CN110317780A - A kind of placenta decidua basalis mescenchymal stem cell preparation method - Google Patents
A kind of placenta decidua basalis mescenchymal stem cell preparation method Download PDFInfo
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- CN110317780A CN110317780A CN201910643009.7A CN201910643009A CN110317780A CN 110317780 A CN110317780 A CN 110317780A CN 201910643009 A CN201910643009 A CN 201910643009A CN 110317780 A CN110317780 A CN 110317780A
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Abstract
The invention discloses a kind of placenta decidua basalis mescenchymal stem cell preparation methods, are related to stem cell preparation field.The present invention provides placenta source decidua basalis mescenchymal stem cell preparation methods to transport including acquisition, separate, change liquid, pass on, freeze, detect, store overall process.The present invention is using placenta decidua basalis as material extraction placenta decidua basalis mescenchymal stem cell, it is prepared using a variety of enzyme-linked conjunction multistep digestion method methods, it is not necessary that animal blood serum is added in extraction process, can be obtained in the shorter time compared with many cells, and cell has good homogeneity, stability.Entire cultivation cycle is short, the mescenchymal stem cell in placenta decidua basalis source needed for acquisition that can be efficient, safe.
Description
Technical field
The present invention relates to the preparation technical fields of stem cell, and in particular to a kind of placenta decidua basalis mescenchymal stem cell packet
Include: acquisition is transported, is separated, changing liquid culture, passage, harvest freeze, detect, store overall process.
Background technique
Stem cell is human body and its various histiocytic initial sources, it can both maintain itself group by cell division
The stabilization of body, and can break up as different type cell, and then constitute the histoorgan of the various complexity of body.For stem cell
Research can be traced in the 1950s, earliest derived to teratoma research.In the 1970s, people have found that one kind
From mesoderm, at the cell of fiber-like adherent growth, referred to as mescenchymal stem cell.
Mescenchymal stem cell is the important member of stem cell line, because having very strong self-renewing and dividing to Various Tissues
The concern changed the potential developed and be increasingly subject to people.At present from groups such as marrow, fat, amnion, placenta, bleeding of the umbilicus and umbilical cords
Mescenchymal stem cell, oriented endothelial cell, osteoblast, cartilage cell, fat cell, sarcoblast and mind are isolated in knitting
Ability through cell differentiation.
Placenta not only plays a role in terms of the development of embryo, nutrition, tolerance, but also has a kind of potential stem cell
Ability.In parent, the mescenchymal stem cell in extraembryonic mesoderm source is constantly divided into the blood vessel of connective tissue, and embryo is complete whereby
At the mass exchange with parent.Decidua basalis constitutes the parent fraction of placenta, accounts for full-term pregnancy placenta small part, bottom is sloughed off
Film surface covers the bottom that intervillous space is collectively formed in one layer of trophocyte and decidua basalis from anchoring villus, referred to as decidua base
Plate stretches out some decidua intervals from this plate to chorion direction, the 2/3 of placenta full thickness is usually no more than, by placenta parent
Face is divided into macroscopic 20 or so parent leaves.Placenta just completes mission after delivery of baby becomes " waste ", but because
It has many advantages, such as ability and itself materials convenience, immunosupress of Multidirectional Differentiation, will become cell therapy, group weaver
Journey and the new research hotspot of genetic engineering field.
United States blood academic year in 2000 can on, Jaroscak etc. report about placenta source attached cell tentatively grinding
Study carefully, and proves that there are mescenchymal stem cell ingredients in placenta；Other researchs also show interstital stem cell rich in placenta,
And find that the mescenchymal stem cell in placenta source can also express certain embryonic stem cell surface markers, as ssEA-4, TRA-1-61,
TRA-1-80, it may be very original cell mass that this result, which prompts the mescenchymal stem cell in placenta source, with other adults
Stem cell is compared, and has wider self-renewing and polyphyly differentiation capability, and materials are convenient, from a wealth of sources, as long as health produces
After woman agrees to, it can be easier to obtain placenta, and its immunogenicity is low, is not related to the advantages such as ethics problem.
Decidua basalis as female and tire interface one of component part, to fetus growth and development from repel play it is most important
Effect.Although decidua basalis derives from parent, there is stronger proliferative capacity, expression compared to fetal origin mescenchymal stem cell
General MSCs fundamental characteristics and mesoderm multi-lineage potential, longterm culture in vitro keep stable karyotype and uniqueness
Immunosuppressive properties be cellular transplantation therapy and organizational engineering good source.And decidua basalis mescenchymal stem cell is as one
The important sources of a self mescenchymal stem cell of mother, energy safety are used to treat the diseases such as the immune system of mother.
Summary of the invention
It is directed to the above problem present in existing common technology, the present invention provides placenta source decidua basalis mesenchymas
Stem cell preparation method includes that acquisition is transported, separated, changes liquid, passes on, freezing, detecting, storing overall process.The present invention is with placenta bottom
Decidua is material extraction placenta decidua basalis mescenchymal stem cell, is prepared using a variety of enzyme-linked conjunction multistep digestion method methods, is extracted
In the process it is not necessary that animal blood serum is added, it can obtain in the shorter time compared with many cells, and cell has good homogeneity, stablize
Property.Entire cultivation cycle is short, the mescenchymal stem cell in placenta decidua basalis source needed for acquisition that can be efficient, safe.
The method includes the steps:
(1) acquisition transport: choosing the placenta of mature childbirth in 38-40 weeks normally produced from hospital, through with puerpera and its
Family members sign informed consent form, and placenta is obtained under delivery room aseptic condition, through meconium on physiological saline cleaning placenta etc. after acquisition
After be put into reservoir bag in the configured protection liquid of α-MEM (containing 100U/ml penicillin and 100U/ml streptomysin), 4-25 DEG C
Laboratory is sent in 24 hours.And it acquires mother's peripheral blood and carries out AIDS, syphilis, hepatitis B, hepatitis, cytomegalovirus detection.
(2) separate: in Biohazard Safety Equipment, the placenta by placenta close to mother face side is put into sterile tray up
In, remove extravasated blood with aseptic nipper and with normal saline flushing mother face, with aseptic operation scissor clip close to parent side
The decidua basalis tissue of rough surface.
(3) it cleans, shred: being quickly transferred to physiology salt after the alcohol that clip decidua basalis tissue is placed in 75% is impregnated 1min
Water is cleaned.Capillary is removed with sterile pincers.Again with the life containing 100U/ml penicillin and 100U/ml streptomysin
Manage salt water impregnate decidua basalis tissue 5min, finally with physiological saline clean decidua basalis 7 times with the penicillin of removal, streptomysin it is residual
It stays.Cleaned decidua basalis tissue block is transferred in 50ml centrifuge tube and is cut with sterile scissors to 0.5-0.8cm3Molecule.
(4) it digests for the first time: 37 DEG C of water-baths (are put according to the mixed enzyme that the volume of decidua basalis prepares digestion for the first time before use
Pot incubates temperature to 37 DEG C), II Collagenase Type of 0.2wt% and 0.4wt% type Ⅳ collagenase are added into centrifuge tube, each enzyme in mixed enzyme
Volume ratio is 1:1, and the volume of mixed enzyme and the volume ratio of decidua basalis tissue block are 1:1.Mixed enzyme and tissue block are mixed, are put into
1h is digested in the shaking table that 37 DEG C of revolving speeds are 120rpm.
(5) it digests for second: the dispase of 0.2 wt % of total volume 1/4 is added after the completion of digestion for the first time, mixes, puts
Enter in the shaking table that 37 DEG C of revolving speeds are 120rpm and digests 15min.
(6) it terminates: isometric complete serum free medium being added after digestion and terminates digestion.1600rpm, 8min from
The heart discards supernatant.
(7) it removes red blood cell: cell is resuspended with physiological saline, 6wt% hydroxyethyl starch (physiological saline and 6% hydroxyl is added
After the volume ratio of hydroxyethyl starch is sufficiently mixed uniformly for 1:5), 150 rpm, 5min centrifugation.It is careful to draw supernatant, then 1200rpm,
6min centrifugation discards supernatant.With brine 2 times.
(8) it counts and surveys living, kind bottle and culture: cell being resuspended with Serum-free complete medium, is counted with trypan exclusion stain
It surveys and lives.The mescenchymal stem cell of acquisition is pressed 2 × 104/cm2Density be inoculated in culture bottle, calculated and connect according to inoculum density
The culture bottle number of kind.With Serum-free complete medium culture medium, corresponding T175 culture is inoculated in after gently blowing and beating resuspension cell
In bottle, progesterone 1.5 × 10 is added in culture medium-9mol/L -2×10-9Mol/L is placed into 37 DEG C, 5%CO2It is cultivated in incubator.
(9) it changes liquid: carrying out replacement Serum-free complete medium within the 3rd day, change liquid every 3 days progress full doses later.
(10) it passes on: when cell grows to degrees of fusion 70-80%, with 10ml physiological saline lotion cell 1 time, being added
0.05% pancreatin room temperature of 10mL digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collects vitellophag.
1200rpm, 6min centrifugation discard supernatant, and cell is resuspended with Serum-free complete medium and carries out passage kind bottle by 1:4.
(11) it harvests, freeze: when cell grows to degrees of fusion 80%, with 10ml physiological saline lotion cell 2 times, being added
0.05% pancreatin room temperature of 10mL digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collect vitellophag into
Row counts.The DMSO that preparation 20% in advance is taken out from refrigerator freezes protection liquid (DMSO: Serum-free complete medium: the white egg of people's blood
White volume ratio is 1:3:1).It is about 5 × 10 by every cryopreservation tube cell number according to cell count6A cell number, calculates cell
The pipe number and volume frozen.It first marks on cryopreservation tube, then is blown and beaten cell constant volume to respective volume with complete medium
It mixes, is slow added into equal volume, prepared 20%DMSO freezes protection liquid (to put refrigerator before use to balance to 4 DEG C), mixing
Cell is dispensed into cryopreservation tube afterwards, every pipe volume is 1mL.
(12) program cooling and preservation: cryopreservation tube is put into the program temperature reduction box of pre-cooling, -80 DEG C of refrigerator overnights are put into
Afterwards, liquid nitrogen is transferred to formally to store.
(13) it detects
<1>cell count and Activity determination: total out in blood counting chamber meter after being dyed using trypan exclusion stain to cell
Cell and dead cell number, according to formula Cell viability=1-(dead cell number/total number of cells) × 100% obtain Cell viability.
<2>flow cytometer detection: collecting cell, and PBS is washed 2 times, and cell suspension, label 1 ~ 6 pipe is made in adjustment cell concentration
Cell suspension is respectively added, makes the cell 1 × 10 of every pipe6It is a, pipe 1 plus the anti-human CD90-FITC of 5 μ L mouse, pipe 2 plus 5 μ L mouse
Antibody, pipe 5 plus 10 μ L hMSC are not added in anti-human CD105-PerCP-Cy 5.5, pipe 3 plus the anti-human CD73-APC of 5 μ L mouse, pipe 4
Positive Isotype control mixture and 10 μ LPE hMSC Negative isotype control mixtures, pipe 6 plus 10 μ L hMSC positive mixtures
It is protected from light with 10 μ LPE hMSC feminine gender mixture room temperatures and is incubated for 30 min, washed repeatedly with PBS two or three times, reduce non-specific knot
It closes；300 μ LPBS are added and mix cell, flow cytometer detects CD90+、CD73+、CD105+With hMSC feminine gender mixture
CD45+、CD34+、CD11b+、CD19+、HLA-DR+The expression of equal major surfaces marker.
<3>mtt assay measures cell growth curve
1, the preparation of MTT: the MTT concentration in this method is 5mg/ml.MTT 5mg is weighed, the phosphate buffer of 1ml is dissolved in
(PBS), 4 DEG C are put and is kept in dark place to be removed the bacterium in solution with 0.22 μm of membrane filtration.
2, the cell for taking P5 generation to prepare, digestion to single cell suspension, and count, with MSC Serum-free complete medium tune
Whole concentration is to 2 × 103/ hole is inoculated in 96 orifice plates, and liquid 200ul is added in every hole, prepares 7 96 orifice plates, overnight incubation altogether.
Next day replaces culture solution, is being incubated for 4h to which 20ul MTT is added in gaging hole every for 24 hours, is discarding MTT liquid after 4h, be added 150ul's
DMSO is protected from light repetition concussion 10min, in microplate reader at 490nm wavelength, detects light absorption value, measures 8 holes every time, continuous to survey
Amount 7 days.By the data creating of detection in 7 days at the MTT growth curve of the cell.With culture " Time (D) " for horizontal axis, " 490nm wave
Strong point absorbance (OD value) " is that the longitudinal axis draws cell growth curve.
<4>induction differentiation capability detection
Osteoinductive differentiation operation
1, plus the gelatin of 300uL0.1% is placed in 24 orifice plates, shakes up liquid, it is made to cover the bottom surface of entire orifice plate；
2, the gelatin for being covered with 0.1% is placed in super-clean bench 30min；
3, gelatin is discarded after 30min, dries 24 orifice plates, cell can be cultivated；
4, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
5, it when cell fusion degree reaches 80%-90%, is digested with pancreatin；
6, by the cell digested by 2 × 104cells/cm2(4 × 104/ hole) cell density be inoculated in 24 orifice plates, often
500uL mesenchyma complete medium is added in hole；
7, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
8, when cell fusion degree reaches 60%-70%, careful siphons away complete medium in hole, is added into 24 orifice plates
The Osteoinductive differentiation culture medium of 500uL；
9, fresh Osteoinductive differentiation culture medium (needing to be preheated to 37 DEG C before use) is used instead every 72h；
10, it after inducing 3 weeks, is dyed with alizarin red；
11, it after there are a large amount of calcium tubercles, changes liquid form and becomes every 48h once half amount changes liquid；
12, after Osteoinductive differentiation, the Osteoinductive differentiation culture medium in 24 orifice plates is siphoned away, is rinsed 2 times with PBS.Every hole is added
The 4% neutral formalin solution of 417uL, fixed 30min；
13, neutral formalin solution is siphoned away, is rinsed 2 times with PBS.208uL alizarin red dye liquor is added in every hole and contaminates 3-5min；
14, alizarin red dye liquor is siphoned away, is rinsed 3 times with PBS；
15, it is placed in microscopically observation skeletonization dyeing effect.
Adipogenic induction differentiation operation
1, induction A liquid and induction B liquid are prepared；
2, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
3, it when cell fusion degree reaches 80%-90%, is digested with pancreatin；
4, by the cell digested by 2 × 104cells/cm2(1.96 × 105/ hole) cell density be inoculated in 24 orifice plates,
1mL mesenchyma complete medium is added in every hole；
5, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
6, every 72h changes liquid, merges until cell fusion degree reaches 100% or crosses；
7, carefully mescenchymal stem cell complete medium is siphoned away, 1mL adipogenic induction culture medium A liquid is added into 24 orifice plates；
8, after inducing 48h, 72h, the A liquid in 24 orifice plates is siphoned away, 1mL adipogenic induction culture medium B liquid is added；
9, after for 24 hours, B liquid is siphoned away, A liquid is gained and continues to induce；
10, A liquid and B liquid alternating action 3-5 times continue to gain B liquid maintaining culture 4-7 days (replacing fresh B liquid every 2-3 days),
Fat drips become larger and are merged into beading；
11, cell culture medium is removed, is washed 2 times with PBS, the fixed 20-30min of ORO Fixative fixer is added；
12, fixer is discarded, with distillation washing 2 times；
13,60% isopropanol is added and embathes 5min；
14, new prepared ORO Stain is added after discarding 60% isopropanol, disseminates 10-20min；
15, dyeing liquor is discarded, is washed 3-5 times, until without extra dye liquor；
16, ORO Buffer 1min is added, discards；
17, distilled water covering cell is added and observes under the microscope.
Break up at chondrocyte induction and operates
1, plus the gelatin of 300uL0.1% is placed in 24 orifice plates, shakes up liquid, it is made to cover the bottom surface of entire orifice plate；
2, the gelatin for being covered with 0.1% is placed in super-clean bench 30min；
3, gelatin is discarded after 30min, dries 24 orifice plates, cell can be cultivated；
4, it is configured to chondrocyte induction culture medium premixed liquid；The TGF-β 3 of 10uL is added at the every 1ml premixed liquid of cartilage differentiation culture medium,
Mix spare (12h domestic demand has used)；
5, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
6, it when cell fusion degree reaches 80%-90%, is digested with pancreatin；
7, by the cell digested by 2 × 104cells/cm2(4 × 104/ hole) cell density be inoculated in 24 orifice plates, often
500uL mesenchyma complete medium is added in hole；
8, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
9, when cell fusion degree reaches 60%-70%, careful siphons away complete medium in hole, is added into 24 orifice plates
500uL at chondrocyte induction differential medium；
10, every 72h more renew at chondrocyte induction culture medium；
11, after breaking up at chondrocyte induction, siphon away in 24 orifice plates at chondrocyte induction differential medium, rinsed 2 times with PBS.Every hole
The 4% neutral formalin solution of 417uL, fixed 30min is added；
12, neutral formalin solution is siphoned away, is rinsed 2 times with PBS.208uL A Li Xinlan's dye liquor is added in every hole and contaminates 30min；
13, A Li Xinlan dye liquor is siphoned away, is rinsed 3 times with PBS；
14, microscopically observation is placed in into cartilage dyeing effect.
<5>other are detected: microorganism detection, fungal detection, endotoxin detection, detection of mycoplasma.
The present invention has the advantages that
(1) it impregnates, greatly reduces using containing dual anti-physiological saline after 75% alcohol soaking disinfection of tissue separating step
The probability of pollution.
(2) clostridiopetidase A that this patent uses one neutral amino acid of proline in main dissociated cells interstitial in physiological conditions
One glycine, one proline polypeptide, clostridiopetidase A only have digestion to cytoplasm, and epithelial cell is influenced less, to be applicable in
Fibroid tissue, epithelial tissue etc. are separated in digestion.Dispase in invention is used to disappear from various different tissues or organ
Change extracellular matrix, discharges and prepare primary unicellular；It can prevent cell accident conglomeration.This patent primary digestion uses when separating
The relatively mild enzyme such as II Collagenase Type, type Ⅳ collagenase, dispase, digests in two steps.Cell yield is high, and activity is high, and cell is pure.
Pancreatin is not used in separating digesting, avoids pancreatin by the mucin between dissociated cells, glycoprotein, influences cytoskeleton, thus
Separate cell, the protein ingredient and epicyte protein to cytoplasm have very strong destruction.
(3) hydroxyethyl starch can make red blood cell form rouleau formation, so that red blood cell be accelerated to settle from cell suspension.
Cooperate hydroxyethyl starch precipitating red blood cell to eliminate most red blood cell after digestion again, avoids between a large amount of red blood cell influences
The adherent effect of mesenchymal stem cells.More compared with conventional lymph separating liquid density-gradient centrifugation method cell yield with hydroxyethyl starch
It is high.Also conventional ammonium chloride splitting erythrocyte change osmotic pressure is avoided, erythrocyte membrane is ruptured, removes red blood cell, but in shadow
While ringing erythrocyte membrane also irreversible damage can be caused to mesenchymal stem cells are asked.
(4) originally culture adds the fast breeding that suitable progesterone promotes decidua basalis mescenchymal stem cell.
Detailed description of the invention
Fig. 1 is P0 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 2 is P1 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 3 is P2 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 4 is P3 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 5 is P4 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 6 is P5 for decidua basalis mescenchymal stem cell aspect graph；
Fig. 7 is stem cell P2 for flow cytometer detection result；
Fig. 8 is P5 for placenta decidua basalis growth of mesenchymal stem cells curve；
Fig. 9 is the Osteoinductive differentiation figure (10x) of decidua basalis mescenchymal stem cell；
Figure 10 is the adipogenic induction differentiation figure (40x) of decidua basalis mescenchymal stem cell；
Figure 11 is to scheme (10x) breaking up at chondrocyte induction for decidua basalis mescenchymal stem cell.
Specific embodiment
Specific embodiment 1
(1) acquisition transport: the tire of the mature childbirth in 38-40 weeks normally produced from Foochow Zong Yuan hospital, Nanjing Military Command is chosen
Disk is signed informed consent form with puerpera and its family members, placenta is obtained under delivery room aseptic condition, clear through physiological saline after acquisition
It washes on placenta and is put into after meconium etc. in the configured protection liquid of α-MEM (containing 100U/ml penicillin and 100U/ml streptomysin)
Reservoir bag, 4-25 DEG C was sent to laboratory in 24 hours.And it acquires mother's peripheral blood and carries out AIDS, syphilis, hepatitis B, hepatitis, big and small
Cellular virus detection.
(2) separate: in Biohazard Safety Equipment, the placenta by placenta close to mother face side is put into sterile tray up
In, remove extravasated blood with aseptic nipper and with normal saline flushing mother face, with aseptic operation scissor clip close to parent side
The basal decidua tissue of rough surface.
(3) clean, shred: by clip decidua basalis tissue be placed in 75% alcohol impregnate 1min after be quickly transferred to physiology salt
Water is cleaned.Capillary is removed with sterile pincers.Again with the life containing 100U/ml penicillin and 100U/ml streptomysin
Manage salt water impregnate decidua basalis tissue 5min, finally with physiological saline clean decidua basalis 7 times with the penicillin of removal, streptomysin it is residual
It stays.Cleaned tissue block is transferred in 50ml centrifuge tube and is cut with sterile scissors to 0.5-0.8cm3Molecule.
(4) it digests for the first time: 37 DEG C of water-baths (are put according to the mixed enzyme that the volume of decidua basalis prepares digestion for the first time before use
Pot incubates temperature to 37 DEG C), 0.2% II Collagenase Type and 0.4% type Ⅳ collagenase are added into centrifuge tube, the volume of each enzyme in mixed enzyme
For 1:1, the volume of mixed enzyme and the volume ratio of decidua basalis tissue block are 1:1.Mixed enzyme and tissue block are mixed, 37 DEG C is put into and turns
1h is digested in the shaking table that speed is 120rpm.
(5) it digests for second: 0.2% dispase of total volume 1/4 is added after the completion of digestion for the first time, mixes, is put into 37
15min is digested in the shaking table that DEG C revolving speed is 120rpm.
(6) terminate: the complete serum free medium that equivalent is added after digestion terminates digestion.1600rpm, 8min centrifugation
It discards supernatant.
(7) it removes red blood cell: cell is resuspended with physiological saline, it is abundant that 6% hydroxyethyl starch (volume ratio 1:5) is added
After mixing, 150 rpm, 5min centrifugation.Careful absorption supernatant, then 1200rpm, 6min centrifugation discard supernatant.Washing 2 times.
(8) it counts and surveys living, kind bottle and culture: cell being resuspended with Serum-free complete medium, is counted with trypan exclusion stain
It surveys and lives.The mescenchymal stem cell of acquisition is pressed 2 × 104/cm2Density be inoculated in culture bottle, calculated and connect according to inoculum density
The culture bottle number of kind.With Serum-free complete medium culture medium, corresponding T175 culture is inoculated in after gently blowing and beating resuspension cell
In bottle, progesterone 1.5 × 10 is added in culture medium-9mol/L -2×10-9Mol/L is placed into 37 DEG C, 5%CO2It is cultivated in incubator.
(9) it changes liquid: carrying out replacement Serum-free complete medium within the 3rd day, change liquid every 3 days progress full doses later.
(10) it passes on: when cell grows to degrees of fusion 70-80%, with 10ml physiological saline lotion cell 1 time, being added
0.05% pancreatin of 10mL digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collects vitellophag.
1200rpm, 6min centrifugation discard supernatant, and cell is resuspended with Serum-free complete medium and carries out passage kind bottle by 1:4.Reach P5
Generation, per generation cell carry out cellular morphology observation with inverted microscope and take pictures.
(11) it harvests: when cell grows to degrees of fusion 80%, with 10ml physiological saline lotion cell 2 times, 10mL is added
0.05% pancreatin digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collects vitellophag
(12) it detects:
Fig. 1-Fig. 6 is P0-P5 for decidua basalis mescenchymal stem cell aspect graph, and cell still shows after repeatedly passing on as can be seen from Fig.
Normal mesenchymal stem cell morphology is shuttle-type fibroblast-like cells, spiral vortex type growth.
Cell detection:
(1) cell harvested after the cell dissociation in P0-P5 generation is counted, surveys work, and the results are shown in Table 1.
Table 1
As it can be seen from table 1 preparation process cell number obtained is stable and cell is more, and cell activity is high.
(2) flow cytometer detection: collecting cell, and PBS is washed 2 times, and cell suspension, label 1 ~ 6 pipe is made in adjustment cell concentration
Cell suspension is respectively added, makes the cell 1 × 10 of every pipe6It is a, pipe 1 plus the anti-human CD90-FITC of 5 μ L mouse, pipe 2 plus 5 μ L mouse
Antibody, pipe 5 plus 10 μ L hMSC are not added in anti-human CD105-PerCP-Cy 5.5, pipe 3 plus the anti-human CD73-APC of 5 μ L mouse, pipe 4
Positive Isotype control mixture and 10 μ LPE hMSC Negative isotype control mixtures, pipe 6 plus 10 μ L hMSC positive mixtures
It is protected from light with 10 μ LPE hMSC feminine gender mixture room temperatures and is incubated for 30 min, washed repeatedly with PBS two or three times, reduce non-specific knot
It closes；300 μ LPBS are added and mix cell, flow cytometer detects CD90+、CD73+、CD105+With hMSC feminine gender mixture
CD45+、CD34+、CD11b+、CD19+、HLA-DR+The expression of equal major surfaces marker.P0-P5 is for flow cytometer detection result
It is shown in Table 2:
Table 2
Obtained different algebra cell streaming phenotypes are uniform as can be known from Table 2, it was demonstrated that cell purity is high.It is thin that cell expresses mesenchyma
Born of the same parents mark CD90 CD73 CD105, do not express hematopoietic cell markers CD45 CD34 CD11b CD19, and it is white thin not express people
Extracellular antigen HLA-DR meets international cell therapy association for standard as defined in mescenchymal stem cell surface marker.It is dry thin
Born of the same parents P2 is shown in Fig. 7 for flow cytometer detection result.
(3) mtt assay measures cell growth curve
1) preparation of MTT: the MTT concentration in this method is 5mg/ml.MTT 5mg is weighed, the phosphate buffer of 1ml is dissolved in
(PBS), 4 DEG C are put and is kept in dark place to be removed the bacterium in solution with 0.22 μm of membrane filtration.
2) cell for taking P5 generation to prepare, digestion to single cell suspension, and count, with MSC Serum-free complete medium tune
Whole concentration is to 2 × 103/ hole is inoculated in 96 orifice plates, and liquid 200ul is added in every hole, prepares 7 96 orifice plates, overnight incubation altogether.
Next day replaces culture solution, is being incubated for 4h to which 20ul MTT is added in gaging hole every for 24 hours, is discarding MTT liquid after 4h, be added 150ul's
DMSO is protected from light repetition concussion 10min, in microplate reader at 490nm wavelength, detects light absorption value, measures 8 holes every time, continuous to survey
Amount 7 days.By the data creating of detection in 7 days at the MTT growth curve of the cell.With culture " Time (tD) " for horizontal axis, " 490nm
Absorbance (OD value) at wavelength " is that the longitudinal axis draws cell growth curve.Cell growth curve is as shown in Figure 8.From Fig. 8 growth curve
Measurement is it can be seen that repeatedly cell maintains stronger proliferation activity after passage.
(4) induction differentiation capability detection
Osteoinductive differentiation, adipogenic induction differentiation are carried out using following table kit, are detected at chondrocyte induction differentiation capability:
Osteoinductive differentiation operation
1, plus the gelatin of 300uL0.1% is placed in 24 orifice plates, shakes up liquid, it is made to cover the bottom surface of entire orifice plate；
2, the gelatin for being covered with 0.1% is placed in super-clean bench 30min；
3, gelatin is discarded after 30min, dries 24 orifice plates, cell can be cultivated；
4, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
5, when cell fusion degree reaches 80%-90%, 3min is digested with 0.05% pancreatin room temperature of 10mL；
6, by the cell digested by 2 × 104cells/cm2(4 × 104/ hole) cell density be inoculated in 24 orifice plates, often
500uL mesenchyma complete medium is added in hole；
7, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
8, when cell fusion degree reaches 60%-70%, careful siphons away complete medium in hole, is added into 24 orifice plates
The Osteoinductive differentiation culture medium of 500uL；
9, fresh Osteoinductive differentiation culture medium (needing to be preheated to 37 DEG C before use) is used instead every 72h；
10, it after inducing 3 weeks, is dyed with alizarin red.
11, it after there are a large amount of calcium tubercles, changes liquid form and becomes every 48h once half amount changes liquid.
12, after Osteoinductive differentiation, the Osteoinductive differentiation culture medium in 24 orifice plates is siphoned away, is rinsed 2 times with PBS.Every hole
The 4% neutral formalin solution of 417uL, fixed 30min is added；
13, neutral formalin solution is siphoned away, is rinsed 2 times with PBS.208uL alizarin red dye liquor is added in every hole and contaminates 3-5min
14, alizarin red dye liquor is siphoned away, is rinsed 3 times with PBS；
15, it is placed in microscopically observation skeletonization dyeing effect.
As a result: osteoblast dyeing is the positive as can be seen from Figure 9, illustrates that the cell of induction differentiation has formed osteocyte
Calcium scoring, it was demonstrated that cell has stronger osteoblast differentiation ability.
Adipogenic induction differentiation operation
1, induction A liquid and induction B liquid are prepared；The wherein formula of A liquid are as follows: people's interstital stem cell adipogenic induction differential medium A
175 mL of liquid-based basal culture medium, people's interstital stem cell adipogenic induction break up dedicated fetal calf serum 20ml, dual anti-2 mL, glutamine
2 mL, 400 μ L of insulin, 200 μ L of 3-isobutyl-1-methylxanthine, 200 μ L of Rosiglitazone, 200 μ L of dexamethasone.B
The formula of liquid are as follows: 175 mL of people's interstital stem cell adipogenic induction differential medium B liquid-based basal culture medium, people's interstital stem cell at
Dedicated 20 mL of fetal calf serum, dual anti-2 mL, 2 mL of glutamine, 400 μ L of insulin are broken up in rouge induction.
2, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
3, when cell fusion degree reaches 80%-90%, 3min is digested with 0.05% pancreatin of 10mL；
4, by the cell digested by 2 × 104cells/cm2(1.96 × 105/ hole) cell density be inoculated in 24 orifice plates,
1mL mesenchyma complete medium is added in every hole；
5, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
6, every 72h changes liquid, merges until cell fusion degree reaches 100% or crosses；
7, carefully mescenchymal stem cell complete medium is siphoned away, 1mL adipogenic induction culture medium A liquid is added into 24 orifice plates；
8, after inducing 48h, 72h, the A liquid in 24 orifice plates is siphoned away, 1mL adipogenic induction culture medium B liquid is added；
9, after for 24 hours, B liquid is siphoned away, A liquid is gained and continues to induce；
10, A liquid and B liquid alternating action 3-5 times continue to gain B liquid maintaining culture 4-7 days (replacing fresh B liquid every 2-3 days),
Fat drips become larger and are merged into beading；
11, cell culture medium is removed, is washed 2 times with PBS, the fixed 20-30min of ORO Fixative fixer is added；
12, fixer is discarded, with distillation washing 2 times；
13,60% isopropanol is added and embathes 5min；
14, new prepared ORO Stain is added after discarding 60% isopropanol, disseminates 10-20min；
15, dyeing liquor is discarded, is washed 3-5 times, until without extra dye liquor；
16, ORO Buffer 1min is added, discards；
17, distilled water covering cell is added and observes under the microscope.
As a result: apparent fat drips occur after inducing as can be seen from Figure 10, oil red O stain is positive, and illustrates that cell has
There is the stronger ability to Adipocyte Differentiation.
Break up at chondrocyte induction and operates
1, plus the gelatin of 300uL0.1% is placed in 24 orifice plates, shakes up liquid, it is made to cover the bottom surface of entire orifice plate；
2, the gelatin for being covered with 0.1% is placed in super-clean bench 30min；
3, gelatin is discarded after 30min, dries 24 orifice plates, cell can be cultivated；
4, it is configured to chondrocyte induction culture medium premixed liquid；The TGF-β 3 of 10uL is added at the every 1ml premixed liquid of cartilage differentiation culture medium,
Mix spare (12h domestic demand has used)；
5, it takes P5 for decidua basalis mescenchymal stem cell, is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
6, when cell fusion degree reaches 80%-90%, 3min is digested with 0.05% pancreatin of 10mL；
7, by the cell digested by 2 × 104cells/cm2(4 × 104/ hole) cell density be inoculated in 24 orifice plates, often
500uL mesenchyma complete medium is added in hole；
8, cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
9, when cell fusion degree reaches 60%-70%, careful siphons away complete medium in hole, is added into 24 orifice plates
500uL at chondrocyte induction differential medium；
10, every 72h more renew at chondrocyte induction culture medium；
11, after breaking up at chondrocyte induction, siphon away in 24 orifice plates at chondrocyte induction differential medium, rinsed 2 times with PBS.Every hole
The 4% neutral formalin solution of 417uL, fixed 30min is added；
12, neutral formalin solution is siphoned away, is rinsed 2 times with PBS.208uL A Li Xinlan's dye liquor is added in every hole and contaminates 30min；
13, A Li Xinlan dye liquor is siphoned away, is rinsed 3 times with PBS；
14, microscopically observation is placed in into cartilage dyeing effect.
As a result: it is stronger to cartilage cell point to illustrate that cell has for alcian blue stained positive as can be seen from Figure 11
The ability of change.The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent
With modification, it is all covered by the present invention.
Claims (7)
1. a kind of placenta decidua basalis mescenchymal stem cell preparation method, which is characterized in that specifically includes the following steps:
(1) placenta acquires: the placenta of the mature childbirth in 38-40 weeks normally produced from hospital is chosen, through physiology after acquisition
It is put into after meconium etc. on salt water cleaning placenta and is protected in liquid with containing the α-MEM of 100U/ml penicillin and 100U/ml streptomysin,
4-25 DEG C of preservation, was sent to laboratory in 24 hours, and acquired mother's peripheral blood and carry out AIDS, syphilis, hepatitis B, hepatitis, giant cell
Viral diagnosis；
(2) decidua basalis tissue separates: in Biohazard Safety Equipment, the placenta by placenta close to mother face side is put into sterile up
In pallet, remove extravasated blood with aseptic nipper and with normal saline flushing mother face, with aseptic operation scissor clip close to parent
The decidua basalis tissue of side rough surface；
(3) it cleans, shred decidua basalis tissue to 0.5-0.8cm with sterile scissors3Molecule；
(4) it digests for the first time: using II Collagenase Type and type Ⅳ collagenase mixture slaking tissue；
(5) it digests for second: being digested using dispase；
(6) it terminates digestion: the complete serum free medium of equivalent being added after digestion and terminates digestion, 1600rpm, 8min centrifugation are abandoned
Remove supernatant；
(7) it removes red blood cell: cell is resuspended with physiological saline, 6wt% hydroxyethyl starch is added and removes red blood cell；
(8) it counts and surveys living, kind bottle and culture；Cell is resuspended with Serum-free complete medium, is counted to survey with trypan exclusion stain and be lived；
The mescenchymal stem cell of acquisition is pressed 2 × 104/cm2Density be inoculated in culture bottle, inoculation is calculated according to inoculum density
Culture bottle number；With Serum-free complete medium culture medium, it is inoculated in corresponding T175 culture bottle after gently blowing and beating resuspension cell,
Progesterone is added in culture medium, is placed into 37 DEG C, 5%CO2It is cultivated in incubator；
(9) it changes liquid: carrying out replacement Serum-free complete medium within the 3rd day, change liquid every 3 days progress full doses later；
(10) it passes on: when cell grows to degrees of fusion 70-80%, with 10ml physiological saline lotion cell 1 time, 10mL is added
0.05% pancreatin room temperature digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collects vitellophag；
1200rpm, 6min centrifugation discard supernatant, and cell is resuspended with Serum-free complete medium and carries out passage kind bottle by 1:4；
(11) it harvests, freeze: when cell grows to degrees of fusion 80%, with 10ml physiological saline lotion cell 2 times, 10mL is added
0.05% pancreatin digests 3min, carries out termination digestion with 10ml Serum-free complete medium, collects vitellophag and is counted；From
The DMSO that preparation 20% in advance is taken out in refrigerator freezes protection liquid；According to cell count, by every cryopreservation tube cell number be about 5 ×
106A cell number calculates the pipe number and volume of cell cryopreservation；It is first marked on cryopreservation tube, then will with complete medium
Cell constant volume is to respective volume, and piping and druming mixing is slow added into equal volume, prepared 20%DMSO freezes protection liquid, mixing
Cell is dispensed into cryopreservation tube afterwards, every pipe volume is 1mL；The 20%DMSO freezes protection liquid will put refrigerator cold-storage before
Balance is to 4 DEG C；
(12) program cooling and preservation: cryopreservation tube is put into the program temperature reduction box of pre-cooling, after being put into -80 DEG C of refrigerator overnights, is turned
Enter liquid nitrogen formally to store；
(13) it detects: stem cell is subjected to cell count, Activity determination, flow cytometer detection, the detection of induction differentiation capability and growth
Curve determination.
2. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (3)
The cleaning, the concrete operation method for shredding tissue are as follows: be placed in clip decidua basalis tissue after 75% alcohol immersion 1min rapidly
Physiological saline is transferred to be cleaned；Capillary is removed with sterile pincers；It uses again and contains 100U/ml penicillin and 100U/
The physiological saline of ml streptomysin impregnates decidua basalis tissue 5min, finally cleans the decidua basalis 7 times moulds with removal with physiological saline
The residual of element, streptomysin；Cleaned decidua basalis tissue block is transferred in 50ml centrifuge tube and is cut with sterile scissors to 0.5-
0.8cm3Molecule.
3. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (4)
The first time digestion step are as follows: the mixed enzyme of digestion for the first time is prepared according to the volume of decidua basalis, mixed enzyme is using preceding putting 37
DEG C water-bath incubates temperature to 37 DEG C, and 0.2 wt %, II Collagenase Type and 0.4 wt % type Ⅳ collagenase are added into centrifuge tube, mixing
The volume ratio of each enzyme is 1:1 in enzyme, and the volume of mixed enzyme and the volume ratio of decidua basalis tissue block are 1:1；By mixed enzyme and tissue
Block mixes, and is put into the shaking table that 37 DEG C of revolving speeds are 120rpm and digests 1h.
4. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (5)
Second of digestion step are as follows: the dispase of 0.2 wt % of total volume 1/4 is added after the completion of digestion for the first time, mixes, puts
Enter in the shaking table that 37 DEG C of revolving speeds are 120rpm and digests 15min.
5. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (7)
The volume ratio of the physiological saline and 6wt% hydroxyethyl starch is 1:5,150 rpm after the two mixes well, 5min centrifugation；Carefully
Supernatant is drawn, then 1200rpm, 6min centrifugation discard supernatant；With brine 2 times.
6. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (8)
The progesterone amount of adding is 1.5 × 10-9mol/L -2×10-9mol/L。
7. a kind of placenta decidua basalis mescenchymal stem cell preparation method according to claim 1, which is characterized in that step (11)
Described 20% DMSO freeze protection liquid be DMSO: Serum-free complete medium: human serum albumin by volume 1:3:1 prepare and
At.
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| CN112011504A (en) * | 2020-09-09 | 2020-12-01 | 江苏育瑞康生物科技有限公司 | Preparation method of placenta decidua mesenchymal stem cells |
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