CN109468273A - A method of obtaining mescenchymal stem cell from umbilical cord - Google Patents
A method of obtaining mescenchymal stem cell from umbilical cord Download PDFInfo
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The method that the invention discloses a kind of to obtain mescenchymal stem cell from umbilical cord.Including with ANER DIAN and/or 75% alcohol disinfecting after umbilical cord washing: removing blood vessel, separation China's Tong Shi glue again；Bed board culture；It changes liquid culture and 14d or cell confluency degree reaches 45-55% harvest: digestion filtering.The method of the invention can more quickly obtain primary mescenchymal stem cell (< 15day), the operation is more convenient, the primary mescenchymal stem cell obtained under equal amount tissue block is more, and can effectively avoid pollution, cell contamination is prevented it is not necessary that antibiotic is added in subsequent culture.
Description
Technical field
The present invention relates to cell preparation field more particularly to a kind of methods that mescenchymal stem cell is obtained from umbilical cord.
Background technique
Mescenchymal stem cell (mesenchymal stem cell, MSC) is derived from mesoderm growing early stage mesoderm and ectoderm
A kind of multipotential stem cell.MSC has immunological regulation, the histoorgan for promoting Radiation in jury, capable of repairing damage or lesion, tool
The functions such as standby multi-lineage potential, therefore be with a wide range of applications.
But prepare the method for mescenchymal stem cell from umbilical cord at present time-consuming, the mescenchymal stem cell quantity of acquisition
It is few, while pollution is easy in operating process.
For example CN105586309A discloses the method that mescenchymal stem cell is prepared from umbilical cord, will add antibiotic
Umbilical cord after cleaning solution cleaning is cut into the segment of about 2cm length, sufficiently shreds into 1-3mm again after treatment3~3mm3Size,
Divide the 175cm for having added complete medium equally2In culture bottle, jiggling makes it be uniformly distributed in bottom of bottle；It is cultivated in incubator,
Obtained cell liquid is centrifuged by the 8th day replacement culture medium, is resuspended, and is cultivated in incubator after paving bottle.The method makes subsequent
Culture often has to prevent from polluting by addition antibiotic, increases the security risk of clinical application.Operating time simultaneously
It is long.Tissue block is excessive, and the culture bottle number that can be spread is few, and cell number is also corresponding less.
Summary of the invention
The method that the purpose of the present invention is to provide a kind of to obtain mescenchymal stem cell from umbilical cord.The method can be more
Primary mescenchymal stem cell (< 15day) is quickly obtained, the operation is more convenient, and primary obtained under equal amount tissue block fills
Matter stem cell population is more, and can effectively avoid pollution, and prevents cell it is not necessary that antibiotic is added in subsequent culture
Pollution.
To achieve the above object, the present invention provides a kind of method that mescenchymal stem cell is obtained from umbilical cord, and feature exists
In, include the following steps,
The washing and disinfection of umbilical cord:
It is immersed into it in ANER DIAN or 75% alcohol after the umbilical cord of health is washed to carry out disinfection, disinfecting time
It must not exceed 30s, cleaning solution be added and cleans 1-2 times；Alternatively,
It is immersed into it in ANER DIAN or 75% alcohol after the umbilical cord of health is washed to carry out disinfection, disinfecting time
It must not exceed 30s, cleaning solution cleaning 1-2 be added after, then be immersed into it in 75% alcohol or ANER DIAN and carry out disinfection, every time
Disinfecting time must not exceed 30s；Cleaning solution is added to clean 1-2 times；
Removal blood vessel, separation China's Tong Shi glue: remove umbilical cord end to end after, remaining umbilical cord is cut into 1-3cm sections；It is preferred that
, remaining umbilical cord is cut into 1.5-2.5cm sections；It is furthermore preferred that remaining umbilical cord is cut into 2cm sections；Appearance is peeled off with tooth tweezer
Skin rejects vein and arteries, removes China's Tong Shi glue therein, magnificent Tong Shi glue is cleaned with cleaning solution；
Bed board culture: being added serum free medium in magnificent Tong Shi glue, it is made just to submerge China's Tong Shi glue, with scissors that China is logical
Family name's glue is shredded to 0.1-1mm3；The tissue block shredded is resuspended in addition serum free medium, retransfers tissue block into culture bottle,
And add serum free medium；It is evenly distributed in tissue block on culture bottle, and culture medium is made to soak entire bottom of bottle, carry out mark
Note is put into incubator and cultivates, in 6-8d, it is preferred that must not move in 7d；Incubator culture；Preferably, condition of culture 37
Spend 5%CO2Saturated humidity culture；
Change liquid culture and harvest: supplemented medium, 10d change liquid in each culture bottle after 7d, suck former culture medium, often
Fresh culture 25mL is added in bottle, continues to cultivate；When 14d or cell confluency degree reaches 45-55%；Preferably, reach 50%
It can harvest；
Digestion filtering: discarding former culture medium, and pancreatin of the addition containing EDTA is incubated for after physiological saline cleaning is added, and becomes to cell
Circle adds pancreatin terminate liquid and carries out cell collection when falling off, the cell of collection is centrifuged, is discarded supernatant, with physiological saline weight
70um cell sieve is crossed after outstanding cell and is filtered to remove tissue block, and supernatant is removed in centrifugation again.
Further, the puerpera of the umbilical cord of the health for the Full term labor caesarean birth of no family history and Genetic history puerpera,
Puerpera is by foetus health after delivery of baby without deformity or congenital disorders.
Further, the umbilical cord after delivery of baby, choose the umbilical cord of middle part 10-20cm, use alcohol gauze by conventional disconnected navel
It is cut after cleaning-sterilizing.
Further, in the washing and sterilisation step of the umbilical cord, cleaning solution is added in umbilical cord, is turned upside down after closeing the lid clear
It washes, repeatedly after 2-4 times, retransfers umbilical cord, ANER DIAN thimerosal is added, so that its is submerged entire umbilical cord, close the lid immediately
Umbilical cord is shifted after turning upside down 2-4 times, adds cleaning solution cleaning, and whole process must not exceed 30s；It is retransferred after cleaning
Umbilical cord is added 75% alcohol and submerges umbilical cord, closes the lid and shift umbilical cord after turning upside down immediately 2-4 times, and whole process must not surpass
30s is crossed, cleaning solution is added and cleans 1-3 times.
Further, the removal blood vessel, separation China's Tong Shi glue step in, after the 0.5-1.5cm each end to end for removing umbilical cord；It is excellent
Choosing, after the 1cm each end to end for removing umbilical cord, remaining umbilical cord is cut into 1-3cm sections；It is furthermore preferred that remaining umbilical cord is cut into
1.5-2.5cm section；It is furthermore preferred that remaining umbilical cord is cut into 2cm sections；Exocuticle is peeled off with tooth tweezer, rejects vein and arterial blood
Pipe removes China's Tong Shi glue therein, magnificent Tong Shi glue is cleaned 1-3 times with cleaning solution；Preferably, it cleans 3 times.
Further, described to change liquid culture and harvest in step: after 7d in each culture bottle supplemented medium；Preferably,
According to the every 10cm of culture bottle floor space2Supplemented medium 0.5-0.8mL, it is furthermore preferred that according to the every 10cm of culture bottle floor space2It mends
Add culture medium 0.6mL；10d changes liquid, sucks former culture medium, every bottle of addition fresh culture；Preferably, according to culture bottle bottom surface
The every 10cm of product2Supplemented medium 0.1-0.5mL, it is furthermore preferred that according to the every 10cm of culture bottle floor space2Supplemented medium 0.3mL after
Continuous culture；When 14d or cell confluency degree reaches 45-55%；Preferably, reaching 50% can harvest:
Further, in the digestion filtration step, former culture medium is discarded, physiological saline cleaning 1-2 is added and contains all over rear addition
The pancreatin of EDTA；Preferably, every 10cm2Add 0.1-0.5mL；It is furthermore preferred that every 10cm2Add 0.3mL；It is incubated at room temperature 3-
6min adds pancreatin terminate liquid when cell rounding falls off；Preferably, every 10cm2Add 0.5-0.8mL；It is furthermore preferred that every
10cm2Add 0.6mL；Cell collection is carried out, the cell of collection carries out centrifugation 300g, 5min, discards supernatant, with physiological saline weight
70um cell sieve is crossed after outstanding cell and is filtered to remove tissue block, again 300g, and supernatant is removed in 5min centrifugation
The umbilical cord after delivery of baby, choose the umbilical cord of middle part 10-20cm, disappeared with alcohol gauze cleaning by conventional disconnected navel
It is cut after poison.This part umbilical cord not vulnerable to pollution, it is in stable condition.
The present invention can also accommodate umbilical cord tissue using the container of other specifications to achieve the purpose that enclosed cleaning.Such as
5mL centrifuge tube, 200mL centrifuge tube, 250mL liquid storage bottle etc..
Difference with the prior art of the present invention:
Existing scheme is only focused on carrying out screening to the infectious disease of puerpera, and few to other aspect concerns.The present invention couple
Puerpera is more concerned with screening heredity medication history and family's medical history other than screening infectious disease, and the health status of fetus is also screening weight
Point, this significantly increases the safety of sample, reduces hidden danger for clinical application from now on.
The cleaning of umbilical cord carries out in culture dish in existing scheme, and open space easily pollutes, and is not easy
It cleans up, cleaning of the invention in the enclosed space, greatly reduces pollution, in the process of entire laboratory treatment
In, it is open operation when only separating and shred magnificent Tong Shi glue, this can be significantly reduced the possibility of pollution.
Existing scheme only achievees the purpose that disinfection using medicinal alcohol wiping when acquiring umbilical cord, remaining is only relied on
Acquisition environment itself reaches what removal umbilical cord itself polluted by the cleaning solution of addition antibiotic to guarantee the sterile of umbilical cord
Purpose, this easily causes the pollution due to caused by sample itself in subsequent cell culture, so that subsequent culture often must not
It does not prevent from polluting by addition antibiotic, increases the security risk of clinical application.Umbilical cord of the invention is disappeared by ANER DIAN
Poison and 75% alcohol disinfecting, ANER DIAN and 75% alcohol are bigger to cellular damage, and will lead to tissue block dehydration, so be easy
The cell death for leading to umbilical cord tissue surface causes culture to fail, and especially adherent tissue block is larger or does not reject umbilical cord table
Pi Shi, since the part cell that umbilical cord surface touches thimerosal is substantially dead, so be not easy to climb out of cell.And the prior art
To prevent from polluting, the antibiotic such as mycillin are added in subsequent culture mostly, can also prevent from polluting without so sterilizing.And
On the one hand this programme eliminates the dead cell of umbilical cord epidermis, another aspect tissue block is cut more broken, and internal intact cell can
Be exposed and carry out adherent, and increase the adherent area of tissue block and culture bottle, so can not only eliminate potential pollution but also
It not will lead to culture failure.2 disinfecting process are greatly avoided since sample itself bring pollutes, and are subsequent thin
Born of the same parents' culture removes pollution hidden trouble, so that no longer needing to addition antibiotic in subsequent cell cultivation process, increases clinical application
Safety.
The present invention limits soaking time and is less than 30 seconds, prevents thimerosal from causing big damage to tissue block.
It needs to increase a 1-2 hours process without culture solution culture during existing scheme plating cells, greatly draw
The operating time is grown, the present invention needs not move through this process, reduces the operating time.
Cell hikes up existing scheme in order to prevent, and tissue block cannot be cut too small, so that the bottle that the tissue block of same amount is spread
Number is less, and the present invention is due to using new culture scheme, and what tissue block can be cut is very small, so as to spread more bottles
Number.As the umbilical cord of a 10cm can only be cut into 1mm according to common scheme3Tissue block, be only capable of 3-5 T175 of paving and cultivate
Bottle, but cultivated using the present invention, such as 10-15 T175 bottles can be spread.The present invention adds in the 7d that tissue block paving bottle starts
The culture base unit weight added is seldom.Existing scheme is directly to add culture medium 20-30mL, is in 7d if the present invention is using T175 bottles
Keep the cultivating system of 10mL, when 7d, adds 5mL again, 10d Shi Zaihuan liquid at 25mL, only so that tissue block be able to maintain it is wet
State, so tissue block will not be hiked up in early period, in 7d liquid feeding, tissue block has been pasted securely due to climbing out of cell, so after
Continuous liquid feeding is hiked up with changing liquid and not will lead to it.
Specific embodiment
The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot
It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, described according to the literature in the art
Technology or conditions or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to
Cross the conventional products of commercially available acquisition.
Embodiment 1:
The screening of puerpera: screening is carried out to the infectious disease testing result and family history of puerpera, Genetic history, selects infectious disease inspection
Survey qualified (including but not limited to hepatitis B, hepatitis, syphilis, AIDS, cytomegalovirus), the Full term labor of no family history and Genetic history
Caesarean birth puerpera, confirmation foetus health is without deformity or congenital disorders after delivery of baby.
Acquire umbilical cord and transport: after delivery of baby, conventional disconnected navel, after the Cord blood of acquisition about 10mL is used for into heparin tube
Continuous detection, the umbilical cord for choosing middle part 10-20cm are put into umbilical cord storage and transportation bottle, storage and transportation with cutting after alcohol gauze cleaning-sterilizing
The DMEM/F12 culture medium of 10mL mycillin containing 100U/mL is added in bottle in advance.Storage and transportation bottle is put into special transport case,
The transport temperature for guaranteeing 2-8 DEG C is prepared in interior transport to laboratory for 24 hours.
The washing and disinfection of umbilical cord: the physiological saline of one bottle of 500mL is opened, mycillin is added wherein, makes physiology salt
Mycillin concentration in water reaches 100U/mL, as cleaning solution, cleans for subsequent umbilical cord.The disinfection of storage and transportation bottle outer surface
After be put into safety cabinet, close the lid after cleaning solution is added and rock cleaning.It shifts in umbilical cord to the centrifuge tube of a 50mL, is added
Cleaning solution, cleaning of turning upside down after closeing the lid repeatedly after 3 times, are shifted in umbilical cord to a new 50mL centrifuge tube,
ANER DIAN thimerosal is wherein added, its is made to submerge entire umbilical cord, closes the lid and shifts umbilical cord after turning upside down 3 times immediately to newly
In centrifuge tube, cleaning solution cleaning is added, whole process must not exceed 30s.It is transferred in new centrifuge tube, is added again after cleaning
75% alcohol submerges umbilical cord, closes the lid and shifts umbilical cord after turning upside down 3 times immediately into new centrifuge tube, whole process must not
More than 30s, cleaning solution is added and cleans 2 times.By disinfecting process twice, can greatly eliminate umbilical cord surface may carry it is thin
Bacterium, to reduce pollution in subsequent culture.
Removal blood vessel, separation China's Tong Shi glue: the umbilical cord after cleaning is relayed in culture dish from centrifuge tube, is cut off end to end
1cm is discarded, and rest part is cut into 2cm sections.One section of umbilical cord is taken, peels off exocuticle with tooth tweezer, rejects vein and arteries, removing
China's Tong Shi glue therein is careful not to stripping to umbilical cord epidermal tissue.Magnificent Tong Shi glue is placed in 50mL centrifuge tube and cleans 2 with cleaning solution
Time.
Magnificent Tong Shi glue is gone in new centrifuge tube, serum free medium is added, it is made just to submerge China's Tong Shi glue, with cutting
Knife protrudes into centrifuge tube and shreds magnificent Tong Shi glue to 0.1-1mm3, pay attention to shredding as far as possible, to obtain more tissue blocks.
Serum free medium is added in the tissue block shredded to be resuspended, and shifts tissue block to 175cm with the pipette of decaptitating
Culture bottle in, and add serum free medium to 10mL/ bottles.
Roll culture bottle, is evenly distributed in tissue block on culture bottle, and culture medium is made to soak entire bottom of bottle, carries out
Label, is put into saturated humidity, CO2It cultivates in 37 degree of incubators that concentration is 5%, must not be moved in 7d.
After 7d in each culture bottle supplemented medium 5mL.Liquid is changed after 10d, sucks former culture medium, and every bottle is added fresh training
Base 25mL is supported, continues to cultivate
It can be harvested when 14d: discard former culture medium, the every bottle of pancreatin of addition containing EDTA after physiological saline cleans one time is added
4mL is incubated at room temperature 4min, and pancreatin terminate liquid 10mL is added when cell rounding falls off, and the cell of collection is transferred to 50mL centrifugation
300g in managing, 5min centrifugation.It discards supernatant, crosses 70um cell sieve after cell is resuspended with physiological saline and be filtered to remove tissue block, then
Secondary 300g, 5min centrifugation.Cell after centrifugation can be frozen or be passed on demand.
Embodiment 2:
The screening of puerpera: with 1 method of embodiment and requirement.
Acquire umbilical cord and transport: with 1 method of embodiment and requirement.
The washing and disinfection of umbilical cord: with 1 method of embodiment and requirement, ANER DIAN thimerosal is only used.
Removal blood vessel, separation China's Tong Shi glue: the umbilical cord after cleaning is relayed in culture dish from centrifuge tube, is cut off end to end
1cm is discarded, and rest part is cut into 2cm sections.One section of umbilical cord is taken, peels off exocuticle with tooth tweezer, rejects vein and arteries, removing
China's Tong Shi glue therein is careful not to stripping to umbilical cord epidermal tissue.Magnificent Tong Shi glue is placed in 50mL centrifuge tube and cleans 2 with cleaning solution
Time.
Magnificent Tong Shi glue is gone in new centrifuge tube, serum free medium is added, it is made just to submerge China's Tong Shi glue, with cutting
Knife protrudes into centrifuge tube and shreds magnificent Tong Shi glue to 0.1-1mm3, pay attention to shredding as far as possible, to obtain more tissue blocks.
Serum free medium is added in the tissue block shredded to be resuspended, and shifts tissue block to 175cm with the pipette of decaptitating
Culture bottle in, and add serum free medium to 10mL/ bottles.
Roll culture bottle, is evenly distributed in tissue block on culture bottle, and culture medium is made to soak entire bottom of bottle, carries out
Label, is put into saturated humidity, CO2It cultivates in 37 degree of incubators that concentration is 5%, must not be moved in 7d.
After 7d in each culture bottle supplemented medium 5mL.Liquid is changed after 10d, sucks former culture medium, and every bottle is added fresh training
Base 25mL is supported, continues to cultivate
Harvest: cell confluency degree, which reaches 50%, to be harvested.Former culture medium is discarded, is added every after physiological saline cleans one time
The pancreatin 4mL containing EDTA is added in bottle, is incubated at room temperature 4min, and pancreatin terminate liquid 10mL is added when cell rounding falls off, collection
Cell is transferred to 300g in 50mL centrifuge tube, 5min centrifugation.It discards supernatant, crosses 70um cell sieve after cell is resuspended with physiological saline
It is filtered to remove tissue block, again 300g, 5min centrifugation.Cell after centrifugation can be frozen or be passed on demand.
Embodiment 3:
The screening of puerpera: with 1 method of embodiment and requirement.
Acquire umbilical cord and transport: with 1 method of embodiment and requirement.
The washing and disinfection of umbilical cord: with 1 method of embodiment and requirement, only with 75% alcohol disinfection solution.
Removal blood vessel, separation China's Tong Shi glue: the umbilical cord after cleaning is relayed in culture dish from centrifuge tube, is cut off end to end
1cm is discarded, and rest part is cut into 2cm sections.One section of umbilical cord is taken, peels off exocuticle with tooth tweezer, rejects vein and arteries, removing
China's Tong Shi glue therein is careful not to stripping to umbilical cord epidermal tissue.Magnificent Tong Shi glue is placed in 50mL centrifuge tube and cleans 2 with cleaning solution
Time.
Magnificent Tong Shi glue is gone in new centrifuge tube, serum free medium is added, it is made just to submerge China's Tong Shi glue, with cutting
Knife protrudes into centrifuge tube and shreds magnificent Tong Shi glue to 0.1-1mm3, pay attention to shredding as far as possible, to obtain more tissue blocks.
Serum free medium is added in the tissue block shredded to be resuspended, and shifts tissue block to 175cm with the pipette of decaptitating
Culture bottle in, and add serum free medium to 10mL/ bottles.
Roll culture bottle, is evenly distributed in tissue block on culture bottle, and culture medium is made to soak entire bottom of bottle, carries out
Label, is put into saturated humidity, CO2It cultivates in 37 degree of incubators that concentration is 5%, must not be moved in 7d.
After 7d in each culture bottle supplemented medium 5mL.Liquid is changed after 10d, sucks former culture medium, and every bottle is added fresh training
Base 25mL is supported, continues to cultivate
Harvest: cell confluency degree, which reaches 55%, to be harvested.Former culture medium is discarded, is added every after physiological saline cleans one time
The pancreatin 4mL containing EDTA is added in bottle, is incubated at room temperature 4min, and pancreatin terminate liquid 10mL is added when cell rounding falls off, collection
Cell is transferred to 300g in 50mL centrifuge tube, 5min centrifugation.It discards supernatant, crosses 70um cell sieve after cell is resuspended with physiological saline
It is filtered to remove tissue block, again 300g, 5min centrifugation.Cell after centrifugation can be frozen or be passed on demand.
Comparative example: it is dry thin that conventional method (referring to CN201610082928.8 namely CN 105586309A) prepares mesenchyma
Born of the same parents
1) in gnotobasis, when third stage of labor, acquires umbilical cord, pays attention to draining the residual blood of umbilical cord arteriovenous and ligature；After acquisition
Umbilical cord disinfection after be placed in the storage and transportation bottle for being added to storage and transportation liquid, be sent to laboratory and carry out separation preparation.Wherein storage and transportation liquid is
α-the MEM of 40ml, and it is added to gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml.Ensure before acquisition
Five detections of pregnant woman's virus are negative, ALT detection qualification.Transport temperature is 4 DEG C, and haulage time is no more than 24 hours.
2) it is put into Biohazard Safety Equipment after umbilical cord bottle alcohol wipe.
3) in 150cm2It in Tissue Culture Dish, is sufficiently washed using cleaning solution umbilical cord 3 times, with tip staight scissors by umbilical cord both ends
1cm length is respectively cut off, is abandoned.It washs again umbilical cord tissue 2 times.Wherein cleaning solution is to be added to 100U/ml gentamicin, 50mg/
Ml penicillin, the medical saline of 5ug/ml amphotericin B.
4) umbilical cord is cut into the segment of about 2cm length with tip staight scissors, is cleaned 3 times.Umbilical cord tissue is shifted to a new training
It supports in ware, in culture dish plus physiological saline is to flooding at umbilical cord tissue 1/2.Vein blood vessel and arteries are removed, tears and takes China logical
Glue.Huatong plastic is placed in the 50ml centrifuge tube for adding a small amount of cleaning solution.
5) huatong plastic is sufficiently washed 3 times with cleaning solution, then with brine 2 times, is transferred to new 50ml centrifuge tube
In, it sufficiently shreds to 1mm3~3mm3Size.
6) the 10ml pipette for spending leading portion divides huatong plastic block equally 4 bottles of 175cm for having added 25ml complete medium2Training
It supports in bottle, jiggling makes it be uniformly distributed in bottom of bottle, wherein complete medium is that 10%FBS and 6ng/ is added in α-MEM
MlEGF+7ng/mlPDGF+5ng/mlTNF- α+5ng/mlIFN- gamma cells combinations of factors.
7) tag-related on culture bottle, including sample bar code, operation item, operating time, operator etc..
8) culture bottle is carefully put into 37 DEG C, 5%CO2In incubator.
9) it cultivates initial 7 days, keeps culture bottle absolute rest.
10) the 8th day according to growing state, according to change liquid, passage, harvest freeze standard practice instructions execution.
Cell passage:
1) it with 75% alcohol disinfecting medium bottle outer wall, is placed into Biohazard Safety Equipment.
2) it is inhaled with decaptitating pipette and abandons old culture medium, replace pipette, 10ml physiological saline is added in acellular culture face.
3) gently the adherent face of tissue is washed in concussion, is discarded, is repeated 2 times.
4) every bottle plus digestive juice 3ml, digestive juice are 0.25% pancreatin+0.03%EDTA, the adherent face of homogeneous immersion cell, 37
DEG C be incubated for 1min.It every bottle plus terminate liquid 10ml after cell rounding, quickly shakes, blows and beats the adherent face of cell with 10ml pipette,
Cell suspension is sucked out into 2 50ml centrifuge tubes, every bottle of each culture bottle plus 10ml physiological saline, purging is primary, import 50ml from
In heart pipe.
5) 1200rpm is centrifuged 6min, abandons supernatant.Merging is precipitated to 1 pipe, adds 40ml physiological saline centrifuge washing again, sinks
It forms sediment and is resuspended with 10ml complete medium, filtered through cell sieve, 5ml complete medium rinses sieve, counts.
Human umbilical cord mesenchymal stem cells are cultivated using the culture medium of different formulations, P0 is counted for cell,
As a result as follows: embodiment 1, embodiment 2, embodiment 3 and the cell number of commercially available MSC culture medium culture are respectively as follows: 1.50 × 106
It is a, 1.10 × 106It is a, 1.08 × 106It is a, 0.65 × 106It is a.
It the results are shown in Table 1.As it can be seen from table 1 method of the invention is shorter than the conventional scheme time, bed board number is more, obtains
Primary cells number it is more, cultivated days are short.
Table 1 is compared result table by taking the umbilical cord of a 10cm long as an example
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (7)
1. a kind of method for obtaining mescenchymal stem cell from umbilical cord, which is characterized in that include the following steps,
The washing and disinfection of umbilical cord:
It is immersed into it in ANER DIAN or 75% alcohol after the umbilical cord of health is washed to carry out disinfection, disinfecting time must not
More than 30s, cleaning solution is added and cleans 1-2 times；Alternatively,
It is immersed into it in ANER DIAN or 75% alcohol after the umbilical cord of health is washed to carry out disinfection, disinfecting time must not
More than 30s, cleaning solution cleaning 1-2 is added after, then is immersed into it in 75% alcohol or ANER DIAN and carries out disinfection, sterilizes every time
Time must not exceed 30s；Cleaning solution is added to clean 1-2 times；
Removal blood vessel, separation China's Tong Shi glue: remove umbilical cord end to end after, remaining umbilical cord is cut into 1-3cm sections；Preferably, will
Remaining umbilical cord is cut into 1.5-2.5cm sections；It is furthermore preferred that remaining umbilical cord is cut into 2cm sections；Exocuticle is peeled off with tooth tweezer, is picked
Except vein and arteries, China's Tong Shi glue therein is removed, magnificent Tong Shi glue is cleaned with cleaning solution；
Bed board culture: being added serum free medium in magnificent Tong Shi glue, it is made just to submerge China's Tong Shi glue, with scissors by magnificent Tong Shi glue
It shreds to 0.1-1mm3；The tissue block shredded is resuspended in addition serum free medium, retransfers tissue block into culture bottle, and mend
Add serum free medium；It is evenly distributed in tissue block on culture bottle, and culture medium is made to soak entire bottom of bottle, mark, put
Enter in incubator and cultivate, in 6-8d, it is preferred that must not be moved in 7d；Incubator culture；Preferably, condition of culture is 37 degree 5%
CO2Saturated humidity culture；
Change liquid culture and harvest: supplemented medium, 10d change liquid in each culture bottle after 7d, suck former culture medium, and every bottle adds
Enter fresh culture 25mL, continues to cultivate；When 14d or cell confluency degree reaches 45-55%；Preferably, reach 50%
Harvest；
Digestion filtering: discarding former culture medium, and pancreatin of the addition containing EDTA is incubated for after physiological saline cleaning is added, de- to cell rounding
Pancreatin terminate liquid is added when falling and carries out cell collection, and the cell of collection is centrifuged, is discarded supernatant, and is resuspended with physiological saline thin
70um cell sieve is crossed after born of the same parents and is filtered to remove tissue block, and supernatant is removed in centrifugation again.
2. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that the umbilical cord of the health
Puerpera for no family history and Genetic history Full term labor caesarean birth puerpera, puerpera is by foetus health after delivery of baby without deformity
Or congenital disorders.
3. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that the umbilical cord is in fetus
After giving birth to, conventional disconnected navel chooses the umbilical cord of middle part 10-20cm, with cutting after alcohol gauze cleaning-sterilizing.
4. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that the washing of the umbilical cord
In sterilisation step, cleaning solution is added in umbilical cord, and cleaning of turning upside down after closeing the lid retransfers navel repeatedly after 2-4 times
ANER DIAN thimerosal is added in band, it is made to submerge entire umbilical cord, closes the lid and shifts umbilical cord after turning upside down immediately 2-4 times, then plus
Enter cleaning solution cleaning, whole process must not exceed 30s；Umbilical cord is retransferred after cleaning, and 75% alcohol is added and submerges umbilical cord, lid
Upper cover shifts umbilical cord after turning upside down immediately 2-4 times, whole process must not exceed 30s, and cleaning solution is added and cleans 1-3 times.
5. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that the removal blood vessel,
It separates in China's Tong Shi glue step, after the 0.5-1.5cm each end to end for removing umbilical cord；Preferably, after the 1cm each end to end for removing umbilical cord,
Remaining umbilical cord is cut into 1-3cm sections；It is furthermore preferred that remaining umbilical cord is cut into 1.5-2.5cm sections；It is furthermore preferred that by remaining
Umbilical cord be cut into 2cm sections；Exocuticle is peeled off with tooth tweezer, rejects vein and arteries, removes China's Tong Shi glue therein, China is logical
Family name's glue is cleaned 1-3 times with cleaning solution；Preferably, it cleans 3 times.
6. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that described to change liquid culture simultaneously
Harvest step in: after 7d in each culture bottle supplemented medium；Preferably, according to the every 10cm of culture bottle floor space2Add training
Base 0.5-0.8mL is supported, it is furthermore preferred that according to the every 10cm of culture bottle floor space2Supplemented medium 0.6mL；10d changes liquid, sucks
Former culture medium, every bottle of addition fresh culture；Preferably, according to the every 10cm of culture bottle floor space2Supplemented medium 0.1-
0.5mL, it is furthermore preferred that according to the every 10cm of culture bottle floor space2Supplemented medium 0.3mL continues to cultivate；When 14d or cell converges
It is right to reach 45-55%；Preferably, reaching 50% can harvest.
7. the method for mescenchymal stem cell is obtained from umbilical cord as described in claim 1, which is characterized in that the digestion filtering step
In rapid, former culture medium is discarded, physiological saline cleaning 1-2 is added all over rear, the pancreatin containing EDTA is added；Preferably, every 10cm2Addition
0.1-0.5mL；It is furthermore preferred that every 10cm2Add 0.3mL；It is incubated at room temperature 3-6min, adds pancreatin when cell rounding falls off
Terminate liquid；Preferably, every 10cm2Add 0.5-0.8mL；It is furthermore preferred that every 10cm2Add 0.6mL；Cell collection is carried out, is collected
Cell carry out centrifugation 300g, 5min, discard supernatant, with physiological saline be resuspended cell after cross 70um cell sieve be filtered to remove tissue
Block, supernatant is removed in 300g, 5min centrifugation again.
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| CN113025568A (en) * | 2021-03-10 | 2021-06-25 | 云南圣衍干细胞技术开发应用股份有限公司 | Culture method and application of umbilical cord mesenchymal stem cells |
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