CN116574677A - Method for separating and extracting amniotic mesenchymal stem cells from human placenta - Google Patents
Method for separating and extracting amniotic mesenchymal stem cells from human placenta Download PDFInfo
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- C12N5/0602—Vertebrate cells
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Abstract
The invention provides a method for separating and extracting amniotic mesenchymal stem cells from human placenta. The method comprises the following steps: 1) Cleaning; 2) Stripping; 3) Digestion; 4) Removing impurities; 5) Wall-attached culture; 6) Differential digestion and passage; 7) Removing impurities and climbing out for the second time; 8) Culturing in a large quantity; step 6) and the P1 generation cells obtained in the step 7) are subjected to differential digestion again, and after inoculation culture, P2 generation amniotic mesenchymal stem cells are obtained; 9) And (5) subculturing. According to the invention, the epithelial cells of the amniotic membrane are digested by trypsin to remove the mixed cells and then are subjected to tissue block culture, so that the damage to primary cells caused by overlong digestion time by a two-step enzyme digestion method can be reduced, and meanwhile, the economic cost brought by using collagenase is reduced; but also can avoid the defects of overlong cell climbing out time and excessive mixed cells in the tissue block culture method, and reduce the time cost; and meanwhile, the tissue blocks are repeatedly utilized by secondary adherence, so that the primary cell obtaining amount is improved.
Description
Technical Field
The invention relates to the technical field, in particular to a method for separating and extracting amniotic mesenchymal stem cells from human placenta.
Background
Mesenchymal stem cells (Mesenchymal Stem cell, MSCs) are adult stem cells with high self-renewal capacity, multipotent differentiation potential, originating from mesoderm, originally found in bone marrow, mainly in connective tissue and organ interstitium, capable of differentiating into various interstitial tissues such as bone, cartilage, adipose tissue, etc. under suitable induction conditions.
Placenta (plamenta) is an important tissue organ for mass exchange between fetus and mother, and is divided into an amniotic membrane layer, a chorion layer and an decidua layer from inside to outside, wherein the amniotic membrane layer is one of important sources of mesenchymal stem cells. The amniotic membrane-derived mesenchymal stem cells have the characteristics of large differentiation potential, strong proliferation capability, low immunogenicity, convenient material acquisition, no limitation of ethic problems, easy industrial preparation and the like, and are likely to become the pluripotent stem cells with the most clinical application prospect.
The amniotic membrane (Amniotic membrane) is positioned at the innermost layer of the placenta, has no distribution of blood vessels, nerves and lymphatic vessels, and comprises the following components from inside to outside: epithelial layer, basement membrane, dense layer, fibroblast layer and sponge layer. Wherein the amniotic mesenchymal stem cells exist in subcutaneous layers such as a compact layer.
At present, the common method for separating and extracting the human amniotic mesenchymal stem cells comprises the following steps: two-step enzymatic digestion and tissue mass adherent culture. The two-step enzyme digestion method is to digest tissues by using pancreatin and collagenase in combination, separate and obtain single cells for culture; the tissue block adherence culture method is to adhere tissue in a culture vessel and to carry out adherence growth after the cells climb out by themselves.
1) The two-step enzyme digestion method has long digestion time, and chemical reagents have great damage to cells, so that the cell morphology is poor, the quality is low, and meanwhile, the cost of the reagents is high, so that the method is not beneficial to large-scale separation culture;
2) The tissue block adherence culture method has long cell climbing-out adherence time, and the number of the harvested primary cells in the culture process is small, the purity is low, and the primary cells need to be purified for multiple times.
Accordingly, there remains a need for those skilled in the art to further improve the need for methods of isolating and extracting human amniotic mesenchymal stem cells.
Disclosure of Invention
The invention aims to provide a method for separating and extracting amniotic mesenchymal stem cells from human placenta, which is used for solving the problems of overlong cell culture period and too slow proliferation speed of the traditional method.
The invention is realized by adopting the following technical scheme.
A method for separating and extracting amniotic mesenchymal stem cells from human placenta, which comprises the following process steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by using hemostats, the surface of the placenta is washed by using physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed;
2) Stripping off
Peeling amniotic membrane from placenta with hook forceps, spreading on 10cm culture dish, washing with physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin to semitransparent state, and cutting into 3-4 cm 2 Is a block of tissue;
3) Digestion
Transferring the amniotic membrane block into a 150mL liquid storage bottle, adding 0.25% trypsin which is 1.5 times of the total volume of the amniotic membrane, putting into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 30min, centrifuging at 1200rpm/min for 5min, and discarding the supernatant; adding trypsin 0.25% which is 1.5 times of the total volume of the amniotic membrane again, digesting for 30min under the same condition, taking out a liquid storage bottle for observation every 10min in the two digestion processes, and shaking vigorously to ensure that the amniotic membrane blocks are fully contacted with liquid; the aim of the operation is to digest the epithelial cells and other miscellaneous cells, improve the cell purity and facilitate the climbing out of the amniotic mesenchymal stem cells.
4) Impurity removal
Filtering the digested tissue mixture with 70 μm cell sieve, removing filtrate, taking out the tissue in the net, placing in 10cm culture dish, rinsing with physiological saline for 2 times, and cutting into 0.3cm pieces with direct shear 2 Left and right tissue blocks.
5) Wall-attached culture
Spreading the chopped tissue blocks in a 10cm culture dish, inversely culturing for 1-2 h until the tissue clings to the culture dish, standing the culture dish, adding 5mL culture medium (friend placenta mesenchymal stem cell culture medium, product number: NC 0103) at 37deg.C, and 5% CO 2 Culturing in incubator, and changing liquid 1 time every 2 days.
6) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach confluence degreeThe culture solution is discarded after 85 percent of culture solution is added with 5mL of PBSPBS buffer (1X), cat No.: PBS-10001) washing culture dish, sucking out, slowly adding 1mL of 0.25% trypsin to digest cells for 2 min, sucking out cell suspension to 50mL centrifuge tube to stop digestion (without washing bottom cells of culture dish), centrifuging at 1200rpm/min for 5min, discarding supernatant, adding 20mL of YOUKANG culture medium, blowing uniformly, inoculating into T182 culture bottle, shaking uniformly by crisscross method, standing at 37deg.C, and standing at 5% CO 2 Culturing in an incubator; the operation is characterized in that the purity of the collected amniotic mesenchymal stem cells is higher by utilizing the digestion time difference.
7) Impurity removal and secondary cell climbing out
Adding 0.25% trypsin to the culture dish containing the adhered amniotic membrane tissue block in the step 6) again for 5 minutes, slightly blowing off cells at the bottom of the culture dish by a pipette, discarding, slowly cleaning the bottom of the culture dish for 2 times by using normal saline, adding 5mL of friendly culture medium to enable the tissue to climb out of mesenchymal stem cells for the second time, when the cell confluency reaches 85%, performing differential digestion according to the step 6), inoculating the cells into a T182 culture bottle, shaking uniformly by a crisscross method, placing at 37 ℃ and placing at 5% CO 2 Culturing in an incubator; the operation is characterized in that the mixed cells which are firstly adhered to the wall are removed, and meanwhile, a large number of primary cells are obtained by repeatedly utilizing tissue blocks.
8) Culturing in large quantities
And 6) carrying out differential digestion on the P1 generation cells obtained in the step 6) and the step 7), and inoculating and culturing to obtain the P2 generation amniotic mesenchymal stem cells.
9) Subculture
After the confluence degree of the P2 generation amniotic mesenchymal stem cells reaches 85%, digesting the cells by using 0.25% trypsin, when the cells are rounded and not falling off, flushing the bottom of a T182 culture bottle by using a pipette to collect the cells, centrifuging at 1200rpm/min for 5min after the digestion is stopped, and carrying out re-suspension seed culture on the cell sediment by using a culture solution to obtain the P3 generation mesenchymal stem cells. And collecting the P3 generation cells after the confluency of the P3 generation mesenchymal stem cells reaches 85 percent.
Preferably, the above is derived from human placentaMethod for separating and extracting amniotic mesenchymal stem cells, in step 6), 3000cells/cm are used 2 Inoculating to 7T 182 culture flasks, adding 20ml of YOUKANG culture medium, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells;
the P1 generation cells cultured in this step were A group, and 4T 182 flasks were numbered A- (1), A- (2), A- (3), A- (4), A- (5), A- (6) and A- (7) in this order.
Preferably, in the above method for separating and extracting amniotic mesenchymal stem cells from human placenta, in step 7), the cells are digested at 3000cells/cm according to step 6) at a differential speed 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells; the time to climb out and the cell confluence were recorded for 85% of the time;
the P1 generation cells cultured in this step were B group, and 7T 182 flasks were numbered as B- (1), B- (2), B- (3), B- (4), B- (5), B- (6) and B- (7) in this order.
Preferably, in the above method for separating and extracting amniotic mesenchymal stem cells from human placenta, in step 8), the ratio of cells/cm is 3000 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P2 generation amniotic mesenchymal stem cells;
the P2 generation cells subjected to one-time adherence culture in the step are C groups, and 7T 182 culture bottles are sequentially numbered as C- (1), C- (2), C- (3), C- (4), C- (5), C- (6) and C- (7);
the P2-generation cells of the secondary adherence culture are D groups, and 7T 182 culture flasks are sequentially numbered as D (1), D- (2), D- (3), D- (4), D- (5), D- (6) and D- (7).
Preferably, in the above method for separating and extracting amniotic mesenchymal stem cells from human placenta, in step 9), the steps of:
the culture solution is discarded after the T182 culture flask with the number of (1) is inoculated with cells for 24 hours, 5mL of PBS is added to wash the culture dish, the culture dish is sucked out, 0.25% trypsin is added to digest the cells, when the cells become round and do not fall off, the bottom of the T182 culture flask is washed by a pipette to collect the cells, and the digestion is stopped, and the number of the cells is calculated by a countstar cell counter.
Then, cells numbered (2) to (6) were collected and counted at 24H intervals as described above.
By means of the technical scheme, the invention has the following beneficial effects:
1) According to the invention, by combining the traditional method, the epithelial cells of the amniotic membrane are digested by trypsin to remove the miscellaneous cells, so that the damage to primary cells caused by overlong digestion time of a two-step enzyme digestion method is reduced, and meanwhile, the economic cost brought by using collagenase is reduced; but also can avoid the defects of overlong cell climbing out time and excessive mixed cells in the tissue block culture method, and reduce the time cost; and meanwhile, the tissue blocks are repeatedly utilized by secondary adherence, so that the primary cell obtaining amount is improved.
2) The amniotic mesenchymal stem cells obtained by separation according to the technical scheme of the invention have the advantages of excellent quality, high purity, large quantity and high proliferation speed.
Drawings
FIG. 1 is a diagram showing the state (x 40) of the amniotic mesenchymal stem cells of the P0 generation in the first embodiment;
FIG. 2 is a graph showing the comparison of the proliferation curves of the cells of the generation P1;
FIG. 3 is a graph showing the comparison of the proliferation curves of the cells of the generation P2;
FIG. 4 is a diagram showing the state (x 40) of amniotic mesenchymal stem cells of example P2 generation;
FIG. 5 is a diagram of the state (x 40) of amniotic mesenchymal stem cells of a P0 generation of control example;
FIG. 6 is a state diagram of a piece of amniotic membrane tissue peeled off in comparative example II;
FIG. 7 is a diagram showing the state of osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation of amniotic mesenchymal stem cells in example III.
Detailed Description
The invention aims to provide a method for separating and extracting amniotic mesenchymal stem cells from human placenta, which is used for solving the problems of overlong cell culture period and too slow proliferation speed of the traditional method. That is, the enzyme digestion method and the tissue climbing method are combined.
The invention provides a method for separating and extracting amniotic mesenchymal stem cells from human placenta. The method comprises the following steps: 1) Cleaning; 2) Stripping; 3) Digestion; 4) Removing impurities; 5) Wall-attached culture; 6) Differential digestion and passage; 7) Removing impurities and climbing out for the second time; 8) Culturing in a large quantity; step 6) and the P1 generation cells obtained in the step 7) are subjected to differential digestion again, and after inoculation culture, P2 generation amniotic mesenchymal stem cells are obtained; 9) And (5) subculturing. According to the invention, the epithelial cells of the amniotic membrane are digested by trypsin to remove the mixed cells and then are subjected to tissue block culture, so that the damage to primary cells caused by overlong digestion time by a two-step enzyme digestion method can be reduced, and meanwhile, the economic cost brought by using collagenase is reduced; but also can avoid the defects of overlong cell climbing out time and excessive mixed cells in the tissue block culture method, and reduce the time cost; and meanwhile, the tissue blocks are repeatedly utilized by secondary adherence, so that the primary cell obtaining amount is improved.
1) Purifying by differential digestion;
the primary cells are damaged when the two-step enzyme digestion method is used for digestion, and the collagenase digestion step in the traditional enzyme digestion method is omitted;
2) The tissue mass secondary adherence method improves the cell quantity;
tissue mass adherent culture process has too many cells, and the invention adds a purification step before the tissue is attached to the incubator.
The invention provides a method for separating and extracting amniotic mesenchymal stem cells from human placenta, which specifically comprises the following process steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by a hemostat, the placenta surface is washed by physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed.
2) Stripping off
Peeling amniotic membrane from placenta with hook forceps, spreading on 10cm culture dish, washing with physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin to semitransparent state, and cutting into 3-4 cm 2 Is included in the block of tissue.
3) Digestion
Transferring the amniotic membrane block into a 150mL liquid storage bottle, adding 0.25% trypsin which is 1.5 times of the total volume of the amniotic membrane, putting into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 30min, centrifuging at 1200rpm/min for 5min, and discarding the supernatant; adding trypsin 0.25% which is 1.5 times of the total volume of the amniotic membrane again, digesting for 30min under the same condition, taking out a liquid storage bottle for observation every 10min in the two digestion processes, and shaking vigorously to ensure that the amniotic membrane blocks are fully contacted with liquid; the aim of the operation is to digest the epithelial cells and other miscellaneous cells, improve the cell purity and facilitate the climbing out of the amniotic mesenchymal stem cells.
4) Impurity removal
Filtering the digested tissue mixture with 70 μm cell sieve, removing filtrate, taking out the tissue in the net, placing in 10cm culture dish, rinsing with physiological saline for 2 times, and cutting into 0.3cm pieces with direct shear 2 Left and right tissue blocks.
5) Wall-attached culture
Spreading the chopped tissue blocks in a 10cm culture dish, inversely culturing for 1-2 h until the tissue clings to the culture dish, standing the culture dish, adding 5mL culture medium (friend placenta mesenchymal stem cell culture medium, product number: NC 0103) at 37deg.C, and 5% CO 2 Culturing in incubator, and changing liquid 1 time every 2 days.
6) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach 85 percent confluence, the culture solution is discarded, and 5mL PBS is addedPBS buffer (1X), cat No.: PBS-10001) washing culture dish, sucking out, slowly adding 1mL of 0.25% trypsin to digest cells for 2 min, sucking out cell suspension to 50mL centrifuge tube to stop digestion (without washing bottom cells of culture dish), centrifuging at 1200rpm/min for 5min, discarding supernatant, adding 20mL of YOUKANG culture medium, blowing uniformly, inoculating into T182 culture bottle, shaking uniformly by crisscross method, standing at 37deg.C, and standing at 5% CO 2 Culturing in an incubator; the operation is characterized in that the purity of the collected amniotic mesenchymal stem cells is higher by utilizing the digestion time difference.
7) Impurity removal and secondary cell climbing out
Adding 0.25% trypsin again to the culture dish containing the adhered amniotic membrane tissue block in the step 6) to digest cells for 5 minutes, slightly blowing off cells at the bottom of the culture dish by a pipette, discarding, slowly cleaning the bottom of the culture dish for 2 times by using normal saline, adding 5mL of friendly culture medium to enable the tissues to climb out of mesenchymal stem cells for the second time, when the cell confluency reaches 85%, carrying out differential digestion according to the step 6), inoculating the cells into a T182 culture bottle, shaking uniformly by a crisscross method, placing the culture dish at 37 ℃ and placing the culture dish at 5% CO 2 Culturing in an incubator; the operation is characterized in that the mixed cells which are firstly adhered to the wall are removed, and meanwhile, a large number of primary cells are obtained by repeatedly utilizing tissue blocks.
8) Culturing in large quantities
And 6) carrying out differential digestion on the P1 generation cells obtained in the step 6) and the step 7), and inoculating and culturing to obtain the P2 generation amniotic mesenchymal stem cells.
9) Subculture
After the confluence degree of the P2 generation amniotic mesenchymal stem cells reaches 85%, digesting the cells by using 0.25% trypsin, when the cells are rounded and not falling off, flushing the bottom of a T182 culture bottle by using a pipette to collect the cells, centrifuging at 1200rpm/min for 5min after the digestion is stopped, and carrying out re-suspension seed culture on the cell sediment by using a culture solution to obtain the P3 generation mesenchymal stem cells. And collecting the P3 generation cells after the confluency of the P3 generation mesenchymal stem cells reaches 85 percent.
Example 1
The embodiment provides a method for separating and culturing amniotic mesenchymal stem cells, which comprises the following steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by a hemostat, the placenta surface is washed by physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed.
2) Stripping off
Peeling amniotic membrane from placenta with hook forceps, spreading on 10cm culture dish, washing with physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin to semitransparent state, and cutting into 3-4 cm 2 Is an amniotic membrane block.
3) Digestion
Transferring the amniotic membrane block into a 150mL liquid storage bottle, adding 0.25% trypsin which is 1.5 times of the total volume of the amniotic membrane, putting into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 30min, centrifuging at 1200rpm/min for 5min, and discarding the supernatant; adding trypsin 0.25% which is 1.5 times of the total volume of the amniotic membrane again, digesting for 30min under the same condition, taking out the liquid storage bottle for observation every 10min in the two digestion processes, and shaking vigorously to ensure that the amniotic membrane is fully contacted with liquid.
4) Impurity removal
Filtering the digested tissue mixture with 70 μm cell sieve, removing filtrate, taking out the tissue in the net, placing in 10cm culture dish, rinsing with physiological saline for 2 times, and cutting into 0.3cm pieces with direct shear 2 Left and right tissue blocks.
5) Wall-attached culture
Spreading the chopped tissue blocks in a 10cm culture dish, inversely culturing for 1-2 h until the tissue clings to the culture dish, standing the culture dish, adding 5mL culture medium (friend placenta mesenchymal stem cell culture medium, product number: NC 0103) at 37deg.C, and 5% CO 2 Culturing in incubator, and changing liquid 1 time every 2 days. Cell climbing out and cell confluency were recorded for 85% of the time, see table 1. After 4d of incubation, it was seen that fibroblasts were climbing out of the tissue mass (FIG. 1), and confluency reached 85% for 8d of incubation.
6) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach 85 percent confluence, the culture solution is discarded, and 5mL PBS is addedPBS buffer (1X), cat No.: PBS-10001) washing the dish, sucking out, slowly adding 1mL of 0.25% trypsin to digest the cells for 2 min, sucking out the cell suspension to a 50mL centrifuge tube to stop digestion (without washing the cells at the bottom of the dish), centrifuging at 1200rpm/min for 5min, discarding the supernatant, and re-suspending the cell pellet at 3000cells/cm 2 Inoculating to 7T 182 culture flasks, adding 20ml of YOUKANG culture medium, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in incubator, and recording as P1 generation sheepMesenchymal stem cells.
The P1 generation cells cultured in this step were A group, and 4T 182 flasks were numbered A- (1), A- (2), A- (3), A- (4), A- (5), A- (6) and A- (7) in this order.
7) Tissue mass secondary adherence and passage
Adding 0.25% trypsin again to the culture dish containing the adhered amniotic membrane tissue block in the step 6) to digest the cells for 5 minutes, slightly blowing off the cells at the bottom of the culture dish by a pipette, discarding the cells, slowly cleaning the bottom of the culture dish for 2 times by using normal saline, adding 5mL of friend culture medium to enable the tissues to climb out of mesenchymal stem cells for the second time, and digesting the cells at a differential speed of 3000cells/cm according to the step 6) when the cell fusion degree reaches 85 percent 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells. The time to climb out was recorded as well as the cell confluence for 85% of the time, see table 1.
The P1 generation cells cultured in this step were B group, and 7T 182 flasks were numbered as B- (1), B- (2), B- (3), B- (4), B- (5), B- (6) and B- (7) in this order.
8) Culturing in large quantities
After the confluence of the P1 generation cells cultured in the step 6) and the step 7) reaches 85%, the cells are subjected to differential digestion again at 3000cells/cm 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P2 generation amniotic mesenchymal stem cells.
The P2 generation cells subjected to one-time adherence culture in the step are C groups, and 7T 182 culture bottles are sequentially numbered as C- (1), C- (2), C- (3), C- (4), C- (5), C- (6) and C- (7);
the P2-generation cells of the secondary adherence culture are D groups, and 7T 182 culture flasks are sequentially numbered as D (1), D- (2), D- (3), D- (4), D- (5), D- (6) and D- (7).
9) Counting
The culture solution is discarded after the T182 culture flask with the number of (1) is inoculated with cells for 24 hours, 5mL of PBS is added to wash the culture dish, the culture dish is sucked out, 0.25% trypsin is added to digest the cells, when the cells become round and do not fall off, the bottom of the T182 culture flask is washed by a pipette to collect the cells, and the digestion is stopped, and the number of the cells is calculated by a countstar cell counter.
Then, cells numbered (2) to (6) were collected and counted at 24H intervals as described above. The results are shown in Table 2 and FIGS. 2 to 4.
Comparative example one
The comparative example uses a two-step enzyme digestion method to separate and culture amniotic mesenchymal stem cells, and the separation culture method comprises the following steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by a hemostat, the placenta surface is washed by physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed.
2) Stripping off
Peeling amniotic membrane from placenta with hook forceps, spreading on 10cm culture dish, washing with physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin to semitransparent state, and cutting into 3-4 cm 2 Is an amniotic membrane block.
3) Pancreatin digestion
Transferring the amniotic membrane block into a 150mL liquid storage bottle, adding 0.25% trypsin which is 1.5 times of the total volume of the amniotic membrane, putting into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 30min, centrifuging at 1200rpm/min for 5min, and discarding the supernatant; adding trypsin 0.25% which is 1.5 times of the total volume of the amniotic membrane again, digesting for 30min under the same condition, taking out the liquid storage bottle for observation every 10min in the two digestion processes, and shaking vigorously to ensure that the amniotic membrane is fully contacted with liquid.
4) Impurity removal
Filtering the digested tissue mixture with 70 μm cell sieve, removing filtrate, taking out the tissue in the net, placing in 10cm culture dish, rinsing with physiological saline for 2 times, and cutting into 0.3cm pieces with direct shear 2 Left and right tissue blocks.
5) Collagenase digestion
Transferring the sheared tissue blocks into a 150mL liquid storage bottle, adding 0.25% type II collagenase into the bottle according to 2 times of the total volume of the amniotic membrane, and putting the liquid storage bottle into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 3 hours until the tissue blocks are completely digested.
6) Filtering and centrifuging
Filtering the digested tissue mixture with a 70 μm cell sieve, taking a cell suspension, adding an equal volume of physiological saline, and centrifuging at 1200rpm/min for 5min.
7) Wall-attached culture
Centrifuging, adding 5ml culture medium (YOUKANG placenta mesenchymal stem cell culture medium, product number: NC 0103) to resuspend cell precipitate (generation P0), inoculating into 10cm culture dish, shaking uniformly by crisscross method, standing at 37deg.C, 5% CO 2 Culturing in incubator, and changing liquid 1 time every 2 days. After 5d of culture, only a small amount of fibroblast-like cells attached to the wall, and the cell purity was low (fig. 5).
8) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach 85% confluence, the culture solution is discarded, 5mL PBS is added to wash the culture dish and then sucked out, 1mL of 0.25% trypsin is slowly added to digest the cells for 2 minutes and then the cell suspension is sucked out to a 50mL centrifuge tube to stop digestion (without flushing the cells at the bottom of the culture dish), the cells are centrifuged at 1200rpm/min for 5 minutes, the supernatant is discarded, and the cell sediment is resuspended at 3000cells/cm 2 Inoculating to 7T 182 culture flasks, adding 20ml of YOUKANG culture medium, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells.
The P1 generation cells cultured in this step were E group, and 7T 182 flasks were numbered E- (1), E- (2), E- (3), E- (4), E- (5), E- (6) and E- (7) in this order.
9) Secondary differential digestion and passaging
Step 8) after the confluence of the E- (7) cultured P1 generation cells reaches 85%, the cells are subjected to differential digestion again, and the cell density is 3000cells/cm 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P2 generation amniotic mesenchymal stem cells.
The P2 cells cultured in this step were F-group, and 7T 182 flasks were numbered F- (1), F- (2), F- (3), F- (4), F- (5), F- (6) and F- (7) in this order.
10 Counting(s)
The culture solution is discarded after the T182 culture flask with the number of (1) is inoculated with cells for 24 hours, 5mL of PBS is added to wash the culture dish, the culture dish is sucked out, 0.25% trypsin is added to digest the cells, when the cells become round and do not fall off, the bottom of the T182 culture flask is washed by a pipette to collect the cells, and the digestion is stopped, and the number of the cells is calculated by a countstar cell counter.
Then, cells numbered (2) to (6) were collected and counted at 24H intervals as described above. The results are shown in Table 2 and FIGS. 2 to 3.
Comparative example two
In this comparative example, amniotic mesenchymal stem cells were isolated and cultured by using a tissue climbing-out method, which comprises the following steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by a hemostat, the placenta surface is washed by physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed.
2) Stripping off
The amniotic membrane is peeled from placenta by hook forceps, spread on a 10cm culture dish, washed to semitransparent form by physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin (figure 6), and cut into 0.3-0.4 cm pieces by direct shearing 2 Is an amniotic membrane block.
3) Wall-attached culture
Spreading the chopped tissue blocks in a 10cm culture dish, inversely culturing for 1-2 h until the tissue clings to the culture dish, standing the culture dish, adding 5mL culture medium (friend placenta mesenchymal stem cell culture medium, product number: NC 0103) at 37deg.C, and 5% CO 2 Culturing in incubator, and changing liquid 1 time every 2 days. Cell climbing out and cell confluency were recorded for 85% of the time, see table 1.
TABLE 1 time to growth of amniotic mesenchymal stem cells (P0 generation) cultured in example one and comparative example two
The cell growth records according to the first, control and control examples described above are shown in Table 1.
The results in Table 1 show that the cell climbing-out speed after secondary adherence is faster than that of primary adherence, and the tissue after primary purification is faster than that of the original tissue adherence, so that the method provided by the invention fully utilizes the tissue mass, is beneficial to the stem cell climbing-out, shortens the primary cell culture period, and obviously improves the cell number.
4) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach 85% confluence, the culture solution is discarded, 5mL PBS is added to wash the culture dish and then sucked out, 1mL of 0.25% trypsin is slowly added to digest the cells for 2 minutes and then the cell suspension is sucked out to a 50mL centrifuge tube to stop digestion (without flushing the cells at the bottom of the culture dish), 1800rpm/min is used for centrifugation for 5 minutes, the supernatant is discarded, and the cell precipitation is resuspended at 3000cells/cm 2 Inoculating to 7T 182 culture flasks, adding 20ml of YOUKANG culture medium, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells.
The P1 generation cells cultured in this step were G group, and 7T 182 flasks were numbered G- (1), G- (2), G- (3), G- (4), G- (5), G- (6) and G- (7) in this order.
5) Secondary differential digestion and passaging
Step 4) after the confluence of the P1 generation cells cultured in the G- (7) reaches 85%, the cells are subjected to differential digestion again, and the cell density is 3000cells/cm 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P2 generation amniotic mesenchymal stem cells.
The P2 cell culture in this step was H group, and 7T 182 flasks were numbered H- (1), H- (2), H- (3), H- (4), H- (5), H- (6) and H- (7) in this order.
6) Counting
The culture solution is discarded after the T182 culture flask with the number of (1) is inoculated with cells for 24 hours, 5mL of PBS is added to wash the culture dish, the culture dish is sucked out, 0.25% trypsin is added to digest the cells, when the cells become round and do not fall off, the bottom of the T182 culture flask is washed by a pipette to collect the cells, and the digestion is stopped, and the number of the cells is calculated by a countstar cell counter.
Then, cells numbered (2) to (6) were collected and counted at 24H intervals as described above. The results are shown in Table 2 and FIGS. 2 to 3.
TABLE 2 number of amniotic mesenchymal stem cells (P1, P2 generation) cells cultured in example one and comparative example
The cell growth records according to the first, control and control examples described above are shown in Table 2.
The cell growth curves are shown in FIG. 2 and FIG. 3, with the days of culture in Table 2 on the abscissa and the cell numbers on the ordinate. The result shows that the proliferation trend of the stem cells with the first and second adherence is similar in passage, which indicates that the invention can obtain more cells in the same time; meanwhile, the proliferation speed of the stem cells in the invention is obviously faster than that of the stem cells in the traditional method, which indicates that the quality of the cells is excellent and the culture time is shorter.
Example two
After the confluence of the primary adherence of example one, the secondary adherence of example one, the P2 generation cells (No. (7)) cultured in the control example one and the control example two reached 85%, the cells were digested by adding 0.25% trypsin, a sufficient amount of cells were sucked into a centrifuge tube in the digestion step, centrifuged at 1200rpm/min for 5min, the supernatant was discarded, and the cells were resuspended by adding PBS, and the cell concentration was adjusted to 3X 10 6 cells/mL。
Detection kit according to cell surface markMesenchymal stem cell (human) surface marker detection kit, cat#: HUXMX-09011), the method performs on-machine detection of surface markers on four cells, respectively.
The results of flow cytometry analysis of the expression of four amniotic mesenchymal stem cell surface antigens CD34, CD45, CD14, CD19, HLA-DR, CD73, CD29 and CD105 show that the four amniotic mesenchymal stem cells all have low expression of CD34, CD45, CD14, CD19, HLA-DR and high expression of CD73, CD29 and CD105, and all meet the characteristics of MSCs, as shown in Table 3.
TABLE 3 results of flow-through detection of amniotic mesenchymal stem cells cultured in example one and comparative example
Example III
After the confluence of the P2 generation cells (number (7)) cultured in the first adherence of the embodiment, the second adherence of the embodiment, the first comparison example and the second comparison example reaches 85%, 0.25% trypsin is added to digest the cells, and sufficient cells are sucked into a centrifuge tube in the digestion step to perform a multi-directional differentiation potential identification experiment corresponding to three differentiation directions of adipogenesis, osteogenesis and chondrogenesis.
Single cell suspension was mixed at 2X 10 4 cells/cm 2 Is inoculated into six-hole plate and usedThe adipogenic differentiation test was performed on human stem cells (cat# HUXXC-90031), and a sufficient amount of lipid droplets of appropriate size were observed to appear, and a staining test was performed using oil red "O".
The results showed that all four amniotic mesenchymal stem cells were seen as red-stained fatty oil droplets (fig. 7- (1)), and all had the ability to differentiate into adipocytes.
Single cell suspension was mixed at 2X 10 4 cells/cm 2 Is inoculated into six-hole plate and usedOsteoinductive differentiation test was performed on human stem cells (HUXXC-90021) and after apparent calcium nodules were observed, the cells were allowed to undergo osteoinductive differentiationStaining experiments were performed with alizarin red.
The results showed that four cases of amniotic mesenchymal stem cells were seen with red-stained calcium deposits (fig. 7- (2)), all of which had the ability to differentiate into bone cells.
Taking 3-4X 10 5 Individual cells were transferred to 15mL centrifuge tubes usingThe chondrogenic differentiation test was performed using a human-related stem cell chondrogenic differentiation kit (cat# HUXXC-90041), and a section staining test was performed using alissine blue after formation of cartilage balls having diameters of 1.5 to 2mm was observed. The results showed that all four amniotic mesenchymal stem cells were seen as blue-stained pellets (fig. 7- (3)), and all had the ability to differentiate into chondrocytes.
The present invention is not limited to the preferred embodiments, but can be modified, equivalent, and modified in any way without departing from the technical scope of the present invention.
Claims (5)
1. A method for separating and extracting amniotic mesenchymal stem cells from human placenta, which is characterized by comprising the following process steps:
1) Cleaning up
After successful delivery of the fetus, placenta tissue is obtained under aseptic conditions, the placenta is taken out into an aseptic tray by using hemostats, the surface of the placenta is washed by using physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin, and residual blood and other impurities are removed;
2) Stripping off
Peeling amniotic membrane from placenta with hook forceps, spreading on 10cm culture dish, washing with physiological saline containing 100U/ml penicillin and 100ug/ml streptomycin to semitransparent state, and cutting into 3-4 cm 2 Is a block of tissue;
3) Digestion
Transferring the amniotic membrane block into a 150mL liquid storage bottle, adding 0.25% trypsin which is 1.5 times of the total volume of the amniotic membrane, putting into a constant temperature shaking table at 37 ℃ and 200rpm/min for digestion for 30min, centrifuging at 1200rpm/min for 5min, and discarding the supernatant; adding trypsin 0.25% which is 1.5 times of the total volume of the amniotic membrane again, digesting for 30min under the same condition, taking out a liquid storage bottle for observation every 10min in the two digestion processes, and shaking vigorously to ensure that the amniotic membrane is fully contacted with liquid;
4) Impurity removal
Filtering the digested tissue mixture with 70 μm cell sieve, removing filtrate, taking out the tissue in the net, placing in 10cm culture dish, rinsing with physiological saline for 2 times, and cutting into 0.3cm pieces with direct shear 2 A left and right tissue block;
5) Wall-attached culture
Spreading the sheared tissue blocks in a 10cm culture dish, inversely culturing for 1-2 h until the tissue clings to the culture dish, erecting the culture dish, adding 5mL of culture medium at 37 ℃ and 5% CO 2 Culturing in an incubator, and changing liquid 1 time every 2 days;
6) Differential digestion and passaging
The tissue primary separation culture mesenchymal stem cells reach 85% confluence, the culture solution is discarded, 5mL PBS is added to wash the culture dish, then the culture dish is sucked out, 1mL of 0.25% trypsin is slowly added to digest the cells, 2 min later, the cell suspension is sucked out to a 50mL centrifuge tube to stop digestion, 1200rpm/min is centrifuged for 5min, the supernatant is discarded, 20mL of YOUKANG culture medium is added to blow evenly, inoculated into a T182 culture bottle and evenly shaken by a crisscross method, and placed at 37 ℃ and 5% CO 2 Culturing in an incubator;
7) Impurity removal and secondary cell climbing out
Adding 0.25% trypsin to the culture dish containing the adhered amniotic membrane tissue block in the step 6) again for 5 minutes, slightly blowing off cells at the bottom of the culture dish by a pipette, discarding, slowly cleaning the bottom of the culture dish for 2 times by using normal saline, adding 5mL of friendly culture medium to enable the tissue to climb out of mesenchymal stem cells for the second time, when the cell confluency reaches 85%, performing differential digestion according to the step 6), inoculating the cells into a T182 culture bottle, shaking uniformly by a crisscross method, placing at 37 ℃ and placing at 5% CO 2 Culturing in an incubator;
8) Culturing in large quantities
Step 6) and the P1 generation cells obtained in the step 7) are subjected to differential digestion again, and after inoculation culture, P2 generation amniotic mesenchymal stem cells are obtained;
9) And (5) subculturing.
2. The method for separating and extracting amniotic mesenchymal stem cells from human placenta according to claim 1, wherein the method comprises the steps of: in step 6), the ratio of 3000cells/cm 2 Inoculating to 7T 182 culture flasks, adding 20ml of YOUKANG culture medium, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells;
the P1 generation cells cultured in this step were A group, and 4T 182 flasks were numbered A- (1), A- (2), A- (3), A- (4), A- (5), A- (6) and A- (7) in this order.
3. The method for separating and extracting amniotic mesenchymal stem cells from human placenta according to claim 1, wherein the method comprises the steps of:
in step 7), cells were digested at a differential speed of 3000cells/cm according to step 6) 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P1 generation amniotic mesenchymal stem cells; the time to climb out and the cell confluence were recorded for 85% of the time;
the P1 generation cells cultured in this step were B group, and 7T 182 flasks were numbered as B- (1), B- (2), B- (3), B- (4), B- (5), B- (6) and B- (7) in this order.
4. The method for separating and extracting amniotic mesenchymal stem cells from human placenta according to claim 1, wherein the method comprises the steps of:
in step 8), the ratio of 3000cells/cm 2 Inoculating to 7T 182 culture flasks, shaking uniformly by crisscross method, standing at 37deg.C, and 5% CO 2 Culturing in an incubator, and marking as P2 generation amniotic mesenchymal stem cells;
the P2 generation cells subjected to one-time adherence culture in the step are C groups, and 7T 182 culture bottles are sequentially numbered as C- (1), C- (2), C- (3), C- (4), C- (5), C- (6) and C- (7);
the P2-generation cells of the secondary adherence culture are D groups, and 7T 182 culture flasks are sequentially numbered as D (1), D- (2), D- (3), D- (4), D- (5), D- (6) and D- (7).
5. The method for separating and extracting amniotic mesenchymal stem cells from human placenta according to claim 1, wherein the method comprises the steps of:
in step 9), counting:
the culture solution is discarded after the T182 culture flask with the number of (1) is inoculated with cells for 24 hours, 5mL of PBS is added to wash the culture dish, the culture dish is sucked out, 0.25% trypsin is added to digest the cells, when the cells become round and do not fall off, the bottom of the T182 culture flask is washed by a pipette to collect the cells, and the digestion is stopped, and the number of the cells is calculated by a Countstar cell counter.
Then, cells numbered (2) to (6) were collected and counted at 24H intervals as described above.
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| 高彦琳;张宁坤;陈厚良;高连如;朱智明;: "改良原代培养体系提高人脐带间充质干细胞的产量", 中国组织工程研究, no. 10, pages 1477 - 1481 * |
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