CN108651441A - A kind of store method of stem cell - Google Patents
A kind of store method of stem cell Download PDFInfo
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- CN108651441A CN108651441A CN201810378604.8A CN201810378604A CN108651441A CN 108651441 A CN108651441 A CN 108651441A CN 201810378604 A CN201810378604 A CN 201810378604A CN 108651441 A CN108651441 A CN 108651441A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
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Abstract
The present invention provides a kind of store methods of stem cell, include the following steps：(1) stem cell primary culture：Stem cell is transferred in complete medium and is cultivated, but when cell fusion degree reaches 80% or more, digestive ferment and physiological saline are added into culture bottle, digestive ferment is allowed to spread even and fine born of the same parents face, after cell rounding, terminates digestion；(2) stem cell secondary culture：Inoculation primary cell is cultivated, and cell density reaches 80% or more, passes on again, reaches for the 3rd 5 generation, is collected cell and is counted；(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in incubator and cultivates.The store method can long-time room temperature preserve umbilical cord mesenchymal stem cells vigor, preserve for 24 hours after cell viability still 80% or more.
Description
Technical field
The present invention relates to biomedical sectors, and in particular to a kind of store method of stem cell more particularly to a kind of preservation
Liquid composition is used for the store method of umbilical cord mesenchymal stem cells.
Background technology
Umbilical cord mesenchymal stem cells (MSCs) have higher differentiation potential, can be broken up to multiple directions.It bone,
There is wide potential applicability in clinical practice in terms of the organizational projects such as cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle.Have
Report isolates MSCs from people's umbilical cord, and cell content, proliferative capacity are better than bone marrow MSCs, and immunogenicity compares bone marrow MSCs
It is low, and have many advantages, such as convenient material drawing, no ethics dispute, therefore increasingly by the concern of research workers.
MSCs, cannot be by the stem cell direct infusion of fresh separated in human body in clinical transplantation treatment, it needs elder generation
It is stored in certain time in the preservation liquid (such as 0.9% sodium chloride injection) that can be transfused, then is transfused in internal.MSCs is in vitro
In the case of vigor can decline gradually, this directly affects the stem cell population and quality of separation, how to maintain the work of in vitro MSCs
Property becomes the task of top priority.Current clinically used preservation liquid has 0.9% sodium chloride injection, 5% glucose injection, and 1%
Human serum albumin liquid etc., but MSCs activity in these preservation liquid can decline, and influence clinical therapeutic effect.
There are the following problems for current umbilical cord store method：1. preserving being made of the culture medium on basis for liquid, nutritional ingredient
Complexity, and the ingredient not necessarily be suitble to umbilical cord under ex vivo needed for；2. the process of preservation is cumbersome, condition is harsh；3. pair
The preservation effect of MSCs is bad.It is a kind of urgent problem to be solved to find a kind of method that can effectively preserve MSCs at present.
Invention content
In order to solve the above technical problems, the present invention provides a kind of store method of stem cell, the preservation of the stem cell
Method can long-time room temperature preserve umbilical cord mesenchymal stem cells vigor, preserve for 24 hours after cell viability still 80% or more.
For this purpose, present invention employs following technical schemes：
In a first aspect, the present invention provides a kind of store method of stem cell, include the following steps：
(1) stem cell primary culture：Stem cell is transferred in complete medium and is cultivated, but cell fusion degree reaches
When 80% or more, digestive ferment and physiological saline are added into culture bottle, digestive ferment is allowed to spread even and fine born of the same parents face, after cell rounding, eventually
Only digest；
(2) stem cell secondary culture：Inoculation primary cell is cultivated, and cell density reaches 80% or more, passes on again,
3-5 generations are reached, cell is collected and are counted；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in incubator and cultivates.
In the present invention, by using specific culture and digestion method, coordinate specific preservation liquid so that stem cell can
It preserves at normal temperatures, and rear stem cell vigor remains to, 80% or more, not generate Stem cell surface marker too big shadow for 24 hours
It rings.
According to the present invention, a concentration of the 1 × 10 of the stem cell5-1×107/ ml, preferably 5 × 105-5×106/ ml,
Such as can be 1 × 105/ml、2×105/ml、3×105/ml、4×105/ml、5×105/ml、6×105/ml、7×105/
ml、8×105/ml、9×105/ml、10×105/ml、20×105/ml、30×105/ml、40×105/ml、50×105/ml、
60×105/ml、70×105/ml、80×105/ml、90×105/ ml or 100 × 105/ ml, preferably 5 × 105-5×106/
Ml, further preferably 2 × 106/ml。
According to the present invention, the temperature of step (1) described culture is 35-40 DEG C, for example, can be 35 DEG C, 36 DEG C, 37 DEG C, 38
DEG C, 39 DEG C or 40 DEG C, preferably 36-38 DEG C.
According to the present invention, the CO of step (1) described culture2A concentration of 3-8%, such as can be 3%, 4%, 5%,
6%, 7% or 8%, preferably 4-6%.
According to the present invention, the digestive ferment is any one or at least two in trypsase, pepsin or carboxypeptidase
The combination of kind, preferably trypsase.
According to the present invention, the mass concentration of the digestive ferment is 0.01-0.1%, for example, can be 0.01%, 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, preferably 0.06-0.08%.
According to the present invention, the density of step (2) the inoculation primary cell is 6500-10000/cm2, such as can be
6500/cm2, 6600/cm2, 6700/cm2, 6800/cm2, 6900/cm2, 7000/cm2, 7200/cm2、
7500/cm2, 7600/cm2, 7800/cm2, 8000/cm2, 8200/cm2, 8500/cm2, 8600/cm2、
8800/cm2, 9000/cm2, 9200/cm2, 9500/cm2, 9800/cm2Or 10000/cm2, preferably 7000-
9000/cm2。
According to the present invention, step (3) the preservation liquid includes following component in parts by weight：Physiological saline 5-15 weight
Part, human serum albumin 3-6 parts by weight and Multiple electrolytes injection 80-95 parts by weight.
In the present invention, pass through the combination of specific physiological saline and human serum albumin so that preserving liquid composition can be
Mescenchymal stem cell is preserved under room temperature, and rear stem cell vigor remains to, 80% or more, not generate Stem cell surface marker for 24 hours
Too much influence.
The parts by weight of the physiological saline be 5-15 parts by weight, such as can be 5 parts by weight, 6 parts by weight, 7 parts by weight,
8 parts by weight, 9 parts by weight, 10 parts by weight, 11 parts by weight, 12 parts by weight, 13 parts by weight, 14 parts by weight or 15 parts by weight, preferably
6-10 parts by weight, preferably 9 parts by weight.
The parts by weight of the human serum albumin are 3-6 parts, such as can be 3 parts by weight, 4 parts by weight, 5 parts by weight or 6
Parts by weight, preferably 4-6 parts by weight, preferably 4 parts by weight.
The parts by weight of the Multiple electrolytes injection are 80-95 parts by weight, such as can be 85 parts by weight, 86 weight
Part, 87 parts by weight, 88 parts by weight, 89 parts by weight, 90 parts by weight, 91 parts by weight, 92 parts by weight, 93 parts by weight, 94 parts by weight or
95 parts by weight, preferably 85-90 parts by weight, preferably 87 parts by weight.
According to the present invention, the temperature of step (1) described incubator is 35-40 DEG C, for example, can be 35 DEG C, 36 DEG C, 37 DEG C,
38 DEG C, 39 DEG C or 40 DEG C, preferably 36-38 DEG C.
According to the present invention, the CO of step (1) described incubator2A concentration of 3-8%, such as can be 3%, 4%, 5%,
6%, 7% or 8%, preferably 4-6%.
As optimal technical scheme, the stem cell store method includes the following steps：
(1) stem cell primary culture：By 1 × 105-1×107/ ml stem cells are transferred in complete medium in 35-40 DEG C, 3-
8%CO2It is cultivated under concentration, but when cell fusion degree reaches 80% or more, it is 0.01- that mass concentration is added into culture bottle
0.1% digestive ferment and physiological saline allows digestive ferment to spread even and fine born of the same parents face, after cell rounding, terminates digestion；
(2) stem cell secondary culture：By 6500-10000/cm of inoculum density2Inoculation primary cell is cultivated, cell
Density reaches 80% or more, passes on again, reaches 3-5 generations, collects cell and counts；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in 35-40 DEG C, 3-8%CO in incubator2It is dense
The lower culture of degree；
Wherein, the preservation liquid includes following component in parts by weight：Physiological saline 5-15 parts by weight, human serum albumin 3-
6 parts by weight and Multiple electrolytes injection 80-95 parts by weight.
Second aspect, the present invention provide store method as described in relation to the first aspect for preserving umbilical cord mesenchymal stem cells
Purposes.
According to the present invention, a concentration of the 1 × 10 of the mescenchymal stem cell5-1×107/ ml, such as can be 1 × 105/
ml、2×105/ml、3×105/ml、4×105/ml、5×105/ml、6×105/ml、7×105/ml、8×105/ml、9×105/
ml、10×105/ml、20×105/ml、30×105/ml、40×105/ml、50×105/ml、60×105/ml、70×105/
ml、80×105/ml、90×105/ ml or 100 × 105/ ml, preferably 5 × 105-5×106/ ml, further preferably 2 ×
106/ml。
Compared with prior art, the present invention at least has the advantages that：
(1) vigor after the method for the present invention stem cell preserves is high, and rear cell viability is still 80% or more for 24 hours, and cell pastes
Wall ability is good, and does not generate large effect to Stem cell surface marker after preserving, and is still mescenchymal stem cell；
(2) the preservation liquid ingredient in method of the invention is simple, and umbilical cord mesenchymal stem cells is suitble to preserve at normal temperatures, protects
It is simple to operation to deposit process.
Figure of description
Fig. 1 (a) is after umbilical cord mesenchymal stem cells preserve 6h using physiological saline, and 96 orifice plates are inoculated with cellular morphology after 6h,
Fig. 1 (b) is after umbilical cord mesenchymal stem cells preserve 6h using preservation liquid, and 96 orifice plates are inoculated with cellular morphology after 6h, and Fig. 1 (c) is navel
After band mescenchymal stem cell preserves 6h using physiological saline, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 1 (d) is umbilical cord mesenchyma
After stem cell is using liquid preservation 6h is preserved, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 1 (e) uses for umbilical cord mesenchymal stem cells
After physiological saline preserves 6h, 96 orifice plates are inoculated with cellular morphology after 30h, and Fig. 1 (f) is that umbilical cord mesenchymal stem cells are protected using liquid is preserved
After depositing 6h, 96 orifice plates are inoculated with cellular morphology after 30h；
Fig. 2 (a) is after umbilical cord mesenchymal stem cells are preserved for 24 hours using physiological saline, and 96 orifice plates are inoculated with cellular morphology after 6h,
Fig. 2 (b) is after umbilical cord mesenchymal stem cells are preserved for 24 hours using preservation liquid, and 96 orifice plates are inoculated with cellular morphology after 6h, and Fig. 2 (c) is navel
After band mescenchymal stem cell is preserved for 24 hours using physiological saline, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 2 (d) fills between umbilical cord
After matter stem cell is using liquid preservation for 24 hours is preserved, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 2 (e) is umbilical cord mesenchymal stem cells
After being preserved for 24 hours using physiological saline, 96 orifice plates are inoculated with cellular morphology after 30h, and Fig. 2 (f) is umbilical cord mesenchymal stem cells using guarantor
After liquid storage preserves for 24 hours, 96 orifice plates are inoculated with cellular morphology after 30h.
Specific implementation mode
Of the invention for ease of understanding, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is only to aid in the understanding present invention, should not be regarded as a specific limitation of the invention.
Experiment material
50ml centrifuge tubes (healthy and free from worry), 0.22um detachable needle syringes (PALL), 60ml syringes (BOON), Dispette
(Axygen), superclean bench (Su Jing is safe and sound), adjustable pipette (Biohit), ordinary optical microscope (Olympus), blood
Ball count version (QIUJING), countess calculating instruments (GIBCO companies)
Cell is P2 for cell, human serum albumin (the blue biology of China), Multiple electrolytes injection (Shanghai Bai Te), physiology salt
Water (the general Ji in Dongguan)
Embodiment 1 preserves the preparation of liquid (I)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
9 parts by weight of physiological saline
4 parts by weight of human serum albumin
87 parts by weight of Multiple electrolytes injection；
The preparation method for preserving liquid, includes the following steps：
By formula ratio by after physiological saline, human serum albumin and Multiple electrolytes injection mixing, filtering is divided in centrifugation
Guan Zhong, packing preserve.
Embodiment 2 preserves the preparation of liquid (II)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
6 parts by weight of physiological saline
6 parts by weight of human serum albumin
90 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 3 preserves the preparation of liquid (III)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
10 parts by weight of physiological saline
4 parts by weight of human serum albumin
85 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 4 preserves the preparation of liquid (IV)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
5 parts by weight of physiological saline
6 parts by weight of human serum albumin
95 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 5 preserves the preparation of liquid (V)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
15 parts by weight of physiological saline
3 parts by weight of human serum albumin
80 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Comparative example 1
Liquid is preserved only with physiological saline, other are same as Example 1.
Comparative example 2
Compared with Example 1, other same as Example 1 in addition to there is no human serum albumin.
Comparative example 3
Compared with Example 1, other same as Example 1 in addition to there is no Multiple electrolytes injection.
Comparative example 4
Compared with Example 1, other same as Example 1 in addition to the parts by weight of human serum albumin are 8 parts by weight.
Comparative example 5
Compared with Example 1, other same as Example 1 in addition to the parts by weight of physiological saline are 3 parts by weight.
Comparative example 6
Compared with Example 1, other same as Example 1 in addition to the parts by weight of physiological saline are 18 parts by weight.
Embodiment 6
The present embodiment provides a kind of store methods of stem cell, are carried out using the preservation liquid of embodiment 1-5 and comparative example 1-6
Cell is preserved, is as follows：
(1) stem cell primary culture：By 2 × 106/ ml stem cells are transferred in complete medium in 37 DEG C, 5%CO2Under concentration
Culture, but when cell fusion degree reaches 80% or more, digestive ferment and physiology salt of the mass concentration for 0.6% are added into culture bottle
Water allows digestive ferment to spread even and fine born of the same parents face, after cell rounding, terminates digestion；
(2) stem cell secondary culture：By 8000/cm of inoculum density2Inoculation primary cell is cultivated, and cell density reaches
It to 80% or more, passes on again, reached for the 3rd generation, collect cell and count；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in 37 DEG C, 5%CO in incubator2Under concentration
Culture；
Embodiment 7
The present embodiment provides a kind of store methods of stem cell, are carried out using the preservation liquid of embodiment 1-5 and comparative example 1-6
Cell is preserved, is as follows：
(1) stem cell primary culture：By 1 × 105/ ml stem cells are transferred in complete medium in 40 DEG C, 8%CO2Under concentration
Culture, but when cell fusion degree reaches 80% or more, digestive ferment and physiology salt of the mass concentration for 0.1% are added into culture bottle
Water allows digestive ferment to spread even and fine born of the same parents face, after cell rounding, terminates digestion；
(2) stem cell secondary culture：By 6500/cm of inoculum density2Inoculation primary cell is cultivated, and cell density reaches
It to 80% or more, passes on again, reached for the 5th generation, collect cell and count；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in 40 DEG C, 8%CO in incubator2Under concentration
Culture；
Embodiment 8
The present embodiment provides a kind of store methods of stem cell, are carried out using the preservation liquid of embodiment 1-5 and comparative example 1-6
Cell is preserved, is as follows：
(1) stem cell primary culture：By 1 × 107/ ml stem cells are transferred in complete medium in 35 DEG C, 3%CO2Under concentration
Culture, but when cell fusion degree reaches 80% or more, mass concentration is added into culture bottle as 0.01% digestive ferment and physiology
Brine allows digestive ferment to spread even and fine born of the same parents face, after cell rounding, terminates digestion；
(2) stem cell secondary culture：By 10000/cm of inoculum density2Inoculation primary cell is cultivated, cell density
Reach 80% or more, pass on again, reached for the 4th generation, collect cell and counts；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in 35 DEG C, 3%CO in incubator2Under concentration
Culture；
Cell performance test
1) cell viability is tested
Specific cell performance test, includes the following steps：
(1) cell of embodiment 6-8 is collected：
Cell is blown and beaten with pipette, cell is all blown down as possible, especially corner, action is soft, avoids
Bubble is generated, is collected in cell suspension to centrifuge tube, 1000rpm/10min is centrifuged；Supernatant is removed after centrifugation, and the life of 5ml is added
Manage brine, cell is blown it is even, add physiological saline arrive 40ml, blow it is even after and draw 1ml count.
(2) cell count
1) corresponding cell is taken out to time point, first first blows and beats 8 mixings with the rifle of 100 μ l, then toward in EP pipes plus
The physiological saline for entering 900 μ l is blown and beaten 10 times with the rifle of 1000 μ l, in the placenta basket for drawing 20 μ l to PE gloves, is mixed well thin
Born of the same parents 8~10 times；
2) it takes the tiling of PE gloves on the table, takes the trypan blue point of 20 μ l on gloves；The method for taking trypan blue：Platform is taken to expect
Before basket should not mixing, and only draw part above, when absorption draws the centre of working part as possible；
3) cell for drawing 10 μ l mixings, enters coverslip edge with tally at oblique 45 degree of subscripts；Another side method is the same,
Add simultaneously；
4) pay attention to when counting：Number is reached the standard grade and left line, does not count offline and right line, the Writing method of counting is per big lattice：
The time of viable count-dead cell number, counting is controlled as possible in 3~4min.
(3) continued to cultivate cell with 96 orifice plates
1) after preserving the half an hour that liquid fixed point counts, while the corresponding cell of physiological saline is taken, preparation continues to cultivate；
2) prepare two pieces of 96 orifice plates every time；
3) physiological saline of 400 μ l is added into per EP pipes in the case where grasping net platform, is diluted to 500 μ l and takes 100 μ l to one pipes new
EP pipes in (altogether about 2 × 105A cell) add the culture solution of 400 μ l to be diluted to 500 μ l (every 100 μ l/40000 cells), it takes
25 μ l are to per hole；
4) cell of each EP pipe adds 4 samples, side to add a blank control, two plate loading methods on each plate
Equally；
5) add after sample side marker it is good which side be to preserve liquid which side is that physiological saline, side sample time point and MTT add
The sample time is finally placed in incubator 37 DEG C, 5%CO2(lower layer being placed on as possible, close to the place of water) is cultivated.
Preservation liquid in embodiment 1-5 and comparative example 1-6 is subjected to cell preservation, 0,6,12,20, for 24 hours respectively to thin
Born of the same parents count, and calculate average viability, the results are shown in Table 1, specific as follows：
Table 1
Conspicuousness p<0.05
As it can be seen from table 1 embodiment 1-5 is after preserving cell for 24 hours, cell viability still has 80% or more, and from implementation
The comparison of example 1-5 finds out that the effect that the embodiment of the present application 1 preserves liquid is best, and cell viability is 83.8% after preserving for 24 hours；
Find out from embodiment and comparative example, comparative example preserves cell vigor after 12h and declines comparatively fast, and rear cell viability only stays for 24 hours
It is even lower to deposit cell viability 50%.
2) adherent experiment
(1) corresponding 96 orifice plate is taken out from incubator, and WST-1 reagents are taken out from refrigerator；
(2) use lancet that the reagent of the WST-1 of 10ul is added into the hole that each is loaded；
(3) tag time is put into incubator and continues culture 4 hours；
(4) proliferation activity is surveyed, i.e., two groups of samples of each period are inoculated with simultaneously in 96 orifice plates (2 pieces), respectively at training totally
After WST-1 reagents incubation 4h is added after foster 6h and 36h, observation is inoculated in the cell in T25 culture bottles, and comparing embodiment 1 (preserves
Liquid group) and comparative example 1 (physiological saline group) group in cell adherent proliferative conditions, as a result such as Fig. 1 (a)-Fig. 1 (f) and Fig. 2 (a)-
Shown in Fig. 2 (f)；
From Fig. 1 (a)-Fig. 1 (f) and Fig. 2 (a)-Fig. 2 (f) as can be seen that either culture 6h still for 24 hours after, embodiment 1
The adherent situation of cell be better than comparative example 1.
3) drain cell detects
After 1 group of embodiment 1 and comparative example are preserved 20h at 4 DEG C, it is carried out at the same time FCM analysis, observation length
Whether phase preservation has an impact the expression of cell surface marker, and the results are shown in Table 2：
Table 2
| CD73 | CD90 | CD105 | CD14 | CD45 | CD34 | HLA-DR | |
| Embodiment 1 | 96.5% | 100% | 96.8% | 0.8% | 1.9% | 0.7% | 0.6% |
| Embodiment 2 | 95.9% | 100% | 96.7% | 0.8% | 1.8% | 0.7% | 0.6% |
| Embodiment 3 | 96.7% | 99.9% | 98.2% | 0.7% | 1.9% | 0.7% | 0.7% |
| Embodiment 4 | 96.4% | 100% | 98.3% | 0.8% | 1.7% | 0.7% | 0.8% |
| Embodiment 5 | 97.2% | 100% | 97.5% | 0.8% | 1.8% | 0.8% | 0.7% |
| Comparative example 1 | 97.8% | 100% | 99% | 0.7% | 1.5% | 0.7% | 0.9% |
| Comparative example 2 | 97.3% | 100% | 98.5% | 0.7% | 1.5% | 0.6% | 0.8% |
| Comparative example 3 | 96.8% | 100% | 97.6% | 0.6% | 1.6% | 0.7% | 0.8% |
| Comparative example 4 | 95.8% | 100% | 96.3% | 0.7% | 1.7% | 0.8% | 0.7% |
| Comparative example 5 | 95.9% | 100% | 96.8% | 0.8% | 1.8% | 0.8% | 0.6% |
| Comparative example 6 | 95.4% | 99.9% | 96.4% | 0.7% | 1.6% | 0.7% | 0.6% |
From table 2 it can be seen that the CD73 in embodiment and comparative example, CD90, CD105 expression in 95% or more, CD14,
CD45, CD34, HLA-DR expression are 2% hereinafter, cell of the explanation after embodiment and comparative example preserves liquid preservation is still
Mescenchymal stem cell.
In conclusion the vigor after the method for the present invention is preserved by preserving liquid is high, for 24 hours after cell viability still 80% with
On, and the adherent ability of cell is good, and large effect is not generated to Stem cell surface marker after preserving, it is still dry thin for mesenchyma
Born of the same parents.
Applicant states that the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment,
But the invention is not limited in above-mentioned detailed process equipment and technological processes, that is, it is above-mentioned detailed not mean that the present invention has to rely on
Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention,
The addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, the selection etc. of concrete mode all fall within the present invention's
Within protection domain and the open scope.
Claims (10)
1. a kind of store method of stem cell, which is characterized in that include the following steps：
(1) stem cell primary culture：Stem cell is transferred in complete medium and is cultivated, but cell fusion degree reach 80% with
When upper, digestive ferment and physiological saline were added into culture bottle, digestive ferment is allowed to spread even and fine born of the same parents face, after cell rounding, terminates digestion；
(2) stem cell secondary culture：Inoculation primary cell is cultivated, and cell density reaches 80% or more, passes on again, reaches
In 3-5 generations, collect cell and count；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in incubator and cultivates.
2. store method according to claim 1, which is characterized in that a concentration of the 1 × 10 of the stem cell5-1×107/
Ml, preferably 5 × 105-5×106/ ml, further preferably 2 × 106/ml。
3. store method according to claim 1 or 2, which is characterized in that the temperature of step (1) described culture is 35-40
DEG C, preferably 36-38 DEG C；
Preferably, the CO of step (1) described culture2A concentration of 3-8%, preferably 4-6%.
4. store method according to any one of claim 1-3, which is characterized in that the digestive ferment be trypsase,
In pepsin or carboxypeptidase any one or at least two combination, preferably trypsase；
Preferably, the mass concentration of the digestive ferment is 0.01-0.1%, preferably 0.06-0.08%.
5. according to the store method described in any one of claim 1-4, which is characterized in that step (2) inoculation is primary thin
The density of born of the same parents is 6500-10000/cm2, preferably 7000-9000/cm2。
6. store method according to any one of claims 1-5, which is characterized in that step (3) the preservation liquid is by weight
It includes following component to measure number：Physiological saline 5-15 parts by weight, human serum albumin 3-6 parts by weight and Multiple electrolytes injection
80-95 parts by weight；
Preferably, the parts by weight of the physiological saline are 6-10 parts by weight, preferably 9 parts by weight；
Preferably, the parts by weight of the human serum albumin are 4-6 parts by weight, preferably 4 parts by weight；
Preferably, the parts by weight of the Multiple electrolytes injection are 85-90 parts by weight, preferably 87 parts by weight.
7. according to the store method described in any one of claim 1-6, which is characterized in that the temperature of step (3) described incubator
Degree is 35-40 DEG C, preferably 36-38 DEG C；
Preferably, the CO of step (2) described incubator2A concentration of 3-8%, preferably 4-6%.
8. according to the store method described in any one of claim 1-7, which is characterized in that include the following steps：
(1) stem cell primary culture：By 1 × 105-1×107/ ml stem cells are transferred in complete medium in 35-40 DEG C, 3-8%
CO2It is cultivated under concentration, but when cell fusion degree reaches 80% or more, it is 0.01-0.1%'s that mass concentration is added into culture bottle
Digestive ferment and physiological saline allow digestive ferment to spread even and fine born of the same parents face, after cell rounding, terminate digestion；
(2) stem cell secondary culture：By 6500-10000/cm of inoculum density2Inoculation primary cell is cultivated, cell density
Reach 80% or more, pass on again, reach 3-5 generations, collect cell and counts；
(3) stem cell is preserved：It takes stem cell to be mixed with the preservation liquid, is placed in 35-40 DEG C, 3-8%CO in incubator2Under concentration
Culture；
Wherein, the preservation liquid includes following component in parts by weight：Physiological saline 5-15 parts by weight, human serum albumin 3-6 weights
Measure part and Multiple electrolytes injection 80-95 parts by weight.
9. store method as described in one of claim 1-8 is used to preserve the purposes of umbilical cord mesenchymal stem cells.
10. purposes as claimed in claim 9, which is characterized in that a concentration of the 1 × 10 of the umbilical cord mesenchymal stem cells5-1×
107/ ml, preferably 5 × 105-5×106/ ml, further preferably 2 × 106/ml。
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| CN111700063A (en) * | 2020-06-29 | 2020-09-25 | 广州瑞铂茵健康管理咨询有限公司 | Preservation method of umbilical cord specimen |
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| CN111700063A (en) * | 2020-06-29 | 2020-09-25 | 广州瑞铂茵健康管理咨询有限公司 | Preservation method of umbilical cord specimen |
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