CN107668025A - A kind of endometrial stem cells protect liquid - Google Patents
A kind of endometrial stem cells protect liquid Download PDFInfo
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- CN107668025A CN107668025A CN201610622766.2A CN201610622766A CN107668025A CN 107668025 A CN107668025 A CN 107668025A CN 201610622766 A CN201610622766 A CN 201610622766A CN 107668025 A CN107668025 A CN 107668025A
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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
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The present invention relates to a kind of endometrial stem cells to protect liquid; the protection liquid mainly forms hypoxemia protective agent, mycoplasma inhibitor and aminoglycoside antibiotics with DMEM/F12 culture medium aqueous dissolutions; every milliliter of DMEM/F12 culture mediums aqueous solution dissolves the 15mg of hypoxemia protective agent 3, the 20mg of mycoplasma inhibitor 1 and the 500U of aminoglycoside antibiotics 100 respectively, and the concentration of the DMEM/F12 culture mediums aqueous solution is 20 45mg/ml.The protection liquid can improve the endometrial stem cells survival rate of storage, improve cell quality.
Description
Technical field
The invention belongs to endometrial stem cells preservation field, and in particular to a kind of endometrial stem cells protect liquid.
Background technology
It is well known that endometrial cell system has very strong self-renewal capacity, can all there is group through the cycle in every month
A large amount of growths with blood vessel are knitted, this propagation process can stop with the end of menstrual cycle.Multinational scientist's current research
It is shown in the endometrial tissue to be come off with women menses, there are a considerable amount of mescenchymal stem cells (stroma stem cell) --
Endometrium (menses) stem cell, quantity are 30 times of derived from bone marrow；These stem cell vigor are stronger, stronger self-renewing and increasing
Value ability, differentiation potential embryonic stem cell.If these stem cells are stored by the method for science, following in women
In life, when the major diseases such as tumour or unexpected grievous injury occurs, so that it may do the transplanting of autologous stem cells immediately, recover
Health.
Having at present through the storage method of blood specimen is obtained after blood specimen, by initial gross separation, is removed most red
Cell, then direct addition frozen stock solution, which freezes, is frozen the endometrial stem cells after culture.Gathered in endometrial stem cells
After need by endometrial stem cells be stored in protection liquid in it is stand-by, the storage Endometrium disclosed in patent CN201110085328 is done
Collection liquid is saved it in after gathering endometrial stem cells described in the method for cell, its collection liquid has re-added antibiotic and liver
Element, the pollution and blood coagulation of stem cell are avoided, improve stem cell motility rate.But endometrial stem cells are protected in the cell-preservation liquid
It is still shorter to hold the time of high motility rate, and as time went on, cell viability declines.
The content of the invention
The defects of it is an object of the invention to overcome in the prior art, design a kind of endometrial stem cells protection liquid, the guarantor
Shield liquid can improve the endometrial stem cells survival rate of storage, improve cell quality.
To achieve the above object, the technical solution adopted in the present invention is that a kind of endometrial stem cells protect liquid, the guarantor
Liquid is protected mainly by hypoxemia protective agent, mycoplasma inhibitor and aminoglycoside antibiotics the DMEM/F12 culture medium aqueous solution
Dissolving forms, and it is former that every milliliter of DMEM/F12 culture mediums aqueous solution dissolves the hypoxemia protective agent 3-15mg, the branch respectively
Body inhibitor 1-35mg and aminoglycoside antibiotics 100-500U, the concentration of the DMEM/F12 culture mediums aqueous solution are
20-45mg/ml。
Preferably, the concentration of the DMEM/F12 culture mediums aqueous solution is 33.8mg/ml.
In the present invention protection further scheme of liquid, the hypoxemia protective agent is cobalt chloride and/or sodium lactate, it is preferable that
The hypoxemia protective agent is that ratio of weight and number is 5:1 cobalt chloride and the mixture of sodium lactate.The present invention selects cobalt chloride and breast
Sour sodium collaboration is used as hypoxemia protective agent, and cobalt chloride and sodium lactate are used in mixed way, inducing hypoxia response, blocks HIF-1 degradeds, and tie up
Hold the excellent for keeping low-oxygen environment.
In a wherein improvement project, every milliliter of DMEM/F12 culture mediums aqueous dissolution mycoplasma inhibitor
10-26mg。
Further to improve, the mycoplasma inhibitor includes one in tetracycline, macrolide, quinolone and fullerene
Kind is several, and the fullerene is the fullerene of empty fullerene or hetero radicals, selected from fullerene C60, fullerene C70, fowler
Alkene C76, fullerene C78, fullerene C80With fullerene C84In one or more.
Preferably described mycoplasma inhibitor includes following components：Fullerene 10-25 parts by weight and quinolone 4-10 weight
Part, the fullerene is that ratio of weight and number is 1.2-3:1 fullerene C60With fullerene C84Mixture, mycoplasma is to cell
Be stored with very big threat effect, it is easy to cause cell infection, so that Apoptosis, said mixture can be protected effectively
The motility rate of endometrial stem cells is held, security context is provided for cell survival and growth.
Further, the mycoplasma inhibitor also includes the component of following parts by weight：Phellodendron extract 5-8 parts, purple
Aster extract 4-8 parts, oxymatrine 3-6 parts.Further added in the present invention in mycoplasma inhibitor phellodendron extract,
Radix Asteris extract and oxymatrine, these three traditional Chinese medicine ingredients can pass through traditional Chinese and western medicine knot on the basis of original western medicine composition
The effective component composition mycoplasma inhibitor of conjunction, effectively suppress and kill the mycoplasma in protection liquid, the action period is shorter, to thin
Born of the same parents have stronger protective effect, prevent cell infection.
In another improvement project, the aminoglycoside antibiotics include streptomysin, gentamicin, kanamycins with
And the one or more in TOB, preferably ratio of weight and number is 1:1.5:2 streptomysin, TOB and gentamicin
Mixture.
It is further to improve, aminoglycoside antibiotics described in every milliliter of DMEM/F12 culture mediums aqueous dissolution
200-400U。
Protection liquid provided by the invention is adapted to the preservation of endometrial cells, the amino of addition closer to human body vivo environment
Glycoside antibiotic plays the effect of sterilization, further improves living environment quality.
Endometrial stem cells protection liquid of the present invention expands the base of target cell further to improve the survival rate of stem cell
Number, in another improvement project, the protection liquid also includes growth accelerator, the growth accelerator by calf serum with it is different anti-bad
Hematic acid, casein sodium and yolk liquid mix, wherein every milliliter of calf serum solution dissolving 1-4mg arabo-ascorbic acid,
1-3mg casein sodium and 0.1-0.3ml yolk liquid；In every milliliter of DMEM/F12 culture mediums aqueous solution described in addition
Growth accelerator 0.05-0.3ml.The growth accelerator maintains the osmotic equilibrium in endometrial stem cells, improves the adherent rate of stem cell,
The viscosity of protection liquid can also be increased simultaneously, improve the stability of the protection liquid.
On the basis of the above, present invention protection liquid further comprises and oestrone, every milliliter of DMEM/F12 culture medium
0.26-0.4 × 10 are dissolved in the aqueous solution-3Mg estradiol and 0.04-0.08 × 10-3Mg oestrone；The addition of a small amount of estrogen
There is obvious facilitation to endometrial stem cells increment.
At one in the preferable concrete scheme of endometrial stem cells protecting effect, every milliliter of DMEM/F12 culture medium
The aqueous solution dissolves cobalt chloride 9mg, macrolide 8mg and gentamicin 200U respectively.
At another in endometrial stem cells protecting effect more preferably concrete scheme, every milliliter of DMEM/F12 is cultivated
The base aqueous solution dissolves 9.5mg cobalt chloride, 1.9mg sodium lactate, 3.5mg fullerene C respectively60, 2mg fullerene C84、
2.2mg quinolone, 2.5mg phellodendron extract, 2.2mg Radix Asteris extract, 2mg oxymatrine, 80U strepto-
Element, 120U TOB, 160U gentamicin, 0.22ml growth accelerator, 0.3 × 10-3Mg estradiol and 0.05 ×
10-3Mg oestrone；
Every milliliter of calf serum solution dissolving 2.5mg arabo-ascorbic acid, 1.4mg caseinic acid wherein in growth accelerator
The yolk liquid of sodium and 0.2ml.
The endometrial stem cells survival rate of the endometrial stem cells protection liquid storage of the present invention is high, and storage time length, is dry
The propagation of cell next step improves sufficiently large radix；Also, the cell quality of proliferation period can be improved, the cell survival after increment
Rate is high.
Brief description of the drawings
Fig. 1 is the influence for preserving liquid to endometrial stem cells growth curve.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
Embodiment 1
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 20mg/ml；
Hypoxemia protective agent：Cobalt chloride 20mg, sodium lactate 10mg；
Mycoplasma inhibitor：Quinolone 10mg；
Aminoglycoside antibiotics：Streptomysin 1000U；
Wherein, DMEM/F12 by by DMEM and F12 powder according to 1:1 ratio mixing, is dissolved with distilled water, added
PH is adjusted to be diluted with 2 times of amount water to after 7.2 constant volumes, produce the DMEM/F12 culture medium aqueous solution after sodium acid carbonate.
The hypoxemia protective agent, mycoplasma inhibitor and aminoglycoside antibiotics of above-mentioned unit quantity are added to DMEM/F12
The culture medium aqueous solution, it is well mixed to produce endometrial stem cells protection liquid.
Embodiment 2
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 45mg/ml；
Hypoxemia protective agent：Cobalt chloride 100mg, sodium lactate 20mg；
Mycoplasma inhibitor：Quinolone 200mg；
Aminoglycoside antibiotics：Streptomysin 1000U, the streptomysin of TOB 1500 and TOB 2000U.
Protect the preparation method preparation method as described in Example 1 of liquid.
Embodiment 3
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 33.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 80mg, sodium lactate 16mg；
Mycoplasma inhibitor：Macrolide 80mg；
Aminoglycoside antibiotics：Gentamicin 2000U.
Protect the preparation method preparation method as described in Example 1 of liquid.
Embodiment 4
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 30mg/ml；
Hypoxemia protective agent：Cobalt chloride 30mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C6050mg, fullerene C8420mg, quinolone 25mg；
Aminoglycoside antibiotics：Streptomysin 700U, TOB 800U, gentamicin 1000U；
Protect the preparation method preparation method as described in Example 1 of liquid.
Embodiment 5
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 30mg/ml；
Hypoxemia protective agent：Cobalt chloride 30mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C6050mg, fullerene C8420mg, quinolone 25mg, phellodendron extract 25mg are purple
Aster extract 20mg, oxymatrine 15mg；
Aminoglycoside antibiotics：Streptomysin 700U, TOB 800U, gentamicin 1000U；Protect the preparation method of liquid such as
Preparation method described in embodiment 1.
Embodiment 6
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 30.2mg/ml；
Hypoxemia protective agent：Cobalt chloride 50mg, sodium lactate 15mg；
Mycoplasma inhibitor：Fullerene C6085mg, fullerene C7045mg, quinolone 50mg, phellodendron extract 40mg are purple
Aster extract 40mg, oxymatrine 30mg；
Aminoglycoside antibiotics：Gentamicin 1000U, kanamycins 1500U；
Growth accelerator：0.5ml, mixed by calf serum and casein sodium, ascorbic acid and yolk liquid, wherein often
Milliliter calf serum solution dissolving 1mg arabo-ascorbic acid, 1mg casein sodium and 0.1ml yolk liquid；
The hypoxemia protective agent of above-mentioned unit quantity, mycoplasma inhibitor, aminoglycoside antibiotics and growth accelerator are added
It is well mixed to produce endometrial stem cells protection liquid to the DMEM/F12 culture medium aqueous solution.
Embodiment 7
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 28mg/ml；
Hypoxemia protective agent：Cobalt chloride 30mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C60100mg, fullerene C84100mg, quinolone 60mg, phellodendron extract 20mg,
Radix Asteris extract 20mg, oxymatrine 20mg；
Aminoglycoside antibiotics：TOB 1600U, gentamicin 1000U；
Growth accelerator：3ml, mixed by calf serum and casein sodium, ascorbic acid and yolk liquid, wherein per milli
Rise calf serum solution dissolving 4mg arabo-ascorbic acid, 3mg casein sodium and 1.3ml yolk liquid.
Protect the preparation method preparation method as described in Example 5 of liquid.
Embodiment 8
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 24.5mg/ml；
Hypoxemia protective agent：Cobalt chloride 46mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C6035mg, fullerene C8424mg, quinolone 22mg, phellodendron extract 35mg are purple
Aster extract 35mg, oxymatrine 25mg；
Aminoglycoside antibiotics：Streptomysin 1000U, gentamicin 1000U；
Growth accelerator：1.8ml, mixed by calf serum with arabo-ascorbic acid, casein sodium, ascorbic acid and yolk liquid
Form, wherein every milliliter of calf serum solution dissolving 1.5mg arabo-ascorbic acid, 2mg casein sodium and 0.9ml yolk
Liquid；
Protect the preparation method preparation method as described in Example 5 of liquid.
Embodiment 9
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 33.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 95mg, sodium lactate 19mg；
Mycoplasma inhibitor：Fullerene C6035mg, fullerene C8424mg, quinolone 22mg, phellodendron extract 25mg are purple
Aster extract 22mg, oxymatrine 20mg；
Aminoglycoside antibiotics：Streptomysin 800U, TOB 1200U, gentamicin 1600U；
Growth accelerator：2.2ml, mixed by calf serum and arabo-ascorbic acid, casein sodium and yolk liquid, wherein
Every milliliter of calf serum solution dissolving 2.5mg arabo-ascorbic acid, 1.4mg casein sodium and 0.2ml yolk liquid；
Estradiol 0.3 × 10-2Mg and oestrone 0.05 × 10-2mg。
By the hypoxemia protective agent of above-mentioned unit quantity, mycoplasma inhibitor, aminoglycoside antibiotics, growth accelerator and female
Hormone is added to the DMEM/F12 culture medium aqueous solution, well mixed to produce endometrial stem cells protection liquid.
Embodiment 10
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 25.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 43mg, sodium lactate 10mg；
Mycoplasma inhibitor：Fullerene C84100mg, quinolone 40mg, phellodendron extract 50mg, Radix Asteris extract 40mg,
Oxymatrine 30mg；
Aminoglycoside antibiotics：Gentamicin 1000U, kanamycins 1000U, TOB 1000U；
Growth accelerator：1.8ml, mixed by calf serum and arabo-ascorbic acid, casein sodium and yolk liquid, wherein
Every milliliter of calf serum solution dissolving 3.0mg arabo-ascorbic acid, 1mg casein sodium and 0.4ml yolk liquid；
Estradiol 0.4 × 10-2Mg and oestrone 0.08 × 10-2mg。
Protect the preparation method preparation method as described in Example 8 of liquid.
Embodiment 11
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 20.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 50mg, sodium lactate 10mg；
Mycoplasma inhibitor：Tetracycline 62mg, quinolone 50mg；
Aminoglycoside antibiotics：Streptomysin 2300U, kanamycins 1500U；
Growth accelerator：2.6ml, mixed by calf serum and arabo-ascorbic acid, casein sodium and yolk liquid, wherein
Every milliliter of calf serum solution dissolving 2.3mg arabo-ascorbic acid, 1.6mg casein sodium and 0.12ml yolk liquid；
Estradiol 0.26 × 10-2Mg and oestrone 0.04 × 10-2mg。
Protect the preparation method preparation method as described in Example 8 of liquid.
Comparative examples 1
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 45mg/ml；
Hypoxemia protective agent：Cobalt chloride 100mg, sodium lactate 20mg；
Mycoplasma inhibitor：Quinolone 200mg；
Aminoglycoside antibiotics：Penicillin 2000U.
Comparative examples 2
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 33.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 80mg；
Mycoplasma inhibitor：Macrolide 80mg；
Aminoglycoside antibiotics：Gentamicin 2000U.
Comparative examples 3
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 30mg/ml；
Hypoxemia protective agent：Cobalt chloride 30mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C6050mg, fullerene C8420mg, quinolone 25mg, phellodendron extract 25mg, oxygen
Change matrine 15mg；
Aminoglycoside antibiotics：Streptomysin 700U, TOB 800U, gentamicin 1000U；Comparative examples 4
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 24.5mg/ml；
Hypoxemia protective agent：Cobalt chloride 46mg, sodium lactate 20mg；
Mycoplasma inhibitor：Fullerene C6035mg, fullerene C8424mg, quinolone 22mg, phellodendron extract 35mg are purple
Aster extract 35mg, oxymatrine 25mg；
Aminoglycoside antibiotics：Streptomysin 1000U, gentamicin 1000U；
Growth accelerator：1.8ml, mixed by calf serum and arabo-ascorbic acid, wherein every milliliter of calf serum solution
Dissolve 1.5mg arabo-ascorbic acid.
Comparative examples 5
The composition of endometrial stem cells protection liquid is as follows：
DMEM/F12 culture mediums aqueous solution 10ml, concentration 33.8mg/ml；
Hypoxemia protective agent：Cobalt chloride 95mg, sodium lactate 19mg；
Mycoplasma inhibitor：Fullerene C6035mg, fullerene C8424mg, quinolone 22mg, phellodendron extract 25mg are purple
Aster extract 22mg, oxymatrine 20mg；
Aminoglycoside antibiotics：Streptomysin 800U, TOB 1200U, gentamicin 1600U；
Growth accelerator：2.2ml, mixed by calf serum and arabo-ascorbic acid, casein sodium and yolk liquid, wherein
Every milliliter of calf serum solution dissolving 2.5mg arabo-ascorbic acid, 1.4mg casein sodium and 0.2ml yolk liquid；
Estradiol 0.3 × 10-2mg。
The specific extracting method of phellodendron extract and Radix Asteris extract is in hypoxemia protective agent in above-described embodiment：Respectively
The golden cypress and aster of formula ratio are weighed, adds 8-10 times of water to measure, is soaked 4 hours, extraction 2 times is decocted, 1.5 hours every time, merges two
Secondary decoction liquor, filtering, it is 1.05-1.10 that filtrate decompression, which is concentrated into relative density, after cooling, adds 60-80% ethanol, stood
At night, take supernatant that ethanol is recovered under reduced pressure, to concentrate without alcohol taste, enter under the conditions of temperature is 50-60 DEG C, pressure is -0.05MPa
Row vacuum drying, is made phellodendron extract and Radix Asteris extract respectively.
Test example preserves influence of the liquid to endometrial stem cells extracorporeal biology characteristic
Survival rate when the preservation liquid of experiment one stores to endometrial stem cells
1. collection tube is grouped
Collection tube is divided into 13 groups of test group, wherein：
The collection tube of test group 1 is equipped with the protection liquid described in embodiment 2；
The collection tube of test group 2 is equipped with the protection liquid described in embodiment 3；
The collection tube of test group 3 is equipped with the protection liquid described in embodiment 4；
The collection tube of test group 4 is equipped with the protection liquid described in embodiment 5；
The collection tube of test group 5 is equipped with the protection liquid described in embodiment 8；
The collection tube of test group 6 is equipped with the protection liquid described in embodiment 9；
The collection tube of test group 7 is equipped with the protection liquid described in comparative examples 1；
The collection tube of test group 8 is equipped with the protection liquid described in comparative examples 2；
The collection tube of test group 9 is equipped with the protection liquid described in comparative examples 3；
The collection tube of test group 10 is equipped with the protection liquid described in comparative examples 4；
The collection tube of test group 11 is equipped with the protection liquid described in comparative examples 5；
The collection tube of test group 12 protects liquid equipped with conventional transplanting, commonly uses transplanting protection liquid to contain penicillin/streptomycin
With the phosphate buffer of heparin；
The collection tube of test group 13 is blank control.
Before starting to gather menses, the collection tube of each test group is waited to put under 4 DEG C of environment.
2. menses are collected
Second day started in volunteer's menstruation, the posture taken semi-crouch or squatted entirely, after menstrual cup doubling twice, is put into
Intravaginal.Carefully take out, the menses in menstrual cup are poured into 12 groups of collection tubes, every 10ml after two hours, protected under the conditions of 4 DEG C
Deposit, the storage life is respectively 12h, 24h and 48h.
3. sterility test
After the collection tube centrifugal treating of test group 13, supernatant is obtained；By supernatant according to blood culture method carry out anaerobic bacteria and
Aerobic bacteria checks, and identifies；If positive findings, then terminate the whole program for storing endometrial stem cells.
4. calculate survival rate
Count the cell number in stem cell supernatant.Method:0.4% of 0.5ml supernatants addition equivalent is taken to expect blue dye
Liquid, fully mix, take it is a little drip in the counting chamber of blood counting chamber, static 2 minutes, micro- Microscopic observation living cell counting with
Dead cell number (cell of all blues is unhealthy or dead cell), calculates cell survival rate.
It the results are shown in Table 1.
The comparison of the survival rate measurement result of the endometrial stem cells storage period different periods of table 1
Note：" -- " represents no motility rate
As seen from Table 1, the passage over time for the endometrial stem cells transplanted in GPF (General Protection False liquid, cell death
Number increase, survival rate are minimum；Add survival rate when protecting the stem cell storage of liquid that the present invention is embodiment 2-5, embodiment 8-9
Height, wherein embodiment 4-5, embodiment 8-9 long-time storage changes are little, and the survival rate for storing 96h is still very high；With GPF (General Protection False
Liquor ratio has highly significant sex differernce (P ＜ 0.001), and embodiment 2-5, embodiment 8-9 protection liquid have to stem cell transplantation
The protection liquid best results of fine protective effect, wherein embodiment 9.Knowable to the comparison of embodiment 2 and comparative examples 1, this hair
After aminoglycoside antibiotics in the protection liquid of bright offer has changed penicillin antibiotic into, the motility rate of stem cell significantly reduces,
And the time-to-live is short.The comparison of embodiment 3 and comparative examples 2 is understood, in the hypoxemia protective agent of protection liquid provided by the invention
After adding sodium lactate, the motility rate of stem cell is higher.The comparison of embodiment 4, embodiment 5 and comparative examples 3 understands that hypoxemia is protected
After agent adds phellodendron extract, Radix Asteris extract and oxymatrine, stem cell motility rate is higher.Embodiment 8 and comparative examples 4
Comparison understand that after growth accelerator adds casein sodium and yolk liquid, the motility rate of stem cell is higher.And embodiment 8 is with implementing
The comparison of example 9 understands that after protection liquid provided by the invention adds estrogen, the motility rate of stem cell is higher.
Experiment two, preserve the survival rate after liquid expands to endometrial stem cells
1. endometrial stem cells are separately cultured
After collection tube sample filtering corresponding to test group 1-12 is obtained, using density-gradient centrifugation method, collection is single can
Cell；
The mononuclearcell of above-mentioned gained is inoculated in Chang complete mediums, the inoculum density of mononuclearcell is
1*105-1*106/ ml, be placed in 37 DEG C, saturated humidity, volume fraction be 5% CO2Cultivated in incubator.
2. cell expands and purifying
After mononuclearcell is inoculated in 4-5 days of beginning cell culture in Chang complete mediums, it is complete to change Chang
Full culture medium, and the non-attached cell of reject；Changing liquid per 3-4 days full doses later, once (depending on cell growth condition, full dose is changed
Liquid, which refers to, changes whole Chang complete mediums)；When cell growth reaches 80-90% fusions, it is with mass concentration
Cell is collected in 0.25% trypsase (trypsase that 1ml is used in the general cell per 1-20ml) digestion, then
By 5000-6000/cm2Density passes on inoculated and cultured, and is designated as P1 generations.3. cell cryopreservation：
After the P1 of step 3 is paved with container bottom for cell, with the Trypsin Induced that weight/mass percentage composition is 0.25%
Collect cell；Cell is resuspended with the frozen stock solution of precooling；The density of cell is maintained at 1-2*106/ml.Frozen stock solution is：In 9 parts by volume
Chang complete mediums in add the DMSO (calbiochem companies) of 1 parts by volume and be prepared and (as contain 10%DMSO
Chang complete mediums).Cell suspension is placed in duroplasts cryopreservation tube, sealed, carries out mark.By every 20 minutes
The speed of decline certain distance while the above-mentioned cell that lands, are finally fully immersed in liquid nitrogen and freeze.
4. cell thaws
Rapid thawing completely is allowed to taking out to put into 42 DEG C of water-baths immediately from liquid nitrogen by cell after freezing two weeks.Immediately
Count the cell number in the cell suspension after melting.Method:0.4% of 0.5ml cell suspensions addition equivalent is taken to expect blue dye liquor,
Fully mix, take it is a little drip in the counting chamber of blood counting chamber, static 2 minutes, micro- Microscopic observation living cell counting with it is dead
Cell number (cell of all blues is unhealthy or dead cell), calculates cell survival rate.
It the results are shown in Table 2.
The comparison of survival rate measurement result after the amplification of the endometrial stem cells of table 2
As seen from Table 2, the survival rate after the endometrial stem cells amplification that GPF (General Protection False liquid is transplanted is minimum, adds
The present invention be embodiment 2-5, embodiment 8-9 protection liquid expansion of stem cells after survival rate it is high, with GPF (General Protection False liquor ratio more all
There is highly significant sex differernce (P ＜ 0.001).Illustrate embodiment 2-5, the protection liquid of embodiment 8-9 components increases to stem cell progeny
The best results of the effect for being improved survival rate, wherein embodiment 9 of value.Knowable to the comparison of embodiment 2 and comparative examples 1,
After aminoglycoside antibiotics in protection liquid provided by the invention has changed penicillin antibiotic into, the motility rate of stem cell significantly drops
It is low, and the time-to-live is short, i.e., aminoglycoside antibiotics has more preferable raising to act on to the value-added survival rate of stem cell progeny.It is real
The comparison for applying example 3 and comparative examples 2 is understood, after adding sodium lactate in the hypoxemia protective agent of protection liquid provided by the invention, is done
The value-added survival rate of cell offspring is higher.The comparison of embodiment 4, embodiment 5 and comparative examples 3 understands that hypoxemia protective agent adds
After entering phellodendron extract, Radix Asteris extract and oxymatrine, the value-added survival rate of stem cell progeny is higher.Embodiment 8 and right
Comparison according to embodiment 4 understands that after growth accelerator adds casein sodium and yolk liquid, the value-added survival rate of stem cell progeny is more
It is high.And the comparison of embodiment 8 and embodiment 9 is understood, after protection liquid provided by the invention adds estrogen, stem cell progeny increment
Survival rate it is higher.
Experiment three, preserve influence of the liquid to endometrial stem cells growth curve
1. collection tube is divided into 2 groups of test group, wherein, the collection tube of test group 1 is equipped with the protection liquid described in embodiment 9；
The collection tube of test group 2 is equipped with the protection liquid described in comparative examples 5.
After the collection of the method for experiment one and experiment two, separation, purifying, increment endometrial stem cells, the He of test group 1 is taken
The P1 of test group 2 is for Endometrial stem cell, with 103Individual/ml density is inoculated in 96 well culture plates, adds Chang and trains completely
Base is supported, 20 plates of every group of cell, second day after adherent starts to do MTT experiments.
Cell growth curve is surveyed in 2.MTT colorimetric tests：Preceding 4h undetermined adds 20 μ L MTT liquid (5mg/ in each hole of cell to be measured
Ml), after culture terminates, nutrient solution is abandoned in suction, adds 150 μ LDMSO, and light absorption value is surveyed at 492nm wavelength.Continue 10d, until cell
Propagation reaches plateau, and daily same time point is carried out；Cell growth curve is drawn, sees Fig. 1；From in figure, adding simultaneously
The protection liquid of estradiol and oestrone has facilitation to the propagation of endometrial stem cells.
Described above is only the preferred embodiment of the present invention, for those skilled in the art, not
On the premise of departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications also should be regarded as this
The protection domain of invention.
Claims (10)
1. a kind of endometrial stem cells protect liquid, it is characterised in that the protection liquid mainly presses down hypoxemia protective agent, mycoplasma
Preparation and aminoglycoside antibiotics are formed with DMEM/F12 culture medium aqueous dissolutions, every milliliter of DMEM/F12 culture
The base aqueous solution dissolves the hypoxemia protective agent 3-15mg, the mycoplasma inhibitor 1-35mg and aminoglycoside antibiosis respectively
Plain 100-500U, the concentration of the DMEM/F12 culture mediums aqueous solution is 20-45mg/ml.
2. endometrial stem cells as claimed in claim 1 protect liquid, it is characterised in that the DMEM/F12 culture mediums aqueous solution
Concentration be 33.8mg/ml.
3. endometrial stem cells as claimed in claim 1 protect liquid, it is characterised in that the hypoxemia protective agent is cobalt chloride
And/or sodium lactate, it is preferable that the hypoxemia protective agent is that ratio of weight and number is 5:1 cobalt chloride and the mixture of sodium lactate.
4. endometrial stem cells as claimed in claim 1 protect liquid, it is characterised in that the mycoplasma inhibitor includes Fourth Ring
One or more in element, macrolide, quinolone and fullerene, the fullerene are the richness of empty fullerene or hetero radicals
Alkene is strangled, selected from fullerene C60, fullerene C70, fullerene C76, fullerene C78, fullerene C80With fullerene C84In one kind or several
Kind.
5. endometrial stem cells as claimed in claim 4 protect liquid, it is characterised in that the mycoplasma inhibitor includes following
Component：Fullerene 10-25 parts by weight and quinolone 4-10 parts by weight, the fullerene are that ratio of weight and number is 1.2-3:1 richness
Strangle alkene C60With fullerene C84Mixture.
6. endometrial stem cells as claimed in claim 5 protect liquid, it is characterised in that the mycoplasma inhibitor also include with
The component of lower parts by weight：Phellodendron extract 5-8 parts, Radix Asteris extract 4-8 parts, oxymatrine 3-6 parts.
7. endometrial stem cells as claimed in claim 1 protect liquid, it is characterised in that the aminoglycoside antibiotics includes
One or more in streptomysin, gentamicin, kanamycins and TOB, preferably ratio of weight and number are 1:1.5:2
Streptomysin, the mixture of TOB and gentamicin.
8. the endometrial stem cells protection liquid as any one of claim 1-7, it is characterised in that the protection liquid also wraps
Growth accelerator is included, the growth accelerator is mixed by calf serum, casein sodium, arabo-ascorbic acid and yolk liquid, wherein
Every milliliter of calf serum solution dissolving 1-3mg casein sodium, 1-4mg arabo-ascorbic acid and 0.1-0.3ml yolk liquid；
The growth accelerator 0.05-0.3ml is added in every milliliter of DMEM/F12 culture mediums aqueous solution.
9. endometrial stem cells as claimed in claim 7 protect liquid, it is characterised in that the Cell protective solutions also include female two
Alcohol and oestrone, every milliliter of DMEM/F12 culture mediums aqueous dissolution 0.26-0.4 × 10-3Mg estradiol and 0.04-0.08
×10-3Mg oestrone.
10. endometrial stem cells as claimed in claim 9 protect liquid, it is characterised in that every milliliter of DMEM/F12 culture
The base aqueous solution dissolves 9.5mg cobalt chloride, 1.9mg sodium lactate, 3.5mg fullerene C respectively60, 2mg fullerene C84、
2.2mg quinolone, 2.5mg phellodendron extract, 2.2mg Radix Asteris extract, 2mg oxymatrine, 80U strepto-
Element, 120U TOB, 160U gentamicin, 0.22ml growth accelerator, 0.3 × 10-3Mg estradiol and 0.05 ×
10-3Mg oestrone；
Every milliliter of calf serum solution dissolving 2.5mg arabo-ascorbic acid wherein in growth accelerator, 1.4mg casein sodium and
0.2ml yolk liquid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610622766.2A CN107668025A (en) | 2016-08-01 | 2016-08-01 | A kind of endometrial stem cells protect liquid |
| PCT/CN2016/094673 WO2018023827A1 (en) | 2016-08-01 | 2016-08-11 | Protective fluid for endometrial stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610622766.2A CN107668025A (en) | 2016-08-01 | 2016-08-01 | A kind of endometrial stem cells protect liquid |
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| CN107668025A true CN107668025A (en) | 2018-02-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610622766.2A Pending CN107668025A (en) | 2016-08-01 | 2016-08-01 | A kind of endometrial stem cells protect liquid |
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| WO (1) | WO2018023827A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109566597A (en) * | 2018-11-05 | 2019-04-05 | 北京世纪劲得生物技术有限公司 | A kind of endometrial stem cells protection liquid and preparation method thereof |
| CN109609442A (en) * | 2019-01-03 | 2019-04-12 | 贵州大学 | A kind of duck uterine epithelial cell isolation and culture method |
| CN113812397A (en) * | 2021-09-30 | 2021-12-21 | 佛山市第一人民医院（中山大学附属佛山医院） | Perfusion preservation liquid |
| CN113826614A (en) * | 2021-09-30 | 2021-12-24 | 佛山市第一人民医院（中山大学附属佛山医院） | Organ preservation solution |
| CN116965403A (en) * | 2023-09-22 | 2023-10-31 | 诺莱生物医学科技有限公司 | Stem cell preservation solution and preparation method and application thereof |
| CN117165534A (en) * | 2023-10-31 | 2023-12-05 | 山东大学齐鲁医院 | Immortalized human gastric cancer fibroblast strain and construction and proliferation strengthening method thereof |
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| CN115386538A (en) * | 2021-05-24 | 2022-11-25 | 上海长一干细胞研究中心有限公司 | Method for separating, extracting and culturing endometrial stem cells from menstrual blood and special culture medium |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109566597A (en) * | 2018-11-05 | 2019-04-05 | 北京世纪劲得生物技术有限公司 | A kind of endometrial stem cells protection liquid and preparation method thereof |
| CN109609442A (en) * | 2019-01-03 | 2019-04-12 | 贵州大学 | A kind of duck uterine epithelial cell isolation and culture method |
| CN113812397A (en) * | 2021-09-30 | 2021-12-21 | 佛山市第一人民医院（中山大学附属佛山医院） | Perfusion preservation liquid |
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| CN113812397B (en) * | 2021-09-30 | 2023-03-14 | 佛山市第一人民医院（中山大学附属佛山医院） | Perfusion preserving fluid |
| CN113826614B (en) * | 2021-09-30 | 2023-03-14 | 佛山市第一人民医院（中山大学附属佛山医院） | Organ preservation solution |
| CN116965403A (en) * | 2023-09-22 | 2023-10-31 | 诺莱生物医学科技有限公司 | Stem cell preservation solution and preparation method and application thereof |
| CN116965403B (en) * | 2023-09-22 | 2023-12-15 | 诺莱生物医学科技有限公司 | Stem cell preservation solution and preparation method and application thereof |
| CN117165534A (en) * | 2023-10-31 | 2023-12-05 | 山东大学齐鲁医院 | Immortalized human gastric cancer fibroblast strain and construction and proliferation strengthening method thereof |
| CN117165534B (en) * | 2023-10-31 | 2024-01-16 | 山东大学齐鲁医院 | Immortalized human gastric cancer fibroblast strain and construction and proliferation strengthening method thereof |
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| WO2018023827A1 (en) | 2018-02-08 |
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Application publication date: 20180209 |