CN109517787A - A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell - Google Patents
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell Download PDFInfo
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- CN109517787A CN109517787A CN201811309102.6A CN201811309102A CN109517787A CN 109517787 A CN109517787 A CN 109517787A CN 201811309102 A CN201811309102 A CN 201811309102A CN 109517787 A CN109517787 A CN 109517787A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The present invention provides a kind of methods of Rapid-Freezing Method and recovery Endometrial stem cell, can reduce the probability of cell damage apoptosis, improve survival rate；The processing of short time low frequency ultrasound is carried out after freezen protective liquid or recovery protection liquid are added into cell; it can make the cell adhered to each other that slight loosening occur, generate gap under the premise of not destroying eucaryotic cell structure, and liquid can be made to be dispersed into droplet, be convenient for penetrating between cell；Cell adhesion molecule can promote to adhere to each other between cell, thus the survival rate for being further reduced individual cells, improving cell；Cell active inhibitors can carry out reversible inhibition to intracellular some physiological activities, improve under the premise of not influencing cell activity and freeze effect.By above-mentioned means, it can reduce that refrigerating process and resuscitation process be physical caused by cell and injury chemically, thus the anabiosis rate for improving cell survival rate and freezing.
Description
Technical field
The invention belongs to tissue cryopreservation resuscitation technical field, in particular to a kind of Rapid-Freezing Method and recovery endometrium
The method of stem cell.
Background technique
Endometrium is one layer for constituting mammality uterus inner wall.Research in endometrial tissue it has proven convenient that exist at present
A certain number of stem cells, stem cell have the initial cell of self-replacation and multi-lineage potential, are the cells of origin of body,
It is the initial cell to form the various histoorgans of human body.Under certain condition, it can be divided into multiple functions cell or tissue
Organ, medical field are known as " general-purpose cell ", the seed cell suitable for injuries of tissues and organs reparation caused by aging and lesion.It is raw
Educate the women of phase, endometrium can acceptor internal hormone influence and generating period regenerate, break up and strip off so that uterus
Internal film tissue has many advantages, such as that reproducibility is strong, materials are convenient, therefore becomes the important sources for obtaining stem cell.Traditional sequencing
It freezes due to being easy that liquid is made to generate crystallization, mechanical injuries often is caused to cell；New-type glass frozen preservation can be complete
Crystallisation problems are avoided entirely, to greatly improve the anabiosis rate of freeze-stored cell.But glass frozen is needed using with cytotoxicity
Dimethyl sulfoxide, be easy cell is caused chemically to injure.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides the sides of a kind of Rapid-Freezing Method and recovery Endometrial stem cell
Method.
Specific technical solution of the present invention is as follows:
One aspect of the present invention provides the method for a kind of Rapid-Freezing Method and recovery Endometrial stem cell, including walks as follows
It is rapid:
S1: endometrial tissue, adhere-wall culture 36h~48h are extracted, and 6~8d of secondary culture is carried out to obtained cell；
Cell clone is saved again at after cell mass, the probability that individual cells are damaged apoptosis can be significantly reduced, thus
Significantly improve the survival rate of cell and the success rate of recovery；
S2: the obtained passage cell of step S1 is placed in DMEM culture medium, is added and is cooled to 2~5 DEG C in advance and freezes protection
Liquid, gently oscillation mixes, the processing of short time low frequency ultrasound, described to freeze in protection liquid added at least one cell adhesion molecule
And at least one cell active inhibitors；
It is added into cell after freezing protection liquid, by handling by the low frequency ultrasound of short time, is not destroying cell knot
Make to freeze in the case where structure inside protection liquid and be dispersed into droplet, to improve permeability of the frozen solution between cell；
Cell adhesion molecule is so that flanking cell surface is had an effect, so that the transmembrane protein adhered to each other, can make list
A cell further adheres to, to prevent from scattering and disappearing；By low-speed centrifugal, cell adhesion molecule is promoted to contact with cell, to improve
The probability that cell adheres to each other；It needs gently to vibrate mixing after centrifugation, be come into full contact with to freeze protection liquid with cell；
Cell active inhibitors can carry out reversible inhibition to intracellular some physiological activities, living not influencing cell
Property under the premise of improve freeze effect；
S3: the obtained cell mixture of step S2 is placed in liquid nitrogen, is cooled at -196 DEG C and is saved；
S4: the cell frozen being taken out, is rapidly heated first to 30~40 DEG C, is then cooled to 2~10 DEG C rapidly, is repeated
The above process 3 times；It is washed 2~4 times after taking-up with PBS buffer solution, is centrifuged, discards cleaning solution；
The cell frozen is needed to be rapidly heated in recovery, be generated during defrosting with freezing the moisture in protection liquid
Ice crystal, damaging cells structure；Meanwhile the recovery of cell activity needs a process when cell recovery, therefore in suitable temperature
It allows cell to heat up repeatedly cooling in range, cell is made gradually to adapt to environment, be ready for recovery；
S5: the recovery for obtaining being added sonicated mistake in advance in cell to step S4 saves liquid, and gently oscillation mixes, and obtains
To cell suspension, the recovery saves in liquid and releases molecule and at least one cell activity added at least one cell adherence
Activator；
By ultrasonic treatment, saves recovery inside liquid and be dispersed into droplet, from
And it improves recovery and saves permeability of the liquid between cell；
Cell adherence release molecule can release it is intercellular adhere to each other, facilitate cell and be separated from each other, so as to subsequent
Use；
Cell activity activator can release the inhibition that cellular physiological activity is subject to, to restore cell activity, improve again
Soviet Union's rate.
Further, the step S1 includes the following steps:
S1.1: acquisition endometrium decidua tissue sample, cleaned up with physiological saline, shred into 1~2mm fragment,
It is laid in Tissue Culture Flask, Type I collagen enzyme is added, primary culture medium is added after concussion digestion 60min and terminates digestion, after centrifugation
Remove supernatant, addition primary culture medium is placed in 5%CO2, cultivated in 37 DEG C of incubator, every primary training of 3~4d replacement
Base is supported, the primary culture medium is the DMEM of the chlorogenic acid of fetal calf serum and 30~50mg/L added with volume fraction 10%
Culture solution；
Chlorogenic acid is a kind of substance extracted from the plants such as Eucommiaceae, Caprifoliaceae, has antibacterial outstanding, disease-resistant
Poison, antioxidation also have good effect for cell growth, wound healing, and will not generate in Rational Dosage thin
Cellular toxicity；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is added secondary culture base, sets after digestion
In 5%CO2, cultivated in 37 DEG C of incubator, cultivate until when cell fusion degree reaches 80~90%, the passage is trained
Support the dense element of mandarin orange that base is fetal calf serum added with volume fraction 15%, the gallic acid of 15~25mg/L and 2~5mg/L
DMEM culture medium.
The dense element of mandarin orange is a kind of natural mixture extracted from orange peel, has the function of extremely strong antibacterial, can be inhibited thin
The growing multiplication of bacterium etc., and have no toxic side effect, the growth of cell will not be impacted, pathogenic microorganism will not be made to generate
Drug resistance.
Further, the step S2 includes the following steps:
S2.1: the obtained passage cell of step S1 being placed in DMEM culture medium and is impregnated, and gently oscillation mixes 1~3 minute,
It is centrifuged 3~5min under the conditions of 1000~1500rpm/min, discards supernatant, repeats aforesaid operations 2~3 times, discards for the last time
DMEM culture medium is added after supernatant；
S2.2: configuration freezes protection liquid, is placed in 5~10min of pre-cooling at 2~5 DEG C；
S2.3: being added into passage cell and what DMEM culture medium was isometric freezes protection liquid, surpasses under the conditions of 1~2.5kHz
5~10s is swung in acoustic shock, is subsequently placed in 2~5 DEG C of refrigerators and is stood 30min；Ultrasound procedure is repeated after taking-up, is subsequently placed in 2~5 DEG C
60min is stood in refrigerator.
Under the low frequency of 1~2.5kHz, the ultrasonic vibration of short time cannot destroy eucaryotic cell structure, but can make to adhere to each other
Cell occurs slight loosening, generates gap, and can make to freeze and liquid is protected to be dispersed into droplet, penetrates into consequently facilitating freezing protection liquid
Between cell.
Further, the ingredient protected in liquid comprising following parts by weight that freezes: 30~40 parts of cryoprotectors,
0.01~0.02 part of cell active inhibitors, 0.02~0.05 part of cell adhesion molecule and 1.5~2.5 parts of hydrophilic colloids；
The permeability cryoprotector include weight fraction ratio be the proline of 2:5:2:1, mannitol, trehalose and
Hydroxyethyl starch；
The cell active inhibitors include Cytochalasin D, nocodazole, tunicamycin, several husband's alkali, brefeldin
A, at least one of CP-10447 and Octreotide；
The cell adhesion molecule include E-Selectin, P- calcium stick element, ICAM-1, peptide F9 and RGD peptide at least one
Kind；
The hydrophilic colloid include tara gum, trigonella bean gum, tragacanth, sesbania gum, tamarind gum, locust bean gum and
At least one of peach gum.
Dimethyl sulfoxide is most common cryoprotector, excellent effect but is more toxic, therefore using upper in the present invention
Combination is stated, to reduce the dosage of dimethyl sulfoxide, to reduce cytotoxicity, while not influencing the effect of cryoprotector；It is hydrophilic
Property colloid can form thixotrope in water, be not required to heating, only by shake etc. mechanical forces gel can be made to become colloidal sol, conveniently make
With；Gel state can be become again from colloidal sol by standing a period of time, make each distributed components in permeability protective agent, and can make
Cell is evenly dispersed wherein, is less prone to precipitating, effectively keep cell vigor and genetic stability, and within easy reach and
Storage.
Further, the cell active inhibitors include the brefeldin A and several husbands that weight fraction ratio is 4:1
Alkali；The cell adhesion molecule is RGD peptide.
Transhipment of the brefeldin A energy blocking protein from endoplasmic reticulum (ER) to golgiosome, and this process is
Reversible；It is responsible for the alpha-Mannosidase of processing I class glycoprotein in several husband's alkali energy selective depression endoplasmic reticulum.Above-mentioned cell activity
Inhibitor can effectively inhibit the physiological activity of cell, and will not influence the activity of cell.
RGD peptide can guide the cell of mediated by integrin viscous on the carriers such as biomaterial, polymer and nano particle
It is attached, adhering to each other between cell can be effectively facilitated, while will not impact to the physiological activity of cell.
Further, the hydrophilic colloid includes the tara gum and sesbania gum that weight fraction ratio is 2:3.
Tara gum and sesbania gum are plant origin, will not generate toxic action to cell, and its main component is all
For polysaccharide, or cell provides certain nutrient.Using said combination, it can effectively keep the activity of cell and heredity steady
It is qualitative, to improve the success rate of anabiosis rate and differentiation.
Further, the step S4 includes the following steps:
S4.1: the cell frozen being taken out, is placed in 40 DEG C of water-baths and places 60s, and taking-up, which is placed in 10 DEG C of water-baths, places
30s；
S4.2: cell is taken out, and is placed in 37 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 5 DEG C of water-baths, places 30s；
S4.3: cell is taken out, and is placed in 30 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 2 DEG C of water-baths, places 30s；
S4.4: cell is taken out, and is gently shaken 1~3min at room temperature, is washed 2~4 times with PBS buffer solution, be centrifuged, discard
Buffer.
Aforesaid operations can make the cell frozen be rapidly heated in recovery, to freeze the moisture in protection liquid in defrosting
Ice crystal, damaging cells structure are generated in the process；Meanwhile the heating cooling repeatedly within the temperature range of 2~40 DEG C, it can also make thin
The physiological activity of born of the same parents slowly restores, and cell is made gradually to adapt to environment, is ready for recovery.
Further, the step S5 includes the following steps:
S5.1: configuration recovery saves liquid, is placed in 5~10min of pre-cooling at 2~5 DEG C；The recovery saves in liquid added with extremely
A kind of few cell adherence releases molecule and at least one cell activity activator；
S5.2: the isometric recovery of protection liquid is added and frozen in the cell obtained to step S4 and saves liquid, 1~2.5kHz
Under the conditions of 5~10s of ultrasonic vibration, be subsequently placed in 2~5 DEG C of refrigerators and stand 30min；Ultrasound procedure is repeated after taking-up, is obtained thin
Born of the same parents' suspension.
Under the low frequency of 1~2.5kHz, the ultrasonic vibration of short time cannot destroy eucaryotic cell structure, but cell mass can be made to send out
Raw slight loosening generates gap, and recovery can be made to save liquid and be dispersed into droplet, consequently facilitating freezing protection liquid infiltration cell mass
In block.
Further, the recovery saves the ingredient that liquid includes following parts by weight:
15~20 parts of plant source recombination human serum albumins, 5~10 parts of lactalbumins, 0.8~1 part of sodium chloride, 0.8~
1.2 parts of glucose, 0.05~0.1 part of lignan, 0.01~0.02 part of cell activity activator and 0.02~0.05 part of cell
Adherency releases molecule；
The cell activity activator includes in taxol, cow-bezoar deoxidation choline, enuatrol, secretin and Endothelin I
At least one；
Cell adherence contact molecule include Calusena lansium element, in RGDS peptide, Fak inhibitor 14 and eptifibatide at least
It is a kind of.
Cell-preservation liquid conventional at present mainly uses fetal calf serum or human serum albumins, cell preciousness degree is higher,
Requirement to nutrition is higher, and the dosage of mentioned component is also higher；Plant source recombination human serum albumin is based on plant production system
Make, be capable of providing with the comparable nutritional ingredient of serum, fetal calf serum can be substituted, so as to reduce cost；To guarantee that cell obtains
Sufficient nutrition is obtained, saves in liquid and also adds lactalbumin, sugar and salinity, in order to provide the nutrition for being similar to human internal environment
Comprehensively, the moderate environment of osmotic pressure, in favor of the survival of cell；Lignan is closest to the plant hormone of human estrogen, energy
The condition of an environment close in human uterine is provided for cell, so that Endometrial stem cell is smoothly recovered.
Further, the cell activity activator is ox sulphur deoxidation choline；It is RGDS that the cell adherence, which releases molecule,
Peptide.
Ox sulphur deoxidation choline can inhibit er stress, make lipid and embrane-associated protein solubilising, can effectively release cell
Physiological activity is by being inhibited, to make cell activity recovery；RGDS peptide can inhibit cell to connect with vitronectin and fiber
The RGD protein binding such as albumen, to release intercellular adhere to each other.
Beneficial effects of the present invention are as follows: the present invention provides the sides of a kind of Rapid-Freezing Method and recovery Endometrial stem cell
Method can reduce the probability of cell damage apoptosis, improve survival rate；After freezen protective liquid or recovery protection liquid are added into cell
The processing of short time low frequency ultrasound is carried out, can make the cell adhered to each other that slight pine occur under the premise of not destroying eucaryotic cell structure
It is dynamic, generate gap, and liquid can be made to be dispersed into droplet, consequently facilitating frozen solution or recovery save liquid penetrate into cell it
Between；By adding cell adhesion molecule, can promote to adhere to each other between cell, to be further reduced individual cells, improve
The survival rate of cell；Cell active inhibitors can carry out reversible inhibition to intracellular some physiological activities, not influence
It is improved under the premise of cell activity and freezes effect.By above-mentioned means, refrigerating process can be reduced and resuscitation process makes cell
At physical and injury chemically, thus the anabiosis rate for improving cell survival rate and freezing, so as to subsequent use.
Specific embodiment
The preparation of main agents and culture medium
A.DMEM culture medium: specific ingredient is as follows:
| Serial number | Compound name | Content (mg/L) | Serial number | Compound name | Content (mg/L) |
| 1 | Anhydrous calcium chloride | 265.00 | 18 | Serine | 42.00 |
| 2 | Ferric nitrate | 0.10 | 19 | L-threonine | 95.00 |
| 3 | Potassium chloride | 400.00 | 20 | L-Trp | 16.00 |
| 4 | Anhydrous magnesium sulfate | 97.67 | 21 | L-tyrosine | 72.00 |
| 5 | Sodium chloride | 6400.00 | 22 | Valine | 94.00 |
| 6 | Anhydrous sodium dihydrogen phosphate | 109.00 | 23 | D-VB5 calcium | 4.00 |
| 7 | Succinic acid | 75.00 | 24 | Choline tartrate | 7.20 |
| 8 | Sodium succinate | 100.00 | 25 | Folic acid | 4.00 |
| 9 | L- R-gene | 84.00 | 26 | Inositol | 7.20 |
| 10 | L- hydrochloric acid cystine | 63.00 | 27 | Niacinamide | 4.00 |
| 11 | Glycine | 30.00 | 28 | Riboflavin | 0.40 |
| 12 | L- histidine monohydrochloride | 42.00 | 29 | Thiamine hydrochloride | 4.00 |
| 13 | L-Isoleucine | 105.00 | 30 | Pyridoxine hydrochloride | 4.00 |
| 14 | L-Leu | 105.00 | 31 | Glucose | 1000.00 |
| 15 | L-Lysine hydrochloride | 146.00 | 32 | Sodium Pyruvate | 110.00 |
| 16 | L-methionine | 30.00 | 33 | It is phenol red | 9.30.00 |
| 17 | L-phenylalanine | 66.00 |
B.PBS buffer:
Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4With 0.24g KH2PO4, it is dissolved in 800mL distilled water, uses
HCl adjusts the pH value of solution to 7.4, and finally plus distilled water is settled to 1L.In 15lbf/in2 (1034 × 105Pa) high pressure
Lower steam sterilization (at least 20 minutes), is stored in room temperature or 4 DEG C of refrigerators.
C. physiological saline:
0.9 gram of sodium chloride is weighed, is dissolved in a small amount of distilled water, is diluted to 100 milliliters.
Below with reference to following embodiment, invention is further described in detail.It should be noted that in following embodiment
The DMEM culture medium is formulated using described in above-mentioned A, on the basis of " also adding " refers to the DMEM culture medium described in A
It is added.DEME culture medium, PBS buffer solution and physiological saline described in following embodiment are all made of above-mentioned formula.
Embodiment 1
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, includes the following steps:
S1: endometrial tissue, adhere-wall culture 48h are extracted, and secondary culture 6d is carried out to obtained cell；
S2: the obtained passage cell of step S1 is placed in DMEM culture medium, is added and is cooled to 2~5 DEG C in advance and freezes protection
Liquid, gently oscillation mixes, the processing of short time low frequency ultrasound, described to freeze in protection liquid added at least one cell adhesion molecule
And at least one cell active inhibitors；
S3: the obtained cell mixture of step S2 is placed in liquid nitrogen, is cooled at -196 DEG C and is saved；
S4: the cell frozen being taken out, is rapidly heated first to 30~40 DEG C, is then cooled to 2~10 DEG C rapidly, is repeated
The above process 3 times；It is washed 2 times after taking-up with PBS buffer solution, is centrifuged, discards cleaning solution；
S5: the pre- recovery isometric with protection liquid is frozen for being cooled to 2~5 DEG C is added in the cell obtained to step S4 and saves
Liquid, short time low speed ultrasonic treatment obtain cell suspension, and the recovery is saved in liquid and released added at least one cell adherence
Molecule and at least one cell activity activator.
Embodiment 2
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, includes the following steps:
S1: endometrial tissue, adhere-wall culture 36h are extracted, and secondary culture 8d is carried out to obtained cell；
S2: the obtained passage cell of step S1 is placed in DMEM culture medium, is added and is cooled to 2~5 DEG C in advance and freezes protection
Liquid, gently oscillation mixes, the processing of short time low frequency ultrasound, described to freeze in protection liquid added at least one cell adhesion molecule
And at least one cell active inhibitors；
S3: the obtained cell mixture of step S2 is placed in liquid nitrogen, is cooled at -196 DEG C and is saved；
S4: the cell frozen being taken out, is rapidly heated first to 30~40 DEG C, is then cooled to 2~10 DEG C rapidly, is repeated
The above process 3 times；It is washed 4 times after taking-up with PBS buffer solution, is centrifuged, discards cleaning solution；
S5: the pre- recovery isometric with protection liquid is frozen for being cooled to 2~5 DEG C is added in the cell obtained to step S4 and saves
Liquid, short time low speed ultrasonic treatment obtain cell suspension, and the recovery is saved in liquid and released added at least one cell adherence
Molecule and at least one cell activity activator.
Embodiment 3
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 1, wherein step
The specific method is as follows by S1:
S1.1: acquisition endometrium decidua tissue sample, cleaned up with physiological saline, shred into 1~2mm fragment,
It is laid in Tissue Culture Flask, Type I collagen enzyme is added, primary culture medium is added after concussion digestion 60min and terminates digestion, after centrifugation
Remove supernatant, addition primary culture medium is placed in 5%CO2, cultivated in 37 DEG C of incubator, every primary training of 3~4d replacement
Base is supported, the primary culture medium is the DMEM culture of the chlorogenic acid of fetal calf serum and 30mg/L added with volume fraction 10%
Liquid；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is added secondary culture base, sets after digestion
In 5%CO2, cultivated in 37 DEG C of incubator, cultivate until when cell fusion degree reaches 80~90%, the passage is trained
Base is supported as the DMEM training of the dense element of mandarin orange of the gallic acid and 2mg/L of fetal calf serum, 25mg/L added with volume fraction 15%
Support base.
Embodiment 4
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 2, wherein step
The specific method is as follows by S1:
S1.1: acquisition endometrium decidua tissue sample, cleaned up with physiological saline, shred into 1~2mm fragment,
It is laid in Tissue Culture Flask, Type I collagen enzyme is added, primary culture medium is added after concussion digestion 60min and terminates digestion, after centrifugation
Remove supernatant, addition primary culture medium is placed in 5%CO2, cultivated in 37 DEG C of incubator, every primary training of 3~4d replacement
Base is supported, the primary culture medium is the DMEM culture of the chlorogenic acid of fetal calf serum and 50mg/L added with volume fraction 10%
Liquid；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is added secondary culture base, sets after digestion
In 5%CO2, cultivated in 37 DEG C of incubator, cultivate until when cell fusion degree reaches 80~90%, the passage is trained
Base is supported as the DMEM training of the dense element of mandarin orange of the gallic acid and 2mg/L of fetal calf serum, 15mg/L added with volume fraction 15%
Support base.
Embodiment 5
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 3, the step
S2 includes the following steps:
S2.1: the obtained passage cell of step S1 being placed in DMEM culture medium and is impregnated, and gently oscillation mixes 1 minute,
It is centrifuged 3min under the conditions of 1500rpm/min, discards supernatant, repeats aforesaid operations 3 times, DMEM is added after discarding supernatant for the last time
Culture medium；
S2.2: configuration freezes protection liquid, is placed at 2~5 DEG C and 5min is pre-chilled；
S2.3: being added into passage cell and what DMEM culture medium was isometric freezes protection liquid, ultrasound shake under the conditions of 1kHz
10s is swung, is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, is subsequently placed in quiet in 2~5 DEG C of refrigerators
Set 60min.
Freeze the ingredient comprising following parts by weight in protection liquid: 30 parts of cryoprotectors, 0.01 part of cell activity inhibit
Agent, 0.02 part of cell adhesion molecule and 1.5 parts of hydrophilic colloids；
Cell active inhibitors include the brefeldin A and several husband's alkali that weight fraction ratio is 4:1；
Cell adhesion molecule is RGD peptide；
Hydrophilic colloid includes the tara gum and sesbania gum that weight fraction ratio is 2:3.
Embodiment 6
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 4, the step
S2 includes the following steps:
S2.1: the obtained passage cell of step S1 being placed in DMEM culture medium and is impregnated, and gently oscillation mixes 3 minutes,
It is centrifuged 5min under the conditions of 1000rpm/min, discards supernatant, repeats aforesaid operations 2 times, DMEM is added after discarding supernatant for the last time
Culture medium；
S2.2: configuration freezes protection liquid, is placed at 2~5 DEG C and 10min is pre-chilled；
S2.3: being added into passage cell and what DMEM culture medium was isometric freezes protection liquid, ultrasound under the conditions of 2.5kHz
5s is shaken, is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, is subsequently placed in 2~5 DEG C of refrigerators
Stand 60min.
Freeze the ingredient comprising following parts by weight in protection liquid: 40 parts of cryoprotectors, 0.02 part of cell activity inhibit
Agent, 0.05 part of cell adhesion molecule and 2.5 parts of hydrophilic colloids；
Cell active inhibitors include the brefeldin A and several husband's alkali that weight fraction ratio is 4:1；
Cell adhesion molecule is RGD peptide；
Hydrophilic colloid includes the tara gum and sesbania gum that weight fraction ratio is 2:3.
Embodiment 7
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 5, the step
S4 includes the following steps:
S4.1: the cell frozen being taken out, is placed in 40 DEG C of water-baths and places 60s, and taking-up, which is placed in 10 DEG C of water-baths, places
30s；
S4.2: cell is taken out, and is placed in 37 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 5 DEG C of water-baths, places 30s；
S4.3: cell is taken out, and is placed in 30 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 2 DEG C of water-baths, places 30s；
S4.4: cell is taken out, and gently shakes 3min at room temperature, is washed 4 times with PBS buffer solution, is centrifuged, is discarded buffering
Liquid.
Embodiment 8
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 6, the step
S4 includes the following steps:
S4.1: the cell frozen being taken out, is placed in 40 DEG C of water-baths and places 60s, and taking-up, which is placed in 10 DEG C of water-baths, places
30s；
S4.2: cell is taken out, and is placed in 37 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 5 DEG C of water-baths, places 30s；
S4.3: cell is taken out, and is placed in 30 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 2 DEG C of water-baths, places 30s；
S4.4: cell is taken out, and gently shakes 1min at room temperature, is washed 2 times with PBS buffer solution, is centrifuged, is discarded buffering
Liquid.
Embodiment 9
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 7, the step
S5 includes the following steps:
S5.1: configuration recovery saves liquid, is placed at 2~5 DEG C and 5min is pre-chilled；
S5.2: the isometric recovery of protection liquid is added and frozen in the cell obtained to step S4 and saves liquid, 1kHz condition
Lower ultrasonic vibration 10s is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, obtains cell suspension.
Recovery saves the ingredient that liquid includes following parts by weight:
20 parts of plant source recombination human serum albumins, 5 parts of lactalbumins, 0.8 part of sodium chloride, 0.8 part of glucose, 0.1 part
Lignan, 0.01 part of cell activity activator and 0.02 part of cell adherence release molecule；
Cell activity activator is ox sulphur deoxidation choline；
It is RGDS peptide that cell adherence, which releases molecule,.
Embodiment 10
A kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, including each step described in embodiment 8, the step
S5 includes the following steps:
S5.1: configuration recovery saves liquid, is placed at 2~5 DEG C and 10min is pre-chilled；
S5.2: the isometric recovery of protection liquid is added and frozen in the cell obtained to step S4 and saves liquid, 2.5kHz item
Ultrasonic vibration 5s under part is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, obtains cell suspension.
Recovery saves the ingredient that liquid includes following parts by weight:
15 parts of plant source recombination human serum albumins, 10 parts of lactalbumins, 1 part of sodium chloride, 1.2 parts of glucose, 0.05 part
Lignan, 0.02 part of cell activity activator and 0.05 part of cell adherence release molecule；
Cell activity activator is ox sulphur deoxidation choline；
It is RGDS peptide that cell adherence, which releases molecule,.
Experimental example 1
Anabiosis rate experiment
The frozen stock solution provided respectively using embodiment 1,3,5,7,9 as experimental group 1~5, with conventional procedural freezing preservation method with
And Conventional vitrification freezing preservation method freezes the endometrial tissue of same source respectively as control group 1~2 respectively, it is multiple
3*10 is pressed after Soviet Union5Inoculum concentration be inoculated into the previously described secondary culture base of 5mL respectively, be placed in 5%CO2, 37 DEG C of incubator
Middle culture 120h, the total number of cells after calculating culture with blood counting chamber, according to total number of cells/(3* after appreciation rate=culture
105) calculate appreciation rate, as anabiosis rate.The results are shown in Table 1.
The comparison of 1 group of cells recovery situation of table
As shown in Table 1, the anabiosis rate of experimental group each group reaches 90% or more, is significantly higher than control group each group.Show this
Conventional method can be effectively reduced caused by cell in the method for inventing the Rapid-Freezing Method and recovery Endometrial stem cell that provide
Injury physically or chemically, and the physiological activity of Endometrial stem cell can be effectively saved, to be conducive to subsequent use.
Experimental example 2
Cell surface immunophenotype genetic experiment
Respectively in the method that embodiment 2,4,6,8,10 provides as experimental group 1~5, with conventional procedural freezing preservation method and often
Glass frozen preservation method is advised respectively as control group 1~2, the endometrial tissue of same source is frozen respectively, after recovery
By 3*105Inoculum concentration be inoculated into the previously described secondary culture base of 5mL respectively, be placed in 5%CO2, train in 37 DEG C of incubator
48h is supported, and flow cytometer detection is carried out to the surface antigen of the cell of acquisition, while using mother cell as positive control.
The experimental results showed that CD14, CD31, CD34 (HSPC and endothelial cell are positive), CD45 (leucocyte sun in each group
Property), CD54 (ICAM-1), CD80 (B7-1), that CD86 (B7-2) and HLA-DR (MHC-II class molecule) etc. do not express hematopoiesis is thin
Cellular surface mark shows as feminine gender, and CD29 and CD44 (receptor of fibrin and transparency grease hydrochlorate, stroma cell expression),
CD73 (SH-3,4), CD105 (SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I class molecule)) and UEA-1
(surface marker of endothelial cell) expression is positive.Compared with positive control, cell surface is immune for experimental group and control group each group
Phenotype type is all the same, and each cell surface antigen ratio is not significantly different.It can thus be appreciated that the cell by originally culture
With atom endometrial stem cells surface antigen having the same, illustrate to be frozen, at recovery using method provided by the invention
The Endometrial stem cell of reason has reproduced the genetic phenotype of parental cell well, and genetic stability is good.
Experimental example 3
Osteoblast Differentiation performance test
Respectively in the method that embodiment 9 and 10 provides as experimental group 1~2, with conventional procedural freezing preservation method and conventional glass
Glass freezing preservation method freezes the endometrial tissue of same source respectively as control group 1~2 respectively, and 3* is pressed after recovery
105Inoculum concentration be inoculated into the previously described secondary culture base of 5mL respectively, be placed in 5%CO2, cultivate in 37 DEG C of incubator,
Cell fusion degree reaches 80~90% after recovering, and centrifuge cell is digested with 0.25% pancreatin, according to 1 × 103Cell/cm2Connect
Kind amount is inoculated in six orifice plates, after complete medium culture for 24 hours is added, Osteogenic Induction Medium is added and continues culture 2~4 weeks,
It changes liquid within every 2~3 days, is formed after culture with alkaline phosphatase staining identification osteoblast, and dyed and identified with Von Kossa
Bone tubercle is formed.
After cultivating 1 week, apparent change occurs for cellular morphology, becomes polygonal from spindle, cell periphery occurs Filamentous
It is prominent, and can extend to surrounding.After culture 2 weeks or more, starting calcified plaque occur in cellular matrix, mineralizer gradually appears, and
And the small junction structure of multilayer is gradually formed, alkaline phosphatase staining at this time is in strong positive reaction, reaches 90% or more；After cultivating 4 weeks,
Visible apparent black calcium scoring is dyed by Von Kossa.Show to be frozen, recovered using method provided by the invention
The Endometrial stem cell of processing is still able to maintain good Osteoblast Differentiation potential.
Experimental example 4
At Adipose Differentiation performance test
Respectively in the method that embodiment 9 and 10 provides as experimental group 1~2, with conventional procedural freezing preservation method and conventional glass
Glass freezing preservation method freezes the endometrial tissue of same source respectively as control group 1~2 respectively, and 3* is pressed after recovery
105Inoculum concentration be inoculated into the previously described secondary culture base of 5mL respectively, be placed in 5%CO2, cultivate in 37 DEG C of incubator,
Cell fusion degree reaches 80~90% after recovering, and centrifuge cell is digested with 0.25% pancreatin, according to 1 × 103Cell/cm2Connect
Kind amount is inoculated in six orifice plates, after complete medium culture for 24 hours is added, Adipogenic induction culture medium is added and continues culture 2~4
It changes liquid in every 2~3 days in week, dyes identification fat drop with oil red after culture and formed.
After culture 3 days, cellular morphology changes, and is gradually tapered up and is shortened by spindle, and 90% or more cell becomes vertical
Rectangular or polygonal；After continuous culture 1 week, there is small fat drop to occur in visible cell under mirror, and fat drop is with culture
The extension of time is gradually expanded, merges；After cultivating 2 weeks, it is seen that merge pockets of fat drop full of entire cell.It is contaminated by oil red
The fat generated in color visible cell dyes red by specificity.Show to be frozen, recovered using method provided by the invention
The Endometrial stem cell of processing is still able to maintain good at Adipose Differentiation potential.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of method of Rapid-Freezing Method and recovery Endometrial stem cell, which comprises the steps of:
S1: endometrial tissue, adhere-wall culture 36h~48h are extracted, and 6~8d of secondary culture is carried out to obtained cell；
S2: the obtained passage cell of step S1 is placed in DMEM culture medium, is added and is cooled to 2~5 DEG C in advance and freezes protection liquid, gently
Light oscillation mixes, the processing of short time low frequency ultrasound, it is described freeze in protection liquid added at least one cell adhesion molecule and
At least one cell active inhibitors；
S3: the obtained cell mixture of step S2 is placed in liquid nitrogen, is cooled at -196 DEG C and is saved；
S4: the cell frozen being taken out, is rapidly heated first to 30~40 DEG C, is then cooled to 2~10 DEG C rapidly, is repeated above-mentioned
Process 3 times；It is washed 2~4 times after taking-up with PBS buffer solution, is centrifuged, discards cleaning solution；
S5: being added the pre- recovery isometric with protection liquid is frozen for being cooled to 2~5 DEG C and save liquid in the cell obtained to step S4,
Short time low speed ultrasonic treatment, obtains cell suspension, and the recovery saves in liquid added at least one cell adherence releasing point
Son and at least one cell activity activator.
2. the method for Rapid-Freezing Method as described in claim 1 and recovery Endometrial stem cell, which is characterized in that the step
S1 includes the following steps:
S1.1: acquisition endometrium decidua tissue sample is cleaned up with physiological saline, shreds into fragment, the tiling of 1~2mm
In Tissue Culture Flask, Type I collagen enzyme is added, primary culture medium is added after concussion digestion 60min and terminates digestion, removes after centrifugation
Supernatant, addition primary culture medium are placed in 5%CO2, cultivated in 37 DEG C of incubator, every primary culture of 3~4d replacement
Base, the primary culture medium are the DMEM training of the chlorogenic acid of fetal calf serum and 30~50mg/L added with volume fraction 10%
Nutrient solution；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is added secondary culture base, is placed in 5% after digestion
CO2, cultivated in 37 DEG C of incubator, cultivate the secondary culture base until when cell fusion degree reaches 80~90%
For the gallic acid of fetal calf serum, 15~25mg/L added with volume fraction 15% and the DMEM of the dense element of the mandarin orange of 2~5mg/L
Culture medium.
3. the method for Rapid-Freezing Method as described in claim 1 and recovery Endometrial stem cell, which is characterized in that the step
S2 includes the following steps:
S2.1: the obtained passage cell of step S1 being placed in DMEM culture medium and is impregnated, and gently oscillation mixes 1~3 minute,
It is centrifuged 3~5min under the conditions of 1000~1500rpm/min, discards supernatant, repeats aforesaid operations 2~3 times, discards for the last time
DMEM culture medium is added after clear；
S2.2: configuration freezes protection liquid, is placed in 5~10min of pre-cooling at 2~5 DEG C；
S2.3: being added into passage cell and what DMEM culture medium was isometric freezes protection liquid, ultrasound shake under the conditions of 1~2.5kHz
5~10s is swung, is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, is subsequently placed in 2~5 DEG C of refrigerators
Middle standing 60min.
4. the method for Rapid-Freezing Method as claimed in claim 3 and recovery Endometrial stem cell, which is characterized in that described to freeze
Protect liquid in include following parts by weight ingredient: 30~40 parts of cryoprotectors, 0.01~0.02 part of cell active inhibitors,
0.02~0.05 part of cell adhesion molecule and 1.5~2.5 parts of hydrophilic colloids；
The permeability cryoprotector includes proline, mannitol, trehalose and the hydroxyl second that weight fraction ratio is 2:5:2:1
Base starch；
The cell active inhibitors include Cytochalasin D, nocodazole, tunicamycin, several husband's alkali, brefeldin A,
At least one of CP-10447 and Octreotide；
The cell adhesion molecule includes at least one of E-Selectin, the glutinous element of P- calcium, ICAM-1, peptide F9 and RGD peptide；
The hydrophilic colloid includes tara gum, trigonella bean gum, tragacanth, sesbania gum, tamarind gum, locust bean gum and peach gum
At least one of.
5. the method for Rapid-Freezing Method as claimed in claim 4 and recovery Endometrial stem cell, which is characterized in that the cell
Activity inhibitor includes the brefeldin A and several husband's alkali that weight fraction ratio is 4:1；The cell adhesion molecule is RGD
Peptide.
6. the method for Rapid-Freezing Method as claimed in claim 4 and recovery Endometrial stem cell, which is characterized in that described hydrophilic
Property colloid include weight fraction ratio be 2:3 tara gum and sesbania gum.
7. the method for Rapid-Freezing Method as described in claim 1 and recovery Endometrial stem cell, which is characterized in that the step
S4 includes the following steps:
S4.1: the cell frozen being taken out, is placed in 40 DEG C of water-baths and places 60s, and taking-up, which is placed in 10 DEG C of water-baths, places 30s；
S4.2: cell is taken out, and is placed in 37 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 5 DEG C of water-baths, places 30s；
S4.3: cell is taken out, and is placed in 30 DEG C of water-baths and is placed 60s, and taking-up, which is placed in 2 DEG C of water-baths, places 30s；
S4.4: cell is taken out, and is gently shaken 1~3min at room temperature, is washed 2~4 times with PBS buffer solution, be centrifuged, discard buffering
Liquid.
8. the method for Rapid-Freezing Method as described in claim 1 and recovery Endometrial stem cell, which is characterized in that the step
S5 includes the following steps:
S5.1: configuration recovery saves liquid, is placed in 5~10min of pre-cooling at 2~5 DEG C；The recovery, which saves, is added at least one in liquid
Kind cell adherence releases molecule and at least one cell activity activator；
S5.2: the isometric recovery of protection liquid is added and frozen in the cell obtained to step S4 and saves liquid, 1~2.5kHz condition
5~10s of lower ultrasonic vibration is subsequently placed in 2~5 DEG C of refrigerators and stands 30min；Ultrasound procedure is repeated after taking-up, and it is outstanding to obtain cell
Liquid.
9. the method for Rapid-Freezing Method as claimed in claim 8 and recovery Endometrial stem cell, which is characterized in that the recovery
Save the ingredient that liquid includes following parts by weight:
15~20 parts of plant source recombination human serum albumins, 5~10 parts of lactalbumins, 0.8~1 part of sodium chloride, 0.8~1.2 part
Glucose, 0.05~0.1 part of lignan, 0.01~0.02 part of cell activity activator and 0.02~0.05 part of cell adherence solution
Except molecule；
The cell activity activator include in taxol, cow-bezoar deoxidation choline, enuatrol, secretin and Endothelin I extremely
Few one kind；
Cell adherence contact molecule includes Calusena lansium element, at least one in RGDS peptide, Fak inhibitor 14 and eptifibatide
Kind.
10. the method for Rapid-Freezing Method as claimed in claim 9 and recovery Endometrial stem cell, which is characterized in that described thin
Cytoactive activator is ox sulphur deoxidation choline；It is RGDS peptide that the cell adherence, which releases molecule,.
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