CN111534477B - Method for culturing primary epithelial stem cell balls of lung tissue of mouse - Google Patents
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Abstract
The invention relates to a method for culturing primary epithelial stem cells of mouse lung tissue, which comprises the steps of digesting a mouse lung lobe tissue block by using preheated collagenase digestion solution, adding an equivalent DMEM/F12 culture medium containing FBS to terminate digestion, and extracting primary epithelial stem cells of the mouse lung tissue to perform suspension culture to form stem cells. The method establishes a primary epithelial stem cell sphere culture method of the mouse lung tissue through a 3D culture model, so as to simulate a lung tissue microenvironment in vitro and provide an effective and reliable research model for clarifying a regulation and control mechanism of proliferation and differentiation of lung epithelial stem cells.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a method for culturing primary epithelial stem cell spheres of mouse lung tissues.
Background
In an organism, almost all cells are in a complex three-dimensional (3D) structure, the life cycle of which is finely controlled by the Extracellular microenvironment, and the cell-cell and cell-Extracellular matrix (ECM) interactions form a 3D communication network that maintains tissue specificity and homeostasis. In traditional two-dimensional (2D) cell adherent culture, in vivo-like tissue structure and connections between cells cannot be achieved, which limits the study on cell morphology, functionality and behavior, and also affects the expression of genes and proteins, predictability of cell-based drug and toxicity screening assays. In most cases, 2D culture models do not simulate well the normal physiological environment of cells in vivo. For example, in 2D culture, cell adsorption occurs on only one side of the cell, whereas in 3D culture it occurs around the entire cell surface, which significantly affects the morphological changes of the cell; meanwhile, part of the primary tissue cells gradually lose the differentiation phenotype when being placed in 2D culture, but restore the in vivo physiological morphology and function again in a 3D culture environment; in addition, it has been found that intracellular signal transduction and secretion of glandular epithelial cells and fibroblasts in a 3D environment more closely approximate to the state of in vivo culture. Therefore, in order to truly mimic the in vivo environment, a large number of 3D cell culture models have been developed in recent years, including suspension culture cell spheres, hydrogels, and solid scaffolds, among others. The 3D cell culture model has stronger representativeness to in vivo microenvironment, shortens the difference between in vitro cell culture and living tissues, and obviously promotes the research and development of tumor biology, tissue engineering and stem cell regenerative medicine.
In an in vitro 3D culture system, suspension culture of cell spheres is one of the most common models, and has strong physiological relevance, and can simulate various characteristics of in vivo tissues and culture environment. Meanwhile, compared with other 3D culture models, cell spheres are easy to analyze through imaging technologies such as optics, fluorescence and confocal imaging, and culture of uniform-size spheres is easy to realize, so that the method is widely applied to research of stem cell regenerative medicine.
It has been shown that human and mouse mammary epithelial stem cells can form mammary epithelial stem cell spheroids in suspension culture. For the respiratory system, lung epithelial stem cells are important cells for maintaining tissue homeostasis and repairing and regenerating tissues after injury. In vitro, the lung epithelial stem cells can form lung spheroids during 3D suspension culture, the lung spheroids simulate lung tissue microenvironment and enhance the inherent dryness of the stem cells, and the lung epithelial stem cells are effective models and tools for researching the biological functions of the stem cells in vivo. However, for the isolation of primary epithelial stem cells in lung tissue, there is no efficient and reliable extraction method because of the lack of specific cell surface markers and distinguishable morphological phenotypes. Some researchers isolated mouse lung epithelial stem cells by Fluorescence Activated Cell Sorting (FACS), but both digestive enzymes and digestion time affect the expression of Cell surface antigens, and thus the isolation effect.
Disclosure of Invention
The invention aims to provide a method for culturing primary epithelial stem cell spheres of mouse lung tissues.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for culturing primary epithelial stem cell balls of mouse lung tissues comprises the following steps:
(1) taking a lung lobe tissue block of the mouse, and placing the lung lobe tissue block into preheated collagenase digestion solution for digestion;
(2) adding an equivalent DMEM/F12 culture medium containing FBS to stop digestion, and after uniform blowing, filtering by using a cell sieve; centrifuging the filtrate, and removing the supernatant;
(3) adding erythrocyte lysate to resuspend cell sediment, incubating at room temperature, adding DMEM/F12 culture medium, mixing, centrifuging, and removing supernatant;
(4) repeating the step (3) until the cell sediment is white;
(5) adding DNA enzyme solution for resuspending cell precipitation, adding DMEM/F12 culture medium, mixing, centrifuging, and removing supernatant;
(6) adding DMEM/F12 culture medium to resuspend the cell precipitate, blowing, beating, mixing, filtering with a cell sieve, centrifuging, and removing supernatant;
(7) adding lung spheroid culture medium for heavy suspension, and performing suspension culture after adjusting the cell density.
Preferably, the mouse is a newborn mouse born 0-1d, the newborn mouse does not need anesthesia, the sterile operation of separating lung tissues is relatively simple, and the possibility of primary cell pollution in the experimental process is reduced; meanwhile, the number of the lung tissue epithelial stem cells of the newborn mouse is large, the proportion is large, the dryness is strong, the activity is high, the yield of the lung epithelial stem cells obtained by separation culture is large, the primary epithelial stem cell ball forming efficiency is higher, the size is larger, and the method is beneficial to large-scale construction of the stem cell balls and efficient implementation of subsequent experiments.
Preferably, in the method of the present invention, the collagenase digestion solution does not contain FBS or insulin, and preferably consists of: DMEM/F12 medium containing collagenase A, trypsin and gentamicin. Insulin, as a polypeptide hormone, can promote the uptake of glucose and amino acid by cells, promote the mitosis and proliferation of cells, reduce the dryness of lung epithelial stem cells and influence the separation effect; FBS is complex in component, can promote differentiation of stem cells and influence maintenance of the dryness of the stem cells according to the prior report, and simultaneously can stop the digestion of collagenase and pancreatin through competitive inhibition, reduce the activity of a collagenase digestion solution and influence the separation effect and yield of the lung epithelial stem cells. The collagenase digestion solution used in the invention does not contain FBS or insulin, and DMEM/F12 culture medium containing FBS is added after collagenase digests tissue blocks to stop digestion, so that collagenase and pancreatin activity are inhibited in time after digestion is finished, the activity of the separated cells is maintained to the maximum extent, meanwhile, the contact time of the cells and the FBS is reduced, and the collagenase digestion solution is beneficial to keeping the inherent characteristics of lung epithelial stem cells and reducing differentiation.
Preferably, in said step (2), the digestion is terminated by adding an equal amount of DMEM/F12 medium containing 10% FBS.
Preferably, in the step (2), the cell sieve is 100 μm.
Preferably, as a further improvement of the present invention, the centrifugation conditions are 700-900 g. The relative centrifugal force in the centrifugation process is increased to 700-900g, so that the cells in the cell suspension are separated and precipitated to the maximum extent on the premise of ensuring the cell viability, the cell yield and the yield are improved, and the culture and the formation of the primary epithelial stem cell ball are facilitated.
Preferably, in the step (6), the cell sieve is 40 μm.
Preferably, the lung spheroid medium is DMEM/F12 medium containing 1 XB-27 additive (without vitamin A), 1 XPicillin/streptomycin solution, 4 μ g/mL heparin, 20ng/mL EGF, 10ng/mL FGF2, 10 μ MY-27632.
Preferably, in the step (7), the cell density is adjusted to 2X 106-3×106/mL。
Preferably, in the step (7), poly-HEMA coated T25 cell culture flasks are used for suspension culture. The poly-HEMA coated T25 cell culture bottle is used for culturing to form primary epithelial stem cell spheres, in the culture process, the interference of the external environment is small, the cells are dispersed more uniformly, the cells can be prevented from being gathered in the center by vortex formed when the pore plate is shaken, the forming efficiency of the cell spheres is higher, and the possibility of pollution in the subsequent culture medium replacement process can be reduced.
The invention establishes a method for culturing the primary epithelial stem cell spheres of the mouse lung tissue through a 3D culture model, so as to simulate the microenvironment of the lung tissue in vitro and provide an effective and reliable research model for clarifying the regulation and control mechanism of proliferation and differentiation of the lung epithelial stem cells. The method of the invention digests lung tissue in collagenase digestive solution without FBS, then adds DMEM/F12 culture medium containing FBS to terminate digestion, maintains the inherent characteristics of lung epithelial stem cells, reduces differentiation, improves centrifugal force, separates and precipitates cells in cell suspension to the maximum extent on the premise of ensuring cell viability, purifies epithelial stem cells by utilizing the cell selection action of lung spheroid culture medium (the lung spheroid culture medium is only suitable for the survival, growth and proliferation of lung epithelial stem cells, other types of cells can not endure the environment of the lung spheroid culture medium and die), improves the forming efficiency of primary epithelial stem cells, simplifies experimental steps, saves time, simplifies experimental steps, shortens experimental time, improves cell yield and reduces the requirements on experimental equipment and conditions as far as possible on the premise of realizing the prior art effect, meanwhile, fluorescent antibody is not needed, and the experiment cost is reduced.
Drawings
FIG. 1 is a comparison of lung tissue primary epithelial stem cell spheres of mice of different weeks of age.
FIG. 2 is the process of primary epithelial stem cell sphere formation in neonatal mouse lung tissue.
FIG. 3 shows immunofluorescence double-standard staining results of primary epithelial stem cell spheres of lung tissues of newborn mice.
FIG. 4 shows the second generation epithelial stem cell pellet experiment results of lung tissue of newborn mice.
FIG. 5 is the 3D-submerged culture induced organoid formation of primary epithelial stem cell spheres from neonatal mouse lung tissue.
FIG. 6 shows the result of adherent culture migration experiment of primary epithelial stem cell spheres in lung tissue of newborn mice.
Detailed Description
The examples used the following main materials and sources:
SPF grade 0-1d C57BL/6 neonatal mice: changzhou Kavens laboratory animals GmbH, animal license number: SCXK (threo) 2016-.
75% of alcohol: lida brand 75% ethanol disinfectant, Nanjing medical products, Inc.
Sterile instruments: the surgical instrument factory of Shanghai medical instrument Limited company is used for sterilization.
Sterile PBS (phosphate buffered saline): beijing Soilebao Tech Co., Ltd., product number P1020.
Cell culture dish: corning (Corning) inc, usa, cat # 430166.
Collagenase digestion solution (2mg/mL collagenase A, 2mg/mL trypsin, 50 μ g/mL gentamicin in DMEM/F12 medium): collagenase A: shanghai Sigma Aldrich trade, Inc., cat # C0130; trypsin: shanghai Sigma Aldrich trade, Inc., Cat # T4799; gentamicin: APExBIO ltd, usa, cat # a 2514; DMEM/F12 medium: saimer Feishale science and technology (China), Inc., cat # 11039021.
FBS (fetal bovine serum): ScienCell, Inc., USA, Cat 0010.
Erythrocyte lysate: shanghai Biyuntian Biotechnology Co., Ltd., cat # C3702.
DNA enzyme: BioFroxx, Germany, cat # 1121.
100 μm cell sieve: hefeisharp Ltd, cat # BS-100-XBS.
40 μm cell sieve: hefeisharp Ltd, cat # BS-40-XBS.
Lung spheroid medium [1 XB-27 (without vitamin A), 1 XPicillin/streptomycin solution, 4 μ g/mL heparin, 20ng/mL EGF, 10ng/mL FGF2, 10 μ MY-27632 DMEM/F12 medium ]: b-27 (50X, without vitamin A): seimer feishell science and technology (china), cat # 12587010; 100 × penicillin/streptomycin solution: ScienCell, Inc., USA, Cat No. 0503; heparin: U.S. APExBIO Inc., cat # B3602; EGF: PeproTech, Inc., USA, Cat 315-09; FGF 2: PeproTech, Inc., USA, Cat 450-33; y-27632: U.S. APExBIO Inc., cat # B1293.
poly-HEMA: shanghai Sigma Aldrich trade, Inc., Cat number P3932;
t25 cell culture flask: corning (Corning) inc, usa, cat # 430639.
Carbon dioxide cell incubator: sammer Feishel technologies (China) Inc., cat # Thermo Forma 311.
Example 1
The method for culturing the primary epithelial stem cell balls of the lung tissue of the mouse comprises the following steps:
(1) thoroughly disinfecting skin of mouse with 75% alcohol, aseptically separating lung tissue, rinsing in precooled sterile PBS (phosphate buffer solution), removing connective tissue and main bronchus in lung, simultaneously separating lung lobe, and cleaning for 2-3 times to remove blood.
(2) Transferring the cleaned lung lobes into a new cell culture dish with sterile forceps, removing residual PBS, and shearing lung tissue to about 1mm with an ophthalmic surgical scissors3Transferring the tissue blocks into preheated collagenase digestion solution, and digesting the tissue blocks for 45 to 60 minutes by a constant temperature shaking table (100 revolutions per minute) at 37 ℃;
(3) adding equivalent DMEM/F12 medium containing 10% FBS to stop digestion, beating and mixing uniformly, and filtering by using a 100-micron cell sieve; centrifuging the filtrate at the temperature of 4 ℃ and the temperature of 700 and 900g for 10 minutes, and removing the supernatant;
(4) adding 2-3mL of erythrocyte lysate to resuspend the cell sediment, incubating for 1-2 minutes at room temperature, adding 6mLDMEM/F12 culture medium, mixing uniformly, centrifuging for 10 minutes at the temperature of 4 ℃ and the temperature of 700-;
(5) repeating the step (4) for 1-2 times until the cell sediment turns white;
(6) adding 4mL of DNase solution (20U/mL) for resuspending cell precipitation, manually shaking at room temperature for 3-5 minutes, adding 6mL of DMEM/F12 culture medium, uniformly mixing, centrifuging at 4 ℃ for 900g for 10 minutes, and removing supernatant;
(7) adding 6ml of MEM/F12 culture medium to resuspend the cell sediment, fully blowing, uniformly mixing, filtering by a 40-micron cell sieve, centrifuging for 10 minutes at 4 ℃ of 700-;
(8) resuspending lung spheroid culture medium, adjusting cell density to 2 × 106-3×106/mL；
(9) Culturing in poly-HEMA-coated T25 cell culture flask with 3-5mL lung spheroid culture medium/flask;
(10) transferred to a carbon dioxide cell incubator (37 ℃, 5% CO)2) The medium was changed every three days and lung spheroids were counted after 10-15 days.
By adopting the method, mice with different ages of weeks, including newborn mice (0-1 day), young mice (4 weeks) and adult mice (8 weeks) are cultured under the same inoculation density and culture conditions, and after 15 days of culture, the formed lung tissue primary epithelial stem cell spheres are larger in size, more regular in shape and more smooth in edges compared with the lung tissue primary epithelial stem cell spheres of the young mice and the adult mice as shown in figure 1.
The process (0-15d) of forming the primary epithelial stem cell spheres of the lung tissue of the newborn mouse is shown in fig. 2, and in the culture process, the volume of the primary epithelial stem cell spheres of the lung tissue of the newborn mouse is gradually increased, the shape of the primary epithelial stem cell spheres is gradually changed regularly, and the edges of the spheres are smoother.
Fig. 3 is an immunofluorescence double-label staining result of primary epithelial stem cell spheres of lung tissue of a newborn mouse, wherein EpCAM: epithelial cell markers, SOX 2: stem cell markers, DAPI: nuclear staining, Merge: EpCAM + SOX2+ DAPI merge. The immunofluorescence double-label staining result proves that a large number of EpCAM and SOX2 double-positive cells exist in the primary epithelial stem cell ball of the lung tissue of the newborn mouse.
FIG. 4 shows the second generation epithelial stem cell pellet experiment results of lung tissue of newborn mice. And (3) continuously performing suspension culture after the primary epithelial stem cell ball of the lung tissue of the mouse is dissociated into a single cell suspension, and forming a second-generation epithelial stem cell ball after 10-15 days.
FIG. 5 shows the 3D-submerged culture of neonatal mouse lung tissue primary epithelial stem cell spheres to induce organoid formation3D Organoid culture techniqueThe primary epithelial stem cell ball of the lung tissue of the mouse is cultured in three dimensions to form a hollow round organoid similar to a terminal airway structure or an alveolar structure so as to simulate the complex spatial morphology of differentiated tissues in vivo.
FIG. 6 shows the adherent culture migration experiment results of primary epithelial stem cell spheres of lung tissue of newborn mice, which induces adherent culture of primary epithelial stem cell spheres of lung tissue of newborn mice, epithelial stem cells at the edge of the spheres migrate radially outwards, and the migrated cells have a typical "paving stone-like" epithelial cell morphology.
Claims (6)
1. A method for culturing primary epithelial stem cell balls of mouse lung tissues is characterized by comprising the following steps:
(1) taking a lung lobe tissue block of the mouse, and placing the lung lobe tissue block into preheated collagenase digestion solution for digestion; the collagenase digestion solution comprises the following components: DMEM/F12 medium containing collagenase A, trypsin and gentamicin;
(2) adding an equivalent DMEM/F12 culture medium containing FBS to stop digestion, and after uniform blowing, filtering by using a cell sieve; centrifuging the filtrate under the conditions of 700 and 900g, and then removing the supernatant;
(3) adding erythrocyte lysate to resuspend cell sediment, incubating at room temperature, adding DMEM/F12 culture medium, mixing uniformly, centrifuging under the condition of 700-900g, and then removing supernatant;
(4) repeating the step (3) until the cell sediment is white;
(5) adding DNA enzyme solution to resuspend cell sediment, adding DMEM/F12 culture medium to mix evenly, centrifuging under the condition of 700-900g, and then removing supernatant;
(6) adding a DMEM/F12 culture medium to resuspend the cell sediment, uniformly blowing and beating, filtering by using a cell sieve, centrifuging under the condition of 700-;
(7) adding a lung spheroid culture medium for resuspension, adjusting the cell density, and then performing suspension culture by using a poly-HEMA coated T25 cell culture bottle; the lung spheroid culture medium is DMEM/F12 culture medium containing 1 Xvitamin A-free B-27 additive, 1 Xpenicillin/streptomycin solution, 4 mu g/mL heparin, 20ng/mL EGF, 10ng/mL FGF2 and 10 mu M Y-27632.
2. The method of claim 1, wherein the mouse is a born 0-1d neonatal mouse.
3. The method of claim 1, wherein digestion is terminated by adding an equal amount of DMEM/F12 medium containing 10% FBS.
4. The method according to claim 1, wherein in the step (2), the cell sieve is 100 μm.
5. The method according to claim 1, wherein in the step (6), the cell sieve is 40 μm.
6. The method of claim 1, wherein the cell density is adjusted to 2 x 106-3×106/mL。
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