CN103013918A - Method for separating and subculturing primary neural stem cell of human embryo midbrain - Google Patents
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Abstract
The invention provides a method for separating and subculturing the primary neural stem cell of the human embryo midbrain. The method is used for separating, culturing and differentiating the primary neural stem cell of the human embryo midbrain in vitro by separating, culturing and identifying the neural stem cell of the human embryo midbrain. The method comprises the following steps: separating and culturing the primary neural stem cell of the human embryo midbrain, subculturing the primary cell, carrying out induced differentiation on the cell, carrying out immunocytochemical staining, and counting the number of cells. According to the method, a neural stem cell is separated from human embryo midbrain successfully by adopting the serum-free culturing and single-cell cloning technology, and the neural stem cell is cultured, subcultured and subjected to adherent differentiation observation, wherein the cell has the continuous cloning capacity and can subculture and express the neural nidogen antigen. The differentiated cell can express the specific antigens of the neuron cells, the colloid cells and oligodendroglia cells.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for separating and subculturing primary cells of human embryonic mesencephalic neural stem cells, which is applied to the in-vitro separation culture and differentiation process of the human embryonic mesencephalic neural stem cells.
Background
The discovery of the neural stem cells is a major breakthrough in the field of neuroscience, and opens up a wide prospect for the research fields of neural development, neural tissue transplantation and the like. A neural stem cell is a cell with self-renewing and extensive differentiation potential, which is in a non-terminal state of differentiation and can eventually give rise to three major cells of the central nervous system, i.e., neuronal cells, astrocytes and oligodendrocytes, by dividing new stem cells or daughter cells with gradually diminishing differentiation potential, either symmetrically or asymmetrically. It is believed that neural stem cells are mainly present in striatum, cerebellar hemisphere, ventricular region, etc. of fetal brain of mammals, and lateral ventricular wall and hippocampus, etc. region of adult mammalian brain. The existence of neural stem cells in particular brain regions in mammalian embryonic stages and adult animals has been continuously demonstrated, but studies on human neural stem cells have been poor. The experiment adopts serum-free culture and single cell cloning technology to try to separate the neural stem cells with self-renewal and multi-differentiation potential from human embryo midbrain, and adopts immunofluorescence staining to identify.
Disclosure of Invention
In order to obtain a large number of stable neural stem cells, the invention provides a method for separating and subculturing primary cells of human embryonic mesencephalic neural stem cells, which is applied to the processes of in vitro separation culture and differentiation of the human embryonic mesencephalic neural stem cells. The in vitro separation culture and differentiation process of the human embryo midbrain neural stem cells is to separate, culture and identify the neural stem cells from the human embryo midbrain. The method comprises the steps of separating cells with single cell cloning capacity in a human embryonic cortex by utilizing serum-free culture and single cell cloning technologies, carrying out culture, passage and adherent differentiation observation, and detecting the expression of a neuronestin antigen of the cloned cells and a specific mature nerve cell antigen after differentiation by adopting indirect immunofluorescence.
Specifically, the method for separating and subculturing the primary cells of the human embryonic mesencephalic neural stem cells comprises the following steps of: the method comprises the following steps of (1) separating and culturing primary midbrain neural stem cells, subculturing the primary midbrain neural stem cells, inducing and differentiating the cells, chemically dyeing immunocytes and counting the cells; the method comprises the following steps of: taking a fresh human embryo induced by water sac with the embryo age of 10-13 weeks, aseptically separating out an embryo midbrain tissue under a microscope, stripping off meninges and surface blood vessels, rinsing the embryo midbrain tissue three times in PBS (phosphate buffer solution), repeatedly blowing and beating the tissue fragments by using a fine suction tube until the tissue fragments are completely dispersed, blowing and beating the tissue fragments after centrifugal washing to prepare a cell suspension, filtering the cell suspension by using a 100-mesh stainless steel filter screen to prepare a single cell suspension, adding a DMEM/F12 serum-free culture medium containing B27, EGF and bFGF, staining and counting live cells by trypan blue, and adjusting the concentration of the live cells to be 1 multiplied by 105Perml, primary cells were plated in 25 cm square flasks at 37 ℃ with 5% CO2Standing and culturing under the condition;
the subculture of the primary cells comprises the following specific steps: digesting the primary clone for 15-30 minutes at 37 ℃ by using 0.25% pancreatin or preparing a single cell suspension by adopting a mechanical cutting separation or blowing beating mode after the primary clone is formed, subculturing the cell suspension into a culture bottle by using a serum-free culture medium containing B27, EGF and bFGF according to the concentration of 1 multiplied by 106/ml, and carrying out 5% CO inoculation at 37 ℃ to obtain a single cell suspension2Standing and culturing under the condition until the generation of secondary clone balls; carrying out passage once every 5-7 days on average;
the steps of inducing the cells to differentiate are as follows: after being cultured in vitro for 7 days, neurospheres of different generations are planted into a culture dish containing a fetal bovine serum coated by polylysine, and immunocytochemical staining is carried out for 3 hours, 10 days and 14 days in vitro.
The step of immunocytochemical staining comprises:
detecting the expression of the nestin in the neurosphere just adhered to the wall for 3 hours by an indirect immunofluorescence method, and detecting the expression of the neurofilament, glial fibrillary acidic protein and galactocerebroside in the differentiated cells 14 days after the neurosphere is differentiated in vitro;
detecting the expression of tyrosine hydroxylase in differentiated cells by using an SABC histochemical staining kit after the neurosphere is differentiated for 10 days;
the cell counting step specifically comprises: 4 independent fields of view are randomly selected for differentiated cells under an inverted microscope of a cell sample after chemical staining to count the number of immune cells and total cells.
Drawings
The foregoing and other objects, features, and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings. Wherein:
FIG. 1 is a schematic flow chart showing the steps of the method for isolating and subculturing primary fine-line of human embryonic mesencephalic neural stem cells of the present invention.
Detailed Description
FIG. 1 is a schematic flow chart of the steps of the method for isolated subculturing primary cells of human embryonic mesencephalic neural stem cells, which is specifically realized by the following steps:
s1: separating and culturing primary midbrain neural stem cells: taking a fresh human embryo induced by a water sac with the embryo age of 10-13 weeks, aseptically separating out an embryo midbrain tissue under a microscope, stripping off a meninges and a surface blood vessel, rinsing the embryo midbrain tissue three times in PBS (phosphate buffer solution), repeatedly blowing and beating the tissue fragments by using a fine suction tube until the tissue fragments are completely dispersed, blowing and beating the tissue fragments after centrifugal washing to prepare a cell suspension, filtering the cell suspension by using a 100-mesh stainless steel filter screen to prepare a single cell suspension, and adding B27 (human leukocyte antigen) containing 1: 50, EGF (epidermal growth factor) containing 20ng/ml and bFGF (fibroblast growth factor) containing 20ng/mlLong factor) DMEM/F12 (1: 1) serum-free medium, trypan blue staining to count viable cells, adjusting the viable cell concentration to 1X 105Perml, the primary cells were plated in 25 cm square flasks, or 8ml cell suspensions, at 37 ℃ in 5% CO2And (5) carrying out static culture under the condition.
S2: subculturing primary cells: digesting the primary clone for 15-30 minutes at 37 ℃ by using 0.25% pancreatin or preparing a single cell suspension by adopting a mechanical cutting separation or blowing beating mode after the primary clone is formed, subculturing the cell suspension into a culture bottle by using a serum-free culture medium containing B27, EGF and bFGF according to the concentration of 1 multiplied by 106/ml, and carrying out 5% CO inoculation at 37 ℃ to obtain a single cell suspension2Standing and culturing under the condition until the generation of secondary clone balls; and carrying out passage once every 5-7 days on average.
S3: cell induction differentiation: after being cultured in vitro for 7 days, neurospheres of different generations are planted in a culture dish containing a polylysine coated fetal bovine serum (DMEM/F12+ B27+ 10% FBS), and immunocytochemical staining is carried out for 3 hours, 10 days and 14 days in vitro.
S4: immunocytochemical staining, in particular
Detecting the expression of Nestin (Nestin) in a neurosphere just adhered to the neurosphere for 3 hours by an indirect immunofluorescence method, and detecting the expression of neurofilament (NF neurofilament), Glial Fibrillary Acidic Protein (GFAP) and Galc (galactocerebroside) in differentiated cells 14 days after the neurosphere is differentiated in vitro; the primary antibody used was: anti-NestinIgG, anti-NF IgG, anti-GFAP IgG and anti-Galc IgG, wherein the secondary antibody is a FITC fluorescent secondary antibody;
expression of Tyrosine Hydroxylase (TH) in differentiated cells 10 days after neurosphere differentiation was examined using SABC (Strept Avidin-Biotin Complex, a convenient and sensitive immunohistochemical staining method showing antigen distribution in tissues and cells) histochemical staining kit. SABC method cell staining the specific staining procedure is described with reference to staining kit.
S5: cell counting: immunocytochemical staining TH (+) cells represent dopaminergic neurons, hematoxylin counterstaining, blue cell nuclei number represents total cell number. 4 independent fields of view were randomly selected for differentiated cells under an inverted microscope (20 ×) to count TH (+) cells and total cell number.
The experimental results and analysis are as follows:
first, primary cells of human embryo midbrain are round and spherical, grow in suspension, more than 80% of living cells are counted by trypan blue staining, the cell number is obviously increased after 7 days, and a plurality of cell masses growing in suspension are visible in a culture bottle to form a sphere, which is called a neurosphere. Such neurosphere-external NESTIN stains strongly positive and can differentiate into NF (+), GFAP (+) and Galc (+) cells in vitro.
Secondly, after the primary neurospheres are digested by 0.25% of pancreatin at 37 ℃, cell suspension is formed, 10% -30% of cells die, and after 2-3 days, a large number of new glomeruli appear in the culture medium and grow like mulberry, and gradually increase to form secondary neurospheres.
And thirdly, the in vitro differentiation of the human embryonic mesencephalic neural stem cells to generate TH (+) cells is gradually reduced along with the increase of the passage times. In primary neural stem cells (P)0) After differentiation for 10 days, immunocytochemical staining showed that TH (+) cells accounted for about 5.65% + -1.39% of the total cells, and P was obtained by passage after 7 days of in vitro culture1The generation of neural stem cells, immunocytochemical staining showed that the proportion of TH (+) cells in the total cells was reduced to 1.56% + -0.37% after 10 days of differentiation. As the culture time and the number of passages in vitro increased, the TH (+) cells also declined rapidly, with less than 0.01% of TH (+) cells after the third generation.
The present invention is not limited to the embodiments described above, and those skilled in the art may make modifications or changes within the scope of the disclosure without departing from the spirit of the present invention, so that the scope of the present invention is defined by the appended claims.
Claims (1)
1. A method for separating and subculturing primary cells of human embryonic mesencephalic neural stem cells, which is characterized by comprising the following steps: the method comprises the following steps of (1) separating and culturing primary midbrain neural stem cells, subculturing the primary midbrain neural stem cells, inducing and differentiating the cells, chemically dyeing immunocytes and counting the cells; wherein,
the steps of the isolation and culture of the primary mesencephalon neural stem cells are specifically as follows: taking a fresh human embryo induced by a water sac with the embryo age of 10-13 weeks, aseptically separating out the midbrain tissue of the embryo under a microscope, stripping off the meninges and the surface blood vessels, rinsing in PBS for three timesRepeatedly blowing and beating the tissue fragments by using a fine suction pipe until the tissue fragments are completely dispersed, centrifugally washing, blowing and beating to prepare a cell suspension, filtering by using a 100-mesh stainless steel filter screen to prepare a single cell suspension, adding a DMEM/F12 serum-free culture medium containing B27, EGF and bFGF, staining and counting living cells by trypan blue, and adjusting the concentration of the living cells to be 1 multiplied by 105Perml, primary cells were plated in 25 cm square flasks at 37 ℃ with 5% CO2Standing and culturing under the condition;
the subculture of the primary cells comprises the following specific steps: digesting the primary clone for 15-30 minutes at 37 ℃ by using 0.25% pancreatin or preparing single cell suspension by adopting a mechanical cutting separation or blowing beating mode after the primary clone is formed, subculturing the cell suspension into a culture bottle by using a serum-free culture medium containing B27, EGF and bFGF according to the concentration of 1 multiplied by 106/ml, and carrying out 5% CO inoculation at 37 ℃2Standing and culturing under the condition until the generation of secondary clone balls; carrying out passage once every 5-7 days on average;
the steps of inducing the cells to differentiate are as follows: after being cultured in vitro for 7 days, neurospheres of different generations are planted into a culture dish containing a fetal calf serum coated by polylysine, and immunocytochemical staining is carried out for 3 hours, 10 days and 14 days in vitro respectively;
the step of immunocytochemical staining comprises: detecting the expression of the nestin in the neurosphere just adhered to the wall for 3 hours by an indirect immunofluorescence method, and detecting the expression of the neurofilament, glial fibrillary acidic protein and galactocerebroside in the differentiated cells 14 days after the neurosphere is differentiated in vitro; and detecting the expression of tyrosine hydroxylase in the differentiated cells by using an SABC histochemical staining kit after the neurosphere is differentiated for 10 days;
the cell counting step specifically comprises: 4 independent fields of view are randomly selected for differentiated cells under an inverted microscope of a cell sample after chemical staining to count the number of immune cells and total cells.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305466A (en) * | 2013-06-09 | 2013-09-18 | 吉林省拓华生物科技有限公司 | Culture method capable of keeping high cell survival rate for neural stem cell |
| CN111411081A (en) * | 2019-01-07 | 2020-07-14 | 北京银丰鼎诚生物工程技术有限公司 | Construction method of human neural stem cell bank |
| CN111808813A (en) * | 2020-08-04 | 2020-10-23 | 西安同袍企业管理集团有限公司 | Material-taking culture method of neural stem cells |
| CN113913383A (en) * | 2021-10-08 | 2022-01-11 | 山东万海生物科技有限公司 | In-vitro culture medium and culture method for embryonic neural stem cells |
-
2012
- 2012-11-30 CN CN 201210505705 patent/CN103013918A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305466A (en) * | 2013-06-09 | 2013-09-18 | 吉林省拓华生物科技有限公司 | Culture method capable of keeping high cell survival rate for neural stem cell |
| CN103305466B (en) * | 2013-06-09 | 2015-02-11 | 吉林省拓华生物科技有限公司 | Culture method capable of keeping high cell survival rate for neural stem cell |
| CN111411081A (en) * | 2019-01-07 | 2020-07-14 | 北京银丰鼎诚生物工程技术有限公司 | Construction method of human neural stem cell bank |
| CN111808813A (en) * | 2020-08-04 | 2020-10-23 | 西安同袍企业管理集团有限公司 | Material-taking culture method of neural stem cells |
| CN113913383A (en) * | 2021-10-08 | 2022-01-11 | 山东万海生物科技有限公司 | In-vitro culture medium and culture method for embryonic neural stem cells |
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