CN101092607B - Method for obtaining cell clone ball from cell line developed along bottom - Google Patents
Method for obtaining cell clone ball from cell line developed along bottom Download PDFInfo
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- CN101092607B CN101092607B CN2007100744986A CN200710074498A CN101092607B CN 101092607 B CN101092607 B CN 101092607B CN 2007100744986 A CN2007100744986 A CN 2007100744986A CN 200710074498 A CN200710074498 A CN 200710074498A CN 101092607 B CN101092607 B CN 101092607B
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- brain glioblastoma
- glioblastoma cell
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Abstract
This invention discloses a method for acquiring cell clone spheres from bottom growth cell line. The method comprises: culturing cells in a serum culture medium, growing at the bottom, collecting suspended cells after 7-20 day culture, centrifuging suspended cells, discarding the supernatant, washing the residue with HBSS solution, placing the residue in a serum-free culture medium added with growth factor, culturing for 1-20 days to obtain clone spheres, separating suspended clone spheres, culturing, while culturing clone spheres and cells adhered to the bottom, replacing the culture medium periodically, and continuously growing new clone spheres. The method has such advantages as simple process, no need for digestive enzyme, and no need for mechanical method. Identical stem cell clone spheres can be obtained continuously, and can be used in theoretical research on stem cells.
Description
Technical field
The present invention relates to a kind of cell clone ball cultural method, be meant that especially a kind of is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth.
Background technology
Cell cultures is the basic step in many biology, medical science and the study of pharmacy, in stem-cell research, wishes to obtain fast a large amount of clone balls sometimes.Present clone from the subsides bottom growth obtains the method for stem cell clone ball must paste floor cells with digestive ferment digestion, make cell suspension, makes single cell suspension with mechanical means again.Its shortcoming is that use digestive ferment and mechanical means in this method all may cause damage by pair cell, and complex steps.
Summary of the invention
Technical problem to be solved by this invention is: providing a kind of is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, and it is easy and simple to handle, and easy damaged cells not, and culture effect is good.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: a kind of is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, comprises the steps:
Step 1, place routine to have blood serum medium to carry out routine brain glioblastoma cell to cultivate, allow brain glioblastoma cell paste bottom growth;
Step 2, cultivation in the brain glioblastoma cell that suspends, when complete single brain glioblastoma cell reaches some amount, were collected the suspension brain glioblastoma cell after 7~20 days;
Step 3, centrifugal treating suspension brain glioblastoma cell, abandoning supernatant is cleaned residuum with HBSS liquid again, and this residuum contains the single brain glioblastoma cell with strong multiplication capacity;
Step 4, the residuum after will cleaning place the serum free medium that is added with somatomedin at least to cultivate;
Step 5, cultivation are after 10~20 days, can obtain diameter at 50~100 microns brain glioblastoma cell clone ball, the brain glioblastoma cell clone ball partly is suspension, part is for pasting bottom growth, the brain glioblastoma cell clone ball that suspends and the brain glioblastoma cell clone ball at the subsides end are separately cultivated with subsides end brain glioblastoma cell, and the periodic replacement substratum is to keep content of effective in the substratum, the cerebral glioma clone ball that promptly can grow out new continuously in the culturing bottle.
Preferably, blood serum medium is arranged is that 5~15% foetal calf serum makes by add mass percent in commercially available basic medium to the used routine of step 1.
Preferably, in the step 3, centrifuge speed is 1500~2000 rev/mins, and the centrifugal treating time is 3~5 minutes.
Preferably, in the step 3, adopt HBSS liquid to clean residuum 2~4 times.
Preferably, in the step 4, described serum free medium is commercially available basic stem cell media.
Preferably, in the step 4, described somatomedin adopts any at least two kinds among bFGF, EGF, the PDGF, and addition is 20~25ug/ml.
Preferably, in the step 5, the brain glioblastoma cell clone ball adopts manual mode to take out.
Preferably, in the step 5, need change a subculture in 5~15 days.
The invention has the beneficial effects as follows: traditional view thinks, pastes the cell of bottom growth if lose and paste end ability, is suspended state, then is considered to dead or will dead cell.And reach after deliberation by repeatedly experiment, find that these suspension cells also not all are dead cells, the cell subsets of high proliferation ability is contained in opposite the inside.And the inventive method is easier than existing stem cell clone ball cultural method, and without digestive ferment, also make single cell suspension without mechanical means, in a culturing bottle, clone ball grows out continuously, thereby can utilize suspension cell in a large number, obtain the on all four stem cell clone ball of genetic background constantly; Can be widely used in fields such as stem cell theoretical investigation, regenerative medicine, new drug development.
Description of drawings
Fig. 1 is the schematic flow sheet of the inventive method.
Below in conjunction with accompanying drawing the present invention is described further.
Embodiment
As shown in Figure 1, the invention provides a kind of is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, and it comprises the steps:
Step 1, place routine to have blood serum medium to carry out routine brain glioblastoma cell to cultivate, cell pastes bottom growth, it is that 5~15% foetal calf serum makes that described routine has blood serum medium can add mass percent in commercially available basic medium, and it is 10% that foetal calf serum preferably adds mass percent;
Step 2, cultivation are after 7~20 days, in the brain glioblastoma cell that suspends, when complete individual cells reaches some amount, collect the suspension brain glioblastoma cell, in the ordinary course of things, these suspension brain glioblastoma cells are considered to lose vigor, cell in heaven or soon dead;
Step 3, centrifugal treating suspension cell, the back abandoning supernatant is cleaned residuum with scavenging solution again behind the centrifugal treating certain hour, and preferred HBSS liquid also cleans 2 times, and centrifuge speed is preferably set to 1500 rev/mins, preferably treatment 5 minutes;
Step 4, the residuum after will cleaning place the serum free medium that is added with somatomedin at least to cultivate, the preferred commercially available basic stem cell media of described serum free medium, described somatomedin preferably adopts any two kinds among bFGF, EGF, the PDGF, addition is 20~25ug/ml, certainly, also can only adopt wherein a kind of according to circumstances or adds three kinds simultaneously, but generally speaking, if only employing is wherein a kind of, effect is not very good, and then expense is too high to adopt three kinds simultaneously.
Step 5, cultivate after 10~20 days, can see diameter at 50~100 microns brain glioblastoma cell clone ball, some is suspension, some is for pasting bottom growth, the clone ball that suspends is separated single culture, and the clone ball at the subsides end, pasting floor cells still cultivates together, and the periodic replacement substratum is to keep content of effective in the substratum, because the suspension clone ball has no chance to grow new clone ball, therefore, emphasis is cultivated clone ball and the cell that pastes the end, and new clone ball continuously can grow out in the former culturing bottle, thereby promptly obtain needed cell clone ball sustainably, can be used for every purposes for taking out.Clone ball can adopt manual mode constantly to take out, and needn't use digestive ferment.Because when the somatomedin in the substratum exhausted, clone ball will be tired gradually, loses three-dimensional shape, therefore need often to change substratum, changed once in general 7~15 days, if clone ball density is very big, as greater than 300/cm
2, even also can promptly change a subculture in 5 days.
Compare below by an application examples of the present invention and a Comparative Examples, further specify the effect of the inventive method.
Application examples
Adopting aforesaid method to cultivate brain glioblastoma cell is U251MG, promptly sees clone ball on the 10th day, and the high-density of clone ball reaches 400/cm
2,, do not see any unusual same culturing bottle cultured continuously 6 months.In these 6 middle of the month, because of other experiment needs, 5 taking-up clone balls, each quantity of taking out is about 500.
Comparative Examples
When adopting ordinary method cultivation brain glioblastoma cell to be the U251MG clone ball, at 10~15 days, have a large amount of necrocytosiss, the work of clear cell debris is more loaded down with trivial details.The density of clone ball is generally at 400/cm
2Below.
Claims (8)
1. one kind is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, it is characterized in that it comprises the steps:
Step 1, place routine to have blood serum medium to carry out routine brain glioblastoma cell to cultivate, allow brain glioblastoma cell paste bottom growth;
Step 2, cultivation in the brain glioblastoma cell that suspends, when complete single brain glioblastoma cell reaches some amount, were collected the suspension brain glioblastoma cell after 7~20 days;
Step 3, the suspension brain glioblastoma cell that will collect are centrifugal, and abandoning supernatant is cleaned residuum with HBSS liquid again, and this residuum contains the single brain glioblastoma cell with strong multiplication capacity;
Step 4, the residuum after will cleaning place the serum free medium that is added with somatomedin at least to cultivate;
Step 5, cultivation are after 10~20 days, can obtain diameter at 50~100 microns brain glioblastoma cell clone ball, the brain glioblastoma cell clone ball partly is suspension, part is for pasting bottom growth, the brain glioblastoma cell clone ball that suspends and brain glioblastoma cell clone ball that pastes the end and subsides end brain glioblastoma cell are separately cultivated, and the periodic replacement substratum is to keep content of effective in the substratum, the brain glioblastoma cell clone ball that promptly can grow out new continuously in the culturing bottle.
2. according to claim 1 a kind of be the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, it is characterized in that: it is that 5~15% foetal calf serum makes by add mass percent in commercially available basic medium that the used routine of step 1 has blood serum medium.
3. a kind of brain glioblastoma cell from the subsides bottom growth according to claim 1 is the method that U251MG obtains cell clone ball, and it is characterized in that: in the step 3, centrifuge speed is 1500~2000 rev/mins, and the centrifugal treating time is 3~5 minutes.
4. a kind of brain glioblastoma cell from the subsides bottom growth according to claim 1 is the method that U251MG obtains cell clone ball, it is characterized in that: in the step 3, adopt HBSS liquid to clean residuum 2~4 times.
5. a kind of brain glioblastoma cell from the subsides bottom growth according to claim 1 is the method that U251MG obtains cell clone ball, and it is characterized in that: in the step 4, described serum free medium is commercially available basic stem cell media.
6. a kind of according to claim 1 or 5 is the method that U251MG obtains cell clone ball from the brain glioblastoma cell that pastes bottom growth, it is characterized in that: in the step 4, described somatomedin adopts any at least two kinds among bFGF, EGF, the PDGF, and addition is 20~25ug/ml.
7. a kind of brain glioblastoma cell from the subsides bottom growth according to claim 1 is the method that U251MG obtains cell clone ball, it is characterized in that: in the step 5, the brain glioblastoma cell clone ball adopts manual mode to take out.
8. be the method that U251MG obtains cell clone ball according to claim 1 or 7 described a kind of brain glioblastoma cells, it is characterized in that: in the step 5, need change a subculture in 5~15 days from the subsides bottom growth.
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CN2007100744986A CN101092607B (en) | 2007-05-17 | 2007-05-17 | Method for obtaining cell clone ball from cell line developed along bottom |
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CN2007100744986A CN101092607B (en) | 2007-05-17 | 2007-05-17 | Method for obtaining cell clone ball from cell line developed along bottom |
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CN101092607A CN101092607A (en) | 2007-12-26 |
CN101092607B true CN101092607B (en) | 2010-08-25 |
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Non-Patent Citations (4)
Title |
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冯若鹏等.胎猪胰岛源胰腺干细胞分离培养与诱导分化试验.中国农业科学40 3.2007,40(3),第582-587页. |
冯若鹏等.胎猪胰岛源胰腺干细胞分离培养与诱导分化试验.中国农业科学40 3.2007,40(3),第582-587页. * |
李茗初等.悬浮法培养C6胶质瘤细胞系和该细胞系中脑肿瘤干细胞的分离.中国现代医学杂志14 2.2004,14(2),57-60. |
李茗初等.悬浮法培养C6胶质瘤细胞系和该细胞系中脑肿瘤干细胞的分离.中国现代医学杂志14 2.2004,14(2),57-60. * |
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