CN114525237B - Method for culturing human upper respiratory tract organoid by using nasal polyp tissue - Google Patents

Method for culturing human upper respiratory tract organoid by using nasal polyp tissue Download PDF

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CN114525237B
CN114525237B CN202210015326.6A CN202210015326A CN114525237B CN 114525237 B CN114525237 B CN 114525237B CN 202210015326 A CN202210015326 A CN 202210015326A CN 114525237 B CN114525237 B CN 114525237B
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culture
differentiation
nasal
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CN114525237A (en
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李刚
汪珂
于言
韩日
王伟佳
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Southern Hospital Southern Medical University
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Abstract

The invention provides a method for culturing human upper respiratory tract organoids by using nasal polyp tissues, which comprises the steps of processing the obtained nasal polyp tissues to obtain cell precipitates, and sequentially culturing the cell precipitates in an amplification culture medium and a differentiation culture medium. The invention successfully establishes a nasal mucosa organoid induced differentiation model to form a standard standardized epithelial organoid culture system. The system can make the nasal mucosa primary epithelial cells obtained by nasal polyp continuously expand and passage in vitro, differentiate and develop for more than 60 days, and become upper respiratory tract organoids with long-term proliferation and multi-cell group differentiation capability.

Description

Method for culturing human upper respiratory tract organoid by using nasal polyp tissue
Technical Field
The invention relates to the technical field of organoid culture, in particular to a method for culturing human upper respiratory organoids by using nasal polyp tissues.
Background
A plurality of scholars at home and abroad discuss the method of primary culture of the upper respiratory tract, but the primary culture of the primary cells can not be cultured in vitro for a long time and the differentiation capability of the primary cells can not be maintained. At present, nasal mucosa in vitro models are mainly divided into 2D primary cell models, 2D tumor cell lines and organ explant models. The 2D primary monolayer epithelial cells lack extracellular matrix, and the intercellular connection is interrupted, so that the differentiation and development of the cells are limited, and the cells do not have the three-dimensional structure of organs and tissues in vivo and various cell types; many signaling pathways in 2D tumor cell lines have been altered and cannot be used as a patient-customized study model; however, the genetic variation and uncontrollable environmental variables in the culture process of the organotypic explant model cause poor repeatability, so the model still has great limitation in the research of the physiological functions of nasal mucosa epithelium and related diseases. Organoids refer to in vitro 3D cultured embryonic stem cells, induced pluripotent stem cells, adult stem cells, and can be cultured in vitro for a long period of time while maintaining genomic and proteomic stability by self-renewal of self-tissue to form a culture with morphological structure, function, and genetic characteristics similar to those of the original organ. The core of organoids is the stem cell population, which has the ability to self-organize, to perform a spontaneous ordering of the cell population by regulating cell-cell and cell-extracellular matrix interactions, to mimic to the greatest extent the fundamental processes of organogenesis development, and to reproduce the spatial structure of the source organ and certain specific functions (e.g., excretion, filtration, neural activity, contraction). Since normal nasal mucosa tissue is not readily available, it may involve medical ethical issues, thus resulting in a lag in studies on nasal mucosa organoids. Nasal polyp is extreme edema in nasal mucosa tissue gaps caused by chronic inflammation, single or multiple polyps are formed in the middle nasal passage, the self-renewal and directional differentiation dry potential of basal cells is not damaged, and a wnt/beta-catenin signal channel is activated in a stem cell culture system for removing inflammatory factors and can be self-assembled to form a normal nasal mucosa structure. The invention aims to realize the in-vitro long-term culture of nasal mucosa organoids by using the residual tissue of nasal polyp operation, and provides a new research idea for the research of the nasal mucosa organoids.
Disclosure of Invention
Aiming at the lag and limitation of the current nasal mucosa organoid research technology, the invention provides a method for culturing human upper respiratory tract organoids by using nasal polyp tissues. The technical scheme of the invention is as follows:
a method for culturing human upper respiratory tract organoid by using nasal polyp tissue includes such steps as treating the tissue of nasal polyp to obtain cell deposit, and sequentially culturing the cell deposit in amplification culture medium and differentiation culture medium.
Further, the method specifically comprises the following steps:
step 1, removing mucus on the surface of the obtained nasal polyp tissue, and then placing the tissue in physiological saline containing 2% (v/v) double antibody for repeated oscillation and washing for several times;
step 2, adding the washed tissue into HBSS buffer solution, and processing the tissue into 1-2mm within 15min 3 The tissue fragment of (a);
step 3, adding a digestive juice I into the tissue fragments, digesting for 1-3 h at the constant temperature of 37 ℃, adding an HBSS buffer solution to stop digestion, and centrifuging to retain precipitates;
step 4, adding a digestive juice II into the precipitate obtained in the step 3, digesting at the constant temperature of 37 ℃ for 8-12 min, adding an HBSS buffer solution to stop digestion, centrifuging and retaining the precipitate;
step 5, adding an HBSS buffer solution into the sediment obtained in the step 4 to resuspend the sediment, filtering by using a 100-micron filter screen and centrifuging to obtain a nasal mucosa epithelial cell sediment;
step 6, adding erythrocyte lysate into the nasal mucosa epithelial cell sediment for cracking, then adding HBSS buffer solution to stop cracking, and obtaining cell sediment after centrifugation;
step 7, adding precooled Advanced DMEM/F12 culture medium into the cell sediment, mixing uniformly, adding matrigel, mixing uniformly, inoculating the mixture into a culture dish after forming gel drops, standing, inverting the mixture at the constant temperature of 37 ℃ for a period of time after solidification, and adding amplification culture medium to the mixture, wherein the amplification culture medium is based on the CO content of 5 ℃ at the temperature of 37 ℃ 2 Carrying out amplification culture for 5-7 days under the condition, and replacing the amplification culture medium every 2-3 days;
and 8, adding a differentiation medium into the system after the amplification culture, and continuously culturing for 13-18 d, wherein the differentiation medium is replaced once every other day to obtain the culture medium.
Further, removing mucus on the surface of the nasal polyp tissue obtained in the step 1, namely placing the nasal polyp in 8-12 mMol/L DTT solution for 10-20 min, and then removing residual mucus on the surface by using a sterile medical cotton swab in a dipping way.
Further, the composition of the digestion solution I in the step 3 according to final concentration is as follows: 100-120U/mL hyaluronidase, 200-250U/mL collagenic III and 10-15% FBS.
Further, the composition of the digestive juice II in the step 4 according to final concentration is as follows: 0.1-0.2 mg/mL deoxyriboneclease, 10-15 ng/mLEGF and 10-15% FBS.
Further, the amplification medium comprises, in terms of final concentration composition: b27 without Vit-A, 0.5-2 ×; 1-3mM of N-acetylystein; EGF,50-500ng/mL; noggin,50-500ng/mL; r-spondin1, 50-1000ng/mL; a83-01, 200-500nM; nicotinamide,10-50mM; glutmax,1-5 ×; gastrin,10nM-50nM; penicillin-streptomycin 100 x solution, 1-3 x; SB202190, 10-50. Mu.M; the above components were dissolved in Advanced DMEM/F12 medium.
Preferably, the amplification medium comprises, in terms of final concentration composition: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 x solution, 1 x; SB202190, 10. Mu.M; the above components were dissolved in Advanced DMEM/F12 medium.
Optionally, the amplification medium further comprises: CHIR99021,1-4uM; FGF4, 100-600ng/mL. Preferably: CHIR99021,2uM; FGF4, 200ng/mL.
Further, the differentiation medium comprises, in terms of final concentration composition: b27 without Vit-A, 0.5-2 ×; 1-3mM of N-acetylystein; EGF, 10-100 ng/mL; noggin,50-500ng/mL; r-spondin1, 50-1000ng/mL; a83-01, 200-500nM; nicotinamide,10-50mM; glutmax,1-5 ×; gastrin,10nM-50nM; penicillin-streptomycin 100 x solution, 1-3 x; SB202190, 10-50. Mu.M; insulin, 2-5 mug/mL; hydro-cortisone, 10-50 nM; transferrin, 1-5 μ g/mL; epinepherine, 1-3 μ M; 0.1 to 0.5 percent of bone bridge extract; retinoic acid, 5-20 nM; the above components were dissolved in DMEM/F12 medium.
Preferably, the differentiation medium comprises, in terms of final concentration composition: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; insulin,2.5 mu g/mL; hydro-cortisone,25nM; transferrin, 2.5. Mu.g/mL; epinepherine, 1.5. Mu.M; 0.2% of Bovine serum extract; retinoic acid,15nM; the above components were dissolved in DMEM/F12 medium.
Optionally, the differentiation medium further comprises: dexamethasone, 50-80 nM;8-bromo-cAMP, 0.1-0.2 mM. Preferably, the following components are used: dexamethasone,60nM;8-bromo-cAMP,0.1mM.
Further, in the step 7, pre-cooled Advanced DMEM/F12 culture medium is added to the cell sediment and mixed uniformly, and matrigel is added and mixed uniformly, specifically: added Advanced DMEM/F12 medium and matrixThe amount of gel was determined from the total cell number of the cell pellet and was 10 5 Ratio of total cell mass/10 μ l matrigel and Advanced DMEM/F12 medium/matrigel =1:1.5 (v/v) ratio meter.
The invention utilizes the residual tissue of nasal polyp operation and adopts an 'amplification-differentiation' sectional culture method to successfully culture a large number of nasal mucosa organoids which can be cultured in vitro for a long time and have the proliferation-differentiation function in a short time, thereby proving that the nasal polyp tissue can culture stably-growing upper respiratory tract organoids in an in-vitro 3D environment. Therefore, the invention successfully establishes a nasal mucosa organoid induced differentiation model to form a standard standardized epithelial organoid culture system. The system can make the nasal mucosa primary epithelial cells obtained by nasal polyp continuously expand and passage in vitro, differentiate and develop for more than 60 days, and become upper respiratory tract organoids with long-term proliferation and multi-cell group differentiation capability. In addition, the method has high stability and strong operability, and most of organoids can still keep good biological characteristics after being continuously cultured for more than 2 months, thereby providing an ideal technical platform for the research on the cilia function, the secretion function, the damage repair mechanism, the stem cell activation, the tumorigenesis mechanism and other mechanisms of the upper respiratory tract and the related diseases of the upper respiratory tract.
Drawings
FIG. 1 is a diagram showing the morphological structure of cells cultured for 6 days in the amplification of the cells in example 1 of the present invention.
FIG. 2 is a structural diagram showing the morphology of cells cultured for 15 days in differentiation in example 1 of the present invention.
FIG. 3 is a structural diagram showing the morphology of cells cultured for 6 days in the amplification reaction in example 2 of the present invention.
FIG. 4 is a structural diagram showing the morphology of cells cultured for 15 days in differentiation mode in example 2 of the present invention.
FIG. 5 is a cell morphology map of 6 days of amplification culture in example 3 of the present invention.
FIG. 6 is a structural diagram showing the morphology of cells cultured for 15 days in differentiation mode in example 3 of the present invention.
FIG. 7 is a graph showing the morphological changes of cells from day 1 to the stage of differentiation and development to day 62 in the amplification culture in example 4 of the present invention.
FIG. 8 is a graph showing the change in the morphological structure of cells of comparative example 1 of the present invention at 6 d.
FIG. 9 is a graph showing the change in the morphological structure of cells over the period from day 1 to day 62 of continuous amplification culture in comparative example 2 of the present invention.
Detailed Description
Matrigel used in the examples of the present invention was purchased from Corning, usa.
Insulin used in the examples of the present invention was purchased from STEMCELL, U.S.A., insulin.
The Hydro-cortisone used in the examples of the present invention was purchased from stem cell, usa, hydrocortisone.
Transferrin, used in the examples of the present invention, was purchased from Invitrogen, usa, i.e., interferon.
Epinepherine, used in the examples of the present invention, was purchased from GIBCO, USA, epinephrine.
The Bovine brain extract used in the present invention was obtained from Lonza, switzerland.
In the description of the present invention, it should be further noted that those who do not specify specific conditions in the examples are performed according to conventional conditions or conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The present embodiment provides a method for tissue culture of human upper airway organoids using nasal polyps, comprising the steps of:
(1) Sterile normal saline is rapidly injected into a specimen bottle filled with a specimen after nasal polyp is cut off in an operation, the tissue needs to be submerged by the saline, a sealing film is pasted after a bottle cover is closed, and the specimen bottle is placed in a specimen transport box filled with a plurality of ice bags in advance and is transported to a laboratory at the highest speed. Physiological saline was changed to tissue preservation solution containing RPMI-1640 medium +2% Penicilin-Streptomyces +2% Amphitericin B +1% FBS in biosafety cabinet, taking care that the liquid should be submerged in the tissue, the specimen bottle was stored in 4 ℃ refrigerator, and sent to the laboratory for pretreatment within 4 hours.
(2) The tissue is clamped by an ophthalmic scissors and placed in a 10mMol/LDTT solution for 10min to remove mucus on the surface of the tissue, then a sterile medical cotton swab is used for dipping to remove residual mucus on the surface (the tissue is not required to be rubbed vigorously and repeatedly by the cotton swab, so that cell injury is easy to occur), the tissue is placed in a 15mL centrifuge tube added with 5mL of 2% double-antibody physiological saline in advance, and the shaking and washing are repeated for several times.
(3) Transferring the washed tissue to 6cm culture dish, adding appropriate amount of HBSS buffer solution, placing ice box under the culture dish, and cutting the tissue to 1-2mm size with sterilized ophthalmic scissors 3 The mincing process should not exceed 15 minutes to avoid tissue drying and cell necrosis.
(4) The minced tissue blocks in the petri dish were transferred to a 15mL centrifuge tube using a pipette gun, and the digest I was added to 2-3mL, followed by 2% double antibody. The composition of the digestive juice I according to the final concentration is as follows: 120U/mL hyaluronidase,250U/mL collagenase III,10% FBS. Placing the tube orifice after being pasted with the sealing film in a constant temperature shaking table at 37 ℃ for digesting for about 2 hours, taking out the centrifuge tube every 30 minutes, blowing digested tissues for a plurality of times in a biological safety cabinet by using a pipette gun, and then continuously placing the tube orifice after being pasted with the sealing film in the constant temperature shaking table at 37 ℃.
(5) Adding HBSS buffer solution in equal proportion after the first digestion is finished to stop digestion, centrifuging at 1200r/5min, and removing the supernatant to leave a precipitate.
(6) Adding 2-3mL of digestive enzyme II into the precipitate, blowing, beating and resuspending, wherein the digestive juice II comprises the following components according to the final concentration: 0.1mg/mL deoxyuberucllease, 10ng/mLEGF,10% FBS. Placing the centrifugal tube into a constant temperature shaker at 37 ℃ for digestion for 12min, adding HBSS buffer solution into 1 to stop digestion, and centrifuging at 1200r/5 min.
(7) After centrifugation, the supernatant was discarded, an appropriate amount of HBSS buffer was added to resuspend the digested tissue pellet, and the tissue mixture was filtered through a 100 μm sieve to obtain a filtrate containing nasal mucosal epithelial cells.
(8) Transferring the filtrate into a 15mL centrifuge tube, and centrifuging at 1100r/3min to obtain the nasal mucosa epithelial cell sediment. Then adding 2mL of erythrocyte lysate, adding 2mL of HBSS for stopping after lysis for 5min, finally centrifuging at 1100r/3min, and removing supernatant to obtain cell sediment.
(9) Measuring total cell number with cell counter, performing whole process on ice at low temperature according to 10 5 Total cell count/10 μ l matrigel ratio and Advanced DMEM/F12 medium/matrigel =1:1.5, according to the cell counting result, adding a calculated amount of precooled Advanced DMEM/F12 culture medium, blowing up the cell sediment, uniformly mixing, and dispersing into single cells as far as possible. Then adding calculated amount of matrigel, mixing until no delamination, and paying attention to avoid generating bubbles as much as possible.
(10) The gel drops were inoculated into a 6cm petri dish using a 200. Mu.l pipette tip (200. Mu.l tip already pre-cooled at 4 ℃), each gel drop being approximately 35. Mu.l in size. Standing the culture dish for 2min, observing whether the gel drops flow, if not, placing the culture dish into a constant temperature incubator at 37 ℃, and inverting for 45min. Add approximately 4mL of pre-warmed nasal mucosa expansion organoid medium. The amplification medium comprises the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; the above components were dissolved in Advanced DMEM/F12 medium.
(11) Observing under a mirror, taking a picture, putting into a constant-temperature incubator at 37 ℃, and replacing the amplification culture medium every 2 days. After 6d, the culture medium is changed to a differentiation medium for culture for 15d. The differentiation medium comprises the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 x solution, 1 x; SB202190, 10. Mu.M; insulin,2.5 mu g/mL; hydro-cortisone,25nM; transferrin, 2.5. Mu.g/mL; epinepherine, 1.5. Mu.M; 0.2% of Bovine serum extract; retinoic acid,15nM; the above components were dissolved in DMEM/F12 medium. The differentiation medium was changed every other day, and subcultured depending on organoid growth. During the amplification period (the use period of the amplification culture medium), the nasal mucosa cells are continuously proliferated, the diameters of organoids are gradually increased, and a compact cell mass is formed (fig. 1 is a cell morphology structure diagram after 6 days of amplification culture); in the differentiation phase (differentiation medium use phase), the degree of cell differentiation and cell polarization is increasing, and organoid portions form vacuolated cell masses with orderly arranged cells and cell polarity (fig. 2 is a cell morphology structure diagram of 15 days of differentiation culture).
Example 2
The present embodiment provides a method for tissue culture of human upper airway organoids using nasal polyps, comprising the steps of:
(1) Sterile normal saline is rapidly injected into a specimen bottle filled with a specimen after nasal polyp is cut off in an operation, the tissue needs to be submerged by the saline, a sealing film is pasted after a bottle cover is closed, and the specimen bottle is placed in a specimen transport box filled with a plurality of ice bags in advance and is transported to a laboratory at the highest speed. Physiological saline was changed to tissue preservation solution containing RPMI-1640 medium +2% Penicilin-Streptomyces +2% Amphitericin B +1% FBS in biosafety cabinet, taking care that the liquid should be submerged in the tissue, the specimen bottle was stored in 4 ℃ refrigerator, and sent to the laboratory for pretreatment within 4 hours.
(2) The tissue is picked up by an ophthalmic scissors and placed in a 10mMol/LDTT solution for 10min to remove mucus on the surface of the tissue, then a sterile medical cotton swab is used for dipping to remove residual mucus on the surface (the tissue is not required to be rubbed back and forth by the cotton swab, so that cell damage is caused), the tissue is placed in a 15mL centrifuge tube in which 5mL of 2% double-antibody-containing physiological saline is added in advance, and the tissue is washed by repeated oscillation for several times.
(3) Transferring the washed tissue to 6cm culture dish, adding appropriate amount of HBSS buffer solution, placing ice box under the culture dish, and cutting the tissue to 1-2mm size with sterilized ophthalmic scissors 3 The mincing process should not exceed 15 minutes to avoid tissue drying and cell necrosis.
(4) The minced tissue pieces in the petri dish were transferred to a 15mL centrifuge tube using a pipette gun, and digest I was added to 2-3mL, followed by 2% diabody. The digestive juice I comprises the following components according to final concentration: 100U/mL hyaluronidase,200U/mL collagenase III,15% FBS. Placing the tube orifice after being pasted with the sealing film in a constant temperature shaking table at 37 ℃ for digesting for about 2 hours, taking out the centrifuge tube every 30 minutes, blowing digested tissues for a plurality of times in a biological safety cabinet by using a pipette gun, and then continuously placing the tube orifice after being pasted with the sealing film in the constant temperature shaking table at 37 ℃.
(5) Adding HBSS buffer solution at equal ratio after the first digestion to terminate the digestion, centrifuging at 1200r/5min, discarding the supernatant and retaining the precipitate,
(6) Adding 2-3mL of digestive enzyme II into the precipitate, beating and resuspending, wherein the digestive juice II comprises the following components according to the final concentration: 0.2mg/mL deoxyuberucllease, 15ng/mLEGF,15% FBS. Placing the centrifugal tube into a constant temperature shaker at 37 ℃ for digestion for 10min, adding HBSS buffer solution to stop digestion 1, and centrifuging at 1200r/5 min.
(7) After centrifugation, the supernatant was discarded, an appropriate amount of HBSS buffer was added to resuspend the digested tissue pellet, and the tissue mixture was filtered through a 100 μm filter to obtain a filtrate containing nasal mucosal epithelial cells.
(8) Transferring the filtrate into a 15mL centrifuge tube, and centrifuging at 1100r/3min to obtain the nasal mucosa epithelial cell sediment. Then 2mL of erythrocyte lysate is added, 2mL of HBSS is added after lysis for 5min to stop, finally, centrifugation is carried out at 1100r/3min, and supernatant is discarded to obtain cell sediment.
(9) Total cell number was determined using a cell counter, all the way operating on low temperature ice, according to 105 total cells/10 μ l matrigel ratio and Advanced DMEM/F12 medium/matrigel =1:1.5, according to the cell counting result, adding a calculated amount of precooled Advanced DMEM/F12 culture medium, blowing off cell precipitates, uniformly mixing, and dispersing into single cells as far as possible. Then adding calculated amount of matrigel, mixing until no layering occurs, and paying attention to the condition that bubbles are not generated as much as possible.
(10) The gel drops were inoculated into 6cm petri dishes using a 200. Mu.l pipette (200. Mu.l pipette tip pre-cooled at 4 ℃) and each gel drop was approximately 35. Mu.l in size. And (3) standing the culture dish for 2min, observing whether the gel drops flow, if not, putting the culture dish into a constant-temperature incubator at 37 ℃, and inverting for 45min. Approximately 4mL of pre-warmed nasal mucosa expanded organoid medium was added. The amplification medium comprises the following components according to final concentration: b27, 2X without Vit-A; n-acetylystein, 3mM; EGF,50ng/mL; noggin,500ng/mL; r-spondin1, 50ng/mL; a83-01, 200nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; CHIR99021,1uM; FGF4, 100ng/mL; the above components were dissolved in Advanced DMEM/F12 medium.
(11) Observing under a mirror, taking a picture, putting into a constant-temperature incubator at 37 ℃, and replacing the amplification culture medium every 2 days. After 6d, the culture medium is changed to a differentiation medium for culture for 15d. Differentiation medium comprised Vit-a free B27,2 ×; n-acetylystein, 3mM; EGF,50ng/mL; noggin,500ng/mL; r-spondin1, 50ng/mL; a83-01, 200nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; insulin,2 mu g/mL; hydro-cortisone,10nM; transferrin, 1. Mu.g/mL; epinepherine, 1.5. Mu.M; 0.1% of Bovine serum extract; retinoic acid,15nM; dexamethasone,30nM;8-bromo-cAMP,0.1mM. The above components were dissolved in DMEM/F12 medium. The differentiation medium was changed every other day. During the expansion period (organoid culture medium using period), the nasal mucosa cells continuously proliferate, the organoid diameter gradually increases, and a compact cell mass is formed (fig. 3 is a cell morphology structure diagram after 6 days of expansion culture); in the differentiation phase (differentiation medium use phase), the degree of cell differentiation and cell polarization is increasing, and organoids gradually form vacuolated cell masses with orderly arranged cells and cell polarity (fig. 4 is a cell morphology structure diagram of differentiation culture for 15 days).
Example 3
The present embodiment provides a method for tissue culture of human upper airway organoids using nasal polyps, comprising the steps of:
(1) Sterile normal saline is rapidly injected into a specimen bottle filled with a specimen after nasal polyp is cut off in an operation, the tissue needs to be submerged by the saline, a sealing film is pasted after a bottle cover is closed, and the specimen bottle is placed in a specimen transport box filled with a plurality of ice bags in advance and is transported to a laboratory at the highest speed. Physiological saline was changed to tissue preservation solution containing RPMI-1640 medium +2% Penicilin-Streptomyces +2% Amphitericin B +1% FBS in biosafety cabinet, taking care that the liquid should be submerged in the tissue, the specimen bottle was stored in 4 ℃ refrigerator, and sent to the laboratory for pretreatment within 4 hours.
(2) The tissue is picked up by an ophthalmic scissors and placed in a 10mMol/LDTT solution for 10min to remove mucus on the surface of the tissue, then a sterile medical cotton swab is used for dipping to remove residual mucus on the surface (the tissue is not required to be rubbed back and forth by the cotton swab, so that cell damage is caused), the tissue is placed in a 15mL centrifuge tube in which 5mL of 2% double-antibody-containing physiological saline is added in advance, and the tissue is washed by repeated oscillation for several times.
(3) Transferring the washed tissue to 6cm culture dish, adding appropriate amount of HBSS buffer solution, placing ice box under the culture dish, and cutting the tissue to 1-2mm size with sterilized ophthalmic scissors 3 The mincing process should not exceed 15 minutes to avoid tissue drying and cell necrosis.
(4) The minced tissue pieces in the petri dish were transferred to a 15mL centrifuge tube using a pipette gun, and digest I was added to 2-3mL, followed by 2% diabody. The composition of the digestive juice I according to the final concentration is as follows: 120U/mL hyaluronidase,250U/mL collagenase III,10% FBS. Placing the tube orifice after being pasted with the sealing film in a constant temperature shaking table at 37 ℃ for digesting for about 2 hours, taking out the centrifuge tube every 30 minutes, blowing digested tissues for a plurality of times in a biological safety cabinet by using a pipette gun, and then continuously placing the tube orifice after being pasted with the sealing film in the constant temperature shaking table at 37 ℃.
(5) Adding HBSS buffer solution in equal proportion after the first digestion is finished to stop the digestion, centrifuging at 1200r/5min, and removing the supernatant to leave a precipitate.
(6) Adding 2-3mL of digestive enzyme II into the precipitate, blowing, beating and resuspending, wherein the digestive juice II comprises the following components according to the final concentration: 0.1mg/mL deoxyuberucllease, 10ng/mLEGF,10% FBS. Placing the centrifugal tube into a constant temperature shaker at 37 ℃ for digestion for 12min, adding HBSS buffer solution into the centrifugal tube 1 to stop digestion, and centrifuging at 1200r/5 min.
(7) After centrifugation, the supernatant was discarded, an appropriate amount of HBSS buffer was added to resuspend the digested tissue pellet, and the tissue mixture was filtered through a 100 μm sieve to obtain a filtrate containing nasal mucosal epithelial cells.
(8) Transferring the filtrate into a 15mL centrifuge tube, and centrifuging at 1100r/3min to obtain the nasal mucosa epithelial cell sediment. Then adding 2mL of erythrocyte lysate, adding 2mL of HBSS for stopping after lysis for 5min, finally centrifuging at 1100r/3min, and removing supernatant to obtain cell sediment.
(9) Total cell number was determined using a cytometric instrument, all the time operating on low temperature ice, according to 105 total cell mass/10 μ l matrigel ratio and Advanced DMEM/F12 medium/matrigel =1:1.5, according to the cell counting result, adding a calculated amount of precooled Advanced DMEM/F12 culture medium, blowing off cell precipitates, uniformly mixing, and dispersing into single cells as far as possible. Then adding calculated amount of matrigel, mixing until no delamination, and paying attention to avoid generating bubbles as much as possible.
(10) The gel drops were inoculated into a 6cm petri dish using a 200. Mu.l pipette tip (200. Mu.l tip already pre-cooled at 4 ℃), each gel drop being approximately 35. Mu.l in size. Standing the culture dish for 2min, observing whether the gel drops flow, if not, placing the culture dish into a constant temperature incubator at 37 ℃, and inverting for 45min. Add approximately 4mL of pre-warmed nasal mucosa expansion organoid medium. The amplification medium comprises the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; CHIR99021,2uM; FGF4, 200ng/mL. The above components were dissolved in Advanced DMEM/F12 medium.
(11) Observing under a mirror, taking a picture, putting into a constant-temperature incubator at 37 ℃, and replacing the amplification culture medium every 2 days. After 6d, the culture medium is changed to a differentiation medium for culture for 15d. The differentiation medium comprises the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; insulin,2.5 mu g/mL; hydro-cortisone,25nM; transferrin, 2.5. Mu.g/mL; epinepherine, 1.5. Mu.M; 0.2% of Bovine serum extract; retinoic acid,15nM; dexamethasone,60nM;8-bromo-cAMP,0.1mM. The above components were dissolved in DMEM/F12 medium. The differentiation medium is replaced once every other day, and passage is carried out according to the growth condition of organoid. During the expansion period (organoid culture medium using period), the nasal mucosa cells continuously proliferate, the organoid diameter gradually increases, and a compact cell mass is formed (fig. 5 is a cell morphology structure diagram after 6 days of expansion culture); in the differentiation phase (differentiation medium use phase), the degree of cell differentiation and cell polarization are increased, and organoids gradually form vacuolated cell masses with orderly arranged cells and cell polarity (fig. 6 is a cell morphology structure chart of 15 days of differentiation culture).
Example 4
This example provides a method for tissue culture of human upper respiratory organoids using nasal polyps, and continuous in vitro expansion, passage, differentiation and development for over 60 days, the specific steps of which differ from those of example 1: differentiation culture was continued for 15 days using a differentiation medium, otherwise the same as in example 1. Fig. 7 provides a diagram of the morphological structure change of the cells from the 1 st day to the 62 nd day of the expansion culture of the present embodiment, the development process of the cells is stable and rapid, and the cells still maintain good biological characteristics by the 62 th day, it is obvious that the method can make the nasal mucosa primary epithelial cells obtained by nasal polyp continuously expand and passage in vitro, and the differentiation and development lasts for more than 60 days, and become the upper respiratory tract organoid with both long-term proliferation and multi-cell group differentiation capability.
Comparative example 1
This comparative example provides a method of upper respiratory organoid culture derived from normal nasal mucosal tissue, which differs from example 1 in that: the material was collected from normal nasal mucosa tissue, otherwise as in example 1. FIG. 8 provides a structural diagram of organoid morphology after 6 days of amplification culture, which shows no obvious difference from the organoid of example 1 after 6 days of amplification under a light microscope (FIG. 8), and illustrates that the method of the present invention is also suitable for the culture of nasal mucosa tissue by taking the material.
Comparative example 2
In the comparative example, nasal polyp tissues are cultured only in an amplification mode until the nasal polyp tissues are cultured for 62 days in an amplification mode, and no differentiation medium culture process is performed, other similar to example 1, the culture result is shown in fig. 9, which shows that the organoids obtained by only adopting amplification culture are cystic structures, the development is slow, the number is small, and the structural morphology is incomplete.
In conclusion, the invention can culture stably-grown upper respiratory tract organoids in an in-vitro 3D environment by using the nasal polyp operation residual tissues. Not only establishes a nasal mucosa organoid induced differentiation model, but also forms a standard and standardized epithelial organoid culture system. Moreover, the system can lead the nasal mucosa primary epithelial cells obtained by nasal polyp to be continuously amplified and passaged in vitro, differentiated and developed for more than 60 days, and become the upper respiratory tract organoid with long-term proliferation and multi-cell group differentiation capability.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims (3)

1. A method of tissue culture of human upper respiratory organoids using nasal polyps, comprising: treating the obtained nasal polyp tissue to obtain cell sediment, and sequentially culturing the cell sediment in an amplification culture medium and a differentiation culture medium;
the method specifically comprises the following steps:
step 1, removing mucus on the surface of the obtained nasal polyp tissue, and then placing the tissue in physiological saline containing 2% (v/v) double antibody for repeated oscillation and washing for several times;
step 2, adding the washed tissue into HBSS buffer solution, and processing the tissue into the tissue with the size of 1 to 2mm within 15min 3 The tissue fragment of (a);
step 3, adding a digestive juice I into the tissue fragments, digesting for 1 to 3 hours at the constant temperature of 37 ℃, adding an HBSS buffer solution to stop digestion, and centrifuging to retain precipitates;
step 4, adding a digestive juice II into the precipitate obtained in the step 3, digesting at the constant temperature of 37 ℃ for 8-12min, adding an HBSS buffer solution to stop digestion, centrifuging and then retaining the precipitate;
step 5, adding an HBSS buffer solution into the sediment obtained in the step 4 to resuspend the sediment, filtering by using a 100-micron filter screen and centrifuging to obtain a nasal mucosa epithelial cell sediment;
step 6, adding erythrocyte lysate into the nasal mucosa epithelial cell sediment for cracking, then adding HBSS buffer solution to stop cracking, and obtaining cell sediment after centrifugation;
step 7, adding precooled Advanced DMEM/F12 culture medium into the cell sediment, mixing uniformly, adding matrigel, mixing uniformly, inoculating the mixture into a culture dish after forming gel drops, standing, inverting the mixture at the constant temperature of 37 ℃ for a period of time after solidification, and adding amplification culture medium to the mixture, wherein the amplification culture medium is based on the CO content of 5 ℃ at the temperature of 37 ℃ 2 Carrying out amplification culture for 5 to 7 days under the condition, and changing the amplification culture medium every 2 to 3 days;
step 8, adding a differentiation medium into the system after the amplification culture to continue culturing for 15d, replacing the differentiation medium every other day, and continuously culturing for more than 60 days by using the differentiation medium after 15 days of differentiation culture to obtain the culture medium;
the digestive juice I in the step 3 comprises the following components according to final concentration: 100 to 120U/mL hyaluronidase,200 to 250U/mL collagenic III and 10 to 15% FBS;
the digestive juice II in the step 4 comprises the following components according to final concentration: 0.1 to 0.2mg/mL deoxyubuconase, 10 to 15ng/mLEGF, and 10 to 15% FBS;
the amplification medium consists of the following components according to final concentration: b27 without Vit-A, 0.5-2 ×; 1-3mM of N-acetylystein; EGF,50-500ng/mL; noggin,50-500ng/mL; r-spondin1, 50-1000ng/mL; a83-01, 200-500nM; nicotinamide,10-50mM; glutmax,1-5 ×; gastrin,10nM-50nM; penicillin-streptomycin 100 x solution, 1-3 x; SB202190, 10-50. Mu.M; CHIR99021,1-4uM; FGF4, 100-600ng/mL; the components are dissolved in Advanced DMEM/F12 culture solution;
the differentiation medium consists of the following components according to final concentration: b27 without Vit-A, 0.5-2 ×; 1-3mM of N-acetylystein; EGF,10 to 100ng/mL; noggin,50-500ng/mL; r-spondin1, 50-1000ng/mL; a83-01, 200-500nM; nicotinamide,10-50mM; glutmax,1-5 ×; gastrin,10nM-50nM; penicillin-streptomycin 100 x solution, 1-3 x; SB202190, 10-50. Mu.M; insulin,2 to 5 mu g/mL; hydro-cortisone,10 to 50nM; transferrin,1 to 5 μ g/mL; epinepherine, 1 to 3 μ M; 0.1 to 0.5 percent of Bovine brand extract; retinoic acid,5 to 20nM; dexamehasone, 50 to 80nM;8-bromo-cAMP,0.1 to 0.2mM; the above components were dissolved in DMEM/F12 medium.
2. The method of tissue culture of human upper airway organoids with nasal polyps according to claim 1, wherein: the amplification culture medium comprises the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 × solution, 1 ×; SB202190, 10. Mu.M; the above components were dissolved in Advanced DMEM/F12 medium.
3. The method of claim 1, wherein said tissue culture of human upper airway organoids using nasal polyps comprises: the differentiation medium consists of the following components according to final concentration: b27, 1X without Vit-A; n-acetylystein, 1mM; EGF,50ng/mL; noggin,100ng/mL; r-spondin1, 250ng/mL; a83-01, 500nM; nicotinamide,10mM; glutmax,1 ×; gastrin,10nM; penicillin-streptomycin 100 x solution, 1 x; SB202190, 10. Mu.M; insulin,2.5 mu g/mL; hydro-cortisone,25nM; transferrin, 2.5. Mu.g/mL; epinepherine, 1.5. Mu.M; 0.2% of Bovine serum extract; retinoic acid,15nM; the above components were dissolved in DMEM/F12 medium.
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CN109679915A (en) * 2019-02-27 2019-04-26 南方医科大学南方医院 A kind of culture of nasopharyngeal carcinoma organoid and identification method
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