CN108865997B

CN108865997B - Culture medium and culture method for in vitro culture of astrocytes

Info

Publication number: CN108865997B
Application number: CN201810569605.0A
Authority: CN
Inventors: 吴冈义; 张靖宜; 王今朝
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2017-12-27
Filing date: 2018-06-05
Publication date: 2022-02-11
Anticipated expiration: 2038-06-05
Also published as: CN108865997A

Abstract

The invention discloses a culture medium and a culture method for in vitro culture of astrocytes, and belongs to the field of cell culture. According to the invention, the epidermal growth factor, the fibroblast growth factor, SAG and Purmorphamine are added into the astrocyte culture medium, so that the in-vitro culture time of astrocytes can be obviously prolonged, the passage times are increased, the purity is improved, and the original characteristics of astrocytes can be maintained. The inventors found that the medium of the present invention can be used at alternate generations and maintained the astrocytes for passage to 13. Culturing until the purity of the astrocytes of 12 generations is more than 95%, and keeping the original characteristics of the astrocytes.

Description

Culture medium and culture method for in vitro culture of astrocytes

Technical Field

The invention belongs to the field of cell culture, and particularly relates to a culture medium and a culture method for in vitro culture of astrocytes.

Background

Astrocytes (Astrocytes) are a type of glial cells that are distributed throughout the central nervous system and perform various complex functions, and they exchange substances with neurons, playing an essential role in the growth of neurons. The primary basis for the identification of astrocytes is their ability to express a collagen fibrillary acidic protein (GFAP). In the central nervous system, stressed astrocytes (Reactive astrocytes) react to various types of injury, eventually forming glial scars. The site of injury to the central nervous system is often determined biologically. Astrocytes participate in the blood cerebrospinal fluid barrier and have a certain regulation effect on the blood flow of the central nervous system, so that the activity of neurons is influenced; astrocytes are connected to synapses and play an important role in neuronal homeostasis (Sofroniew and targets, 2010). Recent research finds that in vitro astrocytes can be converted into neurons under the action of specific small molecular compounds, a new chapter of space (Zhang et al, 2015) is opened for the treatment of diseases such as epilepsy, Alzheimer's disease, Parkinson's disease and the like, and in vitro culture of astrocytes also becomes an indispensable step for researching the conversion of the astrocytes, so that a method for purifying and passaging the astrocytes in vitro is optimized, and a good model is provided for deeply researching the central nervous system.

The existing methods for purifying and passaging the astrocytes still have certain limitations, research results mainly focus on the aspect of purifying the astrocytes, and no new progress is still made in the aspects of culturing and passaging the astrocytes in vitro and keeping better cell activity. The prior art generally cultures in a DMEM/F12 culture medium, cells cannot grow normally after passage to the fourth generation, are quite sparse after adherence, do not form connection, and finally individual cells die. The primary cultured cells can contain 5-10% of fibroblasts and oligodendrocytes, and the cells grow and divide vigorously and are difficult to remove in the later period. Skytt et al, artificially decontaminate such cells, and gradually decrease the concentration of FBS in the culture broth from 20% to 10% over a period of 3 weeks from the primary culture of the cells, resulting in a purity of 95% or more (Skytt et al, 2010). In the case of Liu Xiao Mei, etc., the cells obtained by primary culture are filtered through a stainless steel mesh (. phi.) (200. mu.M), and it generally takes 7 to 8 days after each passage until the cells are confluent to 25cm²The culture flask of (2) has long passage time, the culture medium is replaced for many times, however, the cells can only be transferred to the third generation, and the cost of the experiment is high. In the method for preparing Liu Xiao Mei, the cells are subjected to liquid exchange for many times during the culture process, and the growth environment of the astrocytes is changed for many times, so that the division and differentiation of the cells are not facilitated, and even part of the cells are dead. Numerous studies have shown that addition of 0.25 μ M dibutyryladenosine acid (dBcAMP) in the last week of culture enhances the astrocyte division activity (Lange et al, 2012). However, this method had less effect on cells from multiple passages, and cells that lost division activity during passage could not regain division attributes by addition of dBcAMP. Meanwhile, the culture method consumes higher cost and is in operation flowThe process is complicated.

In conclusion, the existing methods for culturing astrocytes are complex and slow in cell growth, and 75-95% of purity of astrocytes is generally obtained only after the astrocytes are cultured in vitro to the third generation. Operations such as replacing the culture medium for many times in the experimental process not only increase the experimental cost, but also are not beneficial to the growth of cells in a relatively stable environment, and the possibility of bacterial contamination is increased. The dBCcAMP added for keeping the division and differentiation activity of the passage cells has no obvious effect on prolonging the passage of the cells and increasing the available generation number of the passage cells.

At present, astrocytes are the key research points in the field of neuroscience research, and a culture medium and a culture method which can prolong the in vitro culture time of the astrocytes, increase the number of passages, improve the purity and maintain the original characteristics of the astrocytes are urgently needed.

Disclosure of Invention

The invention aims to provide a culture medium and a culture method for in vitro culture of astrocytes and the astrocytes cultured in vitro. The culture of the astrocytes outside the culture substrate can prolong the in-vitro culture time of the astrocytes, increase the passage times, improve the purity and keep the original characteristics of the astrocytes.

The technical scheme adopted by the invention is as follows:

a culture medium for culturing astrocytes in vitro, which is supplemented with Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), a Smo protein agonist Smoothened Aginst (SAG) and a Hh agonist Purmorphamine on the basis of a basal medium.

EGF is an epidermal growth factor, is an effective cell division factor, has high affinity with a receptor on the surface of a cell membrane, and is beneficial to the division and differentiation of cells; FGF is a fibroblast growth factor, has similar action with EGF, and has obvious effect on induction of division and differentiation in the cell growth process. The inventor researches and discovers that the combined action of EGF and FGF can effectively promote the division and differentiation of astrocytes in the in vitro culture process. SAG and Purmorphamine can respectively induce Hh channel activation in cultured cells of miceThe combined action has important effect on the growth and the steady state of the in vitro culture of the cells. The inventors found that astrocytes were very sensitive to SHH agonists. Both Smo Agonists (SAG) and Hedgehog agonists (Purmorphamine) are potent agonists of Smoothened receptors, activating the Hedgehog (Hh) signaling pathway by binding directly to the signal transduction factor Smoothened homolog (Smo) protein of an Hh receptor. The Hh signaling pathway is an important signaling pathway in cells that regulates cell differentiation and cell proliferation. In a typical Hh signaling pathway, the SHH protein binds to a PTCH receptor, and the inhibition of the SMO protein by the PTCH is released, so that downstream signals are triggered to cause gene expression. Importantly, SAG or Purmorphamine were able to significantly induce astrocytes [ Ca ] in vitro culture²⁺]_iUp-regulation of (a), which induces astrocytes to release glial transmitters, such as ATP and glutamate, thereby triggering receptor-regulated neuronal currents. The inventor researches and discovers that addition of Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), a Smo protein agonist Smoothened Aginst (SAG) and an Hh agonist Purmorphamine in a basal medium can obviously prolong the in-vitro culture time of astrocytes, increase the passage frequency, improve the purity of the astrocytes and keep the original characteristics of the astrocytes.

The skilled person can add appropriate amount of Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Smo protein agonist Smoothened Aginst (SAG) and Hh agonist Purmorphamine when culturing astrocytes in vitro, and adjust the content of the added 4 components according to the need or common knowledge.

Both Smoothened Aginst (SAG) and Purmorphamine used in the examples of the present invention were purchased from Invitrogen.

Wherein the basal medium can be selected according to the common general knowledge of the skilled person, or according to the literature of the prior art. The basic culture medium adopted by the invention is as follows: 5% Fetal Bovine Serum (FBS), Gibco MEM serum free Medium GlutaMAX (MEM + GlutaMax), 1M glucose, 50g/L NaHCO ₃1% of cyanThe penicillin antibiotic (P/S) is mixed with the streptomycin, which is a basic culture medium for culturing astrocytes, which is commonly used in the industry at present.

Preferably, the epidermal growth factor is present in the culture medium at a concentration of 5-200 ng/ml.

More preferably, the concentration of Epidermal Growth Factor (EGF) in the medium is 10-20 ng/ml.

Preferably, the concentration of fibroblast growth factor in the culture medium is 5-200 ng/ml.

More preferably, the Fibroblast Growth Factor (FGF) is present in the culture medium at a concentration of 10-20 ng/ml.

When the concentration of EGF and FGF in the culture solution exceeds 20ng/ml, the cultured astrocytes have large morphological change, the cells have slender morphology, the cells are difficult to fill the bottom of the culture flask, and the effect is slightly poor.

Preferably, SAG is present in the culture medium at a concentration of 0.05-1M.

Preferably, the concentration of Purmorphamine in the medium is 0.05-1M.

Preferably, the basal medium comprises the following components: 5% fetal bovine serum (FBS, v/v), Gibco MEM serum free Medium GlutaMAX (MEM + GlutaMax), 1M glucose, 50g/L NaHCO ₃1% penicillin mixed double antibody (P/S, v/v).

Preferably, the medium for culturing astrocytes in vitro (EFPS glial cell culture solution denoted by astrocytes in the examples) comprises the following components: 5% Fetal Bovine Serum (FBS), MEM + GlutaMax, 1M glucose, 50g/L NaHCO ₃1% penicillin mixed double antibody (P/S), 10ng/ml Epidermal Growth Factor (EGF), 10ng/ml Fibroblast Growth Factor (FGF), 0.1. mu.M SAG, 0.1. mu.M Purmorphamine.

Among them, the concentrations of Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), SAG, and Purmorphamine used in the examples of the present invention were half maximal effect concentrations using EC 50.

EC50, half maximal effect concentration (concentration for 50% of maximum effect, EC50) refers to the concentration that causes 50% of the maximal effect. EC50 is a drug safety index. The meaning is as follows: resulting in an effective drug concentration in 50% of the individuals. This fully demonstrates that the addition of 4 components to the medium can produce positive and effective results. Of course, the content of 4 substances can be adjusted as required by those skilled in the art to obtain better culture effect.

Preferably, the concentration of epidermal growth factor in the culture medium is 5-200ng/ml, the concentration of fibroblast growth factor in the culture medium is 5-200ng/ml, the concentration of SAG in the culture medium is 0.05-1M, and the concentration of Purmorphamine in the culture medium is 0.05-1M. Generally, at the level of cultured cells, we limit the above-mentioned EFPS concentration range to this level.

A culture method for culturing astrocytes in vitro, wherein the culture medium of any one of the above is used for replacing a basal culture medium to culture the astrocytes in vitro. For example, after the first generation uses the 4-component medium of the present invention, the second generation uses a basal medium, and the third generation continues to use the 4-component medium of the present invention. And the process is repeated in a reciprocating way.

The inventors found that the medium of the present invention can be used at alternate generations and maintained the astrocytes for passage to 13. Wherein the results of FIG. 3B show that astrocytes that were subcultured to passage 13 using the medium of the present invention are superior to astrocytes that were cultured to passage 5 using only the basal medium in the control. However, the medium of the invention is not recommended for continuous use. If used continuously, astrocytes can only be cultured up to 5-6 passages, and if the culture is continued, astrocytes become very elongated in morphology with fibrillar cytoplasmic branches, similar in morphology to neural stem cells.

Preferably, in each passage of the culture, after digesting the cells with the enzyme solution for 2-5min, shaking the culture vessel (such as a culture bottle) to suspend the oligodendrocytes, and discarding the solution; then adding enzyme solution for digestion to obtain astrocytes for passage.

Preferably, the shaking is carried out vigorously.

Preferably, the enzyme comprises trypsin. Trypsin is currently the most common and other enzymes for digesting proteins may be used as needed or as common knowledge.

Preferably, the enzymatic digestion is preceded by a rinsing step using a medium or buffer.

Preferably, the enzyme solution is added for digestion, and when the cells become round and about 2/3 cells float, the digestion is stopped immediately to obtain astrocytes. The second digestion process is conventional.

The inventor researches and discovers that: astrocytes are relatively tight, while oligodendrocytes are much less adherent. So the double digestion method is adopted. First, a short digestion with an enzyme (e.g., trypsin) is performed, followed by vigorous shaking of the flask to suspend the oligodendrocytes, and the solution is discarded. This process actually achieves a purification effect. The inventors found that vigorous shaking did not result in a significant drop in cell number. Statistics show that the ratio of astrocytes is 72 percent and the ratio of oligodendrocytes is 19 percent without shaking culture of the liquid P2; in the culture of the shaking liquid changing P2, the proportion of astrocytes is 97 percent and the proportion of oligodendrocytes is 1 percent. And shaking to change the DAPI in the liquid P2 culture⁺The cells (DAPI labeled nuclei, which may reflect the total number of cells) of (a) may have very few microglia, pluripotent stem cells, and the like. In the light field observation, oligodendrocytes were evident in both P1-P3 in astrocyte cultures without shaking change (treated only according to the second digestion step), but the P2 field was seen to be essentially astrocytes in astrocyte cultures with shaking change. This fully demonstrates that oligodendrocytes can be removed by shaking to change the fluid, leaving astrocytes. And the interference of oligodendrocyte is reduced, the growth and development of astrocyte are better, the total number of cells is not obviously reduced, and the growth condition of cells is better.

An in vitro cultured astrocyte obtained by passage in vitro culture using the medium according to any one of the above.

An in vitro cultured astrocyte obtained by in vitro culture according to any one of the culture methods described above.

The invention has the beneficial effects that:

the invention adds Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Smo protein agonist Smoothened Agonist (SAG) and Hh agonist Purmorphamine into an astrocyte basal medium. EGF is an epidermal growth factor, is an effective cell division factor, has high affinity with a receptor on the surface of a cell membrane, and is beneficial to the division and differentiation of cells; FGF is a fibroblast growth factor, has similar action with EGF, and has obvious effect on induction of division and differentiation in the cell growth process. The combined action of EGF and FGF can effectively promote the division and differentiation of astrocytes in the in vitro culture process. SAG and Purmorphamine can respectively induce the Hh pathway activation in mouse cultured cells, and the combined action has important effect on the growth and the steady state of the in vitro culture of the cells. The combination of the 4 components can obviously prolong the in-vitro culture time of the astrocytes, increase the passage times, improve the purity and keep the original characteristics of the astrocytes.

The inventors found that the medium of the present invention can be used at alternate generations and maintained the astrocytes for passage to 13. Culturing until the purity of the astrocytes of 12 generations is more than 95%, and keeping the original characteristics of the astrocytes.

Wherein the results of FIG. 3B show that astrocytes that were subcultured to passage 13 using the medium of the present invention are superior to astrocytes that were cultured to passage 5 using only the basal medium in the control. However, the medium of the invention is not recommended for continuous use. If used continuously, astrocytes can only be cultured up to 5-6 passages, and if the culture is continued, astrocytes become very elongated in morphology with fibrillar cytoplasmic branches, similar in morphology to neural stem cells.

The inventor researches and discovers that: astrocytes are relatively tight, while oligodendrocytes are much less adherent. So the double digestion method is adopted. First, a short digestion with an enzyme (e.g., trypsin) is performed, followed by vigorous shaking of the flask to suspend the oligodendrocytes, and the solution is discarded. This process actually achieves a purification effect. The second time is carried out according to the conventional method. Oligodendrocytes can be removed by shaking and changing the liquid, and astrocytes are retained. And the interference of oligodendrocyte is reduced, the growth and development of astrocyte are better, the total number of cells is not obviously reduced, and the growth condition of cells is better.

Drawings

FIG. 1 shows that shaking to change the liquid facilitates astrocyte purification.

FIG. 2 shows the vigorous astrocyte division at the time of passage under EFPS conditions.

FIG. 3 shows the increase in the number of astrocyte subcultures under EFPS conditions.

FIG. 4 is a graph of calcium signaling activity of neurons seeded on astrocytes in experimental groups.

FIG. 5 shows the co-culture of passaged astrocytes with neurons under EFPS conditions.

FIG. 6 shows the morphological effects of continuous and intermittent culture of EFPS medium on astrocytes.

Detailed Description

The present invention is further illustrated by, but is not limited to, the following examples.

Example 1

The first experiment method comprises the following steps:

1. the experimental materials are shown in Table 1.

TABLE 1 Experimental materials

2. The main instruments and equipment are shown in Table 2.

TABLE 2 Main instruments and devices involved in the experiment

3. The software used to record the processed data is shown in table 3.

TABLE 3 software for recording process data

4. Culture medium

The basic culture medium comprises the following components: 5% fetal bovine serum (FBS, v/v), MEM + GlutaMax, 1M glucose, 50g/L NaHCO ₃1% penicillin mixed double antibody (P/S, v/v).

A medium for culturing astrocytes in vitro (EFPS glial cell culture solution denoted as astrocytes in the examples) contains the following components: 5% Fetal Bovine Serum (FBS), Gibco MEM serum free Medium GlutaMAX (MEM + GlutaMax), 1M glucose, 50g/L NaHCO ₃1% penicillin mixed double antibody (P/S), 10ng/ml Epidermal Growth Factor (EGF), 10ng/ml Fibroblast Growth Factor (FGF), 0.1. mu.M SAG, 0.1. mu.M Purmorphamine.

The experimental steps are as follows:

1. primary culture of astrocytes

A newborn 1D mouse is taken under aseptic conditions, the skin is wiped with 75% alcohol, the head bone is carefully cut with an ophthalmic scissors, both brains are taken out of the precooled D-Hanks liquid, the meninges are stripped under a stereoscope, the blood vessels are removed, the cerebral cortex is separated, and the cerebral cortex is transferred to a sterile 15ml centrifuge tube. Then, washing with pre-cooled D-Hanks solution for 2 times, adding 1ml papain (1mg/ml) and 10ul DNase I at a ratio of 1:100, mechanically beating with 1ml tip for 1 time to disperse the tissue, and standing at 37 deg.C with 5% CO₂Digesting in an incubator for 30 min. The digestion was stopped by rinsing 2 times with DMEM basal medium containing 5% fetal bovine serum and 1% P/S, gently pipetting to a non-tissue mass, and centrifuging at 2000r/min for 5 min. 1ml of DMEM containing 5% fetal calf serum and 1% P/S was added,gently pipetting until cells are dispersed, transferring to Matrigel-coated 25cm²After the culture bottle is placed for 5h, the culture bottle is gently shaken, the solution is changed to remove oligodendrocytes with low adherence, and the culture bottle is placed at 37 ℃ for 5% CO₂Culturing in an incubator, and gently changing the culture solution after 24 h. And (4) observing the growth condition of the cells under an inverted microscope, culturing for 8-10 days, and digesting and passaging after the bottom of the culture bottle is fully covered with glial cells.

2. Subculturing of astrocytes

Culturing with astrocyte basal medium. After the primary cultured astrocytes are synthesized into a monolayer and spread on the bottom of a bottle, removing original culture solution in the culture bottle, rinsing with DMEM for 2 times, digesting with 2ml of TrypLE for 2min, shaking the culture bottle vigorously (meanwhile, setting the culture bottle to be a control group without shaking and changing the culture solution), suspending oligodendrocytes, discarding the solution in the culture bottle, rinsing with DMEM for 1 time, adding 2ml of TrypLE for digestion, stopping digestion by adding 1.5 times of the volume of astrocyte basal medium when about 2/3 cells float, transferring to a centrifuge tube of 15ml, centrifuging at 800r/min for 5min, discarding supernatant, adding a small amount of basal medium, blowing to disperse the cells uniformly, transferring to the coated culture bottle, complementing the basal medium to 5ml, and supplementing 5% CO at 37 ℃ for 5 ℃₂Culturing in an incubator. Passages were performed according to this method and recorded by photography under an inverted phase contrast microscope before each passage. Starting from the P4 generation, centrifuging based on the original passage method, discarding the supernatant, adding appropriate amount of EFPS glial cell culture solution containing 10ng/ml EGF, 10ng/ml FGF, 0.1. mu.M Purmorphamine and 0.1. mu.M SAG into the basal medium, pumping to disperse the cells uniformly, and adjusting the density to 1x10⁶Transferring/ml into coated culture flask, supplementing to 5ml with EFPS glial cell culture solution, and standing at 37 deg.C with 5% CO₂Culturing in an incubator for passage, and changing the culture solution of the astrocyte EFPS glial cells into a basic culture medium after 2 d; by density 1x10⁶Inoculating to 24-well plate containing coated cell slide, standing for 1min at 40ul per well, adding 500ul EFPS glial cell culture solution to one group, adding 500ul astrocyte basal culture medium to the other group, and standing at 37 deg.C with 5% CO₂Culturing in an incubator, taking a picture by an inverted phase contrast microscope to record the growth condition of the cells,and (5) after the cells are attached to the wall and overgrown, the cells are used for dyeing identification. Then, after centrifugation, cells are cultured by using EFPS glial cell culture solution for passage (namely, EFPS glial cell culture solution is used for the first generation), and then replaced by a basic culture medium (namely, basic culture medium is used for the second generation), the circulation is repeated, each generation of cells are inoculated to a 24-well plate, and the cells are respectively cultured by using astrocyte EFPS glial cell culture solution (the glial cell culture solution added with EFPS is used for the next generation) and an astrocyte basic culture medium containing 5% fetal bovine serum at 37 ℃ and 5% CO₂Culturing in an incubator.

3. Identification of astrocytes

3.1 immunohistochemical identification of astrocytes

In the stress glial cells, Glial Fibrillary Acidic Protein (GFAP) is abundantly expressed, and purified passaged cells can be identified by immunocytochemical staining or immunofluorescence staining with anti-GFAP antibodies. Cells seeded into 24-well plates for this experiment were used for immunocytochemical staining. After the cell slide was fully grown, the cell slide was taken out and the specimen was washed 3 times at 1xPBS (pH7.4) with shaking for 5min each time, the liquid was aspirated, 4% paraformaldehyde was added to fix the cells for 15min at room temperature with shaking, and then washed 3 times at 1xPBS (pH7.4) with shaking for 5min each time. The liquid was aspirated, 0.05% PBST (1 XPBS in Tween) was added and the membrane was permeabilized at room temperature for 30min in a shaker. Adding 5% donkey serum, sealing for 1 hr, and shaking. Slides were removed, incubated with GFAP monoclonal antibody (prepared from 2% donkey serum), placed in a wet box, overnight at 4 deg.C, washed 3 times with 1XPBS (pH7.4) for 5min each time in a shaker according to immunohistochemical kit instructions. The fluorescent secondary antibody was incubated and placed in a wet box and washed 3 times with 1xPBS (pH7.4) at room temperature for 5min each time in a shaker. Adding DAPI mounting solution, mounting, recording under a fluorescence microscope, and counting the GFAP positive cell rate.

3.2 supporting role for neurons

The most important role of astrocytes in the brain is to support the growth of neurons, and to exchange substances such as lactate, glutamate, and the like with neurons. By this feature, we cultured glial cell species cultured in culture medium supplemented with EGF, FGF, Purmo, SAG in a 24-well plate plated with Matrigel-coated cell slide for 7 days until the glial cells were completely plated with the cell slide and matured, and then seeded into neurons (the growth of glial cells carried after brain was completely inhibited by the addition of cytarabine). In comparison with a group without cultured astrocytes, the calcium current and discharge activity of the neurons are detected by using a calcium imaging and patch clamp method to judge whether the growth of the neurons is supported.

4. Statistical method

All data are expressed as Mean ± SEM. The differences between the experimental and control groups were compared using a one-way anova test analysis, with significant differences between the data when p < 0.05.

II, experimental results:

1. shaking liquid changing to promote purification of astrocytes

The mouse astrocytes are cultured in different culture media, and the mouse astrocytes are subcultured by using an astrocyte basal medium as a control (control) and an EFPS glial cell culture solution containing astrocytes of four growth factors (or called small molecules) as an experimental group. Primary cultures were performed in two cerebral cortex of the same neonatal mouse (P0) and cells from P0 passages were transferred to two flasks (P1). The results are shown in FIG. 1.

The results show that: 3DIV (Days in vitro) immunocytochemical staining of P2 in astrocyte cultures showed a reduction in contaminating cells, with oligodendrocytes being the major contaminating cells, as shown in FIG. 1. Oligodendrocyte bodies were circular or smaller polygons, with major branches of the fibril shape extending from the body, with branches diverging all around the body (fig. 1A, green, O4). While astrocytes lay flat on the bottom, exhibit a flat, sponge-like, stretched morphology, and extend beyond the numerous branches of the network (fig. 1A, red, GFAP), the numerous branched processes are generally not evident with numerous intermediate fibers in fused in vitro cell cultures. In the set of liquid changes without shaking (treated only according to the method of the second digestion step) (FIG. 1A), O4⁺The number of positive cells (oligodendrocytes) was significantly greater than those in the group treated with shaking and fluid change (FIG. 1A), and DAPI was observed⁺Positive cells(DAPI marks the nucleus, which can reflect the total number of cells) showed that the total number of cells was similar, indicating that shaking did not result in a significant decrease in cell number.

Statistics show that the ratio of astrocytes is 72 percent and the ratio of oligodendrocytes is 19 percent without shaking culture of the liquid P2; in the culture with shaking change liquid P2, the ratio of astrocytes to oligodendrocytes was-1% (FIG. 1A, n is 12, P)<0.05, n indicates that 12 fields were randomly selected from 4 mouse cultures). DAPI in P2 culture⁺May also have very few microglia or pluripotent stem cells. In the light field observation, oligodendrocytes were evident in P1-P3 in the astrocyte culture without shaking the exchange solution, but essentially healthy astrocytes were visible in the P2 field by the astrocyte culture with shaking the exchange solution (FIG. 3). FIGS. 1B and C are bar graphs showing GFAP of the non-oscillating group⁺About 76% of the cells, concussion group GFAP⁺Is about 98%. Panel C shows non-oscillating group O4⁺Cell content about 20%, concussion group O4⁺The cells are about 2%.

EFPS glial cell culture conditions promoting cell division differentiation

The cells of the experimental group were then identified by immunofluorescent staining to confirm that the properties of astrocytes were maintained under the conditions of the experimental group.

The results show that: astrocytes cultured in EFPS glial cell culture fluid are vigorous in division and differentiation activity (fig. 2). Ki67 labeled the nucleus of dividing cells (fig. 2A, green), GFAP labeled astrocyte cytoskeleton (fig. 2A, red), and DAPI labeled total nuclei (fig. 2A, blue). For P42 DIV astrocyte culture, the proportion of cells in the Control (Control) division phase is 15%; the proportion of cells in the division phase of the experimental group (EFPS) was 38% (FIG. 2B, n: 17, p)<0.05, n represents that 12 fields are randomly selected from astrocyte culture staining of 4 mouse cerebral cortex), obviously, the cell growth is more vigorous in EFPS glial cell culture solution, and the cell division and differentiation activity is better. Total cell mass in EFPS culture conditions, as seen by immunofluorescence stainingThe number is obviously more than that of the control group, and we can compare the number with that of the control group by using DAPI⁺Counting the cells to obtain DAPI under the control condition⁺494 cells, DAPI in EFPS culture conditions⁺695 cells (FIG. 2C, n is 17, p)<0.05, n indicates that 12 fields were randomly selected from 4 mice cerebral cortex astrocyte culture staining). The results showed that the number of glial cells obtained using EFPS glial cell culture broth increased by 40% compared to the number of glial cells cultured in the control basal medium.

Increasing astrocyte passage number in EFPS glial cell culture conditions

The morphology of the astrocytes was recorded under bright field observation and whether the cells could form a cell monolayer with at least 80% confluence, thereby judging whether to continue passaging the cells.

The results show that: culturing the subcultured astrocytes by using the astrocyte basal medium alone, and carrying out cell culture until P4 (Control in FIG. 3) cells form a fusion monolayer; no confluent monolayers could be formed at P5 and no further passaging was performed. Cells cultured by alternating EFPS glial cell broth with astrocyte basal medium can be passaged to P12 (FIG. 3A, panels P1-P12 of EFPS represent different generation cytograms), and cells cultured in 12mm small disks form a cell monolayer of at least 80% confluent astrocytes (FIG. 3A); no confluent monolayers could be formed at P13 and no further passaging was performed. It is clear that the experimental group (fig. 3EFPS) of cells started from P5, and it is clearly observed that some astrocytes exhibit elongated fibrous branches. The cells of P5 generation in Control and P13 generation in EFPS were scattered, the intercellular space was large, some cells were inoculated into 12mm small disks and were unable to differentiate the astrocyte morphology, exhibited a circular or irregular solid polygon (fig. 3B, arrow), were unable to form an 80% fused cell monolayer, and had no ability to continue passaging (fig. 3B). Statistical analysis was performed on the number of passages of 10 mouse cerebral cortex astrocytes (fig. 3C), the number of passages of control group-4.8 passages, and the number of passages of EFPS-10.5 passages, of which 60% of the cultures could be passed to P12 or more (n ═ 10, P <0.05, n indicates 10 mouse cerebral cortex astrocytes cultures).

Support of neuronal growth by astrocytes in EFPS Medium

Growth of hippocampal neurons co-cultured with astrocytes passaged under EFPS conditions was recorded by calcium imaging and patch clamp methods.

The results show that: the astrocytes subcultured with P12 reached 80% confluence within 4d and were able to spread out essentially over the disc. Two co-cultured 14DIV hippocampal neurons were recorded separately (FIGS. 4A, B, C) and the action potential generated, which resulted in Spontaneous excitatory postsynaptic currents of the neurons (sEPSC). Calcium imaging simultaneously records 6 neurons, neuron generation not only maintains inherent properties of the neurons and generates self-emission, but also is closely connected to form a network, and synchronous activity is generated (fig. 4D and E). The P12 subcultured astrocytes and neurons were co-cultured for 14DIV, and the staining results of immunocytochemistry also show (FIG. 5), that the neuron synapse structure supported by the subcultured astrocytes is in the form of Synapsin⁺And can normally form synapse structure. Synapsin labels neuronal phosphoproteins that coat synaptic vesicles. The activity of the co-cultured neurons is high, which indicates that the subcultured astrocytes cultured in the EFPS glial cell culture solution can well form a connection with the neurons, and the normal activity of the neurons and the formation of a synaptic structure are supported.

In conclusion, the invention can obviously prolong the in vitro culture time of the astrocytes, increase the passage times, improve the purity and keep the original characteristics of the astrocytes by adding the Epidermal Growth Factor (EGF), the Fibroblast Growth Factor (FGF), the SAG and the Purmorphamine into the astrocyte culture solution.

Comparative example 1

The astrocytes were cultured continuously in the EFPS glial cell culture broth according to the same formulation (same as example 1). That is, each generation was cultured with the EFPS glial cell culture broth supplemented with epidermal growth factor, fibroblast growth factor, the Smo protein agonist Smoothened and the Hh agonist Purmorphamine.

The results show that: when the cells were cultured in EFPS medium continuously, at 5 th to 6 th passages, the astrocytes became very slender and branched with fibrous cytoplasm, and the morphology was similar to that of neural stem cells, and it was difficult to normally support the growth of neurons (FIG. 6). Correspondingly, the left panel of FIG. 6 shows that the astrocytes are cultured continuously under EFPS conditions, and the astrocytes are cultured at intervals in which the astrocytes maintain good astrocyte morphology after being passaged to 6.

Comparative example 2

The same as example 1, except that the concentration of EGF and the concentration of FGF in the EFPS glial cell culture broth were 30ng/ml and 30ng/ml, respectively.

The results showed that the cultured astrocytes changed greatly in morphology up to passage 5, and the cells were elongated in morphology and less able to spread over the bottom of the flask, with little effect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A medium for culturing astrocytes in vitro, comprising: the culture medium is characterized in that epidermal growth factor, fibroblast growth factor, a Smo protein agonist Smoothened aginst and an Hh agonist Purmorphamine are added on the basis of a basic culture medium;

in the culture medium, the concentration of the epidermal growth factor is 5-200ng/ml, the concentration of the fibroblast growth factor is 5-200ng/ml, the concentration of a Smo protein agonist Smoothened agonist is 0.05-1m M, and the concentration of an Hh agonist Purmorphamine is 0.05-1 mM;

the basic culture medium comprises the following components: 5% v/v fetal bovine serum, Gibco MEM serum free Medium GlutaMAX, 1M glucose, 50g/L NaHCO₃1% v/v penicillin mixed penicillin double antibody.

2. The culture medium according to claim 1, wherein: in the culture medium, the concentration of the epidermal growth factor is 10-20ng/ml, the concentration of the fibroblast growth factor is 10-20ng/ml, the concentration of a Smo protein agonist Smoothened agonist is 0.05-1mM, and the concentration of an Hh agonist Purmorphamine is 0.05-1 mM.

3. A culture method for culturing astrocytes in vitro, comprising: the use of the medium of claim 1 or 2 for the in vitro culture of astrocytes instead of basal medium.

4. The culture method according to claim 3, wherein: digesting the cells for 2-5min by using enzyme liquid during each passage in the culture, shaking the culture vessel, and removing the solution; then adding enzyme solution for digestion to obtain astrocytes for passage.

5. The culture method according to claim 4, wherein: the shaking process needs violent shaking.