CN107156107A - A kind of human umbilical cord mesenchymal stem cells cryoprotective agent comprising rhMG53 - Google Patents
A kind of human umbilical cord mesenchymal stem cells cryoprotective agent comprising rhMG53 Download PDFInfo
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- CN107156107A CN107156107A CN201710362140.7A CN201710362140A CN107156107A CN 107156107 A CN107156107 A CN 107156107A CN 201710362140 A CN201710362140 A CN 201710362140A CN 107156107 A CN107156107 A CN 107156107A
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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
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- 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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- 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
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Abstract
The invention discloses a kind of human umbilical cord mesenchymal stem cells cryoprotective agent comprising rhMG53, the protective agent is formulated by DMEM/F12 culture mediums, FBS, DMSO and rhMG53, first by DMEM/F12 culture mediums, FBS and DMSO by volume 5:4:1 is configured to mixed liquor；Then rhMG53 is added in 30 μ g/mL ratio in the mixed liquor, human umbilical cord mesenchymal stem cells cryoprotective agent finished product is obtained after being well mixed.Compared with existing mesenchymal stem cell cryopreserving protective agent, clearly, stability is good, safe for protective agent composition of the present invention.For freezing during hUC MSCs, its caryogram and dryness are not influenceed, and can promote the propagation of stem cell, improve its survival rate and into fat, skeletonization, into Neural Differentiation efficiency.The protective agent prepared using the present invention uses cooperatively isopropanol freezing storing box during freezing, simple to operate, preserves conveniently and cost is low.Experiment proves that the cellular transplantation therapy TBI mouse preserved using the present invention can play preferable therapeutic effect, and reliable guarantee is provided for hUC MSCs clinical practice.
Description
Technical field
It is dry more particularly, to a kind of human umbilical cord mesenchymal comprising rhMG53 the present invention relates to the preservation of stem cell cryogenic freezing
Cell cryoprotective agent.
Background technology
Human umbilical cord mesenchymal stem cells（hUC-MSCs）Have the advantages that to obtain simple and be easy to culture, be used as the thin of candidate
Born of the same parents' treatment product is better than embryonic stem cell or induced multi-potent stem cell.Either basic research or clinical practice（As extensively
Degenerative disease and autoimmune disease etc.）, it is required for mescenchymal stem cell that there is stable function and sufficient supply.
It is cell storage mode the most frequently used at present and the cryogenic freezing of cell preserves with the obvious advantage and technology maturation.
During cryogenic freezing, the formation of ice crystal can cause the breakage of cell membrane in intraor extracellular environment, so as to cause
Cell death.This process is the key for determining cell cryopreservation success, and it is that solution is cold to add suitable cryoprotective agent
Freeze and caused during thawing the most effective way of cell membrane damage.
Traditional cell cryopreservation formula of liquid main component is DMSO, but DMSO has cytotoxicity, particularly embryo dry thin
The cryopreservation resuscitation rate of born of the same parents is subject to processing process and the influence of freezing protective agent is larger, because processing mode is different, its cryopreservation resuscitation rate
From 0.4-5%, and different degrees of missing then occurs in stem cell versatility specific markers Oct4 after cell recovery.
Need to the suspend stem cell of culture such as NSC, embryonic stem cell, tumor stem cell then easily causes and freezes rear part stem cell
The reduction of specific activity mark is even lacked.Recent studies have found that, by induced multi-potent stem cell（iPSC）Freeze 10%
In DMSO, the expression of the mark Oct4 of versatility after thawing can be caused almost to completely lose.Hyclone（FBS）It can be cell
Protein, metabolism of lipid and cholesterol etc. are provided during freezing, and can improve and freeze effect, as supplementing or for freezen protective
Basal medium is widely used, but due to its potential biological safety and to acceptor propagate pathogen the problems such as, use
Amount is also unsuitable too high.In addition, trehalose（Trehalose）It is a kind of naturally occurring non-reducing disaccharide, is used as impermeability
Cryoprotective agent, its biological safety is higher, but due to being macromolecular substances, it is impossible to penetration cell film, it is impossible to effectively prevent
The formation of intracellular ice crystal, freezes rear cell survival rate typically relatively low.At this stage, domestic mesenchymal stem cell cryopreserving protective agent is also
There is not the formula of standardization, the effect after recovery is also difficult to control to, it is expensive that external import stem cell correlation freezes reagent, nothing
Method large-scale promotion is in research application.Therefore urgently research and develop it is a kind of with the autonomous property right of China efficiently freeze reagent, it would be highly desirable to open
Send the stem cell cryopreserving pattern of standardization.
MG53 albumen is tripartite motif family (TRIM) egg of the muscle specific expression found in recent years
In vain, family protein Chang Hansan specific block structures, are known respectively as RING and refer to, B-BOX and frizzled domain, and they are common
Effect is combined on cell specific protein, is carried out ubiquitination degraded, is a kind of important cell membrane repair mechanism.Although natural
MG53 albumen is mainly limited to skeletal muscle and cardiac muscular tissue, but newly discovered its is overexpressed MG53 in a variety of nonmuscle cellses
With reduction cellular damage, the effect that apoptosis occurs is reduced.The people source MG53 albumen of genetic recombination（rhMG53）It can be located at damage
Traumatic part position, the damage of the muscle and nonmuscle cells that are caused to mechanical external force, chemical substance or ultraviolet etc. all shows dosage
The protective effect of dependence, biological safety is good.However, about MG53 stem cell cryopreserving damage in repair there is not yet
Research report.
The content of the invention
There is provided one for the problems such as present invention is for the membrane damage caused during the preservation of stem cell cryogenic freezing and low survival rate
The rhMG53 planted in the human umbilical cord mesenchymal stem cells cryoprotective agent for including rhMG53, the cryoprotective agent has repair membrane damage
Effect of wound, can play the effect protected and repaired to the membrane damage during cryopreservation resuscitation.
To achieve the above object, the present invention can take following technical proposals：
Human umbilical cord mesenchymal stem cells cryoprotective agent of the present invention comprising rhMG53, the protective agent is by DMEM/F12
Culture medium, FBS, DMSO and rhMG53 are formulated, first by the DMEM/F12 culture mediums, FBS and DMSO by volume 5:
4:1 is configured to mixed liquor；Then rhMG53 is added in 30 μ g/mL ratio in the mixed liquor, people is obtained after being well mixed
Umbilical cord mesenchymal stem cells cryoprotective agent finished product, i.e. hUC-MSCs cryoprotective agents.
The feature of main component is in the hUC-MSCs hypothermic protective solutions that the present invention is prepared：
1）rhMG53：Due to Mitsugumin53（MG53）Albumen is the Tripartite of a kind of skeletal muscle and myocardium specifically expressing
motif （TRIM）Family protein, Chang Hansan specific structure of the family protein：RING refers to, B-BOX and frizzled domain, they
Collective effect is combined on cell specific protein, is carried out ubiquitination degraded, is a kind of important cell membrane repair mechanism.
2）DMSO（Dimethyl sulfoxide, dimethyl sulfoxide (DMSO)）：With highly polar, higher boiling, heat endurance it is good, with
The miscible characteristic of water, can be dissolved in most of organic matters such as ethanol, propyl alcohol, be described as " alembroth ".
3）DMEM/F12 culture mediums（Dulbecco's modified Eagle medium/F12）：It is hUC-MSCs base
Basal culture medium, is that freezing for stem cell provides isotonic electrolyte.
4）FBS（Fatal bovine serum, hyclone）：Can be offer protein, fat during cell cryopreservation
With cholesterol etc., can improve cell freezes effect.
Compared with existing mesenchymal stem cell cryopreserving protective agent, the protective agent composition that the present invention is prepared is clear and definite, stability
It is good, it is safe.For freezing during hUC-MSCs, its caryogram and dryness are not influenceed, and can promote the propagation of stem cell, improve it
Survival rate and into fat, skeletonization, into Neural Differentiation efficiency.The protective agent prepared using the present invention uses program during freezing
Change slow freezing method and freezen protective is carried out to cell, it is simple to operate, preserve convenient and cost and lower.Experiment is proved, using this hair
The cellular transplantation therapy TBI mouse of bright preservation, can play preferable therapeutic effect, and for hUC-MSCs clinical practice, provide can
The guarantee leaned on.
Brief description of the drawings
Fig. 1 is the streaming figure of hUC-MSCs surface antigens identification.
Fig. 2 is that rhMG53 acts on hUC-MSCs optimum concentration and optimal freezes mode.
Fig. 3 is expression of the protective agent of the present invention to hUC-MSCs dryness gene after recovery.
Fig. 4 is the different influences for freezing scheme to hUC-MSCs caryogram change after recovery.
Fig. 5 is influence of the protective agent of the present invention to hUC-MSCs survival rates.
Fig. 6 is the different influences broken up into fat for freezing scheme to hUC-MSCs after recovery.
Fig. 7 is that difference freezes influence of the scheme to hUC-MSCs Osteoblast Differentiation after recovery.
Fig. 8 is that difference freezes influence into Neural Differentiation of the scheme to hUC-MSCs after recovery.
Fig. 9 be using the present invention freeze the hUC-MSCs after protection liquid Cord blood recovery to TBI model mices body weight and
The influence of mNSS scores.
Figure 10 is to freeze the hUC-MSCs after the Cord blood recovery of protection liquid to the depressed feelings of TBI model mices using the present invention
The influence of condition and cognitive function recovery situation.
Embodiment
More detailed explanation is done to the present invention below by specific embodiment.
The cryopreservation methods that the present embodiment is used in experimental method, unless otherwise noted, are for sequencing slow freezing method
Conventional method.Experiment utensil, instrument, reagent used can be obtained by commercial sources.
First, the human umbilical cord mesenchymal stem cells cryoprotective agent of the present invention comprising rhMG53 is prepared：The protective agent
In include DMEM/F12 culture mediums（HyClone, article No.：SH30023.01B）、FBS（GIBCO, article No.：10270106）、DMSO
（Amresco, article No. 0231）And rhMG53（Novoprotein, article No.：MG53-A-5, by Ohio State Univ-Columbus USA, fiber crops are built
Professor outstanding person provides）Four kinds of components；
First by DMEM/F12 culture mediums, FBS and DMSO by volume 5:4:1 is configured to mixed liquor（Referred to as frozen stock solution A）；Then
RhMG53 is added in 30 μ g/mL ratio in the mixed liquor, hUC-MSCs cryoprotective agents are obtained after being well mixed（Referred to as
Frozen stock solution B）；That is, the cryoprotective agent finished product prepared in the 1mL present invention（B liquid）In, the trainings of DMEM/F12 containing 0.5mL
Support base, 0.4mL FBS, 0.1mL DMSO and 30 μ g rhMG53.
2nd, human umbilical cord mesenchymal stem cells are frozen using the A liquid and B liquid of above-mentioned preparation, step is as follows：
1st, hUC-MSC separation, culture and identification
The acquisition of umbilical cord：The umbilical cord sample in laboratory is obtained from affiliated hospital of Zhengzhou University first, and umbilical cord is in aseptic condition
Under be taken from the Cesarean esction neonate of health, family members know and agree to.
The stripping and culture of jelly of Wharton tissue：In superclean bench, fresh umbilical cord sample is taken out, is positioned over sterile
In culture dish, umbilical cord tissue is rinsed well with the PBS of precooling, using it is ready in advance without mycoderma clamp, scissors and
Tweezers peel off tissue block outer membrane, reject 2 radicular arterieses and 1 radicular vein blood vessel, obtain jelly of Wharton tissue；By jelly of Wharton tissue PBS
After buffer solution for cleaning is clean, is shredded with sterile scissors and be spread evenly across 25cm2Blake bottle bottom of bottle（Density is about 0.2g/
Bottle）, 5min is stood, when tissue block adheres to bottom of bottle completely, 3mL stem cell complete mediums are added into blake bottle.Then,
Tissue Culture Flask is transferred to CO2Cell culture incubator（37 DEG C, 5%CO2Concentration）Middle culture.
HUC-MSCs primary and Secondary Culture：After uterus tissue pieces 4d, the adherent situation of tissues observed block, if it is adherent compared with
Jail, then change fresh culture and continue to cultivate, fresh culture is changed again after otherwise waiting 1 week.Trained by 10d or so tissue block
Support, by microscope it was observed that " climbing " from tissue block in the attached cell of long fusiform, cell now is primary cell, with
Every 2 ~ 3d changes a subculture afterwards.During uterus tissue pieces 2 weeks or so, stem cell can be observed under the microscope in the close of bottom of bottle
Degree is higher（80 ~ 90%）, now, the tissue block in blake bottle is removed with disposable sterilized suction pipe, PBS slowly cleans training
Support bottom of bottle portion；Then, 1mL trypsase is added, 2 ~ 3min is digested under the conditions of 37 DEG C, micro- Microscopic observation cell retraction is rounded
Isometric FBS is added afterwards terminates digestion；Then, cell suspension is slowly blown and beaten several times in bottom of bottle with liquid-transfering gun, treats adherent thin
Born of the same parents are scattered in suspension completely, and supernatant is removed after suspension is moved into 15mL centrifuge tubes, 1000g centrifugations 5min；By the thin of precipitation
Born of the same parents are resuspended with 2mL complete mediums, are dispensed into 2 blake bottles, and supplement complete medium is cultivated to 5mL in incubator, this
When cell marking be P1 generations.
HUC-MSCs's freezes：By the cell in P1 generations, Secondary Culture be P2 for cell after, treat that cell fusion degree reaches 90%
During left and right, Trypsin Induced is centrifuged and collects cell, and cell is divided into 2 groups, is separately added into frozen stock solution A and B, is floated and is adjusted again
Whole cell density is 1 × 106Individual/mL, is dispensed into 2mL cryopreservation tubes, often pipe plus 1ml cell suspensions, carries out and marks and stick envelope
Membrana oralis, is put into the cell cryopreservation box for being placed in 4 DEG C in advance, and freezing storing box is transferred into -80 DEG C of refrigerators, by cryopreservation tube after storing overnight
Move into liquid nitrogen container stored for extended periods.
HUC-MSCs recovery：It is immediately placed in after the P2 frozen in liquid nitrogen is taken out for stem cell in 37 DEG C of water-baths,
1min or so just can melt；In superclean bench, the stem cell suspension after thawing is transferred in 15mL centrifuge tubes, 1000g
Centrifuge 5min；Precipitation is collected, is resuspended with 1mL stem cell complete mediums and adds 25cm2In Tissue Culture Flask, supplemented medium
To 5mL, and by cell suspension it is well mixed after move into CO2Cell culture incubator（37 DEG C, 5%CO2Concentration）Middle culture.After recovery, the
Fresh culture and routine observation cell state are changed to cell within two days.
HUC-MSCs surface antigen identification：By the P2 after above-mentioned recovery for cell, after Secondary Culture, growth conditions are taken
Good P3 is for hUC-MSCs（Follow-up all experiment cells are using P3 for hUC-MSCs）, add the trypsase without EDTA
Digest, centrifuge and collect cell, then rinse cell 2 ~ 3 times with the PBS of precooling, PBS cell is resuspended and adjust its density for 1 ×
106 Individual/mL.100 μ L cell suspensions are taken, 2 μ L CD34-FITC, CD45-FITC, CD44-FITC, CD133-FITC are added respectively
With HLA-ABC-FITC antibody and corresponding control antibodies, 4 DEG C of incubation 30min, are then centrifuged for abandoning supernatant, and add after mixing
Cell, the expression of the above-mentioned cell surface antigen of flow cytomery is resuspended in 0.5mL PBS.
Fig. 1 is the surface marker using flow cytomery hUC-MSCs, is as a result shown, hUC-MSCs is not expressed and made
Blood system cell surface marker CD34, CD45, height expression interstitial cell mark CD44, stem-cell marker CD133, while it is white to express people
Cellular antigens HLA-ABC, meets mescenchymal stem cell characteristic.
2nd, CCK-8 establishes optimal cryoprotective agent component and optimal freezes mode
The establishment of best protection agent component：This experiment is using hUC-MSCs as research object, and the concentration gradient of setting is：0μg/mL
（NC）、15μg/mL、30μg/mL、60μg/mL.Take the good P3 of growth conditions for hUC-MSCs cells, adjustment cell density is：
3×104Individual/mL, is uniformly inoculated in 96 orifice plates, is added per hole and cell patch is cultivated and observed in 100 μ L cell suspensions, incubator
Wall upgrowth situation, when cell fusion degree reaches 50%, sucks the nutrient solution in orifice plate, is separately added into rhMG53 containing various concentrations
Fresh culture, and three multiple holes of every group of setting, in 37 DEG C, 5%CO2Under concentration cultivate, respectively 12h, 24h, 36h, 48h,
At 60h, 72h time point, old culture medium is removed, 90 μ L fresh cultures and 10 μ LCCK-8 are added per hole, be incubated under the conditions of 37 DEG C
2h, the OD450 values of each processing groups of cells are detected using ELIASA.It is repeated 3 times, and draws cell growth curve.
Fig. 2（A）To detect that rhMG53 acts on hUC-MSCs optimum concentration using CCK-8 methods, as a result find, with
The raising of rhMG53 concentration, ability of cell proliferation gets a promotion, compared with normal culture group cell, and rhMG53 concentration is more than 30
The facilitation effect of cell proliferation is obvious during μ g/ml.The dose-effect relationship that rhMG53 acts on cell is considered, with reference to document report
Road, it may be determined that the optimum concentration that rhMG53 is acted on during cell cryopreservation is：30μg/mL（Cell density is 1 × 106Individual/
mL）.
The optimal establishment for freezing mode：Experiment packet situation is normal culture group（Fresh）, conventional cryopreservation group (NC), freeze
Added when depositing addition rhMG53 (Post+rhMG53) when rhMG53 (Whole+rhMG53), recovery are added in recovery, freezing
rhMG53 (Pro+rhMG53).Above-mentioned treated cell is uniformly inoculated in 96 orifice plates（Per 3000, hole cell）, cell training
Support in case and cultivate, remove old culture medium at 12h, 24h, 36h, 48h, 60h, 72h time point respectively, operation and detection method are same
On, draw cell growth curve.
Fig. 2（B）The optimal of hUC-MSCs is acted on for the rhMG53 using the detection of CCK-8 methods and freezes mode, according to result
It has been shown that, considers protectant cost and streamline operation, 30 μ g/mL rhMG53 is added when freezing（That is Pro+
rhMG53）It can be optimal addition manner.I.e. using the cryoprotective agent prepared during cell cryopreservation mode by the present invention（B liquid）For
The protective agent of addition is needed before jelly.
3rd, after fluorescence quantitative PCR detection cryopreservation resuscitation hUC-MSCs dryness genes expression
The extraction of 3.1 cell total rnas：
（1）Cell is collected by centrifugation, 500 μ L RL Solution are added, cell is blown and beaten repeatedly with pipette tips up to whole cell dissolvings,
Whole lysates are drawn in 1.5mL centrifuge tubes；
（2）40 μ L chloroforms are added, are fully mixed, 12000rpm centrifugations 3min；
（3）The μ L of supernatant 200 ~ 300 are drawn in new 1.5mL centrifuge tubes, isometric absolute ethyl alcohol is added, mixed；
（4）Mixing liquid is sucked in Spin column pipes, 12000rpm centrifugation 1min abandon supernatant；
（5）600 μ LWash Buffer, 12000rpm centrifugation 1min are added, filtrate is abandoned；
（6）Repeat step（5）；
（7）Spin column are managed, 12000rpm idle running 1min, thoroughly remove waste liquid；Then put it into new without RNase
In 1.5mL centrifuge tubes；
（8）50 μ LRNas free water are sucked without enzyme pipette tips, room temperature is placed after 1 ~ 2min, 12000rpm centrifugation 1min are collected
Filtrate, obtains total serum IgE, and measure its concentration.
3.2 total serum IgE reverse transcriptions are into cDNA
By TaKaRa reverse transcription reagent box specifications, every group takes 500ngRNA to carry out reverse transcription, operates as follows：
（1）Removal group DNA
By this reaction system be placed in 42 DEG C keep 2min or（5min at room temperature）, to remove a group DNA.
（2）RNA reverse transcriptions synthesize cDNA
This reaction system is put into regular-PCR instrument, setting reaction condition is：" 37 DEG C of 15min, 85 DEG C of 5s ".Reaction terminates
Afterwards, cDNA is collected.
3.3 qRT-PCR detect the expression of related gene
Quantitative PCR reaction system is as follows：
Each sample sets 3 multiple holes, using the Real-Time PCR instruments of Applied Biosystems 7500, by " 95 DEG C
30s,（95 DEG C of 5s, 60 DEG C of 34s）40 circulations " are detected.After PCR terminates, CT values are exported, using comparing CT methods (Δ Δ
CT), according to formula：Relative expression quantity (RQ)=2- Δ Δ CT, calculates the relative expression quantity of each gene, is repeated 3 times and carries out
Data analysis, makes block diagram.
The primer of dryness related gene such as following table：
Fig. 3 is to freeze the influence to hUC-MSCs dryness gene expressions, is as a result shown, conventional cryopreservation and addition rhMG53 freeze dry
Cell, the expression to the dryness gene of stem cell does not make significant difference.
4th, influences of the chromosome karyotype analysis detection rhMG53 to cryopreservation resuscitation hUC-MSCs caryogram
Experimental cell is divided into three groups：Frozen stock solution A groups and frozen stock solution B groups that normal culture group plus the application are prepared.Caryogram is detected
Experimental procedure is as follows：
（1）Take P3 that is in good condition and being in exponential phase for hUC-MSCs cells, digest smear, cell specimen is made, puts
Enter staining jar, dyed 10-15 minutes with Geimsa dyeing liquors；After dyeing terminates, cell specimen is done the wash being filled with water in plastic cup
Several times, hair-dryer air-dries slide, microscopy.
（2）Photomicrography：The slice, thin piece made is placed under digital photography microscope and shot, for analysis.
（3）Karyotyping
Caryogram：One intracellular all chromosome is lined up in certain sequence, the dye of a certain individual ownership cell is represent
Colour solid is constituted, including form, size, number etc., it is divided into the seven groups and one group sex chromosome such as A, B, C, D, E, F, G.
Group type：Caryogram is showed by the form of ideograph, represent the genome of an individual into.
（4）Complete genome analysis.
Fig. 4 is the detection to freezing rear hUC-MSCs caryogram, and the chromosome bar numbers of three groups of cells, form and structure are normal
And it is undistorted, freeze process on conventional cryopreservation protective agent and rhMG53 on hUC-MSC caryogram without influence, illustrate rhMG53 to dry
Cell is safe and nontoxic.
5th, influences of the frozen stock solution A and frozen stock solution B that the Trypan Blue detection present invention is prepared to hUC-MSCs survival rates
Experimental cell is divided into three groups：Frozen stock solution A groups and frozen stock solution B groups that normal culture group plus the application are prepared, freeze to difference
Time（1 month, 6 months and 1 year）Detected.
Normal living cells, after birth structural integrity can repel trypan blue, be allowed to that intracellular can not be entered；And lose and live
Property or the incomplete cell of cell membrane, after birth permeability increase, blueness can be dyed by trypan blue.It has been generally acknowledged that cell membrane is complete
Property lose, you can think that cell is dead, therefore, living cells can be simply and quickly distinguished very much by Trypan Blue
And dead cell.Experimental procedure is as follows：
（1）The cell of difference group, difference freeze the time, are recovered after being taken out from liquid nitrogen, cultivate 6 hours, treat that cell state is steady
After fixed, trypsin digestion and cell collects all cells and prepares single cell suspension, and makees appropriate dilution.
（2）Dyeing：By cell suspension and 0.4% trypan blue solution with 9:1 mixing is mixed, and gently piping and druming is mixed, 3 ~
Plate is dripped after 5min and counts progress cell dyeing.
（3）Microscopic observation is simultaneously counted：The appropriate cell suspension added with Trypan Blue liquid is drawn, cell counting count board meter is added dropwise
Number, dead cell is dyed to the blueness of bright anvil, and living cells is in colorless and transparent.
（4）Count cell survival rate：Living cell rate (%)=total viable cell/(total viable cell+dead cell sum）×
l00%
Trypan blue detects two kinds of frozen stock solutions（The protective agent that the present invention is prepared is B groups, and the mixed liquor without rhMG53 is A groups）It is right
The influence of hUC-MSCs survival rates is as shown in figure 5, result is found, the cell survival rate for the frozen stock solution B groups that the present invention is prepared is higher,
Illustrate that rhMG53, as hUC-MSCs cryoprotective agent particular components, can improve the survival rate of stem cell.
6th, oil red O stain and qRT-PCR detect that hUC-MSCs breaks up situation into fat after cryopreservation resuscitation
Adipogenic induction breaks up：By freeze two groups of cell recoveries and one group of cell normally cultivated（3 groups of cell deriveds are identical, generation
Number is identical）After Secondary Culture, fat inducing culture is replaced with, a fresh culture is changed every 3d, induction differentiation 14d is left
Right observation cellular morphology simultaneously carries out oil red O stain and RNA extraction.
Oil Red O are dyed：Oil red O is a kind of very strong fatsolvent and fat stain, is easier to be combined with triglycerides in small
Fat drips shape, slightly worse with phosphatide adhesion, its dyeing theory is commonly considered as solution effects or suction-operated physically, by means of solution
Effect makes fat stains.Solubility of the dyestuff in the cell in the more former solvent of solubility of lipid is bigger, so being contaminated in dyeing
Material is just transferred to lipid from organic solvent and makes fat stains.Because lipid concentration is different, fatty positive staining result for it is orange extremely
It is red.Experimental procedure is as follows：
Take induction differentiation 14d stem cell digestion centrifugation and with after PBS, adjust cell density, each group takes identical cell
Cell smear is prepared, remaining cell extraction RNA simultaneously carries out subsequent experimental.Poly first by the cell smear prepared 4%
30min is fixed in aldehyde solution；Then, with PBS twice, the oil red O stain liquid of addition 0.5%, dyes 20min；It is put into
20 ~ 30s is rinsed in 60% aqueous isopropanol, flowing water is rinsed, and dries mounting and micro- sem observation.
Into the expression of fat differentiation associated gene：（Experimental method and step are with 3：After fluorescence quantitative PCR detection cryopreservation resuscitation
The expression of hUC-MSCs dryness genes）.Into the primer sequence such as following table of fat differentiation associated gene：
After oil red O stain and qRT-PCR results are as shown in fig. 6, adipogenic induction breaks up 14 days, oil red O dyeing is carried out, it is seen that each
Group cell, which contains, is contaminated fat drips for red compared with standard, is broken up successfully into fat.And contaminated by the cell positive rate that frozen stock solution B freezes
Color is higher, and into fat differentiation associated gene（FABP4、LPL、PPAR-γ）Expression be obviously improved.
7th, alizarin red S dyeing and qRT-PCR detect hUC-MSCs Osteoblast Differentiation situations after cryopreservation resuscitation
Osteoinductive differentiation：By freeze two groups of cell recoveries and one group of cell normally cultivated（3 groups of cell deriveds are identical, generation
Number is identical）After Secondary Culture, Osteogenic Induction Medium is changed, a fresh culture is changed every 3d, induction differentiation 21d is left
Right observation cellular morphology simultaneously carries out alizarin red S dyeing and RNA extraction.
Alizarin red S is dyed：Alizarin red S is a kind of anthraquinone analog compound, alizarin sulfonic acid sodium salt, and it can be with calcium carbonate or phosphoric acid
Calcium salt chelant in calcium forms orange red compound, it is adaptable to the dyeing of a small amount of calcium salt tissue.Staining procedure is as follows：
After stem cell Osteoinductive differentiation 21 days, digestion centrifuges and uses PBS, suitably dilutes and adjusts cell density, takes phase
Cell with quantity prepares cell smear, remaining cell extraction total serum IgE.Paraformaldehyde by the cell smear prepared 4%
30min is fixed in solution；Then, with PBS twice, 1% alizarin red S dyeing liquor is added（PH=8.3）, dye after 10min；
Rinsed 3 times with PBS, dry rear mounting, micro- sem observation is simultaneously taken pictures.
The expression of Osteoblast Differentiation related gene：（Experimental method and step are with 3：After fluorescence quantitative PCR detection cryopreservation resuscitation
The expression of hUC-MSCs dryness genes）.The primer sequence of Osteoblast Differentiation related gene such as following table：
It is visible after alizarin red S is dyed and qRT-PCR results are as shown in fig. 7, Osteoinductive differentiation dyes for 21 days through alizarin red S
A large amount of calcareous tubercles are colored, and illustrate that skeletonization is divided into work(.And the cell positive rate dyeing frozen by frozen stock solution B is higher, and into
Bone differentiation associated gene（ALPL、OSX、RUNX2）Expression be obviously improved.
8th, after Immunofluorescence test cryopreservation resuscitation hUC-MSCs into Neural Differentiation situation
Into Neural Differentiation：By freeze two groups of cell recoveries and one group of cell normally cultivated（3 groups of cell deriveds are identical, algebraically
It is identical）After Secondary Culture, when cell fusion degree is up to 60% or so, the DMEM low sugar medium treatments containing 20%FBS are changed
24h；Change the DMEM low sugar medium treatments 24h of the bFGF containing 10ng/mL；Then, Neural Differentiation inducing culture is changed
（DMEM low sugar culture mediums, 2%DMSO, 100 μM of BHA, the 25mM μ g/ml insulin of KCl, 10 μM of forskolin, 5,1 μM of hydrogen
Change cortisone）, induction differentiation 24h；Replacing Neural Differentiation maintenance inducing culture (DMEM/F12 complete mediums, 10%FBS,
10ng/ml EGF, 10ng/ml bFGF, 1 × N2,1 × B27）, maintain 3-4d.The cell part that differentiation is completed does immune glimmering
Light, another part extracts full RNA and does quantitative fluorescent PCR.
Expression of the Immunofluorescence test into Neural Differentiation associated DC X, NSE, Neun and β-III Tubulin：Differentiation is completed
Each group stem cell, digestion, which is centrifuged, simultaneously collects cell, counts and maintains the inducing culture adjustment cell density to be with Neural Differentiation：
5 × 105/mL, 24 orifice plate bottoms are uniformly taped against by every hole 0.5mL cell suspensions, mixes and carries out after mark, be put into cell
24h is cultivated in incubator.Immunofluorescence test, its step is as follows：
（1）The culture medium in 24 orifice plates is sucked, cell is cleaned with PBST 3 times, the paraformaldehyde for plus 4% fixes 30min；
（2）Fixer is absorbed, PBST cleanings 3 are added in the citrate buffer (PH6.0) containing 0.05%Triton100, water-bath
20min is boiled in pot；
（3）Naturally cool to after room temperature, using PBS 3 times, 0.2mL confining liquid is added dropwise per hole（Containing 3% skimmed milk power,
The TBST solution of 5% lowlenthal serum）Close 1h；
（4）Confining liquid is sucked, (DCX, NSE, Neun and β-III Tubulin are equal for the primary antibody of addition 0.2mL proper proportions dilution per hole
By 1：50 dilutions), 4 DEG C of overnight incubations；
（5）Primary antibody is sucked, is cleaned with PBST 4 times, each 5min.Under the conditions of lucifuge, the corresponding fluorescence secondary antibodies of 0.2mL are added dropwise per hole
（Dilution ratio is：1:1000）, it is incubated 1h；
（6）Secondary antibody is sucked, PBST is cleaned 4 times, each 5min.Lucifuge, 0.2mL DAPI solution is added dropwise per hole（Dilution ratio is
1:2000）Redye nucleus 15min；
（7）DAPI solution is sucked, PBST is cleaned 4 times, and each 5min, fluorescence microscope is simultaneously taken pictures.
The stem cell that two kinds of frozen stock solutions freeze is into Neural Differentiation situation such as Fig. 8, and hUC-MSCs is by one week or so into god
After induction, cellular morphology changes obvious, and multisynaptic neural-like cells form is become by original long shuttle-type, is immunized glimmering
Dyeing and positive cell number statistical result（Fig. 8）Display：Cellular neural differentiation correlation DCX, NeuN and the β frozen by frozen stock solution B-
III Tubulin expression and positive cell number increase are obvious, more efficient into Neural Differentiation.
9th, the hUC-MSCs after Cord blood is to the change of TBI mouse weights and the influence of mNSS scoring events
The foundation of 9.1 brain damage models
This experiment sets up the new brain damage model of C57BL/6 mouse wounds using freely falling body punch method.Use 10% chloraldurate
（3ml/kg）Intraperitoneal injection of mice, after after its anesthesia, in after calvarium portion preserved skin, iodophor disinfection, is fixed on brain solid fixed by mouse
Position instrument, from shape seam midsection 3 ~ 5cm of scalp is lost, the outer manadesma of exposure skull separates manadesma with swab stick tail end, manifests right side cranium
Bone, in about 1.5mm, center line side 1.5mm after bregma, 3mm bone windows is carefully bored out with annular bit, it is ensured that endocranium is complete.Then,
Cranium brain beating device is adjusted, makes striker（Diameter 2.5mm）Just to bone window center, control strike depth be 2mm, by 20g counterweight from
Vertical strike striker, then lifts rapidly striker at 20cm, and modeling is completed.Modeling Success criteria is：After strike, mouse is transient
Can spontaneous remission after tic of limbs, apnea, but several seconds.Postoperation hemostatic, and bone window is closed with bone wax, suture scalp.Operation
Process and it is postoperative should give mouse isothermal holding, 37 ± 0.5 DEG C of anus temperature is kept, until mouse regains consciousness.
9.2 animal and packet
Cleaning grade male C57BL/6 mouse, 10 ~ 12 weeks, body weight 23g ~ 25g, being purchased from Beijing magnificent experimental animal technology of dimension tonneau has
Limit company, adapts to environment after 1 week, modeling is started by the method for [0059].The successful 12 C57BL/6 mouse of modeling are divided at random
For TBI groups（6, postoperative 6 hours, the physiological saline 0.1ml of tail vein injection 0.9%）；TBI+hUC-MSCs groups（6, art
Afterwards after 6 hours, the cell 0.1mL that tail vein injection frozen stock solution B freezes, cell density is：1×107Individual/mL）.1 time a day, even
Continuous injection 3 days.In art, it is postoperative if it find that animal dead, gives supplement immediately, it is ensured that every group of experiment mice 6.
After surgery 0,1,3,7,14, the set time of 21 and 28 days weighs mouse weight and changes and then carry out nervous function
Defect scores（Modified Neurological Severity Score, mNSS scorings）, during which on time supplements and water,
Regularly replace bedding and padding.According to mNSS marking scales, judged using double-blind study and record score value.MNSS standards of grading are from mouse fortune
It is estimated in terms of dynamic, sensation, balanced capacity and reflection case：From 0 point（Normally）- 18 points（Most serious）.
The postoperative body weight situation of change of two groups of mouse is such as（Fig. 9 A）It is shown.After TBI modelings, mouse weight 1 day after surgery, 3 days
Slightly mitigate, the increased weight since 7 days.Comparing different time points body weight can find, two groups of mouse 1 day after surgery, 3 days, 7
My god, body weight is without significant change.After 14 days, hUC-MSCs treatment groups mouse weight is apparently higher than TBI groups.
Fig. 9 B are two groups of mouse mNSS scoring events.Postoperative 1 day, TBI groups and the scoring of TBI+hUC-MSCs groups mouse were distinguished
For（11.3±0.8）With（11.5±0.9）Point, illustrate modeling homogeneity preferably, postoperative 3 days, TBI groups mouse scored on slightly
Rise.After 14 days, hUC-MSCs treatment groups mouse mNSS scores are significantly lower than TBI groups.Illustrate the TBI+hUC- that frozen stock solution B freezes
MSCs can increase the body weight of TBI mouse, reduce the nerve damage degree of TBI mouse.
10th, influences of the hUC-MSCs after Cord blood to TBI mouse Condition of depression, cognitive function recovery situation
Forced swim test：28 days after TBI, two groups are respectively selected 6 mouse, do following experiment：In a cylindrical water receptacle,
It is put into clear water（Highly：25cm, diameter：22cm, water temperature：23±1℃）.Mouse is put into water, because mouse is afraid of water, it will do not stop
It is travelling, with camera record 6 minutes.4 minutes videos after analyzing on computers, record mouse swimming time, floating or only one
Limbs fine motion is not counted in swimming time.Suspension time is obtained using formula：Suspension time（Second）=240 seconds-swimming time.
Syrup preference is tested：Tested within 21 days after TBI, continue one week.Operating procedure is shown in that Zhu etc. reports [48].It is postoperative
21 days, compound concentration was 1% sucrose solution, and pours into drinking bottle.Every mouse to be measured is placed individually into a mouse cage, each
Mouse cage places 2 drinking bottles, allows mouse to adapt to syrup 3 days.Brain trauma the 24th day, is replaced wherein with the drinking bottle containing distilled water
One syrup bottle, and carry out mark.Weigh with scale 2 bottles of weight weights, and labelled Marked Weight.Brain trauma the 28th day, then
2 water bottle weight of secondary weighing, and the consumption of syrup and pure water is calculated, calculate the partially thermophilic degree of two groups of mouse sucrose using formula：Sucrose
Partially thermophilic degree=syrup consumption/（Syrup consumption+pure water consumption）×100%.
New things are recognized（Novel object recognition, NOR）Experiment：27 days after TBI, it will be tested
Mouse is placed in an opaque chest of 50cm × 50 cm × 25cm, is carried out environment and is adapted to 10 minutes.After 24 hours,
The object of 2 same volumes, same color is put into chest（The red cubes of 4cm × 4cm × 4cm）, allow mouse to explore 10
After minute, original cage is put into.After 1 hour, by the spherical object of wherein 1 red cube green（Diameter 4cm）Generation
Replace, mouse is placed into this chest again, allow it to explore 5 minutes, during which explore situation using video recorder record mouse.Now
Red cube is past heritage body, and green spherical object is new object, when analysis mouse explores new, old used for object on computers
Between.Calculate the index of discrimination of mouse, index of discrimination=new time used for object/（During new time used for object+old used for object
Between）×100%.
Figure 10 is to compare two groups of mouse Condition of depression and cognitive function.Forced swim test result is shown：Stem-cell therapy
Group mouse suspension time（125.25±4.55s）Significantly lower than TBI groups（147.45±3.85s）；The experiment display of syrup preference：With
TBI group mouse（49.15 ± 2.32%）Compare, cellular replacement therapy group mouse syrup preference（56.07 ± 2.98%）Have
Improved；In new things identification experiment, the new things index of discrimination of stem-cell therapy group mouse（65.16 ± 1.89%）It is higher than
TBI（53.86 ± 2.12%）.Illustrate that the hUC-MSCs that frozen stock solution B freezes can improve the cognitive function of brain damage mouse, mitigate brain
Damage the Condition of depression of mouse.
Claims (1)
1. a kind of human umbilical cord mesenchymal stem cells cryoprotective agent comprising rhMG53, it is characterised in that：The protective agent by
DMEM/F12 culture mediums, FBS, DMSO and rhMG53 are formulated, and first press the DMEM/F12 culture mediums, FBS and DMSO
Volume ratio 5:4:1 is configured to mixed liquor；Then rhMG53 is added in 30 μ g/mL ratio in the mixed liquor, after being well mixed
Obtain human umbilical cord mesenchymal stem cells cryoprotective agent finished product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109430252A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof |
| WO2020181411A1 (en) * | 2019-03-08 | 2020-09-17 | 玛旺干细胞医学生物科技股份有限公司 | Medical use of mesenchymal stem cells for preventing or treating emotional diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120213737A1 (en) * | 2011-02-18 | 2012-08-23 | University Of Medicine And Dentistry Of New Jersey | Compositions and methods for therapeutic membrane repair |
| CN105145547A (en) * | 2015-10-28 | 2015-12-16 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120213737A1 (en) * | 2011-02-18 | 2012-08-23 | University Of Medicine And Dentistry Of New Jersey | Compositions and methods for therapeutic membrane repair |
| CN105145547A (en) * | 2015-10-28 | 2015-12-16 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109430252A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof |
| WO2020181411A1 (en) * | 2019-03-08 | 2020-09-17 | 玛旺干细胞医学生物科技股份有限公司 | Medical use of mesenchymal stem cells for preventing or treating emotional diseases |
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