CN102399747A - Passage separation method for neural stem cells - Google Patents
Passage separation method for neural stem cells Download PDFInfo
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- CN102399747A CN102399747A CN201110378653XA CN201110378653A CN102399747A CN 102399747 A CN102399747 A CN 102399747A CN 201110378653X A CN201110378653X A CN 201110378653XA CN 201110378653 A CN201110378653 A CN 201110378653A CN 102399747 A CN102399747 A CN 102399747A
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Abstract
The invention relates to a passage separation method for neural stem cells. The method comprises the following steps of: a, centrifuging neural spheres of the neural stem cells grown in a sterile plate containing a Dulbecco modified eagle medium/F 12 (DMEM/F 12) culture medium at a speed of 1,000 revolutions per minute (rpm), extruding and grinding by using ground glass, and filtering the cells by using a sterile stainless steel mesh; and b, diluting the cells until the number is 1*107 cells/milliliter, putting into a 5 percent CO2 culture box for culturing, and culturing continuously for 1 to 2 days to obtain new neural spheres of the neural stem cells. By the passage separation method, the obtained small neural spheres are uniform in sizes; and the damage to the neural stem cells and neural precursor cells is small, the in-vitro passage time is long, and adherently-differentiated neural cells are avoided.
Description
Technical field
The invention belongs to the cell culture technology field, specifically, relate to a kind of going down to posterity and separate the method for the neural ball of NSC.
Background technology
The generation of nervous system disorders and development reduce owing to neurocyte quantity or changing function causes.Can treat the related neural systemic disease through transplanting neural precursor, its mechanism is neurocyte, the secretory nerve protection factor that substitutes minimizing and pathology and regulates the local immunity reaction.Neural precursor can obtain through NSC or embryonic stem cell.
NSC is a kind of multipotential stem cell, under relevant cell factor stimulates, can and form special ball-like structure at in-vitro multiplication, and this ball-like structure is called as neural ball.Neural ball has special microenvironment, can keep survival and the propagation of stem cell in the ball, and gives the potential of NSC to ectoderm cell (neurone and neurogliocyte) differentiation.The preparation of neural ball is the committed step of NSC treatment, and their quality directly has influence on the clinical efficacy of NSC treatment.
Separating the neural ball of NSC, prepare unicellularization method, is the gordian technique that influences the neural ball preparation of high quality, and method commonly used at present comprises enzyme digestion or mechanical piping and druming method.Enzyme digestion is that the method for utilizing independent pancreatin or pancreatin and EDTA to share digests neural ball.Research shows that enzyme digestion can cause stem cell to be undergone mutation, and also has shortcomings such as running time length and cell injury are big.Machinery piping and druming method adopts dropper or transfer pipet that tissue is blown and beaten repeatedly, although the running time is short, same pair cell damage is big.And resulting cell mass size is uneven, causes the partial nerve sphere volume excessive, the survival of the stem cell that affects the nerves, growth and differentiation.
Therefore, the preparation method who sets up a kind of easy, quick, no modificator gene sudden change, the pair cell damage is less and can prepares neural ball of uniform size is present problem demanding prompt solution.
Summary of the invention
The objective of the invention is to; Solve that the NSC that the NSC process of going down to posterity causes is prone to undergo mutation, cell injury is big and the running time long and the uneven first-class problem of neural ball size, a kind of easy, quick, no modificator gene sudden change, the less NSC nerve ball of pair cell damage separation method that goes down to posterity that has is provided.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
The separation method that goes down to posterity of a kind of NSC, its step is following:
A. with the neural ball warp of NSC cross centrifugal after, with obscure glass crush and grind NSC nerve ball, filter through stainless (steel) wire again;
B. cell dilution to 1 * 10 after will filtering
7Individual/mL, place 5%CO
2Continue in the incubator to cultivate, cultured continuously 1-2 days, obtain the neural ball of newborn NSC.
Aforesaid method, preferably, the frosted glass plate of said frosted glass plate for glass plate is prepared from through silicon carbide grinding and hydrofluoric acid corrosion.
Aforesaid method, preferably, the order number of said stainless (steel) wire is 80 orders-200 orders.
Aforesaid method, preferably, the preparation method of said frosted glass plate is:
A. get 2 sheet glass, wherein a sheet glass lies in the container, and the surface evenly is coated with the silicon carbide sand grains above that;
B. the upper surface in sheet glass drips hydrofluoric acid, makes full of liquid between the grains of sand;
C. get another piece sheet glass and be pressed in above the silicon carbide granulosa, push upper glass plate and make annular abrasion with hand;
D. after grinding several minutes, the focussing glass that two mills are good is used flushing with clean water, downcuts from the unfocused surface of two focussing glass respectively with glass cutter, and focussing glass is cut into described frosted glass plate.
Aforesaid method, preferably, the size of said sand grains is 0.1～1 millimeter.
Aforesaid method, preferably, said frosted glass plate is of a size of 10 centimetres of 3 cm x.
Beneficial effect of the present invention is:
Adopt obscure glass and stainless (steel) wire to unite to carry out the neural ball of the NSC separation and Culture that goes down to posterity, substitute in the past pancreatin or mechanical piping and druming method, overcome deficiencies such as cell that previous methods brings is prone to undergo mutation and cell injury is big, the running time is grown.Through the separation of going down to posterity of present method, the neural pelletizing piece size of the filial generation of acquisition is even, and on average can obtain the neural ball of 2-8 filial generation after grinding.Little to NSC and precursor cell damage, NSC is long in the external maintenance vigorous growth generation time, does not have producer sudden change or adherent differentiating phenomenon to take place.
Description of drawings
Fig. 1 amplifies 4 times for the photo of the NSC of slide polishing acquisition.
Fig. 2 a amplifies 4 times for the photo of the neural ball of slide polishing preparation, and Fig. 2 b is the photo of the neural ball of transfer pipet piping and druming method preparation, amplifies 4 times.
Fig. 3 amplifies 200 times for the photo that the NSC marker protein nestin immunofluorescence in the neural ball of slide polishing preparation detects.
Embodiment
Below in conjunction with accompanying drawing, further specify the present invention through embodiment, but not as limitation of the present invention.
The cell culture fluid that contains neural ball described in the present invention can adopt method commonly used in the prior art, and for example enzyme digestion or mechanical piping and druming method are obtained by preparation in the legal aborted fetus of obtaining or other embryos' the cerebral tissue and cultivation.
(1) preparation of frosted glass plate:
Getting 2, each is long 15 centimetres, and wide 15 centimetres, thick is 0.5 centimetre sheet glass, places in the large size porcelain dish; 1 sheet glass is balanced in dish central authorities; Evenly being coated with color in the above is the silicon carbide sand grains of dark red, and the size of sand grains is: 10～20 orders, or 0.1～1 millimeter; Drip hydrofluoric acid again, make full of liquid between the grains of sand.Get an other sheet glass and be pressed in above the silicon carbide granulosa, push upper glass plate and make annular abrasion with hand.After several minutes, two blocks of good obscure glass of mill use flushing with clean water, downcut, obscure glass is cut into the glass bar of 10 centimetres of sizes of 3 cm x, select to grind uniform glass bar and be used for testing with the unfocused surface of glass cutter from glass.
(2) neural ball grinds:
When adopting frosted glass plate polishing of the present invention to grind neural ball; The cell culture fluid that will contain neural ball is after centrifugal; Get its underpart fluid drips at common slide one end, grind gently 1 time with frosted glass plate and common slide, with cell through the aseptic metal mesh filter of 80-200 purpose; After the flushing of cold DMEM/F12 substratum, with cell transfer in the 10mL centrifuge tube.
The result is as shown in Figure 1, and Fig. 1 is after 4 times of amplifications for the photo of the NSC of employing frosted glass plate polishing acquisition of the present invention, the photo that adopts the DMOS camera to take.Visible from figure, adopt the nervous tissue of slide polishing preparation to be homodisperse small cell cluster, help the formation of nervelet ball.
(3) centrifugal:
Above-mentioned cell is centrifugal through 200g, and with the washing of DMEM/F12 substratum once.
(4) cultivate:
In the DMEM/F12 of 10mL substratum, add 20ng/mL EGF and bFGF, B27, cell is suspended with this substratum and is diluted to 1 * 10
7Individual/mL, after cell is placed 5%CO
2Cultivate in the incubator, every day, mirror was observed down and counting.
For identifying that frosted glass plate polishing of the present invention prepares the effect of neural ball, blow and beat method as contrast with transfer pipet of the prior art.When adopting transfer pipet piping and druming method to handle neural ball, adopt electronic pipettor and 10mL transfer pipet, blow and beat fast 10 times.The cell 200g that obtains is centrifugal and with DMEM/F12 substratum washing once, in the DMEM/F12 of 10mL substratum, add 20ng/mL EGF and bFGF, B27, with cell with this substratum suspension.After cell is placed 5%CO
2Cultivate in the incubator, every day, mirror was observed down and counting.
The result is as shown in Figure 2, and among Fig. 2, Fig. 2 a is for adopting the neural ball of frosted glass plate polishing preparation, and Fig. 2 b is the neural ball of transfer pipet piping and druming method preparation, all is the photos under 4 times of amplifications.As seen from the figure, compare with the piping and druming method, the neural nodule number amount that polishing obtains is many, volume is little and the time is short.Above-mentioned experimental result has proved that slide polishing of the present invention is simple to operate, less to the damage of NSC, and the former generation that obtains neural ball quality better.
(5) surface marker of neuronal stem cell detects:
The above-mentioned neural ball continuous passage that obtains through the frosted glass plate polishing was cultivated 50 days, i.e. continuous passage cultivated for 7 generations.1% poly-lysine is coated on the slide glass, after drying, on slide glass, drips neural ball, prepare adherent neural ball.Adherent neural ball warp crosses that 4% Paraformaldehyde 96 is fixed, px passes through and changes and the lowlenthal serum sealing, and sealing is 30 minutes under the room temperature; With the anti-people nestin of rabbit (nestin is the neuronal stem cell marker protein) first antibody, 37 ℃ were reacted 1 hour down respectively; Clean 3 times each 5 minutes with 1 * phosphate buffered saline buffer; Add fluorescent mark goat antirabbit SA again, room temperature lucifuge reaction 30 minutes is cleaned 3 times each 5 minutes with 1 * phosphate buffered saline buffer; Under Laser Scanning Confocal Microscope, detect its result.
The result is as shown in Figure 3, the photo that the amplification of the NSC immunofluorescence check and analysis that the neural ball that Fig. 3 prepares for the slide polishing is interior is 200 times.(see also the referential coloured picture of confession inspector of applying date submission) among Fig. 3, blueness is the nucleus of NSC, and green is neuronal stem cell marker protein nestin.Visible from figure, high expression level NSC surface marker albumen nestin in the neural ball shows the neural ball through the preparation of slide polishing, and the NSC of large number of viable is arranged in its ball.
Claims (6)
1. the separation method that goes down to posterity of a NSC is characterized in that step is following:
A. with the neural ball warp of NSC cross centrifugal after, with obscure glass crush and grind NSC nerve ball, filter through stainless (steel) wire again;
B. cell dilution to 1 * 10 after will filtering
7Individual/mL, place 5%CO
2Continue in the incubator to cultivate, cultured continuously 1-2 days, obtain the neural ball of newborn NSC.
2. the method for claim 1 is characterized in that, the frosted glass plate of said frosted glass plate for glass plate is prepared from through silicon carbide grinding and hydrofluoric acid corrosion.
3. according to claim 1 or claim 2 method is characterized in that the order number of said stainless (steel) wire is 80 orders-200 orders.
4. according to claim 1 or claim 2 method is characterized in that the preparation method of said frosted glass plate is:
A. get 2 sheet glass, wherein a sheet glass lies in the container, and the surface evenly is coated with the silicon carbide sand grains above that;
B. the upper surface in sheet glass drips hydrofluoric acid, makes full of liquid between the grains of sand;
C. get another piece sheet glass and be pressed in above the silicon carbide granulosa, push upper glass plate and make annular abrasion with hand;
D. after grinding several minutes, the focussing glass that two mills are good is used flushing with clean water, downcuts from the unfocused surface of two focussing glass respectively with glass cutter, and focussing glass is cut into described frosted glass plate.
5. method as claimed in claim 4 is characterized in that, the size of said sand grains is 0.1～1 millimeter.
6. method as claimed in claim 4 is characterized in that, said frosted glass plate is of a size of 10 centimetres of 3 cm x.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146641A (en) * | 2013-02-06 | 2013-06-12 | 云南中科灵长类生物医学重点实验室 | Passage method of pluripotent stem cells and application thereof |
| CN105176812A (en) * | 2015-07-28 | 2015-12-23 | 浙江奥瑞健生物技术有限公司 | Stem cell globe cutting and collecting device and stem cell globe cutting and separating passage method |
| CN106619722A (en) * | 2016-12-05 | 2017-05-10 | 上海安集协康生物技术股份有限公司 | Neural stem cell injection for treating brain damage disease |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1458273A (en) * | 2002-01-16 | 2003-11-26 | 中国医学科学院中国协和医科大学血液学研究所 | Separating preparing and use of human fetal dorsal root ganglionic stem cell |
| CN1745168A (en) * | 2002-12-02 | 2006-03-08 | 安增子摩祺株式会社 | Method for culturing neural stem cells using hepatocyte growth factor |
| CN101012449A (en) * | 2006-11-09 | 2007-08-08 | 深圳市达科为生物技术有限公司 | Lymphocyte separation liquid and method of separating spleen lymphocyte |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1458273A (en) * | 2002-01-16 | 2003-11-26 | 中国医学科学院中国协和医科大学血液学研究所 | Separating preparing and use of human fetal dorsal root ganglionic stem cell |
| CN1745168A (en) * | 2002-12-02 | 2006-03-08 | 安增子摩祺株式会社 | Method for culturing neural stem cells using hepatocyte growth factor |
| CN101012449A (en) * | 2006-11-09 | 2007-08-08 | 深圳市达科为生物技术有限公司 | Lymphocyte separation liquid and method of separating spleen lymphocyte |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146641A (en) * | 2013-02-06 | 2013-06-12 | 云南中科灵长类生物医学重点实验室 | Passage method of pluripotent stem cells and application thereof |
| CN103146641B (en) * | 2013-02-06 | 2015-03-25 | 云南中科灵长类生物医学重点实验室 | Passage method of pluripotent stem cells and application thereof |
| CN105176812A (en) * | 2015-07-28 | 2015-12-23 | 浙江奥瑞健生物技术有限公司 | Stem cell globe cutting and collecting device and stem cell globe cutting and separating passage method |
| CN105176812B (en) * | 2015-07-28 | 2017-10-03 | 浙江奥瑞健生物技术有限公司 | A kind of cutting separation propagating method of stem cell sphere cutting collection device and stem cell sphere |
| CN106619722A (en) * | 2016-12-05 | 2017-05-10 | 上海安集协康生物技术股份有限公司 | Neural stem cell injection for treating brain damage disease |
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