CN100494359C - Method for in vitro amplifying and in 3D solid culturing for nerve stem cell - Google Patents
Method for in vitro amplifying and in 3D solid culturing for nerve stem cell Download PDFInfo
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- CN100494359C CN100494359C CNB200610090027XA CN200610090027A CN100494359C CN 100494359 C CN100494359 C CN 100494359C CN B200610090027X A CNB200610090027X A CN B200610090027XA CN 200610090027 A CN200610090027 A CN 200610090027A CN 100494359 C CN100494359 C CN 100494359C
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Abstract
This invention relates to a method for amplifying neural stem cells in vitro by 3-dimensional culture. The method comprises: selecting microcarrier with 3-dimensional environment, pre-treating, coating the microcarrier with 40-60 ng/mL alkaline fibroblast growth factor, 40-60 ng/mL epidermal growth factor, and B27 DMEM/F12 neural stem cell serum-free culture medium, adding 1X105-1X106 neural stem cells into the culture bottle, taking out the microcarrier grown with neural stem cells, removing the microcarrier, and rinsing cells to obtain neural stem cells. The porous microcarrier can enlarge the culture area. The alkaline fibroblast growth factor and epidermal growth factor can promote cell multiple fission and improve cell microenvironment, which is advantageous for multiple fission of neural stem cells.
Description
Technical field
The present invention relates to a kind of cultural method of neural stem cell, particularly related to the method that a kind of neural stem cell 3 D stereo is cultivated amplification in vitro.
Background technology
For a long time, utilize Culture of neural stem cells liquid and cell clone technology, perfect substantially from embryo or tire brain separation and purification of nerve stem cells technology, but external long-term cultivation neural stem cell amplification in vitro problem is still unresolved, adopt the quantity of the neural stem cell that traditional cultural method not only turns out few, and surviving rate is low, is difficult to satisfy Clinical Application.
Summary of the invention
The object of the invention provides the method that a kind of neural stem cell 3 D stereo is cultivated amplification in vitro.
In order to realize this goal of the invention, the invention provides the method that a kind of neural stem cell 3 D stereo is cultivated amplification in vitro, it comprises: the porous microcarrier of selecting to have three-dimensional environment; Microcarrier is carried out pre-treatment, and wherein: it is further comprising the steps of:
(1) the DMEM/F12 neural stem cell serum-free medium bag of Prostatropin (bFGF), 40-60ng/ml epithelical cell growth factor (EGF) and B27 that will contain 40-60ng/ml is made to promote fissional Prostatropin and epithelical cell growth factor to permeate equably in microcarrier by above-mentioned microcarrier;
(2) the above-mentioned microcarrier of handling well is put into Tissue Culture Flask, add above-mentioned neural stem cell serum-free medium, add 1 * 10 at culturing bottle then
5-1 * 10
6Individual neural stem cell after mixing, charges into the carbon dioxide that contains 5% concentration again, and cultivates under the constant temperature about 37 ℃, according to the propagation situation of cell, and per 5-7 days replacing neural stem cell serum-free mediums;
(3) microcarrier that will cover with neural stem cell takes out from culturing bottle, puts into to contain 0.05% tryptic D-hank ' s damping fluid, at room temperature digests 10-30 minutes, removes microcarrier, and the rinsing cell obtains neural stem cell;
The invention provides a kind of neural stem cell 3 D stereo and cultivate the method for amplification in vitro, wherein: the pre-treatment of described microcarrier is 1 part microcarrier to be put into 100 parts PBS damping fluid, at room temperature soaked 20-40 minutes, with often putting upside down microcarrier, microcarrier is fully contacted with damping fluid, then under the pressure about temperature about 120 ℃ and 0.11 MPa, with above-mentioned microcarrier sterilization 15-30 minutes, again above-mentioned microcarrier is used PBS damping fluid washed twice, after serum-free medium washing once, above-mentioned microcarrier is placed refrigerator overnight about 4 ℃;
The invention provides a kind of neural stem cell 3 D stereo and cultivate the method for amplification in vitro, wherein: the pH value of described PBS damping fluid is about 7.4.
The present invention compares with traditional cultural method, the present invention is on the basis of traditional neural stem cell vitro culture technology, use has the porous microcarrier of three-dimensional environment, help the enlarged culturing area like this, and adopt and contain 40～60ng/ml Prostatropin, the DMEM/F12 neural stem cell serum-free medium bag of 40～60ng/ml epithelical cell growth factor and B27 is by microcarrier, can make and promote cell proliferation splitted Prostatropin and epithelical cell growth factor uniformly penetrating in microcarrier, improve the microenvironment of cells survival, help the cell proliferation of nerve cord division, reach the purpose of neural stem cell amplification in vitro.
Description of drawings
Fig. 1 is the micro-enlarged view of the microcarrier of not inoculating cell;
Fig. 2 is the micro-enlarged view of the microcarrier of inoculation neural stem cell;
Fig. 3 is the outside micro-enlarged view of the neural stem cell of propagation in the microcarrier.
Embodiment
The method that neural stem cell 3 D stereo of the present invention is cultivated amplification in vitro comprises:
Selection has the porous microcarrier of three-dimensional environment, for example: the Cytopore that selects Sweden Amersham Biosciences company
TM2 microcarriers;
Microcarrier is carried out pre-treatment, as: the PBS damping fluid of the microcarrier of 1g being put into 100ml, at room temperature soaked 30 minutes, with often putting upside down microcarrier, microcarrier is fully contacted, then under the pressure about temperature about 120 ℃ and 0.11 MPa with damping fluid, with above-mentioned microcarrier sterilization 20 minutes, be about 7.4 PBS damping fluid washed twice again with above-mentioned microcarrier pH value, after serum-free medium washing once, above-mentioned microcarrier placed refrigerator overnight about 4 ℃;
The DMEM/F12 neural stem cell serum-free medium bag of Prostatropin (bFGF), 45ng/ml epithelical cell growth factor (EGF) and B27 (trade name) that will contain 50ng/ml is made to promote fissional Prostatropin and epithelical cell growth factor to permeate equably in microcarrier by above-mentioned microcarrier; The consumption of B27 (trade name) can add according to the introduction of product description.
The above-mentioned microcarrier of handling well is put into Tissue Culture Flask, add above-mentioned neural stem cell serum-free medium, add 1 * 10 at culturing bottle then
5-1 * 10
6Individual neural stem cell after mixing, charges into the carbon dioxide that contains 5% concentration again, and cultivates under the constant temperature about 37 ℃, according to the propagation situation of cell, and per 5-7 days replacing neural stem cell serum-free mediums;
The microcarrier that covers with neural stem cell is taken out from culturing bottle, put into and contain 0.05% tryptic D-hank ' s damping fluid, at room temperature digested 20 minutes, remove microcarrier, the rinsing cell obtains neural stem cell.
Fig. 1 and Fig. 2 are respectively the microcarrier of inoculating cell not and have inoculated the micro-enlarged view of the microcarrier of neural stem cell, from the contrast of these two figure, and the neural stem cell state of in microcarrier, growing as can be seen.From Fig. 3, can more be clear that neural stem cell outside state of propagation in the microcarrier.
More than describing is explanation of the invention, is not that institute of the present invention restricted portion is referring to claim to the qualification of invention, and under the situation of spirit of the present invention, the present invention can do any type of modification.
Claims (3)
1. a neural stem cell 3 D stereo is cultivated the method for amplification in vitro, and it comprises: the porous microcarrier of selecting to have three-dimensional environment; Microcarrier is carried out pre-treatment, it is characterized in that: it is further comprising the steps of:
(1) the DMEM/F12 neural stem cell serum-free medium bag that will contain Prostatropin (bFGF), the 40-60ng/ml epithelical cell growth factor (EGF) of 40-60ng/ml and add B27 is made to promote fissional Prostatropin and epithelical cell growth factor to permeate equably in microcarrier by above-mentioned microcarrier;
(2) the above-mentioned microcarrier of handling well is put into Tissue Culture Flask, add above-mentioned neural stem cell serum-free medium, add 1 * 10 at culturing bottle then
5-1 * 10
6Individual neural stem cell after mixing, charges into the carbon dioxide that contains 5% concentration again, and cultivates under 37 ℃ constant temperature, according to the propagation situation of cell, and per 5-7 days replacing neural stem cell serum-free mediums;
(3) microcarrier that will cover with neural stem cell takes out from culturing bottle, puts into to contain 0.05% tryptic D-hank ' s damping fluid, at room temperature digests 10-30 minutes, removes microcarrier, and the rinsing cell obtains neural stem cell.
2. neural stem cell 3 D stereo as claimed in claim 1 is cultivated the method for amplification in vitro, it is characterized in that: the pre-treatment of described microcarrier is 1 part microcarrier to be put into 100 parts PBS damping fluid, at room temperature soaked 20-40 minutes, with often putting upside down microcarrier, microcarrier is fully contacted with damping fluid, then under the pressure of 120 ℃ temperature and 0.11 MPa, with above-mentioned microcarrier sterilization 15-30 minutes, again above-mentioned microcarrier is used PBS damping fluid washed twice, after serum-free medium washing once, above-mentioned microcarrier placed 4 ℃ refrigerator overnight.
3. the described neural stem cell 3 D stereo of claim 2 is cultivated the method for amplification in vitro, and it is characterized in that: the pH value of described PBS damping fluid is 7.4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNB200610090027XA CN100494359C (en) | 2006-06-23 | 2006-06-23 | Method for in vitro amplifying and in 3D solid culturing for nerve stem cell |
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| CNB200610090027XA CN100494359C (en) | 2006-06-23 | 2006-06-23 | Method for in vitro amplifying and in 3D solid culturing for nerve stem cell |
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| CN101092606A CN101092606A (en) | 2007-12-26 |
| CN100494359C true CN100494359C (en) | 2009-06-03 |
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