectively with 100, every ware
Cell density is inoculated with respectively, and 4% paraformaldehyde is fixed after culture 10 days, Toluidine blue staining, counts clone, as a result such as B in Fig. 2
Shown, B, which can be seen that from the cell in microsphere source, in Fig. 2 has higher clone than the cell in conventional two-dimensional culture source
Forming ability.
Stemness gene expression: by cell inoculation in or without the coated 6 orifice plates of chitosan, culture collected sample after 1,6 day
This, Trizol extracted total RNA after reverse transcription is cDNA, designs the primer of Oct4, Sox2 and Nanog, glimmering by real-time quantitative
Light PCR is quantified, and as a result as shown in C in 2, C can be seen that the cell from microsphere source than conventional two-dimensional culture in Fig. 2
The cell in source has the expression of higher stemness gene.
Embodiment 2
It dissolves the chitosan in the acetic acid of 1mol/L, prepares the chitosan solution of 0.5% (M/M).
According to 0.25ml/cm2Inoculum concentration the acid solution of chitosan is inoculated on culture plate, by culture plate dry (50
Drying and processing 48h at DEG C) after, the NaOH solution that 0.25mol/L is added in the hole of culture plate carries out neutralization reaction 2h, distilled water punching
After washing 3 times, culture plate is soaked in the ethyl alcohol of volume fraction 75%, PBS is rinsed 3 times, and last ultraviolet light irradiation 6h is wrapped
By the culture plate of chitosan coating.
Take the periodontal membrane tissue in root of the tooth, carry out originally culture with tissue block method, by periodontal ligament cell according to 0.5 ×
104/cm2It is inoculated on the culture plate of coating chitosan coating and is trained periodontal ligament cell microsphere.
Embodiment 3
It dissolves the chitosan in the acetic acid of 2mol/L, prepares the chitosan solution of 2% (M/M).
According to 0.25ml/cm2Inoculum concentration the acid solution of chitosan is inoculated on culture plate, by culture plate dry (70
Drying and processing is for 24 hours at DEG C) after, the NaOH solution that 1mol/L is added in the hole of culture plate carries out neutralization reaction 6h, distilled water flushing 3
After secondary, culture plate is soaked in the ethyl alcohol of volume fraction 75%, PBS is rinsed 3 times, and last ultraviolet light irradiation for 24 hours, is coated with
The culture plate of chitosan coating.
The periodontal membrane tissue in root of the tooth is taken, originally culture is carried out with tissue block method, by periodontal ligament cell according to 6 × 104/
cm2It is inoculated on the culture plate of coating chitosan coating and is trained periodontal ligament cell microsphere.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of preparation method of periodontal ligament cell microballoon, which is characterized in that periodontal ligament cell is inoculated in coating chitosan and is applied
Periodontal ligament cell microsphere is trained on the culture plate of film.
2. a kind of preparation method of periodontal ligament cell microballoon according to claim 1, which is characterized in that by the acid of chitosan
Solution inoculum is added aqueous slkali after drying culture plate, in the hole of culture plate and is neutralized, distilled water flushing is dry on culture plate
After net, culture plate is soaked in ethyl alcohol, PBS is rinsed, and last ultraviolet light irradiation obtains the culture plate of coating chitosan coating.
3. a kind of preparation method of periodontal ligament cell microballoon according to claim 2, which is characterized in that the acid of chitosan is molten
Liquid refers to: dissolving the chitosan in the acetic acid of 1-2mol/L, prepares the chitosan solution of 0.5-2% (M/M).
4. a kind of preparation method of periodontal ligament cell microballoon according to claim 2, which is characterized in that according to 0.25ml/
cm2Inoculum concentration the acid solution of chitosan is inoculated in culture plate.
5. a kind of preparation method of periodontal ligament cell microballoon according to claim 2, which is characterized in that carried out to culture plate
The condition of drying is: drying and processing 24-48h at 50-70 DEG C.
6. a kind of preparation method of periodontal ligament cell microballoon according to claim 2, which is characterized in that after drying and processing
The aqueous slkali being added in the hole of culture plate is NaOH solution.
7. a kind of preparation method of periodontal ligament cell microballoon according to claim 6, which is characterized in that NaOH solution it is dense
Degree is 0.25-1mol/L.
8. a kind of preparation method of periodontal ligament cell microballoon according to claim 2, which is characterized in that carry out neutralization reaction
Time be 2-6h.
9. a kind of preparation method of periodontal ligament cell microballoon according to claim 1, which is characterized in that the parodontium is thin
Born of the same parents are derived from the periodontal membrane tissue in root of the tooth, carry out originally culture with tissue block method.
10. a kind of preparation method of periodontal ligament cell microballoon according to claim 1, which is characterized in that the parodontium
Cell is according to 0.5-6 × 104/cm2It is inoculated on the culture plate of coating chitosan coating.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755481A (en) * | 2021-08-31 | 2021-12-07 | 上海市口腔医院(上海市口腔健康中心) | Stentless periodontal ligament stem cell sphere and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101448527A (en) * | 2006-05-19 | 2009-06-03 | 香港大学 | Cell-matrix microspheres, methods for preparation and applications |
| CN103124762A (en) * | 2010-09-29 | 2013-05-29 | 塞恩心血管公司 | Methods for processing microspheres, microspheres processed thereby, and uses thereof |
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2019
- 2019-07-31 CN CN201910698665.7A patent/CN110468096A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101448527A (en) * | 2006-05-19 | 2009-06-03 | 香港大学 | Cell-matrix microspheres, methods for preparation and applications |
| CN101448527B (en) * | 2006-05-19 | 2013-12-18 | 香港大学 | Cell-matrix microspheres, methods for preparation and applications |
| CN103124762A (en) * | 2010-09-29 | 2013-05-29 | 塞恩心血管公司 | Methods for processing microspheres, microspheres processed thereby, and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| X-Z YAN等: "Spheroid formation and stemness preservation of human periodontal ligament cells on chitosan films", 《ORAL DISEASES》 * |
| 闫香珍等: "牙周膜细胞微球体的形成及干性维持研究", 《第十一次全国牙周病学学术会议摘要汇编》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755481A (en) * | 2021-08-31 | 2021-12-07 | 上海市口腔医院(上海市口腔健康中心) | Stentless periodontal ligament stem cell sphere and preparation method and application thereof |
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