CN104928248A - Kit for preparing neural stem cells and method for preparing neural stem cells - Google Patents
Kit for preparing neural stem cells and method for preparing neural stem cells Download PDFInfo
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Abstract
The invention discloses a kit for preparing neural stem cells. The kit for preparing the neural stem cells comprises nervous tissue preserving fluid, nervous tissue washing fluid, nervous tissue decomposing fluid, cell washing fluid, primary cell culturing fluid, cell dissociating fluid, secondary cell culturing fluid and cell cryopreserving fluid. The method for preparing the neural stem cells comprises the steps that cranial nerve tissue is taken and put in the nervous tissue preserving fluid; the cranial nerve tissue is taken out from the nervous tissue preserving fluid and is rinsed by using the nervous tissue washing fluid; the cranial nerve tissue is cut into fragments, and the nervous tissue decomposing fluid is added to dissociate and decompose the fragments; the secondary cell culturing fluid is added into the dissociated and decomposed fluid, mixed fluid passes through a cell strainer, filtering fluid is collected, and centrifugal treatment is performed on the filtering fluid; the centrifuged filtering fluid is added into the primary cell culturing fluid, and the P0-generation neural stem cells are obtained after the culture. According to the kit for preparing the neural stem cells and the method for preparing the neural stem cells, use is easy, the operation is convenient, the cost is low, the culturing process is more stable, and a large number of the neural stem cells can be acquired within a short time.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of test kit for the preparation of neural stem cell and the method preparing neural stem cell.
Background technology
Neural stem cell is the parent cell that a class has division potential and self-renewal capacity, and it can produce the various types of cells of nervous tissue by not reciprocity divisional mode.Sequential according to differentiation potential is divided into: mainly there is the medullary epithelium cell of embryonic stage, radial neuroglia neurone and neuroblast, wherein, radial neuroglia neurone is mainly present in the infancy, can divide and produce neuronal precursor or spongiocyte simultaneously; Neuroblast is mainly present in adult animals body, can produce neural precursor, neurone and all kinds of neurogliocyte.Two classes are mainly divided: stem cell of neural crest and cns stem cell according to position.To have the ability being divided into neurone, astroglia cell and oligodendrocyte at present, energy self is also enough to provide the cell of a large amount of brain tissue cell to be all referred to as neural stem cell.
In the treatment of nervous system disorders, except traditional medicine, operation and rehabilitation, cellular transplantation therapy nervous system disorders research in recent years obtains and extensively carries out, and mainly comprises mesenchymal stem cells MSCs, embryonic stem cell and neural stem cells transplantation etc.2009, human embryo stem cell is successfully expelled in the rat body of Spinal injury by investigator, thus rat is recovered motor capacity, but there is the risk forming tumour in direct injection embryonic stem cell, through experimental results demonstrate, utilizing neural stem cell to carry out cellular transplantation therapy nervous system disorders is effective and the selection of safety, and neural stem cell does not have Tumor formation, has the potential of neuralward unit, astroglia cell and oligodendrocyte differentiation; Be undifferentiated initiating cell, do not express ripe cell antigen, not by immune system recognition, there is low immunogenicity; Its tissue fusion is good, well can merge with the nervous tissue of host, and in host long-term surviving, be optimal Transplanted cells material.Therefore, be devoted in the industry the clinical application research of neural stem cell always.
Neural stem cell can by embryonic stem cell, induced multi-potent stem cells and other differentiation of stem cells.Embryonic stem cell is the totipotent Multifunctional nerve stem cell of one of separating from mammiferous blastaea inner cell mass and original sexual cell preimplantation embryos inner cell mass, this cell can carry out amplification cultivation in vitro, therefore, provide without tumorigenicity and can in vitro fast breeding, stable to go down to posterity, mass propgation neural stem cell becomes the current task of top priority.
The vitro culture of neural stem cell is a more difficult problem always, and its difficult point is 2 points, and one is that the concentration proportioning of required reagent and the factor needs to grope, and takes the formula that a large amount of time and efforts just can reach best; Two is traditional neural stem cell vitro culture is by the neural ball of suspension culture, after observing the multiplication of neural ball quantity, by centrifugal, expand bottle cultivation and reaches the object gone down to posterity.The method of this suspension culture, cell proliferation of nerve cord is slow, and vigor is very poor, is very easy to apoptosis, is difficult to the neural stem cell that results are a large amount of.Therefore this makes the researcher of being correlated be difficult to the research work obtaining a large amount of neural stem cell to carry out being correlated with.
Summary of the invention
By the neural ball of suspension culture for solving traditional neural stem cell vitro culture, after observing the multiplication of neural ball quantity, by centrifugal, expand bottle cultivation and reach the object gone down to posterity, the method for this suspension culture, cell proliferation of nerve cord is slow, and vigor is very poor, be very easy to apoptosis, be difficult to the problems such as a large amount of neural stem cell of results, the object of the present invention is to provide a kind of test kit for the preparation of neural stem cell and prepare the method for neural stem cell.
For achieving the above object, provide a kind of test kit for the preparation of neural stem cell in the embodiment of the present invention, comprise nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 50 ~ 250ug/ml, the bFGF of 10 ~ 20ng/ml, the EGF of 10 ~ 20ng/ml, the glutamine of 0.5 ~ 2.5mmol/ml, the Sodium.alpha.-ketopropionate of 0.5 ~ 2.5mmol/ml.
Further, described nervous tissue conserving liquid is substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, DMEM/F12 containing Streptomycin sulphate 50 ~ 250ug/ml, penicillin 50 ~ 250ug/ml.
Further, nervous tissue washings is the one in physiological saline containing penicillin 50 ~ 250ug/ml, Streptomycin sulphate 50 ~ 250ug/ml, PBS, D-Hanks.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1 ~ 2mg/ml, thymus nucleic acid I0.1 ~ 0.2mg/ml.
Further, cell washing solution is PBS/D-Hanks.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by bFGF, the EGF of 10 ~ 20ng/ml of the basic medium of 1000ml, 10 ~ 20ng/ml, the B27 of 15 ~ 25ug/ml, the N2 of 10 ~ 15ug/ml, the glutamine of 0.5 ~ 2.5mmol/ml, the Sodium.alpha.-ketopropionate of 0.5 ~ 2.5mmol/ml.
Further, cell dissociation buffer is the PBS containing the trypsinase of 1.5 ~ 2.5mg/ml, the EDTA of 0.1 ~ 0.25mg/ml.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO 50 ~ 200mg/ml.
Additionally provide a kind of method preparing neural stem cell in the embodiment of the present invention, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 4 ~ 12 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2-3 times of volume, put into the CO2gas incubator digest and decompose 45 ~ 60 minutes of 35 ~ 40 DEG C simultaneously, took out concussion once every 3 ~ 5 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Further, utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the suspension culture of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the adherent culture of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 ~ 40 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the adherent culture of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 ~ 40 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 1 × 10
5~ 3 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 37 ~ 42 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
Beneficial effect of the present invention is: the cultivation that test kit provided by the invention carries out neural stem cell is convenient, and cost is lower, and the activity of neural stem cell is comparatively strong, and more stable in culturing process, success ratio is higher, can culture of neural stem cells neural in enormous quantities.
In addition, present invention also offers a kind of more stable cell culture processes, by primary neural stem cell suspension culture, reach the object of purifying cells, add serum afterwards and carry out cell attachment cultivation, strengthen the activity of neural stem cell greatly, improve the rate of propagation of neural stem cell.Make the cultivation of neural stem cell more stable, more easily build up corresponding neural stem cell system, a large amount of neural stem cell can be gathered in the crops and there will not be the awkward situation can not carrying out related scientific research work as classical culture protocols because not having enough neural stem cell, can observation of cell be dynamic more intuitively in culturing process, the qualification work that some suspension cells cannot carry out can be done, and preparation method of the present invention is simpler, convenient operation, save a large amount of working hours, method is simple, processing ease, be applicable to very much the use of scientific effort, save the time and efforts that Scientific Research Workers is a large amount of.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
Embodiment 1, a kind of test kit for the preparation of neural stem cell of the present invention, comprises nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 50ug/ml, the glutamine of EGF, 0.5mmol/ml of bFGF, 10ng/ml of 10ng/ml, the Sodium.alpha.-ketopropionate of 0.5mmol/ml.Secondary cells nutrient solution is the neural stem cell adherent culture liquid obtained, and makes neural stem cell be able to adherent culture, has stronger activity.
In the technical program, nervous tissue conserving liquid described is further the substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, the DMEM/F12 containing Streptomycin sulphate 50ug/ml, penicillin 50ug/ml.The effect of nervous tissue conserving liquid ensures nervous tissue activity after sampling and sterilizing.
The present invention has done further restriction to nervous tissue washings, and nervous tissue washings is the one in physiological saline containing penicillin 50ug/ml, Streptomycin sulphate 50ug/ml, PBS, D-Hanks.The effect of nervous tissue washings is washing nervous tissue and removes blood and sterilizing.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1mg/ml, thymus nucleic acid I0.1mg/ml.The effect of nervous tissue decomposed solution is digestion nervous tissue, obtains single cell suspension.
Further, cell washing solution is PBS/D-Hanks.The effect of cell washing solution is that the stem cell that washing obtains removes various liquid residue.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by the glutamine of N2,0.5mmol/ml of B27,10ug/ml of EGF, 15ug/ml of the basic medium of 1000ml, bFGF, 10ng/ml of 10ng/ml, the Sodium.alpha.-ketopropionate of 0.5mmol/ml.Primary cell culture liquid is serum free growth medium, forms neural ball for the neural stem cell that suspends.
Further, cell dissociation buffer is the PBS containing the trypsinase of 1.5mg/ml, the EDTA of 0.1mg/ml.When the effect of cell dissociation buffer is passage, digest adherent neural stem cell.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO 50mg/ml.The effect of cells frozen storing liquid is the neural stem cell of frozen acquisition.
All ingredients selected in test kit provided by the invention is all through that laboratory long-term experiment decides, the invention provides a kind of optimum formula of culture of neural stem cells neural, use this test kit, a large amount of neural stem cell can be obtained in the short period, and there is low cost, easy to operate, efficiency high, to play a significant role in stem cell correlative study and clinical trial thereof, there is extraordinary development prospect.
The test kit that use embodiment 1 provides is when culture of neural stem cells neural, and a kind of method preparing neural stem cell, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 4 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2 times of volumes, put into the CO2gas incubator digest and decompose 45 minutes of 35 DEG C simultaneously, took out concussion once every 3 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the cultivation of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the cultivation of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the cultivation of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 1 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 37 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
The preparation method of the present invention's protection compared with traditional Culture of neural stem cells method, primary cell suspension culture, and after purifying cells, take the mode of adherent culture to obtain neural stem cell.Operation like this makes the activity of neural stem cell greatly strengthen, and makes the culturing process of cell more stable, makes cell can carry out going down to posterity of higher generation and even can form neural stem cell system; A large amount of neural stem cell can be gathered in the crops and there will not be the awkward situation can not carrying out related scientific research work as classical culture protocols because not having enough neural stem cell; Adherent culture makes observation of cell more directly perceived, can do the qualification work that some suspension cells cannot carry out, as immunohistochemical methods etc.; And preparation method of the present invention is more simple, and convenient operation, saves a large amount of working hours.
Learn through overtesting, the test kit using embodiment 1 to provide also uses aforesaid method can obtain following result, about 2cm
3cerebral tissue, can in 3 weeks (21 days) obtain P1 for neural stem cell 2.3 × 10
7individual, can 1 × 10 be obtained to P3 generation
9individual neural stem cell, therefore can show to utilize this test kit and this preparation method can obtain a large amount of neural stem cell at short notice.
Embodiment 2, a kind of test kit for the preparation of neural stem cell of the present invention, comprises nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 100ug/ml, the glutamine of EGF, 2mmol/ml of bFGF, 15ng/ml of 15ng/ml, the Sodium.alpha.-ketopropionate of 2mmol/ml.
Further, described nervous tissue conserving liquid is substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, DMEM/F12 containing Streptomycin sulphate 100ug/ml, penicillin 100ug/ml.
Further, nervous tissue washings is the one in physiological saline containing penicillin 100ug/ml, Streptomycin sulphate 100ug/ml, PBS, D-Hanks.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1.5mg/ml, thymus nucleic acid I0.15mg/ml.
Further, cell washing solution is PBS/D-Hanks.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by the glutamine of N2,2mmol/ml of B27,12.5ug/ml of EGF, 20ug/ml of the basic medium of 1000ml, bFGF, 15ng/ml of 15ng/ml, the Sodium.alpha.-ketopropionate of 2mmol/ml.
Further, cell dissociation buffer is the PBS containing the trypsinase of 2mg/ml, the EDTA of 0.15mg/ml.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO100mg/ml.
The test kit that use embodiment 2 provides is when culture of neural stem cells neural, and a kind of method preparing neural stem cell, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 8 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2.5 times of volumes, put into the CO2gas incubator digest and decompose 53 minutes of 37 DEG C simultaneously, took out concussion once every 4 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Further, utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the cultivation of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the cultivation of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 37 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the cultivation of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 37 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 2 × 10
5individual/milliliter, and 2. take out frozen P2 for Culture of neural stem cells liquid by frozen under the temperature condition of-80 DEG C for nutrient solution, put into and make it melt to the quick concussion of 40 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
Learn through overtesting, the test kit using embodiment 2 to provide also uses aforesaid method can obtain following result, about 2cm
3cerebral tissue, can in 3 weeks (21 days) obtain P1 for neural stem cell 2.7 × 10
7individual, can 1.3 × 10 be obtained to P3 generation
9individual neural stem cell, therefore can show to utilize this test kit and this preparation method can obtain a large amount of neural stem cell at short notice.
Embodiment 3, a kind of test kit for the preparation of neural stem cell of the present invention, comprises nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 200ug/ml, the glutamine of EGF, 1.8mmol/ml of bFGF, 17ng/ml of 17ng/ml, the Sodium.alpha.-ketopropionate of 1.8mmol/ml.
Further, described nervous tissue conserving liquid is substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, DMEM/F12 containing Streptomycin sulphate 200ug/ml, penicillin 200ug/ml.
Further, nervous tissue washings is the one in physiological saline containing penicillin 200ug/ml, Streptomycin sulphate 200ug/ml, PBS, D-Hanks.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1.7mg/ml, thymus nucleic acid I0.17mg/ml.
Further, cell washing solution is PBS/D-Hanks.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by the glutamine of N2,2mmol/ml of B27,14ug/ml of EGF, 22ug/ml of the basic medium of 1000ml, bFGF, 17ng/ml of 17ng/ml, the Sodium.alpha.-ketopropionate of 2.1mmol/ml.
Further, cell dissociation buffer is the PBS containing the trypsinase of 2.3mg/ml, the EDTA of 0.2mg/ml.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO 150mg/ml.
The test kit that use embodiment 3 provides is when culture of neural stem cells neural, and a kind of method preparing neural stem cell, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 10 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2.7 times of volumes, put into the CO2gas incubator digest and decompose 45 ~ 60 minutes of 38 DEG C simultaneously, took out concussion once every 4.2 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Further, utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the cultivation of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the cultivation of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 38 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the cultivation of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 38 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 2.5 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 41 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
Learn through overtesting, the test kit using embodiment 3 to provide also uses aforesaid method can obtain following result, about 2cm
3cerebral tissue, can in 3 weeks (21 days) obtain P1 for neural stem cell 3 × 10
7individual, can 1.7 × 10 be obtained to P3 generation
9individual neural stem cell, therefore can show to utilize this test kit and this preparation method can obtain a large amount of neural stem cell at short notice.
Embodiment 4, a kind of test kit for the preparation of neural stem cell of the present invention, comprises nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 220ug/ml, the glutamine of EGF, 2.0mmol/ml of bFGF, 18ng/ml of 18ng/ml, the Sodium.alpha.-ketopropionate of 2.0mmol/ml.
Further, described nervous tissue conserving liquid is substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, DMEM/F12 containing Streptomycin sulphate 220ug/ml, penicillin 220ug/ml.
Further, nervous tissue washings is the one in physiological saline containing penicillin 220ug/ml, Streptomycin sulphate 220ug/ml, PBS, D-Hanks.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1.8mg/ml, thymus nucleic acid I0.18mg/ml.
Further, cell washing solution is PBS/D-Hanks.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by the glutamine of N2,2.2mmol/ml of B27,15ug/ml of EGF, 23ug/ml of the basic medium of 1000ml, bFGF, 18ng/ml of 18ng/ml, the Sodium.alpha.-ketopropionate of 2.2mmol/ml.
Further, cell dissociation buffer is the PBS containing the trypsinase of 2.4mg/ml, the EDTA of 0.22mg/ml.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO 180mg/ml.
The test kit that use embodiment 4 provides is when culture of neural stem cells neural, and a kind of method preparing neural stem cell, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 11 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2.8 times of volumes, put into the CO2gas incubator digest and decompose 45 ~ 60 minutes of 39 DEG C simultaneously, took out concussion once every 4.5 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Further, utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the cultivation of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the cultivation of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 39 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the cultivation of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 39 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 2.8 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 41 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
Learn through overtesting, the test kit using embodiment 4 to provide also uses aforesaid method can obtain following result, about 2cm
3cerebral tissue, can in 3 weeks (21 days) obtain P1 for neural stem cell 3.2 × 10
7individual, can 2 × 10 be obtained to P3 generation
9individual neural stem cell, therefore can show to utilize this test kit and this preparation method can obtain a large amount of neural stem cell at short notice.
Embodiment 5, a kind of test kit for the preparation of neural stem cell of the present invention, comprises nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 250ug/ml, the glutamine of EGF, 2.5mmol/ml of bFGF, 20ng/ml of 20ng/ml, the Sodium.alpha.-ketopropionate of 2.5mmol/ml.
Further, described nervous tissue conserving liquid is substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, DMEM/F12 containing Streptomycin sulphate 250ug/ml, penicillin 250ug/ml.
Further, nervous tissue washings is the one in physiological saline containing penicillin 250ug/ml, Streptomycin sulphate 250ug/ml, PBS, D-Hanks.
Further, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV2mg/ml, thymus nucleic acid I0.2mg/ml.
Further, cell washing solution is PBS/D-Hanks.
Further, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by the glutamine of N2,2.5mmol/ml of B27,15ug/ml of EGF, 25ug/ml of the basic medium of 1000ml, bFGF, 20ng/ml of 20ng/ml, the Sodium.alpha.-ketopropionate of 2.5mmol/ml.
Further, cell dissociation buffer is the PBS containing the trypsinase of 2.5mg/ml, the EDTA of 0.25mg/ml.
Further, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO 200mg/ml.
The test kit that use embodiment 5 provides is when culture of neural stem cells neural, and a kind of method preparing neural stem cell, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 12 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 3 times of volumes, put into the CO2gas incubator digest and decompose 60 minutes of 40 DEG C simultaneously, took out concussion once every 5 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
Further, utilize in step S5 and cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the cultivation of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the cultivation of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 40 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the cultivation of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 40 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 3 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 42 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
Learn through overtesting, the test kit using embodiment 5 to provide also uses aforesaid method can obtain following result, about 2cm
3cerebral tissue, can in 3 weeks (21 days) obtain P1 for neural stem cell 3.7 × 10
7individual, can 2.3 × 10 be obtained to P3 generation
9individual neural stem cell, therefore can show to utilize this test kit and this preparation method can obtain a large amount of neural stem cell at short notice.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the test kit for the preparation of neural stem cell, it is characterized in that, comprise nervous tissue conserving liquid, nervous tissue washings, nervous tissue decomposed solution, cell washing solution, primary cell culture liquid, cell dissociation buffer, secondary cells nutrient solution, cells frozen storing liquid;
Wherein, secondary cells nutrient solution be neural stem cell containing Serum Growth substratum, this substratum is mixed formed by the basic medium of 1000ml, the foetal calf serum of 50 ~ 250ug/ml, the bFGF of 10 ~ 20ng/ml, the EGF of 10 ~ 20ng/ml, the glutamine of 0.5 ~ 2.5mmol/ml, the Sodium.alpha.-ketopropionate of 0.5 ~ 2.5mmol/ml.
2. the test kit for the preparation of neural stem cell according to claim 1, it is characterized in that, described nervous tissue conserving liquid is the substratum of one or more mixing in RPMI-1640, DMEM, α-MEM, the DMEM/F12 containing Streptomycin sulphate 50 ~ 250ug/ml, penicillin 50 ~ 250ug/ml.
3. the test kit for the preparation of neural stem cell according to claim 1, is characterized in that, nervous tissue washings is the one in physiological saline containing penicillin 50 ~ 250ug/ml, Streptomycin sulphate 50 ~ 250ug/ml, PBS, D-Hanks.
4. the test kit for the preparation of neural stem cell according to claim 1, it is characterized in that, nervous tissue decomposed solution is the substratum containing one or more mixing in DMEM, DMEM/F12, MEM, RPMI-1640 of collagenase I/ collagenase IV1 ~ 2mg/ml, thymus nucleic acid I0.1 ~ 0.2mg/ml.
5. the test kit for the preparation of neural stem cell according to claim 1, is characterized in that, cell washing solution is PBS/D-Hanks.
6. the test kit for the preparation of neural stem cell according to claim 1, it is characterized in that, primary cell culture liquid is the serum free growth medium of neural stem cell, and this substratum is mixed formed by bFGF, the EGF of 10 ~ 20ng/ml of the basic medium of 1000ml, 10 ~ 20ng/ml, the B27 of 15 ~ 25ug/ml, the N2 of 10 ~ 15ug/ml, the glutamine of 0.5 ~ 2.5mmol/ml, the Sodium.alpha.-ketopropionate of 0.5 ~ 2.5mmol/ml.
7. the test kit for the preparation of neural stem cell according to claim 1, is characterized in that, cell dissociation buffer is the PBS containing the trypsinase of 1.5 ~ 2.5mg/ml, the EDTA of 0.1 ~ 0.25mg/ml.
8. the test kit for the preparation of neural stem cell according to claim 1, is characterized in that, cells frozen storing liquid is the neural stem cell foetal calf serum growth medium containing DMSO50 ~ 200mg/ml.
9. prepare a method for neural stem cell, it is characterized in that, the method comprises the steps:
S1, obtain cranial nerve tissue at aseptic condition and put into nervous tissue conserving liquid, and preserve at the temperature condition lower seal of 4 ~ 12 DEG C;
S2, the cranial nerve tissue in step S1 to be taken out from nervous tissue conserving liquid, and use the rinsing of nervous tissue washings;
S3, the cranial nerve tissue after rinsing in step S2 is cut into fragment, and adds the nervous tissue decomposed solution of 2-3 times of volume, put into the CO2gas incubator digest and decompose 45 ~ 60 minutes of 35 ~ 40 DEG C simultaneously, took out concussion once every 3 ~ 5 minutes;
S4, in the liquid after digest and decompose in step S3, add secondary cells nutrient solution, after Homogeneous phase mixing, mixed solution is by 200 object cell sieves, collect filtered liquid, and make filtered liquid under the condition of 1500 revs/min centrifugal 8 minutes, wherein, secondary cells nutrient solution is 2 times of nervous tissue decomposed solution volume;
S5, centrifugal after filtered liquid remove supernatant liquor, add primary cell culture liquid, make the cell concn in solution be 2 × 10
5individual/milliliter, and keep cell suspension culture in solution, namely obtain P0 after cultivation for neural stem cell.
10. the method preparing neural stem cell according to claim 9, is characterized in that, utilizes in step S5 to cultivate the method that the P0 that obtains continues Secondary Culture for neural stem cell and comprise the following steps:
(1) P1 is for the suspension culture of neural stem cell: 1. continue to cultivate P0 for neural stem cell, after P0 is for neural stem cell glomeration, by its under the condition of 1500 revs/min centrifugal 8 minutes; 2. the nutrient solution after centrifugal removes supernatant liquor, adds secondary cells nutrient solution, and makes the cell concn in nutrient solution be 1 × 10
5individual/milliliter, namely obtains P1 for neural stem cell after inoculation culture;
(2) P2 is for the adherent culture of neural stem cell: continue to cultivate P1 for neural stem cell, when P1 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 ~ 40 DEG C was taken out after 30 seconds, namely obtained P2 for neural stem cell after adding the adherent Secondary Culture of secondary cells nutrient solution;
(3) P3 is for the adherent culture of neural stem cell: 1. continue to cultivate P2 for neural stem cell, when P2 reaches 80% for neural stem cell density, secondary cells nutrient solution is poured out, add cell washing solution, after concussion washing, cell washing solution is poured out, after repeated washing 2 times, add cell dissociation buffer, the CO2gas incubator digestion of simultaneously putting into 35 ~ 40 DEG C was taken out after 30 seconds, add secondary cells nutrient solution and make cell suspension, by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, again by nutrient solution under the condition of 1500 revs/min centrifugal 8 minutes, remove supernatant liquor, add cells frozen storing liquid, the cell concn in nutrient solution is made to be 1 × 10
5~ 3 × 10
5individual/milliliter, and nutrient solution is frozen under the temperature condition of-80 DEG C, 2. take out frozen P2 for Culture of neural stem cells liquid, put into and make it melt to the quick concussion of 37 ~ 42 DEG C of water-baths, namely obtain P3 after adding secondary cells nutrient solution adherent culture for neural stem cell.
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