CN108251358A - A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell - Google Patents
A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell Download PDFInfo
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Abstract
The invention discloses a kind of multiple batches of primary separation methods of identical donor source human mesenchymal stem cell, belong to field of cell culture.The method of the present invention is that 1 5mm is obtained after donor tissue is handled3Donor tissue fritter, by fetal calf serum or people's AB serum immersion treatments, then carry out conventional tissue block adherent culture.While harvesting mescenchymal stem cell, flap is carried out to donor tissue block and is continuously cultivated, and the culture supernatant recycling of tissue block is continued to be incubated, realize the high quality of the human mesenchymal stem cell from same donor, multiple batches of stable separation.Form, proliferation and differentiation capability, the immunophenotype of gained cell of the invention remain unchanged between each batch.Compared with traditional tissue block adherent method and enzyme digestion, inventive process avoids the wastes of clinical samples resource, save the manpower and materials of clinical link, also improve the separative efficiency and yield of mescenchymal stem cell.
Description
Technical field
The invention belongs to field of cell culture, and in particular to a kind of identical donor source human mesenchymal stem cell it is multiple batches of
Primary separation method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) has self-renewing and the Multidirectional Differentiation of height
Ability can not only be divided into the interstitial tissues cell such as fat, bone, cartilage, muscle, also with low immunogenicity and immunosupress
Effect is the important seed cell of the treatments such as organizational project and cell replacement therapy, trnasplantion immunity and autoimmune disease.
Last century the seventies, Friedenstein and its colleague have turned out mescenchymal stem cell from marrow first.
In recent years, researcher is again successively from other tissues, such as fat, deciduous teeth and neonatal umbilical cord, placenta, amnion, Cord blood
In isolated MSCs.Wherein, neonatal umbilical cord tissue has rich in MSCs, convenient material drawing and excellent without dispute of ethic etc.
Point, it is considered to be obtain an ideal source of MSCs.
Human umbilical cord mesenchymal stem cells (human umbilical cord-derived mesenchymal stem
Cells, hUC-MSCs) it is the fetal stem cell for being different from embryonic stem cell and adult stem cell, it is a kind of new multipotency interstitial
Stem cell, it is tissue-derived it is original, biological property is stable, Proliferation, Differentiation ability is strong, immunogenicity is low.HUC-MSCs is primarily present
In the magnificent Tong Shi glue (Wharton ' s jelly) and umbilical vein subendothelial layer of umbilical cord, enzyme digestion and tissue block adherent can be passed through
Method obtains.
It is specifically described two methods of enzyme digestion in the prior art and the concrete operation step of tissue block adherent method below.
(1) enzyme digestion (by taking " single enzyme process " as an example)
Umbilical cord is cut into the segment of about 2cm long, is washed repeatedly 2-3 times with containing 1% dual anti-PBS buffer solution, removes umbilical cord
Blood.Umbilical cord is cut off in longitudinal direction, rejects three blood vessels.Remaining umbilical cord tissue is cut into small pieces and is put into small beaker, 1- is cut into ophthalmology
2mm3The tissue fritter of size.Tissue fritter is moved in the 100ml sterile glass vials for including magnetic stir bar, adds in final concentration
For 0.1% clostridiopetidase A II, continuing magnetic force gentle agitation digests 2h under 37 DEG C of water-baths.PBS buffer solution washing collagen is added in,
2000rpm centrifuges 15min, abandons supernatant, stays precipitation.Adding PBS will organize to hang, and with nylon net filter, collect cell filtrate,
1000rpm centrifuges 5min, abandons supernatant.The cell collected is resuspended with DMEM/F12 complete mediums, piping and druming is uniform, counts, inoculation
In Tissue Culture Flask, be placed in 37 DEG C, 5% carbon dioxide, saturated humidity cell incubator in cultivate.It is changed for the first time after for 24 hours
Liquid removes the cell of suspension.A not good liquor is changed per 3d later, after cell is merged up to 80%-90%, carries out routine passage culture.
(2) tissue block adherent method
Umbilical cord is cut into the segment of about 2cm long, is washed repeatedly 2-3 times with containing 1% dual anti-PBS buffer solution, removes umbilical cord
Blood.Umbilical cord is cut off in longitudinal direction, rejects blood vessel.Remaining umbilical cord tissue is cut into smaller tissue fritter (common 1mm3), between certain
Away from evenly laid out in culture vessel, subsequent operation is divided to two kinds of " overturning is dry " method and " Thin cell layer " method：
A. " overturning is dry " method：Culture dish is gently turned, puts 37 DEG C of carbon dioxide incubator cultures 4-6 hours,
It treats that tissue fritter is micro- dry, then slowly culture dish is turned, is slowly added into a small amount of complete medium, ensure that culture solution can
Tissue fritter is not crossed, places back in quiescent culture in incubator；
B. " Thin cell layer " method：The culture dish for being covered with tissue block is just being put, 30 minutes is being stood, tissue block is made preferably to paste
Wall is slowly added to a small amount of culture solution and covers tissue block surface, 37 DEG C of carbon dioxide incubator quiescent cultures.
After completing paving block by A or step B, absolutely stand within the 1st~7 day, every 3 days full doses change liquid, tissues observed block later
Periphery attached cell climbs out of situation, and when operation handles with care.
About 3~4 weeks, when seeing the fusiformis adherent cell growth that is climbed out of around tissue block to 80%- under the microscope
During 90% fusion, add in pancreatin and digest together by cell and tissue block.Mixture slaking liquid is collected, is filtered with 100 mesh filter screens,
Umbilical cord tissue block therein is removed, the cell for collecting filtration continues secondary culture.
The shortcomings that above two method, is：
First：The cell quantity of acquisition is limited.Prior art, either tissue block adherent method or enzyme digestion,
After fresh umbilical cord tissue block is used to obtain a batch hUC-MSCs, i.e., abandoned umbilical cord tissue block as waste.And in fact,
It is limited by factors such as intrinsic culture area, the enzymic digestion efficiency of separation vessel, only through single treatment and culture, in umbilical cord tissue
The mescenchymal stem cell being rich in is difficult to fully, thoroughly be separated.Therefore according to existing technical solution, from clinical acquisitions
The utilization ratio of the umbilical cord sample arrived and the separation yield of hUC-MSCs are relatively low.
Second：Detach the time length (existing tissue block adherent method) needed for hUC-MSCs.Since the umbilical cord of fresh acquisition exists
After tissue block is processed into for the first time, to cultured in vitro environment there are one more slow adaptation process, its institutional framework in addition
It is more solid and fine and close, it is difficult to dissociate individual cells in the short time, therefore umbilical cord tissue block is from first bed board culture to can
It is often longer that progress hUC-MSCs harvests the required time for the first time.Such as：Li little Zhan etc.[1]Block method (i.e. tissue is being planted using tradition
Block adherent method) separation human umbilical cord mesenchymal stem cells when, observe that cell climbs out of within 12 days or so, cell can be only achieved within 16 days or so
80% fusion；Equally it is tissue block adherent method, Li Duo[2]The umbilical cord handled with different time after acquisition carries out mesenchyma and does respectively
The primary separation of cell, find 24 hours in processing group, in 24-48 hour in processing group, 48-72 hours processing group it is primary carefully
Born of the same parents' harvest time that is averaged is respectively 17.33 days, 22.00 days and 21.69 days；What continues clear etc.[3]Report also indicate that, umbilical cord tissue
Block is about 20 days from inoculated and cultured to that can carry out the time that cell passes on for the first time.To sum up, it is obtained using existing tissue block adherent method
The time of hUC-MSCs is obtained generally at 20 days or so, it is difficult to be provided enough for Related Experimental Study or clinical treatment in a short time
Mescenchymal stem cell.
Third：Isolated cell viability is limited and separation costs are high (enzyme digestion).The reagents valency such as clostridiopetidase A, pancreatin
Lattice are expensive, and enzymolysis processing easily causes the decline of mescenchymal stem cell vigor.
Bibliography
[1] research [D] Beijing of Li Duo human umbilical cord mesenchymal stem cells isolated culture method：Beijing Union Medical College blood
Ye Xue research institutes, 2011：20-21.
[2] He Shaoqing, Luo Zhenyu, Liu Qiuying, wait human umbilical cord mesenchymal stem cells be separately cultured and to fat it is thin with skeletonization
Differentiation [J] the China's Tissue Engineering Studies and clinical rehabilitation of born of the same parents, 2010,14 (14)：2492-2496.
[3] Li little Zhan, Ma Hong, Xu Hui, improvement [J] the Jiangsu for waiting human umbilical cord mesenchymal stem cells primary culture methods are big
Learn journal, 2014,25 (5)：375-379.
Invention content
It is an object of the invention to a kind of multiple batches of primary separation methods of identical donor source human mesenchymal stem cell.
The technical solution used in the present invention is：
A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell, includes the following steps：
(1) it aseptic collection donor tissue and is handled；
(2) by treated, donor tissue shreds, and obtains 1-5mm3Donor tissue fritter；
(3) donor tissue fritter 5-20min is impregnated with fetal calf serum or people AB serum；
(4) donor tissue fritter is inoculated into tissue culture plate, and adds appropriate complete medium, cultivated；
(5) when the adherent cell growth that of dissociating is merged to 80%-90%, primary mescenchymal stem cell is harvested；
(6) while harvesting primary mescenchymal stem cell, on the donor tissue fritter in original cuiture system and culture
Carry out recycling reprocessing clearly；Wherein donor tissue fritter repeats above-mentioned fetal calf serum or people AB serum impregnates 5-20min
And subsequent processing, continue to harvest mescenchymal stem cell；Original cuiture supernatant is transferred in new Tissue Culture Flask and is incubated again
Culture continues to harvest mescenchymal stem cell.
Preferably, the donor includes at least one of neonatal umbilical cord, placenta, amnion.
It is multiple batches of primary as donor progress mescenchymal stem cell using neonatal umbilical cord in the method for the present invention embodiment
Separation.And according to the common knowledge of industry technical staff, the method for the present invention can be applicable to its similar to Cord structure
Its compact tissue, such as the separating mesenchymal stem cell from placenta, amnion tissue.And different donors such as placenta, amnion are directed to,
Its processing method and neonatal umbilical cord can difference, please operated according to the conventional method.
Below by donor for neonatal umbilical cord, to further illustrate the present invention taken technical solution：
A kind of multiple batches of primary separation method of identical donor human umbilical cord mesenchymal stem cells, includes the following steps：
(1) aseptic collection neonatal umbilical cord is soaked in buffer solution.
(2) in an aseptic environment, umbilical cord is cut into the segment of 1-5cm long, is fully washed with buffer solution, removal residual umbilical cord
Blood.
(3) umbilical cord segment is longitudinally splitted, tiled, reject blood vessel.
(4) umbilical cord tissue for having removed blood vessel is shredded, obtains 1-5mm3Umbilical cord tissue fritter.
In general, umbilical cord tissue block is cut smaller, it is possible to climb out of the tissue section (artificial shearing causes) of cell just
It is more.So about 1mm is usually used in the prior art3Tissue fritter carry out bed board culture.However rear extended meeting in the present invention
The immersion treatment of 5-20min is carried out to umbilical cord tissue fritter using fetal calf serum or people AB serum, immersion treatment can significantly improve
The adherent viability of tissue block and the separation yield and speed of mescenchymal stem cell.So the body of the invention by umbilical cord tissue block
Product range expands as 1-5mm3.The tissue block of specification of the present invention is not easy to float in follow-up cultivation, and adherent rate or cell are separated into
Power is high, is more suitable for tissue block method's separation of mescenchymal stem cell.
(5) umbilical cord tissue fritter 5-20min is impregnated with fetal calf serum or people AB serum.
The operation of the link can significantly improve the nutrition condition on tissue block periphery in follow-up cultivation microenvironment, reduce tissue block
Floating, improves the adherent viability of tissue block, helps to improve the separation yield and speed of mescenchymal stem cell, be shown in Table 1.
In addition, present invention demonstrates that, employment AB blood serum substitutings fetal calf serum can reach similar separating effect, can obtain
Mescenchymal stem cell product without heterologous animal protein, the technology of the present invention is is provided possibility by this applied to clinic.
(6) umbilical cord tissue fritter is inoculated into tissue culture plate, and adds appropriate complete medium, cultivated.
" overturning is dry " method may be used in present invention culture, can also use " Thin cell layer " method.Can as needed into
Row selection.
(7) when the adherent cell growth that of dissociating is merged to 80%-90%, primary mescenchymal stem cell is harvested.
(8) while harvesting mescenchymal stem cell, the umbilical cord tissue fritter in original cuiture system and culture solution are carried out
Recycling reprocessing；Wherein umbilical cord tissue fritter repeats above-mentioned fetal calf serum or people AB serum and impregnates 5-20min and follow-up
Processing, continue harvest mescenchymal stem cell；Former culture supernatant is transferred in new Tissue Culture Flask is incubated culture again, continues
Harvest mescenchymal stem cell.
The link of the invention realizes umbilical cord tissue block and its repetition of culture supernatant recycles, and greatly improves umbilical cord
The utilization ratio of sample and the hUC-MSCs yield in identical donor source, realize identical donor human umbilical cord mesenchymal stem cells
Multiple batches of primary separation.In the present invention, the umbilical cord tissue fritter being recycled equally receives tire ox before bed board is repeated
The immersion treatment of serum or people's AB serum, the cell that this umbilical cord tissue fritter for repeat to cultivate climbs out of are cultivated more for the first time
Soon, more, reason may be tissue block after an in vitro culture, structure becomes more loose, also more adapts to body
Outer culture environment using after the immersion treatment of fetal calf serum or people's AB serum, increases the nutrition condition on tissue block periphery, so
MSCs can therefrom faster, more separate out.
The present invention has not only recycled umbilical cord tissue block, also achieves the recycling of substance system culture supernatant, leads to
Cross the incubation again to former culture supernatant, can obtain with tissue block single culture quantity quite even more primary cell,
Its reason may be by last round of culture, has part cell and has been directly released into culture solution from tissue block, these are dissipated
Cell wherein be not yet in time for it is adherent, but suspend be present in culture supernatant；In addition, a large amount of adherent thin of clone is formed
Born of the same parents can secrete certain growth factors and wait until in culture supernatant, promote the adherent of above-mentioned free cell and proliferation.
The operation of the method for the present invention is all to carry out in an aseptic environment, buffer solution used need to add in appropriate antibiotic to prevent
Only pollute, in addition the culture environment of human umbilical cord mesenchymal stem cells be 37 DEG C, the cell culture of 5% carbon dioxide, saturated humidity
In case.These are the common knowledge of industry technical staff.
Preferably, it should be immediately soaked in the buffer solution containing antibiotic after aseptic collection neonatal umbilical cord in step (1)；
Such as the HBSS buffer solutions containing 1% blueness/streptomysin or the PBS buffer solution containing 1% blueness/streptomysin.
Preferably, in step (2) buffer solution for the HBSS buffer solutions containing appropriate antibiotic (such as 1% blueness/streptomysin) or
PBS buffer solution.
Preferably, umbilical cord segment is longitudinally splitted in step (3), tiled, reject blood vessel (2 arteries and 1 vein), protected
It stays except extravascular umbilical cord tissue.
Preferably, the umbilical cord tissue for having removed blood vessel is shredded in step (4), obtains 2-5mm3Umbilical cord tissue fritter.
More preferably 3-4mm3Umbilical cord tissue fritter.
In general, tissue block is cut smaller, it is likely that climbs out of the tissue section (artificial shearing causes) of cell more
It is more.But applicants have found that：1mm3Umbilical cord tissue fritter it is too small, when culture, easily floats.It is preferred that 2-5mm3Umbilical cord tissue
Fritter, more preferably 3-4mm3Umbilical cord tissue fritter, the umbilical cord tissue fritter of above-mentioned specification is better than 1mm in adherent effect3
, and it is unlikely to excessive, take into account separation quality and quantity.
Preferably, the concentration of volume percent of fetal calf serum is 10%-100% in step (5).
It is furthermore preferred that the concentration of volume percent of fetal calf serum is 100% in step (5).
Preferably, in step (5) people's AB serum a concentration of 10%-100% of concentration of volume percent.
It is furthermore preferred that in step (5) people's AB serum concentration of volume percent a concentration of 100%.
Using xenogenesis fetal calf serum culture people source MSCs, inevitably by heterologous animal albumen (immunogene or cause of disease)
The cell product for human body therapy is introduced, influences quality and the safety of MSCs clinical practices.The present invention establishes employment AB
Serum immersion treatment human umbilical tissue block and the alternative solution for being separately cultured hUC-MSCs, can avoid with fetal calf serum culture people
The potential risk and drawback of source MSCs is laid a good foundation for the present invention applied to clinical treatment.
The fetal calf serum and people's AB serum of the present invention is commercial product.
Preferably, the middle fetal calf serum of step (5) or people AB serum impregnate umbilical cord tissue fritter 5-20min, more preferably
10-20min。
Further, since what fetal calf serum or people AB serum impregnated is the umbilical cord tissue block of fresh processing rather than " cell " (this
When there are no mescenchymal stem cell therefrom separate outs), along with the time of processing is (5-20 minutes, and inducing cell point very short
The time of change usually by " my god " come in terms of), so the step operation cell will not be caused to break up in advance.In fact, in applicant's research
Identification was all done to the proliferative capacity of every a collection of cell, immunophenotype, into fat and Osteoblast Differentiation ability, was as a result shown really without shadow
It rings.
Preferably, in step (6), umbilical cord tissue fritter is inoculated into tissue culture plate, the thin of tissue fritter will be completed
Born of the same parents' culture plate is in CO2It is buckled in incubator after cultivating 4-6h, then tissue culture plate is righted；It is added into tissue culture plate appropriate
Complete medium continues to cultivate.
In the prior art, the primary separation cell of tissue block method has two kinds of " overturning is dry " method and " Thin cell layer " method.This hair
Bright preferably " overturning is dry " method.Present invention research finds to be tightly attached on culture plate by the umbilical cord tissue fritter impregnated, is falling
Buckle will not fall down when managing.Reason is：First, it is largely magnificent Tong Shi glue to remove the umbilical cord tissue remained after blood vessel
Structure is a kind of very sticky substance, is easily attached to plastic ware surface；Second, it is more that fetal calf serum contains albumen, lipid etc.
Kind nutritional ingredient, pure fetal calf serum has certain viscosity in itself, to impregnate with magnificent Tong Shi glue navel as main component
Band fritter can further increase it and attach power.
After being buckled to culture processing, tissue fritter can be preferably attached on culture plate, then add complete medium
When be not easy float, be conducive to improve hUC-MSCs yield.
Preferably, in step (6), umbilical cord tissue fritter is inoculated into tissue culture plate, is aseptically air-dried (such as
20-35min) to organizing, small block edge is micro- to dry up, then will complete the tissue culture plate back-off culture 4-6h of tissue fritter.
In order to enhance the pasting board effect of tissue block, tissue block falls down when preventing from being buckled in next step, by tissue fritter
Being inoculated into tissue culture plate needs to carry out appropriate air-dry.In addition, air-dried effect also resides in, avoid when next step is buckled to,
The serum that tissue block surface is wrapped in during previous step immersion treatment is flowed on the lid of culture plate, is polluted.
This is an operational details, and many documents do not refer to when delivering, but applicant thinks to increase during practical operation
The step for, be conducive to subsequent operation.
Preferably, in step (6), umbilical cord tissue fritter is inoculated into tissue culture plate, stands 20-40 minutes, then add
Add appropriate complete medium, continue to cultivate.I.e. the present invention can also carry out cell culture using " Thin cell layer " method.
Preferably, complete medium is DMEM/F12 culture mediums, and add 10% fetal calf serum or 10% people AB serum,
1% dual anti-(dual anti-is blueness/streptomysin).
Preferably, cell culture is the CO at 37 DEG C2It is carried out in incubator, every 3 days full doses are changed after absolutely standing 7 days, 7 days
Liquid and tissues observed block periphery attached cell climb out of situation, and when operation handles with care.
Preferably, while harvesting primary mescenchymal stem cell, umbilical cord tissue fritter and culture supernatant are recycled again
Processing；Wherein umbilical cord tissue fritter repeats above-mentioned fetal calf serum or people AB serum impregnates 5-20min and subsequent processing,
Continue to harvest mescenchymal stem cell.The present invention has carried out umbilical cord tissue fritter to repeat culture twice (plus tissue fritter altogether
It is cultivate for the first time, identical to amount to that 3 batches are primary is separately cultured for somatic umbilicus), average each tissue block repeat culture obtain it is primary
Cell number is about 1.6 times that tissue block is cultivated for the first time, and the earliest time of occurrence of the primary cell and generation time is respectively shortened for the first time
It is to average 2.4 days and average 6.9 days (being shown in Table 2,3).
Preferably, while harvesting mescenchymal stem cell, the culture solution in former cultivating system is collected, with 100 mesh
Strainer filtering, removal may remain in microtissue relic therein, then the former culture supernatant after filtering are transferred to new thin
It in born of the same parents' culture bottle, is placed in 37 DEG C of carbon dioxide incubators and cultivates, continue to harvest mescenchymal stem cell.The present invention has carried out two batches
The repetition culture of culture supernatant, the primary cell number that average each culture supernatant culture obtains is about that tissue block is cultivated for the first time
1.5 times, the earliest time of occurrence of primary cell and for the first time generation time be respectively shortened to average 2.5 days and it is 7.3 days average (be shown in Table 2,
3)。
The beneficial effects of the invention are as follows：
The present invention improves the prior art using tissue block adherent method separation hUC-MSCs, is coated with by serum
Continuously culture, uterus tissue pieces supernatant recycle the links such as incubation with umbilical cord tissue block " flap ", solve tissue block method's separation effect
The problems such as rate is low realizes the high quality from same donor hUC-MSCs, multiple batches of stable separation.
Traditional tissue block adherent method is far above using the hUC-MSCs quantity that the method for the present invention obtains.Specifically, using
Pure FBS or people's AB serum immersion treatments, the primary cell single of umbilical cord tissue block can be made, which to detach yield, increases 3-4 times (referring to table
1)；With the single culture Yield comparison of the tissue block of serum immersion treatment, serum impregnates multiple batches of culture (tissue block and the training of joint
Support supernatant), the total output of primary cell can be made to improve 7-8 times (referring to table 2,3)；The list of the tissue block impregnated with unused serum
Secondary culture Yield comparison, serum impregnate the multiple batches of culture (tissue block and culture supernatant) of joint, can make the total output of primary cell
It improves 24-34 times (referring to table 1-3).
The present invention significantly shortens the time of primary separation, when umbilical cord tissue block or former culture supernatant be used to repeat to cultivate
When, cell can be climbed out of in 48-72h.
It is remained unchanged between each batch using form, proliferation and differentiation capability, the immunophenotype of cell obtained by the present invention.
In addition, present invention demonstrates that, employment AB blood serum substitutings fetal calf serum can reach similar separating effect, this is that incite somebody to action this
Invention provides possibility applied to clinic.
To sum up, compared with traditional tissue block adherent method and enzyme digestion, inventive process avoids clinical umbilical cord samples
The waste of resource not only saves the manpower and materials of clinical link, also improves the separative efficiency and yield of hUC-MSCs, can
In a relatively short period of time, the identical donor source mescenchymal stem cell of high quality of quantity abundance is provided for correlative study.
Description of the drawings
Three batches that Fig. 1 is the identical donor hUC-MSCs of om observation detach；
The growth curve (be P4 for cell) that Fig. 2 is the identical donor hUC-MSCs of three batches；
Fig. 3 is the cell cycle of the identical donor hUC-MSCs of three batches；
Fig. 4 is the stream measuring of the identical donor hUC-MSCs immunophenotypes of three batches；
Fig. 5 is that each crowd of hUC-MSCs into fat differentiation capability identifies (× 100)；
The Osteoblast Differentiation ability that Fig. 6 is each crowd of hUC-MSCs identifies (× 100).
Specific embodiment
The fetal calf serum that the present invention uses is purchased from Zhejiang Tian Hang biotech inc (article No. 13011-8611),
People AB serum is purchased from Gemini companies of the U.S. (article No. 100-512).
The technical solution used in the present invention is：
(1) aseptic collection neonatal umbilical cord is soaked in the HBSS bufferings containing 1% dual anti-(dual anti-for blueness/streptomysin) immediately
In liquid.
(2) start super-clean bench, umbilical cord is impregnated with the HBSS buffer solutions containing 1% dual anti-(dual anti-is blueness/streptomysin), with hemostasis
Umbilical cord, the segment of several 1-5cm long is cut into operating scissors, is fully washed in HBSS by one end of the fixed umbilical cord of pincers,
Removal residual Cord blood.
(3) HBSS buffer solutions are replaced, impregnate the umbilical cord segment of previous step；Each umbilical cord segment is taken, first splits its longitudinal direction,
It tiles and removes 3 internal blood vessels, retain except extravascular umbilical cord tissue.
(4) umbilical cord tissue for having removed blood vessel is focused in 10ml small beakers, is shredded into umbilical cord tissue with eye scissors
3-4mm3Tissue fritter.
(5) the umbilical cord tissue fritter 5- of previous step is impregnated with the fetal calf serum of various concentration (FBS) or people AB serum
20min。
(6) umbilical cord tissue fritter is uniformly inoculated into 6 porocyte culture plates, per hole, 5 pieces of placement, sterile in super-clean bench
Air-drying 20-35min, block edge is micro- to dry up to organizing.
(7) culture plate for completing tissue block is buckled to, in 37 DEG C of carbon dioxide incubator (5%CO2, saturated humidity) in
Culture 4-6h is placed, then culture plate is righted.
(8) 2ml DMEM/F12 complete culture solutions are slowly added into each plate hole of 6 well culture plates (containing 10% tire ox blood
It is clear or 10% people AB serum, 1% dual anti-), continue the carbon dioxide incubator (5%CO at 37 DEG C2, saturated humidity) in cultivate,
Absolutely stand 7 days.
Every 3 days full doses change the situation that climbs out of of liquid and tissues observed block periphery attached cell after (9) 7 days, and when operation gently takes light
It puts.
(10) when the attached cell that of dissociating from tissue block visible under mirror, which is grown to 80-90%, merges, to former cultivating system
In umbilical cord tissue fritter and culture solution carry out recycling reprocessing；Wherein umbilical cord tissue fritter repeats above-mentioned fetal calf serum
Or people AB serum impregnates 5-20min and subsequent processing, continues to harvest mescenchymal stem cell；Collect original fluid and with 100 mesh
Culture supernatant is transferred in new Tissue Culture Flask by strainer filtering, puts 37 DEG C of carbon dioxide incubator (5%CO2, saturation it is wet
Degree) in be incubated culture, continue harvest mescenchymal stem cell.
Specific method is：In an aseptic environment, 6 orifice plates are opened, first, take elbow ophthalmic tweezers by the umbilical cord tissue in plate hole
Block carefully presss from both sides one by one from culture plate bottom, to be repeated above-mentioned fetal calf serum or people AB serum and impregnates 5-20min and subsequent
It handles (step 5-10).And then, the old culture solution in former cultivating system is collected, is filtered with 100 mesh filter screens, removal may remain
Umbilical cord tissue relic, then the former culture supernatant after filtering is directly transferred to new Tissue Culture Flask, is placed in 37 DEG C of titanium dioxide
Carbon incubator (5%CO2, saturated humidity) in continue culture and (without adding fresh complete medium, directly trained again after filtering
It supports).After the recycling for completing umbilical cord tissue block and original fluid, digested according to a conventional method in former culture plate with 0.25% pancreatin
Bottom keeps the primary cell of adhered state, completes the harvest of mescenchymal stem cell.
(11) cellular morphology, immunophenotype, proliferation and the differentiation capability of identification each batch hUC-MSCs.Particular content packet
It includes：Observation obtains cellular morphology under inverted microscope, determination of cell count cell growth curve, and Flow cytometry is thin
Born of the same parents' period, Immunophenotyping, to being evaluated into fat and Osteoblast Differentiation ability for each batch of cell.
With reference to specific embodiment, the present invention is described further, but not limited to this.The following embodiments of the present invention
It is middle by organize fritter and former culture supernatant culture or obtain after being incubated, without the mescenchymal stem cell of passage be referred to as original
For cell.
A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell of embodiment 1
Method is as described above, wherein, step (5) is impregnated using the fetal calf serum (FBS) or people AB serum of various concentration
The umbilical cord tissue fritter 15min of one step, primary cell is obtained after bed board culture.
Primary cell (primary culture cell) refers to directly rise in the earliest algebraically of cultured in vitro tissue etc.
Cell.In the present invention by organize fritter and former culture supernatant culture or obtain after being incubated, do without the mesenchyma of passage
Cell is referred to as primary cell.
(successfully dissociate to the earliest time of occurrence of the primary cell of each group, the 1st generation time, uterus tissue pieces success rate
Go out tissue block total number × 100% of tissue block number/inoculation of cell), (umbilical cord per cm is isolated for primary cell number
Cell number) it is for statistical analysis, it the results are shown in Table 1.
Umbilical cord tissue block of the table 1 through the different pretreatments time of adherent separating primary cells and yield for the first time
Compared with serum-free immersion group, * P<0.05, * * P<0.01, * * * P<0.001；Wherein, 100%FBS immersions group vs
Serum-free immersion group, primary cell number are averagely about 3.6 times of serum-free immersion group；100% people AB serum impregnates vs serum-frees
Immersion group, primary cell number are averagely about 4.1 times of serum-free immersion group.
From table 1 it follows that in the case of single culture, using pure FBS or people's AB serum immersion treatment umbilical cord groups
Block is knitted, can significantly shorten the initial time of occurrence and harvest time of primary cell, improves the culture success ratio (tissue of tissue block
Block periphery has cell to swim out of), and primary cell yield is made to increase 3-4 times.Its reason may be：Pure fetal calf serum or people's AB serum
The concentration of the nutriments such as interior attachment proteins, growth factor is significantly higher than the complete medium for containing only 10-50% fetal calf serums, because
This is operationally more meaningful with the clear immersion treatment tissue block of pure blood.
A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell of embodiment 2
Method is as described above, step (5) impregnates the umbilical cord tissue fritter 15min of previous step using 100% fetal calf serum.Into
The comparison of the primary disengaging times of row each batch hUC-MSCs and yield.Wherein, A groups are umbilical cord tissue block culture group for the first time；B-t groups
Umbilical cord tissue block to be recycled after cultivating for the first time cultivates obtained mescenchymal stem cell (primary cell) by the 2nd time；B-s groups are
The mescenchymal stem cell (primary cell) that the supernatant that tissue block is cultivated for the first time obtains after being individually incubated；C-t groups are umbilical cord tissue
Block (being recycled from B-t groups) cultivates obtained mescenchymal stem cell (primary cell) by the 3rd time；C-s groups are the 2nd training of tissue block
The mescenchymal stem cell (primary cell) that foster supernatant (being recycled from B-t groups) obtains after being individually incubated.Count the primary of each group
The earliest time of occurrence of cell, the 1st generation time and primary cell number.The results are shown in Table 2.
The disengaging time and yield of the different batches hUC-MSCs of 2 identical donor source of table△
△In advance through 100% fetal calf serum immersion treatment before tissue block adherent culture.Compared with culture group for the first time, * P<
0.05, * * P<0.01.
In table 2 as can be seen that compared with the umbilical cord tissue block cultivated for the first time, either the umbilical cord tissue block of culture is repeated also
It is culture supernatant, can obtains more primary cell in a relatively short period of time.Specifically, 3 trainings before and after tissue block
The cell supported together with 2 culture supernatant cultures be averaged total output, about tissue block (by FBS immersions) single culture average product
7.5 times and impregnate tissue block single culture average product 27.2 times of (table 1 " serum-free immersion group ") without serum.
3 batch cells have been cultivated repeatedly altogether in the present invention.In theory, 4 batches, 5 batches are also possible to.Culture
3 mainly considerations are in practical application process, with the extension increased and hold time in vitro for repeating incubation times, hair
The probability of raw microbial contamination can increase.
HUC-MSCs primary to the identical donor of three batches of embodiment 2 carries out correlation analysis.As a result it is as follows.
1st, the morphologic detection of the identical donor hUC-MSCs of three batches
The result is shown in Figure 1.In Fig. 1, A groups (tissue block is cultivated for the first time) are the umbilical cord tissue block of first adhere-wall culture to the 7th day
And its fusiformis primary cell that around dissociates；B-t groups (tissue block the 2nd time culture), umbilical cord during the 2nd adhere-wall culture 7 days
Tissue block and surrounding a large amount of primary cells；B-s groups (A group culture supernatants are incubated again), the 1st batch of primary cell of recycling pass for the first time
For when culture supernatant, a large amount of attached cells for obtaining when being individually incubated 7 days；C-t groups (the 3rd culture of tissue block), the 3rd patch
Umbilical cord tissue block and surrounding a large amount of primary cells during wall culture 7 days；C-s groups (B-t group culture supernatants are incubated again), recycling
Culture supernatant when the 2nd batch of culture group (B-t groups) of tissue block is passed on for the first time obtains a large amount of attached cells when being individually incubated 7 days.It is brown
Color lightproof part is umbilical cord tissue block.
It is visible on a small quantity in fusiformis, the patch of rod-short under inverted microscope during inoculated and cultured 1 week during first adhere-wall culture
Parietal cell climbs out of (Fig. 1 .A) from the edge of yellowish-brown umbilical cord tissue fritter.When the 2nd and the 3rd batch repeats to cultivate, tissue block group
It can be obtained with supernatant group when culture was by 1 week and grow to 80% attached cell converged (Fig. 1 .B-t, B-s, C-t, C-s).This
It is special that typical MSCs forms are all presented either in original cuiture system or after passage in the cell in a little umbilical cord sources
Sign：Single cell is into threadiness, and refractivity is strong, and cytoplasm enriches, polarity distribution；It is in arranged in parallel, vortex during cell confluency
Shape is grown.
2nd, the growth curve of the identical donor hUC-MSCs of three batches
Cell growth curve is drawn using cell counting, the results are shown in Figure 2.
As can be seen that the growth curve form of each batch hUC-MSCs is similar in Fig. 2：1-2d cells are in incubation period,
It is proliferated unobvious；3-5d cells enter exponential phase, and cell Proliferation accelerates；Enter plateau after 6d, cell growth is stopped
It is stagnant.
3rd, the cell cycle analysis of the identical donor hUC-MSCs of three batches
By the cell cycle of Flow Cytometry Assay each batch hUC-MSCs, experiment is repeated 4 times, and as a result sees Fig. 3.
Fig. 3 middle and upper parts are divided into representativeness cell cyclic graph (be P4 for cell)；Lower part is divided into statistical analysis figure.Such as Fig. 3
Shown, there was no significant difference for the G0/G1 phases of each group cell or (S+G2/M) phase cell proportion.Illustrate to repeat to cultivate by multiple batches of
The cell of acquisition has normal proliferative capacity.
4th, the immunophenotype of the identical donor hUC-MSCs of three batches
The flow cytometer detection of cell surface marker is carried out for each batch cell to P4.As shown in figure 4, each batch of people's umbilical cord source
Cell equably expresses stroma cell relevant surface markers CD73, CD90, CD105, does not express the surface mark of candidate stem cell
Remember CD34 and human leucocyte common antigen CD45, meet the phenotypic characteristic of mescenchymal stem cell.
5th, the identical donor hUC-MSCs of three batches into fat and Osteoblast Differentiation ability
5.1 detect into fat differentiation capability
Each batch people umbilical cord cells after taking P4 instead of carry out adipogenic induction differentiation, after adding in into fat induction liquid, carefully
Intracellular growth almost stops, and has small fat drips to occur in cytoplasm after 72 hours and gradually increases, mutually merges, cellular morphology is become by fusiform
For round or ellipse, part nucleus deviation.After continuous induction 2~3 weeks, oil red O stain, which is shown into the cell, a large amount of lipids
Precipitation, is as a result shown in Fig. 5.
O is is unstained in Fig. 5；A is the 1st batch of hUC-MSCs oil red O stain；B-t is the 2nd crowd of tissue block source hUC-MSCs
Oil red O stain；B-s is the 1st batch of supernatant source hUC-MSCs oil red O stain；C-t is the 3rd batch of tissue block source hUC-MSCs oil
Red O dyeing；C-s is the 2nd batch of supernatant source hUC-MSCs oil red O stain.Fig. 5 results confirm that multiple batches of be separately cultured does not influence institute
The differentiation into adipocytes in vitro of the hUC-MSCs of acquisition.
5.2 Osteoblast Differentiation abilities detect
Each batch people umbilical cord cells after taking P4 instead of carry out Osteoinductive differentiation.In the process of skeletonization Induction of committed differentiation
In, the volume of cell gradually increases, from threadiness to the Morphological Transitions such as short fusiform, flakey, polygonal, cytoplasm no particulate matter
Gradually increase, cell forms short protrusion and is connected with each other, and is grown in colony sample, has typical osteoblast form.Skeletonization is arrived
The later stage is induced, the cell at cellular nodules center gradually merges and loses eucaryotic cell structure, the visible calcium deposition of iuntercellular, through alizarin red
It takes on a red color after dyeing, as a result sees Fig. 6.
In Fig. 6：O is is unstained；A is the 1st batch of hUC-MSCs Alizarin red staining；B-t is the 2nd crowd of tissue block source hUC-
MSCs Alizarin red stainings；B-s is the 1st batch of supernatant source hUC-MSCs Alizarin red staining；C-t is the 3rd crowd of tissue block source hUC-
MSCs Alizarin red stainings；C-s is the 2nd batch of supernatant source hUC-MSCs Alizarin red staining.Fig. 6 results prove multiple batches of be separately cultured
The external Osteoblast Differentiation ability of hUC-MSCs is not influenced.
A kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell of embodiment 3
Method is as described above, step (5) impregnates the umbilical cord tissue fritter 15min of previous step using 100% people AB serum.Into
The comparison of the primary disengaging times of row each batch hUC-MSCs and yield.Wherein, A groups are umbilical cord tissue block culture group for the first time；B-t groups
Umbilical cord tissue block to be recycled after cultivating for the first time cultivates obtained mescenchymal stem cell (primary cell) by the 2nd time；B-s groups are
The mescenchymal stem cell (primary cell) that the supernatant that tissue block is cultivated for the first time obtains after being individually incubated；C-t groups are umbilical cord tissue
Block (being recycled from B-t groups) cultivates obtained mescenchymal stem cell (primary cell) by the 3rd time；C-s groups are the 2nd training of tissue block
The mescenchymal stem cell (primary cell) that foster supernatant (being recycled from B-t groups) obtains after being individually incubated.Count the primary of each group
The earliest time of occurrence of cell, the 1st generation time and primary cell number.The results are shown in Table 3.
The disengaging time and yield of the different batches hUC-MSCs of 3 identical donor source of table△
| The earliest time of occurrence (d) of primary cell | 1st generation time (d) | Primary cell number (× 104A/cm) | |
| A groups (tissue block is cultivated for the first time) | 7.75±1.71 | 11.8±1.71 | 6.76±0.29 |
| B-t groups (the 2nd culture of tissue block) | 2.25±0.50* | 6.50±0.58* | 10.6±0.77* |
| B-s groups (A group culture supernatants are incubated again) | 1.75±0.50* | 6.75±0.96* | 10.2±0.81* |
| C-t groups (the 3rd culture of tissue block) | 2.50±0.58* | 7.00±0.82* | 10.8±0.82** |
| C-s groups (B-t group culture supernatants are incubated again) | 2.25±0.96* | 6.50±0.58* | 9.86±0.83* |
△In advance through 100% people's AB serum immersion treatments before tissue block adherent culture.Compared with culture group for the first time, * P<
0.05, * * P<0.01.
From table 3 it is observed that (table 2) similar with the result of tissue block impregnated with fetal calf serum, the leaching of employment AB serum
The culture that the umbilical cord tissue block and its culture supernatant steeped can be equally used at least two batches recycles, and primary cell is earliest
Time of occurrence, the 1st generation time, primary cell harvest quantity are equally better than single culture group.Specifically, 3 before and after tissue block
Secondary culture be averaged total output together with the cell of 2 supernatant cultures, and about tissue block (through the immersion of people AB serum) single culture is averaged
7.1 times and 29 times without serum immersion tissue block single culture average product (table 1 " serum-free immersion group ") of yield.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of multiple batches of primary separation method of identical donor source human mesenchymal stem cell, which is characterized in that including following
Step：
Aseptic collection donor tissue is simultaneously handled；
By treated, donor tissue shreds, and obtains 1-5mm3Donor tissue fritter；
Donor tissue fritter 5-20min is impregnated with fetal calf serum or people AB serum；
Donor tissue fritter is inoculated into tissue culture plate, and adds appropriate complete medium, is cultivated；
When the adherent cell growth that of dissociating is merged to 80%-90%, primary mescenchymal stem cell is harvested；
While harvesting primary mescenchymal stem cell, the donor tissue fritter in original cuiture system and culture supernatant are returned
Receive reprocessing；Wherein donor tissue fritter repeats above-mentioned fetal calf serum or people AB serum and impregnates 5-20min and subsequent
Processing continues to harvest mescenchymal stem cell；Original cuiture supernatant is transferred in new Tissue Culture Flask is incubated culture again, continues
Harvest mescenchymal stem cell.
2. multiple batches of primary separation method according to claim 1, it is characterised in that：The donor includes umbilical cord
At least one of band, placenta, amnion.
3. multiple batches of primary separation method according to claim 2, it is characterised in that：When donor is neonatal umbilical cord,
Processing method is as follows：Aseptic collection neonatal umbilical cord is soaked in buffer solution, in an aseptic environment, umbilical cord is cut into 1-5cm long
Segment, fully washed with buffer solution, removal residual Cord blood；Umbilical cord segment is longitudinally splitted, rejects blood vessel.
4. multiple batches of primary separation method according to claim 3, it is characterised in that：Buffer solution is contains appropriate antibiotic
HBSS buffer solutions or PBS buffer solution.
5. multiple batches of primary separation method according to claim 1, it is characterised in that：By treated, donor shreds, and obtains
To 2-5mm3Donor tissue fritter.
6. multiple batches of primary separation method according to claim 1, it is characterised in that：The percent by volume of fetal calf serum is dense
It spends for 10%-100%.
7. multiple batches of primary separation method according to claim 1, it is characterised in that：The percent by volume of people's AB serum is dense
It spends for 10%-100%.
8. multiple batches of primary separation method according to claim 1, it is characterised in that：Donor tissue fritter is inoculated into carefully
In born of the same parents' culture plate, righted after the tissue culture plate back-off culture 4-6h for completing tissue fritter, then by tissue culture plate；To cell
Appropriate complete medium is added in culture plate, continues to cultivate.
9. multiple batches of primary separation method according to claim 7, it is characterised in that：Donor tissue fritter is inoculated into carefully
In born of the same parents' culture plate, aseptically air-dry to organizing small block edge micro- dry, then the tissue culture plate that tissue fritter will be completed
Back-off culture 4-6h.
10. multiple batches of primary separation method according to claim 1, it is characterised in that：Donor tissue fritter is inoculated into
In tissue culture plate, 20-40 minutes are stood, then add appropriate complete medium, cultivated.
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