CN104673752A - Human brain stem cell bank constructing method - Google Patents
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Abstract
The invention relates to a human brain stem cell bank constructing method. The human brain stem cell bank constructing method comprises the following steps: (1) infrastructure construction of a GMP laboratory and a freezer: a brain cell bank consists of the GMP laboratory and the freezer; (2) quality control construction of the GMP laboratory: strictly conforming following neural stem cell culturing operating specifications so as to ensure quality control; (3) a neural stem cell culturing process comprising the following steps: (A) collecting brain cells; (B) separating and subculturing neural stem cells; (C) performing BrdU labelling; (D) performing induced differentiation on the neural stem cells; (E) preparing a conditioned medium; (F) performing induced directional differentiation on the neural stem cells; (G) performing immunocytochemical staining; (H) freezing; (I) registering and storing. By the human brain stem cell bank constructing method, a storage concept is mainly changed, the storage time is prolonged through middle recovery, and the function of the cell bank can be realized if the cell bank can be stored for a long time.
Description
Technical field
The present invention relates to a kind of human brain stem cell bank construction process, belong to stem cells technology field.
Background technology
In current professional stem cell bank, there are hemopoietic stem cell bank, fat stem cell storehouse, immunocyte storehouse etc., also both neural stem cell storehouses, nobody's abr cell storehouse.
Brain cell is the common name of the various kinds of cell forming brain, and brain cell mainly comprises neurone and neurogliocyte.Neurone, also known as neurocyte, is the fundamental unit forming nervous system structures and function.Neurocyte belongs to permanent cell (permanent cells), refers to the cell without regenerative power, namely departs from the cell cycle, forever stop mitotic division after the birth of this type of cell.Be acknowledged as Nobel's physiology of the father of modern neuro science and Medicine winner Ge Erji (Camillo Golgi 1843-1926) illustrated brain first to people Study on Microstructure as far back as 1906, propose the unrenewable theory of mammalian central nervous system.This theory almost shrouds whole 20th century, the yoke progress of repairing of neural injury, the methods for the treatment of of the patient of central nervous system injury is caused only to be confined to rehabilitation training and anti symptom treatment, do not accomplish the renewable reservoir promoting injured nerve, do not treat this disease from the source of damage, therefore curative effect is very unsatisfactory, the treatment of this kind of disease is made to get into a difficult position for a long time.
Until last century Mo, a lot of research proves that central nervous system injury can be repaired and regenerate.Research in recent years finds, transplant ectogenic stem cell except have self, Multidirectional Differentiation and to damage location migration potential except, still can secrete the cytokine of the required nerve growth factor of various brain development and other participation Inflammatory responses, by the cell of alternative damage, improve Growth of Cells microenvironment, activate endogenic neural progenitor cell and build the modes such as new neural network, fundamentally promote the reparation of the cerebral tissue that patient is impaired, to reach the object that nervous function is improved.Therefore, one of the most promising methods for the treatment of of central nervous system disease is treated in stem cell transplantation up to now.
Due to the constraint of medical ethics, transplant allogeneic nerve stem cell to be restricted, and the source of autologous nerve stem cell is unlike hemopoietic stem cell and fat stem cell convenient sources, and neural stem cell is short for storage time under freezing environment, generally can only store about 1 year, and hemopoietic stem cell and fat stem cell can reach 30 years, so, society builds a lot of hemopoietic stem cell bank and fat stem cell storehouse, but never had neural stem cell (abr cell) storehouse.
Summary of the invention
The object of the present invention is to provide a kind of construction process of neural stem cell storehouse, and extended neural stem cell storage time.
To achieve these goals, technical scheme of the present invention is as follows.
The construction process in neural stem cell storehouse, is divided into following step:
(1) capital construction of GMP laboratory and freezer: brain cell storehouse is made up of GMP laboratory and freezer.
(A) GMP laboratory: have the modernization laminar flow laboratory meeting international GMP standard, the total area, more than 600 square metres, is divided into: clear area and non-clean district two portions: (a) non-clean district comprises: Office Area, watchroom, storehouse, scrub disinfection room and prepare between, Air Conditioning Facilities, microorganism detection room; B () clear area comprises: swimming booth, boudoir, and cleanliness factor is 300,000 grades; Clean corridor, cleanliness factor is 100,000 grades; Three independently cell preparation rooms, cleanliness factor is ten thousand grades; Cerebral tissue Chu Cai separate chamber, cleanliness factor is ten thousand grades; Liquor room, cleanliness factor is ten thousand grades; Sensing chamber, cleanliness factor is ten thousand grades; Incubator chamber, cleanliness factor is ten thousand grades.
(B) freezer: freezer scale difference is mainly liquid nitrogen container quantity, a general standard stem cell stores liquid nitrogen container can store 300,000 parts of stem cells, needs usable floor area to be 8 square meters.The brain cell storehouse of a covering province, need to possess 600,000 parts of store contents, generally need freezer 30 square meter, cleanliness factor is ten thousand grades.
(2) Quality Control of GMP laboratory is built: cultivate specification in strict accordance with following neural stem cell operation and carry out, to guarantee quality control: " clinical human's stem cell in vitro preparation quality ensures system and worksheet ", " human nerve stem cell separation and Culture standard operating procedure ", " human nerve stem cell separation and Culture standard operating procedure ", " human nerve stem cell Secondary Culture standard operating procedure ", " human nerve stem cell results standard operating procedure ", " the frozen standard operating procedure of human nerve stem cell ", " human nerve stem cell recovery standard operating procedure ".
(3) Culture of neural stem cells flow process, comprises the following steps:
(A) brain cell is gathered: to the hematencephalon, the patients with cerebral injury that need operation, first hand over to family members, operation can only be carried out stopping blooding, removing the cerebral tissue damaged, but cerebral tissue can not regenerate, how much damage is how many just reduces, on the postoperative impact caused in various degree of patient, certain deformity can be there is in cerebral tissue minimizing.There is stem cells technology at present, brain cell can be bred in vitro, the cerebral tissue of damage is carried out the neural stem cell extracted wherein, carry out external increment, then refrigerated storage, used after treating patient.As families of patients is agreed to, in art, the cerebral tissue of the damage necrosis of removing is retained, leave in sterile bag, air transport is put in storage inspection-free in 36 hours, in order to ensure to never degenerate, cerebral tissue after collection will be delivered to rapidly GMP laboratory, and this city must be delivered within 24 hours, and other provinces and towns transport at most can not more than 36 hours (international standard is 72 hours).In addition, transportation also has strict demand, and cerebral tissue can be placed into a special container, and temperature remains between 4 to 25 degrees Celsius.If transported with aircraft, cerebral tissue is inspection-free on airport, because once irradiate through X-ray, in cerebral tissue, the quality of neural stem cell will be affected.
(B) neural stem cell separation and go down to posterity: rinse cerebral tissue, remove residual blood, after the abundant rinsing of D-Hanks liquid, after cerebral tissue being shredded with eye scissors, transfer to the serum free medium that DMEM/F12 (1:1) adds B27 and bFGF (20ng/ml) again, suction pipe piping and druming mechanical separation makes single cell suspension, cell counting after Trypan Blue, and adjustment cell concn is 5 × 10
4 ~ 5individual/ml, is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l.After neural ball is formed, mechanical separation clone makes single cell suspension, still with 5 × 10 again
4the cell concn of individual/ml is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l.After this every 7d mechanical separation clone is gone down to posterity 1 time, and method is the same.
(C) BrdU mark: BrdU is dissolved in serum free medium, add after filtration sterilization neural ball formed after again mechanical separation clone make single cell suspension in (BrdU final concentration is 5 μm of ol/L), cultivate 7d after new neural ball is formed, being transferred to by neural ball is coated with in the culture plate of poly-lysine in advance, the capable BrdU immunocytochemical stain of adherent 2h.
(D) neural stem cell is differentiation-inducing: selected part is above-mentioned go down to posterity after formed time plant in 24 well culture plates being coated with poly-lysine in advance for neural ball, and add and have blood serum medium (DMEM/F12 containing 10% foetal calf serum), the neural ball of part row Nestin immunocytochemical stain after adherent 2h, the neural ball of another part continues to cultivate, and observes its Growth and Differentiation situation.Separately by secondary for a part make single cell suspension for neural ball mechanical separation after add and have blood serum medium adherent culture, row Tuj1, GFAP, Galc immunocytochemical stain respectively after 7d.
(E) preparation of conditioned medium: the hind leg skeletal muscle tissue getting 1g chicken embryo, adds ice bath homogenate 15min in 9mlD-Hanks liquid, centrifugal acquisition supernatant liquor, and after filtration sterilization, adding two volumes DMEM/F12 substratum, to make conditioned medium for subsequent use.
(F) Induction of committed differentiation of neural stem cell: contrast adherent culture in adding conditioned medium respectively after a part time is made single cell suspension for neural ball mechanical separation and having blood serum medium (DMEM/F12 containing 10% foetal calf serum), equal row ChAT immunocytochemical stain after 7d.
(G) immunocytochemical stain: after culturing cell fixes 30min with 4% paraformaldehyde, the abundant rinsing of PBS, then presses ABC method row mouse-anti Nestin respectively, mouse-anti Tuj1, and the anti-GFAP of rabbit, mouse-anti Galc, mouse-anti Brdu immunocytochemical stain, with DAB and H
2o
2as photoghraphic coupler, the positive is colored as brown color.Concrete grammar is: 1) 0.05M PBS rinsing 10min × 3 time; 2) 0.3%H
2o
2/ 0.05M PBS room temperature 30min; 3) 0.3%Triton-100/0.05M PBS 37 DEG C, 15min; 4) 0.05M PBS rinsing 10min × 3 time; 5) 5% sheep blood serum/0.05M PBS 37 DEG C, 15min; 6) I anti-(2% sheep blood serum/0.05M PBS joins) 4 DEG C, 48h; 7) 0.05M PBS rinsing 10min × 3 time; 8) II anti-(2% sheep blood serum/0.05M PBS joins, 1:200) 37 DEG C, 1.5h; 9) 0.05M PBS rinsing 10min × 3 time; 10) AB mixture (0.05M PBS joins, 1:100) 37 DEG C, 1.5h; 11) 0.05M PBS rinsing 10min × 3 time; 12) Tris-HCl damping fluid 37 DEG C, 15min; 13) DAB colour developing 20min; 14) distilled water rinsing 10min × 3 time.
Then, for proving that the cell of experimental group is cholinergic neuron further, by finish ChAT immunocytochemical stain cell PBS color development stopping and after further closing nonspecific binding site, still use ABC method row Tuj1 immunocytochemical stain, with DAB and H2O2 containing NiCl as photoghraphic coupler, two mark positive cell is colored as black-and-blue.
(H) freezing: strictly to fall anti-stem cell and killed.After neural stem cell is purified out, enters refrigeration chamber at once and carry out program control falling, and add intraor extracellular protective material and deicing fluid, carry out freezing according to regular hour and reduction of speed degree, be temporarily stored in transfer liquid nitrogen container.Freezing must strict bent red control is fallen and under carry out, otherwise stem cell can be killed.
(I) registration stores: according to patient identity card information registering, sticked bar-code is preserved, and setting up can for the cellular informatics archives of retrieval.Preserve for a long time for subzero 196 degrees Celsius, before all links all out of question after, neural stem cell will enter the last freezen protective stage.The neural stem cell be temporarily stored in before this in transfer liquid nitrogen container will be sent to freezer and carry out concentrating storage.
Above-mentioned prolongation neural stem cell method storage time is: neural stem cell comparatively other stem cell is fragile, vitality is poor, generally subzero 196 degrees Celsius of preservation perives are 1 year, in order to extend preservation period, we have invented a kind of method of preserving again of rising in value again of recovering half a year, neural stem cell can be extended, owing to this considerably increases cost storage time, so determine by the economic conditions of patient, generally the longest shelf time can reach 10 years.
This beneficial effect of the invention is: abr cell base construction method of the present invention, mainly changes storage thinking, again recovers and extend storage time in centre, can store the function just realizing cell bank for a long time.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described, better to understand the present invention.
Embodiment
The construction process in the neural stem cell storehouse in the present embodiment, is divided into following step:
(1) capital construction of GMP laboratory and freezer: brain cell storehouse is made up of GMP laboratory and freezer.
(A) GMP laboratory: have the modernization laminar flow laboratory meeting international GMP standard, the total area, more than 600 square metres, is divided into: clear area and non-clean district two portions: (a) non-clean district comprises: Office Area, watchroom, storehouse, scrub disinfection room and prepare between, Air Conditioning Facilities, microorganism detection room; B () clear area comprises: swimming booth, boudoir, and cleanliness factor is 300,000 grades; Clean corridor, cleanliness factor is 100,000 grades; Three independently cell preparation rooms, cleanliness factor is ten thousand grades; Cerebral tissue Chu Cai separate chamber, cleanliness factor is ten thousand grades; Liquor room, cleanliness factor is ten thousand grades; Sensing chamber, cleanliness factor is ten thousand grades; Incubator chamber, cleanliness factor is ten thousand grades.
(B) freezer: freezer scale difference is mainly liquid nitrogen container quantity, a general standard stem cell stores liquid nitrogen container can store 300,000 parts of stem cells, needs usable floor area to be 8 square meters.The brain cell storehouse of a covering province, need to possess 600,000 parts of store contents, generally need freezer 30 square meter, cleanliness factor is ten thousand grades.
(2) Quality Control of GMP laboratory is built: cultivate specification in strict accordance with following mescenchymal stem cell operation and carry out, to guarantee quality control: " clinical human's stem cell in vitro preparation quality ensures system and worksheet ", " human nerve stem cell separation and Culture standard operating procedure ", " human nerve stem cell separation and Culture standard operating procedure ", " human nerve stem cell Secondary Culture standard operating procedure ", " human nerve stem cell results standard operating procedure ", " the frozen standard operating procedure of human nerve stem cell ", " human nerve stem cell recovery standard operating procedure ".
(3) Culture of neural stem cells flow process, comprises the following steps:
(A) brain cell is gathered: to the hematencephalon, the patients with cerebral injury that need operation, first hand over to family members, operation can only be carried out stopping blooding, removing the cerebral tissue damaged, but cerebral tissue can not regenerate, how much damage is how many just reduces, on the postoperative impact caused in various degree of patient, certain deformity can be there is in cerebral tissue minimizing.There is stem cells technology at present, brain cell can be bred in vitro, the cerebral tissue of damage is carried out the neural stem cell extracted wherein, carry out external increment, then refrigerated storage, used after treating patient.As families of patients is agreed to, in art, the cerebral tissue of the damage necrosis of removing is retained, leave in sterile bag, air transport is put in storage inspection-free in 36 hours, in order to ensure to never degenerate, cerebral tissue after collection will be delivered to rapidly GMP laboratory, and this city must be delivered within 24 hours, and other provinces and towns transport at most can not more than 36 hours (international standard is 72 hours).In addition, transportation also has strict demand, and cerebral tissue can be placed into a special container, and temperature remains between 4 to 25 degrees Celsius.If transported with aircraft, cerebral tissue is inspection-free on airport, because once irradiate through X-ray, in cerebral tissue, the quality of neural stem cell will be affected.
(B) neural stem cell separation and go down to posterity: rinse cerebral tissue, remove residual blood, after the abundant rinsing of D-Hanks liquid, after cerebral tissue being shredded with eye scissors, transfer to the serum free medium that DMEM/F12 (1:1) adds B27 and bFGF (20ng/ml) again, suction pipe piping and druming mechanical separation makes single cell suspension, cell counting after Trypan Blue, and adjustment cell concn is 5 × 10
4 ~ 5individual/ml, is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l.After neural ball is formed, mechanical separation clone makes single cell suspension, still with 5 × 10 again
4the cell concn of individual/ml is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l.After this every 7d mechanical separation clone is gone down to posterity 1 time, and method is the same.
(C) BrdU mark: BrdU is dissolved in serum free medium, add after filtration sterilization neural ball formed after again mechanical separation clone make single cell suspension in (BrdU final concentration is 5 μm of ol/L), cultivate 7d after new neural ball is formed, being transferred to by neural ball is coated with in the culture plate of poly-lysine in advance, the capable BrdU immunocytochemical stain of adherent 2h.
(D) neural stem cell is differentiation-inducing: selected part is above-mentioned go down to posterity after formed time plant in 24 well culture plates being coated with poly-lysine in advance for neural ball, and add and have blood serum medium (DMEM/F12 containing 10% foetal calf serum), the neural ball of part row Nestin immunocytochemical stain after adherent 2h, the neural ball of another part continues to cultivate, and observes its Growth and Differentiation situation.Separately by secondary for a part make single cell suspension for neural ball mechanical separation after add and have blood serum medium adherent culture, row Tuj1, GFAP, Galc immunocytochemical stain respectively after 7d.
(E) preparation of conditioned medium: the hind leg skeletal muscle tissue getting 1g chicken embryo, adds ice bath homogenate 15min in 9mlD-Hanks liquid, centrifugal acquisition supernatant liquor, and after filtration sterilization, adding two volumes DMEM/F12 substratum, to make conditioned medium for subsequent use.
(F) Induction of committed differentiation of neural stem cell: contrast adherent culture in adding conditioned medium respectively after a part time is made single cell suspension for neural ball mechanical separation and having blood serum medium (DMEM/F12 containing 10% foetal calf serum), equal row ChAT immunocytochemical stain after 7d.
(G) immunocytochemical stain: after culturing cell fixes 30min with 4% paraformaldehyde, the abundant rinsing of PBS, then presses ABC method row mouse-anti Nestin respectively, mouse-anti Tuj1, and the anti-GFAP of rabbit, mouse-anti Galc, mouse-anti Brdu immunocytochemical stain, with DAB and H
2o
2as photoghraphic coupler, the positive is colored as brown color.Concrete grammar is: 1) 0.05M PBS rinsing 10min × 3 time; 2) 0.3%H
2o
2/ 0.05M PBS room temperature 30min; 3) 0.3%Triton-100/0.05M PBS 37 DEG C, 15min; 4) 0.05M PBS rinsing 10min × 3 time; 5) 5% sheep blood serum/0.05M PBS 37 DEG C, 15min; 6) I anti-(2% sheep blood serum/0.05M PBS joins) 4 DEG C, 48h; 7) 0.05M PBS rinsing 10min × 3 time; 8) II anti-(2% sheep blood serum/0.05M PBS joins, 1:200) 37 DEG C, 1.5h; 9) 0.05M PBS rinsing 10min × 3 time; 10) AB mixture (0.05M PBS joins, 1:100) 37 DEG C, 1.5h; 11) 0.05M PBS rinsing 10min × 3 time; 12) Tris-HCl damping fluid 37 DEG C, 15min; 13) DAB colour developing 20min; 14) distilled water rinsing 10min × 3 time.
Then, for proving that the cell of experimental group is cholinergic neuron further, by finish ChAT immunocytochemical stain cell PBS color development stopping and after further closing nonspecific binding site, still use ABC method row Tuj1 immunocytochemical stain, with DAB and H2O2 containing NiCl as photoghraphic coupler, two mark positive cell is colored as black-and-blue.
(H) freezing: strictly to fall anti-stem cell and killed.After neural stem cell is purified out, enters refrigeration chamber at once and carry out program control falling, and add intraor extracellular protective material and deicing fluid, carry out freezing according to regular hour and reduction of speed degree, be temporarily stored in transfer liquid nitrogen container.Freezing must strict bent red control is fallen and under carry out, otherwise stem cell can be killed.
(I) registration stores: according to patient identity card information registering, sticked bar-code is preserved, and setting up can for the cellular informatics archives of retrieval.Preserve for a long time for subzero 196 degrees Celsius, before all links all out of question after, neural stem cell will enter the last freezen protective stage.The neural stem cell be temporarily stored in before this in transfer liquid nitrogen container will be sent to freezer and carry out concentrating storage.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (1)
1. the construction process in neural stem cell storehouse, is characterized in that: be divided into following step:
(1) capital construction of GMP laboratory and freezer: brain cell storehouse is made up of GMP laboratory and freezer: (A) GMP laboratory: the modernization laminar flow laboratory meeting international GMP standard, the total area, more than 600 square metres, is divided into: clear area and non-clean district two portions: (a) non-clean district comprises: Office Area, watchroom, storehouse, scrub disinfection room and prepare between, Air Conditioning Facilities, microorganism detection room; B () clear area comprises: swimming booth, boudoir, and cleanliness factor is 300,000 grades; Clean corridor, cleanliness factor is 100,000 grades; Three independently cell preparation rooms, cleanliness factor is ten thousand grades; Cerebral tissue Chu Cai separate chamber, cleanliness factor is ten thousand grades; Liquor room, cleanliness factor is ten thousand grades; Sensing chamber, cleanliness factor is ten thousand grades; Incubator chamber, cleanliness factor is ten thousand grades; (B) freezer: freezer scale difference is mainly liquid nitrogen container quantity, a general standard stem cell stores liquid nitrogen container can store 300,000 parts of stem cells, needs usable floor area to be 8 square meters; The brain cell storehouse of a covering province, need to possess 600,000 parts of store contents, generally need freezer 30 square meter, cleanliness factor is ten thousand grades;
(2) Quality Control of GMP laboratory is built: cultivate specification in strict accordance with neural stem cell operation and carry out, to guarantee quality control;
(3) Culture of neural stem cells flow process, comprises the following steps:
(A) brain cell is gathered: to the hematencephalon, the patients with cerebral injury that need operation, first hand over to family members, operation can only be carried out stopping blooding, removing the cerebral tissue damaged, but cerebral tissue can not regenerate, how much damage is how many just reduces, on the postoperative impact caused in various degree of patient, certain deformity can be there is in cerebral tissue minimizing; There is stem cells technology at present, brain cell can be bred in vitro, the cerebral tissue of damage is carried out the neural stem cell extracted wherein, carry out external increment, then refrigerated storage, used after treating patient; As families of patients is agreed to, in art, the cerebral tissue of the damage necrosis of removing is retained, leave in sterile bag, air transport is put in storage inspection-free in 36 hours, in order to ensure to never degenerate, cerebral tissue after collection will be delivered to rapidly GMP laboratory, and this city must be delivered within 24 hours, and other provinces and towns transport at most can not more than 36 hours (international standard is 72 hours); In addition, transportation also has strict demand, and cerebral tissue can be placed into a special container, and temperature remains between 4 to 25 degrees Celsius; If transported with aircraft, cerebral tissue is inspection-free on airport, because once irradiate through X-ray, in cerebral tissue, the quality of neural stem cell will be affected;
(B) neural stem cell separation and go down to posterity: rinse cerebral tissue, remove residual blood, after the abundant rinsing of D-Hanks liquid, after cerebral tissue being shredded with eye scissors, transfer to the serum free medium that DMEM/F12 (1:1) adds B27 and bFGF (20ng/ml) again, suction pipe piping and druming mechanical separation makes single cell suspension, cell counting after Trypan Blue, and adjustment cell concn is 5 × 10
4 ~ 5individual/ml, is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l; After neural ball is formed, mechanical separation clone makes single cell suspension, still with 5 × 10 again
4the cell concn of individual/ml is placed in 24 well culture plates and cultivates, and every hole adds cell suspension 500 μ l; After this every 7d mechanical separation clone is gone down to posterity 1 time, and method is the same;
(C) BrdU mark: BrdU is dissolved in serum free medium, add after filtration sterilization neural ball formed after again mechanical separation clone make single cell suspension in (BrdU final concentration is 5 μm of ol/L), cultivate 7d after new neural ball is formed, being transferred to by neural ball is coated with in the culture plate of poly-lysine in advance, the capable BrdU immunocytochemical stain of adherent 2h;
(D) neural stem cell is differentiation-inducing: selected part is above-mentioned go down to posterity after formed time plant in 24 well culture plates being coated with poly-lysine in advance for neural ball, and add and have blood serum medium (DMEM/F12 containing 10% foetal calf serum), the neural ball of part row Nestin immunocytochemical stain after adherent 2h, the neural ball of another part continues to cultivate, and observes its Growth and Differentiation situation; Separately by secondary for a part make single cell suspension for neural ball mechanical separation after add and have blood serum medium adherent culture, row Tuj1, GFAP, Galc immunocytochemical stain respectively after 7d;
(E) preparation of conditioned medium: the hind leg skeletal muscle tissue getting 1g chicken embryo, adds ice bath homogenate 15min in 9mlD-Hanks liquid, centrifugal acquisition supernatant liquor, and after filtration sterilization, adding two volumes DMEM/F12 substratum, to make conditioned medium for subsequent use;
(F) Induction of committed differentiation of neural stem cell: contrast adherent culture in adding conditioned medium respectively after a part time is made single cell suspension for neural ball mechanical separation and having blood serum medium (DMEM/F12 containing 10% foetal calf serum), equal row ChAT immunocytochemical stain after 7d;
(G) immunocytochemical stain: after culturing cell fixes 30min with 4% paraformaldehyde, the abundant rinsing of PBS, then presses ABC method row mouse-anti Nestin respectively, mouse-anti Tuj1, and the anti-GFAP of rabbit, mouse-anti Galc, mouse-anti Brdu immunocytochemical stain, with DAB and H
2o
2as photoghraphic coupler, the positive is colored as brown color; Concrete grammar is: 1) 0.05M PBS rinsing 10min × 3 time; 2) 0.3%H
2o
2/ 0.05M PBS room temperature 30min; 3) 0.3%Triton-100/0.05M PBS 37 DEG C, 15min; 4) 0.05M PBS rinsing 10min × 3 time; 5) 5% sheep blood serum/0.05M PBS 37 DEG C, 15min; 6) I anti-(2% sheep blood serum/0.05M PBS joins) 4 DEG C, 48h; 7) 0.05M PBS rinsing 10min × 3 time; 8) II anti-(2% sheep blood serum/0.05M PBS joins, 1:200) 37 DEG C, 1.5h; 9) 0.05M PBS rinsing 10min × 3 time; 10) AB mixture (0.05M PBS joins, 1:100) 37 DEG C, 1.5h; 11) 0.05M PBS rinsing 10min × 3 time; 12) Tris-HCl damping fluid 37 DEG C, 15min; 13) DAB colour developing 20min; 14) distilled water rinsing 10min × 3 time;
Then, for proving that the cell of experimental group is cholinergic neuron further, by finish ChAT immunocytochemical stain cell PBS color development stopping and after further closing nonspecific binding site, still use ABC method row Tuj1 immunocytochemical stain, with DAB and H2O2 containing NiCl as photoghraphic coupler, two mark positive cell is colored as black-and-blue;
(H) freezing: strictly to fall anti-stem cell and killed, after neural stem cell is purified out, enters refrigeration chamber at once and carry out program control falling, and add intraor extracellular protective material and deicing fluid, carry out freezing according to regular hour and reduction of speed degree, be temporarily stored in transfer liquid nitrogen container; Freezing must strict bent red control is fallen and under carry out, otherwise stem cell can be killed;
(I) registration stores: according to patient identity card information registering, sticked bar-code is preserved, and setting up can for the cellular informatics archives of retrieval; Preserve for a long time for subzero 196 degrees Celsius, before all links all out of question after, neural stem cell will enter the last freezen protective stage; The neural stem cell be temporarily stored in before this in transfer liquid nitrogen container will be sent to freezer and carry out concentrating storage.
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| CN108823163A (en) * | 2018-06-22 | 2018-11-16 | 安徽 | A kind of preparation and extracting method of human research's abr cell |
| WO2019174535A1 (en) * | 2018-03-14 | 2019-09-19 | 浙江霍德生物工程有限公司 | Method for preparing 3d brain organ |
| CN111004715A (en) * | 2019-12-30 | 2020-04-14 | 安徽惠恩生物科技股份有限公司 | Stem cell collection device |
| CN115690785A (en) * | 2022-12-08 | 2023-02-03 | 广东美赛尔细胞生物科技有限公司 | Temperature control method and system applied to freeze-dried cell storage |
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| CN115690785B (en) * | 2022-12-08 | 2023-06-06 | 广东美赛尔细胞生物科技有限公司 | Temperature control method and system applied to freeze-dried cell storage |
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