CN115786243A - Differentiation medium, culture method and application of lung precursor cells - Google Patents
Differentiation medium, culture method and application of lung precursor cells Download PDFInfo
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Abstract
The invention relates to a differentiation medium of lung precursor cells, a culture method and application thereof, belonging to the technical field of biology. The invention provides a differentiation medium for lung progenitor cells, which comprises a DMEM (DMEM) medium, an F12 medium, levo-glutamine (L-glutamine), insulin, a recombinant epidermal growth factor, a recombinant human fibroblast growth factor-10, a recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid; the differentiation medium is used for differentiating the lung progenitor cells, so that the differentiation time of the lung progenitor cells can be greatly shortened, the detection efficiency of the biological efficacy of the lung progenitor cells is further improved, the blank that an effective and rapid biological efficacy detection means with pertinence is lacked in the field of current lung stem cell/progenitor cell treatment is filled, and the method has great significance for research on regenerative medicine, drug screening and the like aiming at various acute and chronic and degenerative diseases of a respiratory system.
Description
Technical Field
The invention relates to a differentiation medium of lung precursor cells, a culture method and application thereof, belonging to the technical field of biology.
Background
In recent years, various stem/precursor cell-based therapies are steadily advancing at a rapid pace; such cells are expected to replace injured cells of patients, recruit endogenous tissue-specific stem cells to regenerate new tissues or organs, or exert active immunomodulatory effects, etc. after being introduced into the body by various transplantation techniques.
Aiming at various acute and chronic and degenerative diseases of the respiratory system, a mode of repairing tissue organ injuries by using lung tissue specific adult precursor cells (also called adult stem cells, the same applies below) derived from a human body through cell transplantation is expected to fundamentally realize regenerative medical research of major respiratory system diseases (chronic obstructive pulmonary disease, interstitial lung disease, bronchiectasis and the like), and a novel treatment method is provided for a large number of patients; the lung precursor cells which can be used for in vitro separation, amplification and culture and clinical transplantation are mainly bronchial basal layer cells, far-end airway stem cells and the like; such cells, after transplantation back into the human lung, act by differentiating into functional mature bronchial and alveolar epithelial cells.
However, in the development of stem cell/precursor cell drugs, due to various factors such as different genetic and physiopathological backgrounds of donors, heterogeneity of cells, complex and various cell action mechanisms and the like, how to define the quality of cell drugs or preliminarily judge the cell treatment effect is an aspect to be considered; an index that quantifies the quality of cellular drugs is generally referred to as Biological potency (Biological Efficacy), and an assessment of the Biological potency of a cellular drug should primarily observe its ability to differentiate into functional cells.
At present, the conventional method for detecting the biological effectiveness of lung precursor cells comprises a three-dimensional organoid culture and gas-liquid interface differentiation method, and the principle of the method is that the cells form a three-dimensional structure in vitro and are combined with a differentiation culture system to induce the lung precursor cells to differentiate; the method can simulate the physiological structure and characteristics of cells in a human body, but the defect of the method is obvious as a detection means of biological efficacy, the differentiation can be completed in weeks or even longer, the timeliness requirement of the quality inspection of cell treatment drugs is difficult to guarantee, and the standardized quantitative analysis of the cell differentiation efficiency is difficult to perform due to the existence of a three-dimensional structure.
Disclosure of Invention
In order to solve the above problems, the present invention provides a differentiation medium for lung precursor cells, the composition of the differentiation medium comprises a medium matrix and an adjuvant, the composition of the medium matrix comprises a DMEM medium and an F12 medium, and the composition of the medium adjuvant comprises l-glutamine, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid.
In one embodiment of the present invention, in the components of the differentiation medium matrix, the volume ratio of the DMEM medium to the F12 medium is 25 to 75: 25-75, in the ingredients of the differentiation medium adjuvant, the concentration of levoglutamide in a differentiation medium matrix is 2-6 mM, the concentration of insulin in the differentiation medium matrix is 2-15 μ g/mL, the concentration of recombinant epidermal growth factor in the differentiation medium matrix is 0.1-2 μ g/mL, the concentration of recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 25-400 μ g/mL, the concentration of recombinant human hepatocyte growth factor in the differentiation medium matrix is 10-35 μ g/mL, the concentration of adenine in the differentiation medium matrix is 5-50 μ g/mL, the concentration of hydrocortisone in the differentiation medium matrix is 2-20 μ g/mL, and the concentration of retinoic acid in the differentiation medium matrix is 0.1-2nM.
In one embodiment of the invention, the components of the differentiation medium further comprise one or a combination of more than one of ROCK inhibitor, transferrin, albumin and penicillin/streptomycin diabody.
In one embodiment of the present invention, the ROCK inhibitor is Y-27632.
In one embodiment of the present invention, the albumin is Bovine Serum Albumin (BSA).
In one embodiment of the invention, the penicillin/streptomycin diabody is a penicillin/streptomycin diabody solution.
In one embodiment of the invention, the concentration of the penicillin/streptomycin double antibody solution is 5000U/mL.
In one embodiment of the invention, in the components of the differentiation medium adjuvant, the concentration of Y-27632 in a differentiation medium matrix is 0-16 mu M, the concentration of transferrin in the differentiation medium matrix is 0-50 mu g/mL, the concentration of bovine serum albumin in the differentiation medium matrix is 0-400 mu g/mL, and the volume ratio of a penicillin/streptomycin double antibody solution to the differentiation medium matrix is 0-5% (v/v).
In one embodiment of the present invention, the composition of the differentiation medium matrix includes DMEM medium and F12 medium at a volume ratio of 50: in the components of the differentiation medium adjuvant, the concentration of a levoglutamine solution in a differentiation medium matrix is 2mM, the concentration of a penicillin/streptomycin double antibody solution in the differentiation medium matrix is 1% (v/v), the concentration of insulin in the differentiation medium matrix is 5 mu g/mL, the concentration of a recombinant epidermal growth factor in the differentiation medium matrix is 0.5 mu g/mL, the concentration of a recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 150 mu g/mL, the concentration of a recombinant human hepatocyte growth factor in the differentiation medium matrix is 25 mu g/mL, the concentration of adenine in the differentiation medium matrix is 10 mu g/mL, the concentration of hydrocortisone in the differentiation medium matrix is 5 mu g/mL, the concentration of transferrin in the differentiation medium matrix is 1 mu g/mL, the concentration of bovine serum in an albumin medium matrix is 200 mu g/mL, and the concentration of retinoic acid in the differentiation medium matrix is 20% respectively.
The invention also provides a differentiation culture method of the lung precursor cells, which is to perform differentiation culture on the lung precursor cells by using the differentiation culture medium.
In one embodiment of the present invention, the differentiation culture method comprises the steps of:
the method comprises the following steps: inoculating the lung precursor cells into a proliferation culture medium for culturing until the lung precursor cells are attached to the wall;
step two: after the lung precursor cells adhere to the wall, the proliferation culture medium is sucked away, the differentiation culture medium is added into the adherent lung precursor cells for culture until the cell morphology is converted into an elongated filamentous morphology, a thin-layer structure similar to type I alveolar epithelium is formed, and differentiation is completed.
In one embodiment of the present invention, the first step is: lung precursor cells were packed at 1X 10 3 ~5×10 4 Individual cell/cm 2 The cells were cultured in 12-well plates supplemented with proliferation medium until lung precursor cells attached.
In one embodiment of the invention, the components of the proliferation medium comprise 225mL of DMEM medium, 225mL of F12 medium, 20-70mL of Fetal Bovine Serum (FBS), 0.2-2mM of L-glutamine, 1-14ng/mL of insulin, 0.1-1ng/mL of epidermal growth factor, 5-30ug/mL of adenine and 2-20ug/mL of hydrocortisone.
In one embodiment of the invention, the conditions of the lung precursor cell culture are 37 ℃ and 7.5% 2 。
In one embodiment of the present invention, in the second step, the differentiation medium is changed every 2 to 3 days.
In one embodiment of the present invention, in the second step, the morphology of the cells is observed by a microscope.
The invention also provides a method for assessing the biological effectiveness of lung precursor cells, comprising the steps of:
the method comprises the following steps: co-culturing the lung precursor cells and the trophoblast cells to obtain the lung precursor cells after co-culture and amplification;
step two: carrying out differential culture on the lung precursor cells subjected to coculture amplification by using the differential culture method to obtain the lung precursor cells subjected to differential culture;
step three: detecting the lung precursor cells after differentiation culture, and evaluating the biological efficacy of the lung precursor cells according to the detection result.
In one embodiment of the present invention, in the first step, the lung precursor cells and the trophoblast cells are co-cultured in a culture vessel using a proliferation medium.
In one embodiment of the present invention, in the first step, the culture container is further coated with a primer.
In one embodiment of the present invention, in the first step, the substrate glue is corning Matrix glue (Matrigel Matrix).
In one embodiment of the present invention, in the first step, the protein concentration of the corning matrigel is not lower than 1mg/mL.
In one embodiment of the invention, in the first step, the protein concentration of the corning matrix glue is 1.8 to 3mg/mL.
In one embodiment of the present invention, in the first step, the culture container is a T25 flask.
In one embodiment of the present invention, in the first step, the culture vessel is a 6-well plate.
In one embodiment of the present invention, in the first step, the feeder layer cells are seeded in the T25 culture flask at a density of 5 × 10 3 ~10×10 4 Individual cell/cm 2 。
In one embodiment of the present invention, in the first step, the feeder layer cells are seeded in the T25 culture flask at a density of 3 × 10 4 Individual cell/cm 2 。
In one embodiment of the present invention, in the first step, the trophoblast cells are seeded in a 6-well plate at a density of 5 × 10 5 Individual cells/well.
In one embodiment of the present invention, in the first step, the trophoblast cells are irradiated 3T3 cells.
In one embodiment of the present invention, in the first step, the growth medium is replaced every 2 to 3 days.
In one embodiment of the present invention, in said first step, the conditions of the lung precursor cell culture are 37 ℃ and 7.5% 2 。
In one embodiment of the present invention, in the first step, the proliferation medium comprises 225mL of DMEM, 225mL of F12, 20 to 70ml of FBS, 0.2 to 2mm of L-glutamine, 1 to 14ng/mL of insulin, 0.1 to 1ng/mL of epidermal growth factor, 5 to 30ug/mL of adenine, and 2 to 20ug/mL of hydrocortisone.
In one embodiment of the present invention, in the first step, after the lung precursor cells and the trophoblast cells are co-cultured, the method further comprises removing the trophoblast cells.
In one embodiment of the present invention, in the first step, the removing of the trophoblast cells is: washing the cultured cells with PBS buffer solution, incubating at 37 ℃ by using pancreatin, when the trophoblast cells completely fall off and most lung precursor cells are still in an adherent state, sucking supernatant and washing by using the PBS buffer solution, adding the pancreatin until the lung precursor cells completely fall off, adding a digestion stop solution to stop pancreatin digestion, then centrifuging the cell suspension, and collecting cell precipitates to obtain the lung precursor cell suspension from which the trophoblast cells are removed.
In one embodiment of the present invention, in the first step, the concentration of pancreatic enzymes shed from the trophoblast cells is 0.01 to 0.05% (m/v, g/100 mL).
In one embodiment of the present invention, in the first step, the concentration of pancreatin shedding off the lung precursor cells is 0.15 to 0.3% (m/v, g/100 mL).
In one embodiment of the present invention, in the first step, the trophoblast cells are observed to be exfoliated through a microscope.
In one embodiment of the present invention, in the first step, the digestion stop solution is DMEM containing serum not less than 10% (v/v).
In one embodiment of the present invention, in the third step, immunofluorescent staining is performed on the lung precursor cells after differentiation culture, then quantitative detection is performed on the lung precursor cells after immunofluorescent staining by using a fluorescence cell counting instrument or a flow cytometer, and finally the biological efficacy of the lung precursor cells is evaluated according to the detection result.
In one embodiment of the present invention, in the third step, the original differentiation medium is aspirated, all the differentiated lung precursor cells are exfoliated by pancreatin to obtain cell precipitates, the cell precipitates are resuspended and fixed by using a fixing agent, and then the immobilized differentiated and cultured lung precursor cells are subjected to the immunofluorescence staining.
In one embodiment of the present invention, in the third step, the concentration of pancreatin is 0.25% (m/v, g/100 mL).
In one embodiment of the present invention, in the third step, after adding pancreatin, the cells are completely exfoliated by incubating at 37 ℃ for 8 minutes.
In one embodiment of the present invention, in the third step, after all the lung precursor cells are exfoliated, a digestive stop solution is added to terminate the pancreatic digestion.
In one embodiment of the present invention, in the third step, the digestion stop buffer is DMEM containing 10% (v/v) serum.
In one embodiment of the present invention, in the third step, after all the lung precursor cells are exfoliated, the cell suspension is centrifuged to collect cell pellet.
In one embodiment of the present invention, in the third step, the centrifugation is performed at 1200rpm for 3 minutes.
In one embodiment of the present invention, in the third step, the fixing agent is a neutral formalin solution.
In an embodiment of the present invention, in the third step, the neutral formalin is a 3.7% neutral formalin solution.
In one embodiment of the present invention, in the third step, the fixing agent is a fixing buffer (fixation buffer) fixing agent.
In one embodiment of the present invention, in the third step, after resuspending with a fixative, the lung precursor cells are fixed on a shaker at room temperature for 8 to 10 minutes.
In one embodiment of the present invention, in the third step, the incubation is performed on the room temperature shaker at 30 rpm.
In one embodiment of the present invention, in step three, after the lung precursor cells are fixed, the triton X-100 solution and donkey serum are added, and after incubation, the mixture is equally divided into two centrifuge tubes, wherein one centrifuge tube is a control sample tube and the other centrifuge tube is a detection sample tube.
In one embodiment of the invention, in the third step, the cell suspension is centrifuged and the supernatant is aspirated before adding the triton X-100 solution.
In one embodiment of the present invention, in the third step, the triton X-100 solution is 0.2% (v/v) triton X-100 solution.
In one embodiment of the present invention, in the third step, the 0.2% (v/v) triton X-100 solution is prepared as: 2. Mu.L of 10% Triton X-100 was aspirated, diluted with 98. Mu.L of PBS buffer, and then placed on a shaker at room temperature for more than 1 hour, and the solution was observed to be completely transparent and clear before use.
In one embodiment of the present invention, in the third step, after the addition of the triton X-100 solution, the mixture is left for 10 minutes.
In one embodiment of the invention, in the third step, before donkey serum is added, the cell suspension added with the triton X-100 solution is centrifuged, and then supernatant is aspirated.
In one embodiment of the invention, in said step three, the donkey serum is 5% (v/v) donkey serum.
In one embodiment of the present invention, in step three, the 5% (v/v) donkey serum is formulated as: mu.L donkey serum was aspirated, diluted with 95. Mu.L PBS buffer and mixed well.
In one embodiment of the present invention, in step three, the donkey serum is added and then placed for 0.5 hour.
In one embodiment of the present invention, in the third step, the immunofluorescent staining is: centrifuging a control sample tube and a detection sample tube, then absorbing supernatant, adding PBS buffer solution into the control sample tube, adding HOPX primary antibody working solution into the detection sample tube, incubating, centrifuging, absorbing supernatant, adding fluorescent secondary antibody working solution into each tube, and incubating.
In one embodiment of the present invention, in the immunofluorescence staining of step three, the incubation of the HOPX primary antibody working solution is: incubate on the shaker at room temperature for 2 hours.
In one embodiment of the present invention, in the immunofluorescent staining of step three, after the control sample tube and the detection sample tube are centrifuged and the supernatant is discarded, the cells are washed once with PBS buffer.
In one embodiment of the present invention, in the immunofluorescent staining of the third step, the centrifugation is at 1200rpm for 5 minutes.
In an embodiment of the present invention, in the immunofluorescence staining of step three, the incubation of the fluorescent secondary antibody working solution is dark incubation.
In one embodiment of the present invention, in the immunofluorescence staining of step three, the incubation in the dark is incubation for 2 hours in the dark at room temperature.
In one embodiment of the present invention, in the third step, before the quantitative determination, PBS buffer is added to the control sample tube and the detection sample tube, and the cells are washed twice.
In one embodiment of the invention, the immunofluorescent staining selects a type i alveolar epithelial cell HOPX protein as a target.
In one embodiment of the invention, the immunofluorescent staining selects a type i alveolar epithelial cell AQP5 protein as a target.
The invention also provides the application of the differentiation medium or the differentiation culture method or the method for evaluating the biological effectiveness of the lung precursor cells in drug screening aiming at various acute, chronic and degenerative diseases of the respiratory system.
The technical scheme of the invention has the following advantages:
1. the invention provides a differentiation medium for lung progenitor cells, which comprises a DMEM (DMEM) medium, an F12 medium, a levo-glutamine solution, insulin, a recombinant epidermal growth factor, a recombinant human fibroblast growth factor-10, a recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid; the differentiation of the lung precursor cells can be completed in a short time by using the differentiation medium, and the timeliness requirement of the quality inspection of cell therapy medicines is ensured.
2. The invention provides a differentiation culture method of lung precursor cells, which is characterized in that the lung precursor cells are cultured on trophoblast cells, certain nutrition and support can be provided for cell culture, the adherent growth of the cells can be further facilitated by adding a substrate gel, the obtained cell shape is better, and the standardized quantitative analysis of the cell differentiation efficiency is easy to carry out.
3. The invention provides a method for rapidly evaluating the biological effectiveness of lung precursor cells, and as I-type alveolar epithelial cells are alveolar functional cells which bear the blood-gas exchange function, the expression levels of markers AQP5 and HOPX of the I-type alveolar epithelial cells are selected as indexes for evaluating the biological effectiveness of the lung precursor cells, so that the relationship between the cell quality and the dose-effect can be fully and accurately reflected; and the biological efficacy of the lung precursor cells can be quickly, conveniently and stably evaluated by detecting the lung precursor cells after the lung precursor cells are differentiated by using the improved differentiation medium.
Drawings
FIG. 1: morphograms of pre-differentiated lung precursor cells grown on trophoblast cells.
FIG. 2 is a schematic diagram: example 1-1 of the present invention is a morphological diagram of lung precursor cells after differentiation culture.
FIG. 3: comparative example 1-1 of the present invention is a morphological diagram of lung precursor cells after differentiation culture.
FIG. 4: comparative examples 1-2 of the present invention are morphological diagrams of lung precursor cells after differentiation culture.
FIG. 5 is a schematic view of: comparative examples 1-3 of the invention morphograms of lung precursor cells after differentiation culture.
FIG. 6: morphological patterns of lung precursor cells after differentiation culture of comparative examples 1 to 4 of the present invention.
FIG. 7: and (3) identifying AQP5 and HOPX under a fluorescence microscope.
FIG. 8: the results of quantitative assay using a fluorescence cell counter in example 2-1 of the present invention (HOPX as a marker) were plotted, and the RFP value represents the percentage of HOPX-positive cells to total cells tested after differentiation.
FIG. 9: in the result graph of quantitative determination using se:Sub>A flow cytometer in example 2-2 of the present invention (HOPX is used as se:Sub>A marker), the left peak is se:Sub>A negative group (i.e., se:Sub>A control sample), the right peak is se:Sub>A positive group (i.e., se:Sub>A detection sample), and the numbers shown in the FITC-se:Sub>A subset represent the percentage of HOPX-positive cells to the total detection cells after differentiation.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field; the reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
The DMEM medium referred to in the examples below was purchased from Thermo Fisher Scientific, inc. under model number 11965092; the F12 medium was purchased from Saimer Feishell science and technology, model 21765037; l-glutamine (L-glutamine) is purchased from Saimei Feishell science and technology company, and has a model number of A2916801; insulin was purchased from Sigma Aldrich (Sigma-Aldrich) and was designated as I2643; recombinant epidermal growth factor was purchased from sigma aldrich, model SRP3027; recombinant human fibroblast growth factor-10 was purchased from sigma aldrich and is model No. GF172; the recombinant human hepatocyte growth factor is purchased from MCE company and has the model number of HY-P7121; adenine was purchased from sigma aldrich under model a5665; hydrocortisone was purchased from MCE and was designated HY-N0583; retinoic acid is available from sigma aldrich under the type R2625; transferrin is available from sigma aldrich under the type T8158; bovine serum albumin was purchased from tokkris biosciences (TOCRIS) under model number 5217; A5000U/mL penicillin/streptomycin double antibody solution was purchased from Saimer Feishell scientific Inc. under the model number 15070063.
The proliferation medium for culturing lung precursor cells referred to in the following examples had the following composition: 225mL of DMEM medium, 225mL of F12 medium, 50mL of FBS, 1mM of L-glutamine, 10ng/mL of insulin, 1ng/mL of epidermal growth factor, 25. Mu.g/mL of adenine and 10. Mu.g/mL of hydrocortisone.
The 0.2% (v/v) solution of triton X-100 referred to in the examples below was formulated as: 2. Mu.L of 10% Triton X-100 (available from Beijing Soilebao technologies Co., ltd.) was pipetted, diluted with 98. Mu.L of PBS buffer solution, and then allowed to stand on a shaker at room temperature for 1 hour or more, after which the solution was observed to be completely transparent and clear.
The 5% donkey serum referred to in the following examples was formulated as: suck 5 μ L donkey serum, dilute with 95 μ L PBS buffer, mix well for use.
The HOPX primary anti-working solution referred to in the examples below was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology) and was diluted with PBS buffer at the concentration ratios described in the antibody specification.
The fluorescent secondary antibody in example 2-1 was obtained from Alexa Fluor 594 of Saimer Feishel scientific Co., ltd, and was diluted with PBS buffer at the concentration ratio described in the antibody specification.
The fluorescent secondary antibody in example 2-2 was obtained from Alexa Fluor 488, manufactured by Saimer Feishel scientific Co., ltd, and was diluted with PBS buffer at the concentration ratio described in the antibody specification.
Example 1-1: differentiation culture medium for lung precursor cells and culture method thereof
The embodiment provides a differentiation medium for lung precursor cells, the differentiation medium comprises a medium matrix and an adjuvant, the medium matrix consists of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, the adjuvant consists of L-glutamine solution, penicillin/streptomycin double antibody solution, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone, transferrin, bovine serum albumin and retinoic acid, wherein the concentration of the L-glutamine solution in the differentiation medium matrix is 2mM, the concentration of the L-glutamine solution in the differentiation medium matrix is 1% (v/v), the concentration of the insulin in the differentiation medium matrix is 5 [ mu ] g/mL, the concentration of the recombinant epidermal growth factor in the differentiation medium matrix is 0.5 [ mu ] g/mL, the concentration of the recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 150 [ mu ] g/mL, the concentration of the recombinant epidermal growth factor in the differentiation medium matrix is 25 [ mu ] g/mL, the concentration of the recombinant human fibroblast growth factor in the differentiation medium matrix is 10 [ mu ] g/mL, the concentration of the differentiation medium matrix in the differentiation medium matrix is 1 [ mu ] g/mL, the concentration of the recombinant human fibroblast growth factor in the differentiation medium matrix is 10 [ mu ] g/mL, the differentiation medium matrix is 20 [ mu ] g/mL.
The method for differentiating the lung precursor cells comprises the following steps:
the method comprises the following steps: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the lung precursor cell suspension according to the proportion of 10 4 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 24 hours to make the cells adhere to the wall;
step two: after the lung precursor cells were attached to the wall, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the percent CO was 7.5% at 37 ℃ 2 And (5) culturing, and changing the differentiation medium every 2 days.
Comparative example 1-1: differentiation culture medium for lung precursor cells and culture method thereof
The present comparative example provides a differentiation medium for lung precursor cells, comprising a medium matrix consisting of 50% (v/v) DMEM medium and 50% (v/v) F12 medium and an adjuvant consisting of l-glutamine solution, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone, transferrin and bovine serum albumin, wherein the l-glutamine solution has a concentration of 2mM in the differentiation medium matrix, the insulin has a concentration of 5 μ g/mL in the differentiation medium matrix, the recombinant epidermal growth factor has a concentration of 0.5 μ g/mL in the differentiation medium matrix, the recombinant human fibroblast growth factor-10 has a concentration of 150 μ g/mL in the differentiation medium matrix, the recombinant human hepatocyte growth factor has a concentration of 25 μ g/mL in the differentiation medium matrix, the adenine has a concentration of 10 μ g/mL in the differentiation medium matrix, the hydrocortisone has a concentration of 200 μ g/mL in the differentiation medium matrix, the hydrocortisone concentration in the differentiation medium matrix is 1 μ g/mL.
The method for differentiating the lung precursor cells comprises the following steps:
the method comprises the following steps: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the lung precursor cell suspension according to the proportion of 10 4 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 24 hours to make the cells adhere to the wall;
step two: after the lung precursor cells were attached to the wall, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the percent CO was 7.5% at 37 ℃ 2 The culture was continued, and the differentiation medium was changed every 2 days.
Comparative examples 1 to 2: differentiation culture medium for lung precursor cells and culture method thereof
The present comparative example provides a differentiation medium for lung precursor cells, comprising a medium substrate consisting of 50% (v/v) DMEM medium and 50% (v/v) F12 medium and an adjuvant consisting of l-glutamine solution, insulin, recombinant egf, recombinant human fibroblast growth factor-10, adenine, hydrocortisone, transferrin, bovine serum albumin and retinoic acid, wherein l-glutamine solution is at a concentration of 2mM in the differentiation medium substrate, insulin is at a concentration of 5 μ g/mL in the differentiation medium substrate, recombinant egf is at a concentration of 0.5 μ g/mL in the differentiation medium substrate, recombinant human fibroblast growth factor-10 is at a concentration of 1 μ g/mL in the differentiation medium substrate, adenine is at a concentration of 10 μ g/mL in the differentiation medium substrate, hydrocortisone is at a concentration of 5 μ g/mL in the differentiation medium substrate, transferrin is at a concentration of 1 μ g/mL in the differentiation medium substrate, albumin is at a concentration of 20 μ g/mL in the differentiation medium substrate, and retinoic acid is at a concentration of 200 μ g/mL in the differentiation medium substrate.
The method for differentiating the lung precursor cells comprises the following steps:
the method comprises the following steps: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the lung precursor cell suspension according to the ratio of 10 4 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 24 hours to make the cells adhere to the wall;
step two: after the attachment of the lung precursor cells, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added per well, and 7.5% CO was added at 37% 2 The culture was continued, and the differentiation medium was changed every 2 days.
Comparative examples 1 to 3: differentiation culture medium for lung precursor cells and culture method thereof
The present comparative example provides a differentiation medium for lung precursor cells, the differentiation medium comprising a medium substrate and an adjuvant, the medium substrate consisting of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, the adjuvant consisting of l-glutamine solution, insulin, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone, transferrin, bovine serum albumin and retinoic acid, wherein the l-glutamine solution has a concentration of 2mM in the differentiation medium substrate, the insulin has a concentration of 5 μ g/mL in the differentiation medium substrate, the recombinant human fibroblast growth factor-10 has a concentration of 150 μ g/mL in the differentiation medium substrate, the recombinant human hepatocyte growth factor has a concentration of 25 μ g/mL in the differentiation medium substrate, the adenine has a concentration of 10 μ g/mL in the differentiation medium substrate, the hydrocortisone has a concentration of 5 μ g/mL in the differentiation medium substrate, the transferrin has a concentration of 1 μ g/mL in the differentiation medium substrate, and the bovine serum albumin has a concentration of 200 μ g/mL in the differentiation medium substrate.
The method for differentiating the lung precursor cells comprises the following steps:
the method comprises the following steps: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the lung precursor cell suspension according to the ratio of 10 4 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 24 hours to make the cells adhere to the wall;
step two: after the lung precursor cells were attached to the wall, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the percent CO was 7.5% at 37 ℃ 2 The culture was continued, and the differentiation medium was changed every 2 days.
Comparative examples 1 to 4: differentiation culture medium for lung precursor cells and culture method thereof
The present comparative example provides a differentiation medium for lung precursor cells, the differentiation medium comprising a medium substrate and an adjuvant, the medium substrate consisting of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, the adjuvant consisting of l-glutamine solution, insulin, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone, transferrin and bovine serum albumin, wherein the l-glutamine solution has a concentration of 2mM in the differentiation medium substrate, the insulin has a concentration of 5 μ g/mL in the differentiation medium substrate, the recombinant human fibroblast growth factor-10 has a concentration of 150 μ g/mL in the differentiation medium substrate, the recombinant human hepatocyte growth factor has a concentration of 25 μ g/mL in the differentiation medium substrate, the adenine has a concentration of 100 μ g/mL in the differentiation medium substrate, the hydrocortisone has a concentration of 500 μ g/mL in the differentiation medium substrate, the transferrin has a concentration of 1 μ g/mL in the differentiation medium substrate, and the bovine serum albumin has a concentration of 200 μ g/mL in the differentiation medium substrate.
The method for differentiating the lung precursor cells comprises the following steps:
the method comprises the following steps: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the lung precursor cell suspension according to the ratio of 10 4 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 24 hours to make the cells adhere to the wall;
step two: after the lung precursor cells were attached to the wall, the proliferation medium was aspirated, washed once with PBS buffer, and 1mL of the above differentiation culture was added to each wellBased on, 7.5% CO at 37% 2 And (5) culturing, and changing the differentiation medium every 2 days.
Experimental example 1: experiment on differentiation efficiency of Lung precursor cells
This experimental example provides an experiment for the differentiation efficiency of lung precursor cells, the experimental process is as follows:
the differentiation medium described in example 1-1 and comparative examples 1-1 to 1-4 was used to differentiate lung precursor cells, and the differentiation was evaluated by observing the cell morphology using a microscope.
The lung precursor cells originally grown in an epithelial clone state (FIG. 1), and the lung precursor cells differentiated by the differentiation medium of example 1-1 were observed in a microscope for 2 days for cell morphology, as shown in FIG. 2, the cells originally grown in an epithelial clone state were observed to be converted into an elongated filamentous state, a thin layer structure similar to type I alveolar epithelium was formed, and a highly differentiated state was observed; the lung precursor cells differentiated by the differentiation medium of the comparative examples 1-1 and 1-2 are observed for cell morphology in 7 days by a microscope, as shown in figures 3 to 4, most cells are still grown in an epithelial clone shape, a small amount of cells are converted into an elongated filamentous shape, a lamellar structure similar to I-type alveolar epithelium is formed, and the differentiation degree is not high; the lung precursor cells differentiated by the differentiation medium of comparative examples 1-3 were microscopically observed in 5 days for cell morphology, as shown in fig. 5, it was observed that most cells no longer grew in epithelial clones and sporadically distributed in the culture dish, but the cells still remained polygonal lung precursor cells, and many apoptotic vacuolated cells, only a small amount of which was converted into elongated filamentous forms, forming a thin lamellar structure similar to type i alveolar epithelium; the lung precursor cells differentiated by the differentiation medium of comparative examples 1-4 were observed in the form of cell morphology under microscope within 14 days, as shown in fig. 6, it was observed that most of the cells still grew in epithelial clone shape, a small amount of the cells were transformed into elongated filamentous shape, but some of the cells formed fibroblast shape, a marker part of the cells underwent epithelial-mesenchymal transition (EMT) process, and transformed into other forms than expected, and the differentiation efficiency was very low; therefore, compared with comparative examples 1-1 to 1-4, the differentiation medium of example 1-1 saves more than two times of differentiation time, has a better specific differentiation effect towards the alveolar epithelium, and has great application prospects in regenerative medical research, drug screening and the like for the respiratory system.
Example 2-1: method for evaluating biological efficacy of lung precursor cells
This example provides a method for evaluating the biological efficacy of lung precursor cells, as shown in fig. 7, using the expression level of the marker HOPX of type i alveolar epithelial cells as an index for evaluating the biological efficacy of lung precursor cells, since HOPX exhibits a large amount of protein expression under a fluorescence microscope compared to AQP5, the method comprising the steps of:
the method comprises the following steps: selecting the corning matrigel as a substrate gel, and diluting the corning matrigel on ice by using a DMEM (DMEM) culture medium until the protein concentration is 3mg/mL; adding 3mL of diluted corning matrix glue into a T25 culture bottle to enable the diluted corning matrix glue to completely cover the bottom of the T25 culture bottle, placing the T25 culture bottle containing the corning matrix glue at 37 ℃ for incubation for 15 minutes, and then sucking out all corning matrix glue liquid which is not coated on the T25 culture bottle; taking irradiated 3T3 cells as trophoblast cells at 3 × 10 4 Individual cell/cm 2 Inoculating into the above T25 flask at 37 deg.C, 7.5% 2 Culturing for 2 days under the condition; at 10 4 Individual cell/cm 2 Inoculating the revived lung precursor cells onto the cultured trophoblast cells, adding 5mL of lung precursor cell proliferation medium, and adding CO at 37 deg.C and 7.5% 2 Culturing for 3 days under the condition of (1) to obtain lung precursor cells with the cell cloning density of 60 to 80 percent; washing lung precursor cells with cell clone density of 60-80% with PBS buffer solution for 1 time, adding 3mL of pancreatin with concentration of 0.05% (m/v, g/100 mL), incubating at 37 ℃ for 5 minutes, observing that trophoblast cells are completely fallen off and most lung precursor cells are still in an adherent state under a microscope, sucking supernatant, washing for 1 time with PBS buffer solution, adding 3mL of pancreatin with concentration of 0.25% (m/v, g/100 mL), incubating at 37 ℃ for 10 minutes, observing that cells are completely fallen off under a microscope, adding 3mL of DMEM containing 10% (v/v) serum as a digestion stop solution to stop pancreatin digestion, centrifuging cell suspension for 3 minutes at 1200rpm, and collecting cell precipitates, namely collecting cell precipitatesObtaining lung precursor cell suspension with trophoblast cells removed;
step two: taking 12-well plate, adding 1mL proliferation culture medium into each well, and adding the above mentioned lung precursor cell suspension without trophoblast cells according to 10 4 Inoculation of individual cells/well, 37 ℃,7.5% CO 2 Culturing for 24 hours under the condition to make the cells adhere to the wall; 24 hours later, the original lung precursor cell proliferation medium was aspirated, washed once with PBS buffer, 1mL of the differentiation medium of example 1-1 was added to each well, and the content of CO was 7.5% at 37 ℃% 2 Continuously culturing for 48 hours under the condition, and not changing a differentiation culture medium;
step three: after 48 hours, the original differentiation medium was aspirated, washed 1 time with PBS buffer, 3mL of pancreatin with a concentration of 0.25% (m/v, g/100 mL) was added to each well, incubated at 37 ℃ for 8 minutes, cells were observed to completely detach under a microscope, 3mL of DMEM containing 10% (v/v) serum was added to each well as a digestion stop solution to stop the pancreatin digestion, the cells were aspirated several times, and then the cell suspension was centrifuged at 1200rpm for 3 minutes, and cell pellets were collected and transferred to a centrifuge tube; adding 500 μ L of 3.7% (m/v, g/100 mL) neutral formalin solution per tube to resuspend the cell pellet, fixing for 10 minutes at 30rpm on a room temperature shaker, centrifuging the cell suspension for 3 minutes at 1200rpm, then aspirating the supernatant, adding 500 μ L of 0.2% (v/v) Triton X-100 solution per tube, standing for 10 minutes, centrifuging the cell suspension for 3 minutes at 1200rpm, then aspirating the supernatant, adding 500 μ L of 5% (v/v) donkey serum, and incubating for 0.5 hours at 30rpm on a room temperature shaker; fully and uniformly mixing the cell suspension added with donkey serum, subpackaging 50% of the cell suspension by volume into a new centrifugal tube, setting the centrifugal tube as a control sample tube, and setting the centrifugal tube where the rest cells are located as a detection sample tube; centrifuging a control sample tube and a detection sample tube at 1200rpm for 3 minutes, then sucking out supernatant, adding 100 mu L of PBS buffer solution into the control sample tube, adding 100 mu L of HOPX primary anti-working solution into the detection sample tube, incubating for 2 hours at 30rpm on a room temperature shaking bed, and then transferring the incubated samples to a refrigerator at 4 ℃ for incubation for 12 hours; centrifuging the stored control sample tube and the detection sample tube at 1200rpm for 3 minutes, then sucking and removing supernatant, adding a proper amount of PBS (phosphate buffer solution) into each tube to resuspend cell precipitates, blowing and beating for several times, and washing cells; centrifuging the control sample tube and the detection sample tube at 1200rpm for 3 minutes, absorbing and removing supernatant, adding 100 mu L of fluorescent secondary antibody working solution into each tube to resuspend cell precipitates, and incubating for 2 hours at room temperature in a dark place; after incubation is finished, centrifuging the control sample tube and the detection sample tube at 1200rpm for 3 minutes, then sucking and removing supernatant, adding a proper amount of PBS buffer solution into each tube to resuspend cell precipitates, blowing and beating for several times, and washing cells; repeating the washing process for 2 times, re-suspending with 1mL PBS buffer solution for the last time, blowing and beating for several times, and mixing uniformly; loading the washed control sample tube and the washed detection sample tube into a fluorescence cell counter for on-machine detection, during detection, firstly adjusting a threshold value by using the control sample, confirming that the number of negative cells of the control sample is more than 95%, then carrying out on-machine detection on the detection sample, and obtaining the ratio of cells with positive HOPX markers in the detection sample, namely the evaluation result of the biological efficacy of the lung precursor cells, wherein the detection result is shown in figure 8.
As shown in fig. 8, the percentage of HOPX-positive cells to total detected cells (RFP) after differentiation was 72%; the differentiated lung precursor cells show a large amount of HOPX protein expression, which shows that the expression level of a marker HOPX of the type I alveolar epithelial cells is selected as an index for evaluating the biological efficacy of the lung precursor cells, so that the relationship between the cell quality and the dose-effect can be fully and accurately reflected; and the differentiation medium described in example 1-1 is used for differentiation and detection of lung precursor cells, so that the biological efficacy of the lung precursor cells can be rapidly, conveniently and stably evaluated.
Example 2-2: method for evaluating biological efficacy of lung precursor cells
This example provides a method for evaluating the biological efficacy of lung precursor cells, as shown in fig. 7, using the expression level of HOPX, a marker of type i alveolar epithelial cells, as an index for evaluating the biological efficacy of lung precursor cells, since HOPX exhibits a large amount of protein expression compared to AQP5 under a fluorescence microscope, the method comprising the steps of:
the method comprises the following steps: selecting Kangning matrigel as the base gel, and using PBS buffer solutionNingmatrigel: PBS buffer =1:5, and then adding the dilution to a 6-well plate at 500. Mu.L/well so that it completely covers the bottom of the 6-well plate; incubating the 6-well plate containing corning matrigel for 15 minutes at 37 ℃, and sucking out all corning matrigel liquid which is not coated on the 6-well plate; taking irradiated 3T3 cells as trophoblast cells at 2 × 10 4 Individual cell/cm 2 Inoculating into 6-well plate, 7.5% CO at 37% 2 Culturing under the condition; the trophoblast cells were cultured at 1.5X 10 for 12 hours 4 Individual cell/cm 2 Inoculating the revived lung precursor cells onto the trophoblast cells at an inoculation density of 2mL of lung precursor cell proliferation medium, at 37 ℃ and 7.5% CO 2 Continuously culturing for 2 days under the condition of (1) to obtain lung precursor cells with the cell clone density reaching 80 percent confluence; washing the lung precursor cells with the cell clone density reaching 80% confluence by PBS buffer solution for 1 time, adding 750 mu L of pancreatin with the concentration of 0.05% (m/v, g/100 mL) to incubate for 8 minutes at 37 ℃, observing that all trophoblast cells are fallen off and most lung precursor cells are still in an adherent state under a microscope, sucking supernatant and washing for 1 time by using the PBS buffer solution, adding 750 mu L of pancreatin with the concentration of 0.25% (m/v, g/100 mL), incubating for 10 minutes at 37 ℃, observing that all cells are fallen off under the microscope, adding 750 mu L of DMEM containing 10% (v/v) serum as a digestion stop solution to stop pancreatin digestion, then centrifuging the cell suspension for 5 minutes at the speed of 1200rpm, and collecting cell precipitates to obtain the lung precursor cell suspension without trophoblast cells;
step two: taking 12-well plate, adding 1mL proliferation culture medium per well, and removing above mentioned lung precursor cell suspension of trophoblast cells by 5 × 10 3 Inoculation of individual cells/well, 7.5% CO at 37% 2 Culturing for 6 hours under the condition(s) of (1) to allow the cells to adhere to the wall; after 6 hours, the original lung precursor cell proliferation medium was aspirated, washed once with PBS buffer, 1mL of the differentiation medium of example 1-1 was added per well, and the CO content was 7.5% at 37% 2 Continuously culturing for 72 hours without changing the differentiation medium;
step three: after 72 hours, the differentiation medium supernatant was aspirated and washed 1 time with PBS buffer, 750. Mu.L of pancreatin with a concentration of 0.25% (m/v, g/100 mL) was added, incubation was carried out at 37 ℃ for 4min, 750. Mu.L of DMEM containing 10% (v/v) serum was added to each well as a digestion stop solution to stop the trypsinization, and pipetting was carried out several times; then centrifuging the cell suspension for 3 minutes at 1200rpm, collecting cell precipitates, and transferring the cell precipitates to a centrifuge tube; adding 500 μ L of fixation buffer per tube to resuspend the cell pellet, fixing on a shaker at room temperature at 10rpm for 8 minutes, centrifuging the cell suspension at 1200rpm for 5 minutes, then aspirating the supernatant, adding 500 μ L of 0.2% (v/v) Triton X-100 solution per tube, standing for 5 minutes, centrifuging the cell suspension at 1200rpm for 5 minutes, aspirating the supernatant, adding 500 μ L of 5% (v/v) donkey serum, and incubating at 10rpm on a shaker at room temperature for 0.5 hour; fully and uniformly mixing the cell suspension added with donkey serum, subpackaging 50% of cell suspension in a new centrifugal tube, setting the centrifugal tube as a control sample tube, and setting the centrifugal tube in which the rest cells are positioned as a detection sample tube; centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, then sucking out the supernatant, adding 1mL of PBS buffer solution into the control sample tube, adding 500 mu L of HOPX primary anti-working solution into the detection sample tube, incubating for 2 hours at 10rpm on a room temperature shaking table, then centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, then sucking out the supernatant, adding an appropriate amount of PBS buffer solution into each tube, resuspending the cell precipitate, pipetting for several times, and washing the cells; centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, absorbing and removing supernatant, adding 500 mu L of fluorescent secondary antibody working solution into each tube to resuspend cell precipitates, and incubating for 2 hours at room temperature in a dark place; after incubation is finished, centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, then sucking and removing supernatant, adding a proper amount of PBS buffer solution into each tube to resuspend cell precipitates, blowing and beating for several times, and washing cells; repeating the washing process for 2 times, re-suspending with 1mL PBS buffer solution for the last time, blowing and beating for several times, and mixing uniformly; loading the washed control sample tube and the washed detection sample tube to a flow cytometry analyzer for on-machine detection; during detection, the control sample tube is used to adjust the threshold value to confirm that the number of negative cells in the control sample should be kept above 98%, and then the detection sample is subjected to on-machine detection to obtain the ratio of cells positive to the HOPX marker in the detection sample, namely the evaluation result of the biological efficacy of the lung precursor cells, wherein the detection result is shown in FIG. 9.
As shown in fig. 9, the percentage of HOPX-positive cells after differentiation (FITC-se:Sub>A subset) was 61.9% of the total cells tested; the differentiated lung precursor cells show a large amount of HOPX protein expression, which shows that the expression level of a marker HOPX of the type I alveolar epithelial cells is selected as an index for evaluating the biological efficacy of the lung precursor cells, so that the relationship between the cell quality and the dose-effect can be fully and accurately reflected; and the biological efficacy of the lung precursor cells can be rapidly, conveniently and stably evaluated by detecting the lung precursor cells after the differentiation medium is used for differentiating the lung precursor cells as described in the embodiment 1-1.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The differentiation medium for the lung precursor cells is characterized by comprising a medium matrix and an adjuvant, wherein the medium matrix comprises a DMEM medium and an F12 medium, and the medium adjuvant comprises levoglutamine, insulin, a recombinant epidermal growth factor, a recombinant human fibroblast growth factor-10, a recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid.
2. The differentiation medium according to claim 1, wherein the volume ratio of DMEM medium to F12 medium in the composition of the differentiation medium matrix is 25 to 75: 25-75, in the ingredients of the differentiation medium adjuvant, the concentration of levoglutamide in a differentiation medium matrix is 2-6 mM, the concentration of insulin in the differentiation medium matrix is 2-15 μ g/mL, the concentration of recombinant epidermal growth factor in the differentiation medium matrix is 0.1-2 μ g/mL, the concentration of recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 25-400 μ g/mL, the concentration of recombinant human hepatocyte growth factor in the differentiation medium matrix is 10-35 μ g/mL, the concentration of adenine in the differentiation medium matrix is 5-50 μ g/mL, the concentration of hydrocortisone in the differentiation medium matrix is 2-20 μ g/mL, and the concentration of retinoic acid in the differentiation medium matrix is 0.1-2nM.
3. The differentiation medium according to claim 1 or 2, wherein the components of the differentiation medium further comprise one or a combination of more than one of ROCK inhibitor, transferrin, albumin and penicillin/streptomycin diabody.
4. The differentiation medium of claim 3, wherein said ROCK inhibitor is Y-27632.
5. A method for differentiating and culturing lung precursor cells, characterized in that the method comprises differentiating and culturing lung precursor cells using the differentiation medium according to any one of claims 1 to 4.
6. The differentiation culture method according to claim 5, comprising the steps of:
the method comprises the following steps: inoculating the lung precursor cells into a proliferation culture medium for culturing until the lung precursor cells are attached to the wall;
step two: after the lung precursor cells are attached to the wall, sucking the propagation culture medium away, adding the differentiation culture medium of any one of claims 1 to 4 into the attached lung precursor cells, culturing until the cell morphology is converted into an elongated filamentous morphology, forming a thin-layer structure similar to the type I alveolar epithelium, and completing differentiation.
7. A method of assessing the biological effectiveness of a lung precursor cell, the method comprising the steps of:
the method comprises the following steps: co-culturing the lung precursor cells and the trophoblast cells to obtain the lung precursor cells after co-culture and amplification;
step two: differentiating and culturing the lung precursor cells after the co-culture expansion by using the differentiation culture method according to claim 5 or 6 to obtain differentiated and cultured lung precursor cells;
step three: detecting the lung precursor cells after differentiation culture, and evaluating the biological efficacy of the lung precursor cells according to the detection result.
8. The method for evaluating the biological effectiveness of lung precursor cells according to claim 7, wherein in the third step, the differentiated and cultured lung precursor cells are subjected to immunofluorescence staining, then the immunofluorescence stained lung precursor cells are quantitatively detected by using a fluorescence cytometer or a flow cytometer, and finally the biological effectiveness of the lung precursor cells is evaluated according to the detection result.
9. The method of assessing the biological effectiveness of lung precursor cells of claim 8, wherein said immunofluorescent staining selects HOPX protein of type I alveolar epithelial cells as a target.
10. Use of the differentiation medium according to any one of claims 1 to 4, the differentiation culture method according to claim 5 or 6, or the method for evaluating the biological efficacy of a lung precursor cell according to any one of claims 7 to 9 in drug screening for various acute, chronic and degenerative diseases of the respiratory system.
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