CN109251888A - A method of the separating mesenchymal stem cell from umbilical cord tissue - Google Patents
A method of the separating mesenchymal stem cell from umbilical cord tissue Download PDFInfo
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- CN109251888A CN109251888A CN201811033762.6A CN201811033762A CN109251888A CN 109251888 A CN109251888 A CN 109251888A CN 201811033762 A CN201811033762 A CN 201811033762A CN 109251888 A CN109251888 A CN 109251888A
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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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The method of the invention discloses a kind of from umbilical cord tissue separating mesenchymal stem cell, comprising the following steps: umbilical cord acquisition and transport；The pretreatment of umbilical cord tissue；Adhere-wall culture is carried out to umbilical cord Wharton jelly tissue block.The present invention passes through separation method, explore a kind of high-efficiency and continuous, valuable and limited umbilical cord tissue can be utilized, while improving umbilical cord tissue utilization rate and mescenchymal stem cell pick-up rate, purifying rate to greatest extent, shortens method and the path in mescenchymal stem cell acquisition period.
Description
Technical field
The invention belongs to regenerative medicine field of biotechnology, fill specifically, being related to one kind compartment from umbilical cord tissue
The method of matter stem cell.
Background technique
Umbilical cord mesenchymal stem cells (Mesenchymal Stem Cells, MSCs), which refer to, is present in neonatal umbilical cord tissue
One of versatile stem cell, it can be divided into many kinds of histocytes, have wide potential applicability in clinical practice.It is filled between umbilical cord
Matter stem cell (MSCs) differentiation potential with higher, can be broken up to multiple directions.It is in bone, cartilage, muscle, tendon,
Ligament, nerve, liver, the organizational projects aspect such as endothelium and cardiac muscle have wide potential applicability in clinical practice.
The main method of separating mesenchymal stem cell has combination enzyme digestion and tissue block adherent method from umbilical cord at present.Enzyme
Digestion method, which generallys use+0.1% clostridiopetidase A I type of 0.25% trypsase and mixes in equal volume, stays overnight the umbilical cord tissue shredded
Digestion 10-24 hours obtains mescenchymal stem cell matrix composition of layer by centrifuge separation, the culture after being inoculated in inner surface treatment
In consumptive material, suitable stem cell complete medium is added, is put into 5.0%37 DEG C of carbon dioxide incubator cultures；The disadvantage is that navel
Band mescenchymal stem cell is largely digested enzymic digestion cracking, and umbilical cord tissue can only utilize primary, mescenchymal stem cell pick-up rate
It is low, it is very low same in the presence of the primary mescenchymal stem cell purity isolated is caused due to digesting remaining blood vessel endothelium and epidermal tissue
When residual enzyme to cell growth also have biggish inhibiting effect.
Traditional adherent method, using the Cord blood in PBS liquid or the repeated multiple times clean umbilical cord tissue of 0.9% physiological saline
Afterwards, broken using tissue pulverizer or medical tissue instrument shreds, it is directly adherent into adherent treated the culture consumptive material in surface,
Stem cell complete medium is added and is paved with all adherent tissue blocks of encirclement, is put into 5.0%37 DEG C of carbon dioxide incubator cultures, often
A not good liquor was changed every 3-5 days until primary cell covers with and (needs 10-15 days)；It is primary cover with after, by the discarded place of adherent tissue block
Reason, primary cell pass on amplification cultivation；The disadvantage is that due to not having to remove vascular tissue in umbilical cord epidermal tissue and umbilical cord, umbilical cord
Relatively greatly prolong that (at least 10-15 days, primary stem cell can be only achieved passage expansion the period that separation obtains primary mescenchymal stem cell
Surge flat), while primary mescenchymal stem cell is grown along with a large amount of epidermal cell and blood vessel endothelium fibroblast, thus
Keep aim cell purity significantly very low, traditional adherent method it is adherent it is primary after, adherent umbilical cord tissue, which is discarded, also leads to umbilical cord group
It is very low to knit utilization rate, it is difficult to meet the needs of future market.
Summary of the invention
In view of this, the method for the present invention provides a kind of from umbilical cord tissue separating mesenchymal stem cell.
In order to solve the above-mentioned technical problem, the side of the invention discloses a kind of from umbilical cord tissue separating mesenchymal stem cell
Method, comprising the following steps:
Step 1, umbilical cord acquisition and transport；
The pretreatment of step 2, umbilical cord tissue；
Step 3 carries out adhere-wall culture to umbilical cord Wharton jelly tissue block.
Optionally, the umbilical cord acquisition in the step 1 and transport specifically: require acquisition umbilical cord, symbol according to sterile working
It closes umbilical cord donor and acquires standard, length is not less than 15 centimetres, keeps complete, no pinprick and breakage avoid umbilical cord and other as far as possible
Article contact, immerses in the preservation liquid of collecting bottle, and sealing mark, 4 ° of low temperature transport boxes are sent to laboratory as early as possible, and whole process is not
More than 12 hours.
Optionally, the pretreatment of the umbilical cord tissue in the step 2 specifically:
Step 2.1, Biohazard Safety Equipment is switched on by half an hour in advance, and window is adjusted to operating position and green LED is waited " to stablize gas
Stream " lights, and the sterile taking-up from collecting bottle by umbilical cord haemostatic clamp is put into ready glass dish in Biohazard Safety Equipment station
In, 75% alcohol Immersion treatment 2 minutes；
Step 2.2 goes out umbilical cord with hemostasis clamp and is transferred in new ware, the submergence of 0.9% physiological saline cleaning one time, is used in combination
Umbilical cord tissue is cut into 2cm long tissue block by operating scissors, squeezes out remained blood in tissue block with dressing pilers；
Step 2.3, with 0.9% physiological saline cleansing tissue block repeatedly, 2-3 times, until the physiological saline of submergence tissue is clear
It is clear bright；
The epidermal tissue of tissue block and vascular endothelial tissue are rejected tissue clamps completely by step 2.4, use 2, and it is logical to retain China
Glue tissue is put into sterile new ware；Until all tissue blocks are disposed, cut with medical operation, by the huatong plastic of collection
Tissue shear is broken into the huatong plastic block of 2-5mm, in case adhere-wall culture.
Optionally, adhere-wall culture is carried out to umbilical cord Wharton jelly tissue block in the step 3 specifically:
Step 3.1, configuration mescenchymal stem cell complete medium；
Step 3.2 prepares to collect the T75cm to adherent huatong plastic tissue block for step 2.42Culture bottle, by huatong plastic
Tissue block is attached to T75cm by interval 1-2cm spacing2Culture bottle bottom surface is put into 37 DEG C of 5.0%CO2It is incubated for 30 minutes and steams in incubator
Hair falls tissue surface moisture, promotes tissue block adherent not easy to fall off；
Step 3.3 will post the fast T75cm of huatong plastic tissue2Culture bottle takes out from incubator, in Biohazard Safety Equipment
The complete medium configured in electric pipettor aspiration step 3.1, liquid volume added 10ml/ are mixed with the disposable serum pipette of 10ml
T75cm2Culture bottle covers bottle cap, is gently put into 37 DEG C of 5.0%CO2It is cultivated in incubator；
Step 3.4, every 2-3 days, the culture bottle of adhere-wall culture is carried out once partly changing liquid or changes liquid entirely, it is logical to adherent China
The primary growth of mesenchymal stem cells density of glue tissue block reaches 85% or more, is collected passage to primary mescenchymal stem cell and expands
Increase culture, gently clap culture bottle be that adherent tissue block falls off, by culture bottle culture medium and suspended tissue's block be collected into
It is spare in 50ml sterile centrifugation tube；It is further cultured for suitable 0.9% physiological saline is added in bottle and cleans adherent primary cell, repeat
2 times, 1ml0.125% pancreatin is added and digests 2 minutes, fresh complete medium is added and neutralizes, it is effective to be collected in new 50ml centrifugation
0.9% normal saline dilution is centrifuged 1200rpm, 5min abandons supernatant, adds complete medium that passage amplification in proportion is resuspended to 50ml
Culture；
Step 3.5, huatong plastic repeatable adherence culture mescenchymal stem cell primary cell: will be first in step 3.4 centrifuge tube
Secondary adherent primary cell passage treated tissue block, is added 0.9% physiological saline and cleans repeatedly 2 times, tissue block is remaining
Culture solution is cleaned, and is filtered dry moisture with 70um or 100um cell sieve, in case carrying out adherent again；
Step 3.6, by treated in step 3.5 tissue block, according to the requirement repetitive operation of step 3.2-3.5, so
Make every umbilical cord tissue adhere-wall culture at least 3-5 times, i.e. original of the every umbilical cord in different cultivation stage harvest at least 3-5 batches
For mescenchymal stem cell.
Optionally, the configuration mescenchymal stem cell complete medium in the step 3.1 specifically: by 500ml LONZA
Serum free medium, 10ml Pall serum substitute and 5ml 100X L-Glutamine are sufficiently mixed uniformly, 100X, that is, every
100ml culture medium need to only add 1ml L-Glutamine, and mescenchymal stem cell complete medium is prepared.
Optionally, the Pall serum substitute is filtered by 0.2um pin filter.
Compared with prior art, the present invention can be obtained including following technical effect:
1) present invention explores a kind of high-efficiency and continuous by separation method, can utilize valuable and limited umbilical cord tissue, most
While the raising umbilical cord tissue utilization rate and mescenchymal stem cell pick-up rate of limits, purifying rate, it is dry thin to shorten mesenchyma
Born of the same parents obtain method and the path in period.
2) the method for the present invention is combined using physical separation method pretreatment and repeated multiple times adherent method, is utmostly rejected
Epidermal tissue's (outer membrane) of umbilical cord tissue and vascular endothelial tissue (1 radicular vein and 2 radicular arteries) only retain dry thin rich in mesenchyma
Then the huatong plastic tissue of born of the same parents carries out physical treatment to 2-5mm tissue block adherent and adds 37 DEG C of 5.0%CO of full culture medium2
It is incubated in incubator after being covered with to primary interstitial cell, collects adherent huatong plastic tissue block again, cleaned with 0.9% physiological saline
Fall after remaining culture medium adhere-wall culture again, repeatedly 3-5 adhere-wall culture.It is adherent compared to conventional method tissue it is primary just
It does not recycle, for the method for the present invention with the umbilical cord tissue of quantity, the pick-up rate of mescenchymal stem cell improves 3-5 times.
3) due to rejecting the ingredient outside huatong plastic tissue to the greatest extent by physical separation method, it is thin to avoid epidermis
The heteroproteose cells raised growth such as born of the same parents, fibroblast, vascular endothelial cell greatly improves the purifying rate of mescenchymal stem cell, together
When every repeatable adherence it is primary, primary cell grows the period for reaching passage density in obviously successively decreasing contracting, shows that the present invention has and contracts
Short mescenchymal stem cell obtains the advantage in period.
4) in the method for the present invention umbilical cord tissue processing, overall process is not related to the use of any tissue digestion enzyme, guaranteeing
Umbilical cord mesenchymal stem cells obtain the period it is short while, effectively avoid digestive ferment to the growth inhibition effect of primary cell and
Digestive ferment influences the cracking of primary mescenchymal stem cell.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is that MSC-001-1P0 of the present invention is organized adherent 3 days, and primary cell is climbed out of from tissue block；
Fig. 2 is that MSC-001-1P0 of the present invention is organized adherent 7 days, and up to 90%, collection freezes or passes primary cell stand density
Generation；
Fig. 3 is that MSC-001-2P0 of the present invention is organized secondary adherent 2 days, and primary cell starts to climb out of；
Fig. 4 is that MSC-001-2P0 of the present invention is organized secondary adherent 5 days, and up to 95%, collection freezes primary cell stand density
Or passage；
Fig. 5 is MSC-001-3P0 tissue adherent 1 day three times of the present invention, and primary cell climbs out of；
Fig. 6 is MSC-001-3P0 tissue adherent 3 days three times of the present invention, and up to 97%, collection freezes primary cell stand density
Or passage；
Fig. 7 is that MSC-002-1P0 of the present invention is organized adherent 5 days, and primary cell is climbed out of from tissue block；
Fig. 8 is that MSC-002-1P0 of the present invention is organized adherent 9 days, and primary cell stand density is collected jelly close to 85%
It deposits or passes on；
Fig. 9 is that MSC-002-2P0 of the present invention is organized secondary adherent 4 days, and primary cell is climbed out of from tissue block；
Figure 10 is that MSC-002-2P0 of the present invention is organized secondary adherent 7 days, and primary cell stand density is received close to 90%
Collection freezes or passes on；
Figure 11 is that MSC-002-3P0 of the present invention is organized adherent 3 days, and primary cell is climbed out of from tissue block；
Figure 12 is MSC-002-3P0 tissue adherent 5 days three times of the present invention, and primary cell stand density is received close to 93%
Collection freezes or passes on；
Figure 13 is that MSC-003-1P0 of the present invention is organized adherent 4 days, and primary cell is climbed out of from tissue block；
Figure 14 is that MSC-003-1P0 of the present invention is organized adherent 8 days, and primary cell stand density is collected jelly close to 88%
It deposits or passes on；
Figure 15 is that MSC-003-2P0 of the present invention is organized secondary adherent 3 days, and primary cell is climbed out of from tissue block；
Figure 16 is that MSC-003-2P0 of the present invention is organized secondary adherent 6 days, and primary cell stand density is received close to 92%
Collection freezes or passes on；
Figure 17 is MSC-003-3P0 tissue adherent 2 days three times of the present invention, and primary cell is climbed out of from tissue block；
Figure 18 is MSC-003-3P0 tissue adherent 4 days three times of the present invention, and primary cell stand density is received close to 94%
Collection freezes or passes on.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Material used in the present invention is as follows:
PBS sterile tissue saves liquid, umbilical cord acquisition bottle (100ml wide-mouth bottle), heparin tube (parent stays blood sample), acquisition handover
Single, cryogenic thermostat transport box；
0.9% physiological saline, 75% medicinal alcohol, medical operation cut, tissue clamps, dressing pilers, haemostatic clamp, 90mm glass dish
Several (the above article is sterility requirements), Biohazard Safety Equipment；
CO2Incubator, low speed centrifuge, T75cm2Culture bottle (air-permeable surface pretreatment), pipette, centrifuge tube, LONZA
UltraCULTURETM、Pall UltroserTM, L-Glutamine (100x), 0.125% pancreatin.
The method of the invention discloses a kind of from umbilical cord tissue separating mesenchymal stem cell, comprising the following steps:
Step 1, umbilical cord acquisition and transport: acquisition umbilical cord is required (to meet umbilical cord donor acquisition mark in strict accordance with sterile working
It is quasi-), length is not less than 15 centimetres, keeps complete, no pinprick and breakage avoid umbilical cord from contacting with other articles as far as possible, and immersion is adopted
In the preservation liquid for collecting bottle, sealing mark, 4 degree of low temperature transport boxes were sent to laboratory in 2-12 hours as early as possible.
The pretreatment of step 2, umbilical cord tissue --- it is carried out in Biohazard Safety Equipment in GMP standard laminar flow laboratory:
Step 2.1, Biohazard Safety Equipment is switched on by half an hour in advance, and it is green that window is adjusted to operating position (250mm opening) waiting
Color LED " steady air flow " is lighted, the sterile taking-up from collecting bottle by umbilical cord haemostatic clamp, is put into Biohazard Safety Equipment station quasi-
In the glass dish got ready, 75% alcohol Immersion treatment 2 minutes；
Step 2.2 goes out umbilical cord with hemostasis clamp and is transferred in new ware, the submergence of 0.9% physiological saline cleaning one time, is used in combination
Umbilical cord tissue is cut into 1-2cm long tissue block by operating scissors, squeezes out remained blood in tissue block with dressing pilers；
Step 2.3, with 0.9% physiological saline cleansing tissue block repeatedly, 2-3 times, until the physiological saline of submergence tissue is clear
It is clear bright；
Step 2.4, use 2 are tissue clamps by epidermal tissue's (outer membrane) of tissue block and vascular endothelial tissue (2 radicular arteries, 1
Vein) it rejects completely, retain huatong plastic tissue, is put into sterile new ware；Until all tissue blocks are disposed, use is medical
The huatong plastic tissue shear of collection is broken into the huatong plastic block of 0.5cm-2cm by operating scissors, in case adhere-wall culture；
Step 3, the repeated multiple times adhere-wall culture of umbilical cord Wharton jelly tissue block:
Step 3.1, mescenchymal stem cell culture medium prepare: 500ml/ bottles of+10ml Pall blood of LONZA serum free medium
Clear substitute+5ml L-Glutamine (100X) mixes well；100X, that is, every 100ml culture medium need to only add 1ml L- glutamy
Amine；
Step 3.2 prepares to collect the T75cm to adherent huatong plastic tissue block for step 2.42Culture bottle (air-permeable surface
Pretreatment), tissue block is attached to culture bottle bottom surface by interval 0.5cm-5cm spacing, is put into 37 DEG C of 5.0%CO2It is incubated in incubator
It educates 10-30 minutes and evaporates tissue surface moisture, promote tissue block adherent not easy to fall off；
Step 3.3 will post the fast T75cm of huatong plastic tissue2Culture bottle takes out from incubator, in Biohazard Safety Equipment
The culture medium configured in electric pipettor aspiration step 3.1, liquid volume added 10ml/ are mixed with the disposable serum pipette of 10ml
T75cm2Culture bottle (avoiding liquid that tissue block suspends during liquid feeding) covers bottle cap, is gently put into 37 DEG C of 5.0%CO2
It is cultivated in incubator；
Step 3.4, every 2-3 days, the culture bottle of adhere-wall culture is carried out once partly changing liquid or changes liquid entirely, it is logical to adherent China
The primary growth of mesenchymal stem cells density of glue tissue block reaches 85% or more, is collected passage to primary mescenchymal stem cell and expands
Increase culture: gently clap culture bottle be that adherent tissue block falls off, by culture bottle culture medium and suspended tissue's block be collected into
It is spare in 50ml sterile centrifugation tube；It is further cultured for suitable 0.9% physiological saline is added in bottle and cleans adherent primary cell, repeat
2 times, 1ml0.125% pancreatin (rewarming in advance) is added and digests 2 minutes, fresh complete medium is added and neutralizes, is collected in new
50ml is centrifuged effective 0.9% normal saline dilution to 50ml, is centrifuged 800-1200rpm, and 5min abandons supernatant, add culture medium be resuspended by
Ratio passes on amplification cultivation；
Step 3.5, huatong plastic repeatable adherence culture mescenchymal stem cell primary cell: will be first in step 3.4 centrifuge tube
Secondary adherent primary cell passage treated tissue block, is added 0.9% physiological saline and cleans repeatedly 2 times, tissue block is remaining
Culture solution is cleaned, and is filtered dry moisture with 70um or 100um cell sieve, in case carrying out adherent again；
Step 3.6, by treated in step 3.5 tissue block, require to repeat to grasp according to step 3.2,3,4,5 specific steps
Make, can so make every umbilical cord tissue adhere-wall culture at least 3-5 times, i.e., every umbilical cord can harvest at least in different cultivation stages
The primary mescenchymal stem cell of 3-5 batch.
Embodiment design: acquisition prepares a umbilical cord, and ((hepatitis B, hepatitis, syphilis, AIDS etc. are yin for selection blood transfusion five
Property), without heredity medication history, mental disease history etc., collection process sterile working be put into acquisition save liquid), be divided into isometric 3 sections,
Wherein be immediately disconnected processing for 1 section, remaining two sections be placed in 2-8 DEG C of refrigerator save stored respectively 10 hours, 4 hours in liquid after again by institute
The embodiment of design is handled.It is respectively used to the control experiment of embodiment 1, embodiment 2, embodiment 3, reduces umbilical cord source
The influence factor that body difference analyzes experimental result data).
1 umbilical cord of embodiment separates number MSC-001
1. umbilical cord pre-processes: selection transfuses blood five (hepatitis B, hepatitis, syphilis, AIDS etc. are feminine gender), without heredity medication history,
Mental disease history etc., collection process sterile working are put into acquisition and save liquid, and 2-8 DEG C is sent to laboratory in cold chain transportation 2 hours, will
Umbilical cord is divided into 3 sections, wherein 1 section is immediately disconnected processing.
2. umbilical cord separating treatment: after 75% medicinal alcohol immersion treatment, PBS buffer solution or 0.9% brine 2-3
It is secondary, bleeding of the umbilicus in umbilical cord surface and blood vessel is cleaned, medical scissors are cut into 2cm tissue segments, epidermal tissue and vascular endothelial tissue are separated,
Only retain huatong plastic part；
3. huatong plastic tissue PBS buffer solution or 0.9% brine are clean, dry moisture and be cut into 0.5cm*
0.5cm tissue block, it is adherent in culture bottle (inner surface pretreatment) bottom surface, block gap 2cm spacing is organized, 37 DEG C of 5.0%CO are put into2
Incubator is incubated for 30min, promote it is adherent do not fall off, complete medium is added soaking slow tissue block is advisable, and marks: MSC-001-1P0
(the 1st adherent originally culture of MSC-001 umbilical cord) is put into 37 DEG C of 5.0%CO2Incubator culture；
4. after MSC-001-1P0 is covered with, collecting primary cell and freezing or pass on, tissue block carries out secondary adherent MSC-001-
2 P0 (secondary adherent primary cell) are put into 37 DEG C of 5.0%CO2Incubator culture；
5. repeatedly, 4. step can repeatedly adherent 3 times, obtains the primary cell of 3 batches.
Experimental result is shown in Fig. 1-Fig. 6, the umbilical cord tissue in implementation process acquires completion in 2 hours and is sent to laboratory separation
Processing, separation obtains 0.5cm*0.5cm China Tong Shi glue tissue block, in 37 DEG C of 5.0%CO2Incubator is incubated for 30min, carries out 3 times
Adhere-wall culture, experimental result are shown in Table 1, and with the increase of adherent number, primary cell climbs out of time, growth degrees of fusion satisfaction biography
The equal occurrence law of proliferation time of generation or conditions of cryopreservation successively decreases (shortening), while (growth is close for primary cell growth degrees of fusion
Degree) occurrence law raising.
Primary cell climbs out of the variation of time, degrees of fusion time and stand density after 1 embodiment 1 of table is adherent
2 umbilical cord of embodiment separates number MSC-002
1. umbilical cord acquisition: umbilical cord pretreatment: selection blood transfusion five (hepatitis B, hepatitis, syphilis, AIDS etc. are feminine gender), nothings
Heredity medication history, mental disease history etc., collection process sterile working are put into acquisition and save liquid, and 2-8 DEG C is sent in cold chain transportation 2 hours
The umbilical cord tissue of acquisition in 2 hours back is placed in the sterile sealed containers of the liquid containing tissue preserration in 2-8 DEG C of refrigerator by laboratory
Separating treatment is taken out after saving 10 hours；
2. umbilical cord separating treatment: after 75% medicinal alcohol immersion treatment, PBS buffer solution or 0.9% brine 2-3
It is secondary, bleeding of the umbilicus in umbilical cord surface and blood vessel is cleaned, medical scissors are cut into 2cm tissue segments, epidermal tissue and vascular endothelial tissue are separated,
Only retain huatong plastic part；
3. huatong plastic tissue PBS buffer solution or 0.9% brine are clean, dry moisture and be cut into 2cm*0.5cm
Tissue block, it is adherent in culture bottle (inner surface pretreatment) bottom surface, block gap 5cm spacing is organized, 37 DEG C of 5.0%CO are put into2Culture
Case is incubated for 10min, promote it is adherent do not fall off, complete medium is added soaking slow tissue block is advisable, and marks: MSC-002-1P0 (MSC-
The 1st adherent originally culture of 002 umbilical cord) it is put into 37 DEG C of 5.0%CO2Incubator culture；
4. after MSC-002-1P0 is covered with, collecting primary cell and freezing or pass on, tissue block carries out secondary adherent MSC-002-
2P0 (secondary adherent primary cell) is put into 37 DEG C of 5.0%CO2Incubator culture；
5. repeatedly, 4. step can be repeatedly 3-5 times adherent, obtains the primary cell of 3-5 batch.
Experimental result is shown in Fig. 7-Figure 12, umbilical cord tissue in the implementation process laboratory separating treatment after acquisition 12 hours,
Separation obtains 2cm*0.5cm China Tong Shi glue tissue block, and interval 5cm is adherent in 37 DEG C of 5.0%CO2Incubator is incubated for 10min, carries out
During 3 adhere-wall cultures, there is about 30% adherent huatong plastic tissue hydro-planing, float zone primary cell fails to climb out of,
37 DEG C of 5.0%CO when analyzing reason and adherent processing2Incubator incubation time is too short cause it is adherent loosely and tissue block is excessive
There are much relations, as shown in table 2, primary cell climbs out of time and the comparison of embodiment 1 from adherent piece, hence it is evident that the time cycle increases
Add, primary cell growth degrees of fusion also have compared with embodiment 1 be sent to after larger gap analysis reason and umbilical cord acquisition laboratory when
Between too long and adherent piece of excessive, adherent interval larger have direct relation.Following table between correlation data:
2 embodiment 2 of table adherent is compared with embodiment 1 first, second time
3 umbilical cord of embodiment separates number MSC-003
1. umbilical cord acquisition: selection is transfused blood five (hepatitis B, hepatitis, syphilis, AIDS etc. are feminine gender), without heredity medication history, essence
Refreshing history of disease etc., collection process sterile working are put into acquisition and save liquid, and 2-8 DEG C is sent to laboratory in cold chain transportation 2 hours, by 2
The umbilical cord tissue of hour acquisition back, is placed in the sterile sealed containers of the liquid containing tissue preserration after 2-8 DEG C of refrigerator saves 4 hours
Take out separating treatment；
2. umbilical cord separating treatment: after 75% medicinal alcohol immersion treatment, PBS buffer solution or 0.9% brine 2-3
It is secondary, bleeding of the umbilicus in umbilical cord surface and blood vessel is cleaned, medical scissors are cut into 2cm tissue segments, epidermal tissue and vascular endothelial tissue are separated,
Only retain huatong plastic part；
3. huatong plastic tissue PBS buffer solution or 0.9% brine are clean, dry moisture and be cut into 1cm*0.5cm
Tissue block, it is adherent in culture bottle (inner surface pretreatment) bottom surface, block gap 1cm spacing is organized, 37 DEG C of 5.0%CO are put into2Culture
Case is incubated for 20min, promote it is adherent do not fall off, complete medium is added soaking slow tissue block is advisable, and marks: MSC-003-1P0 (MSC-
The 1st adherent originally culture of 003 umbilical cord) it is put into 37 DEG C of 5.0%CO2Incubator culture；
4. after MSC-003-1P0 is covered with, collecting primary cell and freezing or pass on, tissue block carries out secondary adherent MSC-003-
2P0 (secondary adherent primary cell) is put into 37 DEG C of 5.0%CO2Incubator culture；
5. repeatedly, 4. step can be repeatedly 3-5 times adherent, obtains the primary cell of 3-5 batch.
Experimental result is shown in Figure 13-Figure 18, umbilical cord tissue in the implementation process laboratory separating treatment after acquisition 6 hours,
Separation obtains 1cm*0.5cm China Tong Shi glue tissue block, and interval 1cm is adherent in 37 DEG C of 5.0%CO2Incubator is incubated for 20min, carries out
During 3 adhere-wall cultures, there is 5% adherent huatong plastic tissue hydro-planing, float zone primary cell fails to climb out of, and analyzes
37 DEG C of 5.0%CO when reason and adherent processing2Incubator incubation time is insufficient and adherent interval is too low direct relation, primary
Cell from adherent piece climb out of time, adherent time of fusion, cell fusion density and embodiment 1, embodiment 2 comparison be shown in Table 3,
It can be concluded that
First, umbilical cord tissue is logical from the adherent China of the time interval shorter (in 2 hours) and separation for collecting separating treatment
Family name's glue diameter is smaller (0.5*0.5cm), shorter for the primary cell separation and Extraction period, the primary cell stand density cultivated
(degrees of fusion) is also higher；
Second, reasonable spacing distance (2cm), adherent incubation time (30min) are right between adherent umbilical cord China Tong Shi glue
There is facilitation density cycle in the grow period and fusion of primary cell.
3 embodiment 3 of table and embodiment 1, embodiment 2, first, second adherent comparison
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Claims (6)
1. a kind of method of the separating mesenchymal stem cell from umbilical cord tissue, which comprises the following steps:
Step 1, umbilical cord acquisition and transport；
The pretreatment of step 2, umbilical cord tissue；
Step 3 carries out adhere-wall culture to umbilical cord Wharton jelly tissue block.
2. the method according to claim 1, wherein umbilical cord acquisition and transport in the step 1 specifically: press
Acquisition umbilical cord is required according to sterile working, meets umbilical cord donor acquisition standard, length is not less than 15 centimetres, keeps complete, no pinprick
And it is damaged, it avoids umbilical cord to contact with other articles as far as possible, immerses in the preservation liquid of collecting bottle, sealing mark, 4 ° of low temperature transport boxes
It is sent to laboratory as early as possible, whole process is no more than 12 hours.
3. the method according to claim 1, wherein the pretreatment of the umbilical cord tissue in the step 2 specifically:
Step 2.1, Biohazard Safety Equipment is switched on by half an hour in advance, and window is adjusted to operating position and waits green LED " steady air flow "
It lighting, the sterile taking-up from collecting bottle by umbilical cord haemostatic clamp is put into Biohazard Safety Equipment station in ready glass dish,
75% alcohol Immersion treatment 2 minutes；
Step 2.2 goes out umbilical cord with hemostasis clamp and is transferred in new ware, and the submergence of 0.9% physiological saline cleaning one time, and with performing the operation
It cuts and umbilical cord tissue is cut into 2cm long tissue block, squeeze out remained blood in tissue block with dressing pilers；
Step 2.3, with 0.9% physiological saline cleansing tissue block repeatedly, 2-3 times, until the physiological saline clarification of submergence tissue is saturating
It is bright；
Clean, reservation huatong plastic group is rejected by step 2.4, use 2 by tissue clamps by the epidermal tissue of tissue block and vascular endothelial tissue
It knits, is put into sterile new ware；Until all tissue blocks are disposed, cut with medical operation, by the huatong plastic tissue of collection
The huatong plastic block of 2-5mm is shredded into, in case adhere-wall culture.
4. the method according to claim 1, wherein being carried out in the step 3 to umbilical cord Wharton jelly tissue block
Adhere-wall culture specifically:
Step 3.1, configuration mescenchymal stem cell complete medium；
Step 3.2 prepares to collect the T75cm to adherent huatong plastic tissue block for step 2.42Culture bottle, by huatong plastic tissue block
T75cm is attached to by interval 1-2cm spacing2Culture bottle bottom surface is put into 37 DEG C of 5.0%CO2It is incubated for 30 minutes in incubator and evaporates group
Surface moisture is knitted, promotes tissue block adherent not easy to fall off；
Step 3.3 will post the fast T75cm of huatong plastic tissue2Culture bottle takes out from incubator, uses in Biohazard Safety Equipment
The disposable serum pipette of 10ml mixes the complete medium configured in electric pipettor aspiration step 3.1, liquid volume added 10ml/
T75cm2Culture bottle covers bottle cap, is gently put into 37 DEG C of 5.0%CO2It is cultivated in incubator；
Step 3.4, every 2-3 days, the culture bottle of adhere-wall culture is carried out once partly changing liquid or changes liquid entirely, to adherent huatong plastic group
It knits the primary growth of mesenchymal stem cells density of block and reaches 85% or more, passage amplification training is collected to primary mescenchymal stem cell
Support, gently clap culture bottle be that adherent tissue block falls off, by culture bottle culture medium and suspended tissue's block be collected into 50ml without
It is spare in bacterium centrifuge tube；It is further cultured for being added suitable 0.9% physiological saline in bottle and cleans adherent primary cell, repeat 2 times, add
Enter 1ml0.125% pancreatin to digest 2 minutes, fresh complete medium is added and neutralizes, is collected in new 50ml centrifugation effective 0.9%
Normal saline dilution is centrifuged 1200rpm, 5min abandons supernatant, and complete medium resuspension is added to pass on amplification cultivation in proportion to 50ml；
Step 3.5, huatong plastic repeatable adherence culture mescenchymal stem cell primary cell: it will be pasted for the first time in step 3.4 centrifuge tube
Wall primary cell passage treated tissue block, is added 0.9% physiological saline and cleans repeatedly 2 times, by the culture of tissue block remnants
Liquid is cleaned, and is filtered dry moisture with 70um or 100um cell sieve, in case carrying out adherent again；
Step 3.6, by treated in step 3.5 tissue block, according to the requirement repetitive operation of step 3.2-3.5, so make every
Root umbilical cord tissue adhere-wall culture at least 3-5 times, i.e. every umbilical cord harvest primary of at least 3-5 batch in different cultivation stages
Mesenchymal stem cells.
5. according to the method described in claim 4, it is characterized in that, the configuration mescenchymal stem cell in the step 3.1 is complete
Culture medium specifically: fill 500mlLONZA serum free medium, 10mlPall serum substitute and 5ml100XL- glutamine
Divide and be uniformly mixed, 100X, that is, every 100ml culture medium need to only add 1mlL- glutamine, and mescenchymal stem cell is prepared and trains completely
Support base.
6. according to the method described in claim 5, it is characterized in that, the Pall serum substitute is filtered by 0.2um pin
Device filtering.
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