CN104152488A - Construction method of human nerve stem cell bank - Google Patents
Construction method of human nerve stem cell bank Download PDFInfo
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Abstract
The invention discloses a construction method of a human nerve stem cell bank. The method comprises the following steps: carrying out human urine separation to obtain human kidney epithelial cells, subculturing the human kidney epithelial cells, partially cryopreserving the human kidney epithelial cells, inducing the subcultured human kidney epithelial cells to form nerve stem cells, separating the nerve stem cells obtained after induction, carrying out amplification culturing, identifying the nerve stem cells, cryopreserving the confirmed nerve stem cells, coding and warehousing. The method avoids the use of fetal bovine serum in the whole process, eliminates the introduction of foreign proteins, and reduces hidden troubles; the method allows the successfully induced stem cells with activity to be preserved in order to form the bank, and the stem cells can be provided for clinic use within 3d; and a cryopreservation mode in the method is characterized in that nerve bulbs with a certain size are cryopreserved, a protection agent is introduced step by step in the cryopreservation process, and the cooling rate is set in grading, so recovered nerve stem cells still have high cell viability and activity, and have significantly higher than cell viability and activity than the recovered nerve stem cells preserved through current routine slow low-temperature cryopreservation methods.
Description
Technical field
The present invention relates to a kind of construction process of cell bank, relate in particular to the construction process in a kind of human nerve stem cell storehouse.
Background technology
Neural stem cell (Neural Stem Cell, be called for short NSC) be a kind of stem cell being present in the tissue such as embryo or adult brain, spinal cord, be the parent cell that a class has division potential and self-renewal capacity, can be divided into by not reciprocity divisional mode the various types of cells of the nervous tissues such as neurone, astroglia cell, oligodendrocyte.
The characteristic of neuronic renewable reparation is the obstacle that medical science is difficult to surmount always.The fragile rapid wear of central nervous system tissue's structure and its decision intelligency activity, so central nervous system disease and sequela thereof (comprising Parkinson's disease, alzheimer's disease, cerebral apoplexy, glioblastoma and maincenter and peripheral nerve injury etc.) are the persistent ailment that affects human life's health and life quality all the time, all lack for a long time effective treatment means.Routine clinical methods for the treatment of is mainly reduce or prevent that secondary lesion from being main, there is no the possibility of healing.The nineties in last century, the discovery of neural stem cell brought hope to the treatment of above-mentioned nervous system disorders.
At present, people are by constantly having facts have proved the existence of neural stem cell in human's nervous system and having separated successfully, for the cell replacement therapy of nervous system injury reparation and degeneration has brought new hope.Neural stem cell has the characteristic of self, self-replacation.At present in several animal models and human body, attempted neural stem cells transplantation in the spinal cord of the cranial nerve diseased regions such as striatum and damaged, and obtain certain curative effect.
The research of neural stem cell becomes the most positive in stem-cell research field and most active part, and neural stem cell has bright application prospect at clinicing aspect.But application neural stem cells in treating diseases in nervous system widely, source, quantity and store method of neural stem cell etc. are still and restrict the Major Difficulties that it is widely used in clinical treatment field.
By the processing to people's aborted fetus embryo brain, can separate and obtain neural stem cell, but there is serious ethics problem in this method, the neural stem cell that makes to obtain is in this way applied to and clinically can not obtains social approval and permission.
By to embryonic stem cell (Embryonic stem cells, be called for short ES cell) induction, can obtain equally neural stem cell, but itself also there is the ethics problem of destroying embryo in obtaining of ES cell, and because ES cell can cause teratoma, need to remove undifferentiated ES cell completely therefore obtain neural stem cell by induction ES cell, and accomplish that this point not yet realizes at present technically.And the neural stem cell in ES source is all allosome, has rejection when use.
By the induction to versatile stem cell (iPS cell), can obtain equally neural stem cell.IPS cell itself does not have ethics problem, and the problem that does not yet exist allosome to use.But it faces the technical problem that can not accomplish to remove completely undifferentiated iPS cell equally.
Research is in recent years found, by becoming somatic induction, can obtain equally neural stem cell, and its source is for autologous, and in induction without the multipotential stem cell stage.But it is multiple generally to become somatocyte to have, some cell is more difficult to be obtained, and the active procedure of some cell is very painful, and selects suitable one-tenth somatocyte and separation method thereof to become key.
No matter above-mentioned which kind of method obtains neural stem cell, and if quality and quantity can meet Clinical quality requirements, its required time all needs more than 2 months.But some nervosa damage disease uses stem-cell therapy to need optimal treatment " time window "; Such as all needing within a short period of time, Spinal injury, hypoxic ischemic encephalopathy of newborn, craniocerebral injury etc. use stem-cell therapy just can produce effect; Treat these diseases, induce and produce neural stem cell to treat be infeasible if gather clinical samples temporarily.So will induce and the neural stem cell that completes of amplification in vitro is preserved and set up neural stem cell storehouse to meet clinical demand imperative, and there is very important science and clinical value.
In fact,, for the ease of the effective storage of people and using-system and cell resource, various kinds of cell storehouse or tissue bank have worldwide been set up at present, such as umbilical hemopoietic stem cell storehouse, the cell banks such as mesenchyma stem cell.So-called cell bank refers to the place that stores cell and merger associated profiles and collection related data in-196 DEG C of liquid nitrogen being about.But retrieval finds to there is no at present the information that human nerve stem cell cell bank is set up.
Summary of the invention
The object of the invention is to overcome the deficiency of above technology, the construction process in a kind of human nerve stem cell storehouse is provided.
The construction process in human nerve stem cell of the present invention storehouse, step comprises: utilize human urine to separate and obtain people's kidney epithelial cell, the cultivator that goes down to posterity kidney epithelial cell part are frozen, people's kidney epithelial cell of inducing the cultivation of going down to posterity to obtain becomes neural stem cell, separate the neural stem cell that also amplification cultivation induction obtains, identify this neural stem cell, the neural stem cell that frozen qualification is confirmed, coding warehouse-in; It is characterized in that:
The described epithelial method of human urine separation acquisition people's kidney of utilizing is: by centrifugal people's mud-stream urine 400g, abandon supernatant, with containing dual anti-PBS or the resuspended precipitation of physiological saline, 400g is centrifugal again, abandon supernatant, by the resuspended precipitation of kidney epithelial cell substratum, be inoculated in the culturing bottle of the pre-coated processing of gelatin of using 0.1%, be statically placed in 37 DEG C, 5%CO
2incubator in, be cultured to cell attachment occur, between incubation period, incubator oxygen concentration is controlled to 4.0%～7.5% all the time, and adds kidney epithelial cell substratum every day to original bulk, wherein: the formula of described kidney epithelial cell substratum is that every 100ml substratum comprises: 5ml Ultroser G (Pall company, article No. 15950-017), 0.5ml non-essential amino acid (Life technologies company, article No. 11140050), 0.5ml Glutamax (Life technologies company, article No. 35050-061), 1 μ g Urogastron (EGF, R & D), 0.1ml Regular Insulin (Lonza company, article No. CC-4021L), 0.1ml hydrocortisone (Lonza company, article No. CC-4031L), 0.1ml GA-100 (Lonza company, article No. CC-4081L), 0.1ml suprarenin (Lonza company, article No. CC-2221L), 0.1ml T3 (Lonza company, article No. CC-4211L), 0.1ml Transferrins,iron complexes (Lonza company, article No. CC-4205L), 24ml～70ml REBM substratum (Lonza company, article No. CC-3191), 24ml～70ml α-MEM substratum (HyClone company),
Described cultivator kidney epithelial cell the frozen method of part of going down to posterity is: people's kidney epithelial cell growth to be separated out to degree of converging is 80% when above, go down to posterity with 0.01% trysinization, and whenever meeting to go down to posterity while requiring, cell all goes down to posterity, the 5th generation of as many as (P5) of passage; In the time that people's kidney cell is passaged to the s-generation (P2), the cell of getting approximately 10% amount carries out frozen, and frozen storing liquid formula is: the concentration of 10wt%DMSO+10wt% trehalose+10wt% hydroxyethylamyle+20wt% is 20% human serum albumin solution+50wt% people kidney epithelial cell substratum;
Described induction go down to posterity cultivate obtain people's kidney epithelial cell become the method for neural stem cell to be: 1) getting go down to posterity cultivate obtain people's kidney epithelial cell, adjust cell concn be 1 × 10
6individual/ml～1 × 10
7individual/ml, is transferred in aseptic EP pipe, and 250g is centrifugal, abandons supernatant, and collecting cell also proceeds in electric revolving cup, 2) preparation plasmid transformant system: add successively 82 μ l BasicSolution for Mammalian Epithelial Cells and 18 μ l supplement 1 (Lonza company) to mix gently in above-mentioned electric revolving cup, add again 3 μ g pCEP4-Oct4-Sox2-Klf4 plasmids and 2 μ gpCEP4-miR-302 plasmids 3) electric revolving cup is placed in to Amaxa electroporation (Lonza company) above, select T-013 or T-020 program to carry out electricity and turn, 4) cell after electricity is turned is transferred in the T25 culturing bottle that is coated with matrigel (Matrigel), every bottle graft kind 1 × 10
6individual～5 × 10
6individual, then add kidney epithelial cell substratum and be placed in 37 DEG C, 5%CO
2incubator in cultivate 12～18 hours, substratum in culturing bottle is changed into and contain 20ng/ml leukaemia inhibitory factor (LIF afterwards, R & D) nerve stem cell culture medium cultivate again, between incubation period, incubator oxygen concentration is controlled to 4.0%～7.5% all the time, after 10-15 days, be arranged in as seen neural stem cell like cell group neural garland spline structure or that there is polarity arrangement, wherein: the formula of described nerve stem cell culture medium is that every 100ml substratum comprises: 1ml N2 (Life technologies company, article No. 17502-048), 1ml Glutamax (Life technologies company, article No. 35050-061), 1ml non-essential amino acid (Life technologies company, article No. 11140050), 1ml B-27Supplement (Life technologies company, article No. 17504044), 1 μ g Urogastron (EGF, R & D), 1 μ g Prostatropin (bFGF, R & D), 1 μ g TGF-β (BD), 25-75ml Neurobasal substratum (Life technologies company, article No. 21103049), 25-75ml DMEM/F12 substratum (HyClone company),
The method of the neural stem cell that described separation amplification cultivation induction obtain is: the cell mass of the neural stem cell sample that mechanical process picking above-mentioned steps derives, and transfer in the T25 Tissue Culture Flask that nerve stem cell culture medium is housed; Blow and beat gently cell mass with 5ml transfer pipet, cell mass is blown out to fritter; And culturing bottle is put into 37 DEG C, 5%CO
2incubator in cultivate, incubator oxygen concentration continues to be controlled at 4.0%-7.5%.Neural stem cell suspension growth is in culturing bottle after one week, and cell can form sharply marginated neural ball.After this within, every 2-3 days, partly changing liquid cultivates; In the time of Neural Stem Cells ' Growth 5-7 days, neural stem cell ball is enough large, and (the about 200-300 μ of diameter can go down to posterity m) time; The neural stem cell that will go down to posterity is transferred in 50ml centrifuge tube, after neural ball centrifugal (50g, 3-5min), reject supernatant liquor, and add 1ml Accutase (neural stem cell digestive ferment, sigma) in centrifuge tube, make it at 37 DEG C, digest 5-10min.After stopping digesting with DMEM/F12 substratum, the centrifugal 3-5min of 50g; Abandon supernatant, by nerve stem cell culture medium resuspended neural stem cell, go down to posterity after sub-bottle and continue to cultivate according to the ratio of 1:3---1:6, now neural stem cell is designated as P2 generation; The amplification of going down to posterity in the foregoing manner again when every about 5-7 days afterwards and neural stem cell ball are enough large;
The method of described qualification neural stem cell is: the method specifying by industry to amplify through induction obtain neural stem cell identify, mainly identify the indexs such as its phenotype, differentiation capability, security, indices qualified rear just can carry out frozen;
The method of the neural stem cell that described frozen qualification is confirmed is: get the neural stem cell being obtained by the cultivation of going down to posterity after qualification is confirmed, according to ordinary method, it is carried out to had digestive transfer culture cultivation, in the time observing most cells and grow to diameter and be the neural ball of 80～120 μ m, cell is transferred in centrifuge tube and sampling counting, after 50g is centrifugal, discard supernatant, the cell of collection concentration when frozen is 1.5-10 × 10
6individual/ml calculates the frozen storing liquid volume that needs use; First with the 1/3 nerve stem cell culture medium re-suspended cell gently of estimating frozen storing liquid volume, dropwise add again the nerve stem cell culture medium that contains 18wt% trehalose and 18wt% hydroxyethylamyle of 1/3 expectation frozen storing liquid volume, after adding, the centrifuge tube containing above-mentioned solution is put into 4 DEG C of refrigerator 10-30min, make it cooling, dropwise add again afterwards the 20wt% human serum albumin solution that contains 18wt%DMSO that is chilled in advance 4 DEG C of 1/3 expectation frozen storing liquid volume; After completing, mix gently cell, and transfer them in cryopreservation tube; Cryopreservation tube is transferred in programmed cooling instrument, and the rate of temperature fall of programmed cooling instrument is set as: 4 DEG C to～10 DEG C is 0.25 DEG C/min;-10 DEG C to～40 DEG C is 0.5 DEG C/min;-40 DEG C to～80 DEG C is 1 DEG C/min;-80 DEG C is 2 DEG C/min below; After completing, proceeded in liquid nitrogen container in second day;
The method of described coding warehouse-in is: the neural stem cell that frozen people's kidney epithelial cell and frozen induction are obtained is in order with every artificial one group of Unified number, the frozen cell of mark and the liquid nitrogen container depositing in, and will encode and preserve positional information and depositary's information is unified to input computer so that retrieve.Freeze-stored cell information comprises cellular segregation, cultivation, induction, amplification, the operating record such as frozen, and final neural stem cell qualification result, except it is inputted in computer in the lump, also should form written document Unified coding and file.
The present invention compared with prior art, has the following advantages:
The human nerve stem cell storehouse that utilizes the present invention to set up, all motility rates through detecting, after cell recovery and have the cells ratio of complete activity high of the needed cell number of clinical use, cytoactive and security have increased before neural stem cell is frozen, can directly recover to clinical use completely, do not need to carry out again any amplification processing, therefore can 3 days supply with fast clinical use with interior.
The human nerve stem cell storehouse that utilizes the present invention to set up, neural stem cell is to utilize me to form by autologous cell induction, therefore avoided rejection while use completely; And in the time of induction production neural stem cell, cell is the multipotential stem cell stage through inducing not, but is directly induced and generated neural stem cell by kidney epithelial cell, therefore the safety issue of also having avoided multipotential stem cell to use.
The human nerve stem cell storehouse that utilizes the present invention to set up, all operations were process has been avoided use foetal calf serum, has effectively avoided the introducing of foreign protein, has eliminated clinically and has had hidden danger in application.
The human nerve stem cell storehouse that utilizes the present invention to set up, in omnidistance cultivation, incubator environment is set to hypoxemia, oxygen concentration is close to the physiology oxygen concn of human body, not only whole operating process is shortened greatly, and cell more can adapt to internal milieu after being implanted in human body, can obtain more good curative effect.
The human nerve stem cell storehouse that utilizes the present invention to set up; the mode of frozen neural stem cell is frozen a certain size neural ball; and freezing protective agent is that substep imports; and set best rate of temperature fall; make to keep higher Cell viability and activity after neural stem cell recovery, be significantly higher than the method for cryopreservation at a slow speed of current routine.
Brief description of the drawings
Fig. 1: the structure schema in human nerve stem cell storehouse.
Fig. 2: people's kidney epithelial cell form.
Fig. 3: the neural stem cell like cell group that induction generates.
Fig. 4: form before the neural stem cell of induction goes down to posterity.
Fig. 5: 24h form after the neural stem cell of induction goes down to posterity.
Fig. 6: the neural stem cell form before frozen.
Embodiment
The construction process in embodiment 1 human peripheral blood immune cells storehouse
(1), separate people's kidney epithelial cell: get mud-stream urine 150-200ml in sterile chamber, sterile chamber adds dual anti-in right amount in advance, above-mentioned urine is proceeded to 50ml centrifuge tube, the centrifugal 10min of 400g, centrifugal complete, abandon supernatant, with containing dual anti-PBS or physiological saline re-suspended cell, the then centrifugal 10min of 400g, after centrifugal, abandon supernatant, by the resuspended precipitation of kidney epithelial cell substratum 5ml, the above-mentioned cell of collecting is inoculated in the T25 culturing bottle of the pre-coated processing of gelatin of using 0.1%, there is the culturing bottle of cell to be placed in 37 DEG C, 5%CO inoculation
2incubator in, and incubator oxygen concentration is controlled is always 5.0%, wherein: the formula of described kidney epithelial cell substratum is that every 100ml substratum comprises: 5ml Ultroser G (Pall company, article No. 15950-017), 0.5ml non-essential amino acid (Life technologies company, article No. 11140050), 0.5ml Glutamax (Life technologies company, article No. 35050-061), 1 μ g Urogastron (EGF, R & D), 0.1ml Regular Insulin (Lonza company, article No. CC-4021L), 0.1ml hydrocortisone (Lonza company, article No. CC-4031L), 0.1ml GA-100 (Lonza company, article No. CC-4081L), 0.1ml suprarenin (Lonza company, article No. CC-2221L), 0.1ml T3 (Lonza company, article No. CC-4211L), 0.1ml Transferrins,iron complexes (Lonza company, article No. CC-4205L), 24ml～70ml REBM substratum (Lonza company, article No. CC-3191), 24ml～70ml α-MEM substratum (HyClone company), preferably in kidney epithelial cell substratum, the ratio of REBM substratum and α-MEM substratum is 1:1.
(2), people's kidney epithelial cell of cultivating and increase: in step (1), inoculation has the culturing bottle of cell to leave standstill 3 days in incubator; Every day, supplemented medium 1ml was to observing a little cell attachment afterwards; Observed change completely after cell attachment liquid and in after within every two days, change liquid once; When after 6 days, in culturing bottle, cell degree of converging 50% is above, go down to posterity with 0.05% trysinization; Now cell is designated as P1 generation; Reach 80% until cell degree of converging afterwards at every turn and repeat as above method continuous passage amplification when above.
(3), frozen part kidney epithelial cell: be passaged to the s-generation (P2) and cell degree of converging at people's kidney cell in step (2) and reach 80% when above, the cell of getting approximately 10% amount carries out frozen, and frozen density is 0.5 × 10
7individual/ml; Frozen storing liquid formula is: the concentration of 10wt%DMSO+10wt% trehalose+10wt% hydroxyethylamyle+20wt% is 20% human serum albumin solution+50wt% people kidney epithelial cell substratum.
(3), induction kidney epithelial cell becomes neural stem cell: get the P3 to P5 obtaining in step (2) for cell, adjusting cell concn is 0.5 × 10
7individual/ml, is then transferred in the aseptic EP pipe of 1.5ml the centrifugal 8min of 250g, the supernatant in EP pipe is abandoned in suction, and collecting cell also proceeds in electric revolving cup, preparation plasmid transformant system: add successively 82 μ l BasicSolution for Mammalian Epithelial Cells and 18 μ l supplement 1 (Lonza company) to mix gently in above-mentioned electric revolving cup, then add 3 μ g pCEP4-Oct4-Sox2-Klf4 plasmids and 2 μ g pCEP4-miR-302 plasmids, electric revolving cup is placed on Amaxa electroporation (Lonza company), select T-013 (or T-020) program to carry out electricity and turn that (electricity turns condition: pulse voltage (pulsed voltage) 1650V, pulse width (pulse duration) 10ms, pulse width (pulse number) 3), cell after electricity is turned is transferred on the T25 culturing bottle that is coated with matrigel (Matrigel), every bottle graft kind approximately 1 × 10
6individual cell, then adds kidney epithelial cell substratum and is placed in 37 DEG C, 5%CO
2incubator in cultivate 12～18 hours, substratum in culturing bottle is changed into and contain 20ng/ml leukaemia inhibitory factor (LIF afterwards, R & D) nerve stem cell culture medium cultivate again, between incubation period, incubator oxygen concentration is controlled to 5.0% all the time, after electricity turns the 14th day, there is as seen the neural stem cell like cell group that polarity is arranged, wherein: the formula of described nerve stem cell culture medium is that every 100ml substratum comprises: 1ml N2 (Life technologies company, article No. 17502-048), 1ml Glutamax (Life technologies company, article No. 35050-061), 1ml non-essential amino acid (Life technologies company, article No. 11140050), 1ml B-27Supplement (Life technologies company, article No. 17504044), 1 μ g Urogastron (EGF, R & D), 1 μ g Prostatropin (bFGF, R & D), 1 μ g TGF-β (BD), 25-75ml Neurobasal substratum (Life technologies company, article No. 21103049), 25-75ml DMEM/F12 substratum (HyClone company), preferably in nerve stem cell culture medium, the ratio of Neurobasal substratum and DMEM/F12 substratum is 1:1.
Above-mentioned pCEP4 is a kind of fibrocyte expression vector, buys from Invitrogen company; PCEP4-Oct4-Sox2-Klf4 plasmid is the plasmid of Oct4, Sox2, the tri-kinds of versatile stem cell factor nucleotide sequences of Klf4 of containing taking pCEP4 as vector construction; PCEP4-miR-302 plasmid is the plasmid that contains miR302 nucleotide sequence taking pCEP4 as vector construction.
(4), the neural stem cell of separant induction: the cell mass of the neural stem cell sample deriving in mechanical process picking step (3), and transferring in the T25 Tissue Culture Flask that nerve stem cell culture medium is housed; Blow and beat gently cell mass with 5ml transfer pipet, cell mass is blown out to fritter; And culturing bottle is put into 37 DEG C, 5%CO
2incubator in cultivate, incubator oxygen concentration continues to be controlled at 4.0%-7.5%.Neural stem cell suspension growth is in culturing bottle after one week, and cell can form sharply marginated neural ball.After this within, every 2 days, partly changing liquid cultivates; In the time of Neural Stem Cells ' Growth 7 days, neural ball is enough large, and (the about 200-300 μ of diameter can carry out the following amplification of going down to posterity m) time;
(5), the neural stem cell of amplification cultivation induction: the neural stem cell that will go down to posterity is transferred in 50ml centrifuge tube, centrifugal (the 50g of neural ball, 3-5min), absorb supernatant, and add 1ml Accutase cell dissociation buffer in centrifuge tube, make it at 37 DEG C, digest 5-10min.After stopping digesting with DMEM/F12 substratum, the centrifugal 3-5min of 50g; Abandon supernatant, by nerve stem cell culture medium resuspended neural stem cell, go down to posterity after sub-bottle and continue to cultivate according to the ratio of 1:6, now neural stem cell is designated as P2 generation; The amplification of going down to posterity when every about 5-7 days afterwards and neural stem cell ball are enough large;
(6), the neural stem cell of qualification induction: the neural stem cell of getting the induction that step (5) amplifies, the method specifying by industry is identified it, the indexs such as main its phenotype of qualification, differentiation capability, security, indices qualified rear just can carry out frozen;
(7), the neural stem cell of frozen induction: get the neural stem cell (if P4 is for neural stem cell) that goes down to posterity and cultivate in step (5), according to normal running, cell is carried out to had digestive transfer culture; Cultivate after 3 days, in the time observing most cells and grow to diameter and be the neural ball of 80～120 μ m, cell is transferred in 50ml centrifuge tube, first sample 5ml counting, the centrifugal 5min of all the other 50g, discards supernatant after centrifugal, and the cell of collection concentration when frozen is 1.5-10 × 10
6individual/ml calculates the frozen storing liquid volume that needs use; First with the 1/3 nerve stem cell culture medium re-suspended cell gently of estimating frozen storing liquid volume, dropwise add again the nerve stem cell culture medium that contains 18wt% trehalose and 18wt% hydroxyethylamyle of 1/3 expectation frozen storing liquid volume, after adding, the centrifuge tube containing above-mentioned solution is put into 4 DEG C of refrigerator 10-30min, make it cooling, dropwise add again afterwards the 20wt% human serum albumin solution that contains 18wt%DMSO that is chilled in advance 4 DEG C of 1/3 expectation frozen storing liquid volume; After completing, mix gently cell, and transfer them in cryopreservation tube; Cryopreservation tube is transferred in programmed cooling instrument, and the rate of temperature fall of programmed cooling instrument is set as: 4 DEG C to～10 DEG C is 0.25 DEG C/min;-10 DEG C to～40 DEG C is 0.5 DEG C/min;-40 DEG C to～80 DEG C is 1 DEG C/min;-80 DEG C is 2 DEG C/min below; After completing, proceeded in liquid nitrogen container in second day.
(8), coding warehouse-in: the neural stem cell that induction frozen in people's kidney epithelial cell frozen in step (3) and step (7) is obtained is in order with every artificial one group of Unified number, the frozen cell of mark and the liquid nitrogen container depositing in, and will encode and preserve positional information and depositary's information is unified to input computer so that retrieve.Freeze-stored cell information comprises cellular segregation, cultivation, induction, amplification, the operating record such as frozen, and final neural stem cell qualification result, except it is inputted in computer in the lump, also should form written document Unified coding and file.
The neural stem cell of embodiment 2 people inductions frozen
(1), get neural stem cell P4 that the induction that can go down to posterity obtains for cell, be transferred in 50ml centrifuge tube, to its centrifugal (50g, 3-5min), absorb supernatant, and add 1ml Accutase cell dissociation buffer in centrifuge tube, make it at 37 DEG C, digest 5min.
(2), use after DMEM/F12 substratum termination digestion the centrifugal 3-5min of 50g; Abandon supernatant, collecting cell and during according to cell cryopreservation concentration be 5 × 10
6individual cell/ml frozen storing liquid calculates the frozen storing liquid volume that needs use; After having hanged cell with the frozen storing liquid of above-mentioned volume, mix, divide and install in cryopreservation tube; Frozen storing liquid composition is 10%DMSO+90%FBS.
(3), cryopreservation tube is transferred in programmed cooling instrument, the rate of temperature fall of programmed cooling instrument is 1 DEG C/min; After completing, proceeded in liquid nitrogen in second day;
In embodiment 1 and embodiment 2, the neural stem cell of frozen induction is preserved recovery in 6 months, 12 months, 18 months respectively at liquid nitrogen, relatively after motility rate, recovery, cultivates after cell recovery respectively after the ratio, recovery of cell count after 24h cell count when frozen to the time of going down to posterity for the first time and needing; The results are shown in Table 1～table 3, the neural stem cell in the neural stem cell storehouse building with the present invention as seen, after recovery, Cell viability is apparently higher than common neural stem cell cryopreservation methods, and cell that after recovery, 24h obtains is more and can go down to posterity faster.
Table 1: average cell motility rate after neural stem cell recovery
| ? | 6 months | 12 months | 18 months |
| Embodiment 1 | 92.5％ | 90.0％ | 88.6％ |
| Embodiment 2 | 53.3％ | 51.5％ | 49.6％ |
Table 2: the ratio of cultivating cell count after 24h cell count when frozen after neural stem cell recovery:
| ? | 6 months | 12 months | 18 months |
| Embodiment 1 | 95.5％ | 93.3％ | 92.7％ |
| Embodiment 2 | 50.5％ | 48.3％ | 45.5％ |
Table 3: the time of extremely going down to posterity for the first time and needing after recovery
| ? | 6 months | 12 months | 18 months |
| Embodiment 1 | 7 days | 7 days | 7 days |
| Embodiment 2 | 9 days | 9 days | 9 days |
Claims (3)
1. the construction process in a human nerve stem cell storehouse, step comprises: utilize human urine to separate and obtain people's kidney epithelial cell, the cultivator that goes down to posterity kidney epithelial cell part are frozen, people's kidney epithelial cell of inducing the cultivation of going down to posterity to obtain becomes neural stem cell, separate the neural stem cell that also amplification cultivation induction obtains, identify this neural stem cell, the neural stem cell that frozen qualification is confirmed, coding warehouse-in; It is characterized in that:
The described epithelial method of human urine separation acquisition people's kidney of utilizing is: by centrifugal people's mud-stream urine 400g, abandon supernatant, with containing dual anti-PBS or the resuspended precipitation of physiological saline, 400g is centrifugal again, abandon supernatant, by the resuspended precipitation of kidney epithelial cell substratum, be inoculated in the culturing bottle of the pre-coated processing of gelatin of using 0.1%, be statically placed in 37 DEG C, 5%CO
2incubator in, be cultured to cell attachment occur, between incubation period, incubator oxygen concentration is controlled to 4.0%～7.5% all the time, and adds kidney epithelial cell substratum every day to original bulk, wherein: the formula of described kidney epithelial cell substratum is that every 100ml substratum comprises: 5ml Ultroser G (Pall company, article No. 15950-017), 0.5ml non-essential amino acid (Life technologies company, article No. 11140050), 0.5ml Glutamax (Life technologies company, article No. 35050-061), 1 μ g Urogastron (EGF, R & D), 0.1ml Regular Insulin (Lonza company, article No. CC-4021L), 0.1ml hydrocortisone (Lonza company, article No. CC-4031L), 0.1ml GA-100 (Lonza company, article No. CC-4081L), 0.1ml suprarenin (Lonza company, article No. CC-2221L), 0.1ml T3 (Lonza company, article No. CC-4211L), 0.1ml Transferrins,iron complexes (Lonza company, article No. CC-4205L), 24ml～70ml REBM substratum (Lonza company, article No. CC-3191), 24ml～70ml α-MEM substratum (HyClone company),
Described induction go down to posterity cultivate obtain people's kidney epithelial cell become the method for neural stem cell to be: 1) getting go down to posterity cultivate obtain people's kidney epithelial cell, adjust cell concn be 1 × 10
6individual/ml～1 × 10
7individual/ml, is transferred in aseptic EP pipe, and 250g is centrifugal, abandons supernatant, and collecting cell also proceeds in electric revolving cup, 2) preparation plasmid transformant system: add successively 82 μ l BasicSolution for Mammalian Epithelial Cells and 18 μ l supplement 1 (Lonza company) to mix gently in above-mentioned electric revolving cup, then add 3 μ g pCEP4-Oct4-Sox2-Klf4 plasmids and 2 μ gpCEP4-miR-302 plasmids, 3) electric revolving cup is placed in to Amaxa electroporation (Lonza company) above, selects T-013 or T-020 program to carry out electricity and turn, 4) cell after electricity is turned is transferred in the T25 culturing bottle that is coated with matrigel (Matrigel), every bottle graft kind 1 × 10
6individual～5 × 10
6individual, then add kidney epithelial cell substratum and be placed in 37 DEG C, 5%CO
2incubator in cultivate 12～18 hours, substratum in culturing bottle is changed into and contain 20ng/ml leukaemia inhibitory factor (LIF afterwards, R & D) nerve stem cell culture medium cultivate again, between incubation period, incubator oxygen concentration is controlled to 4.0%～7.5% all the time, after 10-15 days, be arranged in as seen neural stem cell like cell group neural garland spline structure or that there is polarity arrangement, wherein: the formula of described nerve stem cell culture medium is that every 100ml substratum comprises: 1ml N2 (Life technologies company, article No. 17502-048), 1ml Glutamax (Life technologies company, article No. 35050-061), 1ml non-essential amino acid (Life technologies company, article No. 11140050), 1ml B-27Supplement (Life technologies company, article No. 17504044), 1 μ g Urogastron (EGF, R & D), 1 μ g Prostatropin (bFGF, R & D), 1 μ g TGF-β (BD), 25-75ml Neurobasal substratum (Life technologies company, article No. 21103049), 25-75ml DMEM/F12 substratum (HyClone company),
The method of the neural stem cell that described frozen qualification is confirmed is: get the neural stem cell being obtained by the cultivation of going down to posterity after qualification is confirmed, according to ordinary method, it is carried out to had digestive transfer culture cultivation, in the time observing most cells and grow to diameter and be the neural ball of 80～120 μ m, cell is transferred in centrifuge tube and sampling counting, after 50g is centrifugal, discard supernatant, the cell of collection concentration when frozen is 1.5-10 × 10
6individual/ml calculates the frozen storing liquid volume that needs use; First with the 1/3 nerve stem cell culture medium re-suspended cell gently of estimating frozen storing liquid volume, dropwise add again the nerve stem cell culture medium that contains 18wt% trehalose and 18wt% hydroxyethylamyle of 1/3 expectation frozen storing liquid volume, after adding, the centrifuge tube containing above-mentioned solution is put into 4 DEG C of refrigerator 10-30min, make it cooling, dropwise add again afterwards the 20wt% human serum albumin solution that contains 18wt%DMSO that is chilled in advance 4 DEG C of 1/3 expectation frozen storing liquid volume; After completing, mix gently cell, and transfer them in cryopreservation tube; Cryopreservation tube is transferred in programmed cooling instrument, and the rate of temperature fall of programmed cooling instrument is set as: 4 DEG C to～10 DEG C is 0.25 DEG C/min;-10 DEG C to～40 DEG C is 0.5 DEG C/min;-40 DEG C to～80 DEG C is 1 DEG C/min;-80 DEG C is 2 DEG C/min below; After completing, proceeded in liquid nitrogen container in second day.
2. the construction process in human nerve stem cell storehouse as claimed in claim 1, is characterized in that: in the formula of described kidney epithelial cell substratum, the blending ratio of REBM substratum and α-MEM substratum is 1:1.
3. the construction process in human nerve stem cell storehouse as claimed in claim 1, is characterized in that: in the formula of described nerve stem cell culture medium, the blending ratio of Neurobasal substratum and DMEM/F12 substratum is 1:1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106417252A (en) * | 2016-09-26 | 2017-02-22 | 北京焕生汇生物技术研究院 | Novel cell cryopreservation reagent |
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| CN112458037A (en) * | 2020-11-25 | 2021-03-09 | 四川大学华西医院 | Urine cell culture method |
| CN112674080A (en) * | 2021-01-29 | 2021-04-20 | 华夏源细胞工程集团股份有限公司 | Programmed cooling method for human neural stem cell working cell bank |
| CN114159473A (en) * | 2022-02-11 | 2022-03-11 | 美亚宜彬生物科技(北京)有限公司 | Compound preparation for improving male reproductive system stem cells and preparation method thereof |
| CN114159473B (en) * | 2022-02-11 | 2022-05-27 | 美亚宜彬生物科技(北京)有限公司 | Compound preparation for improving male reproductive system stem cells and preparation method thereof |
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