CN106399235A - Method for isolating human umbilical cord mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for isolating human umbilical cord mesenchymal stem cells, comprising the steps of acquiring neonatal fresh in-vitro umbilical cord; storing in low-temperature PBS (phosphate buffer solution), and flushing; shearing in low-temperature soak solution; flushing with low-temperature PBS, and treating in vacuum at low temperature; adding PBS for wetting; adding first mixed enzyme solution, and shaking and enzymatically hydrolyzing; adding second mixed enzyme solution, and shaking and enzymatically hydrolyzing; adding fetal bovine serum, PBS and LG-DMEM (low-glucose Dulbecco's modified eagle medium), standing and centrifuging, and removing supernate; adding PBS, standing and centrifuging, and removing supernate; adding fetal bovine serum, PBS and LG-DMEM, standing and centrifuging, and removing supernate; adding fetal bovine serum, PBS and LG-DMEM, and mixing well. The method can provide improved product quality.
Description
Technical field
The present invention relates to the technology of preparing of mescenchymal stem cell, particularly to a kind of separation of human umbilical cord mesenchymal stem cells
Method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is derived from mesoderm growing early stage mesoderm and outer embryo
One class of layer has the pluripotent stem cell of height self-renewal capacity and multi-lineage potential, and it can connect under suitable condition in vitro
Resume culture, frozen.Because MSCs can be divided into different cells under given conditions, therefore it is increasingly becoming cell therapy and base
Cell derived because of practical value great in therapy field.Umbilical cord belongs to tissue outside embryo, and it contains the dry of a large amount of Multidirectional Differentiations
Cell, cell growth amplification rate is fast, and umbilical cord becomes " garbage " after delivery of baby, so drawing materials conveniently, abundance,
More no ethnics Problem, can be used as splendid source for mesenchymal stem cells.
The Chinese patent that application publication number is CN102021144A, Shen Qing Publication day is on April 20th, 2011 discloses one
The method planting separating funicle mesenchyme stem cell, the method comprises the steps：Umbilical cord removes artery and vein, makes tissue pieces
→ adding the cleaning of the phosphate buffer containing penicillin and streptomycin, supernatant → addition 0.1-0.2% collagenase NB4 is abandoned in centrifugation
Digestion → add hyaluronic acid enzymic digestion → addition ethylenediaminetetraacetic acid phosphate solution to mix → use 100-200 mesh filter screen mistake
Filter, collects filtered solution → filtered solution centrifugation, abandoning supernatant, is cleaned 2-3 time with DMEM culture medium, obtain final product.
In cell count assays, research finds, by this method during separating funicle mesenchyme stem cell, its living cells
Rate is put to the test, and batch impact is larger, and product quality is unstable.
Content of the invention
It is an object of the invention to provide a kind of separation method of human umbilical cord mesenchymal stem cells, which solve living cell rate not
Stable problem, has the effect improving product quality.
The above-mentioned technical purpose of the present invention technical scheme is that：
A kind of separation method of human umbilical cord mesenchymal stem cells, comprises the following steps：
I. take the in vitro umbilical cord that neonate is fresh, being placed on temperature control is 2～5 DEG C and penicillin containing 100U/ml and 100U/ml chain
In the PBS liquid of mycin, preserve stand-by；
Ii. take out the umbilical cord processing through step i, be 2～5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
PBS liquid repeatedly rinses, to no bloodstain residual；
Iii. temperature control be 2～5 DEG C soak environment in, shearing washing after umbilical cord, in shear history reject arteriovenous；Institute
Stating umbilical cord and the amount ratio of soak is 1g:10～20ml, described soak include the glucose of 0.9～1.0g/L, 10～
The ethanol of 20g/L, balance of water；
Iv. take the umbilical cord after shearing, the PBS liquid punching being 2～5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
Wash 2～3 times, be placed in after putting it into culture bottle temperature control be -80～-70 DEG C and control vacuum be 10～30Pa in the environment of 5～
20min, removes the solvent on umbilical cord；
V. take culture bottle encapsulation process, be placed in the environment that temperature control is -40～-35 DEG C, treat that the temperature of culture bottle rises to -40
Then culture bottle is transferred in the environment that temperature control is -5～0 DEG C when～-35 DEG C, when the temperature of culture bottle rises to -5～0 DEG C then
Culture bottle is transferred in the environment that temperature control is 37 DEG C, thaws completely to its interior umbilical cord；
Vi. the culture bottle that step v of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin in the inner；
The amount ratio of wherein said umbilical cord and PBS liquid is 1g:0.5～1ml, vibration 1～5min makes umbilical cord and PBS liquid be fully contacted；
Vii. the culture bottle that step vi of learning from else's experience is processed, adds the first mixing enzymatic solution, oscillation treatment, the water-bath of vibration in the inner
Temperature control curve is：0-5min, 15 DEG C of constant temperature → 5.01-20min, are at the uniform velocity warming up to 25 DEG C → 20.01-30min, and 25 DEG C of constant temperature →
30.01-40min, is at the uniform velocity warming up to 37 DEG C → 40.01-75min, 37 DEG C of constant temperature → 75.01-90min, naturally cools to 25 DEG C；
The amount ratio of wherein said umbilical cord and the first mixing enzymatic solution is 1g:2～5ml, described first mixing enzymatic solution includes 1～2mg/
Trypsin-EDTA the Digestive system of g, the collagenase II of 2～3mg/g, the ethanol of 5～10mg/g, balance of water；
Viii. the culture bottle that step vii of learning from else's experience is processed, adds the second mixing enzymatic solution, oscillation treatment, the water of vibration in the inner
Bathing temperature control curve is：0-2min, 25 DEG C of constant temperature → 2.01-10min, are at the uniform velocity warming up to 30 DEG C → 10.01-15min, 30 DEG C of constant temperature
→ 15.01-20min, is at the uniform velocity warming up to 37 DEG C → 20.01-60min, 37 DEG C of constant temperature → 60.01-75min, naturally cools to 25
℃；The amount ratio of wherein said umbilical cord and the second mixing enzymatic solution is 0.5g:2～5ml, described second mixing enzymatic solution includes
The collagenase II of 0.5～1mg/g, 0.5～1mg/g hyaluronidase, the DNA hydrolytic enzyme of 0.2～0.5mg/g, 2～5mg/g
Ethanol, balance of water；
IX. the culture bottle that step viii of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 0.1g/L streptomycin, stands 2～5min simultaneously at 20～25 DEG C
It is centrifuged 5～8min at this temperature；Wherein said umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:
0.2～0.5ml:1～2ml:2～5ml；
X. the culture bottle that step IX of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin, in 20～
Stand 2～5min at 25 DEG C and be centrifuged 5～8min at this temperature, abandon supernatant；Wherein said umbilical cord and PBS liquid amount ratio are
1g:2～5ml；
XI. the culture bottle that step X of learning from else's experience is processed, adds the PBS of hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of liquid, penicillin containing 100U/ml and 100U/ml streptomycin, at 20～25 DEG C standing 2～5min and
It is centrifuged 5～8min at a temperature of this, abandons supernatant, repeat 2～3 times；Wherein said umbilical cord, hyclone, PBS liquid and LG-DMEM training
The amount ratio of foster base is 1g:0.5～1ml:2～5ml:2～5ml；
XII. the culture bottle that step XI of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 100U/ml streptomycin, mixes, obtains final product；Wherein said umbilical cord, tire
The amount ratio of Ox blood serum, PBS liquid and LG-DMEM culture medium is 1g:0.5ml:1ml:1ml.
Cell count assays research finds, the living cell rate of the sample being prepared by technique scheme improve to
More than 97.5% and parallel test result is close, its product quality is relatively stable；
Cell phenotype identification research finds, in amplification cultivation, its cell reaches the sample preparing by technique scheme
Within 80% time of fusion can be reduced to 5 days, increased it and cultivate speed；And the CD73 of its Mus anti-human PE labelling (fills as
The significant antigen of matter stem cell) positive rate improves to more than 90%, improves mescenchymal stem cell purity, product quality obtains
Improve.
More preferably：Umbilical cord after processing through step i uses the process of step ii within 48hr.
More preferably：In step iii, umbilical cord is cut into 1mm × 1mm × 1mm size.
More preferably：In step iii, described soak also includes the L-Glutamic Acid of 0.2～0.5g/L.
More preferably：Described soak includes the glucose of 1.0g/L, the L-Glutamic Acid of 0.4g/L, the second of 18g/L
Alcohol, balance of water.
More preferably：In step vi, the amount ratio of described umbilical cord and PBS liquid is 1g：1ml.
More preferably：In step vii, the amount ratio of described umbilical cord and the first mixing enzymatic solution is 1g:4ml, described
First mixing enzymatic solution includes the trypsin-EDTA Digestive system of 2mg/g, the ethanol of collagenase II, 8mg/g of 3mg/g, surplus
For water；
In step viii, the amount ratio of described umbilical cord and the second mixing enzymatic solution is 0.5g:3.5ml, described second mixed enzyme is molten
Liquid includes collagenase II, 1mg/g hyaluronidase of 0.8mg/g, the DNA hydrolytic enzyme of 0.5mg/g, the ethanol of 5mg/g, balance of
Water.
More preferably：In step IX, described umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium
For 1g:0.4ml:1.5ml:4ml；
In step X, described umbilical cord and PBS liquid amount ratio are 1g:3ml；
In step XI, described umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:0.5ml:2ml:
5ml.
More preferably：The basal medium of described LG-DMEM culture medium includes the glucose of 1000mg/L, 4.0mM
L-Glutamic Acid, 110mg/L Sodium Pyruvate.
More preferably：The basal medium of described LG-DMEM culture medium also includes the HEPES of 25mM.
Cell count assays research finds, the living cell rate of the sample preparing by technique scheme can be further
Improve to more than 99.5% and parallel test result is close, its product quality is more stable；
Cell phenotype identification research finds, in amplification cultivation, its cell reaches the sample preparing by technique scheme
Within 80% time of fusion can be reduced to 3 days further, further increase it and cultivate speed；And its Mus anti-human PE labelling
CD73 (as the significant antigen of mescenchymal stem cell) positive rate improves further to more than 95%, improves mesenchyme further
Stem cell purity, product quality is improved.
In sum, the invention has the advantages that：
1st, cell count assays research finds, the living cell rate of the sample being prepared by the application is improved and parallel examination
The result tested is close, and its product quality is relatively stable, and wherein the living cell rate of sample segment can improve to more than 99%；
2nd, cell phenotype identification research finds, the sample that the application prepares in amplification cultivation, its amplification cultivation speed is fast,
The mescenchymal stem cell purity obtaining is high, and its product quality obtains certain raising, and the cell of wherein sample segment reaches 80%
Time of fusion within 3 days and Mus anti-human PE labelling CD73 positive rate be higher than 93%.
Specific embodiment
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, people in the art
Member can make to the present embodiment after reading this specification as needed does not have the modification of creative contribution, but as long as at this
All protected by Patent Law in the protection domain of invention.
Embodiment 1：The preparation of various raw materials
(1) umbilical cord：From obstetrics and gynecology hospital delivery room collection.
(2) the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin：
Weigh 1.42g Na2HPO4、0.24g KH2PO4, 8g NaCl and 0.2g KCl, add 800ml deionized water stirring molten
Solution, adds 0.06g penicillin sodium and 0.1g streptomycin, adjusts the pH value of solution to 7.4 with HCl, finally plus deionized water constant volume
To 1L, sterilize under High Temperature High Pressure, obtain final product, be stored in room temperature；Penicillin in this solution and streptomycin are converted to potency respectively
For 100U/ml and 100U/ml；
Wherein, penicillin sodium and streptomycin are purchased from Gibco company.
(3) soak：
Weigh a certain amount of glucose, (be partly added with L-Glutamic Acid) ethanol by required, add portions of de-ionized water stirring molten
Solution, last deionized water constant volume, obtain final product；The concrete concentration of each composition is referring to specific embodiment.
(4) first mixing enzymatic solution
Weigh a certain amount of trypsin-EDTA Digestive system, collagenase II, ethanol by required, add portions of de-ionized water stirring
Dissolving, last deionized water constant volume, obtain final product；The concrete concentration of each composition referring to specific embodiment, wherein trypsin-
EDTA Digestive system and collagenase II are purchased from Gibco company.
(5) second mixing enzymatic solution
Required weigh a certain amount of collagenase II, hyaluronidase, DNA hydrolytic enzyme, ethanol, add portions of de-ionized water stirring molten
Solution, last deionized water constant volume, obtain final product；The concrete concentration of each composition is purchased from referring to specific embodiment, wherein collagenase II
Gibco company, hyaluronidase and DNA hydrolytic enzyme are purchased from Amresco company of the U.S..
(6) basal medium of LG-DMEM culture medium
Purchased from Gibco company, and have two types, respectively type containing HEPES and do not contain HEPES type,
Wherein, in HEPES type, glucose is 1000mg/L, and L-Glutamine is 4.0mM, and Sodium Pyruvate is 110mg/L, HEPES
For 25mM；
Without glucose in HEPES type be 1000mg/L, L-Glutamine be 4.0mM, Sodium Pyruvate be 110mg/L and its do not contain
HEPES.
(7) hyclone：Purchased from Hyclone company.
(8) the LG-DMEM culture medium of penicillin containing 100U/ml and 0.1g/L streptomycin or penicillin containing 100U/ml and
The LG-DMEM culture medium of 100U/ml streptomycin
Weigh a certain amount of and (2) source identical penicillin and streptomycin by required, add the LG-DMEM culture medium of (6)
Basal medium is uniformly dispersed and constant volume, obtains final product.
Embodiment 2-4：A kind of separation method of human umbilical cord mesenchymal stem cells, comprises the following steps：
I. take the in vitro umbilical cord that neonate is fresh, being placed on temperature control is 2～5 DEG C and penicillin containing 100U/ml and 100U/ml chain
In the PBS liquid of mycin, preserve stand-by；
Ii. take out the umbilical cord processing through step i, be 2～5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
PBS liquid repeatedly rinses, to no bloodstain residual；
Iii. temperature control be 2～5 DEG C soak environment in, shearing washing after umbilical cord, in shear history reject arteriovenous；Institute
Stating umbilical cord and the amount ratio of soak is 1g:10～20ml, described soak include the glucose of 0.9～1.0g/L, 0.2～
The L-Glutamic Acid of 0.5g/L, the ethanol of 10～20g/L, balance of water；
Iv. take the umbilical cord after shearing, the PBS liquid punching being 2～5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
Wash 2～3 times, be placed in after putting it into culture bottle temperature control be -80～-70 DEG C and control vacuum be 10～30Pa in the environment of 5～
20min, removes the solvent on umbilical cord；
V. take culture bottle encapsulation process, be placed in the environment that temperature control is -40～-35 DEG C, treat that the temperature of culture bottle rises to -40
Then culture bottle is transferred in the environment that temperature control is -5～0 DEG C when～-35 DEG C, when the temperature of culture bottle rises to -5～0 DEG C then
Culture bottle is transferred in the environment that temperature control is 37 DEG C, thaws completely to its interior umbilical cord；
Vi. the culture bottle that step v of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin in the inner；
The amount ratio of wherein said umbilical cord and PBS liquid is 1g:0.5～1ml, vibration 1～5min makes umbilical cord and PBS liquid be fully contacted；
Vii. the culture bottle that step vi of learning from else's experience is processed, adds the first mixing enzymatic solution, oscillation treatment, the water-bath of vibration in the inner
Temperature control curve is：0-5min, 15 DEG C of constant temperature → 5.01-20min, are at the uniform velocity warming up to 25 DEG C → 20.01-30min, and 25 DEG C of constant temperature →
30.01-40min, is at the uniform velocity warming up to 37 DEG C → 40.01-75min, 37 DEG C of constant temperature → 75.01-90min, naturally cools to 25 DEG C；
The amount ratio of wherein said umbilical cord and the first mixing enzymatic solution is 1g:2～5ml, described first mixing enzymatic solution includes 1～2mg/
Trypsin-EDTA the Digestive system of g, the collagenase II of 2～3mg/g, the ethanol of 5～10mg/g, balance of water；
Viii. the culture bottle that step vii of learning from else's experience is processed, adds the second mixing enzymatic solution, oscillation treatment, the water of vibration in the inner
Bathing temperature control curve is：0-2min, 25 DEG C of constant temperature → 2.01-10min, are at the uniform velocity warming up to 30 DEG C → 10.01-15min, 30 DEG C of constant temperature
→ 15.01-20min, is at the uniform velocity warming up to 37 DEG C → 20.01-60min, 37 DEG C of constant temperature → 60.01-75min, naturally cools to 25
℃；The amount ratio of wherein said umbilical cord and the second mixing enzymatic solution is 0.5g:2～5ml, described second mixing enzymatic solution includes
The collagenase II of 0.5～1mg/g, 0.5～1mg/g hyaluronidase, the DNA hydrolytic enzyme of 0.2～0.5mg/g, 2～5mg/g
Ethanol, balance of water；
IX. the culture bottle that step viii of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 0.1g/L streptomycin, stands 2～5min simultaneously at 20～25 DEG C
It is centrifuged 5～8min at this temperature；Wherein said umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:
0.2～0.5ml:1～2ml:2～5ml；
X. the culture bottle that step IX of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin, in 20～
Stand 2～5min at 25 DEG C and be centrifuged 5～8min at this temperature, abandon supernatant；Wherein said umbilical cord and PBS liquid amount ratio are
1g:2～5ml；
XI. the culture bottle that step X of learning from else's experience is processed, adds the PBS of hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of liquid, penicillin containing 100U/ml and 100U/ml streptomycin, at 20～25 DEG C standing 2～5min and
It is centrifuged 5～8min at a temperature of this, abandons supernatant, repeat 2～3 times；Wherein said umbilical cord, hyclone, PBS liquid and LG-DMEM training
The amount ratio of foster base is 1g:0.5～1ml:2～5ml:2～5ml；
XII. the culture bottle that step XI of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 100U/ml streptomycin, mixes, obtains final product；Wherein said umbilical cord, tire
The amount ratio of Ox blood serum, PBS liquid and LG-DMEM culture medium is 1g:0.5ml:1ml:1ml；
Wherein, the design parameter of embodiment 2-4 is as shown in table 1.
The raw material information of table 1 embodiment 2-4
Embodiment 5：A kind of separation method of human umbilical cord mesenchymal stem cells, the difference with embodiment 1 is, step IX and
In step XI, the basal medium of its LG-DMEM culture medium is without HEPES type.
Embodiment 6：A kind of separation method of human umbilical cord mesenchymal stem cells, the difference with embodiment 1 is, step iii
Soak composition in do not contain L-Glutamic Acid.
Embodiment 7：A kind of separation method of human umbilical cord mesenchymal stem cells, the difference with embodiment 1 is, through step i
Umbilical cord after process uses the process of step ii after preserving 100hr.
Embodiment 8：A kind of separation method of human umbilical cord mesenchymal stem cells, the difference with embodiment 1 is, step iii
In, umbilical cord is cut into 3mm × 3mm × 3mm size.
Performance characterization
(1) with reference to the preparation of sample
With reference to sample 1, the difference of its preparation method and embodiment 1 is, in step iii, soak consists of：Glucose 0.45g/
L, ethanol 5g/L, balance of water；And the amount ratio of umbilical cord and soak is 1g:5ml.
With reference to sample 2, the difference of its preparation method and embodiment 1 is, in step iii, soak consists of：Glucose
2g/L, ethanol 40g/L, balance of water；And the amount ratio of umbilical cord and soak is 1g:40ml.
With reference to sample 3, the difference of its preparation method and embodiment 1 is, in step vii, the composition of the first mixing enzymatic solution
For：Trypsin-EDTA Digestive system 0.5mg/g, collagenase II 1mg/g, ethanol 2.5mg/g, balance of water；And umbilical cord and
The amount ratio of one mixing enzymatic solution is 1g:1ml.
With reference to sample 4, the difference of its preparation method and embodiment 1 is, in step vii, the composition of the first mixing enzymatic solution
For：Trypsin-EDTA Digestive system 4mg/g, collagenase II 6mg/g, ethanol 20mg/g, balance of water；And umbilical cord and first mixes
The amount ratio of synthase solution is 1g:10ml.
With reference to sample 5, the difference of its preparation method and embodiment 1 is, in step viii, the composition of the second mixing enzymatic solution
For：Collagenase II 0.25mg/g, hyaluronidase 0.25mg/g, DNA hydrolytic enzyme 0.1mg/g, ethanol 1mg/g, balance of water；
And the amount ratio of umbilical cord and the second mixing enzymatic solution is 0.5g:1ml.
With reference to sample 6, the difference of its preparation method and embodiment 1 is, in step viii, the composition of the second mixing enzymatic solution
For：Collagenase II 2mg/g, hyaluronidase 2mg/g, DNA hydrolytic enzyme 1mg/g, ethanol 10mg/g, balance of water；And umbilical cord and
The amount ratio of the second mixing enzymatic solution is 0.5g:10ml.
With reference to sample 7, the difference of its preparation method and embodiment 1 is, in step IX, umbilical cord, hyclone, PBS liquid,
The amount ratio of LG-DMEM culture medium is 1:0.1:0.25:1.
With reference to sample 8, the difference of its preparation method and embodiment 1 is, in step IX, umbilical cord, hyclone, PBS liquid,
The amount ratio of LG-DMEM culture medium is 1:1:4:10.
With reference to sample 9, the difference of its preparation method and embodiment 1 is, in step X, the amount ratio of umbilical cord and PBS liquid is 1:
1.
With reference to sample 10, the difference of its preparation method and embodiment 1 is, in step X, the amount ratio of umbilical cord and PBS liquid is
1:10.
With reference to sample 11, the difference of its preparation method and embodiment 1 is, in step XI, umbilical cord, hyclone, PBS liquid,
The amount ratio of LG-DMEM culture medium is 1:0.25:1:1.
With reference to sample 12, the difference of its preparation method and embodiment 1 is, in step XI, umbilical cord, hyclone, PBS liquid,
The amount ratio of LG-DMEM culture medium is 1:2:10:10.
With reference to sample 13, the difference of its preparation method and embodiment 1 is, is provided without step v and step vi.
With reference to sample 14, prepare with reference to CN102021144A embodiment 1.
(2) cell counting
Subjects：The suspension being prepared with embodiment 2-8, as test sample, carries out cytometer with reference to sample 1-13 for comparison
Number test.
Content of the test：Take the 1ml sample after mixing, addition 1ml is purchased from 0.4% trypan blue dye liquor of Gibco company (i.e.
Trypan Blue), mix, dye 4min；Take 100 μ L mixed liquor blood counting chambers to count, count dead cell under mirror, live carefully
Born of the same parents' number, wherein dead cell are dyed to significantly blue and living cells refuse dye in colorless and transparent；Thin with living cell rate (%)=work
Born of the same parents' sum/(total viable cell+dead cell sum) × 100% calculating living cell rate.Each sample all parallel preparation 5 times, every time
Preparation sample count 3 times and its living cell rate takes its meansigma methods.
Result of the test：As shown in table 2.Table 2 shows, for comparing reference sample, the work of the suspension that embodiment 2-8 prepares
Cell rate higher (being above 97.5%), and the result of parallel test is close, its product quality is relatively stable, wherein embodiment 2,
3rd, the living cell rate highest of 4 suspensions preparing and parallel test result closest to and the suspension for preparing of embodiment 2
Liquid is best sample.
The result of living cell rate (%) in table 2 cell count assays
(3) cell phenotype identification
Subjects：The suspension being prepared with embodiment 2-8, as test sample, carries out cell table with reference to sample 1-12 for comparison
Type qualification test.
Content of the test：Take the 100 μ L sample (as primary stem cell) after mixing, be placed in sterile culture flask, to
The hyclone (purchased from Hyclone company) of 1ml and the DMEM/F12 culture medium (1 of 10ml is added in each culture bottle:1, it is purchased from
Hyclone company), be placed in 37 DEG C, volume fraction be 5% CO2, humidity be 95%RH environment in quiescent culture；Change after 4 days
Liquid, removes non-attached cell, changes once within every 2 days later；Treat that the cell in culture dish reaches 80% fusion, discard culture fluid,
After PBS rinses 1 time, plus 37 DEG C, trypsin-EDTA Digestive system digestion 2min, gently pat bottle wall and promote cell to take off from bottom of bottle
From being subsequently adding PBS liquid, penicillin containing 100U/ml and the 100U/ml strepto- of penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of element terminates digestion, collects gained cell after piping and druming, centrifugation, abandons supernatant, again mixed with culture fluid, and 1: 2
Ratio carry out passing on inoculation, be placed in 37 DEG C, volume fraction 5%CO2, humidity be 95%RH environment in quiescent culture, every 2 days
Change and once change liquid 1 time, treat that the primary stem cell in culture dish reaches 80% fusion same method had digestive transfer culture；
After subculture 3 generation, trophophase the 4th generation cell of taking the logarithm, it is configured to 1 × 109L-1Suspension, with Mus anti-human PE labelling
CD73 and Isotype control 10 μ L, 4 DEG C of lucifuges are incubated 30min, and PBS liquid washs 3 times, and 10g/L paraformaldehyde is fixed, flow cytometer
Detection and analysis.Each sample is all parallel to be prepared 3 times, every time the sample cell phenotypic evaluation 3 times of preparation and raising flow cytometer
The CD73 positive rate testing and analyzing the Mus anti-human PE labelling obtaining is averaged.
Result of the test：As shown in table 3.Table 3 shows, compares with reference to for sample, the suspension that embodiment 2-8 prepares is in warp
During amplification cultivation, its cell reaches that 80% time of fusion is short, Mus anti-human PE labelling CD73 is (significant as mescenchymal stem cell
Antigen) positive rate is high and result of its parallel test is close, the suspension amplification cultivation speed that this explanation embodiment 2-8 prepares
The mescenchymal stem cell purity hurry up, obtaining is high, and its product quality is higher than with reference to sample, and the suspension that embodiment 2 prepares is
Best sample.
The result of table 3 cell phenotype identification
Claims (10)
1. a kind of separation method of human umbilical cord mesenchymal stem cells is it is characterised in that comprise the following steps：
I. take the in vitro umbilical cord that neonate is fresh, being placed on temperature control is 2 ~ 5 DEG C and penicillin containing 100U/ml and 100U/ml chain
In the PBS liquid of mycin, preserve stand-by；
Ii. take out the umbilical cord processing through step i, be 2 ~ 5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
PBS liquid repeatedly rinses, to no bloodstain residual；
Iii. in the soak environment for 2 ~ 5 DEG C for the temperature control, shear the umbilical cord after washing, in shear history, reject arteriovenous；Institute
Stating umbilical cord and the amount ratio of soak is 1g:10 ~ 20ml, described soak includes the glucose of 0.9 ~ 1.0g/L, 10 ~ 20g/L
Ethanol, balance of water；
Iv. take the umbilical cord after shearing, the PBS liquid punching being 2 ~ 5 DEG C and penicillin containing 100U/ml and 100U/ml streptomycin with temperature control
Wash 2 ~ 3 times, be placed in after putting it into culture bottle temperature control be -80 ~ -70 DEG C and control vacuum be 10 ~ 30Pa in the environment of 5 ~
20min, removes the solvent on umbilical cord；
V. take culture bottle encapsulation process, be placed in the environment that temperature control is -40 ~ -35 DEG C, treat that the temperature of culture bottle rises to -40
Then culture bottle is transferred in the environment that temperature control is -5 ~ 0 DEG C when ~ -35 DEG C, then will train when the temperature of culture bottle rises to -5 ~ 0 DEG C
Foster bottle is transferred in the environment that temperature control is 37 DEG C, thaws completely to its interior umbilical cord；
Vi. the culture bottle that step v of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin in the inner；
The amount ratio of wherein said umbilical cord and PBS liquid is 1g:0.5 ~ 1ml, vibration 1 ~ 5min makes umbilical cord and PBS liquid be fully contacted；
Vii. the culture bottle that step vi of learning from else's experience is processed, adds the first mixing enzymatic solution, oscillation treatment, the water-bath of vibration in the inner
Temperature control curve is：0-5min, 15 DEG C of constant temperature → 5.01-20min, are at the uniform velocity warming up to 25 DEG C → 20.01-30min, and 25 DEG C of constant temperature →
30.01-40min, is at the uniform velocity warming up to 37 DEG C → 40.01-75min, 37 DEG C of constant temperature → 75.01-90min, naturally cools to 25 DEG C；
The amount ratio of wherein said umbilical cord and the first mixing enzymatic solution is 1g:2 ~ 5ml, described first mixing enzymatic solution includes 1 ~ 2mg/g
Trypsin-EDTA Digestive system, the collagenase II of 2 ~ 3mg/g, the ethanol of 5 ~ 10mg/g, balance of water；
Viii. the culture bottle that step vii of learning from else's experience is processed, adds the second mixing enzymatic solution, oscillation treatment, the water of vibration in the inner
Bathing temperature control curve is：0-2min, 25 DEG C of constant temperature → 2.01-10min, are at the uniform velocity warming up to 30 DEG C → 10.01-15min, 30 DEG C of constant temperature
→ 15.01-20min, is at the uniform velocity warming up to 37 DEG C → 20.01-60min, 37 DEG C of constant temperature → 60.01-75min, naturally cools to 25
℃；The amount ratio of wherein said umbilical cord and the second mixing enzymatic solution is 0.5g:2 ~ 5ml, described second mixing enzymatic solution includes 0.5
The collagenase II of ~ 1mg/g, 0.5 ~ 1mg/g hyaluronidase, the DNA hydrolytic enzyme of 0.2 ~ 0.5mg/g, the ethanol of 2 ~ 5mg/g, remaining
Measure as water；
IX. the culture bottle that step viii of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 0.1g/L streptomycin, at 20 ~ 25 DEG C standing 2 ~ 5min and
It is centrifuged 5 ~ 8min at a temperature of this；Wherein said umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:0.2
~0.5 ml:1~2ml:2~5ml；
X. the culture bottle that step IX of learning from else's experience is processed, adds the PBS liquid of penicillin containing 100U/ml and 100U/ml streptomycin, in 20 ~
Stand 2 ~ 5min at 25 DEG C and be centrifuged 5 ~ 8min at this temperature, abandon supernatant；Wherein said umbilical cord and PBS liquid amount ratio are 1g:2
~5ml；
XI. the culture bottle that step X of learning from else's experience is processed, adds the PBS of hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of liquid, penicillin containing 100U/ml and 100U/ml streptomycin, standing 2 ~ 5min here at 20 ~ 25 DEG C
At a temperature of be centrifuged 5 ~ 8min, abandon supernatant, repeat 2 ~ 3 times；Wherein said umbilical cord, hyclone, PBS liquid and LG-DMEM culture medium
Amount ratio be 1g:0.5~1ml:2~5ml:2~5ml；
XII. the culture bottle that step XI of learning from else's experience is processed, adds hyclone, penicillin containing 100U/ml and 100U/ml streptomycin
The LG-DMEM culture medium of PBS liquid, penicillin containing 100U/ml and 100U/ml streptomycin, mixes, obtains final product；Wherein said umbilical cord, tire
The amount ratio of Ox blood serum, PBS liquid and LG-DMEM culture medium is 1g:0.5ml:1ml:1ml.
2. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that through step i
Umbilical cord after process uses the process of step ii within 48hr.
3. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that step iii
In, umbilical cord is cut into 1mm × 1mm × 1mm size.
4. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that step iii
In, described soak also includes the L-Glutamic Acid of 0.2 ~ 0.5g/L.
5. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 4 is it is characterised in that described immersion
Liquid includes the glucose of 1.0g/L, the L-Glutamic Acid of 0.4g/L, the ethanol of 18g/L, balance of water.
6. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that step vi
In, the amount ratio of described umbilical cord and PBS liquid is 1g:1ml.
7. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that step vii
In, the amount ratio of described umbilical cord and the first mixing enzymatic solution is 1g:4ml, described first mixing enzymatic solution includes the pancreas egg of 2mg/g
White enzyme-EDTA Digestive system, the ethanol of collagenase II, 8mg/g of 3mg/g, balance of water；
In step viii, the amount ratio of described umbilical cord and the second mixing enzymatic solution is 0.5g:3.5ml, described second mixed enzyme is molten
Liquid includes collagenase II, 1mg/g hyaluronidase of 0.8mg/g, the DNA hydrolytic enzyme of 0.5mg/g, the ethanol of 5mg/g, balance of
Water.
8. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that step IX
In, described umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:0.4 ml:1.5ml:4ml；
In step X, described umbilical cord and PBS liquid amount ratio are 1g:3ml；
In step XI, described umbilical cord, the amount ratio of hyclone, PBS liquid and LG-DMEM culture medium are 1g:0.5ml:2ml:
5ml.
9. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 1 is it is characterised in that described LG-
The basal medium of DMEM culture medium includes the glucose of 1000mg/L, the L-Glutamic Acid of 4.0mM, 110mg/L Sodium Pyruvate.
10. a kind of separation method of human umbilical cord mesenchymal stem cells according to claim 9 is it is characterised in that described LG-
The basal medium of DMEM culture medium also includes the HEPES of 25mM.
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| CN107988152A (en) * | 2017-11-10 | 2018-05-04 | 武汉北度生物科技有限公司 | The separation method of oxidation-resistant active ingredient in human umbilical cord mesenchymal stem cells nutrient solution |
| CN109628393A (en) * | 2019-01-22 | 2019-04-16 | 汇麟生物科技(北京)有限公司 | A kind of method and its digestive juice for separating funicle mesenchyme stem cell |
| CN110452877A (en) * | 2018-05-07 | 2019-11-15 | 北京吉尚立德生物科技有限公司 | A kind of cultural method of lung cancer solid tumor primary cell |
| CN110684726A (en) * | 2018-07-06 | 2020-01-14 | 上海中溢精准医疗科技有限公司 | Method for separating and culturing umbilical cord mesenchymal stem cells |
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| CN102021144A (en) * | 2009-09-18 | 2011-04-20 | 上海市第一人民医院 | Method for separating funicle mesenchyme stem cell |
| CN103396990A (en) * | 2013-08-22 | 2013-11-20 | 顺昊细胞生物技术（天津）有限公司 | Method for preparing mesenchymal stem cells |
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| CN102021144A (en) * | 2009-09-18 | 2011-04-20 | 上海市第一人民医院 | Method for separating funicle mesenchyme stem cell |
| CN101974484A (en) * | 2010-11-03 | 2011-02-16 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
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| CN107988152A (en) * | 2017-11-10 | 2018-05-04 | 武汉北度生物科技有限公司 | The separation method of oxidation-resistant active ingredient in human umbilical cord mesenchymal stem cells nutrient solution |
| CN110452877A (en) * | 2018-05-07 | 2019-11-15 | 北京吉尚立德生物科技有限公司 | A kind of cultural method of lung cancer solid tumor primary cell |
| CN110684726A (en) * | 2018-07-06 | 2020-01-14 | 上海中溢精准医疗科技有限公司 | Method for separating and culturing umbilical cord mesenchymal stem cells |
| CN109628393A (en) * | 2019-01-22 | 2019-04-16 | 汇麟生物科技(北京)有限公司 | A kind of method and its digestive juice for separating funicle mesenchyme stem cell |
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