CN115627251A - Method for constructing in-vitro pulmonary fibrosis organoid model - Google Patents
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Abstract
A method for constructing an in vitro pulmonary fibrosis organoid model comprises the following steps: (1) suspension culture of lung organoids: (2) co-culturing fibroblasts with lung organoids: (3) Constructing a pulmonary fibrosis model and detecting the drug effect of the anti-pulmonary fibrosis drug. The pulmonary fibrosis organoid model constructed by the invention has simple operation method and wide application range, can effectively simulate the pathological characteristics of pulmonary fibrosis and be used for drug detection, and has the following specific characteristics: the suspension culture mode greatly enhances the interaction among lung histiocytes, between lung histiocyte-interstitial cells and between interstitial cells; the suspension culture mode greatly reduces the dosage of matrigel required in the traditional organoid culture and greatly reduces the economic cost of modeling; the pulmonary fibrosis model constructed by the method is simple to operate and short in time period, and a series of indications of pulmonary fibrosis diseases are obviously reproduced.
Description
Technical Field
The invention relates to a method for constructing an in-vitro pulmonary fibrosis organoid model.
Background
Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive interstitial lung disease with unknown etiology, the disease course is mostly chronic and progressive, the lesion mostly occurs under the pleura and at the base, HRCT can see grid-shaped shadow, honeycomb-shaped change or not pulling bronchiectasis, and the Idiopathic Pulmonary Fibrosis (IPF) is better to be developed in middle-aged men. According to statistics, the incidence rate of IPF is 2-29/10 ten thousand, and the IPF is increased by 11% per year, the average survival time of patients is only 3-5 years, and the survival time of 5 years is only 30%. At present, the treatment of the disease cannot effectively reverse or prevent the development of pulmonary fibrosis, and two major treatment drugs recommended in the recommendation guidelines for clinical treatment of idiopathic pulmonary fibrosis: nintedanib and pirfenidone can only delay the disease progression of patients with mild-moderate IPF to a limited extent, and the two drugs have certain adverse reactions.
The development of new drugs for developing IPF through intensive research on the mechanism and treatment of pulmonary fibrosis diseases to promote the development of new drugs is the central focus of the field of treatment of the diseases at present. The construction of in vitro disease models plays a crucial role for the above studies. The traditional new drug research and development process is usually as long as 10-20 years, the success rate, the time period and the capital cost are strictly limited by models, the traditional pulmonary fibrosis model is mostly an animal model (such as a bleomycin-treated mouse model) treated by different compounds, and the model has the limitations of long molding time, large difference, high cost and obvious species difference; although the cell line model cultured in 2D has low cost and greatly simplifies the operation difficulty, the application of the cell line model is also greatly limited because only cells with single components are difficult to restore the real pathological state of the cells in vivo.
Organoids (Organoids) cultured in 3D can combine the advantages of animal and cell models, not only enrich the cell composition in the model, reconstruct part of the structure and function of the tissue, but also greatly reduce the in vitro modeling time and capital cost, effectively overcome a series of problems caused by species differences, and can be efficiently applied to in vitro disease modeling and drug development.
Recently, reports of pulmonary fibrosis disease simulation by using a 3D organoid model are gradually increased, for example, when lung organoids induced by hiPSCs are used for pulmonary fibrosis disease modeling, the disease characterization can be reproduced to a certain extent, but the maturity of the model and normal lung tissues have a certain difference, and the induction period of the method is long (2-3 months); adult stem cell derived lung organoids have brought new hopes for the simulation of lung disease due to the short culture period and the great reduction of the structure and molecular phenotype of donor tissues. The application provides a method for constructing an in vitro pulmonary fibrosis model by utilizing a lung organoid model derived from mouse or human lung adult cells.
Disclosure of Invention
The invention aims to provide a method for constructing an in vitro pulmonary fibrosis organoid model, which co-cultures a lung organoid and fibroblasts by using a suspension and oscillation culture mode, can effectively enhance the interaction among cells, simulate the disease characterization of patients with pulmonary fibrosis, and can be used for drug effect detection and drug screening of anti-pulmonary fibrosis drugs.
The method of the invention comprises the following steps:
1. suspension culture of lung organoids:
(1) culturing lung organoids by using a glue drop embedding mode, sucking a culture medium in a culture dish when the lung organoids grow to be passable, and adding trypLE Express digestive juice; after the glue drops are blown away, digesting;
(2) after the lung organoid is digested into small cell masses, adding a DMEM/F12 culture medium into the small cell masses to stop digestion, and obtaining digested cells;
(3) collecting the digested cells, performing centrifugal operation, removing culture medium supernatant, and performing cell counting to obtain centrifuged cells;
(4) resuspending the centrifuged cells with a lung organoid culture medium according to the cell density of 10-30 ten thousand per milliliter, transferring the cells into a low-adsorption 6-pore plate, and performing suspension culture;
(5) after culturing for 3-5 days, replacing the lung organoid culture medium with a lung organoid culture medium containing TGF-beta 1 or containing bleomycin, and continuously culturing for 2-3 days to obtain a suspension culture lung organoid for subsequent co-culture;
2. co-culturing fibroblasts with lung organoids:
(1) culturing until the adherent growth of cells covers fibroblasts with an area 80-90% of the area of the culture dish, sucking the culture medium away, washing the fibroblasts with PBS, and adding TrypLE Express digestive juice for digestion;
(2) beating the culture dish to separate the digested cells from the bottom of the plate, and adding a fibroblast culture medium with the volume 3-5 times that of the digestion solution into the culture dish to terminate digestion; collecting cells, centrifuging, removing culture medium supernatant, and counting cells to obtain digested fibroblasts;
(3) collecting suspension-cultured lung organoids, centrifuging, removing culture medium supernatant, adding TrypLE Express digestive juice to resuspend the suspension-cultured lung organoids, and adding DMEM/F12 culture medium to stop digestion to obtain secondarily digested lung organoids;
(4) collecting secondarily digested lung organoids, performing centrifugal operation, removing culture medium supernatant, and performing cell counting to obtain digested lung organoids;
(5) co-culturing the digested fibroblasts with the digested lung organoids: resuspending fibroblasts and lung organoid with lung organoid culture medium respectively; transferring the resuspended fibroblasts and the resuspended lung organoids to the same low adsorption culture dish in sequence, supplementing a lung organoid culture medium, adding Matrigel glue into the lung organoid culture medium, continuously carrying out co-culture on the cells for 3-5 days, and observing the co-culture state of the cells to obtain co-culture cells;
3. pulmonary fibrosis model construction and drug effect detection of anti-pulmonary fibrosis drugs:
(1) preparing a pulmonary fibrosis induction culture medium: adding TGF-beta 1 into a lung organoid culture medium;
(2) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 1: adding TGF-beta 1 and nintedanib into the lung organoid culture medium;
(3) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 2: adding TGF-beta 1 and pirfenidone to a lung organoid culture medium;
(4) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 3: adding TGF-beta 1, nintedanib and pirfenidone into a lung organoid culture medium;
(5) by using the fibroblast-lung organoid co-culture system constructed above, 1 blank control group and 4 experimental groups were set: replacing the culture medium in the blank control group with fresh lung organoid culture medium; the culture medium in the experimental group 1 is replaced by a pulmonary fibrosis induction culture medium; the culture medium in the experimental group 2 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 1; the culture medium in the experimental group 3 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 2; the culture medium in the experimental group 4 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 3;
(6) and (5) continuing culturing, and collecting the cultured cells for fibrosis markers identification and anti-pulmonary fibrosis drug effect test.
In the step 1, the culture in the step (1) is carried out for 7 to 10 days; the digestion is to continue digesting for 3 to 10 minutes in an incubator at 37 ℃; 1ml of TrypLE Express digest was added to 1 6cm dish for digestion.
In the step 1, the small cell masses in the step (2) have 1 to 10 cells per mass; when the digestion is stopped, DMEM/F12 culture medium with the volume 3-5 times that of the digestion solution is added.
In the step 1, the centrifugation in the step (3) is carried out at 1000 to 1200rpm for 3 minutes.
In the step 1, the specific suspension culture mode of the step (4) is as follows: adding Matrigel glue with the volume ratio of 0.5-1% into a culture medium, and placing a cell plate in a shaking table of an incubator at 37 ℃, wherein the rotation speed of the shaking table is 80-120 rpm; during cell culture, organoid status was observed every 3 days and lung organoid medium was changed.
In the step 1, the concentration of TGF-beta 1 in the step (5) in the lung organoid culture medium is 20-50 ng/ml, and the concentration of bleomycin in the lung organoid culture medium is 10-30 ug/ml.
In the step 2, when the TrypLE Express digestive juice is added in the step (1), 1ml of TrypLE Express digestive juice is added into 1 vessel with 6 cm; digestion was performed by incubation in an incubator at 37 ℃ for 3 minutes.
In the step 2, the centrifugation in the step (2) is carried out at 1000 to 1200rpm for 3 minutes.
In the step 2, the centrifugation in the step (3) is carried out at 1000 to 1200rpm for 3 minutes.
In the step 2, when the TrypLE Express digestive juice is added in the step 3, the adding amount is 1-2 ml; digesting for 5-10 minutes in an incubator at 37 ℃.
In the step 2, the collection and suspension culture of lung organoids in the step (3) is performed by using a cut 1ml pipette tip.
In the step 2, the volume of the DMEM/F12 medium in the step (3) is 3 to 5 times that of the digestion solution.
In the step 2, the centrifugation in the step (4) is carried out at 1000 to 1200rpm for 3 minutes.
In the step 2, the Matrigel gel of the step (5) is added in an amount of 0.5 to 1% by volume of the lung organoid medium.
In the step 2, when the fibroblast and the lung organoid are co-cultured in the step (5), the cell plate is placed in a shaking table of an incubator at 37 ℃, and the rotation speed of the shaking table is 80-120 rpm.
In the step 2, when the fibroblasts and the lung organoids are co-cultured in the step (5), and the fibroblasts and the lung organoids are transferred to the same low-adsorption culture dish, the cell ratio of the fibroblasts to the lung organoids is (1-2): 1; taking 1 well of a 6-well plate as an example, the total amount of cells is 20 to 50 ten thousand.
In the step 3, 20 to 50ng/ml of TGF-beta 1 is added in the step (1).
In the step 3, 20-50 ng/ml of TGF-beta 1 and 3-8 uM of nintedanib are added in the step (2).
In the step 3, 20-50 ng/ml of TGF-beta 1 and 20-100 ug/ml of pirfenidone are added in the step (3).
In the step 3, the step (4) is added with 20 to 50ng/ml of TGF-beta 1,3 to 8uM of nintedanib and 20 to 100ug/ml of pirfenidone.
In the above step 3, the continuous culture of the step (5) is carried out in an incubator at 37 ℃ for 3 days with shaking.
The pulmonary fibrosis organoid model constructed by the invention has the advantages of simple operation method and wide application range, can effectively simulate the pathological characteristics of pulmonary fibrosis and is used for drug detection, and has the following specific characteristics: the suspension culture mode greatly enhances the interaction among lung histiocytes, between lung histiocyte-interstitial cells and between interstitial cells; the suspension culture mode greatly reduces the dosage of matrigel required in the traditional organoid culture and greatly reduces the economic cost of modeling; the pulmonary fibrosis model constructed by the method is simple to operate and short in time period, and a series of indications of pulmonary fibrosis diseases are obviously reproduced.
Drawings
FIG. 1 is a graph showing the results of photoscopy of murine lung organoids cultured in example 1: left panel, budding lung organoids; right panel, vesicular organoids;
fig. 2 is a light mirror result graph of the murine pulmonary fibrosis model constructed in example 1: left panel, control group lung organoid morphology culture in suspension; right panel, lung organoid-fibroblast coculture after fibrosis induction;
FIG. 3 is a graph showing the results of the a-SMA histochemical assay for murine pulmonary fibrosis model in example 2: left panel, control group suspension culture lung organoid a-SMA staining; right drawing: co-culturing lung organoid-fibroblast, and staining a-SMA after fibrosis induction;
FIG. 4 is a graph of the results of RT-qPCR identification of pulmonary fibrosis marker expression in the murine pulmonary fibrosis model in example 3;
fig. 5 is a light microscope result chart of the mouse pulmonary fibrosis model constructed in example 4 for detecting the drug effect of pulmonary fibrosis: left panel, control group lung organoid morphology culture in suspension; in the middle figure, the lung organoid-fibroblast coculture is in a form after fibrosis induction; right panel: treating pulmonary fibrosis organs with anti-fibrosis drug;
FIG. 6 is a graph of the results of the human lung organoid photomasks cultured in example 5: on the left, human lung organoid morphology was cultured in suspension in control group; right panel, morphology of human lung organoid-human fibroblasts after coculture and induction of fibrosis.
Detailed Description
In the present embodiment, the lung organoid medium is a medium for culturing lung organoids, and comprises DMEM/F12 basal medium, 100 XN 2, 50 XB 27, 1% P/S by volume, 1 to 5mM Glutamax,0.2 to 1. Mu.M monothioglycerol,1 to 10. Mu.M CHIR99021, 100 to 500ng/ml R-spondin-1,1 to 50ng/ml human FGF10,1 to 50ng/ml human KGF,0.05 to 0.3mM 8-bromo-cAMP (cyclic phosphate) and 0.05 to 0.3mM IBMX (3-isobutyl-1-methylxanthine) and 20 to 100nM all-trans-retinic acid.
In the present example, PBS is phosphate-balanced physiological saline.
Example 1
Murine lung organoids were used:
1. suspension culture of lung organoids:
(1) culturing lung organoids by using a glue drop embedding mode, sucking the culture medium in a culture dish when the lung organoids grow to passage after 7 days of culture, and adding TrypLE Express digestive juice; after the glue drops are blown away, continuously digesting for 3 minutes in an incubator at 37 ℃; 1ml of TrypLE Express digestive juice is added into 1 vessel with 6 cm;
(2) after the lung organoid is digested into small cell masses, adding DMEM/F12 culture medium with the volume 3 times that of the digestive juice into the small cell masses to stop digestion, and obtaining digested cells; the small cell masses have 1-10 cells per mass;
(3) collecting the digested cells, centrifuging for 3 minutes at 1000 rpm; then removing culture medium supernatant, and counting cells to obtain centrifuged cells;
(4) suspending the centrifuged cells by using lung organoid culture medium according to the cell density of 10-30 ten thousand per milliliter, transferring the centrifuged cells into a low-adsorption 6-pore plate, and performing suspension culture; during the cell culture, the organoid status was observed every 3 days and the lung organoid medium was changed, and the lung organoid morphology is shown in FIG. 1 (left, budding lung organoids; right, alveolar lung organoids), as can be seen from the figure: the suspension cultured lung organoids have two forms, i.e., bud-like form and vesicle-like form.
(5) Adding 20ug/ml bleomycin into lung organoid culture medium after culturing for 3 days, and continuously culturing for 2 days for 5 days to obtain suspension culture lung organoids; the specific suspension culture mode is as follows: adding 0.5% Matrigel gel into the culture medium, placing the cell plate in a shaking table of an incubator at 37 ℃, wherein the rotation speed of the shaking table is 80rpm;
2. co-culturing fibroblasts with lung organoids:
(1) culturing until the adherent growth of cells covers fibroblasts with an area of 80-90% of the area of the culture dish, sucking the culture medium away, washing the fibroblasts with PBS, adding TrypLE Express digestive juice, incubating for 3 minutes in an incubator at 37 ℃, and adding 1ml of TrypLE Express digestive juice into 1 6cm dish;
(2) beating the culture dish to separate the digested cells from the bottom of the plate, and adding a fibroblast culture medium with the volume 3 times that of the digestion solution into the culture dish to terminate digestion; cells were harvested and centrifuged at 1000rpm for 3 minutes; then removing culture medium supernatant, and counting cells to obtain well digested fibroblasts;
(3) collecting suspension culture lung organoids, centrifuging for 3 minutes at 1000rpm, then removing culture medium supernatant, adding TrypLE Express digestive juice, resuspending the suspension culture lung organoids, digesting for 5 minutes in a 37 ℃ incubator, adding DMEM/F12 culture medium with the volume 3 times of the volume of the digestive juice to stop digestion, and obtaining secondarily digested lung organoids; when TrypLE Express digestive juice is added, the adding amount is 1ml; collecting lung organoids in suspension culture by using a cut 1ml gun head;
(4) collecting secondarily digested lung organoids, centrifuging for 3 minutes at 1000rpm, removing culture medium supernatant, and counting cells to obtain digested lung organoids;
(5) co-culturing the digested fibroblasts with the digested lung organoids: resuspending fibroblasts and lung organoid with lung organoid culture medium respectively; transferring the resuspended fibroblasts and the resuspended lung organoids to the same low-adsorption culture dish in sequence, supplementing a lung organoid culture medium, adding 0.5% Matrigel gel glue into the culture medium, placing the cell plate in a shaking table of a 37 ℃ incubator, wherein the rotation speed of the shaking table is 80rpm; continuously carrying out co-culture for 3 days, and observing the co-culture state of the cells to obtain co-culture cells; when the fibroblast and the lung organs are co-cultured, when the fibroblast and the lung organs are transferred to the same low-adsorption culture dish, the ratio of the fibroblast to the lung organ cells is 2: 1; taking 1 hole of a 6-hole plate as an example, the total amount of the cells is 20-50 ten thousand;
3. constructing a pulmonary fibrosis model:
(1) preparing a pulmonary fibrosis induction culture medium: adding 25ng/ml of TGF-beta 1 into the lung organoid culture medium;
(2) the fibroblast-lung organoid co-culture system constructed above is utilized to set 1 blank control group and 1 experimental group: replacing the culture medium in the blank control group with fresh lung organoid culture medium; replacing the culture medium in the experimental group with a pulmonary fibrosis induction culture medium;
(3) after shaking culture in an incubator at 37 ℃ for 3 days, organoid morphological changes in pulmonary fibrosis models were observed (the results are shown in FIG. 2, the left panel shows the morphology of lung organoids cultured in suspension in the control group; the right panel shows the morphology of lung organoids-fibroblasts after co-culture and induction of fibrosis), as can be seen from the following figures: after a lung organoid-fibroblast coculture system is treated by TGF-beta 1, the form is obviously changed: organoids transform from a vesicular morphology to a solid structure.
Example 2
The identification of the pulmonary fibrosis model constructed in the embodiment 1 is provided, the pulmonary fibrosis organs are subjected to paraffin embedding and slicing, and then the a-SMA immunohistochemical staining observation is carried out, and the specific steps are as follows:
1) Organoid collection and fixation: the mixture was put into a prepared fixing solution (4% formaldehyde fixation) and fixed for 2 hours. Centrifuging at 1200rpm for 5min after fixation is finished, and discarding formalin fixing solution;
2) Gradient dehydration: sequentially immersing the fixed organoids in 85% alcohol, 95% alcohol and 100% alcohol for 30min respectively;
3) Transparent wax dipping: adding xylene, immersing organoid, and treating for 20min twice; then adding paraffin wax, and soaking the paraffin wax for 1.5h at 60 ℃;
4) Embedding the section: wrapping the organoid with an embedding mould, then slicing into sections of 4-6 μm with a slicer, and attaching the sections to an anti-falling glass slide;
5) Baking slices: placing the glass slide on a glass slide frame, placing the glass slide on an oven, and baking water and paraffin on the glass slide for 30min at 65 ℃;
6) Dewaxing: dewaxing with xylene three times for 10min each time; then dipping and washing the fabric with 100 percent alcohol for three times, 1 minute each time; finally, soaking and washing for 1min by using running water;
7) a-SMA dyeing: sealing with 3% hydrogen peroxide at room temperature for 10min, washing with running water for 5min, and repairing antigen; performing primary antibody overnight incubation at 4 ℃, PBS washing, secondary antibody room temperature incubation for 1h, PBS washing, DAB color development, flowing water washing, hematoxylin dyeing for 1min and flowing water washing in sequence;
8) Fixing after dyeing: sequentially soaking in 95% ethanol and 100% ethanol twice for 2min;
9) And (3) transparent and sealing: using dimethylbenzene for transparence for 2min, taking out, airing and sealing with neutral gum;
10 Observation of a-SMA staining under a normal light microscope (results shown in fig. 3: left panel, control group suspension culture lung organoid a-SMA staining; right drawing: lung organoid-fibroblast coculture after fibrosis induction a-SMA staining). As can be seen from the figure: after the lung organoid-fibroblast coculture system is treated by TGF-beta 1, the a-SMA expression is obviously increased.
Example 3
The identification of the pulmonary fibrosis model constructed in the embodiment 1 is provided, and the RT-qPCR detection of pulmonary fibrosis markers is carried out on pulmonary fibrosis organs, and the specific steps are as follows:
pulmonary fibrosis organoids are collected, RNA is extracted, reverse transcription and RT-qPCR experiments are carried out, the expressions of fibrosis markers a-SMA, col1A1 and MMP2 are detected, GAPDH is used as an internal reference (the result is shown in figure 4), and compared with the control organoids, the expression of the markers in the fibrotic lung organoids is obviously increased.
Example 4
Murine lung organoids were used:
1. suspension culture of lung organoids:
(1) culturing lung organoids by using a glue drop embedding mode, sucking a culture medium in a culture dish after culturing for 7 days until the lung organoids grow to be passable, and adding trypLE Express digestive juice; after the glue drops are blown away, continuously digesting for 5 minutes in an incubator at 37 ℃; 1ml of TrypLE Express digestive juice is added into 1 6cm dish;
(2) after the lung organoid is digested into small cell masses, adding DMEM/F12 culture medium with the volume 3 times that of the digestive juice into the small cell masses to stop digestion, and obtaining digested cells; the small cell masses have 1-10 cells per mass;
(3) collecting the digested cells, performing centrifugation operation, and centrifuging for 3 minutes at 1100 rpm; then removing culture medium supernatant, and counting cells to obtain centrifuged cells;
(4) resuspending the centrifuged cells by using a lung organoid culture medium according to the cell density of 10-30 ten thousand per milliliter, transferring the centrifuged cells into a low-adsorption 6-pore plate, and performing suspension culture; in the process of culturing cells, observing the organoid state every 3 days and replacing a lung organoid culture medium; the suspension culture mode is as follows: adding 0.8% Matrigel gel into a culture medium, and placing a cell plate in a shaking table of an incubator at 37 ℃, wherein the rotating speed of the shaking table is 80-120 rpm;
(5) after 5 days of culture, the lung organoid culture medium is replaced by a lung organoid culture medium containing 25ng/ml TGF-beta 1, and the culture is continued for 2 days to obtain a suspension culture lung organoid for subsequent co-culture;
2. co-culturing fibroblasts with lung organoids:
(1) culturing fibroblasts until the coverage area of adherent growth of the cells is 80-90% of the area of a culture dish, sucking a culture medium away, washing the fibroblasts by using PBS, adding trypLE Express digestive juice, and incubating for 3 minutes in an incubator at 37 ℃; when TrypLE Express digestive juice is added, 1ml of TrypLE Express digestive juice is added into 1 6cm dish;
(2) beating the culture dish to separate the digested cells from the bottom of the plate, and adding a fibroblast culture medium with the volume 3 times that of the digestion solution into the culture dish to terminate digestion; cells were harvested and centrifuged at 1100rpm for 3 minutes; then removing culture medium supernatant, and counting cells to obtain well digested fibroblasts;
(3) collecting the suspension culture lung organoids, centrifuging for 3 minutes at 1100rpm, then removing culture medium supernatant, adding TrypLE Express digestive juice to resuspend the suspension culture lung organoids, digesting for 5 minutes in an incubator at 37 ℃, adding DMEM/F12 culture medium with the volume 3 times that of the digestive juice to stop digestion, and obtaining secondarily digested lung organoids; when TrypLE Express digestive juice is added, the adding amount is 2ml; collecting lung organoids in suspension culture by using a cut 1ml gun head;
(4) collecting secondarily digested lung organoids, centrifuging for 3 minutes at 1100rpm, removing culture medium supernatant, and counting cells to obtain digested lung organoids;
(5) co-culturing the digested fibroblasts with the digested lung organoids: resuspending fibroblasts and lung organoid with lung organoid culture medium respectively; transferring the resuspended fibroblasts and the resuspended lung organoids to the same low adsorption culture dish in sequence, supplementing a lung organoid culture medium, adding 1% Matrigel gel glue into the culture medium, and placing the cell plate in a shaking table of a 37 ℃ incubator at the rotating speed of 120rpm; continuously carrying out co-culture for 3 days, and observing the co-culture state of the cells to obtain co-culture cells; when the fibroblast and the lung organs are co-cultured, when the fibroblast and the lung organs are transferred to the same low-adsorption culture dish, the ratio of the fibroblast to the lung organ cells is 2: 1; taking 1 hole of a 6-hole plate as an example, the total amount of the cells is 20-50 ten thousand;
3. constructing a pulmonary fibrosis model:
(1) preparing a pulmonary fibrosis induction culture medium: adding 30ng/ml of TGF-beta 1 into the lung organoid culture medium;
(2) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium: adding 30ng/ml of TGF-beta 1,5uM of nintedanib and 40ug/ml of pirfenidone to the lung organoid medium;
(3) by using the fibroblast-lung organoid co-culture system constructed above, 1 blank control group and 2 experimental groups were set: replacing the culture medium in the blank control group with fresh lung organoid culture medium; the culture medium in the experimental group 1 is replaced by a pulmonary fibrosis induction culture medium; the culture medium in the experimental group 2 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium;
(4) continuously culturing in an incubator at 37 ℃ for 3 days by shaking, and observing the morphological change of the pulmonary fibrosis organoid (the result is shown in figure 5, the left figure shows the morphology of the pulmonary organoid cultured in a suspension way by a control group, the middle figure shows the morphology of the pulmonary organoid-fibroblast coculture after the fibrosis induction, and the right figure shows the morphology of the pulmonary organoid treated by anti-fibrosis drugs); as can be seen from the figure: after the lung organoid-fibroblast coculture system is treated by TGF-beta 1, the morphology is obviously changed: organoids change from a vesicular morphology to a solid structure; after the lung fibrosis organoids are treated by the anti-pulmonary fibrosis drugs, the organoid morphology is restored to a vesicle-shaped structure from a solid structure.
Example 5
Suspension culture of human lung organoids:
1. suspension culture of lung organoids:
(1) culturing lung organoids by using a glue drop embedding mode, sucking a culture medium in a culture dish after culturing for 8 days until the lung organoids grow to be passable, and adding trypLE Express digestive juice; after the glue drops are blown away, continuously digesting for 10 minutes in an incubator at 37 ℃; 1ml of TrypLE Express digestive juice is added into 1 6cm dish;
(2) after the lung organoid is digested into small cell masses, adding DMEM/F12 culture medium with the volume 3 times that of the digestive juice into the small cell masses to stop digestion, and obtaining digested cells; the small cell mass is that each mass has 1-10 cells;
(3) collecting the digested cells, performing centrifugation operation, and centrifuging for 3 minutes at 1200 rpm; then removing culture medium supernatant, and counting cells to obtain centrifuged cells;
(4) resuspending the centrifuged cells with a lung organoid culture medium according to the cell density of 10-30 ten thousand per milliliter, transferring the cells into a low-adsorption 6-pore plate, and performing suspension culture; in the process of culturing cells, observing the organoid state every 3 days and replacing a lung organoid culture medium; the suspension culture mode is as follows: adding 1% Matrigel gel into a culture medium, and placing a cell plate in a shaking table of an incubator at 37 ℃, wherein the rotation speed of the shaking table is 80-120 rpm;
(5) after culturing for 6 days, replacing the lung organoid culture medium with a lung organoid culture medium containing 25ng/ml TGF-beta 1, and continuously culturing for 3 days to obtain a suspension culture lung organoid for subsequent co-culture;
2. co-culturing fibroblasts with lung organoids:
(1) culturing until the adherent growth of cells covers fibroblasts with an area 80-90% of the area of the culture dish, sucking the culture medium away, washing the fibroblasts with PBS, adding trypLE Express digestive juice, and incubating for 3 minutes in an incubator at 37 ℃; when TrypLE Express digestive juice is added, 1ml of TrypLE Express digestive juice is added into 1 6cm dish;
(2) beating the culture dish to separate the digested cells from the bottom of the plate, and adding a fibroblast culture medium with the volume 5 times that of the digestion solution into the culture dish to terminate digestion; cells were harvested and centrifuged at 1200rpm for 3 minutes; then removing culture medium supernatant, and counting cells to obtain well digested fibroblasts;
(3) collecting the suspension culture lung organoids, centrifuging for 3 minutes at 1200rpm, then removing culture medium supernatant, adding TrypLE Express digestive juice to resuspend the suspension culture lung organoids, digesting for 7 minutes in an incubator at 37 ℃, adding DMEM/F12 culture medium with the volume 3 times that of the digestive juice to stop digestion, and obtaining secondarily digested lung organoids; when TrypLE Express digestive juice is added, the adding amount is 2ml; collecting lung organoids in suspension culture by using a cut 1ml gun head;
(4) collecting secondarily digested lung organoids, centrifuging for 3 minutes at 1200rpm, removing culture medium supernatant, and counting cells to obtain digested lung organoids;
(5) co-culturing the digested fibroblasts with the digested lung organoids: resuspending fibroblasts and lung organoid with lung organoid culture medium respectively; transferring the resuspended fibroblasts and the resuspended lung organoids to the same low adsorption culture dish in sequence, supplementing a lung organoid culture medium, adding 1% Matrigel gel glue into the culture medium, and placing the cell plate in a shaking table of a 37 ℃ incubator at the rotating speed of 90rpm; continuously carrying out co-culture for 4 days, and observing the co-culture state of the cells to obtain co-culture cells; when the fibroblasts and the lung organoids are cultured together, the ratio of the fibroblasts to the lung organoids is 2: 1 when the fibroblasts and the lung organoids are transferred to the same low-adsorption culture dish; taking 1 well of a 6-well plate as an example, the total amount of cells is 20 to 50 ten thousand.
3. Constructing a pulmonary fibrosis model:
(1) preparing a pulmonary fibrosis induction culture medium: adding 25ng/ml of TGF-beta 1 into the lung organoid culture medium;
(2) the fibroblast-lung organoid co-culture system constructed above is utilized to set 1 blank control group and 1 experimental group: replacing the culture medium in the blank control group with fresh lung organoid culture medium; replacing the culture medium in the experimental group with a pulmonary fibrosis induction culture medium;
(3) continuously culturing in 37 deg.C incubator for 3 days, observing organoid morphological change in pulmonary fibrosis model (the result is shown in figure 6, left figure, human lung organoid morphology cultured in suspension in control group; right figure, morphology after human lung organoid-human fibroblast coculture and fibrosis induction); as can be seen from the figure: after a human lung organoid-human fibroblast coculture system is treated by TGF-beta 1, the morphology is obviously changed: organoids transform from a vesicular morphology to a solid structure.
Claims (10)
1. A method for constructing an in vitro pulmonary fibrosis organoid model is characterized by comprising the following steps:
(1) Suspension culture of lung organoids:
(1) culturing lung organoids by using a glue drop embedding mode, sucking a culture medium in a culture dish when the lung organoids grow to be passable, and adding trypLE Express digestive juice; after the glue drops are blown away, digesting;
(2) after the lung organoid is digested into small cell masses, adding a DMEM/F12 culture medium into the small cell masses to stop digestion, and obtaining digested cells;
(3) collecting the digested cells, performing centrifugal operation, removing culture medium supernatant, and performing cell counting to obtain centrifuged cells;
(4) resuspending the centrifuged cells with a lung organoid culture medium according to the cell density of 10-30 ten thousand per milliliter, transferring the cells into a low-adsorption 6-pore plate, and performing suspension culture;
(5) after culturing for 3-5 days, replacing the lung organoid culture medium with a lung organoid culture medium containing TGF-beta 1 or containing bleomycin, and continuously culturing for 2-3 days to obtain a suspension culture lung organoid for subsequent co-culture;
(2) Co-culturing fibroblasts with lung organoids:
(1) culturing fibroblasts until the coverage area of adherent growth of the cells is 80-90% of the area of a culture dish, sucking the culture medium away, washing the fibroblasts by PBS, and adding trypLE Express digestive juice for digestion;
(2) beating the culture dish to separate the digested cells from the bottom of the plate, and adding a fibroblast culture medium with the volume 3-5 times that of the digestion solution into the culture dish to terminate digestion; collecting cells, centrifuging, removing culture medium supernatant, and counting cells to obtain digested fibroblasts;
(3) collecting the suspension culture lung organoids, centrifuging, removing the supernatant of the culture medium, adding TrypLE Express digestive juice to resuspend the suspension culture lung organoids, and adding a DMEM/F12 culture medium to stop digestion to obtain secondarily digested lung organoids;
(4) collecting secondarily digested lung organoids, performing centrifugal operation, removing culture medium supernatant, and performing cell counting to obtain digested lung organoids;
(5) co-culturing the digested fibroblasts with the digested lung organoids: resuspending fibroblasts and lung organoid with lung organoid culture medium respectively; transferring the resuspended fibroblasts and the resuspended lung organoids to the same low-adsorption culture dish in sequence, supplementing a lung organoid culture medium, adding Matrigel glue into the lung organoid culture medium, continuously co-culturing the cell plate for 3-5 days, and observing the co-culture state of the cells to obtain co-cultured cells;
(3) Constructing a pulmonary fibrosis model and detecting the drug effect of the anti-pulmonary fibrosis drug;
(1) preparing a pulmonary fibrosis induction culture medium: adding TGF-beta 1 into a lung organoid culture medium;
(2) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 1: adding TGF-beta 1 and nintedanib into the lung organoid culture medium;
(3) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 2: adding TGF-beta 1 and pirfenidone to a lung organoid culture medium;
(4) preparing an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 3: adding TGF-beta 1, nintedanib and pirfenidone into a lung organoid culture medium;
(5) by using the fibroblast-lung organoid co-culture system constructed above, 1 blank control group and 4 experimental groups were set: replacing the culture medium in the blank control group with fresh lung organoid culture medium; the culture medium in the experimental group 1 is replaced by a pulmonary fibrosis induction culture medium; the culture medium in the experimental group 2 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 1; the culture medium in the experimental group 3 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 2; the culture medium in the experimental group 4 is replaced by an anti-pulmonary fibrosis drug-pulmonary fibrosis induction culture medium 3;
(6) and (5) continuing culturing, and collecting the cultured cells for fibrosis markers identification and anti-pulmonary fibrosis drug effect test.
2. The method for constructing the in vitro pulmonary fibrosis organoid model according to claim 1, wherein the organoid used for constructing the pulmonary fibrosis model in step (1) is a suspension-cultured lung organoid, and the culture in step (1) is carried out for 7-10 days; the digestion is carried out in an incubator at 37 ℃ for 3 to 10 minutes; 1ml of TrypLE Express digest was added to 1 6cm dish for digestion.
3. The method for constructing an in vitro pulmonary fibrosis organoid model according to claim 1, wherein in step (1), the small cell masses in step (2) have 1-10 cells per mass; when the digestion is stopped, DMEM/F12 medium with the volume 3-5 times of the volume of the digestion solution is added.
4. The method for constructing in vitro pulmonary fibrosis organoid model according to claim 1, wherein in step (1), the specific suspension culture mode of step (4) is: adding 0.5-1% Matrigel into a culture medium, and placing a cell plate in a shaking table of an incubator at 37 ℃, wherein the rotation speed of the shaking table is 80-120 rpm; during cell culture, organoid status was observed every 3 days and lung organoid medium was changed.
5. The method for constructing an in vitro pulmonary fibrosis organoid model according to claim 1, wherein the lung organoids in step (1) need to be pretreated with TGF- β 1 or bleomycin before co-culturing to cause the organoids to be damaged to some extent, the concentration of TGF- β 1 in the lung organoid medium in step (5) is 20-50 ng/ml, and the concentration of bleomycin in the lung organoid medium is 10-30 ug/ml.
6. The method for constructing in vitro pulmonary fibrosis organoid model according to claim 1, wherein in the step (2), when the TrypLE Express digestive juice is added in the step (1), 1ml of TrypLE Express digestive juice is added in 1 6cm dish; digestion was performed by incubation in an incubator at 37 ℃ for 3 minutes.
7. The method for constructing in vitro pulmonary fibrosis organoid model according to claim 1, wherein in the step (2), the addition amount is 1-2 ml when TrypLE Express digestive juice is added in the step (3); digesting for 5-10 minutes in an incubator at 37 ℃; collecting suspension cultured lung organoids by using a cut 1ml gun head; the volume of the DMEM/F12 culture medium is 3-5 times of the volume of the digestive juice.
8. The method for constructing an in vitro pulmonary fibrosis organoid model according to claim 1, wherein the culture medium for lung organoid-fibroblast coculture in step (2) is a lung organoid culture medium, and the Matrigel gel of step (5) is added in an amount of 0.5 to 1% by volume of the lung organoid culture medium.
9. The method for constructing in vitro pulmonary fibrosis organoid model according to claim 1, wherein in the step (2), when the fibroblast and the lung organoid are co-cultured in the step (5), the cell plate is placed in a shaking table of an incubator at 37 ℃, and the rotation speed of the shaking table is 80-120 rpm; when the fibroblast and the lung organoid are cultured together, when the fibroblast and the lung organoid are transferred to the same low adsorption culture dish, the cell ratio of the fibroblast to the lung organoid is (1-2): 1; taking 1 well of a 6-well plate as an example, the total cell amount is 20 to 50 ten thousand.
10. The method for constructing an in vitro pulmonary fibrosis organoid model according to claim 1, wherein in step (3), the further culture in step (5) is a shaking culture in an incubator at 37 ℃ for 3 days.
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Cited By (2)
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|---|---|---|---|---|
| CN115786244A (en) * | 2023-02-13 | 2023-03-14 | 淇嘉科技(天津)有限公司 | Anti-pulmonary fibrosis drug effect screening process |
| CN115838684A (en) * | 2023-02-13 | 2023-03-24 | 淇嘉科技(天津)有限公司 | In-vitro accurate simulation method for pulmonary fibrosis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876386A (en) * | 2020-08-10 | 2020-11-03 | 上海市第一人民医院 | Method for culturing breast cancer organoid and co-culturing tumor-associated fibroblast |
| CN112852709A (en) * | 2021-01-25 | 2021-05-28 | 南京医科大学 | Method for culturing mouse lung organoid |
| CN114891720A (en) * | 2022-04-25 | 2022-08-12 | 创芯国际生物科技(广州)有限公司 | Method for constructing pulmonary fibrosis in-vitro model |
| CN115094022A (en) * | 2022-05-31 | 2022-09-23 | 创芯国际生物科技(广州)有限公司 | Construction method of lung cancer fibroblast and lung cancer organoid co-culture model |
-
2022
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876386A (en) * | 2020-08-10 | 2020-11-03 | 上海市第一人民医院 | Method for culturing breast cancer organoid and co-culturing tumor-associated fibroblast |
| CN112852709A (en) * | 2021-01-25 | 2021-05-28 | 南京医科大学 | Method for culturing mouse lung organoid |
| CN114891720A (en) * | 2022-04-25 | 2022-08-12 | 创芯国际生物科技(广州)有限公司 | Method for constructing pulmonary fibrosis in-vitro model |
| CN115094022A (en) * | 2022-05-31 | 2022-09-23 | 创芯国际生物科技(广州)有限公司 | Construction method of lung cancer fibroblast and lung cancer organoid co-culture model |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115786244A (en) * | 2023-02-13 | 2023-03-14 | 淇嘉科技(天津)有限公司 | Anti-pulmonary fibrosis drug effect screening process |
| CN115838684A (en) * | 2023-02-13 | 2023-03-24 | 淇嘉科技(天津)有限公司 | In-vitro accurate simulation method for pulmonary fibrosis |
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