CN112680412B - Animal source-free passage method for passage of stem cells - Google Patents
Animal source-free passage method for passage of stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, relates to an animal source-free passage method for passage of stem cells, and in particular relates to a clinical stem cell digestive juice without animal source components and digestive terminationAnd (3) preparing a liquid and carrying out animal-source-free passage on stem cells. An animal-source-free passaging method for passaging clinical-grade stem cells comprising the steps of: extracting human amniotic membrane and umbilical cord mesenchymal stem cells, conventionally culturing to the third generation, and carrying out cell identification; inoculating third generation human amniotic membrane and umbilical cord mesenchymal stem cells to 75cm 2 Culturing in a culture flask until the confluence degree reaches 80-90%; and (3) using digestive juice without animal source components and digestive terminator solution to passaging the human amniotic membrane and umbilical cord mesenchymal stem cells. The animal source-free passage method for passaging the stem cells, provided by the invention, meets the animal source component-free standard, and can effectively digest the stem cells to a single cell form, so that the stem cells are uniformly inoculated, and the optimal culture state is achieved.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to an animal source-free passage method for passaging stem cells, and in particular relates to a preparation method of a clinical stem cell animal source-free component digestive juice and digestive terminator solution and an animal source-free passage method for stem cells.
Background
Stem cells are a class of cells with self-renewing and multipotent differentiation potential. Research shows that stem cells can induce and differentiate into various functional cells forming human body under certain conditions, are seed cells forming various tissues and organs of the human body, and are secret place for regenerating and repairing organs of the human body.
The culture medium without animal source components is a common culture medium for extraction and culture in the production process of clinical stem cells, and can effectively avoid foreign protein allergy and foreign pathogen infection because the culture medium is not contacted with any component containing an extract source in the production process. At present, a plurality of domestic and foreign brands of culture mediums without animal source components are obtained by FDA certification in the United states, and a new way is provided for stem cell culture and clinical application.
Most of the stem cells found at present are adherent cells, the growth of which depends on a cell culture flask, and the adherent cells need to be digested from the cell culture flask in the process of cell proliferation for further operation. Traditional trypsin is extracted from porcine pancreas, and is difficult to avoid foreign protein allergy and porcine pathogen infection during application. The digestion liquid such as the mild digestive enzyme and the mild cell dissociation reagent can replace trypsin to carry out passage digestion, but due to lack of related components of trypsin, defects such as prolonged digestion process, uneven digestion and the like are caused, cell masses are easy to form, and the stem cell culture state is seriously influenced.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a preparation method of a clinical stem cell non-animal-source component digestive juice and digestive terminator solution and a stem cell non-animal-source passage method.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A digestive juice without animal source components for digesting stem cells is composed of the following components in concentration unit mg/L: 100 to 150 portions of EDTA-2Na, 50 to 100 portions of recombinant human trypsin, 50 to 70 portions of dipotassium hydrogen phosphate, 350 to 450 portions of potassium chloride, 300 to 400 portions of sodium bicarbonate and 7000 to 9000 portions of sodium chloride.
A digestion stop solution for stopping stem cell digestion, comprising the following components in concentration unit mg/L: 7000 to 9000 parts of sodium chloride, 100 to 150 parts of disodium hydrogen phosphate dodecahydrate, 350 to 450 parts of potassium chloride, 50 to 70 parts of potassium dihydrogen phosphate, 80 to 100 parts of magnesium sulfate, 100 to 150 parts of calcium chloride, 900 to 1100 parts of D-glucose, 300 to 400 parts of sodium bicarbonate and 350 to 450 parts of human serum albumin.
An animal-origin-free passaging method for passaging stem cells specifically comprises the following steps.
Step 1, taking out stem cells cultured by using a culture medium without animal source components, discarding the culture medium, washing the cells for 2 times by using PBS, and sucking out liquid in a culture bottle.
Step 2, pressing 25cm 2 and/mL adding the digestion solution without animal source components into a culture flask, completely immersing cells into the digestion solution, and discarding the redundant digestion solution.
Step 3, placing at 37 ℃ and saturated humidity with 5% CO by volume 2 The incubator is filled for 1 to 2 minutes.
Step 4, taking out the culture flask according to V Digestion stop solution :V Digestion liquid without animal source component =1: 1 volume addThe above digestion stop solution was mixed and the cell suspension was gently collected with a pipette.
Step 5, centrifugation at 1000rpm for 5 minutes, and discarding the supernatant.
And 6, adding a culture medium without animal source components, uniformly mixing the cell suspension, and inoculating the cell suspension into a culture bottle.
The beneficial effects of the invention are as follows.
The animal source-free passage method for passaging the stem cells, provided by the invention, meets the animal source component-free standard, and can effectively digest the stem cells to a single cell form, so that the stem cells are uniformly inoculated, and the optimal culture state is achieved.
Drawings
FIG. 1 shows umbilical cord mesenchymal stem cells and amniotic mesenchymal stem cells in culture in a medium without animal origin components. Wherein A is umbilical cord mesenchymal stem cells cultured by a culture medium without animal source components; and B is amniotic mesenchymal stem cells cultured in a culture medium without animal-derived components.
FIGS. 2-1 to 2-6 are cell surface marker assays of umbilical cord mesenchymal stem cells in culture in animal-derived component free medium. Wherein 2-1 to 2-3 are umbilical cord mesenchymal stem cell surface markers cultured by a culture medium without animal source components; 2-4 to 2-6 are amniotic mesenchymal stem cell surface markers cultured in a culture medium without animal-derived components.
FIG. 3 shows the measurement of the differentiation capacity of umbilical cord mesenchymal stem cells and amniotic mesenchymal stem cells in culture in a medium without animal origin. Wherein A is the detection of the differentiation capacity of umbilical cord mesenchymal stem cells cultured by a culture medium without animal source components; b is the detection of the cell differentiation capacity of the amniotic mesenchymal stem cells cultured by the culture medium without animal source components.
FIG. 4 is a diagram of the digestion of umbilical cord mesenchymal stem cells by a passaging method without animal origin. Within 1 minute, the cells become round and fall off, namely, are digested into single cell suspension, and the cells in the state of no adhesion are observed under a microscope.
Fig. 5 is a graph of mild digestive enzyme digestion of umbilical cord mesenchymal stem cells. After 5 minutes of digestion, the cells become round and fall off, and a small amount of adherent cells remain under microscopic observation.
FIG. 6 shows results of two passaging methods for digesting umbilical cord mesenchymal stem cells. Umbilical cord mesenchymal stem cells digested by the animal-origin-free passage method are in a uniform single-cell suspension state, and cell masses exist. The umbilical cord mesenchymal stem cell mass digested by mild digestive enzymes is more, as shown by the arrow in the figure.
Fig. 7 is a diagram of the digestion of amniotic mesenchymal stem cells by a passaging method without animal origin. Within 2 minutes, the cells become round and fall off, namely, the cells are digested into single cell suspension, and cells in an adherent state are observed under a microscope.
Fig. 8 is a graph of mild digestive enzyme digestion of amniotic mesenchymal stem cells. After digestion for 10 minutes, the cells become round and fall off, and a small amount of adherent cells remain under microscopic observation.
Fig. 9 is the results of two passaging methods for digesting amniotic mesenchymal stem cells. The amniotic mesenchymal stem cells digested by the animal-origin-free passage method are in a uniform single-cell suspension state, and cell aggregates exist. The amniotic mesenchymal stem cell mass digested by mild digestive enzyme is more, as shown by the arrow in the figure.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments and drawings, which are given by way of example only and are not intended to limit the scope of the present application. Various modifications and variations may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
The invention relates to a suitable clinical stem cell passage method which is applied to the passage of cells in the process of culturing human stem cells without animal sources.
An animal-origin-free passaging method for passaging clinical-grade stem cells specifically comprises the following steps.
Step 1, extracting human amniotic membrane and umbilical cord mesenchymal stem cells, conventionally culturing to the third generation, and carrying out cell identification.
Step 2, inoculating third generation human amniotic membrane and umbilical cord mesenchymal stem cells to 75cm 2 Culturing in culture flask until confluence reaches 80-90%.
And 3, passaging the human amniotic membrane and umbilical cord mesenchymal stem cells by using a digestive juice without animal source components and a digestive terminator solution.
The digestive juice without animal source components is composed of the following components in concentration unit mg/L: 100 to 150 portions of EDTA-2Na, 50 to 100 portions of recombinant human trypsin, 50 to 70 portions of dipotassium hydrogen phosphate, 350 to 450 portions of potassium chloride, 300 to 400 portions of sodium bicarbonate and 7000 to 9000 portions of sodium chloride.
The digestion stopping solution is composed of the following components in concentration unit mg/L: 7000 to 9000 parts of sodium chloride, 100 to 150 parts of disodium hydrogen phosphate dodecahydrate, 350 to 450 parts of potassium chloride, 50 to 70 parts of potassium dihydrogen phosphate, 80 to 100 parts of magnesium sulfate, 100 to 150 parts of calcium chloride, 900 to 1100 parts of D-glucose, 300 to 400 parts of sodium bicarbonate and 350 to 450 parts of human serum albumin.
Example 1 human amniotic membrane and umbilical cord mesenchymal stem cells were extracted, cultured conventionally to the third generation, and cell identification was performed.
1. Materials and methods.
1. A material.
DMEM/F12 medium (Hyclone, usa); elite GRO TM Advanced (GMP degree) (daceae, china); PBS buffer (Biological Industries, israel); collagenase IV (Thermofisher, USA); mesenCult TM Osteogenic Differentiation Kit (Human) (Stemcell, usa); mesenCult TM Adipogenic Differentiation Kit (Human) (Stemcell, usa);
MesenCult TM ACF Chondrogenic Differentiation Kit (Stemcell, usa); alizarin Red S (racing organism, china); oil Red O (racing organism, china); alcian Blue (racing biology, china); CD34 FITC direct antibody (biolegend, USA); CD73FITC direct antibody (biolegend, USA); CD90 FITC direct antibody (biolegend, USA); HLA-DRFITC direct antibody (biolegend, USA).
Digestion solution without animal source components: the composition, in terms of concentration units mg/L, is composed of the following components in concentration: EDTA-2Na100, recombinant human trypsin 50, dipotassium hydrogen phosphate 60, potassium chloride 400, sodium bicarbonate 350 and sodium chloride 8000.
Digestion stop solution: the composition, in terms of concentration units mg/L, is composed of the following components in concentration: 8000 sodium chloride, 125 disodium hydrogen phosphate dodecahydrate, 400 potassium chloride, 60 potassium dihydrogen phosphate, 90 magnesium sulfate, 125 calcium chloride, 1000D-glucose, 350 sodium bicarbonate, 400 human serum albumin.
2. The method.
Step one: extraction, culture and identification of human umbilical cord and amniotic mesenchymal stem cells.
1) Isolation and culture of human umbilical cord mesenchymal stem cells.
The parturient with a caesarean section who had normal physical examination and had no HIV, HBV, HCV treponema pallidum infection was selected. The fetal umbilical cord was collected within 10min of delivery of the fetus and immersed in sterile PBS. Transferring the umbilical cord into a biosafety cabinet, removing residual blood, umbilical cord epidermis, umbilical vein and umbilical artery, and extracting the umbilical cord Wharton's jelly. Cutting Wharton's jelly into 1X 1mm pieces 2 Uniformly sticking the mixture on the lower surface of a cell culture dish, placing the cell culture dish upside down into a 37 ℃ and 5% CO2 incubator for culturing for 2 hours, turning the culture dish, and adding a culture medium without animal source components. 37 ℃ and 5% CO 2 Culturing in an incubator, labeled P0. Observing the cell density of the adherent cells, adding 1mL of digestion solution without animal source components into a culture dish for digestion for 1min after the cell fusion reaches 80%, stopping digestion by using digestion stop solution, centrifuging for 5min at 800 rpm, discarding the supernatant, adding culture medium without animal source components for resuspension, and carrying out the following steps: 3 are inoculated in a new culture bottle, liquid is changed every other day, and the culture is carried out until the culture reaches P3 after full passage, and the cell morphology is shown in figure 1A.
2) Isolation and culture of human amniotic mesenchymal stem cells.
The parturient with a caesarean section who had normal physical examination and had no HIV, HBV, HCV treponema pallidum infection was selected. The fetal amniotic membrane was collected within 10 minutes of delivery of the fetus. The amniotic membrane was cleaned with PBS to remove chorion and residual blood stain, and was cut to a size of 10X 7.5 cm. Subpackaging the digestion solution without animal source component into T75 culture flask, adding 1 amniotic membrane 10-15mL each flask, digesting at 37deg.C for 30min, discarding supernatant, and repeating for 3 times. Collecting digested tissue, cutting, and adding into final productThe digestion was stopped by adding 10 volumes of DMEM/F12 medium to a concentration of 1mg/ml collagenase IV for 60min at 37 ℃. Tissue debris was removed using a 60 μm pore size cell sieve. Centrifuging the filtered liquid at 800 rpm for 5min, and discarding the supernatant. Cell pellet was resuspended in medium without animal origin. 37 ℃ and 5% CO 2 Culturing in an incubator, labeled P0. Observing the cell density of the adherent cells, adding 1mL of digestion solution without animal source components into a culture dish for digestion for 1min after the cell fusion reaches 80%, stopping digestion by using digestion stop solution, centrifuging for 5min at 800 rpm, discarding the supernatant, adding culture medium without animal source components for resuspension, and carrying out the following steps: 3 are inoculated in a new culture bottle, liquid is changed every other day, and the culture is carried out until the culture reaches P3 after full passage, and the cell morphology is shown in figure 1B.
3) And (5) flow cytometry identification.
Taking P3 generation conventionally cultured human umbilical cord and amniotic mesenchymal stem cells, and re-suspending in PBS after pancreatin digestion to adjust the cell density to 1×10 7 100uL of cell suspension is taken and added with 5 uL of PE marked CD34, CD73, CD90 and HLA-DR antibody respectively, incubated on ice for 30min, and after PBS is washed and resuspended, analysis is carried out by a flow cytometer, as shown in figures 2-1 to 2-6, human umbilical cord and amniotic mesenchymal stem cells positively express CD73 and CD90 and do not express CD34 and HLA-DR.
4) Cell differentiation experiments.
Human umbilical cord and amniotic mesenchymal stem cells are inoculated into a six-hole plate for conventional culture until the generation of P3, and MesenCurt is respectively applied according to the specification TM Osteogenic Differentiation Kit(Human)、MesenCult TM Adipogenic Differentiation Kit (Human) and MesenCurt TM -ACF Chondrogenic Differentiation Kit performing osteogenic, adipogenic and chondrogenic induced differentiation on human umbilical cord and amniotic mesenchymal stem cells, and staining by using Alizarin Red S, oil Red O and Alcian Blue after the induced differentiation is finished. As shown in FIG. 3, human umbilical cord and amniotic mesenchymal stem cells have osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation capacity under the culture of an induced differentiation medium.
Example 2.
Inoculating third generation human amniotic membrane and umbilical cord mesenchymal stem cells to 75cm 2 Culturing in culture flask until confluence reaches 80-90%.
1. Materials and methods.
1. A material.
DMEM/F12 medium (Hyclone, usa); elite GRO TM Advanced (GMP degree) (daceae, china); PBS buffer (Biological Industries, israel).
2. The method.
Culture of hAMSCs cells: taking third generation human amniotic membrane and umbilical cord mesenchymal stem cells cultured in culture medium without animal source component according to 5×10 4 /cm 2 Inoculating to 75cm 2 The cell density of the adherent cells is observed after the inoculation of the culture flask, and the cell confluence reaches 80-90%.
Example 3 human amniotic membrane and umbilical cord mesenchymal stem cells were passaged using a digestion solution free of animal-derived components and a digestion stop solution.
1. Materials and methods.
1. A material.
PBS buffer (Biological Industries, israel); mild digestive enzymes (you kang, china); no animal source component digestive juice; digestion stop solution; DMEM/F12 medium (Hyclone, usa); elite GRO TM Advanced (GMP degree) (daceae, china).
Digestion solution without animal source components: the composition, in terms of concentration units mg/L, is composed of the following components in concentration: EDTA-2Na100, recombinant human trypsin 50, dipotassium hydrogen phosphate 60, potassium chloride 400, sodium bicarbonate 350 and sodium chloride 8000.
Digestion stop solution: the composition, in terms of concentration units mg/L, is composed of the following components in concentration: 8000 sodium chloride, 125 disodium hydrogen phosphate dodecahydrate, 400 potassium chloride, 60 potassium dihydrogen phosphate, 90 magnesium sulfate, 125 calcium chloride, 1000D-glucose, 350 sodium bicarbonate, 400 human serum albumin.
2. The method.
1) And (3) using digestive juice without animal source components and digestive terminator solution to passaging the human amniotic membrane and umbilical cord mesenchymal stem cells.
Take 75cm 2 Human amniotic membrane and umbilical cord with confluence of 80-90% cultured in culture flaskMesenchymal stem cells. The PBS buffer solution is used for washing the cells twice, the rest PBS is discarded, 1mL of digestion solution without animal source components is added into a culture flask, the cells are observed and photographed under a microscope, and when the cells become round and fall off, namely, the cells are digested into single cell suspension, the cell suspension is collected. Digestion was stopped using a digestion stop solution and 45mL of a solution containing 5% EliteGRO was added TM Advanced DMEM/F12 medium re-suspension of cell suspension at 1:3 are inoculated in a new culture flask for culture until the next day of photographing. As a result, it was found that umbilical cord mesenchymal stem cells were digested by using an animal-origin-free passaging method. Within 1 minute, the cells were rounded off, i.e., digested into a single cell suspension, and cells in an adherent-free state were observed under a microscope, as shown in fig. 4 and 6A. The amniotic mesenchymal stem cells are digested by using an animal-source-free passage method. Within 2 minutes, the cells were rounded off, i.e., digested into a single cell suspension, and cells in an adherent-free state were observed under a microscope, as shown in fig. 7 and 9A.
2) The human amniotic membrane and umbilical cord mesenchymal stem cells were passaged using mild digestive enzymes.
Take 75cm 2 The confluence degree of the human amniotic membrane and umbilical cord mesenchymal stem cells cultured in the culture flask reaches 80-90%. The cells were washed twice with PBS buffer, the remaining PBS was discarded, 1mL of mild digestive enzyme was added to the flask, the flask was photographed under microscopic observation, and when the cells became round and shed, i.e., digested into single cell suspension, the cell suspension was collected. 2mL of a solution containing 5% EliteGRO was added TM Advanced DMEM/F12 medium stopped digestion, 200g, 5min centrifuged cell suspension. The supernatant was discarded, cells were collected and 45mL of a solution containing 5% EliteGRO was added TM Advanced DMEM/F12 medium re-suspension of cell suspension at 1:3 are inoculated in a new culture flask for culture until the next day of photographing. As a result, it was found that umbilical cord mesenchymal stem cells were digested with mild digestive enzymes. After 5 minutes of digestion, the cells become round and fall off, and a small amount of cells in an adherent state are observed under a microscope, as shown in fig. 5 and 6B. The amniotic mesenchymal stem cells are digested with mild digestive enzymes. After digestion for 10 minutes, the cells became round and shed, and a small amount of adherent cells were observed under a microscope, as shown in fig. 8 and 9B.
Claims (1)
1. An animal-origin-free passage method for passage of stem cells, which is characterized by comprising the following steps:
step 1, taking out stem cells cultured by using a culture medium without animal source components, discarding the culture medium, washing the cells for 2 times by using PBS, and sucking out liquid in a culture bottle;
step 2, pressing 25cm 2 Adding animal source component-free digestive juice into a culture flask to completely immerse cells in the digestive juice, and discarding excessive digestive juice;
step 3, placing at 37 ℃ and saturated humidity with 5% CO by volume 2 Culturing in an incubator for 1-2 minutes;
step 4, taking out the culture flask, adding the digestion stopping solution, uniformly mixing, and gently collecting the cell suspension by using a suction tube;
step 5, centrifuging at 1000rpm for 5 minutes, and discarding the supernatant;
step 6, adding a culture medium without animal source components, uniformly mixing the cell suspension, and inoculating the cell suspension into a culture bottle;
the digestive juice without animal source components is composed of the following components in concentration unit mg/L: 100 to 150 parts of EDTA-2Na, 50 to 100 parts of recombinant human trypsin, 50 to 70 parts of dipotassium hydrogen phosphate, 350 to 450 parts of potassium chloride, 300 to 400 parts of sodium bicarbonate and 7000 to 9000 parts of sodium chloride;
the digestion stopping solution is composed of the following components in concentration unit mg/L: 7000 to 9000 parts of sodium chloride, 100 to 150 parts of disodium hydrogen phosphate dodecahydrate, 350 to 450 parts of potassium chloride, 50 to 70 parts of potassium dihydrogen phosphate, 80 to 100 parts of magnesium sulfate, 100 to 150 parts of calcium chloride, 900 to 1100 parts of D-glucose, 300 to 400 parts of sodium bicarbonate and 350 to 450 parts of human serum albumin;
the volume ratio of the digestion stopping solution to the digestion solution without animal source components is 1:1.
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| CN107058219A (en) * | 2017-04-13 | 2017-08-18 | 上海莱馥生命科学技术有限公司 | A kind of method that application stem cell self-characteristic prepares dental pulp stem cell |
| CN107090427A (en) * | 2017-06-23 | 2017-08-25 | 友康恒业生物科技（北京）有限公司 | A kind of genetic recombination trypsase cell dissociation buffer |
| CN109439621A (en) * | 2018-11-12 | 2019-03-08 | 王晓柯 | A kind of mild enzyme cell dissociation buffer of stem cell |
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| CN107058219A (en) * | 2017-04-13 | 2017-08-18 | 上海莱馥生命科学技术有限公司 | A kind of method that application stem cell self-characteristic prepares dental pulp stem cell |
| CN107090427A (en) * | 2017-06-23 | 2017-08-25 | 友康恒业生物科技（北京）有限公司 | A kind of genetic recombination trypsase cell dissociation buffer |
| CN109439621A (en) * | 2018-11-12 | 2019-03-08 | 王晓柯 | A kind of mild enzyme cell dissociation buffer of stem cell |
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