CN113913383A - In-vitro culture medium and culture method for embryonic neural stem cells - Google Patents
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Abstract
The invention discloses an in vitro culture medium and a culture method for embryonic neural stem cells, wherein the in vitro culture medium for the embryonic neural stem cells comprises a serum-free culture medium, EGF and bFGF, and the concentration of the EGF and the bFGF in the in vitro culture medium for the embryonic neural stem cells are 15-25ng and 5-15ng respectively. The in vitro culture method of the embryonic neural stem cells comprises the following steps of S1, inoculating the embryonic neural stem cells into an in vitro culture medium of the embryonic neural stem cells, and adding CO2Culturing in a cell culture box at 5 × 105Placing each 5ml of the culture solution in a T25 culture flask; and S2, supplementing liquid into the T25 culture bottle at intervals of 2-3 days, shaking and then continuing culturing. The embryonic neural stem cell in-vitro culture medium and the culture method can well perform in-vitro amplification and purification on the neural stem cells by culturing the neural stem cells by using the cell stimulating factors and the serum-free culture medium.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an embryonic neural stem cell in-vitro culture medium and a culture method.
Background
The neural stem cell is an adult stem cell derived from the central nervous system and has self-renewal capacity for life; also pluripotent stem cells, can be induced to differentiate into various types of mature neural cells, such as neurons, astrocytes and oligodendrocytes. The developing and mature central nervous system is distributed with neural stem cells. The neural stem cells are in the stage of rapid growth and development in the embryonic stage, and the parts of the neural stem cells in the embryonic stage mainly comprise cerebral cortex, striatum, hippocampus, olfactory bulb, cerebellum, spinal cord and other regions. The neural stem cells with proliferation and differentiation capacity still exist in the areas of the lateral ventricle wall, the hippocampus and the like of the adult brain. Neural stem cells in the lower hippocampus, lateral subventricular zone, etc. of the brain tissue of adult mammals can activate neural stem cells in a resting state or inactivate inhibitory factors under certain stimulatory factors. The second aspect is the retrograde differentiation of mature neural cells. Under the action of epidermal growth factor and fibroblast growth factor, the neural stem cells can maintain an undifferentiated state and proliferate in a large amount.
Currently, the criteria for identifying neural stem cells are the following: firstly, nerve cells can be observed to grow into clusters under a light microscope to form suspended nerve spheres; secondly, smearing a single cell suspension, and detecting that the Nestin expression is positive by an immunofluorescence staining method; thirdly, neural stem cells have the potential to differentiate into neurons and glial cells. And (3) morphological observation: the primary culture is generally carried out for about 7 days, most of neural stem cells grow into balls, and the neural balls with different sizes can be seen to float in a culture medium under a microscope. Neurospheres are characteristic growth characteristics of neural stem cells, and neurospheres have been observed to be one of the essential conditions for identifying neural stem cells. Detecting the surface specific molecular markers of the neural stem cells: nestin is highly expressed from generation 1 to generation 10. From the 3 rd generation, the expression rate of the cell marker tends to be uniform, and the proliferative capacity of the cell tends to be stable. High expression of CD29 and CD90 from generation 3 to generation 9, and low expression of CD45 and CD 34. Induced differentiation experiment: the neural stem cells have the capacity of multi-directional differentiation, but the differentiation direction is neural tissue cells including neurons and glial cells. After being induced by a culture medium containing serum, the neural stem cells can be differentiated into neurons and glial cells. Immunofluorescence results show that the neuron cell highly expresses MAP2, and the glial cell highly expresses APC.
The stem cells play an important role in nervous system diseases, and firstly, the damaged nerve cells are replaced in a targeted mode and nerve connection is reconstructed by planting the nerve stem cells; secondly, nerve pathway remodeling: by activating endogenous neural progenitor cells, the endogenous neural progenitor cells are proliferated in a large quantity and migrate to damage focuses, various cell types including neurons, astrocytes and oligodendrocytes are generated, the neural connection of the brain is recovered, and the functional recovery is promoted; thirdly, angiogenesis: maintenance of neural function is dependent on local blood flow, and thus promotion of angiogenesis is a key factor in the treatment of neurological diseases; fourthly, inhibiting inflammation: relieving related inflammatory reaction, and protecting nerve cells; fifthly, secretion of trophic factors: through secreting trophic factors, nerve cell apoptosis is reduced. Indications are as follows: senile dementia, Parkinson, cerebral palsy, apoplexy, spinal cord injury and other nervous system diseases.
The application approach of the neural stem cells is mainly to perform lumbar puncture subarachnoid injection in principle, and patients with operation indications can perform local in-situ transplantation and targeted site-specific transplantation at the same time. However, the culture of the neural stem cells has the following difficulties that the neural stem cells grow slowly, the nutrition requirement is high, the inoculation density is preferably increased during the culture, and the balling growth of the neural stem cells is facilitated. The primary culture is generally carried out for about 7 days, and most of neural stem cells grow into balls. The growth of the neural stem cells is slow in the culture process, the culture period is long, the conditions are strict, and the number of passages is generally 1: 2. Secondly, materials are taken during primary culture, the pia mater is required to be stripped off, and because the pia mater is rich in blood vessels, if the pia mater is mixed into brain tissues, certain components in the blood can induce the brain tissues to be differentiated into mature neurons or glial cells. Thirdly, the content of the neural stem cells in brain tissues is extremely low, and the number of the neural stem cells is smaller and the capability of multidirectional differentiation is smaller along with the increase of gestational age. The growth speed of the neural stem cells is slow, the adaptability is poor, and the inoculation density should be selected to be higher during primary culture and subculture, so that a better effect can be obtained.
In the prior art, the brain tissue of 8-12 weeks old aborted fetuses is mostly taken, and is centrifuged and layered by utilizing different cell specific gravities through rotating speed centrifugation, so that the purification purpose is achieved, but the method has obvious defects, low purification efficiency and small collection quantity; and the existing culture medium and culture method can not well culture the embryonic neural stem cells in vitro.
Disclosure of Invention
Therefore, the invention provides an in vitro culture medium and a culture method for embryonic neural stem cells, and aims to solve the problems that in the prior art, the neural stem cells are low in culture and purification efficiency and cannot be well cultured in vitro due to simple rotating speed centrifugation.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to a first aspect of the invention, the invention provides an embryonic neural stem cell in-vitro culture medium, which comprises a serum-free culture medium, EGF and bFGF, wherein the concentration of the EGF in the embryonic neural stem cell in-vitro culture medium is 15-25ng, and the concentration of the bFGF in the embryonic neural stem cell in-vitro culture medium is 5-15 ng.
Further, the serum-free medium is DMEM/F12.
Further, in the in vitro culture medium of the embryonic neural stem cells, the concentration of EGF is 20ng, and the concentration of bFGF is 10 ng.
According to a second aspect of the present invention, the present invention provides an in vitro culture method of embryonic neural stem cells based on the in vitro culture medium of embryonic neural stem cells, comprising the following steps:
s1, inoculating embryonic neural stem cells into an in-vitro culture medium of the embryonic neural stem cells, and adding CO2Culturing in a cell culture box at 5 × 105Placing each 5ml of the culture solution in a T25 culture flask;
and S2, supplementing liquid into the T25 culture bottle at intervals of 2-3 days, shaking and then continuing culturing.
Further, the in vitro culture method of the embryonic neural stem cells further comprises the following steps before the step S1:
taking a human embryo with the gestational age of 6-10 weeks of abortion, separating tissues from the fetal cerebral cortex, and sequentially carrying out trypsin digestion and mechanical blow beating on the tissues to obtain the embryonic neural stem cells in the form of single cell suspension.
Further, in step S1The temperature of the culture and the culture in the step S2 is 36.5-37.5 ℃, and CO is2The concentration of (A) is 4.5-5.5%.
Further, the temperature of the cultivation in step S1 and the cultivation in step S2 was 37 ℃ CO2The concentration of (2) is 5%.
Further, in performing step S2, from the CO2And taking out the T25 culture bottle from the cell culture box and placing the T25 culture bottle in a super clean bench for the fluid replacement.
Further, the total time of the culturing in step S1 and the culturing in step S2 is 7 to 10 days.
The invention has the following advantages:
the in vitro culture medium of the embryonic neural stem cells comprises a serum-free culture medium, EGF and bFGF, wherein the EGF can promote a single neural stem cell to generate microspheres in vitro, and the bFGF can promote the proliferation of forebrain stem cells and can induce the proliferation of neuroblasts and neuron/astrocyte precursors; the culture method of the invention utilizes the cell stimulating factor and the serum-free culture medium to culture the neural stem cells, and can well perform in-vitro amplification and purification on the neural stem cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so that those skilled in the art can understand and read the present invention, and do not limit the conditions for implementing the present invention, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the functions and purposes of the present invention, should still fall within the scope of the present invention.
FIGS. 1 to 5 are optical inverted microscope photographs of embryonic neural stem cells cultured according to example 10 of the present invention, which were cultured up to day 5, day 8, day 13, day 15 and day 18, respectively;
FIGS. 6 to 10 show the flow cytometry results of the embryonic neural stem cells cultured in example 10 of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides an in vitro embryonic neural stem cell culture medium, which comprises a serum-free culture medium, EGF and bFGF, wherein the concentration of the EGF in the in vitro embryonic neural stem cell culture medium is 15-25ng, and the concentration of the bFGF in the in vitro embryonic neural stem cell culture medium is 5-15 ng.
The in vitro culture medium of the embryonic neural stem cells comprises a serum-free culture medium, EGF and bFGF, wherein the serum-free culture medium can avoid immune reaction caused by serum protein; the recombinant factors (EGF and bFGF) can stimulate the proliferation of the neural stem cells, so that the neural stem cells can grow in an adherent manner in the proliferation process, other impurities or other cells can be removed better, and the obtained neural stem cells are purer.
The in-vitro culture medium of the embryonic neural stem cells, which is composed of the serum-free culture medium, the EGF and the bFGF, limits the concentration of the EGF to be 15-25ng and the concentration of the bFGF to be 5-15ng, and the in-vitro culture medium of the embryonic neural stem cells in the concentration range can well culture the embryonic neural stem cells in vitro and obtain good culture effect.
Example 2
The in vitro culture medium for the embryonic neural stem cells as described in example 1, wherein the serum-free culture medium is DMEM/F12.
Serum-free media and reagents are widely used to culture mammalian and invertebrate cells to produce monoclonal antibodies, viral antigens, recombinant proteins, and the like. Most serum-free media contain transferrin, which transports ions into cells, and insulin, which regulates the uptake of glucose, as well as some proteins and albumins, fibrin, fetuin, etc., which perform various functions in cell culture, such as providing a matrix for cell attachment, anti-bioreactor shear forces, as a carrier for lipids and other growth differentiation factors, etc.
There are many kinds of Serum-free media, including UltraCULTURETM medium, PC-1. sup. TM. medium, UltraMEM Reduced Serum medium, UltraCHOTM medium, and the like. In the in vitro culture medium for the embryonic neural stem cells, DMEM/F12 is taken as a serum-free culture medium to be compounded with EGF and bFGF to form the in vitro culture medium for the embryonic neural stem cells, and the in vitro culture medium for the embryonic neural stem cells compounded with DMEM/F12, EGF and bFGF can obtain a good culture effect when the embryonic neural stem cells are cultured in vitro.
Example 3
The in vitro embryonic neural stem cell culture medium according to example 1 or 2, wherein the concentration of EGF is 20ng and the concentration of bFGF is 10 ng.
The in vitro culture medium of the embryonic neural stem cells is a preferred in vitro culture medium of the embryonic neural stem cells, and particularly the culture medium consisting of DMEM/F12, EGF (with the concentration of 20ng) and bFGF (10ng) can obtain better culture effect when being used for in vitro culture of the embryonic neural stem cells. EGF, bFGF and DMEM/F12 are well synergistic to each other at the concentration ranges specified in this example.
Example 4
The present embodiment provides an in vitro culture method of embryonic neural stem cells based on the in vitro culture medium of embryonic neural stem cells according to any one of embodiments 1 to 3, comprising the following steps:
s1, inoculating embryonic neural stem cells to in-vitro culture of the embryonic neural stem cellsAdding CO into nutrient medium2Culturing in a cell culture box at 5 × 105Placing each 5ml of the culture solution in a T25 culture flask;
and S2, supplementing liquid into the T25 culture bottle at intervals of 2-3 days, shaking and then continuing culturing.
The culture method of this example was carried out by culturing the embryonic neural stem cells in vitro using the medium for in vitro culture of embryonic neural stem cells of examples 1 to 3, and the concentration of the embryonic neural stem cells was defined to be 5X 105The seed cell density is 5ml, and the embryonic neural stem cells can obtain good culture effect at the seed density.
Example 5
The in vitro culture method of embryonic neural stem cells according to embodiment 4, further comprising the following steps before step S1:
taking a human embryo with the gestational age of 6-10 weeks of abortion, separating tissues from the fetal cerebral cortex, and sequentially carrying out trypsin digestion and mechanical blow beating on the tissues to obtain the embryonic neural stem cells in the form of single cell suspension.
In the in vitro culture method of the embryonic neural stem cells, human embryos aborted for 6-10 weeks are taken, tissues are separated from fetal brain cortex, and the tissues obtained by separation are sequentially subjected to trypsin digestion and mechanical blowing, so that the embryonic neural stem cells in the form of single cell suspension are obtained; the proportion of the protoneural stem cells obtained by adopting trypsin digestion is higher, the purification efficiency is high, and the number of the collected embryonic neural stem cells is large.
Example 6
The in vitro culture method of embryonic neural stem cells according to example 4, wherein the temperature of the culture in step S1 and the temperature of the culture in step S2 are 36.5-37.5 ℃, and CO is added2The concentration of (A) is 4.5-5.5%. Temperature range and CO defined in this example2Within the concentration range, the embryonic neural stem cells can well grow and reproduce.
Example 7
The in vitro culture method of embryonic neural stem cells according to example 6, step S1The temperature of the culture in (1) and the culture in step S2 was 37 ℃ and CO was2The concentration of (2) is 5%.
Temperature and CO as defined in the example2Concentration is a more preferred condition, temperature and CO in this example2At the concentration, the embryonic neural stem cells can further better grow and reproduce.
Example 8
The method for in vitro culturing embryonic neural stem cells according to example 4, wherein the CO is used in step S22And taking out the T25 culture bottle from the cell culture box and placing the T25 culture bottle in a super clean bench for the fluid replacement. The in vitro culture medium of the embryonic neural stem cells defined in the above examples 1 to 3 was supplemented with the supplementary solution. The introduction of foreign matter to cause adverse effects can be avoided by performing the fluid replacement in the clean bench.
Example 9
The method for in vitro culturing embryonic neural stem cells according to example 4, wherein the total time of the culturing in step S1 and the culturing in step S2 is 7-10 days. The embryonic neural stem cells can obtain good growth results within the total time defined in the present example.
Example 10
Taking human embryo with 6-10 weeks gestational age of abortion, separating tissue from fetal cerebral cortex, and performing in vitro culture by using single cell suspension obtained by trypsin digestion and mechanical blow beating.
The cells were stimulated to proliferate by adding EGF at a final concentration of 20ng and bFGF at a final concentration of 10ng in serum-free medium (DMEM/F12), placed in a cell culture chamber, and incubated at 37 deg.C with 5% CO2Concentration, seeding density of viable cells 5X 1055ml/T25 culture bottles. Supplementing liquid according to the growth condition of cells and the appearance of the culture solution, if obvious yellowing is found, supplementing liquid, taking out a T25 culture bottle from a carbon dioxide incubator every 2-3 days, supplementing liquid in a super clean bench, and slightly shaking from different directions to uniformly distribute the cells. 37 ℃ and 5% CO2And (5) continuing culturing. Culturing the neural stem cells for 7-10 days, observing the growth state of the cells, photographing, and observing the cells under an optical inverted microscope (OLYMPUS) mirror to obtain the primary neural stem cells.
Test example 1
The cells cultured in example 10 were observed by an optical inverted microscope (OLYMPUS), and the optical inverted microscope photographs of the cells cultured up to day 5, day 8, day 13, day 15 and day 18 are shown in FIGS. 1 to 5, respectively.
As can be seen from the graphs in FIGS. 1-5, the growth state of the neural stem cells at the 5 th day can be seen that the neural stem cells begin to stick to the bottom of the culture bottle and change from a round shape to a strip shape, wherein the strip shape cells are the neural stem cells; the growth state of the neural stem cells at day 8, it can be seen that the neural stem cells have formed sheet-like colonies.
Test example 2
The cells cultured in example 10 were detected by flow cytometry on the surface markers CD73+, CD90+, CD105+, CD45+, CD34+, CD11b +, CD19+, HLA-DR +, and CD14+, and the flow reports obtained by detection are shown in fig. 6-10, and the expression rates are as follows: CD73 +: 99.46%, CD90 +: 99.81%, CD105 +: 87.89%, CD45 +: 0.2%, CD34 +: 0.0%, CD11b +: 0.0%, CD19 +: 0.0%, HLA-DR +: 0.0% CD14 +: 0.2 percent. The results of this test example show that it is possible to culture neural stem cells by the method of example 10.
As a result: cells isolated from human embryonic brain form large numbers of neural stem cell spheres in serum-free medium containing EGF and bFGF, which can be expanded and subcultured in vitro. And identifying most of the neural stem cell balls as Nestin expression positive cells by an immunocytochemistry and immunocytofluorescence method.
Cells positive for CD73, CD90, and CD105 expression can appear following adherent differentiation of these neural stem cell spheres. The fetal brain cells after being frozen can also culture neural stem cells with the same characteristics. The results of the flow cytometry and the cell growth curve show that the neural stem cells have stronger proliferation capacity under the condition of adding EGF and bFGF in serum-free culture in vitro.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. An in vitro embryonic neural stem cell culture medium, which is characterized by comprising a serum-free culture medium, EGF and bFGF, wherein the concentration of the EGF in the in vitro embryonic neural stem cell culture medium is 15-25ng, and the concentration of the bFGF in the in vitro embryonic neural stem cell culture medium is 5-15 ng.
2. The in vitro culture medium for the embryonic neural stem cells as claimed in claim 1, wherein the serum-free culture medium is DMEM/F12.
3. The in vitro embryonic neural stem cell culture medium of claim 1 or 2, wherein the concentration of EGF is 20ng and the concentration of bFGF is 10ng in the in vitro embryonic neural stem cell culture medium.
4. An in vitro culture method of embryonic neural stem cells based on the in vitro culture medium of embryonic neural stem cells according to any one of claims 1 to 3, comprising the following steps:
s1, inoculating embryonic neural stem cells into an in-vitro culture medium of the embryonic neural stem cells, and adding CO2Culturing in a cell culture box at 5 × 105Placing each 5ml of the culture solution in a T25 culture flask;
and S2, supplementing liquid into the T25 culture bottle at intervals of 2-3 days, shaking and then continuing culturing.
5. The in vitro culture method of embryonic neural stem cells according to claim 4, further comprising the following steps before step S1:
taking a human embryo with the gestational age of 6-10 weeks of abortion, separating tissues from the fetal cerebral cortex, and sequentially carrying out trypsin digestion and mechanical blow beating on the tissues to obtain the embryonic neural stem cells in the form of single cell suspension.
6. The method for in vitro culturing embryonic neural stem cells according to claim 4, wherein the temperature of the culturing in step S1 and the culturing in step S2 is 36.5-37.5 ℃, CO2The concentration of (A) is 4.5-5.5%.
7. The method for in vitro culturing embryonic neural stem cells according to claim 6, wherein the temperature of the culturing in step S1 and the culturing in step S2 is 37 ℃ and CO2The concentration of (2) is 5%.
8. The method for in vitro culturing embryonic neural stem cells according to claim 4, wherein step S2 is performed by using the CO2And taking out the T25 culture bottle from the cell culture box and placing the T25 culture bottle in a super clean bench for the fluid replacement.
9. The method for in vitro culturing embryonic neural stem cells according to claim 4, wherein the total time of the culturing in step S1 and the culturing in step S2 is 7 to 10 days.
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