CN109251889A - A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon - Google Patents
A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon Download PDFInfo
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- CN109251889A CN109251889A CN201811085961.1A CN201811085961A CN109251889A CN 109251889 A CN109251889 A CN 109251889A CN 201811085961 A CN201811085961 A CN 201811085961A CN 109251889 A CN109251889 A CN 109251889A
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- stem cell
- dental pulp
- mescenchymal stem
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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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
- C12N2513/00—3D culture
Abstract
The present invention discloses a kind of preparation system of transplanting dental pulp mescenchymal stem cell microballoon.The preparation system using can agitating device be placed in cell in dimensional culture environment, can promote dental pulp mescenchymal stem cell formed 3 D stereo chondritic, preferably keep culture cell stemness, play a role in the transplanting of tissue damage reparation conducive to it.
Description
Technical field
The invention belongs to stem cells technology field, it is related to preparation and its transplanting side of a kind of dental pulp mescenchymal stem cell microballoon
Method.
Background technique
Stem cell is a kind of initial cell with self-renewal capacity and polyphyly differentiation potential, is the ideal of cell transplantation
Cell.In ontogenetic process, there are various forms of stem cells, such as embryonic stem cell, multipotential stem cell and various groups
Committed stem cell such as candidate stem cell, neural stem cell in knitting etc..But the challenge of ethics is faced in practical application or is taken
The limitation of material.
Mescenchymal stem cell (MSC) is a kind of Subaerial blue green algae group being present in tissue with polyphyly differentiation potential,
It is divided into Various Tissues cell because it can induce in certain circumstances, has immunogenicity low, can be formed after transplanting stable chimeric
The features such as, therefore can be used as one kind preferably for the cell of heteroplastic transplantation.Dental pulp mescenchymal stem cell (dental pulp stem
Cells, DPSCs) it is that isolated one kind from pulp tissue has high proliferation ability, height self-renewal capacity, multidirectional
The cell of differentiation capability.Have found within 2000 dental pulp mescenchymal stem cell for the first time, the discovery such as Gronthos in vitro deciduous teeth and
All there is dental pulp mescenchymal stem cell in wisdom tooth.The mankind have 20 deciduous teeth, can gradually fall off naturally in the newborn permanent teeth alternating phase, dental pulp
It is tissue-derived to be easy to get very much, and hurtless measure.And also have the advantage that pulp tissue derived from autologous tissue, no immunological rejection
Reaction can be used for tissue and neomorph and repair；Lesion itself is cured with stimulation pathological tissues；With immunological regulation and resist
Scorching characteristic；DPSCs can in vitro it is stable proliferation and decline rate it is low；A small amount of tissue can obtain a large amount of stem cells, be suitable for big rule
Mould culture, it is small to body injury the advantages that.Existing research successfully constructs dental pulp, dentine and set of teeth using the cell
It knits, recent studies have shown that, dental pulp mescenchymal stem cell has apparent advantage for the treatment of inflammatory bowel disease.Dental pulp mesenchyma
Stem cell separation, improving for culture technique will push it to carry out disease treatment.
Currently, dental pulp mescenchymal stem cell is is carried out by the dental pulp mesenchymal stem cell transplantation method that uses of clinical trial in vitro
It is prepared into single cell suspension after culture, amplification, single cell suspension is then injected to affected part, such method is single due to injection
Cell suspension is injected into unicellular other positions for being easy to be distributed to body of patient's body；In addition to this, there are also a kind of at present
The preparation method of stem cell microballoon, the method, which generally spreads one layer of Ago-Gel in the bottom of culture bottle or culture dish, makes cell
It is unable to adherent growth and forms microsphere, but the method is cumbersome, and is not suitable for large-scale production.
To overcome disadvantages mentioned above, the present invention, which provides one kind, can preferably keep stem cell biology characteristic, and easy to operate, suitable
Close the preparation method of the dental pulp mescenchymal stem cell microballoon of large-scale production and for being transplanted to patient's body, the cell after transplanting
Microsphere is not easy to diffuse to other positions of body, is convenient for clinical manipulation, has in the reparation and dental tissue engineering of pulp tissue
Wide application prospect, and be possible to replace the treatment method of existing tooth defect or missing.
Summary of the invention
The object of the present invention is to provide a kind of transplanting preparation methods of dental pulp mescenchymal stem cell microballoon.Use Nostoc commune Vanch
Dental pulp mescenchymal stem cell culture to 1-6 generation is carried out secondary culture, passage training when cell fusion degree reaches 70 ~ 90% by method
Feeding cell is washed, is seeded in new Tissue Culture Flask or Tissue Culture Dish after digestion, and the training of new passage cell will be housed
It supports bottle or culture dish is put into the dental pulp stem cell three-dimensional cultivation device that revolving speed is 50 ~ 300rpm, make to be placed on culture bottle thereon
Or culture dish can constant speed wave, wave culture apparatus and be put into 37 DEG C, in 5% CO2 cell incubator.It is dry thin to dental pulp mesenchyma
When born of the same parents' microsphere diameter is up to 40 ~ 200um, drawn with pipette into centrifuge tube, with centrifuge with 300 ~ 800rpm revolving speed centrifugation 2 ~
10min is resuspended dental pulp mescenchymal stem cell microballoon with 0.9% physiological saline of 0.1-30 milliliter afterwards and precipitates, then with 0.5-3mm pin hole
After syringe sucks microsphere suspension, it is injected into affected part.Entire invention includes 2 steps:
1) preparation of dental pulp mescenchymal stem cell microballoon
In the above method, the dental pulp mescenchymal stem cell algebra cultivated was the 1st ~ 6 generation.
In the above method, secondary culture is carried out when the dental pulp mescenchymal stem cell degrees of fusion cultivated reaches 70-90%.
In the above method, the cell of secondary culture is washed, after digestion with 4 × 104~1×106A density is inoculated in new thin
In born of the same parents' culture bottle or culture dish.
In the above method, the culture bottle that new passage cell is housed or culture dish are put into stem cell dimensional culture system.
In the above method, stem cell cultivating system is waved with a certain revolving speed constant speed that revolving speed is 50 ~ 300rpm, makes to be placed on thereon
Culture bottle or culture dish can constant speed wave.
In the above method, stem cell cultivating system is put into 37 DEG C, in 5% CO2 cell incubator.
The transplanting of dental pulp mescenchymal stem cell microballoon
It 2) is 40 ~ 200um by dental pulp mescenchymal stem cell microballoon culture to diameter in the above method.
In the above method, 40 ~ 100um dental pulp mescenchymal stem cell microsphere suspensions are drawn with pipette into centrifuge tube.
In the above method, by the dental pulp mescenchymal stem cell microballoon centrifuge in centrifuge tube with 300 ~ 800rpm revolving speed centrifugation 2 ~
10min。
In the above method, dental pulp mescenchymal stem cell microballoon precipitating is resuspended with 0.9% physiological saline of 0.1-30 milliliter.
In the above method, after microsphere being sucked with 0.5-3mm pin hole syringe, be injected into affected part.
Detailed description of the invention
Fig. 1 is 24 hours aspect graphs (10 ×) of dental pulp mescenchymal stem cell microballoon culture
Fig. 2 is dental pulp mescenchymal stem cell adhere-wall culture and microballoon culture stemness factor expression situation map
Fig. 3 is FCM analysis figure after dental pulp microballoon mescenchymal stem cell culture 3 days, CD34 (-), CD45 (-), CD90 (+),
HLA-DR(-)
Fig. 4 is to carry out within dental pulp microballoon mescenchymal stem cell culture 3 days 21 days into rouge osteogenic induction.A, cell are induced to differentiate into fat
Cell, oil red O stain；B, cell are induced to differentiate into osteocyte, Alizarin red staining.
Specific embodiment
Experimental method in following embodiments is unless otherwise instructed conventional method.
The preparation and transplanting of embodiment 1, people's dental pulp mescenchymal stem cell microballoon
Reagent
The reagent of culture stem cell in the present invention is conventional commercial product, for example, stem cell factor is purchased from
Peprotech, trypsase are purchased from Gibco, and fetal calf serum is purchased from Gibco, complete medium :+10% body of F12 basal medium
Fetal calf serum+stem cell factor of product percentage.
The preparation of people's dental pulp mescenchymal stem cell microballoon
With the primary dental pulp mescenchymal stem cell of commonsense method culture to the 1st ~ 6 generation, when the when progress that reaches 70-90% of cell fusion degree
Secondary culture, cell is washed, is seeded in new culture bottle or culture dish after digestion, and culture bottle or culture dish is put into dry
This device is waved with a certain revolving speed constant speed that revolving speed is 40 ~ 200um, makes to be placed on culture thereon by three-dimensional cell cultivation system
Bottle or culture dish can constant speed wave, be put into cell incubator, cultivation temperature be 37 DEG C, CO2 concentration be 5%.
Dental pulp mescenchymal stem cell can balling-up growth, such as Fig. 1, growth diameter be 40 ~ 200um.
The dental pulp mescenchymal stem cell of balling-up growth embodies better stemness and maintains, such as the dental pulp mesenchyma of Fig. 2 and non-balling-up
Stem cell is compared, and the expression of the stemness factor Oct-4, Sox2, Nanog are increased.
The dental pulp mescenchymal stem cell of balling-up growth is able to maintain the characteristic of mescenchymal stem cell, as Fig. 3 shows balling-up culture
Dental pulp mescenchymal stem cell flow cytometry, CD34 (-), CD44 (+), CD90 (+), HLA-DR(-)；As Fig. 4 is shown as
The dental pulp mescenchymal stem cell of ball culture, which can induce differentiation, becomes fat cell, osteocyte.
The transplanting of people's dental pulp mescenchymal stem cell microballoon
When it is 40-200um that cell microspheres, which are expanded to diameter, drawn with pipette into centrifuge tube, with centrifuge with 300 ~
800rpm revolving speed is centrifuged 2 ~ 10min, and dental pulp mescenchymal stem cell microballoon precipitating is resuspended with 0.9% physiological saline of 0.1-30 milliliter afterwards,
After again microsphere suspension being sucked with 1mm pin hole syringe, be injected into Periodontitis Model mouse affected part；Meanwhile by same concentrations
0.1-30 milliliter dental pulp mescenchymal stem cell single cell suspension be injected into control group Periodontitis Model mouse affected part.
Dental pulp stem cell microballoon can be used for the transplanting of dental pulp stem cell.
Claims (10)
1. the preparation of people's dental pulp mescenchymal stem cell uses a kind of three-dimensional oscillating cultivating system.
2. dental pulp mescenchymal stem cell microballoon according to claim 1 waves cultivating system, it is characterised in that the system
It can make dental pulp mescenchymal stem cell not adherent growth, and three-dimensional globular stereochemical structure can be formed.
3. dental pulp mescenchymal stem cell microballoon according to claim 2 waves cultivating system, it is characterised in that the system
Include 1-10 horizontal positioned rocking troughs, multiple culture bottles or culture dish can be horizontally placed in the rocking trough.
4. the cultivating system of dental pulp mescenchymal stem cell microballoon according to claim 3, it is characterised in that the system can
The revolving speed kept constant, is waved in certain space, and the range of speeds is 50 ~ 300rpm.
5. the cultivating system of dental pulp mescenchymal stem cell microballoon according to claim 4, it is characterised in that the system can
It is compatible with cell incubator.
6. the preparation method of dental pulp mescenchymal stem cell microballoon according to claim 5 is inoculated in Tissue Culture Flask or thin
In born of the same parents' culture dish, the density of dental pulp mescenchymal stem cell is 4 × 104~1×106It is a.
7. the preparation system of dental pulp mescenchymal stem cell microballoon according to claim 6, by the 1st ~ 6 of conventional method culture the
It is passaged to new culture bottle or culture dish for dental pulp mescenchymal stem cell, these culture bottles or culture dish are put into dental pulp stem cell
In dimensional culture system, and this device is put into cell incubator, condition of culture is 37 DEG C of temperature, and CO2 concentration is 5%.
8. the preparation method of dental pulp mescenchymal stem cell microballoon according to claim 7, is expanded to directly to cell microspheres
When diameter is 40 ~ 200um, the cultivating system containing cell ball is removed, cell ball is collected by centrifugation using 300-800rpm.
9. the cell ball of collection is used 0.9% physiological saline by the stem cell sphere of three-dimensional configuration according to claim 8
Washing 3-6 times is carried out, the stem cell sphere of three-dimensional configuration is collected, is resuspended using 0.1-30 milliliters of volumes, for use.
10. three-dimensional stem cell sphere suspension according to claim 9, microsphere is sucked out using 0.5-3mm pin hole syringe,
It is injected into corresponding transplanting target tissue.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811085961.1A CN109251889A (en) | 2018-09-18 | 2018-09-18 | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon |
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| CN201811085961.1A CN109251889A (en) | 2018-09-18 | 2018-09-18 | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408592A (en) * | 2019-07-25 | 2019-11-05 | 四川大学 | For the regenerated multipotential stem cell MDPSCs of dental pulp and its isolated culture method and application |
| CN110684728A (en) * | 2019-11-13 | 2020-01-14 | 青岛奥克生物开发有限公司 | Preparation method of cell microspheres of adherent cells |
| CN112220966A (en) * | 2020-11-23 | 2021-01-15 | 北京大学口腔医学院 | Injection, preparation method of injection and application of injection in dental pulp regeneration |
| CN112294846A (en) * | 2020-10-22 | 2021-02-02 | 清华大学深圳国际研究生院 | Stem cell microsphere and application thereof |
| CN112442477A (en) * | 2019-09-04 | 2021-03-05 | 三顾股份有限公司 | High proliferative activity 3D structure cell sphere, manufacturing method and application thereof |
| CN112522192A (en) * | 2020-12-23 | 2021-03-19 | 重庆市博康生物工程技术有限公司 | Separation culture method of dental pulp mesenchymal stem cells |
| CN113201525A (en) * | 2021-04-12 | 2021-08-03 | 清华大学深圳国际研究生院 | Stem cell microsphere group, stem cell in-vitro amplification method and application |
| CN113621570A (en) * | 2021-08-26 | 2021-11-09 | 浙江大学 | Tissue engineering dental pulp based on digital optical processing printing technology and preparation method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408592A (en) * | 2019-07-25 | 2019-11-05 | 四川大学 | For the regenerated multipotential stem cell MDPSCs of dental pulp and its isolated culture method and application |
| CN112442477A (en) * | 2019-09-04 | 2021-03-05 | 三顾股份有限公司 | High proliferative activity 3D structure cell sphere, manufacturing method and application thereof |
| CN110684728A (en) * | 2019-11-13 | 2020-01-14 | 青岛奥克生物开发有限公司 | Preparation method of cell microspheres of adherent cells |
| CN112294846A (en) * | 2020-10-22 | 2021-02-02 | 清华大学深圳国际研究生院 | Stem cell microsphere and application thereof |
| CN112294846B (en) * | 2020-10-22 | 2023-03-21 | 清华大学深圳国际研究生院 | Stem cell microsphere and application thereof |
| CN112220966A (en) * | 2020-11-23 | 2021-01-15 | 北京大学口腔医学院 | Injection, preparation method of injection and application of injection in dental pulp regeneration |
| CN112522192A (en) * | 2020-12-23 | 2021-03-19 | 重庆市博康生物工程技术有限公司 | Separation culture method of dental pulp mesenchymal stem cells |
| CN112522192B (en) * | 2020-12-23 | 2022-11-29 | 重庆市博康生物工程技术有限公司 | Separation culture method of dental pulp mesenchymal stem cells |
| CN113201525A (en) * | 2021-04-12 | 2021-08-03 | 清华大学深圳国际研究生院 | Stem cell microsphere group, stem cell in-vitro amplification method and application |
| CN113201525B (en) * | 2021-04-12 | 2023-03-21 | 清华大学深圳国际研究生院 | Stem cell microsphere group, stem cell in-vitro amplification method and application |
| CN113621570A (en) * | 2021-08-26 | 2021-11-09 | 浙江大学 | Tissue engineering dental pulp based on digital optical processing printing technology and preparation method |
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