CN106434546A - Method for preparing mesenchymal stem cell by fully utilizing umbilical cord resources - Google Patents
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Abstract
A method for preparing mesenchymal stem cells by fully utilizing umbilical cord resources includes the steps of washing off most of residual blood on an umbilical cord; preparing the umbilical cord into tissue fragments, and digesting the tissue fragments by collagenase II; adding a tissue block obtained from collagenase digestion into an eluent to centrifuge so as to separate unpurified primary umbilical cord mesenchymal stem cells; grafting the obtained primary stem cells to a plastic culturing container by 5.0x104/cm<2>-1.0x105/cm<2>; digesting after the cells attaching to the bottom of the plastic culturing container is fused by more than 80%; purifying after digestion and dispersion according to needs. The method has the advantages that the limited umbilical cord resources can be fully made use of, unnecessary waste can be reduced, prepared tissue volume can be increased, the umbilical cord mesenchymal stem cells with effective bioactivity can be extracted by making full use of all mesenchymal stem sources, and high-yielding mesenchymal stem cells contained in the umbilical cord can be obtained.
Description
Technical field
The present invention relates to a kind of method of stem cell preparation, more particularly, to one kind prepares mesenchyme using umbilical cord resource completely
The method of stem cell, to improve the utilization ratio to umbilical cord resource.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cell, MSCs) derives from the mesoderm of mesoderm growing early stage and outer embryo
Layer, is present in whole body connective tissue and organ-tissue, is the adult that a class has height self renewal and multi-lineage potential
Stem cell.MSCs initially finds in bone marrow, because it has multi-lineage potential, hematopoiesis support and promotes stem cell implantation, exempt from
Epidemic disease regulation and control and receiving publicity the features such as self replication.Due to, under MSCs in vivo or in vitro specific inductive condition, can break up
For the Various Tissues cell such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle and endothelium, continuous passage culture and
Still there is after freezen protective multi-lineage potential, the old and feeble histoorgan causing with pathological changes can be used for as preferable seed cell
The reparation damaging.MSCs can secrete produce multiple promote Growth and Differentiation cytokines, including TGF-β, EGF, SDF-1, HGF,
The cytokines such as VEGF and IL-6, have and avoid adjusting function.Therefore, its have in the field such as biological engineering and cell therapy wide
Application prospect.
MSCs is initially to find in bone marrow, finds that umbilical cord, umbilical blood, Placenta Hominiss and some other tissue there is also later
MSCs.But in Adult Human Bone Marrow MSCs negligible amounts, be easily infected, have the factors such as stronger immunogenicity to limit it
Clinical practice.The MSCs in people's umbilical cord source from cell content, ability of cell proliferation, immunogenicity is low, draw materials convenient etc. in terms of all
Better than bone marrow MSCs.The MSCs in umbilical cord source is the ideal substitute of bone marrow MSCs.Extract from umbilical cord mesenchyme dry so that umbilicuss
Band can be turned waste into wealth.
Although umbilical cord MSCs is intended to the approval by Hospital Ethical Committee using no ethics dispute, umbilical cord acquisition,
Could gather after multipara and its family members' informed consent.
The mescenchymal stem cell that the impact of the individual variations such as the umbilical cord length of collection, thickness, color and luster, acquisition time obtains
Quantity is different, and ability of cell proliferation is also different.
From umbilical cord, 2 tremulous pulsies and 1 vein, the arteriovenous mucoprotein sample connective tissue of wrapping umbilical cord are magnificent Tong Shi glue
Mesenchyme can separately be obtained in (Wharton ' s Jelly), umbilical vein endothelial, subendothelial layer and tissues surrounding vascular do
Cell.
When mescenchymal stem cell separates in vitro and expands, add certain density hyclone to provide the cell growth must
The nutrition of palpus；Associated proteins, Detoxication are provided；It is cell attachment, spread over required Factor Source on culture matrix；Rise
Buffer acts on；Protease inhibitor is provided, makes to make remaining trypsin inactivation in passage, protect cell not injured
Evil.Human serum, platelet-derived thing, platelet lysates liquid, blood cell lysate and umbilical sera etc. is used to add mesenchyme to do at present thin
To provide more preferable cell proliferation environment more and more interested to researchers in the cultivating system of born of the same parents.
Cell-seeding-density (Incxulum Density) affects cell growth and existence to a great extent.Plant concentration relatively
When low, cell growth breeding is relatively slow, and final cell yield is also low, especially attached cell, faces to a certain when inoculum density is low
During dividing value, cell will not even extend on medium side, also results in the waste of culture medium.In umbilical cord mesenchymal stem cells cyton
In outer incubation, find the most suitable cell implantation concentrations, be the key obtaining good stability and the high stem cell products of yield
Factor.
The mode of umbilical cord mesenchymal stem cells acquisition at present is common enzymic digestion intravenouss embrane method, Wharton ' s Jelly
Tissue adherent method, collagenase and pancreatin simultaneous digestion method.
Enzymic digestion intravenouss embrane method：Folder closes umbilical vein one end, pours into collagen glue enzyme, is placed in containing in dual anti-PBS, 37 DEG C of rings
Border is incubated 0.5 hour, collects enzyme liquid, with PBS lavation repeatedly, collects irrigating solution, centrifuge washing is abandoned after supernatant simultaneously with enzyme liquid,
Resuspended with complete medium, inoculated and cultured, after 3 days, full dose changes liquid, changes weekly liquid twice later, observes adherent growth situation.This
Method complex operation, the initial mescenchymal stem cell order of magnitude that obtains is low, and epithelial cell accounting profit is high.
Wharton ' s Jelly organizes adherent method：Umbilical cord is placed in containing in dual anti-PBS, clean remaining blood presses blood vessel spiral shell
Rotation is walked formula and is rejected two tremulous pulsies and a vein, with toothed forcepss strip off peplos, Wharton ' s Jelly is torn, is soaked with DPBS
After foam washing is washed, umbilical cord tissue is shredded to 1mm3～3mm3Size, after DPBS washing, carries out adhere-wall culture.Typically after about 5-7 days
It can be seen that the single strip spindle cell of adherent growth climbs out of from tissue, take within two weeks paste block can obtain MSCs.The method separates
Mescenchymal stem cell complex operation, the primary cell time of acquisition is long.
Patent CN101608174 B discloses a kind of collagenase and pancreatin simultaneous digestion method：Umbilical cord tissue is cut into small pieces
It is placed in collagenase digested, after washing and filtering, resuspended with complete medium, inoculated and cultured, change liquid after 2 days and abandon non-adherent growth carefully
Born of the same parents, converge situation according to cell growth later, carry out passing on changing liquid using the cell of the elution adherent growth containing pancreatin.
This method cell harvesting amount is high, keeps the biological originality of cell, and cultivation cycle is short.
Content of the invention
Further object is that providing a kind of method preparing mescenchymal stem cell using umbilical cord resource completely,
To reduce umbilical cord length, thickness, color and luster, acquisition time, to prepare the impact of the individual variations such as ability of cell proliferation, obtain more
Primary cell quantity.
A kind of method preparing mescenchymal stem cell using umbilical cord resource completely, comprises the steps：
Step one, the remained blood of most of umbilical cord is gone in cleaning, and retains the internal 10v/v%～2v/v%'s of artery and vein
Remain non-solidificating blood；
Step 2, umbilical cord is made 2mm3～5mm3Size tissue pieces are (such as：2.5mm3), blend compounds protoenzyme II digests
(such as：Add the 0.2w/v% collagenase II of 2～3 times of tissue pieces volumes)；
Step 3, adds DMEM culture medium to carry out 300r/min10 minute centrifugation, extracts remaining tissue pieces and upper completely
The colloidal liquid that layer liquid has a common boundary, collects upper liquid；
Step 4, adds eluent in upper liquid again, and resuspended rear 1800r/min is centrifuged 15 minutes；
Step 5, collects precipitation after abandoning upper liquid, adds eluent resuspended, 1000r/min is centrifuged 10 minutes, repeated washing
Twice, that is, obtain the mescenchymal stem cell containing inside umbilical cord；
Step 6, by 5.0 × 104/cm2～1.0 × 105/cm2The primary umbilical cord mesenchymal stem cells obtaining are inoculated in and contain
Have in the LG-DMEM culture medium of 10v/v% hyclone (FBS), 37 DEG C of the rearmounted temperature of mixing, CO2Concentration 5v/v% titanium dioxide
Carbon incubator is cultivated, and changes complete medium for the first time, discards non-adherent cell, change and cultivate completely after every 2 days later after 3 days
Base, after attached cell fusion rate reaches more than 80%, digestion dispersion attached cell, is purified on demand.
The method of the present invention, goes the remained blood of most of umbilical cord using buffer solution for cleaning, and buffer is phosphate-buffered
Liquid (pH7.20, ionic strength I=0.05) contains penicillin 100,000 units/L, streptomycin 100mg/L, 3.00mmol/L reduced form
Glutathione and 2v/v% mercaptoethanol.
The method of the present invention, it digests scattered attached cell, adds serum-free cryoprotective agent to carry out subpackage.
The method of the present invention, the utilization constant-temperature table of its step 2 improves digestive efficiency and stable environment, after obtaining digestion
Organize remaining tissue pieces and suspension mixture.
The beneficial effect that technical solution of the present invention is realized：
The method that the present invention provides make use of whole collection umbilical cords to prepare umbilical cord mesenchymal stem cells, makes full use of limited
Umbilical cord tissue resource, reduces and causes unnecessary waste, increased the tissue volume for preparation, and it is complete to make full use of collection
Portion's umbilical cord resource, at utmost Shangdi extraction has the umbilical cord mesenchymal stem cells of effective biological activity, is conducive to the high place of production to obtain
Obtain and contain mescenchymal stem cell inside umbilical cord.
Using 0.2w/v% collagenase II digestion, and the presence of constant-temperature table makes Wharton ' s Jelly and other group
Knit and more expose, increase the contact area of tissue and enzyme, primary mescenchymal stem cell can be improved within the identical time
Yield.
Add reduced glutathion and mercaptoethanol in buffer, accurately adjust its pH value, Fresh is to keep thin
Extracellular environment, it is to avoid or the infringement of minimizing cell, increase cytoactive.
Fall umbilical cord major part remained blood with wash buffer, retain the non-solidification blood of residual of 10v/v%～20v/v%
Liquid, in culture systems, remained blood can provide the micro blood factor that the external world cannot obtain, and promotes cell growth, shortens umbilical cord
Incubation time under the conditions of non-human for the mescenchymal stem cell.
By 5.0 × 104/cm2～1.0 × 105/cm2Inoculating cell, promotes that the cell attachment time is short, growth rate fast, raw
Long dimensionally stable, cell survival rate are high, it is to avoid the impact of the cell amplification that umbilical cord individual variation causes.
Brief description
The P0 of Figure 1A embodiment 1 carries out microscopy figure for umbilical cord mesenchymal stem cells growth；
The P1 of Figure 1B embodiment 1 carries out microscopy figure for the growth of example umbilical cord mesenchymal stem cells；
Fig. 2 is P1, P2, P3, P4 and P5 growth curve figure for umbilical cord mesenchymal stem cells of embodiment 4, in figure transverse axis
For cell algebraically, the longitudinal axis is cell quantity；Sample 1, sample 2 and sample 3 are three parallel cell samples with passing on；
Fig. 3 be embodiment 5 part using umbilical cord with completely using umbilical cord prepare umbilical cord mesenchymal stem cells quantitative comparison figure；
Fig. 4 A cultivates for embodiment 1 and makes cell suspension to P5 for cell and carry out flow cytomery, and negative right
According to scatterplot；
Fig. 4 B cultivates for embodiment 1 and makes cell suspension to P5 for cell and carry out flow cytomery, and strongly expressed
Scatterplot；
Fig. 5 A cultivates for embodiment 1 and makes cell suspension to P5 for cell, carry out CD34 obtained by flow cytomery,
CD45, CD73, CD90 and CD105 negative control figure；
Fig. 5 B cultivates for embodiment 1 and makes cell suspension to P5 for cell, carry out CD34 obtained by flow cytomery,
CD45, CD73, CD90 and CD105 strongly expressed curve chart.
Specific embodiment
Describe technical scheme below in conjunction with accompanying drawing in detail.The embodiment of the present invention is only in order to illustrate the skill of the present invention
Art scheme and unrestricted, although being described in detail to the present invention with reference to preferred embodiment, those of ordinary skill in the art
It should be appreciated that the technical scheme of invention can be modified or equivalent, without deviating from the essence of technical solution of the present invention
God and scope, it all should be covered in scope of the presently claimed invention.
Used by following examples of the present invention, reality and instrument see below table 1, if the reagent unexplained reference used by other, all
Purchased from healthy and free from worry (Corning) company.
Table 1
The preparation method of embodiment of the present invention umbilical cord mesenchymal stem cells is as follows：
Embodiment 1
(1) prepare umbilical cord cleaning buffer solution：1.00v/v% penicillin/streptomycin, 3.00mmol/L reduced form gluathione
Peptide, the phosphate buffer (pH7.20, I=0.05) of 2w/w% mercaptoethanol, penicillin/streptomycin mother liquid concentration is penicillin
(10,000units/ml)/streptomycin (10,000 μ g/ml)；
(2) gather the umbilical cord of newborn baby according to national regulation, rinsed with cleaning buffer solution and remove major part inside and outside umbilical cord
Remained blood (retains the non-solidificating blood of residual of the internal 10v/v～20v/v% of arteriovenous), all shreds the whole umbilical cord groups of acquisition
Knit fragment；
(3) the 0.2w/v% collagenase II of 2～3 times of tissue volume is added to disappear in the obtained human umbilical tissue of step (2)
Change is processed, and digests 2 hours under the conditions of 37 DEG C of ambient temperature, improves digestive efficiency and stable environment using constant-temperature table, and acquisition disappears
Remaining tissue pieces and suspension mixture is organized after change；
(4) add DMEM culture to mix based in the postdigestive mixture of step (3), 300r/min centrifugation 10 minutes；
(5) abandon step (4) centrifuged deposit, collect upper liquid, remaining tissue pieces need to be extracted completely and upper liquid is had a common boundary
Colloidal liquid；
(6) add eluent in the upper liquid that step (5) obtains, resuspended rear 1800r/min is centrifuged 15 minutes；
(7) step (6) collects precipitation after abandoning upper liquid, adds piece of tissue eluent to carry out resuspended, 1000r/min centrifugation 10
Minute, repeat the above steps are cleaned twice, that is, obtain the mescenchymal stem cell containing inside umbilical cord；
(8) human umbilical cord mesenchymal stem cells obtaining step (7) are by 5.0 × 104/cm2～1.0 × 105/cm2Concentration connects
Plant in the plastic culture vessel of LG-DMEM complete medium filling 10v/v%FBS, 37 DEG C of the rearmounted temperature of mixing, CO2Dense
Degree 5v/v% CO2 gas incubator culture.Change complete medium for the first time after 3 days, discard non-adherent cell, after every 2 days later
Change complete medium, carry out on demand purifying after attached cell fusion rate reaches more than 80%, digest dispersion attached cell,
Serum-free cryoprotective agent is added to carry out subpackage.
Table 2
Embodiment 2
Step (2) in embodiment 1 is rinsed with cleaning buffer solution remove most of remained blood inside and outside umbilical cord compare into clear
The whole blood of the inside and outside residual of umbilical cord is gone in eccysis, takes a case result as shown in table 3, with respect to the result of table 2, has longer
Growth cycle and less upper Dongcheng quantity.
Table 3
Embodiment 3
By step (2) in embodiment 1 shred acquisition whole umbilical cord tissue fragments compare into shred acquisition part umbilical cord tissue
Fragment, takes a case result as shown in table 4, reduces with respect to the result preparation quantity of table 2, frozen negligible amounts.
Table 4
Embodiment 4
Step in embodiment 1 is tested in three umbilical cord samples, is expanded to P5 generation.
Embodiment 5
Step in embodiment 1 is tested in three umbilical cord samples, obtains preparing primary mescenchymal stem cell quantity to according to the facts
Example one prepares quantity.
The growth of Example 1 umbilical cord mesenchymal stem cells carries out microscopy.Just detached umbilical cord mesenchymal stem cells cell is in
Spherical suspending is in culture fluid, and is mixed with certain erythrocyte.12 hours～24 hours inner cells of inoculation are adherent and stretch, 36h
After～48h cell attachment stretch expand and be mixed with non-become fiber shape feature heteroproteose cell, cell wears after propagation with spindle cell
Based on it is seen that polygon, star, refractivity good (see Figure 1A).The proliferative cell of primary umbilical cord mesenchymal stem cells skewness
Race, the inoculum density preferably going out just can reach more than 80% fusion in 5d～7d.Secondary Culture to P1 generation, passage cell still with
Based on fusiformis, heteroproteose cell gradually decreases, and cell ratio is more uniform, and merging completely is in parallel vortex shape, more primary bigger (see Figure 1B).
The P1 of Example 1 utilizes after the dyeing of trypan blue exclusion principle for cell, Life TechnologiesAutomated cell calculating instrument carries out analysis of accounts.The inventive method obtain each for mescenchymal stem cell cell according to refusing
Dye assay activity all can reach 97.00%～100.00%.
Cell P1, P2, P3, P4 and P5 of Example 4 is verified (ginseng for the proliferative conditions of umbilical cord mesenchymal stem cells
See Fig. 2), as seen from Figure 3, with the increase passing on algebraically, cell quantity is in that multiple increases.In theory through passing on for 4 times as thin
16 times of the former quantity of born of the same parents, stretches extension because cell can increase with algebraically, cell quantity during actual P5 is 15 times of left sides of P1
Right.
Example 5 cell using umbilical cord and prepares umbilical cord mesenchymal stem cells quantitative comparison using umbilical cord through part completely
It can be seen that, due to making full use of umbilical cord, increase preparation source using umbilical cord completely, obtain primary cell compared with part using umbilical cord many
(referring to Fig. 3)
Cultivate to P5 for cell by embodiment 1, washed with PBS after being dispersed into individual cells 2 times～3 times, make 1.0 ×
106Cell suspension, 5 surface markers such as flow cytometer detection CD34, CD45, CD73, CD90 and CD105 are (referring to Fig. 5 A and figure
5B).It is analyzed with flow cytometer showed software Flow Jo, mescenchymal stem cell gradually purification in succeeding generations, heteroproteose cell amount is more next
Fewer.When P5, P6 harvest, strongly expressed labelling cell reaches more than 95%, and express cell is not less than 2% (referring to Fig. 4 A and Fig. 4 B),
Meet national issuing standard requirement it was demonstrated that the immunophenotype of cell is highly stable it is ensured that the umbilical cord mesenchyma prepared is done carefully
Born of the same parents' cell safety.
Claims (7)
1. a kind of prepare the method for mescenchymal stem cell it is characterised in that comprising the steps using umbilical cord resource completely：
Step one, the remained blood of most of umbilical cord is gone in cleaning, and retains the residual of the internal 10v/v%～20v/v% of artery and vein
Non- solidificating blood；
Step 2, umbilical cord is made tissue pieces, and blend compounds protoenzyme II digests；
Step 3, adds DMEM culture medium to carry out 300r/min10 minute centrifugation, extracts remaining tissue pieces and upper liquid completely
The colloidal liquid having a common boundary, collects upper liquid；
Step 4, adds eluent in upper liquid again, and resuspended rear 1800r/min is centrifuged 15 minutes；
Step 5, collects precipitation after abandoning upper liquid, adds eluent resuspended, 1000r/min is centrifuged 10 minutes, repeated washing two
Secondary, that is, obtain the mescenchymal stem cell containing inside umbilical cord；
Step 6, the primary umbilical cord mesenchymal stem cells of described acquisition are inoculated in the LG-DMEM containing 10v/v% hyclone
Cultivate in culture medium, change complete medium for the first time after 3 days, discard non-adherent cell, change after every 2 days later and cultivate completely
Base, carries out on demand purifying, digests dispersion attached cell.
2. according to claim 1 prepare the method for mescenchymal stem cell it is characterised in that institute using umbilical cord resource completely
In the step one stated, go the remained blood of most of umbilical cord using buffer solution for cleaning, described buffer is phosphate buffer
Containing penicillin 100,000 units/L, streptomycin 100mg/L, 3.00mmol/L reduced glutathion and 2v/v% mercaptoethanol.
3. according to claim 2 prepare the method for mescenchymal stem cell it is characterised in that institute using umbilical cord resource completely
The pH of buffer 7.20 stated, ionic strength 0.05.
4. according to claim 1 prepare the method for mescenchymal stem cell it is characterised in that institute using umbilical cord resource completely
The collagenase II stating digests the 0.2w/v% collagenase II of 2～3 times of tissue pieces volumes of addition.
5. according to claim 1 prepare the method for mescenchymal stem cell it is characterised in that institute using umbilical cord resource completely
37 DEG C of the cultivation temperature of the step 6 stated, CO2Concentration 5v/v%.
6. according to claim 1 prepare the method for mescenchymal stem cell it is characterised in that institute using umbilical cord resource completely
The primary umbilical cord mesenchymal stem cells of the acquisition stated are with concentration 5.0 × 104/cm2～1.0 × 105/cm2Inoculation.
7. according to claim 1 completely using umbilical cord resource prepare mescenchymal stem cell method it is characterised in that step
Digestion described in rapid two uses constant-temperature table to obtain and organizes remaining tissue pieces and suspension mixture.
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| CN111492051A (en) * | 2017-12-22 | 2020-08-04 | 奇斯药制品公司 | Mesenchymal stromal cells and method for obtaining mesenchymal stromal cells from umbilical cord |
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| CN111492051B (en) * | 2017-12-22 | 2024-02-23 | 奇斯药制品公司 | Mesenchymal stromal cells and method for obtaining mesenchymal stromal cells from umbilical cord |
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