CN111139221A - Culture and cryopreservation method of amniotic mesenchymal stem cells - Google Patents
Culture and cryopreservation method of amniotic mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a culture and cryopreservation method of amniotic mesenchymal stem cells, which comprises amniotic tissue separation, amniotic mesenchymal stem cell separation, P0 generation amniotic mesenchymal stem cell culture and amplification culture. According to the culture method of the amniotic mesenchymal stem cells, in the separation stage of the amniotic mesenchymal stem cells, the amniotic mesenchymal stem cells can be more effectively separated from the amniotic tissues by digesting with a special mixed enzyme digestive juice system (the final concentration of each component in the digestive juice is 1.5-2U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV), so that the yield of the P0 generation cells is obviously improved.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a culture and cryopreservation method of amniotic mesenchymal stem cells.
Background
With the intensive research on the functional characteristics of the mesenchymal stem cells in recent years, the mesenchymal stem cells become a hot research point of cell replacement therapy. The human amniotic mesenchymal stem cells have the characteristics of multidirectional differentiation potential, immunoregulation, hematopoietic support and the like with the bone marrow mesenchymal stem cells with wide application, and also have the advantages of wide sources, convenient material taking, no ethical limitation, strong cell proliferation capacity and the like. The survival rate is high in various tissues and organs, and the survival time is long. The amnion mesenchyme stem cell originated from the placenta amnion tissue is one kind of adult stem cell with obvious plasticity and multidirectional differentiation potential, and may be differentiated into different tissue cell types originated from 2 germ layers under the regulation of different growth factors, such as fat cell, osteoblast, liver cell, cardiac muscle cell, glia cell, etc. HAMSC expresses no major histocompatibility class II antigen and micro-expression major histocompatibility class I antigen, and has low immunogenicity, small transplantation rejection and immunoregulation effect.
The prior amnion mesenchymal stem cell extraction method separates amnion from chorion, digests hAEC from basement membrane by pancreatin with different concentrations, neutral protease or other digestive enzymes for different times, and then digests and separates most hAEC by collagenase, and has the disadvantages of complex process, long operation time and low yield.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for culturing and cryopreserving amnion mesenchymal stem cells. According to the culture method, in the separation stage of the amniotic mesenchymal stem cells, the amniotic mesenchymal stem cells can be more effectively separated from the amniotic tissues by digesting through a special mixed enzyme digestive juice system, so that the yield of the P0 generation cells is obviously improved.
The technical scheme of the invention is as follows:
a culture method of amnion mesenchyme stem cell includes the following steps:
(1) separating amniotic membrane tissues: soaking and washing placenta tissue, and separating to obtain amnion tissue;
(2) separating the amniotic mesenchymal stem cells: adding a mixed enzyme digestive juice into the amniotic tissue for digestion, filtering and collecting filtrate, centrifuging the filtrate, and removing supernatant to obtain a precipitate;
(3) culturing the P0 generation amniotic mesenchymal stem cells: adding a serum-free culture medium into the precipitate obtained in the step (2) for resuspension to obtain a cell suspension, inoculating, culturing, and adding trypsin for digestion, wherein according to different tolerance of the epithelial cells and the interstitial cells to the trypsin, the interstitial cells are usually firstly detached from the wall when the cells are digested, and the epithelial cells are detached from the wall after being digested for a long time. Digesting the cells for 2-3min, wherein the mesenchymal stem cells can be digested, collecting the digested cells, centrifuging, discarding the supernatant, and obtaining the precipitate which is the P0 generation amniotic mesenchymal stem cells;
(4) amplification culture: the amnion mesenchymal stem cells of the P0 generation in the step (3) are seeded into a new serum-free culture medium for subculture, and are harvested when the amnion mesenchymal stem cells are transferred to the P3 generation after 2-3 passages to obtain purified amnion mesenchymal stem cells;
the mixed enzyme digestive juice is prepared by the following method: dissolving neutral protease, deoxyribonuclease I and collagenase IV in DME/F12 to obtain the product; the final concentration of each component in the mixed enzyme digestive juice is as follows: 1.5-2U/mL neutral protease, 0.5mg/mL DNase I, 1mg/mL collagenase IV.
Preferably, in step (1), the washing is performed by soaking in PBS buffer for 2-3min, then soaking in 75% alcohol for 1-2min, and then using physiological saline.
Preferably, in the step (2), the volume ratio of the amniotic membrane tissue to the mixed enzyme digestive juice is 1:0.8-1.2, and the digestion is carried out at 37 ℃ for 90-120 min; the filtration is carried out by adopting a filter screen with 250 meshes of 150-.
Preferably, in step (3), the composition of the serum-free medium is: 90% of alpha-MEM, 5% of platelet lysate, 10ng/ml of epidermal growth factor, 10ng/ml of recombinant basic fibroblast growth factor and 2mmol/L L-glutamine, wherein the inoculation density is 1-3 multiplied by 104Per cm2The culture is carried out at 37 +/-0.5 ℃ and 75 +/-5% RH and 5 +/-0.5% CO2The culture is carried out in the incubator until the cell growth reaches 80-90% fusion; the digestion is carried out for 2-3min at the temperature of 37 ℃ until the cells are in a spherical shape; the rotation speed of the centrifugation is 1000-1500rpm, and the time of the centrifugation is 4-6 min.
Preferably, in the step (4), the seed separation rate is 1:3-1:6, and the subculture is carried out at 37 +/-0.5 ℃, 75 +/-5% RH and 5 +/-0.5% CO2The incubator of (2) for cultivation.
The amnion mesenchymal stem cells prepared by the method.
The cryopreservation method of the amniotic mesenchymal stem cells comprises the following steps:
(1) adding trypsin into the amniotic mesenchymal stem cells for digestion, centrifuging after the digestion is finished, removing a supernatant, adding a serum-free freezing medium for resuspending cell precipitation, and adjusting the cell density to obtain a cell suspension;
(2) and subpackaging the cell suspension into freezing tubes, cooling, and transferring to liquid nitrogen for storage.
Preferably, in step (1), the digestion is carried out at 37 ℃ for 2-3min, and the centrifugation is carried out at 4 ℃ and 1200rpm for 3-7 min.
Preferably, in step (1), the adjustment is made to a cell density of 0.5 to 1.5X 107one/mL.
Preferably, in the step (2), the temperature is reduced from room temperature to 4 ℃ for 20min, then reduced to-20 ℃ for 30min, and then reduced to-80 ℃ for 24-72 h.
The invention has the beneficial effects that:
1. the culture method of the amniotic mesenchymal stem cells comprises the steps of digesting by adopting a special mixed enzyme digestive fluid system (the final concentration of each component in the digestive fluid is 1.5-2U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV) at the separation stage of the amniotic mesenchymal stem cells, wherein the added deoxyribonuclease I can disperse the adhered cells to form single cells, the cells can easily permeate a filter screen, the yield is higher, the amniotic mesenchymal stem cells can be more effectively separated from amniotic tissues, the yield of P0 generation cells is further obviously improved, furthermore, the method adopts a further digestion method, compared with the traditional two-step digestion, the operation is more convenient, the digestion time is shortened, and the survival rate of the P0 generation cells obtained by the method is more than 95 percent, the flow detection and analysis results of the P3 generation cells obtained by the culture method show, the amniotic mesenchymal stem cells cultured by the culture method have the characteristics of good activity, high yield, high purity and good repeatability, namely, the amniotic mesenchymal stem cells cultured by the culture method have the advantages that the CD73, the CD90 and the CD105 are positive, the expression rate is more than 90%, the CD11b, the CD19, the CD34, the CD45 and the HLA-DR are negative, and the expression rates are less than 2%.
2. According to the culture method of the amniotic mesenchymal stem cells, a serum-free culture medium (consisting of 90% a-MEM, 5% platelet lysate, 10ng/ml epidermal growth factor, 10ng/ml recombinant basic fibroblast growth factor and 2mmol/L L-glutamine) is adopted in both the culture stage and the amplification culture stage of the P0 generation amniotic mesenchymal stem cells, so that the introduction of animal-derived components is completely avoided, the risk of virus, fungus, mycoplasma and other microbial pollution caused by serum is reduced, and the probability of variation is reduced. The serum-free culture system can stably maintain the normal biology characteristics and functions of the human amniotic mesenchymal stem cells, the quality of the human amniotic mesenchymal stem cells cultured by the culture system is stable and controllable, the preparation standard of clinical-grade stem cells in accordance with the guiding principle of cell therapy product research and evaluation technology is met, and the safety of clinical application of the stem cells is ensured.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1: morphological picture (40 times magnification) of P3 generation amniotic mesenchymal stem cells obtained in example 1 of the invention
FIG. 2: detecting the expression result of the surface marker CD90 of the P3 generation amniotic mesenchymal stem cells obtained in the embodiment 1 of the invention by a flow cytometer;
FIG. 3: detecting the expression result of the surface marker CD105 of the P3 generation amniotic mesenchymal stem cell obtained in the embodiment 1 of the invention by a flow cytometer;
FIG. 4: detecting the expression result of the surface marker CD73 of the P3 generation amniotic mesenchymal stem cells obtained in the embodiment 1 of the invention by a flow cytometer;
FIG. 5: detecting a negative phenotype NegCKTL (comprising CD11b, CD19, CD34, CD45 and HLA-DR) of the P3 generation amniotic mesenchymal stem cells obtained in the embodiment 1 of the invention by using a flow cytometer;
wherein 1 is isotype control, and 2 is expression result of amnion mesenchymal stem cell surface marker CD90 (figure 2) \ CD105 (figure 3) \ CD73 (figure 4) \ NegCKTL (figure 5).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention. In the following examples, platelet lysates were obtained from David and serum-free frozen stocks were obtained from BI (Cat. No.: 05-712-1E).
Example 1
The embodiment provides a culture method of amniotic mesenchymal stem cells, which comprises the following steps:
(1) separating amniotic membrane tissues: soaking the placenta tissue in PBS buffer solution, taking blood water as a sample source microorganism detection sample, then soaking the placenta tissue for 2min, then soaking the placenta tissue in 75% alcohol for about 1min, repeatedly washing the placenta tissue with normal saline, lightly tearing the amniotic tissue with tissue forceps, soaking the amniotic tissue in PBS for separation of amniotic mesenchymal stem cells, and finally sampling the normal saline for washing the placenta to detect microorganisms;
(2) separating the amniotic mesenchymal stem cells: cutting amnion tissue into 1-2mm2Small pieces, add to 50mL sterile centrifuge tube and add to the aliquotsDigesting the accumulated mixed enzyme digestive juice (which is obtained by dissolving neutral protease, deoxyribonuclease I and collagenase IV in DME/F12 to obtain the mixture with the final concentration of 1.5U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV) at 37 ℃ for 90min, filtering all tissues and the digestive juice by a 200-mesh filter screen, collecting the filtrate, transferring the filtrate into a 50-mL centrifuge tube, centrifuging at the rotating speed of 1800rpm for 8min, and carefully sucking and discarding the supernatant to obtain a precipitate;
(3) culturing the P0 generation amniotic mesenchymal stem cells: adding a serum-free medium (consisting of 90% a-MEM, 5% platelet lysate, 10ng/ml epidermal growth factor, 10ng/ml recombinant basic fibroblast growth factor and 2mmol/L L-glutamine) into the precipitate obtained in the step (2), and resuspending the mixture in a ratio of 1X 104Per cm2The density of (2) was inoculated into a T75 flask containing 15mL of serum-free medium at 37 ℃ and 75% RH and 5% CO2The incubator of (1) for primary cell culture. Culturing for 48h, observing the cell adherence condition under a microscope, and replacing fresh culture medium for the cells. When the cells are cultured until the cells grow to be 80% fused, washing the cells twice by using a DPBS washing solution, adding 2mL of trypsin into each T75 culture bottle to digest the cells, digesting the cells for 2min at 37 ℃, observing the cells to be round under a microscope, gently beating the cells from the bottom of the bottle, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS into each T75 culture bottle to wash the cells twice, adding the washing solution into the collected cell suspension, and centrifuging the cells for 5min at 4 ℃ and 1200 rpm. Discarding the supernatant to obtain cell sediment which is the P0 generation amniotic mesenchymal stem cells, adding a serum-free culture medium to mix the cell sediment uniformly, counting 100 mu L to obtain the number of cells which is the harvest number of the P0 generation amniotic mesenchymal stem cells;
(4) amplification culture: inoculating the obtained P0 generation amnion mesenchymal stem cells into culture bottles with new serum-free culture medium according to the seed separation rate of 1:3 for culture, adding 15ml serum-free culture medium into each T75 culture bottle for culture, placing at 37 ℃, 75% RH and 5% CO for culture2Culturing in incubator until cell growth reaches 80% fusion, washing with DPBS lotion twice, adding 2mL trypsin to each T75 culture flask to digest cells, digesting at 37 deg.C for 2min, and taking microscopeAnd after cell rounding is observed, gently beating the cells from the bottom of the bottle, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL PBS into each T75 culture bottle for cleaning twice, adding the cleaning solution into the collected cell suspension, centrifuging for 5min at 4 ℃ and 1200rpm, obtaining cell sediment, namely the P1 generation amniotic mesenchymal stem cells, transferring the P1 generation amniotic mesenchymal stem cells to the P3 generation by the same method, and obtaining the purified amniotic mesenchymal stem cells.
The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:
(1) replacing a fresh serum-free culture medium 24 hours before freezing and storing the P3 generation amniotic mesenchymal stem cells to enable the cells to be in a logarithmic growth phase all the time, adding 2mL of trypsin to digest the cells when the cells grow to 80% fusion, digesting the cells at 37 ℃ for 2min, observing the cells under a microscope, slightly beating the cells from the bottom of a bottle when the cells are in a spherical state, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS into each T75 culture bottle to wash the cells twice, adding the washing solution into the collected cell suspension, centrifuging the cells at 4 ℃, 1200rpm and 5min, discarding the supernatant, adding a serum-free freezing solution to resuspend the cell sediment, and adjusting the cell density to be 1 x 10 to adjust the cell density7one/mL.
(2) And subpackaging the cell suspension into freezing tubes, wherein each tube contains 1mL of the cell suspension, cooling from room temperature to 4 ℃ for 20min, cooling to-20 ℃ for 30min, cooling to-80 ℃ for 24h, and transferring to liquid nitrogen for storage.
The cell morphology of the P0 generation amniotic mesenchymal stem cells is identified to be fusiform and polygonal, the P0 generation amniotic mesenchymal stem cells are approximately 80% fused in about 8 days, the purity of the cells after passage is improved, the morphology is uniform compared with the primary cells, and the cells mainly grow in parallel arrangement or grow in a vortex shape (see figure 1); the flow detection and analysis results of the P3 generation amniotic mesenchymal stem cells show that the expression rates of CD73, CD90 and CD105 are positive and are more than 90%, the expression rates of CD11b, CD19, CD34, CD45 and HLA-DR are negative and are less than 2%, and the characteristics of the mesenchymal stem cells are met (see fig. 2-5).
Example 2
The embodiment provides a culture method of amniotic mesenchymal stem cells, which comprises the following steps:
(1) separating amniotic membrane tissues: soaking the placenta tissue in PBS buffer solution, taking blood water as a sample source microorganism detection sample, then soaking the placenta tissue for 3min, then soaking the placenta tissue in 75% alcohol for about 2min, repeatedly washing the placenta tissue with normal saline, lightly tearing the amniotic tissue with tissue forceps, soaking the amniotic tissue in PBS for separation of amniotic mesenchymal stem cells, and finally sampling the normal saline for washing the placenta to detect microorganisms;
(2) separating the amniotic mesenchymal stem cells: cutting amnion tissue into 1-2mm2Adding small pieces into a 50mL sterile centrifuge tube, adding a mixed enzyme digestive solution (which is obtained by dissolving neutral protease, deoxyribonuclease I and collagenase IV in DME/F12 and has a final concentration of 2U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV) with the volume of 0.8 times of the amniotic tissue volume, digesting for 120min at 37 ℃, filtering all tissues and the digestive solution through a 150-mesh filter screen, collecting filtrate, transferring the filtrate into a 50mL centrifuge tube, centrifuging for 10min at a rotating speed of 1500rpm, carefully sucking and discarding supernatant to obtain a precipitate;
(3) culturing the P0 generation amniotic mesenchymal stem cells: adding a serum-free medium (consisting of 90% a-MEM, 5% platelet lysate, 10ng/ml epidermal growth factor, 10ng/ml recombinant basic fibroblast growth factor and 2mmol/L L-glutamine) into the precipitate obtained in the step (2), and resuspending the mixture in a ratio of 3X 104Per cm2The density of (2) was inoculated into a T75 flask containing 15mL of serum-free medium at 36.5 ℃ and 70% RH and 4.5% CO2The incubator of (1) for primary cell culture. Culturing for 48h, observing the cell adherence condition under a microscope, and replacing fresh culture medium for the cells. When the cells are cultured until the cells grow to 90% fusion, washing the cells twice by using a DPBS washing solution, adding 2mL of trypsin into each T75 culture bottle to digest the cells, digesting the cells at 37 ℃ for 3min, observing the cells to be round under a microscope, gently beating the cells from the bottom of the bottle, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS into each T75 culture bottle to wash the cells twice, adding the washing solution into the collected cell suspension, and centrifuging the cells for 6min at 4 ℃ and 1000 rpm.Discarding the supernatant to obtain cell sediment which is the P0 generation amniotic mesenchymal stem cells, adding a serum-free culture medium to mix the cell sediment uniformly, counting 100 mu L to obtain the number of cells which is the harvested number of the P0 generation cells;
(4) amplification culture: inoculating the obtained P0 generation amnion mesenchymal stem cells into culture bottles with new serum-free culture medium according to the seed separation rate of 1:6 for culture, adding 15ml serum-free culture medium into each T75 culture bottle for culture, placing at 36.5 deg.C, 70% RH, 4.5% CO2Culturing in an incubator until the cell growth reaches 90% fusion, washing twice with DPBS washing liquid, adding 2mL of trypsin to digest the cell in each T75 culture bottle, digesting for 3min at 37 ℃, observing the cell to become round under a microscope, gently beating the cell from the bottle bottom, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS to each T75 culture bottle, washing twice, adding the washing liquid into the collected cell suspension, centrifuging for 4min at 4 ℃, 1200rpm, and obtaining cell sediment, namely the P1 generation amniotic mesenchymal stem cell, transferring the P1 generation amniotic mesenchymal stem cell to P3 generation by the same method, and obtaining the purified amniotic mesenchymal stem cell.
The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:
(1) replacing a fresh serum-free culture medium 24 hours before freezing and storing the P3 generation amniotic mesenchymal stem cells to enable the cells to be in a logarithmic growth phase all the time, adding 2mL of trypsin to digest the cells when the cells grow to 90% of fusion, digesting the cells at 37 ℃ for 3min, observing the cells under a microscope, slightly beating the cells from the bottom of a bottle when the cells are in a spherical state, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS into each T75 culture bottle to wash the cells twice, adding the washing solution into the collected cell suspension, centrifuging the cells at 4 ℃, 1200rpm and 3min, discarding the supernatant, adding a serum-free freezing solution to resuspend the cell sediment, and adjusting the cell density to be 0.5 multiplied by 10 to adjust the cell density to be 0.5 multiplied by 10 to resuspend7one/mL.
(2) And subpackaging the cell suspension into freezing tubes, wherein each tube contains 1mL of the cell suspension, cooling from room temperature to 4 ℃ for 20min, cooling to-20 ℃ for 30min, cooling to-80 ℃ for 72h, and transferring to liquid nitrogen for storage.
The identification shows that the flow detection and analysis results of the P3 generation amniotic mesenchymal stem cells show that CD73, CD90 and CD105 are positive, the expression rate is more than 90%, CD11b, CD19, CD34, CD45 and HLA-DR are negative, the expression rates are less than 2%, and the characteristics of the mesenchymal stem cells are met.
Example 3
The embodiment provides a culture method of amniotic mesenchymal stem cells, which comprises the following steps:
(1) separating amniotic membrane tissues: soaking the placenta tissue in PBS buffer solution, taking blood water as a sample source microorganism detection sample, then soaking the placenta tissue for 3min, then soaking the placenta tissue in 75% alcohol for about 2min, repeatedly washing the placenta tissue with normal saline, lightly tearing the amniotic tissue with tissue forceps, soaking the amniotic tissue in PBS for separation of amniotic mesenchymal stem cells, and finally sampling the normal saline for washing the placenta to detect microorganisms;
(2) separating the amniotic mesenchymal stem cells: cutting amnion tissue into 1-2mm2Adding the small pieces into a 50mL sterile centrifuge tube, adding a mixed enzyme digestive solution (which is obtained by dissolving neutral protease, deoxyribonuclease I and collagenase IV in DME/F12 and has a final concentration of 1.5U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV) with the volume of 1.2 times that of the amniotic membrane tissue, digesting for 100min at 37 ℃, filtering all tissues and the digestive solution through a 250-mesh filter screen, collecting filtrate, transferring the filtrate into a 50mL centrifuge tube, centrifuging for 6min at the rotating speed of 2000rpm, carefully sucking and discarding supernatant to obtain a precipitate;
(3) culturing the P0 generation amniotic mesenchymal stem cells: adding a serum-free medium (consisting of 90% a-MEM, 5% platelet lysate, 10ng/ml epidermal growth factor, 10ng/ml recombinant basic fibroblast growth factor and 2mmol/L L-glutamine) into the precipitate obtained in the step (2), and resuspending the mixture in a ratio of 2X 104Per cm2The medium was inoculated into a T75 flask containing 15mL of serum-free medium at 37.5 ℃ and 80% RH and 5.5% CO2The incubator of (1) for primary cell culture. Culturing for 48h, observing the cell adherence condition under a microscope, and replacing fresh culture medium for the cells. Culturing the cells until the cells growWhen 80% of fusion is achieved, after washing twice with DPBS washing liquid, 2mL of trypsin is added into each T75 culture bottle to digest cells for 3min at 37 ℃, after cell rounding is observed under a microscope, the cells are gently flapped from the bottom of the bottle, the cell suspension is sucked into a 50mL centrifuge tube, 10mL of PBS is added into each T75 culture bottle to be washed twice, and the washing liquid is added into the collected cell suspension together and centrifuged for 4min at 4 ℃ and 1500 rpm. Discarding the supernatant to obtain cell sediment which is the P0 generation amniotic mesenchymal stem cells, adding a serum-free culture medium to mix the cell sediment uniformly, counting 100 mu L to obtain the number of cells which is the harvested number of the P0 generation cells;
(4) amplification culture: inoculating the obtained P0 generation amnion mesenchymal stem cells into culture bottles with new serum-free culture medium according to the seed separation rate of 1:3 for culture, adding 15ml serum-free culture medium into each T75 culture bottle for culture, placing at 37.5 ℃, 80% RH, 5.5% CO2Culturing in an incubator until the cells grow to be 80% fused, washing twice by using DPBS washing liquid, adding 2mL of trypsin to digest the cells in each T75 culture bottle, digesting for 3min at 37 ℃, observing the cells to be round under a microscope, gently beating the cells from the bottle bottom, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS to each T75 culture bottle, washing twice, adding the washing liquid into the collected cell suspension, centrifuging for 6min at 4 ℃, 1200rpm, and obtaining cell sediment, namely the P1 generation amniotic mesenchymal stem cells, transferring the P1 generation amniotic mesenchymal stem cells to P3 generation by the same method, and obtaining the purified amniotic mesenchymal stem cells.
The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:
(1) replacing a fresh serum-free culture medium 24 hours before freezing and storing the P3 generation amniotic mesenchymal stem cells to enable the cells to be in a logarithmic growth phase all the time, adding 2mL of trypsin to digest the cells when the cells grow to 80% fusion, digesting the cells at 37 ℃ for 3min, observing the cells under a microscope, slightly beating the cells from the bottom of a bottle when the cells are in a spherical state, sucking the cell suspension into a 50mL centrifuge tube, adding 10mL of PBS into each T75 culture bottle to wash the cells twice, adding the washing solution into the collected cell suspension, centrifuging the cells at 4 ℃, 1200rpm and 7min, discarding the washing solution, and removing the cellsRemoving supernatant, adding serum-free freezing medium to resuspend cell precipitate, and adjusting cell density to 1.5 × 107one/mL.
(2) And subpackaging the cell suspension into freezing tubes, wherein each tube contains 1mL of the cell suspension, cooling from room temperature to 4 ℃ for 20min, cooling to-20 ℃ for 30min, cooling to-80 ℃ for 48h, and transferring to liquid nitrogen for storage.
The identification shows that the flow detection and analysis results of the P3 generation amniotic mesenchymal stem cells show that CD73, CD90 and CD105 are positive, the expression rate is more than 90%, CD11b, CD19, CD34, CD45 and HLA-DR are negative, the expression rates are less than 2%, and the characteristics of the mesenchymal stem cells are met.
Comparative example 1
The difference from example 1 is that the composition of the mixed enzyme digest was different, specifically, the composition of the mixed enzyme digest of this comparative example was: 1.5-2U/mL neutral protease, 0.5mg/mL hyaluronidase, and 1mg/mL collagenase IV.
Comparative example 2
The difference from example 1 is that DNase I in the digestion solution of the mixed enzyme was removed.
TABLE 1 detection result of harvest quantity of P0 generation amnion mesenchymal stem cells
The results show that the culture method of example 1 can harvest the most P0 generation cells, and then example 3, example 2, comparative example 1 and comparative example 2; the amniotic mesenchymal stem cells harvested in example 1 are identified, and the results show that the cells of the P0 generation are fusiform and polygonal in morphology, approximately 80% of the cells are fused in about 8 days, the purity of the cells after passage is improved, the forms are more uniform than primary cells, the cells mainly grow in parallel arrangement or grow in a vortex shape (see figure 1), and the flow detection analysis results of the cells of the P3 generation show that CD73, CD90 and CD105 are positive, the expression rate is more than 90%, CD11b, CD19, CD34, CD45 and HLA-DR are negative, the expression rates are less than 2%, and the cells accord with the characteristics of the mesenchymal stem cells (see figures 2-5). Further analysis proves that the mixed digestive enzyme system adopted by the mixed enzyme digestive juice can more effectively separate the amniotic mesenchymal stem cells from the amniotic tissues, so that the yield of the P0 generation cells is obviously improved, and the P3 generation amniotic mesenchymal stem cells harvested by the method have good activity and high purity.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A culture method of amnion mesenchyme stem cell is characterized by comprising the following steps:
(1) separating amniotic membrane tissues: soaking and washing placenta tissue, and separating to obtain amnion tissue;
(2) separating the amniotic mesenchymal stem cells: adding a mixed enzyme digestive juice into the amniotic tissue for digestion, filtering and collecting filtrate, centrifuging the filtrate, and removing supernatant to obtain a precipitate;
(3) culturing the P0 generation amniotic mesenchymal stem cells: adding a serum-free culture medium into the precipitate obtained in the step (2) for resuspension to obtain a cell suspension, inoculating and culturing, adding trypsin for digestion, then centrifuging, and removing the supernatant to obtain the precipitate, namely the P0 generation amniotic mesenchymal stem cells;
(4) amplification culture: the amnion mesenchymal stem cells of the P0 generation in the step (3) are seeded into a new serum-free culture medium for subculture, and are harvested when the amnion mesenchymal stem cells are transferred to the P3 generation, so that purified amnion mesenchymal stem cells are obtained;
the mixed enzyme digestive juice is prepared by the following method: dissolving neutral protease, deoxyribonuclease I and collagenase IV in DME/F12 to obtain the product; the final concentration of each component in the mixed enzyme digestive juice is as follows: 1.5-2U/mL neutral protease, 0.5mg/mL DNase I, 1mg/mL collagenase IV.
2. The method for culturing amniotic mesenchymal stem cells according to claim 1, wherein in the step (1), the amniotic mesenchymal stem cells are first soaked in PBS buffer solution for 2-3min, then soaked in 75% alcohol for 1-2min, and then washed with physiological saline.
3. The method for culturing amnion mesenchymal stem cells according to claim 1, wherein in the step (2), the volume ratio of the amnion tissue to the mixed enzyme digestive fluid is 1:0.8-1.2, and the digestion is performed at 37 ℃ for 90-120 min; the filtration is carried out by adopting a filter screen with 250 meshes of 150-.
4. The method for culturing amnion mesenchymal stem cells according to claim 1, wherein in step (3), the composition of the serum-free medium is: 90% of alpha-MEM, 5% of platelet lysate, 10ng/ml of epidermal growth factor, 10ng/ml of recombinant basic fibroblast growth factor and 2mmol/L L-glutamine, wherein the inoculation density is 1-3 multiplied by 104Per cm2The culture is carried out at 37 +/-0.5 ℃ and 75 +/-5% RH and 5 +/-0.5% CO2Culturing in the incubator until the cell growth reaches 80-90% fusion; the digestion is carried out for 2-3min at the temperature of 37 ℃ until the cells are in a spherical shape; the rotation speed of the centrifugation is 1000-1500rpm, and the time of the centrifugation is 4-6 min.
5. The method for culturing amnion mesenchymal stem cells according to claim 1, wherein in step (4), the differentiation rate is 1:3 to 1:6, and the subculture is performed at 37 ± 0.5 ℃, 75 ± 5% RH, 5 ± 0.5% CO2The incubator of (2) for cultivation.
6. The amniotic mesenchymal stem cells prepared by the method of any one of claims 1-5.
7. The cryopreservation method of the amniotic mesenchymal stem cells of claim 6, comprising the following steps:
(1) adding trypsin into the amniotic mesenchymal stem cells for digestion, centrifuging after the digestion is finished, removing a supernatant, adding a serum-free freezing medium for resuspending cell precipitation, and adjusting the cell density to obtain a cell suspension;
(2) and subpackaging the cell suspension into freezing tubes, cooling, and transferring to liquid nitrogen for storage.
8. The cryopreservation method of amniotic mesenchymal stem cells according to claim 7, wherein in the step (1), the digestion is carried out at 37 ℃ for 2-3min, and the centrifugation is carried out at 4 ℃ and 1200rpm for 3-7 min.
9. The cryopreservation method of amniotic mesenchymal stem cells according to claim 7, wherein in the step (1), the adjustment is performed until the cell density is 0.5-1.5 x 107one/mL.
10. The cryopreservation method of amniotic mesenchymal stem cells according to claim 7, wherein in the step (2), the temperature is reduced from room temperature to 4 ℃ for 20min, then reduced to-20 ℃ for 30min, and then reduced to-80 ℃ for 24-72 h.
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