CN110592006A - Human mesenchymal stem cell bank and construction method thereof - Google Patents
Human mesenchymal stem cell bank and construction method thereof Download PDFInfo
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Abstract
The application provides a method for constructing a human mesenchymal stem cell bank, which comprises the following steps: separating and purifying the umbilical cord tissue to obtain umbilical cord mesenchymal stem cells; separating and purifying the placenta tissue to obtain placenta mesenchymal stem cells; and (3) performing amplification, cryopreservation and/or identification on the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells. The construction method of the source mesenchymal stem cell bank can establish the human source mesenchymal stem cell bank with rich mesenchymal stem cell sources, can realize the large-scale culture of the mesenchymal stem cells, and provides sufficient guarantee for the clinical application of the mesenchymal stem cells.
Description
Technical Field
The application relates to the field of stem cells, in particular to a human mesenchymal stem cell bank and a construction method thereof.
Background
The mesenchymal stem cells exist in various tissues, have the potential of differentiating into adipogenic, osteogenic, chondrogenic, adipogenic, neurogenic, bone marrow stroma, even liver cells and the like, and are important seed cells and gene therapy vectors in cell therapy and tissue engineering research. The mesenchymal stem cells provide a new treatment way for various clinical refractory diseases, such as wound healing, cartilage repair, bone injury, nephropathy, diabetes, nervous system diseases, autoimmune diseases, malignant tumors and the like, and make the production of various tissues and organs in a laboratory possible.
In the prior art, clinically used mesenchymal stem cells are mainly subjected to cell culture as required, however, the cell culture efficiency is low, the success rate is not high, the large-scale culture of the mesenchymal stem cells is difficult to realize, and the clinical use is greatly limited.
Disclosure of Invention
The purpose of the application is to provide a human mesenchymal stem cell bank capable of realizing large-scale culture of mesenchymal stem cells and a construction method.
In order to achieve the above object, the present application provides the following technical solutions:
a method for constructing a human mesenchymal stem cell bank comprises the following steps:
separating and purifying the umbilical cord tissue to obtain umbilical cord mesenchymal stem cells;
separating and purifying the placenta tissue to obtain placenta mesenchymal stem cells;
and (3) performing amplification, cryopreservation and/or identification on the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells.
Preferably, the umbilical cord tissue is separated and purified by a tissue block adherence method.
Preferably, the step of separating and purifying the umbilical cord tissue by tissue block adherence comprises:
removing the accessory tissue of the umbilical cord and processing the umbilical cord into a strip-shaped umbilical cord tissue;
washing the umbilical cord tissue with a flushing agent;
adding a first buffer solution, and processing the umbilical cord tissue into particles with a preset size;
adding a second buffer solution, placing the granular umbilical cord tissues into a culture vessel in which a matrix is embedded in advance, adding a culture medium, and placing the umbilical cord tissues into an incubator for standing culture;
harvesting the dissociated cells when it is observed that the cells around the umbilical cord tissue are dissociated by a predetermined width;
subculturing the harvested free cells to obtain stable umbilical cord mesenchymal stem cells.
Preferably, the first buffer is a phosphate buffered saline solution.
Preferably, the step of treating the umbilical cord tissue to be in the form of particles of a predetermined size comprises: the umbilical cord tissue was stirred with a hand-held electric homogenizer.
Preferably, the second buffer is a phosphate buffered saline solution containing 1% antibiotic.
Preferably, the step of placing the umbilical cord tissue in a culture vessel pre-embedded with a matrix comprises:
spreading the umbilical cord tissue on a substrate of a culture vessel with a density of sheets per cm2Placing a piece of said umbilical cord tissue.
Preferably, the step of harvesting the dissociated cells when it is observed that the cells around the umbilical cord tissue are dissociated by a predetermined width comprises:
specifically, when the width of the umbilical cord tissue edge cells freed is observed to be greater than or equal to 0.5m, labeling is performed and the freed cells are harvested.
Preferably, the placental tissue comprises at least one of an amniotic membrane, a chorionic smooth muscle layer, a chorionic trophoblast, and a periostracum layer.
Preferably, the placenta tissue is separated and purified by an enzyme digestion method.
Preferably, the step of separately purifying the placenta tissues by enzyme digestion comprises:
processing said placental tissue into strips of clumpy tissue;
washing the tissue strip with a irrigant;
adding a third buffer solution, and respectively processing the tissue strips into granular tissue blocks with preset sizes;
adding trypsin into the tissue blocks for carrying out first digestion to obtain first digestion liquid;
carrying out tissue block adherent culture on the aggregated cells in the first digestive juice;
performing secondary digestion on the cells subjected to tissue block adherent culture to obtain a second digestion solution;
and (3) carrying out adherent culture on the free cells of the second digestive juice to a logarithmic growth phase, and harvesting cell products containing the mesenchymal stem cells, or carrying out subculture on the cell products containing the mesenchymal stem cells.
Preferably, the third buffer is a phosphate buffered saline solution.
Preferably, in the step of adding trypsin to the tissue mass for the first digestion to obtain the first digestion solution, the amount of the trypsin is equal to the volume of the tissue mass, and the concentration of the trypsin is 0.25%.
Preferably, the step of adding trypsin to the tissue mass for the first digestion to obtain a first digestion solution further comprises: shaking at 37 deg.C for 60-90min until the tissue mass is viscous.
Preferably, the step of performing tissue block adherent culture on the aggregated cells in the first digestive fluid comprises:
centrifuging the first digestive juice for the first time to obtain a first supernatant;
centrifuging the first supernatant for a second time to obtain the aggregated cells as a precipitate;
and performing adherent culture on the aggregated cells.
Preferably, the step of performing a second digestion on the cells subjected to the tissue block adherent culture to obtain a second digestion solution further includes: trypsin was added for a second digestion.
Preferably, in the step of adding trypsin for the second digestion, the amount of the trypsin is equal to the volume of the cells after the tissue block adherent culture is carried out, and the concentration of the trypsin is 0.25%.
Preferably, after the step of performing adherent culture on the free cells of the second digestion solution, the method further comprises:
when the adherence of free cells of the second digestive juice is observed, periodically observing and replacing the culture solution by half amount;
monitoring the confluent state of the growth of the free cells of the second digestive juice when the free cells of the second digestive juice are observed to be aggregated.
Preferably, before the separation and purification of the umbilical cord tissue and the placenta tissue, the method comprises the following steps:
human umbilical cord and placenta were washed with a physiological saline containing 1% penicillin streptomycin.
Preferably, the method for constructing the source mesenchymal stem cell bank of the applicant further comprises: respectively inducing the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells to differentiate.
Preferably, the umbilical cord mesenchymal stem cells and the placental mesenchymal stem cells are induced to differentiate into at least one of osteoblasts, adipocytes or neural cells, respectively.
Preferably, the method for constructing the source mesenchymal stem cell bank of the applicant further comprises: and performing information construction and management on the human mesenchymal stem cell bank.
The application also provides a human mesenchymal stem cell bank which is constructed by adopting the construction method of the human mesenchymal stem cell bank.
Compared with the prior art, the scheme of the application has the following advantages:
1. in the construction method of the source mesenchymal stem cell bank, the umbilical cord tissue and the placenta tissue are separated and purified to obtain mesenchymal stem cells with different sources, and a human source mesenchymal stem cell bank with rich mesenchymal stem cell sources can be established; after the human-derived mesenchymal stem cell bank is established, the mesenchymal stem cells in the cell bank are amplified, so that the large-scale culture of the mesenchymal stem cells can be realized, and sufficient guarantee is provided for the clinical application of the mesenchymal stem cells.
2. According to the construction method of the source mesenchymal stem cell bank, the umbilical cord tissue is cultured by a tissue block adherence method, and in the culture process, the umbilical cord tissue only needs to be placed in a culture vessel embedded with a matrix in advance for culture without spreading matrix glue, so that the culture steps are simplified, the cultured cells are good in adhesion and strong in activity, and high-quality mesenchymal stem cells are provided for establishing the human mesenchymal stem cell bank.
3. In the construction method of the source mesenchymal stem cell bank, the placenta tissue is cultured by an enzyme digestion method, and during the culture process, only a single enzyme with stable property, namely trypsin, is adopted, and other enzymes which are easily influenced by external factors and have changed properties, such as collagenase, hyaluronic acid and the like, are not adopted, so that the stability of the digestion process is fundamentally ensured.
4. In the construction method of the source mesenchymal stem cell bank, the placenta tissue comprises at least one of amnion, chorion smooth muscle layer, chorion trophoblast and periostracum layer, so that the source of the mesenchymal stem cells is greatly enriched, and the human source mesenchymal stem cell bank with more comprehensive functions can be established so as to be suitable for various medical purposes.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
FIG. 1 is a flow chart of a method of one embodiment of the method of constructing a source mesenchymal stem cell bank of the present applicant;
FIG. 2 is a flow chart of another embodiment of the method for constructing the source mesenchymal stem cell bank of the present applicant;
FIG. 3 is a flow chart of another embodiment of the method for constructing the source mesenchymal stem cell bank of the present applicant;
FIG. 4 is a diagram of the growth state of umbilical cord mesenchymal stem cells obtained by the method for constructing source mesenchymal stem cell bank of the applicant;
FIG. 5 is a diagram showing the growth state of placental mesenchymal stem cells obtained by the method for constructing the source mesenchymal stem cell bank of the present applicant;
fig. 6 is a graph showing the results of osteogenic differentiation, adipogenic differentiation and neurogenic differentiation of mesenchymal stem cells obtained by the method for constructing the source mesenchymal stem cell bank of the present applicant.
Detailed Description
The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application. In addition, if a detailed description of the known art is not necessary to show the features of the present application, it is omitted.
In the present example, the maternal placenta (with 25-35cm umbilical cord retained) was obtained at Guangzhou Hospital department of obstetrics and gynecology, under written informed consent approved by the ethical Committee of medical center of Hospital, Guangzhou, who was within 35 years of age, negative for infectious disease, 35 weeks of gestational age, no more than 18 hours ruptured fetal membranes, and regular birth tests, without abnormalities in birth, infection, or other diseases that could interfere with placenta viability.
The method comprises the steps of primarily cleaning blood stains on the surfaces of an umbilical cord and a placenta by using physiological saline containing 1% of streptomycin, and transporting the blood stains to a laboratory within 3 hours at the temperature of 2-8 ℃ for treatment by a professional researcher.
The human umbilical cord is composed of two arteries and a vein which are respectively called as an umbilical artery and an umbilical vein, colloidal mesenchyme also exists in the gap between the umbilical artery and the umbilical vein, and the umbilical cord tissue adopted by the construction method of the source mesenchyme stem cell bank of the applicant is the colloidal mesenchyme in the gap between the umbilical artery and the umbilical vein.
The human placenta consists of the amnion (amniotic membrane), the phylliform chorion (chorion villus) and the decidua basalis. The placenta tissues adopted by the construction method of the source mesenchymal stem cell bank of the applicant comprise an Amnion (AM), a chorionic smooth muscle layer (CM), a chorionic trophoblast (CV) and an Decidua (DP).
Referring to fig. 1, an embodiment of the present application provides a method for constructing a human mesenchymal stem cell bank, including the following steps:
s11, separating and purifying the umbilical cord tissue to obtain umbilical cord mesenchymal stem cells;
s12, separating and purifying the placenta tissue to obtain placenta mesenchymal stem cells;
s13, expanding, freezing and/or identifying the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells.
In another embodiment of the present application, the umbilical cord tissue is separated and purified by a tissue block adherence method, and the placenta tissue is separated and purified by an enzyme digestion method. Specifically, referring to fig. 2, a method for constructing a human mesenchymal stem cell bank provided in another embodiment of the present application includes the following steps:
s21, separating and purifying the umbilical cord tissue by adopting a tissue block adherence method to obtain umbilical cord mesenchymal stem cells;
s22, separating and purifying the placenta tissue by adopting an enzyme digestion method to obtain placenta mesenchymal stem cells;
s23, expanding, freezing and/or identifying the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells.
In another embodiment of the present application, the umbilical cord tissue is isolated and purified by a tissue patch adherence method, and at least one of an amniotic membrane, a chorionic smooth muscle layer, a chorionic trophoblast layer, and a periostracum layer of the placenta tissue is isolated and purified by an enzyme digestion method. Specifically, referring to fig. 3, a method for constructing a human mesenchymal stem cell bank provided in another embodiment of the present application includes the following steps:
s31, separating and purifying the umbilical cord tissue by adopting a tissue block adherence method to obtain umbilical cord mesenchymal stem cells;
s32, separating and purifying at least one of amnion, chorionic smooth muscle layer, chorionic trophoblast and periostracum layer in the placenta tissue by adopting an enzyme digestion method to obtain placenta mesenchymal stem cells;
s33, expanding, freezing and/or identifying the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells.
Specifically, the method for separating and purifying the umbilical cord tissue comprises the following steps:
removing the accessory tissue of the umbilical cord and processing the umbilical cord into a strip-shaped umbilical cord tissue;
washing the umbilical cord tissue with a flushing agent;
adding a first buffer solution, and processing the umbilical cord tissue into particles with a preset size;
adding a second buffer solution, placing the granular umbilical cord tissues into a culture vessel in which a matrix is embedded in advance, adding a culture medium, and placing the umbilical cord tissues into an incubator for standing culture;
harvesting the dissociated cells when it is observed that the cells around the umbilical cord tissue are dissociated by a predetermined width;
subculturing the harvested free cells to obtain stable umbilical cord mesenchymal stem cells.
Specifically, the umbilical cord collateral tissues refer to two umbilical arteries and one umbilical vein of the umbilical cord, and the umbilical cord tissues adopted by the construction method of the source mesenchymal stem cell bank of the applicant are colloidal mesenchyme in the gap between the umbilical artery and the umbilical vein. The umbilical cord tissue is processed into a strip-shaped umbilical cord tissue, in particular to a strip-shaped umbilical cord tissue with the width of 1 cm.
In one embodiment of the present application, the flushing agent is preferably physiological saline; the first buffer solution is preferably Phosphate Buffered Saline (PBS); the second buffer solution adopts phosphate buffered saline solution containing 1% of antibiotic, and preferably phosphate buffered saline solution containing 1% of double-chain mycin. The culture vessel used for the umbilical cord tissue is preferably a culture flask, which is pre-embedded with a matrix.
The step of processing the umbilical cord tissue into a particulate form of a predetermined size comprises: stirring the umbilical cord tissue by adopting a handheld electric homogenizer, and processing the umbilical cord tissue into granules with the diameter of 1-2 mm.
A step of placing the umbilical cord tissue in a culture vessel pre-embedded with a matrix, comprising: spreading the umbilical cord tissue on a substrate of a culture vessel with a density of sheets per cm2Placing a piece of said umbilical cord tissue.
The step of adding the culture medium and placing the culture medium in an incubator for static culture comprises the following steps: at each timeDropping a drop of culture medium on the umbilical cord tissue, and placing at 37 deg.C and 5% CO2And standing in a carbon dioxide incubator with 95% humidity for 4-6 hours. Further, after 4 to 6 hours of stationary culture, 2 to 4ml of a medium was supplemented to submerge the umbilical cord tissue, and the temperature was continued at 37 ℃ with 5% CO2And standing and culturing in a carbon dioxide incubator with 95% humidity. Ensuring that the culture vessel is still in place within the first 7 days of culture; on day 7 of culture, fresh medium was replaced so that at least the medium in 2/3 in the culture vessel was replaced. After 7 days of culture, regular observations were made.
When the cells around the umbilical cord tissue are dissociated to a preset width, specifically, the dissociated width of the cells at the edge of the umbilical cord tissue is greater than or equal to 0.5m, which means that the cells enter a dissociation phase, the cell gap is increased, and then the dissociated cells are marked and harvested. Specifically, the step of harvesting the dissociated cells comprises: marking is carried out under a microscope, the umbilical cord tissue is stirred away by a gun head or a pipette so as to be separated from the surface of the culture medium, the umbilical cord tissue floats in a culture vessel, and all umbilical cord tissue floating in the culture vessel is removed before the mesenchymal stem cells are harvested so as to harvest the free mesenchymal stem cells.
The umbilical cord tissue is cultured by a tissue block adherence method, in the culture process, the umbilical cord tissue only needs to be placed in a culture vessel embedded with a matrix in advance for culture, and matrix glue does not need to be paved, so that the culture steps are simplified, the cultured cells are good in adhesion and strong in activity, and high-quality mesenchymal stem cells are provided for establishing a human mesenchymal stem cell bank.
In embodiments of the present application, the placental tissue is isolated and purified using enzymatic digestion, and preferably, the placental tissue comprises at least one of an amniotic membrane, a chorionic smooth muscle layer, a chorionic trophoblast, and a periostracum layer. In an embodiment of the present application, the placenta tissue includes an amnion, a chorion smooth muscle layer, a chorion trophoblast, and a periostracum layer, and by extracting mesenchymal stem cells from placenta tissue of various sources, not only are the sources of the mesenchymal stem cells enriched, but also a human-derived mesenchymal stem cell bank with more comprehensive functions can be established, and more importantly, the placenta tissue can be applied to various medical purposes.
In the embodiment of the present application, the step of separately purifying the placenta tissue by using the enzymatic digestion method comprises:
processing said placental tissue into strips of clumpy tissue;
washing the tissue strip with a irrigant;
adding a third buffer solution, and respectively processing the tissue strips into granular tissue blocks with preset sizes;
adding trypsin into the tissue blocks for carrying out first digestion to obtain first digestion liquid;
carrying out tissue block adherent culture on the aggregated cells in the first digestive juice;
performing secondary digestion on the cells subjected to tissue block adherent culture to obtain a second digestion solution;
and (3) carrying out adherent culture on the free cells of the second digestive juice to a logarithmic growth phase, and harvesting cell products containing the mesenchymal stem cells, or carrying out subculture on the cell products containing the mesenchymal stem cells.
In the step of separating and purifying the placenta tissue, the third buffer solution is preferably phosphate buffered saline solution.
Specifically, the placenta tissue is processed into a strip-shaped massive tissue strip, specifically a tissue strip processed into a strip-shaped massive tissue strip with the width of 1 cm.
Respectively processing the tissue strips into granular tissue blocks with preset sizes, comprising: stirring the placenta tissue by adopting a handheld electric homogenizer, and processing the placenta tissue into granules with the diameter of 1-2 mm.
Wherein, in the step of adding trypsin into the tissue block for the first digestion to obtain a first digestion solution, the dosage of the trypsin is equal to the volume of the tissue block, and the concentration of the trypsin is 0.25%.
Specifically, the step of adding trypsin to the tissue mass for the first digestion to obtain a first digestion solution further comprises: shaking at 37 deg.C for 60-90min until the tissue mass is viscous.
Preferably, the step of performing tissue block adherent culture on the aggregated cells in the first digestive fluid comprises:
centrifuging the first digestive juice for the first time to obtain a first supernatant;
centrifuging the first supernatant for a second time to obtain the aggregated cells as a precipitate;
and performing adherent culture on the aggregated cells.
Specifically, the first digestive juice is supplemented with a complete culture medium to 50ml, and first centrifugation is carried out to obtain a first supernatant; centrifuging the first supernatant at 1500r/min for 5min, discarding the supernatant, adding 20ml of complete culture medium, harvesting the aggregated cells at the bottom, transferring the aggregated cells to a culture bottle, and adding 5% CO2And (5) standing and culturing in an incubator.
Preferably, the step of performing a second digestion on the cells subjected to the tissue block adherent culture to obtain a second digestion solution further includes: trypsin was added for a second digestion. The dosage of the trypsin is equal to the volume of the cells after the tissue block adherent culture, and the concentration of the trypsin is 0.25 percent.
The step of taking the free cells of the second digestive juice for adherent culture comprises the following steps: supplementing complete culture medium to 50ml to the second digestive juice, mixing, filtering to remove tissue mass in the second digestive juice, centrifuging the filtrate at 1500r/min for 5min, discarding supernatant, adding 20ml for full culture, collecting bottom precipitate as free cell, transferring the free cell to culture bottle, and adding 5% CO2And (5) standing and culturing in an incubator.
In the construction method of the source mesenchymal stem cell bank, the placenta tissue is cultured by an enzyme digestion method, and during the culture process, only a single enzyme with stable property, namely trypsin, is adopted, and other enzymes which are easily influenced by external factors and have changed properties, such as collagenase, hyaluronic acid and the like, are not adopted, so that the stability of the digestion process is fundamentally ensured.
In an embodiment of the present application, the step of expanding the umbilical cord mesenchymal stem cells and the placental mesenchymal stem cells comprises:
in the step of separating and purifying umbilical cord mesenchymal stem cells, when the cells around the umbilical cord tissue are observed to be dissociated to a preset width, harvesting the dissociated cells, and performing subculture on the harvested dissociated cells, wherein the step specifically comprises the following steps: when the growth of the dissociated cells in the culture vessel to 85% -90% confluency (log phase growth) was observed, the dissociated cells were subcultured by digesting with trypsin for 10-50s, while retaining 1/3. Preferably, the time for digestion with trypsin is 30 s.
In the step of separating and purifying placenta mesenchymal stem cells, after the step of performing adherent culture on the free cells of the second digestive juice, the method further comprises the following steps: when the adherence of free cells of the second digestive juice is observed, periodically observing and replacing the culture solution by half amount; monitoring the confluent state of the growth of the free cells of the second digestive juice when the free cells of the second digestive juice are observed to be aggregated. Specifically, when free cells were observed to grow to 80% -90% confluency (log phase) or when growth inhibition occurred and cells were no longer significantly expanded, the free cells were maintained 1/3 and subcultured using trypsin digestion.
In the embodiment of the application, the steps of cryopreserving the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells are as follows: and (3) performing subculture, centrifuging, uniformly mixing the precipitated cells and the cell serum-free freezing solution, transferring into a freezing tube, temporarily storing in a refrigerator at the temperature of-80 ℃, editing and storing the freezing position, and transferring into liquid nitrogen for long-term storage.
Further, the method for constructing the source mesenchymal stem cell bank of the applicant further comprises the following steps: respectively inducing the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells to differentiate. Preferably, the umbilical cord mesenchymal stem cells and the placental mesenchymal stem cells are induced to differentiate into at least one of osteoblasts, adipocytes or neural cells, respectively.
Further, the method for constructing the source mesenchymal stem cell bank of the applicant further comprises the following steps: and performing information construction and management on the human mesenchymal stem cell bank. The method specifically comprises the following steps: the information of the patient is recorded, and the information of the generation number, the cell amount, the storage and the like of each cell is recorded.
In the embodiment of the application, the identification steps of the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells are as follows:
the biological characteristics of the mesenchymal stem cells obtained by the method for constructing the source mesenchymal stem cell bank are detected by morphological observation, cell immunophenotyping and mesenchymal stem cell adipogenic, osteogenic and neuro-differentiation potential experiments.
1. Morphological observation of mesenchymal stem cells:
the culture was kept static for 7 days after inoculation, and thereafter the morphology and growth of mesenchymal stem cells were observed under a microscope every day and recorded and photographed.
As for the observation result of umbilical cord mesenchymal stem cells obtained by the tissue block adherence method, referring to FIG. 4, in FIG. 4, a is the growth of umbilical cord mesenchymal stem cells in 3-10 days of static culture, and it can be seen that partially adherent cells are generated in 3-10 days of static culture. Panel b shows the local, dense growth centered on the tissue mass after cell culture. The c picture shows that after 10-15 days, the cells are integrally fused and spread to the bottom of the bottle, and simultaneously, the cells are fibroblast-like and grow in parallel or vortex. d, the figure shows the umbilical cord mesenchymal stem cells obtained after the third-generation culture of the cells obtained in the tissue block adherence method, and the figure shows that the cells have uniform shape after adherence, long prismatic shape, parallel or vortex growth and rapid cell growth after passage.
Referring to fig. 5, a graph a in fig. 5 shows the growth of the placental mesenchymal stem cells after 3 to 4 days of static culture, and it can be seen that partially adherent cells are generated after 3 to 4 days of static culture. Panel b shows that adherent cells increase and form colonies 7-10 days after culture, each colony has 1-2 germinal centers, and grows densely locally. The cells around the colony are in a fusiform shape with cytoplasm protrusions and are in a fibroblast-like shape, the cells in the center of the colony are round, the cytoplasm is rich, the nucleus is large, the cells grow in a vortex shape, and the cells in the center of the vortex are distributed in multiple layers. Panel c shows the presence of a small number of epithelial cells, which grow rapidly after passage, spreading the whole flask over 3-4 days. And d, the diagram shows the placenta mesenchymal stem cells obtained after the cells obtained by the enzyme digestion method are subjected to third-generation culture, and the diagram shows that the cells are uniform in shape, long prismatic, parallel or spiral in growth after being attached to the wall, and the cells grow rapidly after passage.
2. And (3) identifying the cellular immune phenotype:
digesting the near-fused third-generation mesenchymal stem cells by trypsin, centrifuging and washing to prepare 1 × 107The method comprises the following steps of/mL cell suspension, respectively subpackaging 200ul cell suspension in 7 flow tubes, respectively adding Fitc mouse anti-human CD 905 ul, PE mouse anti-human CD 445 ul, Percp-CyTM5.5 mouse anti-human CD 1055 ul, Apc mouse anti-human CD735ul, mesenchymal stem cell (hMSC) positive isotype control cocktail 20ul, PE hMSC negative isotype cocktail 20ul, hMSC positive cocktail 20ul and PE hMSC negative cocktail 20ul into the 7 flow tubes, respectively carrying out dark incubation for 30min, adding 1mL PBS, centrifuging at a rotating speed of 1500r/min for 5min, discarding supernatant, repeating the steps of centrifuging and discarding supernatant twice, re-suspending cells in 500 mu L, and detecting the surface marker of the cells by using a flow cytometer.
Referring to table 1, flow analysis results showed that all cells highly expressed the following proteins: CD73, CD90, CD44, CD105, flow analysis results also showed that all cells underexpressed the following proteins: CD34, CD19, CD11b, CD45, HLA-DR. Therefore, it is demonstrated that the primary cultured cells have surface markers of mesenchymal stem cells, and the cells obtained by isolation are placental mesenchymal stem cells. Meanwhile, the expression rate of the positive molecules is higher than 95%, the expression rate of the negative molecules is lower than 2%, and the expression rate can reach the minimum standard of International Society for Cell Therapy (ISCT), namely, the mesenchymal stem cells which are separated by the method for separating the mesenchymal stem cells are qualified mesenchymal stem cells.
TABLE 1 statistical table of expression rates of P3 generation mesenchymal stem cell surface antibodies
Inducing differentiation and staining results of umbilical cord mesenchymal stem cells and placenta mesenchymal stem cells:
fig. 6 shows the results of differentiation induction and staining of umbilical cord mesenchymal stem cells and placental mesenchymal stem cells. It is shown that the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells induced by osteogenesis are transformed from long fusiform to cubic shape and are changed like paving stones. Alizarin red staining shows that cells grow in colonies and appear calcium nodules, compact opaque masses are formed among the cells, large flaky red staining areas appear, the staining area is wide, the area is large, and the cells show obvious osteogenic activity.
The morphology of umbilical cord mesenchymal stem cells and placenta mesenchymal stem cells cultured by adipogenic induction begins to change on day 7, the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells gradually change into mast-like cells from slender fusiform, lipid droplets are formed in cytoplasm of a small part of mesenchymal stem cells after 7-8 days, and a large amount of fat cells are formed after induction for 11 days. As shown in the figure, the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells cultured by adipogenic induction are stained positively with oil red O, and the formation of lipid droplets in the induced cells is confirmed.
As shown in the figure, after 1 day of the neural induction culture, the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells can be seen to be round in cell morphology and granular in shape, the cells are seen to be light blue in Nisshi staining, and blue-black Nisshi corpuscles can be seen in the cells.
The method for constructing the source mesenchymal stem cell bank of the applicant is adopted to extract the output condition of the mesenchymal stem cells:
table 2 shows the extraction of mesenchymal stem cells from umbilical cord tissue and placental tissue, and it is known from Table 2 that the extraction rate of mesenchymal stem cells from umbilical cord tissue is 96.67%, and the yield is (30.84 + -1.82) × 105A plurality of; in the placenta tissue, the extraction rate of the amnion mesenchymal stem cells is 86.67%, and the yield is (33.64 +/-6.57) multiplied by 105The success rate of extracting the mesenchymal stem cells of the chorionic smooth muscle layer is 96.67 percent, and the yield is (22.13 +/-4.15) multiplied by 105Mesenchyme of chorionic trophoblastThe success rate of stem cell extraction is 100%, and the yield is (15.28 +/-4.72) multiplied by 105The extraction success rate of the mesenchymal stem cells of the decidua layer is 93.33 percent, and the yield is (9.46 +/-2.22) multiplied by 105And (4) respectively.
TABLE 2 statistical table of mesenchymal stem cell extraction conditions of umbilical cord tissue and placenta tissue
From the above, the method for extracting the umbilical cord mesenchymal stem cells by adopting the tissue mass adherence method and the method for extracting the amniotic mesenchymal stem cells, the chorionic smooth muscle interlayer mesenchymal stem cells, the chorionic trophoblast interlayer mesenchymal stem cells and the decidua interlayer mesenchymal stem cells by adopting the enzyme digestion method have the advantages of high success rate, short first passage time, high extraction efficiency and high yield, and are ideal sources for clinically culturing the mesenchymal stem cells in a large scale. In addition, the umbilical cord mesenchymal stem cells obtained by adopting the tissue block adherence method have high purity, can retain the cell activity to the maximum extent, thereby reducing the possibility of pollution, and simultaneously, the proliferation activity and the form of the cells are not obviously changed after continuous transmission for 10 generations. The cell viability of the amniotic mesenchymal stem cells, the chorionic smooth muscle mesenchymal stem cells, the chorionic trophoblast mesenchymal stem cells and the decidual mesenchymal stem cells extracted by adopting an enzyme digestion method is kept at a higher level.
The application also provides a human mesenchymal stem cell bank which is constructed by adopting the construction method of the human mesenchymal stem cell bank. By adopting the mesenchymal stem cells with different sources, the function of the human-derived mesenchymal stem cell bank with the mesenchymal stem cell source can be greatly enriched, and the mesenchymal stem cells in the cell bank can be amplified, so that the large-scale culture of the mesenchymal stem cells can be realized, and sufficient guarantee is provided for the clinical application of the mesenchymal stem cells.
The foregoing is only a partial embodiment of the present application, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present application, and these modifications and decorations should also be regarded as the protection scope of the present application.
Claims (23)
1. A method for constructing a human mesenchymal stem cell bank is characterized by comprising the following steps:
separating and purifying the umbilical cord tissue to obtain umbilical cord mesenchymal stem cells;
separating and purifying the placenta tissue to obtain placenta mesenchymal stem cells;
and (3) performing amplification, cryopreservation and/or identification on the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells.
2. The method for constructing the human mesenchymal stem cell bank according to claim 1, wherein the umbilical cord tissue is separated and purified by a tissue block adherence method.
3. The method for constructing the human mesenchymal stem cell bank according to claim 2, wherein the step of separating and purifying the umbilical cord tissue by using a tissue block adherence method comprises the following steps:
removing the accessory tissue of the umbilical cord and processing the umbilical cord into a strip-shaped umbilical cord tissue;
washing the umbilical cord tissue with a flushing agent;
adding a first buffer solution, and processing the umbilical cord tissue into particles with a preset size;
adding a second buffer solution, placing the granular umbilical cord tissues into a culture vessel in which a matrix is embedded in advance, adding a culture medium, and placing the umbilical cord tissues into an incubator for standing culture;
harvesting the dissociated cells when it is observed that the cells around the umbilical cord tissue are dissociated by a predetermined width;
subculturing the harvested free cells to obtain stable umbilical cord mesenchymal stem cells.
4. The method for constructing a human mesenchymal stem cell bank according to claim 3, wherein the first buffer solution is a phosphate buffered saline solution.
5. The method for constructing a human mesenchymal stem cell bank according to claim 3, wherein the step of processing the umbilical cord tissue into granules of a predetermined size comprises: the umbilical cord tissue was stirred with a hand-held electric homogenizer.
6. The method for constructing a human mesenchymal stem cell bank according to claim 3, wherein the second buffer solution is a phosphate buffered saline solution containing 1% antibiotic.
7. The method for constructing a human mesenchymal stem cell bank according to claim 3, wherein the step of placing the umbilical cord tissue in a culture vessel in which a matrix is pre-embedded comprises:
spreading the umbilical cord tissue on a substrate of a culture vessel with a density of sheets per cm2Placing a piece of said umbilical cord tissue.
8. The method for constructing a human mesenchymal stem cell bank according to claim 3, wherein the step of harvesting the dissociated cells when the cells around the umbilical cord tissue are observed to be dissociated by a predetermined width comprises:
when the width of the cell liberation at the edge of the umbilical cord tissue is observed to be greater than or equal to 0.5m, labeling is performed and the liberated cells are harvested.
9. The method of constructing a human mesenchymal stem cell bank according to claim 1, wherein the placental tissue comprises at least one of an amniotic membrane, a chorionic smooth muscle layer, a choriotrophoblast, and a periostracum layer.
10. The method for constructing a human mesenchymal stem cell bank according to claim 1, wherein the placental tissue is isolated and purified by an enzymatic digestion method.
11. The method for constructing the human mesenchymal stem cell bank according to claim 10, wherein the step of separately purifying the placenta tissues by enzymatic digestion comprises:
processing said placental tissue into strips of clumpy tissue;
washing the tissue strip with a irrigant;
adding a third buffer solution, and respectively processing the tissue strips into granular tissue blocks with preset sizes;
adding trypsin into the tissue blocks for carrying out first digestion to obtain first digestion liquid;
carrying out tissue block adherent culture on the aggregated cells in the first digestive juice;
performing secondary digestion on the cells subjected to tissue block adherent culture to obtain a second digestion solution;
and (3) carrying out adherent culture on the free cells of the second digestive juice to a logarithmic growth phase, and harvesting cell products containing the mesenchymal stem cells, or carrying out subculture on the cell products containing the mesenchymal stem cells.
12. The method for constructing a human mesenchymal stem cell bank according to claim 11, wherein the third buffer solution is phosphate buffered saline.
13. The method for constructing the human mesenchymal stem cell bank according to claim 11, wherein in the step of adding trypsin to the tissue mass for the first digestion to obtain the first digestion solution, the amount of the trypsin is equal to the volume of the tissue mass, and the concentration of the trypsin is 0.25%.
14. The method for constructing the human mesenchymal stem cell bank according to claim 11, wherein the step of adding trypsin to the tissue mass for the first digestion to obtain a first digestion solution further comprises: shaking at 37 deg.C for 60-90min until the tissue mass is viscous.
15. The method for constructing the human mesenchymal stem cell bank according to claim 11, wherein the step of performing tissue block adherent culture on the aggregated cells in the first digestive fluid comprises:
centrifuging the first digestive juice for the first time to obtain a first supernatant;
centrifuging the first supernatant for a second time to obtain the aggregated cells as a precipitate;
and performing adherent culture on the aggregated cells.
16. The method for constructing a human mesenchymal stem cell bank according to claim 11, wherein the step of performing the second digestion on the cells subjected to the tissue block adherence culture to obtain the second digestion solution further comprises: trypsin was added for a second digestion.
17. The method for constructing the human mesenchymal stem cell bank according to claim 16, wherein in the step of adding trypsin for the second digestion, the amount of the trypsin is equal to the volume of the cells after the tissue block adherent culture, and the concentration of the trypsin is 0.25%.
18. The method for constructing a human mesenchymal stem cell bank according to claim 11, wherein the step of performing adherent culture on the free cells of the second digestive fluid further comprises:
when the adherence of free cells of the second digestive juice is observed, periodically observing and replacing the culture solution by half amount;
monitoring the confluent state of the growth of the free cells of the second digestive juice when the free cells of the second digestive juice are observed to be aggregated.
19. The method for constructing the human mesenchymal stem cell bank according to claim 1, wherein before the separation and purification of the umbilical cord tissue and the placenta tissue, the method comprises the following steps:
human umbilical cord and placenta were washed with a physiological saline containing 1% penicillin streptomycin.
20. The method for constructing a human mesenchymal stem cell bank according to claim 1, further comprising: respectively inducing the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells to differentiate.
21. The method for constructing a human mesenchymal stem cell bank according to claim 20, wherein: inducing the umbilical cord mesenchymal stem cells and the placenta mesenchymal stem cells to differentiate into at least one of osteoblasts, adipocytes or neural cells, respectively.
22. The method for constructing a human mesenchymal stem cell bank according to claim 1, further comprising: and performing information construction and management on the human mesenchymal stem cell bank.
23. A human mesenchymal stem cell bank, which is constructed by the method for constructing a human mesenchymal stem cell bank according to any one of claims 1 to 22.
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Non-Patent Citations (1)
| Title |
|---|
| 侯宗柳等: "《围产期成体干细胞基础与临床应用》", 31 August 2016 * |
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