CN111254115A - Preparation method of culture medium suitable for large-scale culture of clinical-grade neural stem cells - Google Patents
Preparation method of culture medium suitable for large-scale culture of clinical-grade neural stem cells Download PDFInfo
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Abstract
The invention designs a preparation method of a culture medium suitable for large-scale culture of clinical-grade neural stem cells, which comprises the following steps: DMEM/F12, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, insulin, hydrocortisone, WNT3a, Notch1, bovine serum albumin, heparin sodium, putrescine, sodium selenite, Glutamax-I, endothelial growth factor, progesterone, HEPES, NaHCO 3. The invention does not contain animal-derived additives, cell components and conditioned medium, and has the following advantages: providing a stem cell culture medium which is free of animal-derived components and has definite chemical components; the quality problem of product batches can be effectively guaranteed, standardization is facilitated, proliferation of stem cells can be well supported, characteristics of the stem cells can be kept, and meanwhile, a suspension culture mode also provides guarantee for batch use of the stem cells in clinical experiments.
Description
Technical Field
The invention relates to a culture method of neural stem cells, in particular to a preparation method of a culture medium suitable for large-scale culture of clinical neural stem cells.
Background
The neural stem cells refer to cell groups in an early development stage in a nervous system, and can self-replicate through two division modes of self symmetry or asymmetry to generate cells in different development stages and different differentiation directions in a nervous tissue, and finally generate three types of cells such as neuroblasts, astrocytes, oligodendrocytes and the like with certain cell number and various cell types. The neural stem cells have direct or indirect treatment effects on injuries of a nervous system and neurodegenerative diseases, so that the culture medium for clinically culturing the neural stem cells on a large scale has great application value.
The neural stem cells are cultured mainly in two modes, namely monolayer culture and neurosphere suspension culture. The suspension poly-sphere culture mode can well support the proliferation of stem cells and maintain the characteristics of the stem cells, and meanwhile, the suspension culture mode also makes the mass use of the stem cells in clinical experiments possible.
Disclosure of Invention
The invention provides a neural stem cell culture medium which can be used for in-vitro long-term mass culture and amplification of human neural stem cells.
The invention also provides a cell cultured by the method for culturing and amplifying the human neural stem cells in vitro for a long time by using the neural stem cell culture medium, which has stable performance and keeps higher dryness.
The invention also provides a method for carrying out in-vitro long-term culture and amplification on the human neural stem cells by using the neural stem cell culture medium, which can solve the problem of batch production of the clinical stem cells.
A neural stem cell culture medium, which comprises the following components: DMEM/F12, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, insulin, hydrocortisone, WNT3a, Notch1, bovine serum albumin, heparin sodium, putrescine, sodium selenite, Glutamax-I, endothelial growth factor, progesterone, HEPES, NaHCO 3.
Preferably, the neural stem cell culture medium comprises the following components in volume ratio: DMEM/F1298 ml, non-essential amino acids 1ml, Glutamax-I1 ml.
Preferably, the amount of the recombinant human epidermal growth factor in the neural stem cell culture medium is 0.5-1 μ g.
Preferably, the amount of the recombinant human basic fibroblast growth factor in the neural stem cell culture medium is 0.5-1 μ g.
Preferably, the amount of recombinant human insulin in the neural stem cell culture medium is 1-2.5 mg.
Preferably, the amount of hydrocortisone in the neural stem cell culture medium is 100-.
Preferably, the amount of WNT3a in the neural stem cell culture medium is 10-50 nmol/L.
Preferably, the amount of Notch1 in the neural stem cell culture medium is 10-50 nmol/L.
Preferably, the amount of heparin sodium in the neural stem cell culture medium is 10-50 μ g.
Preferably, the amount of sodium selenite in the neural stem cell culture medium is 5-10 μ g.
Preferably, the amount of the recombinant human endothelial growth factor in the neural stem cell culture medium is 0.5-2 μ g.
Preferably, the amount of xanthone in the neural stem cell culture medium is 1-2. mu.g.
Preferably, it is used. The neural stem cell culture medium maintains the pH value of the culture medium by using HEPES and NaHCO3 together.
Preferably, the pH value of the neural stem cell culture medium is 7.0-7.2.
The research shows that the method greatly maintains the cell viability rate (> 95%).
The invention selects commercial DMEM/F12 culture solution as the basic culture medium for the neural stem cell culture;
the mechanism of action of the specific components is illustrated: heparin has the following specific effects: 1) can be combined with a plurality of proteins to enhance the protein function; 2) promoting the proliferation of stem cells; 3) can indirectly inhibit neurons, indirectly achieve the effect of inhibiting the differentiation of neural stem cells, and is beneficial to long-term neural stem cell culture. WNT3 a; notch 1; insulin acts by promoting the uptake of glucose and amino acids by cells, thereby promoting cell division and growth. Hydrocortisone promotes the growth and development of neural stem cells; putrescine can stimulate and induce the growth and proliferation of neural stem cells. The sodium selenite promotes and participates in the metabolism of the neural stem cells; l-glutamine can promote the synthesis of nucleic acid and protein during the development and growth of neural stem cells; the human transferrin can combine with iron ions, so that the iron ions are reasonably utilized by the neural stem cell cells, the toxicity to the cells is reduced, the growth of the fiber cells is inhibited, and the increase of the neural stem cells is facilitated; progesterone has effects of promoting growth and regulating nerve stem cells. HEPES and NaHCO3 are responsible for maintaining the acid-base equilibrium in the cell sap.
The neural stem cell culture medium provided by the invention can ensure that human neural stem cells are cultured and amplified in vitro for a long time (>30 generations), and the performance of the neural stem cells after a large amount of amplification is stable; the neural stem cells cultured by the invention highly express specific markers on the surfaces of the neural stem cells such as nestin (nestin), musashi, CD133 and the like; the cultured stem cells can be further induced to differentiate into neurons, astrocytes and oligodendrocytes. The invention well solves the technical problems of easy differentiation and difficult long-term culture and amplification of the human neural stem cells in vitro culture, thereby providing a large amount of neural stem cells with unified sources and standards for clinical application. The invention solves the problems of neural stem cells and the sources of cells clinically used by the neural stem cells, and makes the neural stem cells possible to be applied to the treatment of nervous system injury and degenerative diseases, the directional differentiation and toxicology of the stem cells, pharmacology and other functions.
Drawings
FIG. 1 is a photomicrograph (100 magnification) of a human neural stem cell pellet cultured for 7 days;
FIG. 2 is a fluorescent labeling photomicrograph (200 Xmagnification) of Nestin of a 7-day-old section of human neural stem cells;
FIG. 3 is a Musashi fluorescence labeling micrograph of a 7-day-old human neural stem cell section (200-fold magnification);
FIG. 4 is a photomicrograph (200 Xmagnification) of a CD133 fluorescence-labeled human neural stem cell section cultured for 7 days;
FIG. 5 is a graph showing astrocytes (200-fold magnification) after differentiation of human neural stem cells cultured for 7 days, using a GFAP antibody for fluorescent labeling;
FIG. 6 is a graph showing astrocytes (200-fold magnification) after 7 days of culture of human neural stem cells after differentiation using TUJ-1 antibody fluorescence labeling;
FIG. 7 is a graph showing astrocytes (200-fold magnification) after 7 days of culture for differentiation of human neural stem cells, labeled with PDGF antibody fluorescence. (ii) a
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1 culture expansion of neural Stem cells
(1) Passage amplification mode: adopting the neural stem cell culture medium for culture and amplification, placing a cell bottle in a 5% CO2 and 37 ℃ temperature incubator for culture, and replacing the neural stem cell culture medium for half a day every 3-4 days according to the cell proliferation condition; continuously culturing the neural stem cells in the neural stem cell culture medium, digesting the neural stem cells into single cells at 37 ℃ by using Accutase when the diameter of the stem cell sphere of most of the neural stem cells is 500-700 microns, removing the single cells after centrifugation, adding a half of newly prepared neural stem cell culture medium and the other half of used neural stem cell culture medium, mixing the two half of newly prepared neural stem cell culture medium and the other half of used neural stem cell culture medium, and performing bottle distribution and continuous subculture according to the proportion of 1:2 or 1: 4. The human neural stem cells can be continuously cultured and amplified for more than 30 generations according to the passage amplification mode.
Example 2 neural Stem cell sphere sections and marker protein identification
① observing cell ball, selecting neurosphere with diameter of 200-;
② slicing at-21 deg.C to-19 deg.C with thickness of 4.5 um;
③ the slices were placed in a 60 oven overnight;
④ taking out the slices, and freezing at-20 deg.C;
⑤ frozen section, acetone (cold acetone) fixed at room temperature for 10min, PBS wash 10min × 3;
⑥ rinsing with buffer solution for 3 times, and blocking with DPBS (containing 0.1% Triton X-100) containing 5% normal goat serum at 37 deg.C for 30min or overnight at 4 deg.C;
⑦ slicing in a wet box, adding dropwise primary antibody at 37 deg.C overnight at 4 deg.C, washing with PBS for 10min × 3;
⑧ adding secondary antibody at 37 deg.C for 30min, washing with PBS for 10min × 3;
⑨ staining with 1ug/ml DAPI for 10min, washing with PBS for 10min × 3;
⑩ the film was mounted on a mounting liquid and observed under a fluorescent microscope.
Example 3 differentiation identification of neural Stem cells
The neural stem cell can be differentiated into a neuron, an astrocyte and an oligodendrocyte, wherein the neuron contains β -TublinIII antigen, the cell is fluorescently labeled by β -TublinIII antibody, whether the neuron is detected, the astrocyte contains GFAP antigen protein, the cell is fluorescently labeled by the GFAP antibody, whether the astrocyte is detected, whether the oligodendrocyte contains (O4) antigen protein, the cell is fluorescently labeled by the O4 antibody, and whether the oligodendrocyte is detected.
Experimental methods
(1) Directly beating the neural stem cell ball cultured for 7 days, or digesting the neural stem cell ball with ACCUTASE solution and beating to obtain single cell suspension.
(2) Cells were cultured at 3X104The cells were seeded on polylysine (100mg/ml) coated glass cover slips at a density, 1% Fetal Bovine Serum (FBS), B27 supplement (1: 50 dilution) were added to DMEM/F12 medium, and growth factors (EGF, bFGF) were removed for differentiation culture.
(3) After 3 days of culture, staining with immunocytochemistry
① fixing single cell cover glass of neural stem cell cultured for 3 days with 4% paraformaldehyde + 0.3% triton-x100 for 15 min;
② rinsing with DPBS for 3 times, and blocking with DPBS containing 5% normal goat serum (containing 0.1% Triton X-100) at 37 deg.C for 30 min;
③ adding anti- β III Tubulin (β -Tubulin III) monoclonal antibody, anti-collagen-derived acidic protein (GFAP) monoclonal antibody and anti-oligodendrocyte protein (O4) antibody on a cover glass sheet with cells, and reacting at 4 deg.C overnight;
④ rinsing with DPBS containing 0.1% TritonX-100 for 3 times, each for 10 min;
⑤ adding secondary antibody at 37 deg.C for 30min, washing with PBS for 10min × 3;
⑥ staining with 1ug/ml DAPI for 10min, washing with PBS for 10min × 3;
⑦ the film was mounted on a mounting liquid and observed under a fluorescent microscope.
Example 4 neural Stem cell Gene identification (STR assay)
Neural Stem Cells (NSCs) not only have self-renewal capacity, but also differentiate into neurons, astrocytes and oligodendrocytes. When the neural stem cells are cultured for a long time, because the neural stem cells may be mutated or genetically unstable due to various influencing factors such as microenvironment, etc., it is necessary to determine whether the neural stem cells are genetically stable during the long-term culture process by using a more advanced analysis method.
Short Tandem Repeat (STR) analysis method is also called microsatellite DNA, the repeat unit is 2-6 bp, the repeat frequency is 10-60 times, and the gene fragment is below 400 bp. STRs are distributed in chromosomes, are located near non-coding regions, can also be located in introns, promoters and Ali sequences, have great variation in people due to the variation of the number of core sequence units, constitute the genetic polymorphism of STRs, and nowadays STR analysis methods are widely applied to many research fields such as gene localization, forensic identification, anthropology, diagnosis of genetic diseases and the like. We used STR analysis to know if we cultured neural stem cells for a long time and genetic stability.
Experimental methods
2) STR kit
STR kits are manufactured by Promega corporation, kit name: 16 HSSystemThe HSSystemis. Numbering: DC 2101.
3) Extraction of total DNA from human neural stem cells
(1) Human neural stem cell culture
(2) Taking 1.5-1.6X106 neural stem cells from each sample, digesting with trypsin at 37 ℃ for 5min, centrifuging at 1000rpm for 5min, discarding supernatant, adding D-Hank's solution to suspend and precipitate cells, and repeating for 1 time; centrifuging at 5000g for 5min, and removing supernatant; adding 500 μ l cell lysate (containing protenase K5 μ l), reversing 20 times, water bathing at 50 deg.C for 3h, and shaking once in half an hour; cooling to room temperature, adding 500 μ l of balance phenol, rapidly reversing for 1min, and mixing; centrifuging for 15min at 5000g, sucking the upper aqueous phase and adding into an EP tube; adding 450 μ l chloroform/isoamyl alcohol (24: 1) into the water phase, mixing, and centrifuging at 5000g for 15 min; sucking the upper water phase and new EP tube, adding 10 μ l RNAaseA diluent (1 μ l RNAaseA +9 μ l buffer), and water-bathing at 37 deg.C for 30 min; adding 50 μ l of 3mol/L NaAC (1/10 vol), adding absolute ethanol (2.5 times of volume) into EP tube, mixing, and standing at-20 deg.C for 30 min; centrifuging at 10000g for 15min, and discarding the supernatant; washing with 70% cold ethanol for 1-2 times, washing with anhydrous ethanol, centrifuging at 12000g for 10min, removing supernatant, and air drying; add 10. mu.l TE buffer (1. mu.l + 9. mu.l sterile water) and store at-20 ℃.
4) Agarose gel electrophoresis
(1) DNAmarker: 5 μ l DL15,000; (2) electrolyte solution: TAE diluent;
(3) electrophoretic voltage: 170V; (4) electrophoresis time: 30min
5) STR reaction system
DNA: the maximum sample loading amount is 17.5 mu l;
5×MasterMix：5μl；
10×PrimerPairMix：2.5μl；
deionized water: make up to 25. mu.l.
6) PCR reaction
(1) The reaction conditions are 96 ℃ and 2 minutes; circulating 10 times at 94 ℃ for 30 seconds; circulating for 10 times at 60 ℃ for 30 seconds;
circulating 10 times at 70 ℃ for 45 seconds; circulating for 22 times at 90 ℃ for 30 seconds; 30 seconds at 60 ℃ and 22 times of circulation; circulating for 22 times at 70 ℃ for 45 seconds; the mixture was stored at 60 ℃ for a minute and 4 ℃.
Example 5 neural stem cell telomerase identification
Telomeres are structures essential for the protection of chromosome ends for stability. Maintenance of telomere length requires the presence of telomerase activity. Immortalized cells and tumor cells can survive for a long time, and telomerase plays an important role. Telomerase has a very close relationship with the proliferation, differentiation and immobility of cells, and the obvious increase of telomerase has a positive relationship with the canceration of cells, the apoptosis or the aging of cells.
Stem cells are cells with proliferative and differentiative potential, have the ability to self-renew and replicate, and are capable of producing highly differentiated functional cells. However, in the in vitro long-term culture, the division frequency is large, and the possibility of canceration of cells exists, so that the safety detection of the cells is extremely important in the long-term culture process. The experiment judges whether the neural stem cells cultured for a long time are possible to become cancerated cells and apoptosis by detecting the activity of telomerase.
Experimental methods
1) Grouping: culturing 7 days human neural stem cells (generation 4, generation 10, and generation 30), using Hela cells as positive control, and fibroblasts as negative control;
2) a kit:
kit manufactured by Roche company, named telotagggtelomericepcr ELISA kit No.: 11854666910.
3) preparing the cells
(1) Collecting human Neural Stem Cell (NSC) balls cultured for 2-24 months continuously, discarding supernatant, and washing with D-Hanks solution for 2 times;
(2) 0.25% trypsin (1: 5D-Hanks liquid dilution) was digested for 5min at 37 deg.C (tumour cells IMR-32 removed this step) and transferred to a 15ml centrifuge tube;
(3) centrifuging at 4 deg.C 955g for 5min, removing supernatant, and adding DMEM culture solution to disperse;
(4) trypan blue staining (20. mu.l cells + 80. mu.l staining) was diluted 1: 10 and counted;
(5) taking about 2x105 cells in a 1.5ml EP tube, centrifuging at 4 ℃ 3000g for 5min, and removing supernatant;
(6) centrifuging at 3000g for 5min, discarding supernatant, adding D-Hank's solution to suspension precipitate cells, and repeating for 1 time; centrifuging at 3000g for 5min, and removing supernatant;
4) extraction of RNA and PCR
(1) The cell pellet was resuspended in 200. mu.l Lysis reagent (Lysis reagent, 1 liquid), pre-cooled on ice and pipetted at least 3 times, and incubated in ice for 30 min. If frozen cell pellets are used for extraction, the cell pellets are thawed on ice before adding the lysis solution.
(2) Centrifuging at 16000g for 20min at 4 deg.C, and transferring the supernatant into a centrifuge tube;
(3) mu.l of the Reaction complex (Reaction mix, 2 liquid) was added to a tube suitable for PCR amplification, and 3. mu.l of cell extract (corresponding to 3X103 cells or 50ug total protein) was added. Adding sterile water to the total volume of 50ul, and performing all the sucking steps on ice;
(4)TRAP-PCR。
5) ELISA detection
(1) Adding 20 μ l of Denaturation reagent (liquid 3) into a centrifuge tube (if a large amount of samples exist, a microplate MTP without nuclease coating is recommended), adding 5 μ l of PCR amplification product, and incubating for 10 minutes at 20 ℃;
(2) add 225. mu.l hybridization buffer (hybridization buffer, lane 4) and mix well with shaker;
(3) adding 100 mul of the mixture into an MTP microporous plate, covering with metal paper, and incubating for 2h in an oscillator at 37 ℃ and 300 rpm;
(4) discarding all the liquid, Washing 3 times with Washing buffer (Washing buffer, 5 liquid, kit 10 ×, diluted 1 ×, when used), 250 μ l per well for at least 30 seconds, discarding the Washing liquid;
(5) adding 100 μ l Anti-DIG-POD working solution (Anti-DIG-POD, 6 solution, diluted 50 times when in use, to prepare working solution), covering with metal paper, and incubating for 2 hr at 20 deg.C in 300rpm oscillator;
(6) discarding the liquid, rinsing with 250 μ l of washing buffer per well for 5 times, each time for at least 1.5min, carefully discarding the washing buffer;
(7) adding 100 μ l of TMB substrate solution (liquid 8), covering with metal paper, and incubating at 20 deg.C for 20min in a shaker at 300 rpm;
(8) add 100. mu.l Stop solution (Stop reagent, solution 9) to Stop the reaction;
(9) when the color of the POD substrate is changed from blue to yellow, absorbance values of 450nm and 690nm are respectively detected within 30 min.
Claims (15)
1. A preparation method of a culture medium suitable for large-scale culture of clinical-grade neural stem cells is characterized in that the neural stem cell culture medium comprises the following components: DMEM/F12, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, insulin, hydrocortisone, WNT3a, Notch1, bovine serum albumin, heparin sodium, putrescine, sodium selenite, Glutamax-I, endothelial growth factor, progesterone, HEPES, NaHCO 3.
2. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the neural stem cell culture medium comprises the following components in percentage by volume: DMEM/F1298 ml, non-essential amino acids 1ml, Glutamax-I1 ml.
3. The method of claim 1, wherein the amount of recombinant human epidermal growth factor in the culture medium is 0.5-1 μ g.
4. The method of claim 1, wherein the amount of the recombinant human basic fibroblast growth factor in the neural stem cell culture medium is 0.5-1 μ g.
5. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the amount of recombinant human insulin in the neural stem cell culture medium is 1-2.5 mg.
6. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells as claimed in claim 1, wherein the amount of hydrocortisone in the neural stem cell culture medium is 100-1000 μ g.
7. The method for preparing culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the amount of WNT3a in the neural stem cell culture medium is 10-50 nmol/L.
8. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the amount of Notch1 in the neural stem cell culture medium is 10-50 nmol/L.
9. The method according to claim 1, wherein the amount of heparin sodium in the neural stem cell culture medium is 10-50 μ g.
10. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the amount of sodium selenite in the neural stem cell culture medium is 5-10 μ g.
11. The method of claim 1, wherein the amount of the recombinant human endothelial growth factor in the neural stem cell culture medium is 0.5-2 μ g.
12. The method of claim 1, wherein the amount of xanthone in the neural stem cell culture medium is 1-2 μ g.
13. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the neural stem cell culture medium is maintained at the pH value of the culture medium by using HEPES and NaHCO3 together.
14. The method of claim 1, wherein the neural stem cell culture medium has a pH of 7.0-7.2.
15. The method for preparing a culture medium suitable for large-scale culture of clinical-grade neural stem cells according to claim 1, wherein the method is found to greatly maintain the cell viability (> 95%) through research.
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