CN113249317A - Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells - Google Patents
Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a method and a system for separating, culturing and amplifying umbilical cord mesenchymal stem cells, which comprises the following steps: sample pretreatment, step two: obtaining the Walton gel, shearing the Walton gel into small pieces of tissues, adding filtered erythrocyte lysis buffer solution with the volume 3.5 times of the volume of the small pieces of tissues, and performing a third step: collagenase digestion, the digestion solution being a balance comprising 1% collagenase type IV, 0.5% hyaluronidase, 300U/ml DNase, and 2% serum replacementA salt solution; step four: separating and culturing primary cells, adding a serum-free culture medium of the mesenchymal stem cells to obtain a cell suspension, and adding a primary cell culture solution special for the umbilical cord mesenchymal stem cells to perform primary culture; step five: subculturing the cells, collecting the cells at a concentration of 5000/cm using trypsin at 0.215% by mass2Inoculating; step six: and collecting the umbilical cord mesenchymal stem cells, and performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a separation culture amplification method and a separation culture amplification system for human umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) refer to a class of adult stem cells with self-renewal and multipotentiality, mainly derived from mesoderm tissue. Since the first discovery and successful isolation of MSCs in bone marrow in the last 60 th century, subsequent researchers isolated MSCs from tissues such as fat, placenta, umbilical cord, dental pulp, and the like. MSCs are capable of differentiating into multiple blastoderm multi-type cells, such as epiblast-like cells of the ectoderm; adipocytes, chondrocytes, osteocytes of mesoderm, etc.; endodermal insulin-secreting cells, and the like. The MSC has various sources, easy separation, amplification and culture, low immunogenicity, strong updating and differentiation capacity, wide treatment range and the like.
Particularly, umbilical cord mesenchymal stem cells (hUC-MSCs) which are separated, amplified and cultured from human umbilical cord Wharton's jelly are derived from clinically discarded umbilical cord tissues of pregnant women, so that the umbilical cord mesenchymal stem cells are free from the disputes and requirements of ethics, morals and laws, are convenient to obtain materials and wide in source, and are convenient to collect, store, transport and the like. In addition, the hUC-MSCs have rich content, stronger proliferation and differentiation capacity than common adult stem cells, can be quickly separated, cultured and amplified in vitro, and can be passaged for many times to keep dry. The hUC-MSCs have lower immunogenicity, low expression HLA-DR and low allograft immune rejection, and are favorable for long-term storage and used for body immune regulation or disease treatment.
There are many mature methods for separating and extracting the hUC-MSCs, but how to rapidly and sufficiently extract the hUC-MSCs in the Wharton jelly and keep the cells well dry is still the key and difficult point of the current research and is also the key of the later clinical storage, application and industrialization transformation. At present, the commonly used separation culture methods of the hUC-MSCs are an enzyme digestion method and a tissue block adherent culture method. Although the enzyme digestion method can obtain a large amount of primary cells for the first time, the cell adherence effect and the form are poor due to the double destruction of mechanical shearing force and digestive enzyme in the extraction process, the digestion time of a tissue block is difficult to be uniformly quantified in the large-scale production process, in addition, the separation and extraction cost is high, and the processing time is relatively long. The primary culture time of the conventional tissue block culture method is generally long, about 15 days are needed, and the separation and extraction efficiency is influenced mainly by the size of the sheared Wharton's jelly tissue block and the first adherence treatment.
Disclosure of Invention
In order to solve the problems that the prior art has long time, low efficiency and small cell harvest amount of primary cell isolation and culture, thereby shortening the culture days of the primary cells, improving the cell harvest amount, and improving the cell passage times, purity and multidirectional differentiation capacity, the invention provides a human umbilical cord mesenchymal stem cell isolation, culture and amplification method, which comprises the following steps:
the method comprises the following steps: pretreating a sample, cleaning blood and impurities attached to an aseptically obtained umbilical cord, soaking and disinfecting the umbilical cord in 75% alcohol for 1min, cleaning the umbilical cord for 3 times by using cleaning solution, cutting off the umbilical cord along the inner sides of ligation ends at two sides, discarding an edema or blood coagulation part, cutting the rest umbilical cord into 6cm +/-2 cm in length, extruding the blood in each section of umbilical cord, and transferring the umbilical cord into a new culture dish filled with the cleaning solution;
step two: obtaining Wharton's jelly, taking out all Wharton's jelly in umbilical cord, washing with normal saline solution for 3 times, and shearing Wharton's jelly into 2-4mm pieces3The left and right small blocks are organized; filtering the erythrocyte lysis buffer solution by using a 0.22 mu m microfiltration membrane at room temperature, adding the filtered erythrocyte lysis buffer solution with the volume 3.5 times that of the small tissue, and treating the small tissue for 7-9 minutes; the treated pieces of tissue were collected by centrifugation and washed 2-3 times with PBS; the erythrocyte lysis buffer comprises 5g/L NH4Cl and 0.1mMol Na2-EDTA，pH 7.2-7.4；
Step three: digesting with collagenase, adding collagenase digestion solution 2-3 times of the volume of the small tissue, transferring to 5% CO at 37 deg.C2After 8-12 hours, the culture box is transferred to a clean workbench and diluted by adding PBS; filtering with a sterile sieve, collecting the filtrate into a 50mL centrifuge tube, washing with PBS, and centrifuging to remove residual enzyme; the digestion solution is a solution containing 1% type IV gumA balanced salt solution of proenzyme, 0.5% hyaluronidase, 300U/ml dnase and 2% serum replacement;
step four: performing isolation culture on the primary cells, namely flicking the cell sediment at the bottom of a centrifugal tube in the third step, adding a serum-free culture medium of the mesenchymal stem cells to obtain a cell suspension, paving the cell suspension in a cell culture bottle, placing the cell suspension in a cell culture box, and adding the umbilical cord mesenchymal stem cells to perform primary culture by using a primary cell culture solution after the tissue is attached to the bottom of the bottle; the serum-free culture medium of the mesenchymal stem cells comprises 0.1 volume part of beta-mercaptoethanol, 1 volume part of non-essential amino acid aqueous solution, 10 volume parts of serum substitute, 89 volume parts of alpha-MEM/DMEM-F12 and 10ng/ml of b-FGF;
step five: subculturing the cells, using trypsin with a mass percent concentration of 0.215% after the cell fusion degree reaches about 80%, tapping the side wall of the culture flask during digestion, collecting the cells by centrifugation, and collecting the collected cells at a rate of 5000 cells/cm2Inoculating;
step six: collecting the umbilical cord mesenchymal stem cells harvested in the fifth step, and then carrying out osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments; the osteogenic differentiation is cultured by using an osteogenic induction liquid, and the osteogenic induction liquid culture comprises the following steps: 20 mu g/ml of insulin, 0.1 mu g/ml of transferrin, 40.6 ng/ml of BMP, 5ng/ml of growth hormone, 100 mu g/ml of glutathione, 1.2mM of L-glutamine, 60 mu M of beta-mercaptoethanol, 0.12mM of non-essential amino acid aqueous solution, 15 mu M of nano ferric oxide and nitrogen-doped graphene mixture and DMEM as a culture medium; the adipogenic differentiation adopts adipogenic induction liquid, and the adipogenic induction liquid comprises: 10mol/L dexamethasone, 5mg/L insulin, 0.5 mmol/L3-isobutylmethylxanthine, 60 mu mol/L indomethacin, 10% volume fraction FBS, and DMEM as culture medium; the chondrogenic differentiation adopts chondrogenic induction culture solution, and the chondrogenic induction culture solution adopts a DMEM-F12 culture medium containing 50mg/ml of mudsnail polypeptide, 6.5mg/L of insulin, 6.5mg/L of transferrin, 20 mu g/L of transforming growth factor beta 1, 0.5 mu mol/L of dexamethasone, 50mg/L of vitamin C, 6 mu M of isoalizarin, 5% of FBS and 1% of double antibody for induction culture.
Further, in the sixth step, in the chondrogenic differentiation experiment, the culture medium is changed every 5 days, after 14 days of culture, the chondrogenic induction culture medium is removed, and 500 μ L of RNA extraction reagent is added until a clear and non-viscous liquid is formed; adding 180 mu L of chloroform, shaking vigorously and mixing uniformly, and standing at room temperature; after centrifugation, transferring the upper layer transparent RNA aqueous phase into a new RNA enzyme-free EP tube; adding isopropanol with the same volume, mixing uniformly, centrifuging, removing supernatant, and keeping precipitate; adding 60 μ L of 50% ethanol, centrifuging, removing supernatant, drying at room temperature for 4min, extracting RNA, and detecting RNA quality and concentration; and the extracted RNA was reverse-transcribed to obtain a cDNA sample, which was then amplified by PCR, stained with 1% alistic blue glacial acetic acid solution at room temperature for 30min, and then subjected to agarose gel electrophoresis, and the result was observed by an electrophoresis gel imager.
Further, in the sixth step, in the adipogenic differentiation experiment, the cells with good growth of P5 generation were selected to be 2X 104Individual cell/cm2Is inoculated into a 6-well cell culture plate, and when 100% fusion is reached, the adipogenic induction liquid is added into each well to start induction; after 3 days, the original adipogenic induction liquid was replaced with a fresh adipogenic induction liquid and the culture was maintained for 24 hours, and when many and small fat droplets appeared, the culture was maintained for 7 days with the adipogenic induction liquid; after induction, cells are fixed by 4% paraformaldehyde, stained by oil red O, and the transcription level of the adipogenic marker gene PPAR-gamma is detected by RT-PCR.
Further, in the sixth step, in the osteogenic differentiation experiment, the cells with good growth of P5 generation were selected to be 3 × 104Individual cell/cm2The cell density of (a) was inoculated into 6-well cell culture plates, after 24 hours, an osteogenic induction solution was added to each well for culture, and the primary osteogenic induction solution culture was replaced with a fresh osteogenic induction solution culture every 3 days, after 2 weeks, the cells were fixed with paraformaldehyde and stained with alizarin red for 3-5 minutes, and the expression level of the osteogenic marker gene OPN was identified by RT-PCR.
Further, the culture solution for umbilical cord mesenchymal stem cells in the fourth step does not contain animal-derived components and human platelet lysate.
Further, in the fourth step, when the tissue is placed in a cell culture box to be jointedAfter the bottom of the bottle, adding a primary cell culture solution for umbilical cord mesenchymal stem cells, the volume of which is 3-5 times of the volume of the small tissue, and adding 5% CO at 37 DEG C2Performing primary culture under the condition; replacing half of the original primary cell culture solution with fresh primary cell culture solution after 2 days, and removing small tissue blocks when the umbilical cord mesenchymal stem cells climb out of the umbilical cord tissue blocks after attaching to the wall for about 4 days; the original primary cell culture fluid was replaced with fresh primary cell culture fluid every 3-4 days.
And further, a quality inspection step is included, and the umbilical cord mesenchymal stem cells harvested in the step five are subjected to quality inspection, including microbial contamination, cell number, cell activity and safety evaluation.
Further, the method also comprises a step of analyzing a cell surface marker, wherein the umbilical cord mesenchymal stem cells harvested in the step five are collected and then subjected to flow analysis on the cell surface marker; selecting P5 generation well-growing cells, digesting and centrifuging when the cells reach 80-90% fusion state under observation to prepare single cell suspension, respectively adding CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19, incubating, centrifuging, washing and resuspending, and detecting the expression level of each antigen marker by using a flow cytometer.
The invention also provides a system for realizing the isolated culture and amplification method of the human umbilical cord mesenchymal stem cells, which comprises the following steps:
the cell collection system is used for pretreating the umbilical cord sample and obtaining Wharton's jelly in the umbilical cord;
the separation culture system is used for performing collagenase digestion on the Wharton's jelly of the small tissues, and performing primary cell separation culture and subculture;
and the multidirectional differentiation detection system is used for carrying out osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments on the harvested cells.
Further, still include: a flow cytometry detection system for detecting the expression level of each antigen marker by using a flow cytometer; the quality inspection system is used for performing quality inspection on the harvested umbilical cord mesenchymal stem cells, and comprises microbial contamination, cell number, cell activity and safety evaluation; and the marking system is used for collecting the umbilical cord mesenchymal stem cells and then carrying out flow analysis on the cell surface markers.
Drawings
FIG. 1 is a picture of an umbilical cord after pretreatment;
FIG. 2 is a drawing showing the peeling of the Walton's gum;
FIG. 3 is a wall sticking diagram of a tissue block of Wharton's jelly;
FIG. 4 is a climbing out of umbilical cord mesenchymal stem cells from a tissue mass;
FIG. 5 is a photograph of the morphology of cells at passage P1;
FIG. 6 is a picture of the flow detection result of umbilical cord mesenchymal stem cells of generation P5;
FIG. 7 is a drawing of experimental results of multidirectional differentiation of umbilical cord mesenchymal stem cells;
FIG. 8 is a graph of the growth curve of umbilical cord mesenchymal stem cells.
Detailed Description
The umbilical cord mesenchymal stem cell separation culture amplification method provided by the invention specifically comprises the following steps:
step one, sample pretreatment: taking out the aseptically obtained umbilical cord from the super clean bench, cleaning attached blood and sundries, transferring the umbilical cord to 75% alcohol for soaking and sterilizing for 1min, cleaning the sterilized umbilical cord for 3 times by using cleaning solution, then cutting off along the inner sides of the ligation ends at two sides of the umbilical cord by using tissue forceps and scissors, discarding the edematous or blood coagulation part, cutting the rest umbilical cord into the length of 6cm +/-2 cm, extruding the blood in each section of umbilical cord by using the tissue forceps, then transferring the umbilical cord to a new culture dish filled with the cleaning solution, and carrying out pretreatment on the umbilical cord, wherein the picture is shown in figure 1.
Preferably, the washing solution is a physiological saline solution containing penicillin, streptomycin and amphotericin.
Step two, obtaining the Walton's gum: tearing the amniotic membrane along the torsion of umbilical cord, taking out all Wharton's jelly, washing with physiological saline solution for 3 times, and cutting into pieces of 2-4mm3The left and right small blocks are organized; adding a volume of red blood cell lysis buffer 3.5 times the volume of the small piece of tissue, and treating the small piece of tissue with the buffer at room temperature for 7-9 minutes; the treated pieces were collected by centrifugation and washed 2-3 times with PBS; wherein the centrifugation is carried outPreferably 1300rpm, 5 ℃ for 10 minutes.
Preferably, the physiological saline solution contains penicillin, streptomycin and amphotericin. The erythrocyte lysis buffer contained 5g/L NH4Cl and 0.1mM Na2EDTA, pH 7.2-7.4. Before use, the erythrocyte lysis buffer was filtered through a 0.22 μm microfiltration membrane and equilibrated to room temperature.
Step three, adding a digestion solution containing collagenase IV 2-3 times of the volume of the small tissue, transferring to 37 ℃ and 5% CO2After 8-12 hours, the culture box is transferred to a clean workbench and diluted by adding PBS; and filtered through a 100 or 200 mesh sterile screen, the filtrate was collected into a 50mL centrifuge tube, washed with PBS, and centrifuged to remove residual enzyme;
the digestion solution was a balanced salt solution containing 1% collagenase type IV, 0.5% hyaluronidase, 300U/ml dnase and 2% serum replacement.
Step four, separating and culturing primary cells: flicking the cell sediment at the bottom of the centrifuge tube in the third step, adding a serum-free culture medium of the mesenchymal stem cells to obtain a cell suspension, transferring the cell suspension to a 100mm culture bottle, paving the culture bottle in the cell culture bottle, placing the culture bottle in a cell culture box, and adding the umbilical cord mesenchymal stem cells to perform primary culture by using a primary cell culture solution after the tissue is attached to the bottom of the bottle as shown in figure 3; the culture solution for the umbilical cord mesenchymal stem cells does not contain animal-derived components and human platelet lysate.
Preferably, after the tissue is placed in a cell culture box to be attached to the bottom of a bottle, the primary cell culture solution for the umbilical cord mesenchymal stem cells with the volume being 3-5 times of the volume of the small piece of tissue is added, and the mixture is subjected to 5% CO at 37 ℃ to obtain the umbilical cord mesenchymal stem cells2Performing primary culture under the condition; replacing half of the original primary cell culture solution with fresh primary cell culture solution after 2 days, and removing small tissue blocks when umbilical cord mesenchymal stem cells climb out of the umbilical cord tissue blocks after the umbilical cord mesenchymal stem cells adhere to the walls for about 4 days as shown in figure 4; the original primary cell culture fluid was replaced with fresh primary cell culture fluid every 3-4 days.
The serum-free culture medium of the mesenchymal stem cells comprises 0.1 volume part of beta-mercaptoethanol, 1 volume part of non-essential amino acid aqueous solution, 10 volume parts of serum substitute, 89 volume parts of alpha-MEM/DMEM-F12 and b-FGF with the concentration of 10 ng/ml.
Step five, cell subculturing: observing the state of cells around the tissue block in the culture bottle, carrying out subculture amplification after the cell fusion degree reaches about 80%, using trypsin with the mass percentage concentration of 0.215% in the subculture process, tapping the side wall of the culture bottle during digestion, collecting the cells by centrifugation, and centrifuging at 1300rpm and 6 ℃ for 9 minutes; collecting the cells at a density of 5000 cells/cm2Inoculating, the cell can reach 90% fusion degree after 4 days, and the extracted umbilical cord mesenchymal stem cell can stably transmit for more than 20 generations. Wherein, 2.4 multiplied by 10 can be harvested in the P0 generation7Cells, the P1 generation, were harvested at 3.7X 108Cells, and the P5 generation can be harvested at 7.9X 1012A cell. The figure 5 shows a picture of the morphology of the P1 generation cells, and the figure 8 shows a picture of the growth curve of the umbilical cord mesenchymal stem cells.
Cell doubling time
And t is the culture time,
to initiate the number of cells after seeding,
the number of cells after t hours of culture.
And step six, in order to detect the multidirectional differentiation potential of the umbilical cord mesenchymal stem cells, performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments on the cells harvested in the step five respectively.
Osteogenic differentiation experiment
Well-growing cells from P5 generation were selected at 3X 104Individual cell/cm2Was seeded into 6-well cell culture plates. After 24 hours, an osteogenic induction liquid was added to each well for culture, and the osteogenic induction liquid was replaced with fresh osteogenic induction liquid every 3 days. The osteogenesis inducing liquid comprises: insulin 20 mug/ml, transferrin 0.1 mug/ml, BMP-40.6 ng/ml,5ng/ml of growth hormone, 100 mu g/ml of glutathione, 1.2mM of L-glutamine, 60 mu M of beta-mercaptoethanol, 0.12mM of non-essential amino acid, 15 mu M of a mixture of nano ferric oxide and nitrogen-doped graphene, and a culture medium of DMEM. Preferably, the mixture of the nano ferric oxide and the nitrogen-doped graphene is prepared from 16 parts by mass of the nano ferric oxide, 8 parts by mass of the modified graphene oxide, 12 parts by mass of urea and DMEM as a matrix.
After 2 weeks, cells were fixed with paraformaldehyde and stained with alizarin red for 3-5 minutes. And simultaneously, identifying the expression level of osteopontin OPN (osteopontin) of the osteogenesis marker gene on the transcription level by using an RT-PCR (reverse transcription-polymerase chain reaction) technology. Wherein A in FIG. 7 is a photograph of alizarin red staining after osteogenic differentiation, and alizarin red reacts with calcium nodules during osteogenic period to generate deep red color after two weeks of osteogenic induction of umbilical cord mesenchymal stem cells obtained by the method of the present invention; in addition, the osteogenic marker gene OPN also showed differential expression levels before and after induction.
Adipogenic differentiation experiment
Well-growing P5 generation cells were selected at 2X 104Individual cell/cm2Was seeded into 6-well cell culture plates. When 100% fusion is achieved, adding DMEM adipogenic induction liquid containing 10mol/L dexamethasone, 5mg/L insulin, 0.5 mmol/L3-isobutylmethylxanthine, 60 mu mol/L indomethacin and 10% FBS in volume fraction into each well to start induction; after 3 days, the original adipogenic induction liquid was replaced with a fresh adipogenic induction liquid, and the culture was maintained for 24 hours. When many and small fat drops appear, maintaining the culture for 7 days by using a adipogenesis induction liquid; after induction, cells are fixed by 4% paraformaldehyde, stained by oil red O, and the transcription level of the adipogenic marker gene PPAR-gamma is detected by RT-PCR.
B in FIG. 7 is a photograph of oil red O staining after adipogenic differentiation, and oil red O staining of adipogenic cells was evident two weeks after adipogenic induction of HUC-MSCs obtained by the method of the present invention. In addition, the adipogenesis marker gene PPAR- γ also showed differential expression levels before and after induction.
Chondrogenic differentiation test
Well-growing P3 generation cells were selected at 5X 106Individual cell/cm2Is inoculated into a T25 cell culture flaskIn (1). After 5 days, when 100% fusion was achieved, chondrogenic induction medium, preferably DMEM/F12 medium containing 50mg/ml mudsnail polypeptide, 6.5mg/L insulin, 6.5mg/L transferrin, 20. mu.g/L transforming growth factor beta 1, 0.5. mu. mol/L dexamethasone, 50mg/L vitamin C, 6. mu.M isoalizarin, 5% FBS and 1% double antibody, was added separately to induce culture.
Changing the culture solution every 5 days, culturing for 14 days, removing the culture solution into cartilage induction culture solution, and adding 500 mu L of RNA extraction reagent Trizol until clear and non-viscous liquid is formed; adding 180 mu L of chloroform, shaking vigorously and mixing uniformly, and standing at room temperature; after centrifugation, transferring the upper layer transparent RNA aqueous phase into a new RNA enzyme-free EP tube; adding isopropanol with the same volume, mixing uniformly, centrifuging, removing supernatant, and keeping precipitate; adding 60 μ L of 50% ethanol, centrifuging, removing supernatant, drying at room temperature for 4min, extracting RNA, and detecting RNA quality and concentration; and the extracted RNA was reverse-transcribed to obtain a cDNA sample, which was then amplified by PCR, stained with 1% alistic blue glacial acetic acid solution (pH 2.5) for 30min at room temperature, and then subjected to agarose gel electrophoresis, and the result was observed by an electrophoresis gel imager.
C in FIG. 7 is a photograph of Alisine blue staining after chondrogenic differentiation. The result shows that the umbilical cord mesenchymal stem cells can well realize three-dimensional differentiation.
In a preferred embodiment, the method further comprises a quality inspection step, wherein the umbilical cord mesenchymal stem cells harvested in the step five are subjected to quality inspection, including microbial contamination, cell number, cell activity and safety evaluation.
In a preferred embodiment, the method further comprises a step of analyzing the cell surface marker, wherein the cell surface marker is analyzed by flow after the umbilical cord mesenchymal stem cells are collected,
selecting P5 generation well-grown cells, observing that the cells reach 80% -90% fusion state, digesting and centrifuging to prepare single cell suspension (concentration is 1 × 10)6And/ml), adding CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19 respectively, incubating, centrifuging, washing, suspending, and detecting the expression level of each antigen marker by using a flow cytometer.
The results show that umbilical cord mesenchymal stem cells have better homogeneity, all express CD73, CD90 and CD105, and do not express CD34, CD45, HLA-DR, CD11b and CD 19. The results are shown in FIG. 6.
The invention also provides a system for realizing the umbilical cord mesenchymal stem cell separation culture amplification method, which comprises the following steps:
the cell collecting system is used for pretreating the umbilical cord sample and obtaining the Wharton's jelly in the umbilical cord,
the separation culture system is used for performing collagenase digestion on the Wharton's jelly of the small tissues, and performing primary cell separation culture and subculture;
and the multi-directional differentiation detection system is used for carrying out osteogenic, adipogenic and chondrogenic differentiation experiments on the harvested cells.
A flow cytometry detection system for detecting the expression level of each antigen marker by using a flow cytometer;
in a preferred embodiment, the kit further comprises a flow cytometry detection system for detecting the expression level of each antigen marker by using a flow cytometer; the quality inspection system is used for performing quality inspection on the harvested umbilical cord mesenchymal stem cells, and comprises microbial contamination, cell number, cell activity and safety evaluation; and the marking system is used for collecting the umbilical cord mesenchymal stem cells and then carrying out flow analysis on the cell surface markers.
The method and the system for obtaining the mesenchymal stem cells by the tissue mass separation method are simple, the cost is low, the cell activity is good, the purity is high, the concentration of the finally obtained cells is high, the method for obtaining the mesenchymal stem cells is simple, the MSC can be separated quickly and efficiently, the culture time is shortened, the cells can be seen to creep out of the tissue mass after being cultured for about 4 days, the obtaining rate of primary cells and the cell creep-out time are obviously improved, the concentration of the finally obtained cells is high, and the whole culture process of the umbilical cord mesenchymal stem cells can be standardized, programmed and standardized.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A method for separating, culturing and amplifying human umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
the method comprises the following steps: pretreating a sample, cleaning blood and impurities attached to an aseptically obtained umbilical cord, soaking and disinfecting the umbilical cord in 75% alcohol for 1min, cleaning the umbilical cord for 3 times by using cleaning solution, cutting off the umbilical cord along the inner sides of ligation ends at two sides, discarding an edema or blood coagulation part, cutting the rest umbilical cord into 6cm +/-2 cm in length, extruding the blood in each section of umbilical cord, and transferring the umbilical cord into a new culture dish filled with the cleaning solution;
step two: obtaining Wharton's jelly, taking out all Wharton's jelly in umbilical cord, washing with normal saline solution for 3 times, and shearing Wharton's jelly into 2-4mm pieces3The left and right small blocks are organized; filtering the erythrocyte lysis buffer solution by using a 0.22 mu m microfiltration membrane at room temperature, adding the filtered erythrocyte lysis buffer solution with the volume 3.5 times that of the small tissue, and treating the small tissue for 7-9 minutes; the treated pieces of tissue were collected by centrifugation and washed 2-3 times with PBS; the erythrocyte lysis buffer comprises 5g/L NH4Cl and 0.1mMol Na2-EDTA，pH 7.2-7.4；
Step three: digesting with collagenase, adding collagenase digestion solution 2-3 times of the volume of the small tissue, transferring to 5% CO at 37 deg.C2After 8-12 hours, the culture box is transferred to a clean workbench and diluted by adding PBS; filtering with a sterile sieve, collecting the filtrate into a 50mL centrifuge tube, washing with PBS, and centrifuging to remove residual enzyme; the digestion solution is a balanced salt solution containing 1% collagenase type IV, 0.5% hyaluronidase, 300U/ml dnase and 2% serum replacement;
step four: performing separation culture on primary cells, namely flicking the cell sediment at the bottom of a centrifugal tube in the third step, adding a serum-free culture medium of the mesenchymal stem cells to obtain a cell suspension, paving the cell suspension in a cell culture bottle, placing the cell suspension in a cell culture box, and after tissue is attached to the bottom of the bottle, adding umbilical cord mesenchymal stem cells into the cell culture box to perform primary culture by using a primary cell culture solution, wherein the culture solution for the umbilical cord mesenchymal stem cells does not contain animal-derived components and human platelet lysate; the serum-free culture medium of the mesenchymal stem cells comprises 0.1 volume part of beta-mercaptoethanol, 1 volume part of non-essential amino acid aqueous solution, 10 volume parts of serum substitute, 89 volume parts of alpha-MEM/DMEM-F12 and 10ng/ml of b-FGF;
step five: subculturing the cells, using trypsin with a mass percent concentration of 0.215% after the cell fusion degree reaches about 80%, tapping the side wall of the culture flask during digestion, collecting the cells by centrifugation, and collecting the collected cells at a rate of 5000 cells/cm2Inoculating;
step six: a multidirectional differentiation potential detection step, namely collecting the umbilical cord mesenchymal stem cells harvested in the step five, and then performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments; the osteogenic differentiation is cultured by using osteogenic induction liquid, and the osteogenic induction liquid comprises: 20 mu g/ml of insulin, 0.1 mu g/ml of transferrin, 40.6 ng/ml of BMP, 5ng/ml of growth hormone, 100 mu g/ml of glutathione, 1.2mM of L-glutamine, 60 mu M of beta-mercaptoethanol, 0.12mM of non-essential amino acid aqueous solution, 15 mu M of nano ferric oxide and nitrogen-doped graphene mixture and DMEM as a culture medium; the adipogenic differentiation adopts adipogenic induction liquid, and the adipogenic induction liquid comprises: 10mol/L dexamethasone, 5mg/L insulin, 0.5 mmol/L3-isobutylmethylxanthine, 60 mu mol/L indomethacin, 10% volume fraction FBS, and DMEM as culture medium; the chondrogenic differentiation adopts chondrogenic induction culture solution, the chondrogenic induction culture solution uses a DMEM-F12 culture medium containing 50mg/ml mud snail polypeptide, 6.5mg/L insulin, 6.5mg/L transferrin, 20 mu g/L transforming growth factor beta 1, 0.5 mu mol/L dexamethasone, 50mg/L vitamin C, 6 mu M isoalizarin, 5% FBS and 1% double antibody to induce and culture, the solution is changed once every 5 days, after 14 days of culture, the chondrogenic induction culture solution is removed, 500 mu L RNA extraction reagent is added until clear and non-viscous liquid is formed; adding 180 mu L of chloroform, shaking vigorously and mixing uniformly, and standing at room temperature; after centrifugation, transferring the upper layer transparent RNA aqueous phase into a new RNA enzyme-free EP tube; adding isopropanol with the same volume, mixing uniformly, centrifuging, removing supernatant, and keeping precipitate; adding 60 μ L of 50% ethanol, centrifuging, removing supernatant, drying at room temperature for 4min, extracting RNA, and detecting RNA quality and concentration; and the extracted RNA was reverse-transcribed to obtain a cDNA sample, which was then amplified by PCR, stained with 1% alistic blue glacial acetic acid solution at room temperature for 30min, and then subjected to agarose gel electrophoresis, and the result was observed by an electrophoresis gel imager.
2. The method for separating, culturing and expanding the human umbilical cord mesenchymal stem cells according to claim 1, wherein in the sixth step, in the adipogenic differentiation experiment, cells with good growth of P5 generation are selected to be 2 x 104Individual cell/cm2Is inoculated into a 6-well cell culture plate, and when 100% fusion is reached, the adipogenic induction liquid is added into each well to start induction; after 3 days, the original adipogenic induction liquid was replaced with a fresh adipogenic induction liquid and the culture was maintained for 24 hours, and when many and small fat droplets appeared, the culture was maintained for 7 days with the adipogenic induction liquid; after induction, cells are fixed by 4% paraformaldehyde, stained by oil red O, and the transcription level of the adipogenic marker gene PPAR-gamma is detected by RT-PCR.
3. The method for separating, culturing and expanding human umbilical cord mesenchymal stem cells according to claim 1, wherein in the sixth step, in the osteogenic differentiation experiment, cells with good growth of P5 generation are selected to be 3 x 104Individual cell/cm2Is inoculated into a 6-well cell culture plate, after 24 hours, osteogenic induction liquid is added into each well for culture, and the primary osteogenic induction liquid is replaced by fresh osteogenic induction liquid every 3 days for culture, after 2 weeks, cells are fixed by paraformaldehyde and are stained by alizarin red for 3-5 minutes, and the expression level of the osteogenic marker gene OPN is identified by RT-PCR.
4. The isolated culture and amplification method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: in the fourth step, after the tissue is placed in a cell culture box to be attached to the bottom of the bottle, adding the primary cell culture solution for the umbilical cord mesenchymal stem cells, the volume of which is 3-5 times of the volume of the small tissue, into the cell culture box, and performing cell culture at 37 ℃ and 5% CO2Performing primary culture under the condition; after 2 days useReplacing half of the original primary cell culture solution with fresh primary cell culture solution, and removing small tissue blocks when the umbilical cord mesenchymal stem cells climb out of the umbilical cord tissue blocks after attaching to the wall for about 4 days; the original primary cell culture fluid was replaced with fresh primary cell culture fluid every 3-4 days.
5. The isolated culture and amplification method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: and a quality inspection step, wherein the umbilical cord mesenchymal stem cells harvested in the step five are subjected to quality inspection, and the quality inspection comprises microbial contamination, cell number, cell activity and safety evaluation.
6. The isolated culture and amplification method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: the step of analyzing the cell surface marker is also included, and the cell surface marker is analyzed in a flow mode after the umbilical cord mesenchymal stem cells harvested in the step five are collected; selecting P5 generation well-growing cells, digesting and centrifuging when the cells reach 80-90% fusion state under observation to prepare single cell suspension, respectively adding CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19, incubating, centrifuging, washing and resuspending, and detecting the expression level of each antigen marker by using a flow cytometer.
7. A system for implementing the isolated culture and expansion method of human umbilical cord mesenchymal stem cells according to any one of claims 1 to 6, which comprises:
the cell collection system is used for pretreating the umbilical cord sample and obtaining Wharton's jelly in the umbilical cord;
the separation culture system is used for performing collagenase digestion on the Wharton's jelly of the small tissues, and performing primary cell separation culture and subculture;
and the multidirectional differentiation detection system is used for carrying out osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments on the harvested cells.
8. The system of claim 7, wherein: further comprising: a flow cytometry detection system for detecting the expression level of each antigen marker by using a flow cytometer; the quality inspection system is used for performing quality inspection on the harvested umbilical cord mesenchymal stem cells, and comprises microbial contamination, cell number, cell activity and safety evaluation; and the marking system is used for collecting the umbilical cord mesenchymal stem cells and then carrying out flow analysis on the cell surface markers.
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