CN115786243B - 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 of lung precursor cells, wherein the differentiation medium comprises DMEM medium, F12 medium, L-glutamine, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid; the differentiation medium is used for differentiating the lung precursor cells, so that the differentiation time can be greatly shortened, the detection efficiency of the biological efficacy is further improved, the gap that the existing lung stem cells/precursor cells treatment field lacks an effective and rapid detection means with targeted biological efficacy is filled, and the method has great significance for research on regenerative medicine, drug screening and the like of various acute and chronic 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 cell/precursor cell-based therapies are being steadily advanced at a rapid pace; such cells, after being introduced into the body by various transplantation techniques, are expected to replace damaged cells of a patient or regenerate new tissues or organs by recruiting endogenous tissue-specific stem cells, or exert positive immune regulation, etc.
In the method for repairing tissue organ injury by cell transplantation by utilizing lung tissue specific adult precursor cells (also called adult stem cells, hereinafter the same) derived from human bodies for various acute, chronic and degenerative diseases of respiratory systems, the method is expected to fundamentally realize the regenerative medical research of serious respiratory system diseases (chronic obstructive pulmonary disease, interstitial lung disease, bronchiectasis and the like) and provide a new treatment method for patients; wherein, the lung precursor cells which can be used for in vitro separation, expansion culture and clinical transplantation are mainly bronchial basal layer cells, distal 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, how to define the quality of cell drugs or to primarily judge the therapeutic effect of cells is an important consideration because of various factors such as different genetic and physiological and pathological backgrounds of donors, heterogeneity of cells themselves, complex and diverse cell action mechanisms and the like; an index that quantifies the quality of a cytosol is generally referred to as biological potency (Biological Efficacy), and assessment of the biological potency of a cytosol should primarily be viewed for its ability to differentiate into functional cells.
Currently, conventional methods for detecting the biological efficacy of lung precursor cells include three-dimensional organoid culture and gas-liquid interface differentiation methods, which are based on the principle that lung precursor cells are induced to differentiate by allowing the cells to form a three-dimensional structure in vitro, in combination with a differentiation culture system; the method can simulate the physiological structure and characteristics of cells in a human body, but has the defects that the method is particularly obvious as a detection means of biological efficacy, can complete differentiation in a few weeks or more, is difficult to ensure the timeliness requirement of quality inspection of cell therapeutic drugs, and is difficult to perform standardized quantitative analysis on the cell differentiation efficiency 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 comprising a medium matrix comprising DMEM medium and F12 medium and an adjuvant comprising 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 DMEM medium to F12 medium is 25 to 75: 25-75 parts of a differentiation medium adjuvant, wherein the concentration of L-glutamine in a differentiation medium matrix is 2-6 mM, the concentration of insulin in the differentiation medium matrix is 2-15 mug/mL, the concentration of recombinant epidermal growth factor in the differentiation medium matrix is 0.1-2 mug/mL, the concentration of recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 25-400 mug/mL, the concentration of recombinant human hepatocyte growth factor in the differentiation medium matrix is 10-35 mug/mL, the concentration of adenine in the differentiation medium matrix is 5-50 mug/mL, the concentration of hydrocortisone in the differentiation medium matrix is 2-20 mug/mL, and the concentration of retinoic acid in the differentiation medium matrix is 0.1-20 nM.
In one embodiment of the invention, the components of the differentiation medium further comprise one or more combinations of ROCK inhibitors, transferrin, albumin and penicillin/streptomycin diabodies.
In one embodiment of the invention, the ROCK inhibitor is Y-27632.
In one embodiment of the 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 penicillin/streptomycin diabody solution has a concentration of 5000U/mL.
In one embodiment of the invention, the concentration of Y-27632 in the 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 the penicillin/streptomycin diabody solution to the differentiation medium matrix is 0% -5% (v/v).
In one embodiment of the present invention, the volume ratio of DMEM medium to F12 medium in the components of the differentiation medium matrix is 50:50, wherein the concentration of the L-glutamine solution in the differentiation medium matrix is 2mM, the concentration of the 5000U/mL 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 μg/mL, the concentration of the recombinant epidermal growth factor in the differentiation medium matrix is 0.5 μg/mL, the concentration of the recombinant human fibroblast growth factor-10 in the differentiation medium matrix is 150 μg/mL, the concentration of the recombinant human hepatocyte growth factor in the differentiation medium matrix is 25 μg/mL, the concentration of adenine in the differentiation medium matrix is 10 μg/mL, the concentration of hydrocortisone in the differentiation medium matrix is 5 μg/mL, the concentration of transferrin in the differentiation medium matrix is 1 μg/mL, the concentration of bovine serum albumin in the differentiation medium matrix is 200 μg/mL, and the concentration of retinoic acid in the differentiation medium matrix is 20nM.
The invention also provides a differentiation culture method of the lung precursor cells, which is to use the differentiation culture medium to conduct differentiation culture on the lung precursor cells.
In one embodiment of the present invention, the differentiation culture method comprises the steps of:
step one: inoculating lung precursor cells into a proliferation culture medium for culturing until the lung precursor cells adhere to the wall;
step two: after the lung precursor cells are attached, sucking away the proliferation culture medium, adding the differentiation culture medium into the attached lung precursor cells for culturing until the cell morphology is converted into an elongated filament morphology, forming a lamellar structure similar to the type I alveolar epithelium, and completing differentiation.
In one embodiment of the present invention, the first step is: lung precursor cells were packed at 1×10 3 ~5×10 4 Individual cells/cm 2 Is inoculated in a 12-well plate added with proliferation medium until lung precursor cells adhere.
In one embodiment of the invention, the proliferation medium comprises 225mL of DMEM medium, 225mL of F12 medium, 20-70 mL of Fetal Bovine Serum (FBS), 0.2-2 mM of L-glutamine, 1-14 ng/mL of insulin, 0.1-1 ng/mL of epidermal growth factor, 5-30 ug/mL of adenine and 2-20 ug/mL of hydrocortisone.
In one embodiment of the invention, the lung precursor cell culture conditions are 37℃and 7.5% CO 2 。
In one embodiment of the present invention, in the second step, the differentiation medium needs to be replaced every 2 to 3 days.
In one embodiment of the present invention, in the second step, the cell morphology is observed by a microscope.
The invention also provides a method of assessing the biological efficacy of a lung precursor cell, the method comprising the steps of:
step one: co-culturing lung precursor cells and trophoblast cells to obtain lung precursor cells after co-culture expansion;
step two: performing differential culture on the lung precursor cells subjected to co-culture expansion by using the differential culture method to obtain the differential cultured lung precursor cells;
step three: the differentiated cultured lung precursor cells are tested and the biological efficacy of the lung precursor cells is assessed based on the test results.
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 base gel is corning Matrigel (Matrigel Matrix).
In one embodiment of the present invention, in the first step, the protein concentration of the corning matrigel is not less than 1mg/mL.
In one embodiment of the present invention, in the first step, the protein concentration of the corning matrigel is 1.8-3 mg/mL.
In one embodiment of the present invention, in the first step, the culture vessel 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 trophoblast cells are seeded at a density of 5X 10 in a T25 flask 3 ~10×10 4 Individual cells/cm 2 。
In one embodiment of the present invention, in the first step, the trophoblast cells are seeded at a density of 3X 10 in a T25 flask 4 Individual cells/cm 2 。
In one embodiment of the present invention, in the first step, the trophoblast cells are seeded at a density of 5X 10 in a 6-well plate 5 Individual cells/wells.
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 proliferation medium is replaced every 2 to 3 days.
In one embodiment of the present invention, in the first step, the lung precursor cell culture conditions are 37℃and 7.5% CO 2 。
In one embodiment of the present invention, in the first step, the proliferation medium comprises 225mL of DMEM, 225mL of F12, 20-70 mL of FBS, 0.2-2 mM of L-glutamine, 1-14 ng/mL of insulin, 0.1-1 ng/mL of epidermal growth factor, 5-30 ug/mL of adenine and 2-20 ug/mL of hydrocortisone.
In one embodiment of the present invention, in the first step, after the lung precursor cells are co-cultured with the trophoblast cells, the method further comprises removing the trophoblast cells.
In one embodiment of the present invention, in the first step, the removing the trophoblast cell is: washing cultured cells with PBS buffer solution, incubating at 37 ℃, sucking supernatant and washing with PBS buffer solution when most of lung precursor cells are still in an adherent state, adding pancreatin until the lung precursor cells are completely shed, adding digestion stop solution to stop pancreatin digestion, centrifuging the cell suspension, and collecting cell precipitate to obtain the lung precursor cell suspension without trophoblast cells.
In one embodiment of the present invention, in the first step, the concentration of pancreatin in the exfoliated trophoblast cells is 0.01-0.05% (m/v, g/100 mL).
In one embodiment of the present invention, in the first step, the concentration of pancreatin in the exfoliated 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 dropping of the trophoblast cells is observed by a microscope.
In one embodiment of the present invention, in the first step, the digestion terminator is DMEM containing not less than 10% (v/v) serum.
In one embodiment of the present invention, in the third step, the differentiated lung precursor cells are subjected to immunofluorescent staining, and then the immunofluorescent stained lung precursor cells are quantitatively detected by a fluorescent cytometer 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, the differentiated lung precursor cells are all exfoliated using pancreatin to obtain a cell pellet, the cell pellet is resuspended and fixed using a fixative, and the fixed differentiated lung precursor cells are subjected to the immunofluorescent 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 incubation at 37 ℃ for 8 minutes.
In one embodiment of the present invention, in the third step, after the lung precursor cells are completely shed, a digestion stop solution is added to stop the pancreatin digestion.
In one embodiment of the present invention, in the third step, the digestion terminator is DMEM containing 10% (v/v) serum.
In one embodiment of the invention, in step three, after the lung precursor cells are all shed, the cell suspension is centrifuged to collect the cell pellet.
In one embodiment of the present invention, in the third step, the centrifugation is performed at a speed of 1200rpm for 3 minutes.
In one embodiment of the present invention, in the third step, the fixative is a neutral formalin solution.
In one 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 fixative is a fixation buffer (fixative).
In one embodiment of the present invention, in the third step, after the step of resuspension with the fixative, the lung precursor cells are fixed by placing on a shaking table at room temperature for 8 to 10 minutes.
In one embodiment of the invention, in said step three, incubation is performed on said room temperature shaker at 30 rpm.
In one embodiment of the present invention, in the third step, after fixing the lung precursor cells, the triton X-100 solution and donkey serum are added, and after incubation, the cells are split into two centrifuge tubes on average, wherein one centrifuge tube is a control sample tube, and the other centrifuge tube is a detection sample tube.
In one embodiment of the present invention, in the third step, the supernatant is removed after centrifuging the cell suspension prior to adding the triton X-100 solution.
In one embodiment of the present invention, in the third step, the solution of triton X-100 is a 0.2% (v/v) solution of triton X-100.
In one embodiment of the present invention, in the third step, the 0.2% (v/v) solution of triton X-100 is formulated as: after absorbing 2. Mu.L of 10% triton X-100 and diluting with 98. Mu.L of PBS buffer, the solution was allowed to stand on a shaker at room temperature for more than 1 hour, and the solution was observed to be completely transparent and clear and then ready for use.
In one embodiment of the present invention, in the third step, the solution of triton X-100 is added and then left for 10 minutes.
In one embodiment of the present invention, in the third step, the supernatant is removed by centrifugation of the cell suspension to which the triton X-100 solution is added, before donkey serum is added.
In one embodiment of the present invention, in said step three, said donkey serum is 5% (v/v) donkey serum.
In one embodiment of the present invention, in said step three, said 5% (v/v) donkey serum is formulated as: mu.L donkey serum was aspirated, diluted with 95. Mu.L PBS buffer, and thoroughly mixed.
In one embodiment of the present invention, in said step three, after adding said donkey serum, the donkey serum is left 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, absorbing supernatant, adding PBS buffer solution into the control sample tube, adding HOPX primary antibody working solution into the detection sample tube, centrifuging after incubation, absorbing supernatant, adding fluorescent secondary antibody working solution into each tube, and incubating.
In one embodiment of the present invention, in the immunofluorescent staining of the third step, the incubation of the HOPX primary antibody working solution is: incubate on the room temperature shaker for 2 hours.
In one embodiment of the present invention, in the immunofluorescent staining of the third step, after centrifuging the control sample tube and the test sample tube and discarding the supernatant, the cells are washed once with PBS buffer.
In one embodiment of the present invention, in the immunofluorescent staining of step three, the centrifugation is at 1200rpm for 5 minutes.
In one embodiment of the present invention, in the immunofluorescent staining of the third step, the incubation of the fluorescent secondary antibody working solution is incubation in a dark place.
In one embodiment of the present invention, in the immunofluorescent staining of step three, the incubation in the dark is for 2 hours at room temperature.
In one embodiment of the present invention, in the third step, the PBS buffer is added to the control sample tube and the test sample tube and the cells are washed twice before the quantitative detection.
In one embodiment of the invention, the immunofluorescent staining selects as a target the HOPX protein of type i alveolar epithelial cells.
In one embodiment of the invention, the immunofluorescent staining selects the AQP5 protein of type i alveolar epithelial cells as a target.
The invention also provides application of the differentiation culture medium or the differentiation culture method or the method for evaluating biological efficacy of lung precursor cells in drug screening for various acute and chronic degenerative diseases of respiratory system.
The technical scheme of the invention has the following advantages:
1. the invention provides a differentiation medium of lung precursor cells, wherein the differentiation medium comprises DMEM medium, F12 medium, L-glutamine solution, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone and retinoic acid; the differentiation culture medium can complete the differentiation of lung precursor cells in a short time, and ensures the timeliness requirement of the quality inspection of cell therapeutic drugs.
2. The invention provides a differentiation culture method of lung precursor cells, which cultures lung precursor cells on trophoblast cells, can provide certain nutrition and support for cell culture, is further beneficial to the adherent growth of cells by adding base adhesive, has better cell morphology, and is easy to perform standardized quantitative analysis on cell differentiation efficiency.
3. The invention provides a method for rapidly evaluating biological efficacy of lung precursor cells, because the type I alveolar epithelial cells are alveolar functional cells bearing blood-gas exchange function, the expression levels of the markers AQP5 and HOPX of the type I alveolar epithelial cells are selected as indexes for evaluating the biological efficacy of the lung precursor cells, and the relationship between cell quality and 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 using the improved differentiation medium to differentiate the lung precursor cells and then detecting the lung precursor cells.
Drawings
Fig. 1: morphology of lung precursor cells grown on trophoblast cells prior to differentiation.
Fig. 2: morphology of lung precursor cells after differentiation culture according to example 1-1 of the present invention.
Fig. 3: comparative example 1-1 morphology of lung precursor cells after differentiation culture.
Fig. 4: comparative examples 1-2 morphology of lung precursor cells after differentiation culture.
Fig. 5: morphology of lung precursor cells after differentiation culture according to comparative examples 1-3 of the present invention.
Fig. 6: morphology of lung precursor cells after differentiation culture according to comparative examples 1-4 of the present invention.
Fig. 7: and (3) identifying results of the AQP5 and HOPX under a fluorescence microscope.
Fig. 8: in example 2-1 of the present invention, the result of quantitative measurement using a fluorescent cytometer (HOPX as a marker) shows that RFP values represent the percentage of cells positive for HOPX after differentiation to total cells detected.
Fig. 9: in the result graph of quantitative detection using se:Sub>A flow cytometer (HOPX as se:Sub>A marker) in example 2-2 of the present invention, 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 indicated by FITC-se:Sub>A subset represent the percentage of cells positive for HOPX after differentiation to total detected cells.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by routine experimental procedures or conditions described in the literature in this field; the reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
DMEM medium referred to in the examples below was purchased from sammer femto technology (Thermo Fisher Scientific), model 11965092; f12 medium was purchased from Siemens technologies under model 21765037; l-glutamine was purchased from Semerle Feishul technologies under the model A2916801; insulin was purchased from Sigma Aldrich, model I2643; recombinant epidermal growth factor was purchased from sigma aldrich company under the model SRP3027; recombinant human fibroblast growth factor-10 was purchased from sigma aldrich under the model GF172; recombinant human hepatocyte growth factor is purchased from MCE company and is model HY-P7121; adenine was purchased from sigma aldrich under model a5665; hydrocortisone available from MCE company under the model HY-N0583; retinoic acid is available from sigma aldrich under the model R2625; transferrin was purchased from sigma aldrich under the model T8158; bovine serum albumin was purchased from tokriles bioscience (tocis), model 5217;5000U/mL penicillin/streptomycin diabody solution was purchased from Semerle Feishmania technology, model 15070063.
The components of the proliferation medium for culturing lung precursor cells referred to in the following examples are: 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 following examples was formulated as: mu.L of 10% triton X-100 (available from Beijing Soy Corp.) was pipetted, diluted with 98. Mu.L of PBS buffer, and placed on a room temperature shaker for 1 hour or more, and the solution was completely clear after observation.
The 5% donkey serum referred to in the examples below was formulated as: mu.L donkey serum was aspirated, diluted with 95. Mu.L PBS buffer, and mixed well for use.
The working solution for the first HOPX antibody in the following examples was purchased from san krus biotechnology (Santa Cruz Biotechnology), and the first HOPX antibody was diluted with PBS buffer at the concentration ratio described in the antibody specification.
The secondary fluorescent antibody in example 2-1 was Alexa Fluor ™ 594 available from Semer Feier technology, and was diluted with PBS buffer at the concentration ratio described in the antibody specification.
The secondary fluorescent antibody in example 2-2 was Alexa Fluor ™ 488 available from Semer Feiche technologies, inc., and was diluted with PBS buffer at the concentration ratios described in the antibody specification.
Example 1-1: differentiation medium for lung precursor cells and culture method thereof
The embodiment provides a differentiation medium for lung precursor cells, wherein the differentiation medium comprises a medium matrix and an adjuvant, the medium matrix consists of 50% (v/v) of DMEM medium and 50% (v/v) of F12 medium, the adjuvant consists of a L-glutamine solution, a 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, the L-glutamine solution has a concentration of 2mM in the medium matrix of differentiation medium, the concentration of 5000U/mL of penicillin/streptomycin double-antibody solution has a concentration of 5 mug/mL in the medium matrix of differentiation medium, the concentration of recombinant human fibroblast growth factor-10 has a concentration of 150 mug/mL in the medium matrix of differentiation medium, the recombinant human hepatocyte growth factor has a concentration of 25 mug/mL in the medium of serum albumin, the concentration of 200 mug/mL in the medium of differentiation medium, the concentration of the serum albumin/mL has a concentration of 200 mug/mL in the medium of differentiation medium matrix of differentiation medium, and the differentiation medium has a concentration of 200 mug/mL in the medium of serum.
The differentiation method of the lung precursor cells comprises the following steps:
step one: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing lung precursor cell suspension according to 10 4 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 24 hours to adhere cells;
step two: after attachment of lung precursor cells, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the mixture was incubated at 37℃with 7.5% CO 2 Culture under, change differentiation medium every 2 days.
Comparative examples 1-1: differentiation 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 matrix composed of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, and an adjuvant composed of a 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 medium matrix, the insulin has a concentration of 5 μg/mL in the medium matrix, the recombinant human fibroblast growth factor-10 has a concentration of 150 μg/mL in the medium matrix, the recombinant human hepatocyte growth factor has a concentration of 25 μg/mL in the medium matrix, the adenine has a concentration of 10 μg/mL in the medium matrix, the hydrocortisone has a concentration of 5 μg/mL in the medium matrix, the serum albumin has a concentration of 200 μg/mL in the medium matrix.
The differentiation method of the lung precursor cells comprises the following steps:
step one: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing lung precursor cell suspension according to 10 4 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 24 hours to adhere cells;
step two: after attachment of lung precursor cells, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the mixture was incubated at 37℃with 7.5% CO 2 Culture under, change differentiation medium every 2 days.
Comparative examples 1-2: differentiation 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 matrix composed of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, and an adjuvant composed of a l-glutamine solution, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, adenine, hydrocortisone, transferrin, bovine serum albumin and retinoic acid, wherein the l-glutamine solution has a concentration of 2mM in the medium matrix, insulin has a concentration of 5 μg/mL in the medium matrix, recombinant human fibroblast growth factor-10 has a concentration of 1 μg/mL in the medium matrix, adenine has a concentration of 10 μg/mL in the medium matrix, hydrocortisone has a concentration of 5 μg/mL in the medium matrix, transferrin has a concentration of 1 μg/mL in the medium matrix, and retinoic acid has a concentration of 200 μg/mL in the medium matrix.
The differentiation method of the lung precursor cells comprises the following steps:
step one: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing lung precursor cell suspension according to 10 4 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 24 hours to adhere cells;
step two: lung precursor fineAfter cell attachment, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the mixture was incubated at 37℃with 7.5% CO 2 Culture under, change differentiation medium every 2 days.
Comparative examples 1-3: differentiation 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 matrix composed of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, and an adjuvant composed of a 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 medium matrix of differentiation medium, insulin has a concentration of 5 μg/mL in the medium matrix of differentiation medium, recombinant human fibroblast growth factor-10 has a concentration of 25 μg/mL in the medium matrix of differentiation medium, adenine has a concentration of 10 μg/mL in the medium matrix of differentiation medium, hydrocortisone has a concentration of 5 μg/mL in the medium matrix of differentiation medium, transferrin has a concentration of 1 μg/mL in the medium matrix of serum albumin and retinoic acid has a concentration of 200 μg/mL in the medium of differentiation medium.
The differentiation method of the lung precursor cells comprises the following steps:
step one: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing lung precursor cell suspension according to 10 4 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 24 hours to adhere cells;
step two: after attachment of lung precursor cells, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the mixture was incubated at 37℃with 7.5% CO 2 Culture under, change differentiation medium every 2 days.
Comparative examples 1 to 4: differentiation 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 matrix composed of 50% (v/v) DMEM medium and 50% (v/v) F12 medium, and an adjuvant composed of a 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 matrix, insulin has a concentration of 5 μg/mL in the differentiation medium matrix, recombinant human fibroblast growth factor-10 has a concentration of 150 μg/mL in the differentiation medium matrix, recombinant human hepatocyte growth factor has a concentration of 25 μg/mL in the differentiation medium matrix, adenine has a concentration of 100 μg/mL in the differentiation medium matrix, hydrocortisone has a concentration of 500 μg/mL in the differentiation medium matrix, transferrin has a concentration of 1 μg/mL in the differentiation medium matrix, and bovine serum albumin has a concentration of 200 μg/mL in the differentiation medium matrix.
The differentiation method of the lung precursor cells comprises the following steps:
step one: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing lung precursor cell suspension according to 10 4 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 24 hours to adhere cells;
step two: after attachment of lung precursor cells, the proliferation medium was aspirated, washed once with PBS buffer, 1mL of the above differentiation medium was added to each well, and the mixture was incubated at 37℃with 7.5% CO 2 Culture under, change differentiation medium every 2 days.
Experimental example 1: differentiation efficiency experiment of lung precursor cells
The experimental example provides an experiment for differentiation efficiency of lung precursor cells, and the experimental process is as follows:
lung precursor cells were differentiated and cultured by the differentiation method using the differentiation medium described in example 1-1 and comparative example 1-4, and the differentiation was judged by observing the cell morphology using a microscope.
The lung precursor cells were originally grown in an epithelial clonal shape (FIG. 1), and the lung precursor cells differentiated by the differentiation medium of example 1-1 were observed in a microscope for 2 days to observe the cell morphology, and as shown in FIG. 2, the cells originally grown in an epithelial clonal shape were observed to be transformed into an elongated filament-like morphology, forming a lamellar structure similar to the type I alveolar epithelium, and being in a higher differentiated morphology; the lung precursor cells differentiated by the differentiation medium of comparative examples 1-1 and 1-2 were observed in the cell morphology within 7 days under the microscope, as shown in fig. 3-4, and it was observed that most cells still grew in the epithelial clone shape, and a small amount was converted into the elongated filament morphology, forming a lamellar structure resembling the type i alveolar epithelium, and it was seen that the degree of differentiation was not high; the lung precursor cells differentiated by the differentiation medium of comparative examples 1-3 were observed for cell morphology within 5 days under a microscope, as shown in fig. 5, it was observed that most cells were no longer in epithelial clonal growth, but the cells remained in a polygonal morphology of the lung precursor cells, and the apoptotic vacuolated cells were more, only a small amount was converted into an elongated filament morphology, forming a lamellar structure resembling the type i alveolar epithelium; the lung precursor cells differentiated by the differentiation medium of comparative examples 1-4 were observed for cell morphology within 14 days under a microscope, as shown in fig. 6, and most cells were observed to be still in epithelial clonal growth, and a small amount was converted into an elongated filiform morphology, but some cells were formed into a fibroblast morphology, and a marker part of cells were transformed into other morphologies than expected by the epithelial-mesenchymal transition (EMT) process, and differentiation efficiency was very low; compared with comparative examples 1-1 to 1-4, the differentiation medium of example 1-1 saves more than twice the differentiation time, has better effect of specific differentiation in the direction of alveolar epithelium, and has great application prospects for research on respiratory regenerative medicine, drug screening and the like.
Example 2-1: method for evaluating biological efficacy of lung precursor cells
This example provides a method for assessing the biological efficacy of lung precursor cells, as shown in FIG. 7, since HOPX shows a higher amount of protein expression than AQP5 under a fluorescence microscope, the expression level of the marker HOPX of type I alveolar epithelial cells is used as an index for assessing the biological efficacy of lung precursor cells, comprising the steps of:
step one: selecting corning matrigel as base gel, and diluting the corning matrigel to protein concentration of 3mg/mL on ice by using DMEM culture medium; adding 3mL of diluted corning matrigel into a T25 culture flask to completely cover the bottom of the T25 culture flask, incubating the T25 culture flask containing the corning matrigel at 37 ℃ for 15 minutes, and sucking all corning matrigel liquid which is not coated on the T25 culture flask; taking irradiated 3T3 cells as trophoblast cells at a ratio of 3×10 4 Individual cells/cm 2 Is inoculated in the T25 culture flask at 37 ℃ and 7.5 percent CO 2 Culturing for 2 days under the condition; at 10 4 Individual cells/cm 2 Inoculating resuscitated lung precursor cells to cultured trophoblast cells, adding 5mL of lung precursor cell proliferation medium at 37deg.C and 7.5% CO 2 Culturing for 3 days under the condition of (1) to obtain lung precursor cells with cell clone density reaching 60-80%; washing lung precursor cells with a cell clone density of 60-80% by using PBS buffer solution for 1 time, adding 3mL of pancreatin with a concentration of 0.05% (m/v, g/100 mL), incubating at 37 ℃ for 5 minutes, observing that all trophoblast cells fall off and most lung precursor cells are still in an adherent state under a microscope, sucking supernatant and washing by using PBS buffer solution for 1 time, adding 3mL of pancreatin with a concentration of 0.25% (m/v, g/100 mL), incubating at 37 ℃ for 10 minutes, observing all cells fall off under a microscope, adding 3mL of DMEM containing 10% (v/v) serum as a digestion stop solution to stop pancreatin digestion, then centrifuging the cell suspension at 1200rpm for 3 minutes, and collecting cell sediment to obtain lung precursor cell suspension from which the trophoblast cells are removed;
step two: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the above lung precursor cell suspension without trophoblast according to 10 4 Individual cell/well seeding, 37 ℃,7.5% co 2 Culturing for 24 hours under the condition to enable the cells to adhere to the wall; after 24 hours, the original lung precursor cell proliferation medium was aspirated, washed once with PBS buffer, and 1mL of differentiation medium of example 1-1 was added to each well at 37℃with 7.5% CO 2 Continuously culturing for 48 hours under the condition without changing the differentiation medium;
step three: after 48 hours, the original differentiation medium was aspirated, washed 1 time with PBS buffer, 3mL of pancreatin at a concentration of 0.25% (m/v, g/100 mL) was added to each well, incubated at 37℃for 8 minutes, total shedding of cells was observed under a microscope, digestion with pancreatin was terminated by adding 3mL of DMEM containing 10% (v/v) serum as a digestion terminator to each well, and the cell suspension was centrifuged at 1200rpm for 3 minutes, and cell pellet was collected and transferred to a centrifuge tube; mu.L of 3.7% (m/v, g/100 mL) neutral formalin solution was added per tube to resuspend the above cell pellet, the pellet was fixed on a room temperature shaking table at 30rpm for 10 minutes, then the supernatant was removed after centrifugation of the cell suspension at 1200rpm for 3 minutes, 500. Mu.L of 0.2% (v/v) triton X-100 solution was added per tube, left for 10 minutes, the supernatant was removed after centrifugation of the cell suspension at 1200rpm for 3 minutes, 500. Mu.L of 5% (v/v) donkey serum was added, and the pellet was incubated on a room temperature shaking table at 30rpm for 0.5 hours; fully and uniformly mixing the cell suspension added with donkey serum, taking the cell suspension with the volume ratio of 50% and sub-packaging into a new centrifuge tube, setting the tube as a control sample tube, and setting a centrifuge tube in which the rest cells are located as a detection sample tube; centrifuging a control sample tube and a detection sample tube at a speed of 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 antibody working solution into the detection sample tube, incubating for 2 hours at a speed of 30rpm on a room temperature shaking table, and then transferring the incubated sample into 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 out supernatant, adding a proper amount of PBS buffer into each tube to resuspend cell sediment, blowing for several times, and washing cells; centrifuging a control sample tube and a detection sample tube at 1200rpm for 3 minutes, then sucking out supernatant, adding 100 mu L of fluorescent secondary antibody working solution into each tube to resuspend cell sediment, and incubating for 2 hours at room temperature in a dark place; after the incubation is finished, centrifuging the control sample tube and the detection sample tube at 1200rpm for 3 minutes, then sucking out supernatant, adding a proper amount of PBS buffer into each tube to resuspend cell sediment, blowing for several times, and washing cells; repeating the above washing process for 2 times, re-suspending with 1mL PBS buffer solution for the last time, and mixing by blowing for several times; and loading the washed control sample tube and the washed detection sample tube into a fluorescent cell counter for on-machine detection, wherein during detection, the control sample is firstly used for adjusting a threshold value, the number of negative cells of the control sample is confirmed to be more than 95%, then the on-machine detection is carried out on the detection sample, the cell ratio positive to the HOPX marker in the detection sample is obtained, namely the evaluation result of the biological efficacy of the lung precursor cells is obtained, and the detection result is shown in figure 8.
As shown in fig. 8, the percentage of cells positive for HOPX after differentiation (RFP) was 72% of the total detected cells; the differentiated lung precursor cells show a large amount of HOPX protein expression, which shows that the expression level of the marker HOPX of the I-type 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 using the differentiation medium described in the example 1-1 to differentiate the lung precursor cells and then detecting the differentiated lung precursor cells.
Example 2-2: method for evaluating biological efficacy of lung precursor cells
This example provides a method for assessing the biological efficacy of lung precursor cells, as shown in FIG. 7, since HOPX shows a higher amount of protein expression than AQP5 under a fluorescence microscope, the expression level of the marker HOPX of type I alveolar epithelial cells is used as an index for assessing the biological efficacy of lung precursor cells, comprising the steps of:
step one: the corning matrigel is selected as the base gel, and PBS buffer solution is used for preparing the corning matrigel according to the following steps: PBS buffer = 1:5, and then adding the diluted solution into a 6-well plate at a volume ratio of 500 mu L/well so as to completely cover the bottom of the 6-well plate; incubating the 6-well plate containing the corning matrigel at 37 ℃ for 15 minutes, and sucking all corning matrigel liquid which is not coated on the 6-well plate; taking irradiated 3T3 cells as trophoblast cells at a ratio of 2×10 4 Individual cells/cm 2 Is inoculated in 6-well plate at 37 ℃ and 7.5% CO 2 Culturing under the condition; after 12 hours of culture of trophoblast cells, 1.5X10 4 Individual cells/cm 2 Will resuscitate according to the inoculation density of (a)Is inoculated on trophoblast cells, 2mL of lung precursor cell proliferation medium is added, and the temperature is 37 ℃ and 7.5 percent CO 2 Continuously culturing for 2 days under the condition of (2) to obtain lung precursor cells with cell clone density reaching 80% confluence; washing the lung precursor cells with PBS buffer solution until the cell clone density reaches 80% confluence for 1 time, adding 750 mu L of pancreatin with the concentration of 0.05% (m/v, g/100 mL), incubating for 8 minutes at 37 ℃, observing that all trophoblast cells fall off and most lung precursor cells are still in an adherent state under a microscope, sucking the supernatant and washing with PBS buffer solution for 1 time, adding 750 mu L of pancreatin with the concentration of 0.25% (m/v, g/100 mL), incubating for 10 minutes at 37 ℃, observing all cells fall off under a microscope, adding 750 mu L of DMEM with 10% (v/v) serum as digestion stopping solution to stop digestion of pancreatin, and centrifuging the cell suspension at 1200rpm for 5 minutes, and collecting cell sediment to obtain lung precursor cell suspension from which trophoblast cells are removed;
Step two: taking 12-well plate, adding 1mL proliferation culture medium into each well, and mixing the above lung precursor cell suspension without trophoblast at a ratio of 5×10 3 Individual cells/wells were seeded at 37 ℃,7.5% co 2 Culturing for 6 hours under the condition of (2) to enable the cells to adhere to the wall; after 6 hours, the original lung precursor cell proliferation medium was aspirated, washed once with PBS buffer, and 1mL of differentiation medium of example 1-1 was added to each well at 37℃with 7.5% CO 2 Continuously culturing for 72 hours under the condition of not 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 at a concentration of 0.25% (m/v, g/100 mL) was added, incubated at 37℃for 4min, and 750. Mu.L of DMEM containing 10% (v/v) serum was added per well as a digestion stop solution to terminate pancreatin digestion, and the whole was blown several times; subsequently centrifuging the cell suspension at 1200rpm for 3 minutes, collecting cell pellets, and transferring the cell pellets into a centrifuge tube; adding 500 mu L of a fixing buffer per tube to resuspend the cell pellet, fixing the cell pellet on a room temperature shaking table at a speed of 10rpm for 8 minutes, centrifuging the cell suspension at a speed of 1200rpm for 5 minutes, then removing the supernatant by pipetting, adding 500 mu L of 0.2% (v/v) triton X-100 solution per tube, standing for 5 minutes, centrifuging the cell suspension at a speed of 1200rpm for 5 minutes, removing the supernatant by pipetting, adding 500 mu L of 5% (v/v) donkey serum, and incubating at a speed of 10rpm for 0.5 hours on the room temperature shaking table; fully and uniformly mixing the cell suspension added with donkey serum, taking the cell suspension with the volume ratio of 50% and sub-packaging into a new centrifuge tube, setting the tube as a control sample tube, and setting a centrifuge tube in which the rest cells are located as a detection sample tube; centrifuging a control sample tube and a detection sample tube at 1200rpm for 5 minutes, then sucking out supernatant, adding 1mL of PBS buffer into the control sample tube, adding 500 mu L of HOPX primary antibody working solution into the detection sample tube, incubating for 2 hours at 10rpm on a room temperature shaking table, centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, then sucking out supernatant, adding an appropriate amount of PBS buffer into each tube, resuspending cell sediment, blowing for several times, and washing cells; centrifuging a control sample tube and a detection sample tube at 1200rpm for 5 minutes, then sucking out supernatant, adding 500 mu L of fluorescent secondary antibody working solution into each tube to resuspend cell sediment, and incubating for 2 hours at room temperature in a dark place; after the incubation is finished, centrifuging the control sample tube and the detection sample tube at 1200rpm for 5 minutes, then sucking out supernatant, adding a proper amount of PBS buffer into each tube to resuspend cell sediment, blowing for several times, and washing cells; repeating the above washing process for 2 times, re-suspending with 1mL PBS buffer solution for the last time, and mixing by blowing for several times; loading the washed control sample tube and the washed detection sample tube to a flow cytometry analyzer for on-machine detection; during detection, a control sample tube is used for adjusting a threshold value, the negative cell number of the control sample is confirmed to be kept above 98%, then the detection sample is subjected to on-machine detection, and the cell ratio positive to the HOPX marker in the detection sample is obtained, namely the evaluation result of the biological effectiveness of the lung precursor cells, and the detection result is shown in figure 9.
As shown in fig. 9, the percentage of HOPX positive cells after differentiation (FITC-se:Sub>A subset) was 61.9% of total detected cells; the differentiated lung precursor cells show a large amount of HOPX protein expression, which shows that the expression level of the marker HOPX of the I-type 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 using the differentiation medium described in the example 1-1 to differentiate the lung precursor cells and then detecting the differentiated lung precursor cells.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (4)
1. The differentiation medium for the lung precursor cells is characterized in that the components of the differentiation medium consist of a medium matrix and an adjuvant, the components of the medium matrix consist of a DMEM medium and an F12 medium, and the volume ratio of the DMEM medium to the F12 medium is 25-75: 25-75% of a culture medium adjuvant, wherein the culture medium adjuvant comprises the components of L-glutamine, insulin, recombinant epidermal growth factor, recombinant human fibroblast growth factor-10, recombinant human hepatocyte growth factor, adenine, hydrocortisone, retinoic acid, Y-27632, transferrin, albumin and penicillin/streptomycin double antibody, the concentration of L-glutamine in the culture medium matrix is 2-6 mM, the concentration of insulin in the culture medium matrix is 2-15 mug/mL, the concentration of recombinant epidermal growth factor in the culture medium matrix is 0.1-2 mug/mL, the concentration of recombinant human fibroblast growth factor-10 in the culture medium matrix is 25-400 mug/mL, the concentration of adenine in the culture medium matrix is 5-50 mug/mL, the concentration of hydrocortisone in the culture medium matrix is 2-20 mug/mL, the concentration of retinoic acid in the culture medium matrix is 0.1-20 mug/mL, the concentration of albumin in the culture medium matrix is 0.1-0.0% to 0 mug/mL, the concentration of the culture medium is 10-35 mug/mL, the concentration of the recombinant human hepatocyte growth factor in the culture medium matrix is 5-50 mug/mL, the concentration of adenine in the culture medium matrix is 5-50 mug/mL, the concentration of the culture medium matrix is 2-20 mug/mL, the concentration of the serum is 0-0% of the culture medium, the concentration of the serum is 0-0% of the medium and the concentration of the serum is 0-0 to 0% of 50 mug/mL.
2. A method for differentiating and culturing lung precursor cells, wherein the method comprises differentiating and culturing lung precursor cells using the differentiation medium according to claim 1.
3. The differentiation culture method according to claim 2, wherein the differentiation culture method comprises the steps of:
step one: inoculating lung precursor cells into a proliferation culture medium for culturing until the lung precursor cells adhere to the wall;
step two: after the lung precursor cells are attached, sucking away the proliferation medium, adding the differentiation medium as described in claim 1 into the attached lung precursor cells for culturing until the cell morphology is changed into an elongated filament morphology, forming a lamellar structure similar to the type I alveolar epithelium, and completing differentiation.
4. Use of the differentiation medium according to claim 1 or the differentiation culture method according to claim 2 or 3 for drug screening against various acute and chronic degenerative diseases of the respiratory system.
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