CN112553155A - Umbilical cord blood mesenchymal stem cell culture method - Google Patents
Umbilical cord blood mesenchymal stem cell culture method Download PDFInfo
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- CN112553155A CN112553155A CN202011578876.6A CN202011578876A CN112553155A CN 112553155 A CN112553155 A CN 112553155A CN 202011578876 A CN202011578876 A CN 202011578876A CN 112553155 A CN112553155 A CN 112553155A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- C—CHEMISTRY; METALLURGY
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The umbilical cord blood mesenchymal stem cell culture method provided by the invention comprises the steps of collecting and accepting umbilical cord blood, washing umbilical cord blood, preparing and activating autologous umbilical cord blood platelet-rich plasma, separating umbilical cord blood mononuclear cells, culturing umbilical cord blood mesenchymal stem cells after pretreatment of a Transwell chamber, and purifying and amplifying the umbilical cord blood mesenchymal stem cells. The method solves various defects caused by the fact that fetal calf serum with certain concentration is required to be used for culture in the prior art, and the stored platelet-rich plasma is used for culturing cells after being activated, so that the method is high in efficiency, safer and easy to accept. The whole process omits fussy steps such as adding various factors, is simple and convenient to operate, and can greatly save the culture cost.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a method for culturing umbilical cord blood mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) can be obtained from a variety of tissues, such as bone marrow, umbilical cord, etc., and not only have multipotent differentiation potential, but also can modulate the immunological activities of T cells, B cells, natural killer cells, dendritic cells, etc. In recent years, various research results show that umbilical cord blood contains mesenchymal stem cells with multidirectional differentiation functions, the expansion capacity of the mesenchymal stem cells is dozens of times of that of autologous bone marrow stem cells, and the cells are named as umbilical cord blood mesenchymal stem cells. Human umbilical cord blood-derived mesenchymal stem cells have advantages over stem cells derived from bone marrow, umbilical cord or other tissue sources, including the following: (1) the umbilical cord blood collection is convenient, the pain of the donor is avoided, and the umbilical cord blood is collected in a non-invasive manner. (2) The immune system in umbilical cord blood is in the original stage, and the incidence and the severity of rejection reaction after transplantation are low. (3) The chance of infection by various viruses in the cord blood is relatively small. But the content of mesenchymal stem cells in the umbilical cord blood is very little, and the frequency of MSCs in each umbilical cord blood sample is 0-2.3 cell clones/108MNCs, have certain difficulty in extraction and separation. At present, an in vitro extraction culture system generally contains fetal calf serum with a certain concentration, belongs to animal source components, and may have potential virus or mycoplasma pollution carried by animals. In addition, in the process of culturing the umbilical cord blood mesenchymal stem cells, growth factors such as bFGF, EGF, SCF and the like are generally added into the culture medium, and the growth factors are expensiveThe culture cost is greatly increased. Therefore, the method has important research significance on how to separate and purify the mesenchymal stem cells which can be stably passaged and are safe and high in quality.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a solution of a method for culturing umbilical cord blood mesenchymal stem cells, which comprises the following steps:
a culture method of umbilical cord blood mesenchymal stem cells comprises the following operation steps:
(1) collecting umbilical cord blood: placing the collected umbilical cord blood into umbilical cord blood preservation solution for preservation within 24 hours, wherein the preservation temperature is 3-5 ℃;
(2) washing of cord blood: taking the preservation solution with the umbilical cord blood, mixing and stirring uniformly, then carrying out centrifugal treatment, then adding cleaning solution into the centrifugal precipitate, mixing and stirring uniformly, and then carrying out centrifugal treatment again to obtain supernatant and precipitate;
(3) preparing and activating the umbilical cord blood platelet-rich plasma: continuously centrifuging the supernatant obtained in the step (2), removing the serum at the upper part, taking the middle and bottom precipitates as platelet rich plasma, continuously adding a calcium chloride solution into the obtained platelet rich plasma, activating, and centrifuging to obtain activated platelet rich plasma, wherein the activating is carried out by incubating at 36.8-37.2 ℃ for 25-35min or freezing in a refrigerator at 3-5 ℃ for 15-17 h;
(4) separation of cord blood mononuclear cells: adding a Du's phosphate buffer solution into the sediment obtained in the step (2), uniformly mixing, adding into a lymphocyte separation solution, sucking leucocyte layer cells, namely mononuclear cells, sucking the mononuclear cells into another clean centrifugal tube, adding the Du's phosphate buffer solution, repeatedly centrifuging and washing for 2-3 times, collecting the bottom mononuclear cell sediment, and obtaining umbilical cord blood mononuclear cells;
(5) culturing umbilical cord blood mesenchymal stem cells in a pretreated Transwell chamber: subjecting the cord blood mononuclear cells obtained in the step (4) to a reaction at a temperature of 1X 106/cm2The culture medium is inoculated on the lower chamber surface of the pretreated Transwell chamber in density to culture umbilical cord blood mesenchymal stem cells;
(6) purifying and amplifying umbilical cord blood mesenchymal stem cells: and replacing the complete culture medium after 5-7 days of cell culture to discard non-adherent cells, replacing the medium half a day every 3-4 days, finding sporadic cell colonies in 8-12 days, and amplifying the cells by using the complete culture medium to obtain the purified umbilical cord blood mesenchymal stem cells.
Specifically, in the step (1), 1.8 to 2.2g of trisodium citrate, 0.6 to 1.0g of citric acid, 2.1 to 2.6g of glucose, 0.2 to 0.5g of adenine and 2.8 to 3.4g of sodium dihydrogen phosphate are contained in 100mL of the preservation solution, and the preservation solution is sterilized before use.
Specifically, in the step (2), the washing solution is a physiological saline solution containing 0.5% by mass of human serum albumin or a duller's phosphate buffer solution containing 0.5% by mass of human serum albumin.
Specifically, in the step (2), during the centrifugation treatment, the centrifugal force is 550g and 450-; in the step (3), the centrifugal force is 1100-1300g and the centrifugation time is 9-10min during the first centrifugation, and the centrifugal force is 2500-3500g and the centrifugation time is 15-25min during the second centrifugation.
Specifically, in the step (3), the mass fraction of calcium chloride in the calcium chloride solution is 3-5%.
Specifically, in the step (3), the pretreated Transwell chamber is obtained by the following method: 24h in advance according to 1 × 103Per cm2The umbilical cord mesenchymal stem cells are inoculated at the density of (a), the upper chamber surface of the bottom membrane of the six-hole Transwell chamber is coated, and the used culture medium is a complete culture medium.
Specifically, the complete culture medium is a basal medium DMEM/F12 which contains activated platelet-rich plasma with the mass fraction of 4-6%.
Specifically, in the step (5), the environment for culturing the umbilical cord blood mesenchymal stem cells is CO with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent2An incubator.
According to the technical scheme, the beneficial effects of the invention are as follows:
the umbilical cord blood mesenchymal stem cell culture method provided by the invention solves various defects caused by the necessity of culturing fetal bovine serum with certain concentration in the prior art by steps of collecting and accepting umbilical cord blood, washing umbilical cord blood, preparing and activating autologous umbilical cord blood platelet-rich plasma, separating umbilical cord blood mononuclear cells, culturing umbilical cord blood mesenchymal stem cells after pretreatment of a Transwell chamber and purifying and amplifying the umbilical cord blood mesenchymal stem cells, has high efficiency, safety and easy acceptance by culturing own cells after activating platelet-rich plasma stored by the own, takes activated platelet-rich plasma (aPRP) as an autologous umbilical cord blood extract, obtains a platelet concentrate through gradient centrifugation separation, adds an activating agent to activate platelets to obtain activated aPRP, can release various cell culture factors, such as PDGF, TGF-beta, bFGF, EGF, VEGF and the like, and simultaneously utilizes the characteristics of an upper chamber and a lower chamber of a Transwell chamber to inoculate the umbilical cord mesenchymal stem cells in the upper layer of the chamber in advance, the umbilical cord mesenchymal stem cells can continuously release cell growth factors in the culture process, so that the adherent growth of the umbilical cord blood mesenchymal stem cells is facilitated, and the purposes of separation and purification are further achieved. The whole process omits fussy steps such as adding various factors, is simple and convenient to operate, and can greatly save the culture cost.
Drawings
FIG. 1 is a morphological diagram of umbilical cord blood mesenchymal stem cells after culture according to an embodiment of the present invention.
FIG. 2 is a surface marker of mesenchymal stem cells of umbilical cord blood cultured in the embodiment of the present invention.
FIG. 3 shows differentiation-induced adipocytes of umbilical cord blood mesenchymal stem cells cultured according to an embodiment of the present invention.
FIG. 4 shows osteoblasts induced by mesenchymal stem cells of umbilical cord blood cultured according to an embodiment of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Examples
A culture method of umbilical cord blood mesenchymal stem cells comprises the following operation steps:
(1) collecting umbilical cord blood: storing the collected umbilical cord blood in an umbilical cord blood storage solution within 24 hours at the storage temperature of 3-5 ℃, wherein each 100mL of the storage solution contains 1.8-2.2g of trisodium citrate, 0.6-1.0g of citric acid, 2.1-2.6g of glucose, 0.2-0.5g of adenine and 2.8-3.4g of sodium dihydrogen phosphate, and the storage solution is sterilized before use;
(2) washing of cord blood: taking a preservation solution with umbilical cord blood, mixing and stirring uniformly, then carrying out centrifugal treatment, wherein the centrifugal force is 550g and the centrifugal time is 10min, then adding a cleaning solution into the centrifugal precipitate, mixing and stirring uniformly, and then carrying out centrifugal treatment again to obtain a supernatant and a precipitate, wherein the cleaning solution is physiological saline containing 0.5% by mass of human serum albumin or a Du's phosphate buffer containing 0.5% by mass of human serum albumin;
(3) preparing and activating the umbilical cord blood platelet-rich plasma: continuously centrifuging the supernatant obtained in the step (2), removing the serum at the upper part, continuously adding a calcium chloride solution with the mass fraction of 3-5% into the obtained platelet-rich plasma, activating, and centrifuging to obtain activated platelet-rich plasma, wherein the activating treatment is performed by incubating at 36.8-37.2 ℃ for 25-35min or freezing in a refrigerator at 3-5 ℃ for 15-17h, in the step, the centrifugal force is 1100-1300g during the first centrifugation, the centrifugal time is 9-10min, and the centrifugal force is 2500-3500g during the second centrifugation, the centrifugal time is 15-25 min;
(4) separation of cord blood mononuclear cells: adding a Du's phosphate buffer solution into the sediment obtained in the step (2), uniformly mixing, adding into a lymphocyte separation solution, sucking leucocyte layer cells, namely mononuclear cells, sucking the mononuclear cells into another clean centrifugal tube, adding the Du's phosphate buffer solution, repeatedly centrifuging and washing for 2-3 times, collecting the bottom mononuclear cell sediment, and obtaining umbilical cord blood mononuclear cells;
(5) pretreated TraCulturing umbilical cord blood mesenchymal stem cells in an nswell chamber: subjecting the cord blood mononuclear cells obtained in the step (4) to a reaction at a temperature of 1X 106/cm2The culture medium is inoculated on the lower chamber surface of a pretreated Transwell chamber, and is used for culturing umbilical cord blood mesenchymal stem cells, wherein the culture environment of the umbilical cord blood mesenchymal stem cells is a CO2 culture box with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent, and the pretreated Transwell chamber is obtained by adopting the following method: inoculating umbilical cord mesenchymal stem cells at the density of 1 × 103/cm 2 24h in advance, coating the upper chamber face of the bottom membrane of the six-hole Transwell chamber, and using a complete culture medium which is a basal medium DMEM/F12 and contains activated platelet-rich plasma with the mass fraction of 4-6%;
(6) purifying and amplifying umbilical cord blood mesenchymal stem cells: and replacing the complete culture medium after 5-7 days of cell culture to discard non-adherent cells, replacing the medium half a day every 3-4 days, finding sporadic cell colonies in 8-12 days, and amplifying the cells by using the complete culture medium to obtain the purified umbilical cord blood mesenchymal stem cells.
The umbilical cord blood-derived mesenchymal stem cells cultured in the above example are identified, and the identification result is as follows:
first, morphological characteristics
As shown in figure 1, the cultured umbilical cord blood-derived mesenchymal stem cells are observed under an inverted microscope, and the cells are in a short spindle shape, uniform in size and good in cell growth situation.
Surface marker of mesenchymal stem cells in umbilical cord blood
As shown in figure 2, the cultured second generation umbilical cord blood mesenchymal stem cells (P2-MSCs) are subjected to phenotype analysis by a flow cytometry method, FITC marked anti-CD44, anti-CD45, PE marked anti-CD34, anti-CD105 and anti-HLA-DR are combined with a cell surface Marker, and the result is shown in figure 2, the fusiform cells express the surface markers CD105 and CD44 of the mesenchymal stem cells and do not express CD34, CD45 and HLA-DR, and the cells obtained by separation are determined to be the umbilical cord blood mesenchymal stem cells and have stem cell characteristics.
Thirdly, identification of differentiation function of umbilical cord blood mesenchymal stem cells to adipocytes
As shown in FIG. 3, the cultured second-generation umbilical cord blood mesenchymal stem cells (P2-MSCs) were digested with pancreatin at 10%4/cm2The cells were seeded in a six-well plate previously coated with polylysine and cultured for 1-2 days at 37 degrees under 5% CO2 conditions. When the cells reach 100% confluency, the culture medium is carefully replaced by the ScienCell adipogenic differentiation induction culture medium MADM. Fresh medium was then replaced every 4-5 days. Fat droplets were first observed at D6 when the induction medium was changed, and after 18-21 days of induced differentiation, the cells could be stained with oil red O. The result shows that the cord blood mesenchymal stem cell subset can be differentiated to the fat cell after being induced in vitro.
Fourth, identification of osteogenic differentiation function of umbilical cord blood mesenchymal stem cells
As shown in FIG. 4, the cultured second-generation umbilical cord blood mesenchymal stem cells (P2-MSCs) were digested with pancreatin at 10%4/cm2The cells were seeded in a six-well plate previously coated with polylysine and cultured for 1-2 days at 37 degrees under 5% CO2 conditions. When the cells reach 100% confluence, the culture medium is carefully replaced by a ScienCell osteogenic differentiation induction culture medium MODM. Fresh medium was then replaced every 4-5 days. The brown color (4X) or the white color of the clone can be observed by microscopic examination of the cultured D6-D8, and the clone can be stained by alizarin red solution after the induction culture is carried out for 14-16 days. The result shows that the cord blood mesenchymal stem cell subset can be differentiated to osteoblast after being induced in vitro.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.
Claims (8)
1. A culture method of umbilical cord blood mesenchymal stem cells is characterized by comprising the following operation steps:
(1) collecting umbilical cord blood: placing the collected umbilical cord blood into umbilical cord blood preservation solution for preservation within 24 hours, wherein the preservation temperature is 3-5 ℃;
(2) washing of cord blood: taking the preservation solution with the umbilical cord blood, mixing and stirring uniformly, then carrying out centrifugal treatment, then adding cleaning solution into the centrifugal precipitate, mixing and stirring uniformly, and then carrying out centrifugal treatment again to obtain supernatant and precipitate;
(3) preparing and activating the umbilical cord blood platelet-rich plasma: continuously centrifuging the supernatant obtained in the step (2), removing the serum at the upper part, taking the middle and bottom precipitates as platelet rich plasma, continuously adding a calcium chloride solution into the obtained platelet rich plasma, activating, and centrifuging to obtain activated platelet rich plasma, wherein the activating is carried out by incubating at 36.8-37.2 ℃ for 25-35min or freezing in a refrigerator at 3-5 ℃ for 15-17 h;
(4) separation of cord blood mononuclear cells: adding a Du's phosphate buffer solution into the sediment obtained in the step (2), uniformly mixing, adding into a lymphocyte separation solution, sucking leucocyte layer cells, namely mononuclear cells, sucking the mononuclear cells into another clean centrifugal tube, adding the Du's phosphate buffer solution, repeatedly centrifuging and washing for 2-3 times, collecting the bottom mononuclear cell sediment, and obtaining umbilical cord blood mononuclear cells;
(5) culturing umbilical cord blood mesenchymal stem cells in a pretreated Transwell chamber: inoculating the umbilical cord blood mononuclear cells obtained in the step (4) on the lower chamber surface of the pretreated Transwell chamber at the density of 1 × 106/cm2, and culturing umbilical cord blood mesenchymal stem cells;
(6) purifying and amplifying umbilical cord blood mesenchymal stem cells: and replacing the complete culture medium after 5-7 days of cell culture to discard non-adherent cells, replacing the medium half a day every 3-4 days, finding sporadic cell colonies in 8-12 days, and amplifying the cells by using the complete culture medium to obtain the purified umbilical cord blood mesenchymal stem cells.
2. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 1, wherein in the step (1), 1.8 to 2.2g of trisodium citrate, 0.6 to 1.0g of citric acid, 2.1 to 2.6g of glucose, 0.2 to 0.5g of adenine and 2.8 to 3.4g of monosodium phosphate are contained in 100mL of the preservation solution, and the preservation solution is sterilized before use.
3. The method of claim 1, wherein the washing solution in step (2) is a physiological saline solution containing 0.5% by mass of human serum albumin or a Duchen phosphate buffer solution containing 0.5% by mass of human serum albumin.
4. The method for culturing mesenchymal stem cells of umbilical cord blood as claimed in claim 1, wherein in the step (2), the centrifugal force is 550g and the centrifugation time is 10min when the centrifugation is performed; in the step (3), the centrifugal force is 1100-1300g and the centrifugation time is 9-10min during the first centrifugation, and the centrifugal force is 2500-3500g and the centrifugation time is 15-25min during the second centrifugation.
5. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 1, wherein in the step (3), the mass fraction of calcium chloride in the calcium chloride solution is 3-5%.
6. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 1, wherein in the step (3), the pretreated Transwell chamber is obtained by the following method: umbilical cord mesenchymal stem cells were seeded at a density of 1X 103/cm 2 24h in advance, coating the upper compartment face of the bottom membrane of the six-well Transwell chamber, and the medium used was complete medium.
7. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 6, wherein the complete culture medium is a basal medium DMEM/F12 containing activated platelet rich plasma with a mass fraction of 4-6%.
8. The method of claim 1, wherein in the step (5), the mesenchymal stem cells of umbilical cord blood are culturedThe environment of (A) is CO with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent2An incubator.
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| CN115531297A (en) * | 2022-10-28 | 2022-12-30 | 东莞市东南部中心医院 | Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof |
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