CN110317780B - Preparation method of placenta membrane mesenchymal stem cells - Google Patents
Preparation method of placenta membrane mesenchymal stem cells Download PDFInfo
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- CN110317780B CN110317780B CN201910643009.7A CN201910643009A CN110317780B CN 110317780 B CN110317780 B CN 110317780B CN 201910643009 A CN201910643009 A CN 201910643009A CN 110317780 B CN110317780 B CN 110317780B
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Abstract
The invention discloses a preparation method of placenta membrane mesenchymal stem cells, and relates to the field of stem cell preparation. The invention provides a preparation method of placenta-derived decidua basalis mesenchymal stem cells, which comprises the whole processes of collection, transportation, separation, liquid change, passage, cryopreservation, detection and storage. The invention takes the placenta decidua basalis as a material to extract the mesenchymal stem cells of the placenta decidua basalis, and the mesenchymal stem cells are prepared by adopting a multi-enzyme combined multi-step digestion method, animal serum is not required to be added in the extraction process, more cells can be obtained in a short time, and the cells have good uniformity and stability. The whole culture period is short, and the needed mesenchymal stem cells from the decidua basalis of the placenta can be efficiently and safely obtained.
Description
Technical Field
The invention relates to the technical field of stem cell preparation, in particular to a placenta ghost membrane mesenchymal stem cell, which comprises the following components: collecting, transporting, separating, changing liquid, culturing, subculturing, harvesting, freezing, detecting and storing.
Background
Stem cells are the original source of human body and its various tissue cells, it can not only maintain the stability of its own population through cell division, but also differentiate into different types of cells, and further constitute various complex tissues and organs of the body. Stem cell studies dates back to the 50 s of the 20 th century, originally from teratoma studies. In the 70's of the 20 th century, cells derived from mesoderm and fibroblast-like adherent growth were discovered and called mesenchymal stem cells.
Mesenchymal stem cells are important members of the stem cell family, and are increasingly receiving attention due to the strong potential of self-renewal and differentiation and development to various tissues. Mesenchymal stem cells have been isolated from tissues such as bone marrow, fat, amnion, placenta, cord blood, and umbilical cord, and have the ability to differentiate into endothelial cells, osteoblasts, chondrocytes, adipocytes, myoblasts, and neural cells.
The placenta not only plays a role in the development, nutrition and tolerance of the embryo, but also has a potential stem cell capacity. In the mother body, the ectoderm-derived mesenchymal stem cells are continuously differentiated into blood vessels of connective tissues, whereby the embryo completes the material exchange with the mother body. The decidua basalis forms the maternal part of the placenta, which occupies a very small part of the placenta in term pregnancy, and the surface of the decidua basalis is covered with a layer of trophoblast cells from fixed villi and forms a bottom of villus gap together with the decidua basalis, called decidua basal plate, from which decidua intervals protrude towards the direction of the chorion, generally not exceeding 2/3 of the whole thickness of the placenta, and the maternal surface of the placenta is divided into about 20 maternal leaves visible to the naked eye. The placenta becomes waste after the delivery of the fetus, and becomes a new research hotspot in the fields of cell therapy, tissue engineering and genetic engineering due to the multidirectional differentiation capability, the convenient self-drawing of materials, immunosuppression and the like.
On the annual meeting in american hematology in 2000, jaroschak et al reported a preliminary study on placenta-derived adherent cells and demonstrated the presence of mesenchymal stem cell components in the placenta; other researches show that the placenta contains abundant mesenchymal stem cells, and the result shows that the placenta-derived mesenchymal stem cells can also express certain embryonic stem cell surface markers, such as sseA-4, TRA-1-61 and TRA-1-80, and the result suggests that the placenta-derived mesenchymal stem cells can be very primitive cell groups, have the advantages of wider self-renewal and multi-differentiation capacity compared with other adult stem cells, convenient material taking, wide source, easy placenta acquisition after the healthy puerperae agrees, low immunogenicity, no ethical problem and the like.
The decidua basalis, one of the constituents of the maternal-fetal interface, plays a crucial role in protecting the fetus from rejection during growth and development. Although the decidua basalis is derived from the mother, compared with the fetal mesenchymal stem cells, the decidua basalis has stronger proliferation capacity, expresses the basic characteristics of general MSCs and mesoderm multidirectional differentiation potential, and the chromosome karyotype and the unique immunosuppressive characteristic which are stably maintained by in vitro long-term culture are good sources for cell transplantation treatment and tissue engineering. And the decidua basalis mesenchymal stem cells are used as an important source of autologous mesenchymal stem cells of a mother, and can be safely used for treating diseases of the immune system of the mother and the like.
Disclosure of Invention
Aiming at the problems in the prior common technology, the invention provides a preparation method of placenta-derived basolateral decidua mesenchymal stem cells, which comprises the whole processes of collection, transportation, separation, liquid exchange, passage, cryopreservation, detection and storage. The invention takes the placenta decidua basalis as a material to extract the mesenchymal stem cells of the placenta decidua basalis, and the mesenchymal stem cells are prepared by adopting a multi-enzyme combined multi-step digestion method, animal serum is not required to be added in the extraction process, more cells can be obtained in a short time, and the cells have good uniformity and stability. The whole culture period is short, and the needed mesenchymal stem cells from the decidua basalis of the placenta can be efficiently and safely obtained.
The method comprises the following steps:
(1) collecting and transporting: selecting placenta from normal delivery of 38-40 weeks and full term in hospital, signing with informed consent of parturient and family members, collecting placenta under aseptic condition, cleaning meconium on placenta with normal saline, placing into protective solution (containing 100U/ml penicillin and 100U/ml streptomycin) prepared from alpha-MEM, storing in storage bag, and delivering to laboratory at 4-25 deg.C for 24 hr. Collecting mother peripheral blood for detecting AIDS, syphilis, hepatitis B, hepatitis C, and cytomegalovirus.
(2) Separation: in the biological safety cabinet, putting the placenta face of the side of the placenta close to the mother face upwards into a sterile tray, removing extravasated blood with sterile forceps, flushing the mother face with normal saline, and shearing decidua basalis tissue close to the rough face of the mother face with sterile operation curved scissors.
(3) Cleaning and cutting: soaking the decidua tissue in 75% alcohol for 1min, and quickly transferring to normal saline for cleaning. The capillaries were removed with sterile toothed forceps. Then, the decidua basalis tissue is soaked for 5min by using normal saline containing 100U/ml penicillin and 100U/ml streptomycin, and finally, the decidua basalis is washed by using the normal saline for 7 times to remove the residue of the penicillin and the streptomycin. Transferring the cleaned decidua basalis tissue block to a 50ml centrifuge tube, and shearing the decidua basalis tissue block to 0.5-0.8cm by using sterile scissors3And (3) fine particles.
(4) First digestion: preparing mixed enzyme for the first digestion according to the volume of the bottom decidua (incubating to 37 ℃ in a 37 ℃ water bath before use), adding 0.2wt% of collagenase II and 0.4wt% of collagenase IV into a centrifuge tube, wherein the volume ratio of the enzymes in the mixed enzyme is 1:1, and the volume ratio of the mixed enzyme to the bottom decidua tissue mass is 1: 1. Mixing the mixed enzyme with the tissue block, and digesting in a shaker at 37 deg.C and 120rpm for 1 h.
(5) And (3) second digestion: after the first digestion is completed, 0.2wt% of dispase in total volume of 1/4 is added, mixed evenly and put into a shaker with 37 ℃ and 120rpm for digestion for 15 min.
(6) And (4) terminating: after digestion was complete, digestion was stopped by adding an equal volume of complete serum-free medium. The supernatant was discarded by centrifugation at 1600rpm for 8 min.
(7) Removing red blood cells: resuspend the cells in normal saline, add 6wt% hydroxyethyl starch (the volume ratio of normal saline to 6% hydroxyethyl starch is 1: 5) and mix well, centrifuge at 150 rpm for 5 min. The supernatant was carefully aspirated, centrifuged at 1200rpm for 6min and discarded. Washed 2 times with physiological saline.
(8) Counting, measuring activity, seed bottle and culturing: cells were resuspended in serum-free complete medium and counted for viability using trypan blue exclusion. The obtained mesenchymal stem cells are arranged in a 2 x 10 mode4/cm2The density of (2) was inoculated into a culture flask, and the number of the inoculated culture flasks was calculated from the inoculation density. Using serum-free complete medium culture medium, lightly blowing and beating resuspension cells, then inoculating the resuspension cells into corresponding T175 culture bottles, and supplementing progesterone into the culture medium by 1.5X 10-9mol/L -2×10-9mol/L, standing at 37 deg.C and 5% CO2Culturing in an incubator.
(9) Liquid changing: serum-free complete medium was changed on day 3, followed by a full change every 3 days.
(10) Passage: when the cells grow to 70-80% confluence, the cells are washed with 10mL of normal saline for 1 time, 10mL of 0.05% pancreatin is added for digestion at room temperature for 3min, the digestion is stopped with 10mL of serum-free complete medium, and the digested cells are collected. 1200rpm, 6min centrifugation to discard the supernatant, resuspend cells with serum-free complete medium and subculture flasks at 1: 4.
(11) Harvesting and freezing: when the cells grew to 80% confluence, the cells were washed 2 times with 10ml of physiological saline, and added10mL of 0.05% trypsin was digested at room temperature for 3min, the digestion was stopped with 10mL of serum-free complete medium, and the digested cells were collected and counted. The pre-prepared 20% DMSO cryopreservation solution (DMSO: serum-free complete medium: human serum albumin volume ratio 1:3: 1) was taken out of the refrigerator. According to the cell count, the number of cells per cryopreserved tube is about 5X 106And counting the number of cells, and calculating the number and volume of the frozen cells. Marking on a freezing tube, then fixing the volume of the cells to a corresponding volume by using a complete culture medium, blowing and uniformly mixing, slowly adding a prepared 20% DMSO freezing protective solution with the same volume (balancing to 4 ℃ by using a refrigerator before use), and subpackaging the cells into the freezing tube after uniformly mixing, wherein the volume of each tube is 1 mL.
(12) And (3) programmed cooling and storing: putting the freezing tube into a pre-cooled programmed cooling box, putting the box into a refrigerator at minus 80 ℃ for one night, and then transferring the box into liquid nitrogen for formal storage.
(13) Detection of
<1> cell count and Activity assay: total and dead cells were counted on a blood count plate after staining the cells by trypan blue exclusion, and the cell viability was obtained according to the formula cell viability =1- (number of dead cells/total number of cells) × 100%.
<2>Flow detection: collecting cells, washing with PBS for 2 times, adjusting cell concentration to prepare cell suspension, marking 1-6 tubes, adding cell suspension to make cell of each tube 1 × 106Each tube 1 was incubated with 5 μ L of mouse anti-human CD90-FITC, tube 2 with 5 μ L of mouse anti-human CD105-PerCP-Cy 5.5, tube 3 with 5 μ L of mouse anti-human CD73-APC, tube 4 without antibody, tube 5 with 10 μ L of hMSC positive isotype control mixture and 10 μ L of LPE hMSC negative isotype control mixture, tube 6 with 10 μ L of hMSC positive mixture and 10 μ L of LPE hMSC negative mixture for 30min at room temperature in the dark, and washed with PBS for two or three repetitions to reduce non-specific binding; adding 300 mu LPBS to mix the cells evenly, and detecting CD90 by an up-flow cytometer+、CD73+、CD105+And hMSC negative mixture CD45+、CD34+、CD11b+、CD19+、HLA-DR+Etc. of the major surface markers.
<3> MTT method for determining cell growth curve
1. Preparation of MTT: the concentration of MTT in this method was 5 mg/ml. MTT 5mg was weighed, dissolved in 1ml of Phosphate Buffered Saline (PBS), filtered through a 0.22 μm filter to remove bacteria from the solution, and stored at 4 ℃ in the dark.
2. Taking P5 generation prepared cells, digesting to single cell suspension, counting, adjusting concentration to 2 × 10 with MSC serum-free complete medium3Per well was inoculated into 96-well plates, 200ul of liquid was added to each well, and a total of 7 96-well plates were prepared and incubated overnight. Changing the culture solution the next day, adding 20ul MTT into the hole to be measured every 24h, incubating for 4h, discarding the MTT solution after 4h, adding 150ul DMSO, repeatedly shaking for 10min in dark, detecting the light absorption value at 490nm wavelength on the microplate reader, measuring 8 holes each time, and continuously measuring for 7 days. The data from the 7 day assay was generated as a MTT growth curve for the cells. The cell growth curve was plotted with the horizontal axis of "time (D)", and the vertical axis of "absorbance (OD value) at 490nm wavelength".
<4> detection of differentiation-inducing ability
Osteogenic induced differentiation procedure
1. Adding 300uL0.1% gelatin into a 24-well plate, and shaking to make the liquid cover the whole bottom surface of the well plate;
2. placing 0.1% gelatin on a super clean bench for 30 min;
3. removing gelatin after 30min, and air drying 24-well plate to obtain cultured cells;
4. taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
5. when the cell fusion degree reaches 80% -90%, digesting with pancreatin;
6. digesting the cells to 2X 104cells/cm2（4×104Per well) was inoculated in a 24-well plate, 500uL of mesenchymal complete medium was added per well;
7. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
8. when the cell fusion degree reaches 60-70%, carefully sucking away the complete culture medium in the hole, and adding 500uL osteogenesis induced differentiation culture medium into the 24-hole plate;
9. replacing with fresh osteogenesis inducing differentiation medium every 72h (preheating to 37 deg.C before use);
10. after 3 weeks of induction, staining was performed with alizarin red;
11. after a large number of calcium nodules appear, the liquid change form is changed into half liquid change every 48 hours;
12. after osteogenic induction differentiation, the osteogenic induction differentiation medium in the 24-well plate was aspirated and washed 2 times with PBS. 417uL of 4% neutral formaldehyde solution is added into each hole, and the mixture is fixed for 30 min;
13. the neutral formaldehyde solution was aspirated and washed 2 times with PBS. Adding 208uL alizarin red dye solution into each hole for dyeing for 3-5 min;
14. sucking away alizarin red dye solution, and washing with PBS 3 times;
15. the resultant was observed under a microscope to observe the staining effect of the bone.
Adipogenic induced differentiation procedure
1. Preparing an induction A liquid and an induction B liquid;
2. taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
3. when the cell fusion degree reaches 80% -90%, digesting with pancreatin;
4. digesting the cells to 2X 104cells/cm2（1.96×105Per well) was inoculated in a 24-well plate, 1mL of mesenchymal complete medium was added per well;
5. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
6. changing liquid every 72h until the cell fusion degree reaches 100% or the cell is over-fused;
7. carefully sucking away the complete culture medium of the mesenchymal stem cells, and adding 1mL of adipogenic induction culture medium A into a 24-pore plate;
8. after 48h and 72h of induction, sucking away the solution A in the 24-well plate, and adding 1mL of adipogenic induction culture medium solution B;
9. after 24h, sucking away the solution B, and replacing the solution A for continuous induction;
10. alternately acting liquid A and liquid B for 3-5 times, continuously replacing liquid B for maintaining culture for 4-7 days (replacing fresh liquid B every 2-3 days), enlarging lipid droplets, and combining into bead-like shape;
11. removing cell culture medium, washing with PBS for 2 times, and fixing with ORO Fixative fixing solution for 20-30 min;
12. discarding the stationary liquid, and washing with distilled water for 2 times;
13. adding 60% isopropanol, and soaking and washing for 5 min;
14. discarding 60% isopropanol, adding newly prepared ORO Stain, and dip-dyeing for 10-20 min;
15. discarding the dyeing solution, and washing with water for 3-5 times until no excess dyeing solution exists;
16. adding ORO Buffer for 1min, and discarding;
17. distilled water was added to cover the cells and observed under a microscope.
Chondrogenic induced differentiation procedure
1. Adding 300uL0.1% gelatin into a 24-well plate, and shaking to make the liquid cover the whole bottom surface of the well plate;
2. placing 0.1% gelatin on a super clean bench for 30 min;
3. removing gelatin after 30min, and air drying 24-well plate to obtain cultured cells;
4. preparing a cartilage induction culture medium premix; adding 10uL of TGF-beta 3 into each 1ml of the chondrogenic differentiation culture medium, and uniformly mixing for later use (the mixture needs to be used up within 12 hours);
5. taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
6. when the cell fusion degree reaches 80% -90%, digesting with pancreatin;
7. digesting the cells to 2X 104cells/cm2（4×104Per well) was inoculated in a 24-well plate, 500uL of mesenchymal complete medium was added per well;
8. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
9. when the cell fusion degree reaches 60-70%, carefully sucking away the complete culture medium in the hole, and adding 500uL chondrogenesis induced differentiation culture medium into the 24-pore plate;
10. replacing new chondrogenic induction medium every 72 h;
11. after chondrogenic induction differentiation, the chondrogenic induction differentiation medium in the 24-well plate was aspirated and washed 2 times with PBS. 417uL of 4% neutral formaldehyde solution is added into each hole, and the mixture is fixed for 30 min;
12. the neutral formaldehyde solution was aspirated and washed 2 times with PBS. Adding 208uL alisin blue staining solution into each hole for staining for 30 min;
13. sucking away the aliskiren blue dye solution, and washing with PBS 3 times;
14. the cartilage staining effect is observed under a microscope.
<5> other tests: microorganism detection, fungus detection, endotoxin detection and mycoplasma detection.
The invention has the advantages that:
(1) the tissue separation step is soaked in 75% alcohol for disinfection and then soaked in physiological saline containing double antibodies, so that the pollution probability is greatly reduced.
(2) The collagenase adopted by the method mainly dissociates proline-neutral amino acid-glycine-proline polypeptide in intercellular substance under physiological condition, has digestion function only to the intercellular substance, has little influence to epithelial cells, and is suitable for digesting and separating fibrous tissues, epithelial tissues and the like. The dispase in the invention is used for digesting extracellular matrix from various tissues or organs, releasing and preparing primary single cells; can prevent the cells from being accidentally agglomerated. According to the method, mild enzymes such as collagenase II, collagenase IV and dispase are adopted in primary digestion and separation, and digestion is carried out in two steps. High cell yield, high activity and pure cells. Pancreatin is not used in separation and digestion, so that the influence of the pancreatin on cytoskeleton by dissociating intercellular mucin and glycoprotein is avoided, cells are separated, and protein components of intercellular substance and cell membrane protein are damaged strongly.
(3) Hydroxyethyl starch can cause the erythrocytes to form rouleaux aggregates, thereby accelerating the sedimentation of the erythrocytes from the cell suspension. Most of red blood cells are removed by being matched with hydroxyethyl starch to precipitate red blood cells after digestion, so that the phenomenon that a large amount of red blood cells influence the adherence effect of the mesenchymal stem cells is avoided. The cell yield is higher by using hydroxyethyl starch compared with the conventional lymph separation liquid density gradient centrifugation method. The conventional method that ammonium chloride is used for cracking erythrocytes to change osmotic pressure, so that erythrocyte membranes are broken and erythrocytes are removed is also avoided, but irreversible damage can be caused to mesenchymal stem cells while the erythrocyte membranes are influenced.
(4) The addition of a proper amount of progesterone in primary culture promotes the rapid proliferation of decidua basalis mesenchymal stem cells.
Drawings
FIG. 1 is a morphogram of decidua mesenchyma stem cell generation P0;
FIG. 2 is a morphogram of decidua mesenchymal stem cell generation P1;
FIG. 3 is a morphogram of decidua mesenchymal stem cell generation P2;
FIG. 4 is a morphogram of decidua mesenchymal stem cell generation P3;
FIG. 5 is a morphogram of decidua mesenchymal stem cell generation P4;
FIG. 6 is a morphogram of decidua mesenchymal stem cell generation P5;
FIG. 7 shows the results of flow-through assay of stem cell P2;
FIG. 8 is a growth curve of mesenchymal stem cells of P5 generation placental decidua;
fig. 9 is an osteogenic induced differentiation map (10 x) of decidua basalis mesenchymal stem cells;
fig. 10 is a adipogenic induced differentiation diagram (40 x) of decidua basalis mesenchymal stem cells;
fig. 11 is a graph (10 x) of chondrogenic induced differentiation of decidua mesenchymal stem cells.
Detailed Description
Detailed description of the preferred embodiment 1
(1) Collecting and transporting: selecting placenta of 38-40 weeks term delivery from Fuzhou general college hospital in south Jing military region, signing with informed consent of puerpera and family members, collecting placenta under aseptic condition, cleaning placenta with normal saline, placing into protective solution (containing 100U/ml penicillin and 100U/ml streptomycin) prepared from alpha-MEM, storing in a bag, and delivering to laboratory at 4-25 deg.C within 24 hr. Collecting mother peripheral blood for detecting AIDS, syphilis, hepatitis B, hepatitis C, and cytomegalovirus.
(2) Separation: in a biological safety cabinet, putting the placenta face of the side of the placenta close to the mother face upwards into a sterile tray, removing extravasated blood by using sterile forceps, flushing the mother face by using normal saline, and shearing decidua basalis tissue close to the rough face of the mother face by using sterile operation curved scissors.
(3) Cleaning and cutting: the decidua tissue cut from the bottom is soaked in 75% alcohol for 1min and then is quickly transferred to normal saline for cleaning. The capillaries were removed with sterile toothed forceps. Then, the decidua basalis tissue is soaked for 5min by using normal saline containing 100U/ml penicillin and 100U/ml streptomycin, and finally, the decidua basalis is washed by using the normal saline for 7 times to remove the residue of the penicillin and the streptomycin. Transferring the cleaned tissue block to a 50ml centrifuge tube, and shearing to 0.5-0.8cm with sterile scissors3And (3) fine particles.
(4) First digestion: preparing mixed enzyme for the first digestion according to the volume of the bottom decidua (incubating to 37 ℃ in a 37 ℃ water bath before use), adding 0.2% type II collagenase and 0.4% type IV collagenase into a centrifuge tube, wherein the volume of each enzyme in the mixed enzyme is 1:1, and the volume ratio of the mixed enzyme to the bottom decidua tissue mass is 1: 1. Mixing the mixed enzyme with the tissue block, and digesting in a shaker at 37 deg.C and 120rpm for 1 h.
(5) And (3) second digestion: after the first digestion is completed, 0.2% of dispase of total volume 1/4 is added, mixed evenly and digested for 15min in a shaker with 37 ℃ and 120 rpm.
(6) And (4) terminating: after digestion is complete, digestion is terminated by adding an equal amount of complete serum-free medium. The supernatant was discarded by centrifugation at 1600rpm for 8 min.
(7) Removing red blood cells: resuspend the cells in normal saline, add 6% hydroxyethyl starch (volume ratio 1: 5), mix well, centrifuge at 150 rpm for 5 min. The supernatant was carefully aspirated, centrifuged at 1200rpm for 6min and discarded. Washing is carried out for 2 times.
(8) Counting, measuring activity, seed bottle and culturing: cells were resuspended in serum-free complete medium and counted for viability using trypan blue exclusion. The obtained mesenchymal stem cells are processed into2×104/cm2The density of (2) was inoculated into a culture flask, and the number of the inoculated culture flasks was calculated from the inoculation density. Using serum-free complete medium culture medium, lightly blowing and beating resuspension cells, then inoculating the resuspension cells into corresponding T175 culture bottles, and supplementing progesterone into the culture medium by 1.5X 10-9mol/L -2×10-9mol/L, standing at 37 deg.C and 5% CO2Culturing in an incubator.
(9) Liquid changing: serum-free complete medium was changed on day 3, followed by a full change every 3 days.
(10) Passage: when the cells grow to 70-80% confluence, the cells are washed with 10mL of normal saline for 1 time, 10mL of 0.05% pancreatin is added for digestion for 3min, the digestion is stopped with 10mL of serum-free complete medium, and the digested cells are collected. 1200rpm, 6min centrifugation to discard the supernatant, resuspend cells with serum-free complete medium and subculture flasks at 1: 4. The cells were transferred to P5 passage, and the cells were examined by inverted microscope for morphology and photographed.
(11) Harvesting: when the cells grow to the fusion degree of 80%, the cells are washed 2 times by 10mL of physiological saline, 10mL of 0.05% pancreatin is added for digestion for 3min, the digestion is stopped by 10mL of serum-free complete medium, and the digested cells are collected.
(12) And (3) detection:
fig. 1-6 are morphological diagrams of decidua mesenchymal stem cells at P0-P5 generations, and it can be seen from the diagrams that the cells still show normal mesenchymal stem cell morphology after multiple passages, and are spindle-shaped fibroblast-like cells and grow in a vortex mode.
Cell detection:
(1) the cells harvested after digestion from the P0-P5 passages were counted and assayed for viability, and the results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the number of cells obtained by this preparation process was stable and many, and the cell activity was high.
(2) Flow detection: cells were harvested and washed with PBSAdjusting the cell concentration to prepare cell suspension 2 times, marking 1-6 tubes, and adding the cell suspension to make the cell of each tube 1 × 106Each tube 1 was incubated with 5 μ L of mouse anti-human CD90-FITC, tube 2 with 5 μ L of mouse anti-human CD105-PerCP-Cy 5.5, tube 3 with 5 μ L of mouse anti-human CD73-APC, tube 4 without antibody, tube 5 with 10 μ L of hMSC positive isotype control mixture and 10 μ L of LPE hMSC negative isotype control mixture, tube 6 with 10 μ L of hMSC positive mixture and 10 μ L of LPE hMSC negative mixture for 30min at room temperature in the dark, and washed with PBS for two or three repetitions to reduce non-specific binding; adding 300 mu LPBS to mix the cells evenly, and detecting CD90 by an up-flow cytometer+、CD73+、CD105+And hMSC negative mixture CD45+、CD34+、CD11b+、CD19+、HLA-DR+Etc. of the major surface markers. The results of the flow-type detection of P0-P5 are shown in Table 2:
TABLE 2
As can be seen from Table 2, the obtained different generations of cell flow phenotypes were homogeneous, demonstrating high cell purity. The cells express the mesenchymal cell marker CD90 CD73 CD105, do not express the hematopoietic cell marker CD45 CD34 CD11b CD19, and do not express the human leukocyte antigen HLA-DR, meeting the criteria specified by the International society for cell therapy for mesenchymal stem cell surface markers. The results of flow-through assay of stem cell P2 are shown in FIG. 7.
(3) MTT method for determining cell growth curve
1) Preparation of MTT: the concentration of MTT in this method was 5 mg/ml. MTT 5mg was weighed, dissolved in 1ml of Phosphate Buffered Saline (PBS), filtered through a 0.22 μm filter to remove bacteria from the solution, and stored at 4 ℃ in the dark.
2) Taking P5 generation prepared cells, digesting to single cell suspension, counting, adjusting concentration to 2 × 10 with MSC serum-free complete medium3Per well was inoculated into 96-well plates, 200ul of liquid was added to each well, and a total of 7 96-well plates were prepared and incubated overnight. Changing the culture solution the next day, adding 20ul MTT into the wells to be tested every 24h for incubationAnd (4) discarding the MTT solution after 4h, adding 150ul of DMSO, repeatedly oscillating for 10min in a dark place, detecting the light absorption value at 490nm wavelength on a microplate reader, measuring 8 holes each time, and continuously measuring for 7 days. The data from the 7 day assay was generated as a MTT growth curve for the cells. The cell growth curve was plotted with the culture "time (tD)" as the horizontal axis and the absorbance (OD value) at 490nm as the vertical axis. The cell growth curve is shown in FIG. 8. From the growth curve measurements in fig. 8, it can be seen that the cells maintain strong proliferation activity after multiple passages.
(4) Detection of differentiation-inducing ability
The following kits are used for carrying out osteogenic induced differentiation, adipogenic induced differentiation and chondrogenic induced differentiation capacity detection:
osteogenic induced differentiation procedure
1. Adding 300uL0.1% gelatin into a 24-well plate, and shaking to make the liquid cover the whole bottom surface of the well plate;
2. placing 0.1% gelatin on a super clean bench for 30 min;
3. removing gelatin after 30min, and air drying 24-well plate to obtain cultured cells;
4. taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
5. when the cell fusion degree reaches 80-90%, digesting with 10mL of 0.05% pancreatin at room temperature for 3 min;
6. digesting the cells to 2X 104cells/cm2（4×104Per well) was inoculated in a 24-well plate, 500uL of mesenchymal complete medium was added per well;
7. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
8. when the cell fusion degree reaches 60-70%, carefully sucking away the complete culture medium in the hole, and adding 500uL osteogenesis induced differentiation culture medium into the 24-hole plate;
9. replacing with fresh osteogenesis inducing differentiation medium every 72h (preheating to 37 deg.C before use);
10. after 3 weeks of induction, staining was performed with alizarin red.
11. After a large number of calcium nodules appeared, the change pattern was changed to half a change every 48 h.
12. After osteogenic induction differentiation, the osteogenic induction differentiation medium in the 24-well plate was aspirated and washed 2 times with PBS. 417uL of 4% neutral formaldehyde solution is added into each hole, and the mixture is fixed for 30 min;
13. the neutral formaldehyde solution was aspirated and washed 2 times with PBS. Adding 208uL alizarin red dye solution into each hole for dyeing for 3-5min
14. Sucking away alizarin red dye solution, and washing with PBS 3 times;
15. the resultant was observed under a microscope to observe the staining effect of the bone.
As a result: it can be seen from fig. 9 that osteoblasts were stained positively, indicating that the cells induced to differentiate had formed osteocyte calcified nodules, demonstrating that the cells had a strong osteoblast differentiation capacity.
Adipogenic induced differentiation procedure
1. Preparing an induction A liquid and an induction B liquid; wherein the formula of the solution A is as follows: 175 mL of A liquid basal medium of a human mesenchymal stem cell adipogenic induction differentiation medium, 20mL of special fetal bovine serum for human mesenchymal stem cell adipogenic induction differentiation, 2 mL of double antibody, 2 mL of glutamine, 400 mu L of insulin, 200 mu L of 3-isobutyl-1-methylxanthine, 200 mu L of rosiglitazone and 200 mu L of dexamethasone. The formula of the solution B is as follows: 175 mL of B liquid basal medium of the human mesenchymal stem cell adipogenic induced differentiation medium, 20mL of special fetal bovine serum for the human mesenchymal stem cell adipogenic induced differentiation, 2 mL of double antibodies, 2 mL of glutamine and 400 mu L of insulin.
2. Taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
3. when the cell fusion degree reaches 80% -90%, digesting with 10mL of 0.05% pancreatin for 3 min;
4. digesting the cells to 2X 104cells/cm2（1.96×105Per well) was inoculated in a 24-well plate, 1mL of mesenchymal complete medium was added per well;
5. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
6. changing liquid every 72h until the cell fusion degree reaches 100% or the cell is over-fused;
7. carefully sucking away the complete culture medium of the mesenchymal stem cells, and adding 1mL of adipogenic induction culture medium A into a 24-pore plate;
8. after 48h and 72h of induction, sucking away the solution A in the 24-well plate, and adding 1mL of adipogenic induction culture medium solution B;
9. after 24h, sucking away the solution B, and replacing the solution A for continuous induction;
10. alternately acting liquid A and liquid B for 3-5 times, continuously replacing liquid B for maintaining culture for 4-7 days (replacing fresh liquid B every 2-3 days), enlarging lipid droplets, and combining into bead-like shape;
11. removing cell culture medium, washing with PBS for 2 times, and fixing with ORO Fixative fixing solution for 20-30 min;
12. discarding the stationary liquid, and washing with distilled water for 2 times;
13. adding 60% isopropanol, and soaking and washing for 5 min;
14. discarding 60% isopropanol, adding newly prepared ORO Stain, and dip-dyeing for 10-20 min;
15. discarding the dyeing solution, and washing with water for 3-5 times until no excess dyeing solution exists;
16. adding ORO Buffer for 1min, and discarding;
17. distilled water was added to cover the cells and observed under a microscope.
As a result: from FIG. 10, it can be seen that after induction, very distinct lipid droplets appeared, and the staining of oil red O was positive, indicating that the cells had a strong ability to differentiate into adipocytes.
Chondrogenic induced differentiation procedure
1. Adding 300uL0.1% gelatin into a 24-well plate, and shaking to make the liquid cover the whole bottom surface of the well plate;
2. placing 0.1% gelatin on a super clean bench for 30 min;
3. removing gelatin after 30min, and air drying 24-well plate to obtain cultured cells;
4. preparing a cartilage induction culture medium premix; adding 10uL of TGF-beta 3 into each 1ml of the chondrogenic differentiation culture medium, and uniformly mixing for later use (the mixture needs to be used up within 12 hours);
5. taking P5 generation decidua mesenchymal stem cell, placing at 37 deg.C and 5% CO2Culturing in an incubator;
6. when the cell fusion degree reaches 80% -90%, digesting with 10mL of 0.05% pancreatin for 3 min;
7. digesting the cells to 2X 104cells/cm2（4×104Per well) was inoculated in a 24-well plate, 500uL of mesenchymal complete medium was added per well;
8. the cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator;
9. when the cell fusion degree reaches 60-70%, carefully sucking away the complete culture medium in the hole, and adding 500uL chondrogenesis induced differentiation culture medium into the 24-pore plate;
10. replacing new chondrogenic induction medium every 72 h;
11. after chondrogenic induction differentiation, the chondrogenic induction differentiation medium in the 24-well plate was aspirated and washed 2 times with PBS. 417uL of 4% neutral formaldehyde solution is added into each hole, and the mixture is fixed for 30 min;
12. the neutral formaldehyde solution was aspirated and washed 2 times with PBS. Adding 208uL alisin blue staining solution into each hole for staining for 30 min;
13. sucking away the aliskiren blue dye solution, and washing with PBS 3 times;
14. the cartilage staining effect is observed under a microscope.
As a result: from FIG. 11, it can be seen that Alicinblue staining was positive, indicating that the cells have a strong ability to differentiate into chondrocytes. The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. A preparation method of mesenchymal stem cells of placental decidua is characterized by comprising the following steps:
(1) placenta collection: selecting placenta from normal delivery of 38-40 weeks and full term in hospital, collecting, cleaning meconium on placenta with normal saline, adding into alpha-MEM protective solution containing 100U/ml penicillin and 100U/ml streptomycin, storing at 4-25 deg.C, delivering to laboratory within 24 hr, and collecting mother peripheral blood for detecting AIDS, syphilis, hepatitis B, hepatitis C, and cytomegalovirus;
(2) separating decidua basalis tissue: in a biological safety cabinet, putting the placenta face of one side of the placenta close to the mother face upwards into a sterile tray, removing extravasated blood by using sterile forceps, flushing the mother face by using normal saline, and shearing bottom decidua tissues close to the rough face of one side of the mother body by using sterile operation bent scissors;
(3) cleaning, cutting decidua tissue with sterile scissors to 0.5-0.8cm3Micro particles;
(4) first digestion: mixing collagenase type II and collagenase type IV to digest tissues;
(5) and (3) second digestion: digesting by adopting dispase;
(6) termination of digestion: adding an equivalent complete serum-free culture medium to stop digestion after digestion is finished, centrifuging at 1600rpm for 8min, and removing a supernatant;
(7) removing red blood cells: resuspending the cells in normal saline, adding 6wt% hydroxyethyl starch to remove erythrocytes;
(8) counting, measuring the survival rate, culturing in a seed bottle; suspending the cells by serum-free complete culture medium, and counting and determining the activity by trypan blue exclusion; the obtained mesenchymal stem cells are arranged in a 2 x 10 mode4/cm2The density of (2) is inoculated in a culture bottle, and the number of the inoculated culture bottles is calculated according to the inoculation density; using serum-free complete medium culture medium, lightly blowing and beating resuspension cells, inoculating in corresponding T175 culture bottle, supplementing progesterone in the culture medium, placing at 37 deg.C and 5% CO2Culturing in an incubator;
(9) liquid changing: replacing the serum-free complete culture medium on day 3, and then replacing the whole culture medium every 3 days;
(10) passage: when the cells grow to 70-80% fusion degree, washing the cells with 10mL of normal saline for 1 time, adding 10mL of 0.05% pancreatin, digesting for 3min at room temperature, stopping digestion with 10mL of serum-free complete medium, and collecting the digested cells; centrifuging at 1200rpm for 6min, discarding supernatant, and resuspending cells with serum-free complete medium according to a ratio of 1:4 for subculturing a seed bottle;
(11) harvesting and freezing: when the cells grow to the fusion degree of 80%, washing the cells for 2 times by using 10mL of normal saline, adding 10mL of 0.05% pancreatin for digestion for 3min, stopping digestion by using 10mL of serum-free complete medium, collecting the digested cells and counting; taking out the DMSO cryopreservation protective solution prepared in advance from the refrigerator; according to the cell count, the number of cells per cryopreserved tube was 5X 106Counting the number of cells, and calculating the number and volume of the tubes for freezing the cells; marking on a freezing tube, then fixing the volume of cells to a corresponding volume by using a complete culture medium, blowing, uniformly mixing, slowly adding 20% DMSO (dimethyl sulfoxide) freezing protection solution with the same volume, uniformly mixing, and subpackaging the cells into the freezing tube, wherein the volume of each tube is 1 mL; before the 20% DMSO frozen protective solution is used, the protective solution is put into a refrigerator to be refrigerated and balanced to 4 ℃;
(12) and (3) programmed cooling and storing: putting the freezing tube into a precooled program cooling box, putting the box into a refrigerator at minus 80 ℃ for overnight, and then transferring the box into liquid nitrogen for formal storage;
(13) and (3) detection: carrying out cell counting, activity detection, flow detection, induced differentiation capacity detection and growth curve determination on the stem cells;
the supplementary progesterone amount in the step (8) is 1.5 multiplied by 10-9mol/L -2×10-9mol/L；
The 20% DMSO cryopreservation protective solution in the step (11) is DMSO: serum-free complete medium: the human serum albumin is prepared according to the volume ratio of 1:3: 1.
2. The method for preparing mesenchymal stem cells from placental decidua according to claim 1, wherein the specific operation method for cleaning and shearing the tissue in step (3) comprises: soaking the decidua tissue in 75% alcohol for 1min, and rapidly transferring to normal saline for cleaning; removing the capillary vessel with aseptic toothed forceps; then, the bottom decidua tissue is soaked in normal saline containing 100U/ml penicillin and 100U/ml streptomycin for 5min, and finally, the bottom decidua is washed by the normal saline for 7 times to remove the residue of the penicillin and the streptomycin; will be cleanedTransferring the tissue block with decidua from bottom to 50ml centrifuge tube, and shearing to 0.5-0.8cm with sterile scissors3And (3) fine particles.
3. The method for preparing mesenchymal stem cells of placental decidua according to claim 1, wherein the first digestion step of step (4) is: preparing mixed enzyme for first digestion according to the volume of the bottom decidua, putting the mixed enzyme in a water bath kettle at 37 ℃ before use, incubating to 37 ℃, adding 0.2wt% of type II collagenase and 0.4wt% of type IV collagenase into a centrifuge tube, wherein the volume ratio of each enzyme in the mixed enzyme is 1:1, and the volume ratio of the mixed enzyme to the bottom decidua tissue mass is 1: 1; mixing the mixed enzyme with the tissue block, and digesting in a shaker at 37 deg.C and 120rpm for 1 h.
4. The method for preparing mesenchymal stem cells of placental decidua according to claim 1, wherein the second digestion step of step (5) is: after the first digestion is completed, 0.2wt% of dispase in total volume of 1/4 is added, mixed evenly and put into a shaker with 37 ℃ and 120rpm for digestion for 15 min.
5. The method for preparing mesenchymal stem cells of placental decidua according to claim 1, wherein the volume ratio of the physiological saline and 6wt% hydroxyethyl starch in step (7) is 1:5, and the two are fully mixed and centrifuged at 150 rpm for 5 min; carefully sucking the supernatant, centrifuging at 1200rpm for 6min, and removing the supernatant; washed 2 times with physiological saline.
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| CN111575229B (en) * | 2020-04-13 | 2021-04-30 | 广东华夏健康生命科学有限公司 | Separation method of placenta decidua stem cells |
| 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 |
| CN112442480A (en) * | 2020-11-17 | 2021-03-05 | 焕生汇生物基因技术(北京)有限公司 | Preparation method and application of exosome derived from human basal decidua mesenchymal stem cells |
| CN112852723A (en) * | 2020-12-31 | 2021-05-28 | 广东唯泰生物科技有限公司 | Method for processing and culturing primary placental wall periostracum mesenchymal stem cells |
| WO2023044902A1 (en) * | 2021-09-27 | 2023-03-30 | 金涌长生医学生物科技股份有限公司 | Decidual placental mesenchymal stem cell and use thereof in preparation of pharmaceutical composition for promoting angiogenesis |
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