CN105420179A - Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues - Google Patents
Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues Download PDFInfo
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Abstract
The invention relates to a method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues. The method comprises the steps of (1) sampling umbilical cord and placenta amnia and inoculating; (2) performing amplifying and passage on mesenchymal stem cells and amnion epithelial cells in a culture flask; (3) identifying the mesenchymal stem cells and the amnion epithelial cells: 1) when culture cells is transferred to the third generation, identifying the purity of culture cells after trypsinization; 2) verifying the cell purity to be 90 percent or more, namely the standard is met, and a mixture of the epithelial cells and the mesenchymal stem cells is obtained. The method is low in cost, simple and rapid, umbilical cord and placenta amnia are cut into tiny pieces by using a tissue cutting machine, which is conducive to swimming out of the tissues after the cells are directly inoculated in the culture flask; the cost is greatly lowered due to the fact that no proteinase reagent is used, and the preparation time is reduced, so that large-scale cell preparation in short time is realized; the method uses cell culture mediums being added with growth factors and other nutrients, and serum is saved.
Description
Technical field
The invention belongs to technical field of cell culture, especially a kind of method organizing extraction epithelial cell and mescenchymal stem cell simultaneously from umbilical cord and placenta amnion.
Background technology
Umbilical cord and placenta belong to embryo's surrounding tissue, and umbilical cord tissue is the source of mescenchymal stem cell (mesenchymalstemcells, MSCs).Placenta is made up of placenta amnion, chorion and basal decidua.Placenta contains a large amount of multiple stem cells, placenta amnion epithelial cell (amnioticepithelialcells, AECs) there is the characteristic of multiple stem cell, wherein there are some Subaerial blue green algae, its characteristic is embryonic stem cell (embryonicstemcell, ESCs) closely.Placenta mesenchyma stem cell is then present under amniotic epithelial cells layer.Amnion originates from the epithelial lining of embryo, its occur early than interior, in, outer three germinal layers, therefore, the differentiation capability of the sub-totipotent cell of placenta is better than the stem cell originating from other three germinal layers.Many experiments prove that they are more easily induced to differentiate the various cells becoming body, and the potential therefore for clinical treatment is just larger, also just has larger market.Compared to umbilical cord, it is fewer that the work obtaining stem cell from placenta is also carried out, and therefore the meaning of this development project is just more great.Chorionic villi of placenta is also containing a large amount of mescenchymal stem cells and hemopoietic stem cell (Hematopoieticstemcells, HSCs); But because its volume is very large, be not easy to draw materials, so be the last selection utilizing placenta tissue.
Human mesenchymal stem cell (MSC) and epithelial stem cell (ESC) are implanted in treatment various diseases and comprise treatment degenerative neuropathy, newborn baby ischemic and Adult Human Brain apoplexy, and have in cancer therapy and study widely, and obtain clinical application in multiple field.
By retrieval, find following several sections of patent publication us relevant to present patent application:
1, Human plactnta Subaerial blue green algae and stem cell bank construction process (CN103966159A) thereof, provide a kind of Human plactnta Subaerial blue green algae, it derives from human placenta's amnion of stripping, for the cellular features contained by the epithelial lining of amnion and mesenchyme layer, have employed the method be progressively separated, decrease the pollution of amnion mesenchymal confluent monolayer cells to the sub-totipotent cell of placenta to greatest extent, effectively improve the dryness of the sub-totipotent cell of Human plactnta.This invention additionally provides a kind ofly to be prepared Human plactnta Subaerial blue green algae from placenta amnion and sets up the method for stem cell bank, this invention is after peeling off human placenta's amnion and be cut into 5 × 5cm tissue block, with mesenchyme layer, the method be progressively separated be have employed to the epithelial lining of amnion, first protein enzyme solution digestion is carried out, its protein enzyme solution used contains trypsinase, Collagenase II, and EDTA.Then use Ficoll lymphocyte separation medium gradient centrifugation further, gather cell between two-layer liquid level to reach the object of a kind of Human plactnta Subaerial blue green algae of separation and Culture.
The step of the separate tissue of foregoing invention method is many, and a large amount of a variety of reagent be used to comprise expensive collagenase II.
2, a kind of Isolation and ldentification method (CN104450612A) of human amnion mesenchymal stem cell, disclose a kind of Isolation and ldentification method of human amnion mesenchymal stem cell, tissue explants adherent method is adopted to isolate amnion mesenchymal stem cell and Secondary Culture, P1-P3 is carried out to the observation of cellular form for cell, six well plate method are adopted to detect cell proliferative conditions with the positive molecule of flow cytomery surface of cell membrane and negative molecule, it is hAMSCs that cell is isolated in this experiment from placenta amnion tissue, for studying biology and the immunological characteristic of hAMSCs further, differentiation capability, karyotypic stability, and it can be used as the seed cell etc. of clinical application to provide the foundation.This invention, after stripping human placenta amnion, is cut into very tiny fritter (about 1-2cm) with eye scissors, then gripping be laid in Tissue Culture Flask, make cell vacillate out to realize adherent growth and propagation from tissue block in cultivation.When Growth of Cells merges in blocks, wash away amnion tissue block by sterile phosphate buffered saline (PBS).
Use a large amount of reagent to comprise expensive collagenase although aforesaid method avoids, the tissue block of plantation is still larger, and in-house cell can not be allowed the most effectively to vacillate out.And manual to shear and lay also very consuming time, be used in placenta amnion draw materials fashion can, but being used in umbilical cord draws materials just unactual, because the amount of umbilical cord tissue is much bigger, and very hard.
Through technical comparison, present patent application and above-mentioned two sections of patent publication us exist larger different.
Summary of the invention
The object of the invention overcomes the deficiencies in the prior art part, provides a kind of low cost, organizes the method simultaneously extracting epithelial cell and mescenchymal stem cell quickly and easily from umbilical cord and placenta amnion.
To achieve these goals, the technical solution adopted in the present invention is as follows:
Organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, step is as follows:
(1) umbilical cord and placenta amnion are drawn materials and are inoculated:
1. high-temperature sterilization instruments and large glass dish, simultaneously disinfection by ultraviolet light Bechtop;
2. substratum is prepared: the composition of perfect medium is: DMEM/F12 adds the mixing additive of the foetal calf serum of perfect medium cumulative volume 5%, the Regular Insulin of perfect medium cumulative volume 1%, siderophilin and selenium, in perfect medium, final concentration is that in the Urogastron of 20 ngs/ml and perfect medium, final concentration is the basic fibroblast growth factor of 20 ngs/ml, sterile filtration, 4 DEG C of preservations;
In described DMEM/F12, the volume ratio of DMEM:F12 is 1:1; Described mixing additive and the final concentration in mixing additive are Regular Insulin 5 mcg/ml, siderophilin 10 mcg/ml and Sodium Selenite 5 ngs/ml;
3. strictly screen donor, blood testing gets rid of transmissible disease;
4. umbilical cord and placenta is transported in low temperature ice box: after mechanically peel placenta amnion, umbilical cord and placenta amnion are placed in large glass dish respectively, following steps are all aseptically carried out, umbilical cord is cut into the segment that 1cm is long, placenta amnion is cut into the square that about 5X5cm is long, with the Streptomycin sulphate cleansing tissue twice of the penicillin+cumulative volume 1% of phosphate-buffered saline+cumulative volume 1%, the blood of wash clean umbilical cord;
5. umbilical cord and placenta amnion tissue are placed in the phosphate-buffered saline of 4 DEG C, cell survival is had influence on to prevent from being organized in overheated when machinery shreds, using-system cutting machine is broken into the fragment of 1-3mm respectively, use low speed disrupting tissue, keep cytoactive simultaneously, obtain broken umbilical cord and amnion tissue;
6. the umbilical cord of fragmentation and amnion tissue are transferred in T175 Tissue Culture Flask respectively, and with organizing scraper to be laid at the bottom of culturing bottle, carefully add a small amount of perfect medium, make nutrient solution can cover broken umbilical cord and amnion tissue block, be unlikely to again to make it floating, be placed in 37 DEG C, saturated humidity 5%CO
2cultivate in incubator;
(2) mescenchymal stem cell and amniotic epithelial cells increase and go down to posterity in culturing bottle:
1. observe visible cell in culturing process to vacillate out gradually from tissue block, and realize adherent growth and propagation further, cultivate 3-4 days, when Growth of Cells merge in blocks time, wash away remaining tissue by sterile phosphate buffered saline, be all changed to fresh complete medium continue cultivate;
2. within after this every 4 days, change a nutrient solution until cell covers with 90-100% culturing bottle floorage, just carry out trysinization and go down to posterity;
3. trysinization is gone down to posterity: abandon nutrient solution, cell is washed 2 times with PBS, every T175 is that the pancreas enzyme-EDTA of 0.25% was 37 DEG C of digestion 10 minutes with 10 milliliters of mass percents, after cell suspension each T175 add 1 milliliter of foetal calf serum with in and pancreatin reaction, 1000 revs/min centrifugal 10 minutes, settling flux, in perfect medium, goes down to posterity in new T175 culturing bottle in 1:4 cell count ratio;
(3) the qualification of mescenchymal stem cell and amniotic epithelial cells:
1. when the third generation is passed in trysinization, the purity with flow cytometry culturing cell:
The marker of amniotic epithelial cells comprises: Keratin sulfate CK19, CD29, and CD34; The marker of placenta and umbilical cord mesenchymal stem cells comprises: CD73, CD90, and CD105;
2. flow cytometer checking cell purity reaches more than 90%, and common transmissible disease is got rid of in chemical examination, and gets rid of bacterium, mycoplasma contamination, is namely conformance with standard, obtains the mixture of epithelial cell and mescenchymal stem cell.
And, described step (1) 3. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis; Or, described step (3) 2. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis.
And, described step (1) 5. in low speed be 400-800 rev/min.
And, the freezen protective of the mixture of described epithelial cell and mescenchymal stem cell and method for resuscitation, step is as follows:
(1) get the mixture of epithelial cell and mescenchymal stem cell, wash twice to wash FBS off with PBS, 0.25% pancreas enzyme-EDTA digestion, every T175 with 10 milliliters, 37 DEG C, 10 minutes, after cell suspension each T175 add 1 milliliter of FBS with in and pancreatin react.Centrifugal 10 minutes at 1000 revs/min;
(2) press cell density (1-3) X10 again
7/ ml is suspended in cells frozen storing liquid, is dispensed in 2ml cryopreservation tube, uses cell cryopreservation box frozen at-80 DEG C of refrigerator slow coolings, transfers to that liquid nitrogen container is medium-term and long-term to be saved backup after one day;
Described cells frozen storing liquid composition: the DMSO of total mass 20%, the human serum albumin of total mass 20%, the 60%M199 substratum of total mass;
(3), before using cell, thawed rapidly in 37 DEG C of water-baths by cell in cryopreservation tube, centrifugal 10 minutes at 1000 revs/min, settling flux is in PBS, and being prepared into cell density is 1x10
8the cell suspension of/ml, to be implanted in interior experiment and clinical application as comprising, is kept at 4 DEG C before use always.
The advantage that the present invention obtains and positively effect are:
Present method cost is low, simple and quick, umbilical cord and placenta amnion are shredded into minimum fragment (being less than 2-3mm) by using-system cutting machine, much smaller than method any at present, be very beneficial to cell after being directly inoculated into culturing bottle and vacillate outside tissue, thus realize the growth of note wall; Do not use any protease reagent, thus reduce costs significantly, reduce preparation time, increase yield, prepared by mass cell become a reality; The method uses adds somatomedin and other nutraceutical complete cell culture medium, thus reduce foetal calf serum (FBS) concentration to 5%, but obtain the culture effect using 10%FBS at the same level and better with routine, save serum, but unlike most of serum-free culture, cause cell proliferation to slow down or differentiation ahead of time.
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
The reagent used in the present invention, if no special requirements, is the common agents in this area; The method used in the present invention, if no special requirements, is the ordinary method in this area.
Organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, step is as follows:
(1) umbilical cord and placenta amnion are drawn materials and are inoculated:
1. high-temperature sterilization instruments and large glass dish, simultaneously disinfection by ultraviolet light Bechtop;
2. substratum is prepared: the composition of perfect medium is: DMEM/F12 adds the mixing additive of the foetal calf serum of perfect medium cumulative volume 5%, the Regular Insulin of perfect medium cumulative volume 1%, siderophilin and selenium, in perfect medium, final concentration is that in the Urogastron of 20 ngs/ml and perfect medium, final concentration is the basic fibroblast growth factor of 20 ngs/ml, sterile filtration, 4 DEG C of preservations;
In described DMEM/F12, the volume ratio of DMEM:F12 is 1:1; Described mixing additive and the final concentration in mixing additive are Regular Insulin 5 mcg/ml, siderophilin 10 mcg/ml and Sodium Selenite 5 ngs/ml;
3. strictly screen donor, blood testing is got rid of and is comprised acquired immune deficiency syndrome (AIDS), first, second, hepatitis C, the transmissible diseases such as syphilis;
4. umbilical cord and placenta is transported in low temperature ice box: after mechanically peel placenta amnion, umbilical cord and placenta amnion are placed in large glass dish respectively, following steps are all aseptically carried out, by the segment of umbilical cord scissors into about 1cm length, placenta amnion is cut into the square that about 5X5cm is long, with the Streptomycin sulphate cleansing tissue twice of the penicillin+cumulative volume 1% of phosphate-buffered saline+cumulative volume 1%, the blood of wash clean umbilical cord as far as possible;
5. umbilical cord and placenta amnion tissue are placed in the PBS of 4 DEG C, cell survival is had influence on to prevent from being organized in overheated when machinery shreds, using-system cutting machine is broken into minimum piece (fragment of about 1-3mm) respectively, use low speed (such as, 400-800 rev/min) disrupting tissue, keep cytoactive simultaneously, obtain broken umbilical cord and amnion tissue;
6. the umbilical cord of fragmentation and amnion tissue are transferred in T175 Tissue Culture Flask respectively, and with organizing scraper to be laid at the bottom of culturing bottle, carefully add a small amount of perfect medium, make nutrient solution can cover broken umbilical cord and amnion tissue block, be unlikely to again to make it floating, be placed in 37 DEG C, saturated humidity 5%CO
2cultivate in incubator;
(2) mescenchymal stem cell and amniotic epithelial cells increase and go down to posterity in culturing bottle:
1. observe visible cell in culturing process to vacillate out gradually from tissue block, and realize adherent growth and propagation further, cultivate 3-4 days, when Growth of Cells merge in blocks time, wash away remaining tissue with aseptic PBS, be all changed to fresh complete medium continue cultivate;
2. within after this every 4 days, change a nutrient solution until cell covers with 90-100% culturing bottle floorage, just carry out trysinization and go down to posterity;
3. trysinization is gone down to posterity: abandon nutrient solution, washes cell 2 times with PBS, and 0.25% pancreas enzyme-EDTA (every T175 is with 10 milliliters) was 37 DEG C of digestion 10 minutes.After cell suspension each T175 add 1 milliliter of FBS with in and pancreatin reaction, 1000 revs/min are centrifugal 10 minutes, and settling flux, in perfect medium, goes down to posterity in new T175 culturing bottle in 1:4 cell count ratio;
(3) the qualification of mescenchymal stem cell and amniotic epithelial cells:
1., when the third generation is passed in trysinization, the purity of culturing cell is identified with flow cytometer (FACS):
The marker of amniotic epithelial cells comprises: Keratin sulfate CK19, CD29, and CD34; The marker of placenta and umbilical cord mesenchymal stem cells comprises: CD73, CD90, and CD105;
2. flow cytometer checking cell purity reaches more than 90%, and chemical examination eliminating comprises acquired immune deficiency syndrome (AIDS), first, second, the transmissible disease that hepatitis C etc. are common, and gets rid of bacterium, mycoplasma contamination, is namely conformance with standard, obtains the mixture of epithelial cell and mescenchymal stem cell.
Umbilical cord cells cultivates the mescenchymal stem cell of generation more than 90%, and amnion cell cultivates generation mescenchymal stem cell and epithelial cell (wherein containing the sub-totipotent cell of part), because these two kinds of cells are play synergy in most of clinical application, and be combined utilization, so there is no need to be separated, as long as it is conformance with standard that the summation of these two kinds of cells accounts for more than 90% of total cell count, what net result obtained is purer mescenchymal stem cell and mescenchymal stem cell and epithelial cell mixed culture, can according to clinical needs choice for use.
The freezen protective of the mixture of above-mentioned epithelial cell and mescenchymal stem cell and method for resuscitation, step is as follows:
(1) get the mixed culture of epithelial cell and mescenchymal stem cell, twice is washed to wash FBS off, 0.25% pancreas enzyme-EDTA digestion (every T175 is with 10 milliliters), 37 DEG C with PBS, 10 minutes, after cell suspension each T175 add 1 milliliter of FBS with in and pancreatin reaction.1000 revs/min centrifugal 10 minutes;
(2) press cell density (1-3) X10 again
7/ ml is suspended in cells frozen storing liquid, is dispensed in 2ml cryopreservation tube, uses cell cryopreservation box frozen at-80 DEG C of refrigerator slow coolings, transfers to that liquid nitrogen container is medium-term and long-term to be saved backup after one day;
Described cells frozen storing liquid composition: the DMSO of total mass 20%, the human serum albumin of total mass 20%, the 60%M199 substratum of total mass;
(3), before using cell, thawed rapidly in 37 DEG C of water-baths by cell in cryopreservation tube, centrifugal 10 minutes at 1000 revs/min, settling flux is in PBS, and being prepared into cell density is 1x10
8the cell suspension of/ml, to be implanted in interior experiment and clinical application as comprising, is kept at 4 DEG C before use always.
Claims (4)
1. organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, it is characterized in that: step is as follows:
(1) umbilical cord and placenta amnion are drawn materials and are inoculated:
1. high-temperature sterilization instruments and large glass dish, simultaneously disinfection by ultraviolet light Bechtop;
2. substratum is prepared: the composition of perfect medium is: DMEM/F12 adds the mixing additive of the foetal calf serum of perfect medium cumulative volume 5%, the Regular Insulin of perfect medium cumulative volume 1%, siderophilin and selenium, in perfect medium, final concentration is that in the Urogastron of 20 ngs/ml and perfect medium, final concentration is the basic fibroblast growth factor of 20 ngs/ml, sterile filtration, 4 DEG C of preservations;
In described DMEM/F12, the volume ratio of DMEM:F12 is 1:1; Described mixing additive and the final concentration in mixing additive are Regular Insulin 5 mcg/ml, siderophilin 10 mcg/ml and Sodium Selenite 5 ngs/ml;
3. strictly screen donor, blood testing gets rid of transmissible disease;
4. umbilical cord and placenta is transported in low temperature ice box: after mechanically peel placenta amnion, umbilical cord and placenta amnion are placed in large glass dish respectively, following steps are all aseptically carried out, umbilical cord is cut into the segment that 1cm is long, placenta amnion is cut into the square that about 5X5cm is long, with the Streptomycin sulphate cleansing tissue twice of the penicillin+cumulative volume 1% of phosphate-buffered saline+cumulative volume 1%, the blood of wash clean umbilical cord;
5. umbilical cord and placenta amnion tissue are placed in the phosphate-buffered saline of 4 DEG C, cell survival is had influence on to prevent from being organized in overheated when machinery shreds, using-system cutting machine is broken into the fragment of 1-3mm respectively, use low speed disrupting tissue, keep cytoactive simultaneously, obtain broken umbilical cord and amnion tissue;
6. the umbilical cord of fragmentation and amnion tissue are transferred in T175 Tissue Culture Flask respectively, and with organizing scraper to be laid at the bottom of culturing bottle, carefully add a small amount of perfect medium, make nutrient solution can cover broken umbilical cord and amnion tissue block, be unlikely to again to make it floating, be placed in 37 DEG C, saturated humidity 5%CO
2cultivate in incubator;
(2) mescenchymal stem cell and amniotic epithelial cells increase and go down to posterity in culturing bottle:
1. observe visible cell in culturing process to vacillate out gradually from tissue block, and realize adherent growth and propagation further, cultivate 3-4 days, when Growth of Cells merge in blocks time, wash away remaining tissue by sterile phosphate buffered saline, be all changed to fresh complete medium continue cultivate;
2. within after this every 4 days, change a nutrient solution until cell covers with 90-100% culturing bottle floorage, just carry out trysinization and go down to posterity;
3. trysinization is gone down to posterity: abandon nutrient solution, cell is washed 2 times with PBS, every T175 is that the pancreas enzyme-EDTA of 0.25% was 37 DEG C of digestion 10 minutes with 10 milliliters of mass percents, after cell suspension each T175 add 1 milliliter of foetal calf serum with in and pancreatin reaction, 1000 revs/min centrifugal 10 minutes, settling flux, in perfect medium, goes down to posterity in new T175 culturing bottle in 1:4 cell count ratio;
(3) the qualification of mescenchymal stem cell and amniotic epithelial cells:
1. when the third generation is passed in trysinization, the purity with flow cytometry culturing cell:
The marker of amniotic epithelial cells comprises: Keratin sulfate CK19, CD29, and CD34; The marker of placenta and umbilical cord mesenchymal stem cells comprises: CD73, CD90, and CD105;
2. flow cytometer checking cell purity reaches more than 90%, and common transmissible disease is got rid of in chemical examination, and gets rid of bacterium, mycoplasma contamination, is namely conformance with standard, obtains the mixture of epithelial cell and mescenchymal stem cell.
2. according to claim 1ly organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, it is characterized in that: described step (1) 3. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis; Or, described step (3) 2. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis.
3. according to claim 1ly organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, it is characterized in that: described step (1) 5. in low speed be 400-800 rev/min.
4. the method organizing extraction epithelial cell and mescenchymal stem cell simultaneously from umbilical cord and placenta amnion according to any one of claims 1 to 3, it is characterized in that: the freezen protective of the mixture of described epithelial cell and mescenchymal stem cell and method for resuscitation, step is as follows:
(1) get the mixture of epithelial cell and mescenchymal stem cell, wash twice to wash FBS off with PBS, 0.25% pancreas enzyme-EDTA digestion, every T175 with 10 milliliters, 37 DEG C, 10 minutes, after cell suspension each T175 add 1 milliliter of FBS with in and pancreatin react.Centrifugal 10 minutes at 1000 revs/min;
(2) press cell density (1-3) X10 again
7/ ml is suspended in cells frozen storing liquid, is dispensed in 2ml cryopreservation tube, uses cell cryopreservation box frozen at-80 DEG C of refrigerator slow coolings, transfers to that liquid nitrogen container is medium-term and long-term to be saved backup after one day;
Described cells frozen storing liquid composition: the DMSO of total mass 20%, the human serum albumin of total mass 20%, the 60%M199 substratum of total mass;
(3), before using cell, thawed rapidly in 37 DEG C of water-baths by cell in cryopreservation tube, centrifugal 10 minutes at 1000 revs/min, settling flux is in PBS, and being prepared into cell density is 1x10
8the cell suspension of/ml, to be implanted in interior experiment and clinical application as comprising, is kept at 4 DEG C before use always.
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