CN117757720A - Culture medium and method for efficiently constructing rabbit-derived liver organoids - Google Patents
Culture medium and method for efficiently constructing rabbit-derived liver organoids Download PDFInfo
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Abstract
The invention discloses a culture medium and a method for efficiently constructing rabbit liver organoids. Comprises washing fresh rabbit liver tissue with PBS, and cutting; adding tissue digestion liquid into the tissue fragments for digestion, and collecting suspension and cell/tissue sediment after multiple short-time digestion; centrifuging to collect digested cell/tissue pellet; inoculating tissue cells to a prefabricated matrigel top (On-top), and culturing by using rabbit-derived liver organoid culture medium; transferring to gel drop for embedding culture (suspension method) after 2 days, increasing On-top during culture, and culturing for about 7 days from material collection to organoid culture passage, wherein conventional embedding method is basically 14-20 days, thereby shortening culture period. The success rate of the rabbit source liver organoid culture is over 95 percent, which is obviously improved compared with the success rate of the culture of other animal source liver organoids in the market, and the growth speed of organoids in primary culture is accelerated.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium and a method for efficiently constructing rabbit liver organoids.
Background
Organoids (organoids) are in vitro 3D cultured biological models capable of forming three-dimensional tissue structures, can be used as in vitro models for diagnosis and treatment of various diseases, and have wide application prospects in various aspects such as stem cells and development, regenerative medicine, tumor precise medical treatment, etiology and pathology, drug development and the like. The organoids are used as a new 'model organism', and because the composition, structure and function of the organoids are very close to those of organs in the body, the organoids can provide a brand new tool for exploring the development of human bodies, the occurrence mechanism of diseases, screening high-flux medicines and the like.
The liver is an important metabolic regulation center of human body, and participates in synthesis and decomposition of saccharides, fat and amino acids; as a detoxification center, it can degrade various drugs and molecules such as ethanol; the liver performs exocrine functions by synthesizing and secreting bile salts and bile acids, etc. However, the liver may be simultaneously damaged by a variety of causes including viral infection, excessive drinking, inflammation, metabolic factors, genetic variation and autoimmune changes, and malignant tumors. The incidence and mortality of liver disease is rising year by year, with about 200 tens of thousands of people dying from liver failure worldwide. To date, studies of liver disease have relied primarily on cell lines and animal models. Due to limitations of conventional two-dimensional (2D) cell culture systems, liver parenchymal cells cultured in vitro often lack certain liver-specific genes and specific biological functions, such as maintenance of cytochrome P450, specific connection between cells, etc., especially lack interactions between different types of cells, between cells and extracellular matrix, and thus do not reproduce well the heterogeneity and complex structural features of liver cells. Animal models for liver disease research have advantages over 2D cultured cells including functional vasculature, matrix and immune components, but animal models are limited in their different physiological, pharmaceutical metabolic characteristics and different phenotypes for diseases caused by genetic variation. The liver organoids of small animals such as mice and rats which are mainly used at present have low formation rate and slow growth rate, and the application is limited, so that a method for efficiently constructing liver organoids of large animals such as rabbit sources is urgently needed to be studied.
Disclosure of Invention
Aiming at the problems that the liver animal model is mainly small animals such as mice and rats, has low formation rate and slow growth rate and has limitation in application, the invention provides a method for efficiently constructing liver organs of large animals such as rabbit sources.
The invention firstly provides a culture medium for efficiently constructing rabbit liver organoids, which comprises the following components: wnt3a conditioned medium, R-spondin1 conditioned medium, noggin, HEPES, 1X GlutaMax, penicillin/streptomycin, N cell culture additive, B27 cell culture additive, MEM nonessential amino acid solution (NEAA), nicotinamide, N-acetylcystein, A8301, human EGF, human HGF, human FGF10, gastrin1, forskolin, and advanced DMEM/F12 containing Y27632.
The preferred culture medium of the invention comprises the following components: 40-60% (V/V) Wnt3a conditioned medium, 9-11% (V/V) R-spondin1 conditioned medium, 10-30ng/mL Noggin, 4-6mmol/mL HEPES, 1 XGlutaMax, 1 Xpenicillin/streptomycin, 1-2% (V/V) N2 cell culture additive, 1-2% (V/V) B27 cell culture additive, 1-2% (V/V) MEM nonessential amino acid solution (NEAA), 1-3mmol/mL icotinamide, 1-2mmol/mL N-acetylcysteine, 4-6umol/mlA8301, 40-60ng/mL human EGF, 40-60ng/mL human HGF, 40-60ng/mL human FGF10, 9-11nmol/mL gamin 1, 9-11umol/mL Forskolin, DMEM 12 containing 9-11 umol/Y27vance EM.
Further preferred media of the invention comprise the following components: 50% (V/V) Wnt3a conditioned medium, 10% (V/V) R-spondin1 conditioned medium, 20ng/mL Noggin, 5mmol/mL HEPES, 1 XGlutaMax, 1 Xpeniilin/streptomyin, 1% (V/V) N2 cell culture additive, 1% (V/V) B27 cell culture additive, 1% (V/V) MEM nonessential amino acid solution (NEAA), 2mmol/mL icotinamide, 1mmol/mL N-acetylcysteine, 5umol/mlA8301, 50ng/mL human EGF, 50ng/mL human HGF, 50ng/mL human FGF10, 10nmol/mL Gastin 1, 10umol/mL 27. Mu.mol/mL Forskolin, and advance EM/F12 containing 10umol/mL Y632.
The invention further provides a method for efficiently constructing rabbit liver organoids, which comprises the following steps:
step one: digestion and separation of rabbit liver tissue:
step two: inoculating and culturing rabbit liver tissue organoids;
step three: subculturing rabbit liver tissue organoids;
the step of inoculating and culturing the rabbit liver tissue organoid comprises the following steps:
1) Cell inoculation: adding Advanced DMEM/F12 (containing 1%V/V penicillin-streptomycin and gentamicin) culture solution into the separated primary cells, uniformly mixing, and centrifuging; discarding the supernatant, re-suspending the collected cell/tissue sediment with the rabbit liver organoid culture medium according to any one of claims 1-3, adding the suspension into the top of the prefabricated matrigel, and naturally dispersing the suspension to obtain cell clusters;
2) Organoid culture-On-top method: inoculating the cell mass into a prefabricated matrigel, placing the matrigel in an environment with 37 ℃ and carbon dioxide content of 5% for culturing for 3-5 days, changing liquid every 2-3 days in the process, and when a culture medium is added, a suction head faces to the side wall to avoid damaging the matrigel;
3) Organoid culture-embedding method: transferring the cultured organoids into gel drops for embedding culture, sucking the culture supernatant, adding 3mL of DISPASE II (disperse enzyme II) into each hole, blowing off the gel drops by using a Pasteur dropper, incubating for 15-20 minutes at 37 ℃, collecting suspension, and blowing for 10-20 times by using a 1mL suction head; centrifuging at 4deg.C under 250g for 5min, removing supernatant, adding diluted matrigel into cell precipitate, and mixing, wherein the volume ratio of rabbit source organoid culture medium to matrigel is 1:2; inoculating into cell culture plate according to volume of 25-40 μl per drop, placing the culture plate in CO2 incubator at 37deg.C, heating for 30min, taking out the culture plate after colloid is solidified, adding preheated organoid culture solution 600 μl/hole, adding appropriate amount of sterile PBS or double distilled water into peripheral holes, reducing culture solution evaporation, changing culture solution for 3-4 days, and adding culture solution with suction head facing side wall to avoid damaging matrigel.
Step one, digestion and separation of rabbit liver tissue comprises the following steps:
1) Tissue cleaning: taking fresh rabbit source tissue, placing the fresh rabbit source tissue into a centrifuge tube, adding 10 times of PBS (phosphate buffered saline) for shaking and cleaning, and discarding the supernatant;
2) Tissue cutting: transferring the cleaned tissue into a 10cm culture dish, and cutting the tissue into small pieces of 0.5mm3 by using a surgical knife;
3) Tissue digestion: placing the tissue fragments into a centrifuge tube, adding at least 5 times of tissue digestion liquid, and performing digestion treatment at 200rpm at 37 ℃ in a constant-temperature shaking table; observing while digesting, and digesting by using a short-time and multiple-time method, wherein the time of each digestion is controlled to be 3-5 minutes, and repeating the steps until most of tissues are digested, and only a small amount of tissue fragments are left;
4) Cell collection: collecting the upper suspension by natural sedimentation, adding a large proportion of PBS, placing on ice for termination, centrifuging at 4 ℃ and 300g for 5 minutes, centrifuging to remove the supernatant, collecting digested cell/tissue sediment, and re-suspending cells by using sterile PBS to prepare a cell suspension;
5) Cell sieving: filtering the cell suspension with a 100 μm sieve, filtering the cell suspension into a new 15ml centrifuge tube, and washing the sieve with sterile PBS;
6) Single cell resuspension: the centrifuge tube containing the cell filtrate was centrifuged at 4℃for 300g for 5min, the supernatant was carefully removed, and an appropriate amount of Advanced DMEM/F12 (Dulbecco' sModified Eagle Medium/Nutrient Mixture F-12) (containing 1%V/V penicillin-streptomycin and gentamicin, product name: penicillin-streptomycin-gentamicin mixed solution (100 Xthree antibodies, alias: three antibodies)) was added to the cell pellet, gently beaten into a single cell suspension,
7) Lysing erythrocytes: if more red blood cells are observed in the cell sediment, the following steps of red blood cell lysis are carried out, after centrifugation, supernatant is removed, 1ml of red blood cell lysate is added to resuspend the cell sediment, the mixture is blown and uniformly mixed, the mixture is placed on a shaking table at 4 ℃,120rpm is carried out, 5min is oscillated, the temperature is 300g, the mixture is centrifuged for 5min, supernatant is removed, and 10ml of Advanced DMEM/F12 (containing 1%V/V penicillin-streptomycin and gentamicin) is added to resuspend the cell sediment to obtain primary cells.
Step three, subculturing rabbit source liver tissue organoids comprises the following steps:
1) And (3) observation: when the diameter of the organoid reaches 200-300 mu m, the organoid can be passaged under the observation under the mirror, and the organoid can be passaged generally every 7-14 days;
2) Cleaning: removing the culture solution, adding 1ml of PBS, and cleaning for 1 time, wherein the matrigel is not damaged;
3) And (3) glue melting: removing the old culture medium, adding 3mL of DISPASE II (disperse enzyme II) into a pore plate, blowing off Matrigel, transferring the organoid mixture to a 15mL centrifuge tube, digesting for 15-20min on a constant temperature oscillator at 37 ℃, observing the organoid state under a microscope, and centrifugally collecting;
4) Digestion: the supernatant was discarded and TrypLE was added to the centrifuge tube TM Dispersing matrigel by using a pipettor, digesting for 3-5 min on a constant temperature oscillator at 37 ℃, observing under a mirror until the organoid is digested into small cell mass, adding 1ml of Advanced DMEM/F12 culture medium to terminate digestion, transferring cells to a 15ml centrifuge tube, centrifuging for 5min at 4 ℃ and 300g, and removing supernatant;
5) Cleaning: removing supernatant, stopping digestion of Advanced DMEM/F12 medium, transferring cells to a 15ml centrifuge tube, centrifuging at 4 ℃ for 5min at 300g, removing supernatant, washing, centrifuging and collecting cells;
6) And (3) seed glue: adding Matrigel according to the amount of precipitated cells, fully and uniformly mixing, planting in a 24-hole plate, and after the completion of the plating, inversely placing the culture plate in an incubator for 30min, solidifying the Matrigel, taking out the culture plate, adding 600 mu l/hole of preheated rabbit source liver organoid culture solution, adding a proper amount of sterile PBS or double distilled water into peripheral holes, and reducing the evaporation of a culture medium;
7) And (3) liquid adding: adding corresponding organoid culture medium, observing the density and state of cells after passage under a microscope, placing the culture dish into a CO2 incubator at 37 ℃ for continuous culture, replacing the culture medium for 3-4 days, and when the culture medium is added, the suction head faces the side wall to avoid damaging matrigel.
In the cell inoculation step, the prefabricated matrigel is prepared by mixing matrigel and advanced DMEM/F12 according to a volume ratio of 2:1, adding the mixture into a 6-hole plate, and incubating at 37 ℃ to solidify the mixture to form a gel layer with the thickness of 1 mm.
The rabbit liver tissue organoid culture can realize the autonomous assembly of cells in the early stage, the recovery of the signal transduction among the cells, antagonism damage and activation of apoptosis related signal paths caused by the loss of nest, so that more cells survive and are assembled to form organoids, the step of increasing On-top in the culture process is carried out, the time from the material taking to the organoid culture passage is about 7 days, and the conventional embedding method is basically about 14-20 days, thereby shortening the culture period and obviously improving the application feasibility. On the other hand, the success rate of the rabbit source liver organoid culture is over 95 percent by using a company self-grinding culture medium, which is obviously improved compared with the 70 percent culture success rate of other animal source liver organoids in the market, and the growth speed of organoids in primary culture is accelerated. The rabbit source liver in-vitro organoid research model is established, so that the rabbit source liver in-vitro organoid research model can be differentiated into cell types specific to the source organs, is a physiological high-correlation system, and has application value compared with the characteristics of single function, high animal model cost and long period of a cell line model. The liver organoid model obtained by the method is applied to pharmacological, pharmacodynamic and toxicological analysis.
Drawings
FIG. 1 is a photomicrograph of rabbit liver organoids grown on days 1-9.
FIG. 2 is a statistical plot of the number of organoids formed in 7 days of culture using the medium of the invention as compared to other small animal liver organoids.
FIG. 3 is a statistical plot of organoids formed using the method of the present invention versus conventional embedding.
FIG. 4 is a graph showing the morphology of the organoids cultured on day 7 according to example 2 of the present invention and comparative example 3.
FIG. 5 is a graph showing the comparison of the number of organoids formed on day 7 in the culture of example 2 of the present invention with that of control 3.
FIG. 6 is a graph showing a comparison of diameters formed on day 7 of organoids cultured in example 2 of the present invention and in comparative example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description. It should be understood that the detailed description is presented merely to illustrate the invention, and is not intended to limit the invention.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
The sources of the products used in the embodiment of the invention are as follows:
rabbit source tissue cleaning liquid: 1 XPBS (containing 1-2 xpencilin/streptomycin diabodies) (Thermo, 15140122);
rabbit tissue digest: 200-500 μg/mL Collagenase II (Sigma, C-6885), 10-20 μg/mL DNAse I (Soxhaust, D8071) and 1 Xpenicilin/streptomycin (Thermo, 15140122) of advanced DMEM/F12 medium;
rabbit-derived organoid medium: wnt3a conditioned Medium (ATCC, CRL-2647), R-spondin1 conditioned Medium3710-001-01), noggin (Peprotech, 120-10 c), HEPES (Thermo, 15630080), 1 XGlutaMax (Thermo, 35050061), 1 Xpenicillin/streptomycin (Thermo, 15140122), N2 (Gibco, 17502048), B27 (Gibco, 17504044), MEM non-essential amino acid solution (NEAA) (Procell/pramoxine, PB 180424), nicotinamide (Sigma, N0636), N-acetylcysteine (Sigma, A9165), 5umol/mlA8301 (MCE, HY-10432), human EGF (Peprotech, AF-100-15), human HGF (Peprotech, 100-39), human FGF10 (Peprotech, 100-26), gastin 1 (MCE, HY-P1097), forskolin (MCE, 15371), DMEM containing 10/Y (MCE, A91632-Y) (MCE, HY-78) and DMEM (100-78).
Organoid digests:
1x TrypLE TM express enzyme (1X), phenol red (Gibco)，12605028)
The rabbit organ comes from the donation of a scientific research institution.
Example 1
Preparing a rabbit-derived liver organoid medium comprising the following components: 50% (V/V) Wnt3a conditioned medium, 10% (V/V) R-spondin1 conditioned medium, 20ng/mL Noggin, 5mmol/mL HEPES, 1 XGlutaMax, 1 Xpeniilin/streptomyin, 1% (V/V) N2 cell culture additive, 1% (V/V) B27 cell culture additive, 1% (V/V) MEM nonessential amino acid solution (NEAA), 2mmol/mL icotinamide, 1mmol/mL N-acetylcysteine, 5umol/mlA8301, 50ng/mL human EGF, 50ng/mL human HGF, 50ng/mL human FGF10, 10nmol/mL Gastin 1, 10umol/mL 27. Mu.mol/mL Forskolin, and advance EM/F12 containing 10umol/mL Y632.
Example 2
A method for efficiently constructing rabbit liver organoids, comprising the following steps:
step one: digestion and separation of rabbit liver tissue:
1) Tissue cleaning: taking fresh rabbit source tissue, placing the fresh rabbit source tissue into a centrifuge tube, adding 10 times of PBS (phosphate buffered saline) for shaking and cleaning, and discarding the supernatant;
2) Tissue cutting: the washed tissue was transferred to a 10cm dish and minced to 0.5mm using a scalpel 3 Is a small block of (2);
3) Tissue digestion: placing the tissue fragments into a centrifuge tube, adding at least 5 times of tissue digestion liquid, and performing digestion treatment at 200rpm at 37 ℃ in a constant-temperature shaking table; observing while digesting, and digesting by using a short-time and multiple-time method, wherein the time of each digestion is controlled to be 3-5 minutes, and repeating the steps until most of tissues are digested, and only a small amount of tissue fragments are left;
4) Cell collection: the suspension was collected by natural sedimentation and stopped by adding a large proportion of PBS on ice, centrifugation was performed for 5 minutes at 4℃and 300g, the supernatant was removed by centrifugation, the digested cell/tissue pellet was collected and the cells were resuspended using sterile PBS to make a cell suspension.
5) Cell sieving: filtering the cell suspension with a 100 μm sieve, filtering the cell suspension into a new 15ml centrifuge tube, and washing the sieve with sterile PBS;
6) Single cell resuspension: the above centrifuge tube containing the cell filtrate was centrifuged at 300g at 4℃for 5min and the supernatant carefully removed. Adding appropriate amount of Advanced DMEM/F12 (Dulbecco' sModified Eagle Medium/Nutrient Mixture F-12) (containing 1%V/V penicillin-streptomycin and gentamicin) into the cell pellet, and gently beating to prepare single cell suspension;
7) Lysing erythrocytes: if more erythrocytes are observed in the cell pellet, the following erythrocyte lysis step is performed. After centrifugation, the supernatant was removed, 1ml of red blood cell lysate was added to resuspend the cell pellet, the mixture was blown and mixed well, the mixture was placed on a 4℃shaking table at 120rpm, shaking was performed for 5min,4℃and 300g, and centrifugation was performed for 5min, the supernatant was removed, and 10ml of Advanced DMEM/F12 (containing 1%V/V penicillin-streptomycin and gentamicin) was added to resuspend the cell pellet.
Step two: inoculating and culturing rabbit liver tissue organoids;
1) Cell inoculation: adding culture solution of Advanced DMEM/F12 (containing 1%V/V penicillin-streptomycin and gentamicin) into the separated primary cells, mixing, centrifuging at 4 ℃ for 300g and 5 min; discarding the supernatant, re-suspending the collected cell/tissue sediment with rabbit liver organoid culture medium (determining matrigel amount according to the number of living cells obtained by separation, dividing the living cell amount after trypan blue staining by 5 as stem cell number, and planting one gel drop in each 5000 stem cells), adding the prefabricated matrigel top to naturally disperse; the prefabricated matrigel is prepared by mixing matrigel and advanced DMEM/F12 according to a volume ratio of 2:1, adding the mixture into a 6-hole plate, and incubating at 37 ℃ to solidify the mixture to form a gel layer with the thickness of 1 mm;
2) Organoid culture-On-top method: inoculating the cell mass into a matrigel prepared by a 6-pore plate, culturing for 3-5 days in an environment with 37 ℃ and carbon dioxide content of 5%, changing liquid every 2-3 days in the process, and adding a culture medium with a suction head facing the side wall to avoid damaging the matrigel;
3) Organoid culture-embedding method: transferring the cultured organoids into gel drops for embedding culture, specifically sucking the culture supernatant, adding 3mL of DISPASE II (disperse enzyme II) into each hole, blowing off the gel drops by using a Pasteur dropper, incubating for 15-20 minutes at 37 ℃, collecting suspension, and blowing for 10-20 times by using a 1mL suction head; centrifuging at 4deg.C under 250g for 5min, removing supernatant, adding diluted matrigel into cell precipitate, and mixing, wherein the volume ratio of rabbit source organoid culture medium to matrigel is 1:2; inoculating into cell culture plate according to volume of 25-40 μl per drop, placing the culture plate in CO2 incubator at 37deg.C, heating for 30min, taking out the culture plate after colloid is solidified, and adding 600 μl/well of preheated organoid culture solution. Proper amount of sterile PBS or double distilled water is added into the peripheral holes, so that the evaporation of the culture solution is reduced. The culture medium was changed for 3-4 days. When the culture solution is added, the suction head faces to the side wall, so that the matrigel is prevented from being damaged.
Step three: subculturing rabbit liver tissue organoids;
1) And (3) observation: when the organoids grow to a certain number and size, the organoids can be passaged: when the diameter of the organoid reaches 200-300 mu m, the organoid can be passaged under the observation under the mirror, and the organoid can be passaged generally every 7-14 days;
2) Cleaning: removing the culture solution, adding 1ml of PBS, and cleaning for 1 time, wherein the matrigel is not damaged;
3) And (3) glue melting: removing the old culture medium, adding 3mL of DISPASE II (disperse enzyme II) into a pore plate, blowing off Matrigel, transferring the organoid mixture to a 15mL centrifuge tube, digesting for 15-20min on a constant temperature oscillator at 37 ℃, observing the organoid state under a microscope, and centrifugally collecting;
4) Digestion: discarding supernatant, adding proper amount of TrypLE into centrifuge tube TM Express Enzyme (24 well plate 800-1000. Mu.l), matrigel was dispersed with a pipette. Digestion is carried out on a constant temperature oscillator at 37 ℃ for 3-5 min, and observation is carried out under a lens until the organoid is digested into small cell clusters (3-8 cells) under the lens. Adding 1ml of Advanced DMEM/F12 culture medium to stop digestion, transferring the cells to a 15ml centrifuge tube, centrifuging at 4 ℃ for 5min at 300g, and removing the supernatant;
5) Cleaning: removing supernatant, stopping digestion of Advanced DMEM/F12 medium, transferring cells to a 15ml centrifuge tube, centrifuging at 4 ℃ for 5min at 300g, removing supernatant, washing, centrifuging and collecting cells;
6) And (3) seed glue: according to the amount of the precipitated cells, a proper amount of Matrigel is added, fully mixed (a large amount of bubbles are avoided from being generated by blowing) and planted in a 24-well plate, and 40 mu l/well. After the completion of the plating, the culture plate is placed in an incubator upside down for 30min, and the glue can be solidified. The plates were removed and 600. Mu.l/well of pre-heated rabbit liver organoid culture medium was added. Adding a proper amount of sterile PBS or double distilled water into the peripheral holes, and reducing the evaporation of the culture medium;
7) And (3) liquid adding: adding corresponding organoid culture medium, observing the density and state of cells after passage under a mirror, and placing the culture dish into a CO2 incubator at 37 ℃ for continuous culture. The medium was changed for 3-4 days. When the culture medium is added, the suction head faces to the side wall, so that matrigel is prevented from being damaged.
Comparative example 1
Description: before performing the experiment, the digestion reagent, the culture solution and PBS were left to warm at room temperature, matrigel was thawed overnight in a refrigerator at 4deg.C, and the tip in contact with Matrigel was frozen at-20deg.C for 1 hour using the culture medium Hepaticult TM Organoid Growth Medium(Mouse)/HepatiCult TM Liver organoid growth medium (mouse) brand: STEMCELL Technologies/catalyst #060301Kit.
1. Treatment of tissue:
1) Tissue cleaning
In an ultra clean bench, the tissue of genus source was taken out from the primary tissue preservation solution, placed in a 100mm sterile petri dish, and aspirated and washed 3 times with PBS containing double antibody (for tissue with more bacteria, repeated washing is required).
2) Mechanical separation of tissue
Mechanically separating the specimen using sterile scissors and a scalpel to separate the bulk tissue into approximately 1mm 3 Fine pieces or paste-like.
3) Tissue digestion
The mechanically digested tissue was completely transferred into a 15ml sterile centrifuge tube according to 1:4, adding a proper amount of tissue digestion liquid, lightly blowing the tissue with a 1ml gun head, and fully dispersing the tissue; the centrifuge tube containing the digestion solution was placed on a shaking table of a constant temperature air bath and digested and separated for 30min at 37℃at 120 rpm. Each 5min in the middle was removed, the separation effect was observed under a mirror until most of the tissue was stopped after separation and digestion, and stopped with sterile PBS.
4) The cell suspension was filtered through a 100 μm sieve, the cell suspension was filtered into a new 15ml centrifuge tube, and the sieve was washed with sterile PBS.
5) The above centrifuge tube containing the cell filtrate was centrifuged at 1200g at 4℃for 5min, and the supernatant was carefully removed. Washing the precipitated cells with PBS once; adding a proper amount of cell culture solution into the cell sediment, and lightly blowing to prepare single-cell suspension.
2. Organoid inoculation culture
1) Adding Advanced DMEM/F12 culture solution into the separated primary cells, uniformly mixing, centrifuging at the temperature of 300g and 5min and at the temperature of 4 ℃; the supernatant was discarded and approximately 100. Mu.L of liquid was retained.
2) The tube was placed in ice and cooled for 1min, 100 μl of pre-melted matrigel (the amount of matrigel was determined based on the number of living cells isolated, the amount of living cells after trypan blue staining was generally divided by 5 as the number of stem cells, one droplet per 5000 stem cells) was added to the tube, and matrigel and cell pellet were slowly mixed with a pre-chilled gun head.
3) A mixture of the cell suspension and Matrigel was dropped on a pre-heated 24-well low-adsorption culture plate at about 40. Mu.l per drop. The culture plate was placed at 37℃CO 2 The incubator was heated for 30min, after which the plates were removed and 600 μl/well of pre-heated liver organoid growth medium (mice) was added. Proper amount of sterile PBS or double distilled water is added into the peripheral holes, so that the evaporation of the culture solution is reduced.
4) And replacing the culture solution for 3-4 days. When the culture solution is added, the suction head faces to the side wall, so that the matrigel is prevented from being damaged.
The number of organoids formed after 7 days of culture was significantly greater than that of control 1 in example 2 of the present invention, as compared to example 2 of rabbit liver organoids formed in comparison to that of FIG. 2.
Comparative example 2
Referring to the culture method of example 2, in which the organoid culture-On-top method was omitted in the second step, only organoid culture-embedding method was used for culture.
The organoid formation diameter was measured and compared with the organoid formation diameter pair of example 2 such as shown in FIG. 3, the organoid formation diameter of the culture method of the present invention was much larger than that of comparative example 2.
Comparative example 3
Using the same culture method as in example 2, in which the rabbit-derived organoid medium was replaced with the liver organoid growth medium of comparative example 1, the rabbit-derived liver organoids were cultured and observed, and the organoids were compared with those cultured in example 2, the morphology of the organoids was shown in FIG. 4, the number of organoids formed was shown in FIG. 5, and the diameters of the organoids formed were shown in FIG. 6, as can be seen from FIGS. 4 to 6, and the organoids cultured in example 2 were significantly superior to those cultured in comparative example 3, both in terms of the morphology of the organoids, the number of organoids formed, and the diameters of the organoids formed.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (7)
1. A culture medium for efficiently constructing rabbit liver organoids is characterized by comprising the following components: wnt3a conditioned medium, R-spondin1 conditioned medium, noggin, HEPES, 1X GlutaMax, penicillin/streptomycin, N cell culture additive, B27 cell culture additive, MEM nonessential amino acid solution (NEAA), nicotinamide, N-acetylcystein, A8301, human EGF, human HGF, human FGF10, gastrin1, forskolin, and advanced DMEM/F12 containing Y27632.
2. The medium for efficiently constructing rabbit liver organoids according to claim 1, comprising the following components: 40-60% (V/V) Wnt3a conditioned medium, 9-11% (V/V) R-spondin1 conditioned medium, 10-30ng/mL Noggin, 4-6mmol/mL HEPES, 1 XGlutaMax, 1 Xpenicillin/streptomycin, 1-2% (V/V) N2 cell culture additive, 1-2% (V/V) B27 cell culture additive, 1-2% (V/V) MEM nonessential amino acid solution (NEAA), 1-3mmol/mL icotinamide, 1-2mmol/mL N-acetylcysteine, 4-6umol/mlA8301, 40-60ng/mL human EGF, 40-60ng/mL human HGF, 40-60ng/mL human FGF10, 9-11nmol/mL gamin 1, 9-11umol/mL Forskolin, DMEM 12 containing 9-11 umol/Y27vance EM.
3. The medium for efficiently constructing rabbit liver organoids according to claim 1, comprising the following components: 50% (V/V) Wnt3a conditioned medium, 10% (V/V) R-spondin1 conditioned medium, 20ng/mL Noggin, 5mmol/mL HEPES, 1 XGlutaMax, 1 Xpeniilin/streptomyin, 1% (V/V) N2 cell culture additive, 1% (V/V) B27 cell culture additive, 1% (V/V) MEM nonessential amino acid solution (NEAA), 2mmol/mL icotinamide, 1mmol/mL N-acetylcysteine, 5umol/mlA8301, 50ng/mL human EGF, 50ng/mL human HGF, 50ng/mL human FGF10, 10nmol/mL Gastin 1, 10umol/mL 27. Mu.mol/mL Forskolin, and advance EM/F12 containing 10umol/mL Y632.
4. A method for efficiently constructing rabbit liver organoids, said method comprising the steps of:
step one: digestion and separation of rabbit liver tissue;
step two: inoculating and culturing rabbit liver tissue organoids;
step three: subculturing rabbit liver tissue organoids;
the step of inoculating and culturing the rabbit liver tissue organoid comprises the following steps:
1) Cell inoculation: adding Advanced DMEM/F12 (containing penicillin-streptomycin and Primocin) culture solution into the separated primary cells, mixing well, and centrifuging; discarding the supernatant, re-suspending the collected cell/tissue sediment with the rabbit liver organoid culture medium according to any one of claims 1-3, adding the suspension into the top of the prefabricated matrigel, and naturally dispersing the suspension to obtain cell clusters;
2) Organoid culture-On-top method: inoculating the cell mass into a prefabricated matrigel, placing the matrigel in an environment with 37 ℃ and carbon dioxide content of 5% for culturing for 3-5 days, changing liquid every 2-3 days in the process, and when a culture medium is added, a suction head faces to the side wall to avoid damaging the matrigel;
3) Organoid culture-embedding method: transferring the cultured organoids into gel drops for embedding culture, sucking the culture supernatant, adding 3mL of DISPASE II (disperse enzyme II) into each hole, blowing off the gel drops by using a Pasteur dropper, incubating for 15-20 minutes at 37 ℃, collecting suspension, and blowing for 10-20 times by using a 1mL suction head; centrifuging at 4deg.C under 250g for 5min, removing supernatant, adding diluted matrigel into cell precipitate, and mixing, wherein the volume ratio of rabbit source organoid culture medium to matrigel is 1:2; inoculating into cell culture plate according to volume of 25-40 μl per drop, placing the culture plate in CO2 incubator at 37deg.C, heating for 30min, taking out the culture plate after colloid is solidified, adding 600 μl/hole of preheated rabbit liver organoid culture solution, adding appropriate amount of sterile PBS or double distilled water into peripheral holes, reducing culture solution evaporation, changing culture solution for 3-4 days, and introducing culture solution with suction head facing side wall to avoid damaging matrigel.
5. The method of claim 4, wherein the step of digestion and separation of rabbit liver tissue comprises the steps of:
1) Tissue cleaning: taking fresh rabbit source tissue, placing the fresh rabbit source tissue into a centrifuge tube, adding 10 times of PBS (phosphate buffered saline) for shaking and cleaning, and discarding the supernatant;
2) Tissue cutting: the washed tissue was transferred to a 10cm dish and minced to 0.5mm using a scalpel 3 Is a small block of (2);
3) Tissue digestion: placing the tissue fragments into a centrifuge tube, adding at least 5 times of tissue digestion liquid, and performing digestion treatment at 200rpm at 37 ℃ in a constant-temperature shaking table; observing while digesting, and digesting by using a short-time and multiple-time method, wherein the time of each digestion is controlled to be 3-5 minutes, and repeating the steps until most of tissues are digested, and only a small amount of tissue fragments are left;
4) Cell collection: collecting the upper suspension by natural sedimentation, adding a large proportion of PBS, placing on ice for termination, centrifuging at 4 ℃ and 300g for 5 minutes, centrifuging to remove the supernatant, collecting digested cell/tissue sediment, and re-suspending cells by using sterile PBS to prepare a cell suspension;
5) Cell sieving: filtering the cell suspension with a 100 μm sieve, filtering the cell suspension into a new 15ml centrifuge tube, and washing the sieve with sterile PBS;
6) Single cell resuspension: the above centrifuge tube containing cell filtrate was centrifuged at 4℃for 5min at 300g, the supernatant carefully removed, and an appropriate amount of Advanced DMEM/F12 (Dulbecco' sModified Eagle Medium/Nutrient Mixture F-12) (containing 1%V/V penicillin-streptomycin-gentamicin) was added to the cell pellet, gently blown into a single cell suspension,
7) Lysing erythrocytes: if more red blood cells are observed in the cell sediment, the following steps of red blood cell lysis are carried out, after centrifugation, supernatant is removed, 1ml of red blood cell lysate is added to resuspend the cell sediment, the mixture is blown and uniformly mixed, the mixture is placed on a shaking table at 4 ℃,120rpm is carried out, 5min is oscillated, the temperature is 300g, the mixture is centrifuged for 5min, supernatant is removed, and 10ml of Advanced DMEM/F12 (containing 1%V/V penicillin-streptomycin and gentamicin) is added to resuspend the cell sediment to obtain primary cells.
6. The method for efficiently constructing rabbit liver organoids according to claim 4, wherein said step three rabbit liver tissue organoids subculture comprises the steps of:
1) And (3) observation: when the diameter of the organoid reaches 200-300 mu m, the organoid can be passaged under the observation under the mirror, and the organoid can be passaged generally every 7-14 days;
2) Cleaning: removing the culture solution, adding 1ml of PBS, and cleaning for 1 time, wherein the matrigel is not damaged;
3) And (3) glue melting: removing the old culture medium, adding 3mL of DISPASE II (disperse enzyme II) into a pore plate, blowing off Matrigel, transferring the organoid mixture to a 15mL centrifuge tube, digesting for 15-20min on a constant temperature oscillator at 37 ℃, observing the organoid state under a microscope, and centrifugally collecting;
4) Digestion: discard supernatant, centrifugeTrypLE is added into the tube TM Dispersing matrigel by using a pipettor, digesting for 3-5 min on a constant temperature oscillator at 37 ℃, observing under a mirror until the organoid is digested into small cell mass, adding 1ml of Advanced DMEM/F12 culture medium to terminate digestion, transferring cells to a 15ml centrifuge tube, centrifuging for 5min at 4 ℃ and 300g, and removing supernatant;
5) Cleaning: removing supernatant, stopping digestion of Advanced DMEM/F12 medium, transferring cells to a 15ml centrifuge tube, centrifuging at 4 ℃ for 5min at 300g, removing supernatant, washing, centrifuging and collecting cells;
6) And (3) seed glue: adding Matrigel according to the amount of precipitated cells, fully and uniformly mixing, planting in a 24-hole plate, and after the completion of the plating, inversely placing the culture plate in an incubator for 30min, solidifying the Matrigel, taking out the culture plate, adding 600 mu l/hole of preheated rabbit source liver organoid culture solution, adding a proper amount of sterile PBS or double distilled water into peripheral holes, and reducing the evaporation of a culture medium;
7) And (3) liquid adding: adding corresponding organoid culture medium, observing cell density and state after passage under a microscope, placing the culture dish into CO at 37deg.C 2 The incubator continues to cultivate, the culture medium is replaced for 3-4 days, and when the culture medium is added, the suction head faces the side wall, so that matrigel is prevented from being damaged.
7. The method of claim 4, wherein the step of inoculating the cells comprises mixing the matrigel with the advanced DMEM/F12 at a volume ratio of 2:1, adding the mixture to a 6-well plate, and incubating at 37 ℃ to solidify the mixture to form a gel layer of 1 mm.
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| CN202311796177.2A CN117757720A (en) | 2023-12-25 | 2023-12-25 | Culture medium and method for efficiently constructing rabbit-derived liver organoids |
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