CN114149977A - Method for immortalizing mouse lung microvascular endothelial cells to obtain extracellular vesicles - Google Patents
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Abstract
The invention discloses a method for immortalizing mouse lung microvascular endothelial cells to obtain extracellular vesicles, which comprises the following steps: s1, separation of primary mouse pulmonary microvascular endothelial cells: separating peripheral lung tissue of newborn mouse, cutting the tissue into small pieces with ophthalmic scissors, preparing single cell suspension, and removing CD45 by magnetic bead negative selection+、CD90.2+And CD326+Screening endothelial cell colonies for in vitro proliferation; s2, endothelial cell immortalization: transfecting six kinds of lentiviruses, screening, and selecting an immortalized cell strain according to the growth rate and the morphology of the cell; s3, vesicle extraction: extracting vesicle by ultrasonic centrifugation. According to the invention, by means of magnetic bead negative selection and selection, damage of cells extracted by traditional magnetic bead positive selection is avoided, and loss of endothelial cells is greatly improved;according to the invention, the immortalized cell is constructed by transfecting the lentivirus to the primary cell, and the immortalized cell secretory vesicle keeps the characteristic of the primary cell secretory vesicle, so that the vesicle extraction cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles.
Background
Pulmonary Microvascular Endothelial Cells (PMECs) are an important component of the alveolar-capillary barrier and participate in gas exchange. They play an important role in pulmonary homeostasis and inflammatory responses. In view of the important role of PMECs, culturing PMECs in vitro is crucial for elucidating the molecular and cellular mechanisms of pulmonary diseases. The study of mouse lung microvascular endothelial cells (pMPMECs) is limited by cell culture. The most common methods for isolating pMPMECs that have been published to date include: enzymolysis of lung tissue and magnetic bead immunobinding of pMPMECs to CD31 or CD 102. However, positive magnetic bead selection for pmpmpmecs results in early cell senescence and low proliferation capacity. Furthermore, non-specific binding of magnetic beads often leads to contamination of other cells (e.g. fibroblasts, smooth muscle cells, epithelial cells) in lung tissue.
Extracellular vesicles secreted by PMECs (PMEC-EVs) are secreted directly into the bloodstream and can interact with circulating leukocytes to modulate local immune responses. More and more researches show that PMEC-EVs are related to the pathophysiology of inflammatory lung diseases, and the research on the specific action mechanism of PMEC-EVs mediated lung diseases is still in an exploring stage. Due to the limited ability of pMPMECs to secrete EVs, and the lack of reliable pMPMECs cell lines. Instead of PMEC-EVs, many researchers used umbilical vein endothelial cell-derived vesicles in the study. However, vesicles are highly heterogeneous, with the properties of vesicles from different sources varying widely. Therefore, the use of other endothelial-derived vesicles to explore the role of PMECs-EVs in pulmonary disease may lead to research bias.
Disclosure of Invention
The invention aims to provide a method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles, which solves the technical problems, avoids the damage of the traditional magnetic bead positive selection cell extraction and greatly improves the problems of endothelial cell loss and cell aging; the immortalized cell is constructed by transfecting lentivirus to the primary cell, and the immortalized cell secretory vesicle keeps the characteristic of the primary cell secretory vesicle, thereby greatly reducing the extraction efficiency and the cost of the vesicle.
The purpose of the invention can be realized by the following technical scheme:
a method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles, comprising the steps of:
s1 separation of primary cells of mouse pulmonary microvascular endothelium
1) Preparation of mouse limbal Single cell suspension
Separating mouse lung tissue, fragmenting the tissue and preparing single cell suspension;
2) mouse lung margin single cell culture
Adding the single cell suspension into a complete culture medium to culture until the cell fusion degree is more than 90%;
3) separation and purification of mouse lung microvascular endothelial cells
Negative selection of resuspended single cells: removing lymphocytes, fibroblasts and epithelial cells by using anti-mouse CD45 magnetic beads, anti-mouse CD90.2 magnetic beads and anti-mouse CD326 magnetic beads respectively;
s2 immortalization of endothelial cells
S2.1 plating 500ul of mouse lung microvascular endothelial primary cell suspension in collagen-coated 24-well plates (3X 10)4Individual cells/well), cultured for 24 h.
S2.2 lentivirus containing GFP (1X 10)7IU/mL) was thawed in a 37 ℃ water bath and removed from the water bath immediately after thawing. Then 30, 60, 90, 120 and 150 μ L of lentiviral stock solution (containing 5 μ LViralEntry) were added to establish different MOIs (MOI 10,20,30,40, 50). The volume was adjusted to 500. mu.L using mPMEs medium.
S2.324 well plates 4000 rpm, centrifuged for 30 minutes to increase transduction efficiency, and then incubated in an incubator (37 ℃, 5% CO)2) And (5) incubating for 72 h.
S2.4 inverted fluorescence microscope photographs of cells 36, 48, 60, 72h at different MOIs were taken to assess the virus transduction rates at different MOIs and times.
S2.5 lentivirus (Lenti-SV40, Lenti-SV40T, Lenti-SV40T (puro)), Lenti-RasV12, Lenti-hTERT, Lenti-HPV-16E6/E7(puro)) was thawed in a water bath at 37 ℃ and removed from the water bath immediately after thawing. Then, an appropriate amount of lentiviral particles was added to the medium of mPMECs containing 5. mu.L ViralEntry.
S2.6 after incubating the mPMEs medium containing ViralEntry and lentivirus in S2.5 in an incubator for 48h, the mPMEs medium was changed.
S2.7 when cells 90-95% confluent, the cells were digested. The first transduced pmpmpmecs were reseeded on collagen coated 24-well plates, medium was changed every 2 days, S2.5-S2.6 repeated lentiviral transduction was repeated, and cell growth rate and morphology were monitored to select immortalized candidate cells.
S3, extracting mouse lung microvascular endothelial cell vesicles.
Further, the concrete operation steps of the preparation of the mouse lung limbus single cell suspension are as follows:
taking out two lungs of a mouse from an aseptic culture dish, peeling off visceral pleura, cutting off lung margins on two sides, placing the mouse in a DMEM-F12 basal medium, rinsing for 3 times, transferring the mouse into a centrifuge tube with the specification of 5ml, and adding a proper amount of type I collagenase working solution.
Shearing the tissue, filling collagenase working solution to 10ml, transferring the mixed solution into a tube C, and placing the tube C in a tissue grinder for treatment.
And after treatment, placing the C tube in a constant-temperature horizontal shaking table to vibrate for 30min, then placing the C tube in a tissue grinder to treat, filtering the obtained suspension through a 70-micron nylon filter screen, centrifuging, washing a DMEM-F12 basic culture medium for 3 times, adding an erythrocyte lysate to fully crack, stopping, centrifuging, and then calculating the number of living cells to obtain the lung limbus single cell suspension.
Further, the mouse lung limbus single cell culture step is as follows:
adding the single cell suspension into a complete culture medium, then re-suspending, adjusting the cell density, placing the mixture into a T-75 culture flask for culturing for 24 hours, then removing the supernatant, replenishing the complete culture medium again, changing the culture medium every two days, and observing until the cell fusion degree is more than 90%.
Further, the separation and purification steps of the mouse lung microvascular endothelial cells comprise:
after the lung limbal single cells were digested and resuspended in a single cell suspension, lymphocytes, fibroblasts and epithelial cells were removed using anti-mouse CD45 magnetic beads, anti-mouse CD90.2 magnetic beads and anti-mouse CD326 magnetic beads, respectively.
And (4) culturing the rest cells subjected to negative selection in the culture dish again, replacing the culture medium once in 2 days, observing by using a light mirror, and selecting endothelial cell colonies by using a cloning ring after the edge of the endothelial cell colonies is circled by using a marker pen.
Further, 7-14 days old newborn mice are selected in the step 1).
Further, the collagenase I working solution is prepared by dissolving collagenase I in a complete culture medium containing serum.
Further, the addition of 10 volumes of complete medium to the red blood cell lysate was terminated.
Further, the complete medium contained 92mg/L D-valine, 100IU/ml heparin, 1% ECGS.
Further, ViralEntry was used as the transfection assisting reagent in S2.
The lentiviruses include Lenti-SV40, Lenti-SV40T, Lenti-SV40T (puro), Lenti-RasV12, Lenti-hTERT, Lenti-HPV-16E6/E7 (puro).
Further, the extraction of the vesicles adopts an ultracentrifugation method. The endothelial cell colony is detected by means of flow cytometry and immunofluorescence.
The invention has the beneficial effects that:
1. according to the invention, by means of magnetic bead negative selection and selection, damage of cells extracted by traditional magnetic bead positive selection is avoided, and the problems of endothelial cell loss and cell aging are greatly improved;
2. according to the invention, the immortalized cell is constructed by transfecting the lentivirus to the primary cell, and the immortalized cell secretory vesicle keeps the characteristic of the primary cell secretory vesicle, so that the vesicle extraction efficiency and the vesicle extraction cost are greatly reduced.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of the extraction of the lung microvascular endothelial cells of the mouse of the present invention;
FIG. 2 is a light microscope image of the lung microvascular endothelial cells of the mouse of the present invention;
FIG. 3 is a diagram showing the identification of the mouse pulmonary microvascular endothelial cell surface molecule of the present invention;
FIG. 4 is a graph showing the identification of the tube-forming ability and phagocytic ability of the lung microvascular endothelial cells of the mouse of the present invention;
FIG. 5 is a flow chart of the mouse lung microvascular endothelial cell immortalization construction process of the present invention;
FIG. 6 is a morphological diagram of the mouse lung microvascular endothelial cells of the present invention transfected with different lentiviruses;
FIG. 7 is a diagram showing the identification of molecules on the surface of the immortalized pulmonary microvascular endothelial cells of the present invention;
FIG. 8 is a graph showing the identification of the tube-forming ability and phagocytic ability of the immortalized pulmonary microvascular endothelial cells of the present invention;
FIG. 9 is a graph comparing gene expression and proliferation of primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention;
FIG. 10 is a graph comparing the permeability function of primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention;
FIG. 11 is a graph comparing inflammatory stimuli responses of primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention;
FIG. 12 is a graph comparing the morphology of vesicles secreted by primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention;
FIG. 13 is a graph comparing the size distribution of vesicles secreted by primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention;
FIG. 14 is a graph comparing the size distribution of vesicles secreted by primary pulmonary microvascular endothelial cells (pMPMECs) and immortalized pulmonary microvascular endothelial cells (iMPMECs) of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
S1 separation of primary cells of mouse pulmonary microvascular endothelium
S1.1 preparation of mouse limbal Single cell suspension
Taking out two lungs of a mouse from an aseptic culture dish after the mouse is sacrificed, cutting off lung margins on two sides after a visceral pleura is stripped, placing the lung margins in a DMEM-F12 basal medium, rinsing for 3 times, transferring the lung margins into a centrifuge tube (Eppendorf tube) with the specification of 5ml, and adding a proper amount of collagenase I working solution (collagenase I is dissolved in a complete culture medium containing serum);
fully shearing the tissue by using an ophthalmic scissors, filling collagenase working solution to 10ml, transferring the mixed solution into a GentleMecACS C tube (called as a C tube for short) of MeitianNi, then placing the tissue into a GentleMecACS tissue grinder of MeitianNi and operating the program (m-lung-01-02).
After the program is finished, the tube C is directly placed in a constant-temperature horizontal shaking table (37 ℃ at 200rpm) to be shaken for 30min, and then placed in a GentleMeC ACS tissue grinder to run the program. Filtering the obtained suspension through a 70um nylon filter screen, centrifuging, washing the DMEM-F12 basic culture medium for 3 times, adding erythrocyte lysate to fully lyse, stopping, centrifuging, and calculating the number of living cells to obtain the lung limbus single cell suspension.
S1.2 mouse lung limbus single cell culture: and adding the single cell suspension obtained in the S1.1 into a complete culture medium, then carrying out heavy suspension, adjusting the cell density, placing the suspension into a T-75 culture flask for culturing for 24 hours, then discarding the supernatant, and replenishing the fresh complete culture medium again. And changing the liquid every two days, and observing until the cell fusion degree is more than 90%.
Complete medium was supplemented with 92mg/L D-valine, 100IU/ml heparin and 1% ECGS in basal medium. D-valine is used for inhibiting excessive proliferation of fibroblasts; the heparin protects endothelial cell glycocalyx, and is helpful for maintaining the normal structure and function of endothelial cells; the ECGS contains various trace elements and mineral substances required by the growth and the reproduction of endothelial cells, and can maintain the activity and the proliferation of the endothelial cells to the maximum extent.
S1.3, separation and purification of mouse lung microvascular endothelial cells: after the lung margin single cells are digested and resuspended into single cell suspension, carrying out negative selection on the resuspended single cells: lymphocytes, fibroblasts, and epithelial cells were removed using anti-mouse CD45 magnetic beads, anti-mouse CD90.2 magnetic beads, and anti-mouse CD326 magnetic beads, respectively.
The remaining cells from the negative selection were placed in a petri dish for culture and the medium was changed once 2 days and observed by light microscopy. The fused lung microvascular endothelial cells were cobblestone-like, and after the endothelial cell colony edges were circled with a marker pen, endothelial cell colonies (endothelial primary cells) were selected with a cloning loop, as shown in fig. 1. The cells obtained can be detected by means of flow cytometry, immunofluorescence, etc., and the results are shown in fig. 2 to 4.
S2 immortalization of endothelial cells
S2.1 plating 500ul of mouse lung microvascular endothelial primary cell suspension in collagen-coated 24-well plates (3X 10)4Individual cells/well), cultured for 24 h.
S2.2 lentivirus containing GFP (1X 10)7IU/mL) was thawed in a 37 ℃ water bath and removed from the water bath immediately after thawing. Then 30, 60, 90, 120 and 150 μ L of lentivirus stock (containing 5 μ LViralEntry) were added to establish different MOIs (multiplicities of infection) (MOI 10,20,30,40, 50). The volume was adjusted to 500. mu.L using mPMEs medium.
The traditional lentivirus transfer promoter polybrene has low transfection efficiency and high toxicity to primary cells, and ViralEntry utilizes the latest polymerization technology to furthest protect the primary cells while improving the transfection efficiency and ensure that the death and subsequent change of the primary cells cannot be caused during transfection.
S2.324 well plates 4000 rpm, centrifuged for 30 minutes to increase transduction efficiency, and then incubated in an incubator (37 ℃, 5% CO)2) And (5) incubating for 72 h.
S2.4 inverted fluorescence microscope photographs of cells 36, 48, 60, 72h at different MOIs were taken to assess the virus transduction rates at different MOIs and times.
S2.5 lentivirus (Lenti-SV40, Lenti-SV40T, Lenti-SV40T (puro)), Lenti-RasV12, Lenti-hTERT, Lenti-HPV-16E6/E7(puro)) was thawed in a water bath at 37 ℃ and removed from the water bath immediately after thawing. Then, an appropriate amount of lentiviral particles, preferably 30 MOI in the present invention, was added to the medium of mPMECs containing 5. mu.L ViralEntry.
S2.6 after incubation of the mPMEs medium containing ViralEntry and lentivirus in S2.5 in an incubator for 48h, the medium was replaced with fresh mPMEs medium.
S2.7 when cells 90-95% confluent, the cells were digested. The first transduced pmpmpmecs were reseeded on collagen coated 24-well plates, medium was changed every 2 days, S2.5-S2.6 repeated lentiviral transduction was repeated, and cell growth rate and morphology were monitored to select immortalized candidate cells, and the construction flow is shown in fig. 5.
FIG. 6 is a morphological diagram of mouse lung microvascular endothelial cells transfected with different lentiviruses; FIGS. 7 to 9 show the morphology and function of successfully constructed immortalized cells.
S3 extraction of mouse lung microvascular endothelial cell vesicles
Extracting vesicles by ultracentrifugation and identifying vesicle properties. The vesicle characteristics secreted by primary and immortalized cells are shown in fig. 12 to 14.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (10)
1. A method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles, comprising the steps of:
s1 separation of primary cells of mouse pulmonary microvascular endothelium
1) Preparation of mouse limbal Single cell suspension
Separating mouse lung tissue, fragmenting the tissue and preparing single cell suspension;
2) mouse lung margin single cell culture
Adding the single cell suspension into a complete culture medium to culture until the cell fusion degree is more than 90%;
3) separation and purification of mouse lung microvascular endothelial cells
Negative selection of resuspended single cells: removing lymphocytes, fibroblasts and epithelial cells by using anti-mouse CD45 magnetic beads, anti-mouse CD90.2 magnetic beads and anti-mouse CD326 magnetic beads respectively;
s2 immortalization of endothelial cells
Inoculating the mouse pulmonary microvasculature into a 24-well plate according to a proportion, adding lentivirus and a transfection assisting reagent, culturing, carrying out a second round of transfection, and continuously subculturing the obtained cells until cells capable of maintaining the shape are selected, namely the immortalization success is achieved;
s3, extracting mouse lung microvascular endothelial cell vesicles.
2. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 1, wherein the specific operation steps for preparing the mouse pulmonary limbus single cell suspension are as follows:
taking out two lungs of a mouse from an aseptic culture dish, peeling off visceral pleura, cutting off lung margins on two sides, placing the mouse in a DMEM-F12 basal culture medium, rinsing for 3 times, transferring the mouse into a centrifuge tube with the specification of 5ml, and adding a proper amount of type I collagenase working solution;
shearing the tissue, filling collagenase working solution to 10ml, transferring the mixed solution into a tube C, and placing the tube C in a tissue grinder for treatment;
and after treatment, placing the C tube in a constant-temperature horizontal shaking table to vibrate for 30min, then placing the C tube in a tissue grinder to treat, filtering the obtained suspension through a 70-micron nylon filter screen, centrifuging, washing a DMEM-F12 basic culture medium for 3 times, adding an erythrocyte lysate to fully crack, stopping, centrifuging, and then calculating the number of living cells to obtain the lung limbus single cell suspension.
3. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 2, wherein the mouse pulmonary limbal single cell culture step is:
adding the single cell suspension into a complete culture medium, then re-suspending, adjusting the cell density, placing the mixture into a T-75 culture flask for culturing for 24 hours, then removing the supernatant, replenishing the complete culture medium again, changing the culture medium every two days, and observing until the cell fusion degree is more than 90%.
4. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 3, wherein the step of isolating and purifying the mouse pulmonary microvascular endothelial cells comprises:
after the lung limbus single cells are digested and resuspended into single cell suspension, lymphocytes, fibroblasts and epithelial cells are respectively removed by using anti-mouse CD45 magnetic beads, anti-mouse CD90.2 magnetic beads and anti-mouse CD326 magnetic beads;
and (4) culturing the rest cells subjected to negative selection in the culture dish again, replacing the culture medium once in 2 days, observing by using a light mirror, and selecting endothelial cell colonies by using a cloning ring after the edge of the endothelial cell colonies is circled by using a marker pen.
5. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 1, wherein 7-14 days old suckling mice are selected in the step 1).
6. The method for immortalizing mouse lung microvascular endothelial cells to obtain extracellular vesicles according to claim 2, wherein the collagenase type I working solution is prepared by dissolving collagenase type I in a complete culture medium containing serum.
7. The method of claim 2, wherein the erythrocyte lysate is terminated by adding 10 times of volume of complete medium.
8. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 3, wherein the complete medium comprises 92mg/L D-valine, 100IU/ml heparin, 1% ECGS.
9. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 1, wherein the transfection-assisting reagent in S2 is ViralEntry;
the lentiviruses include Lenti-SV40, Lenti-SV40T, Lenti-SV40T (puro), Lenti-RasV12, Lenti-hTERT, Lenti-HPV-16E6/E7 (puro).
10. The method for immortalizing mouse pulmonary microvascular endothelial cells to obtain extracellular vesicles according to claim 1, wherein the vesicles are extracted by ultracentrifugation; the endothelial cell colony is detected by means of flow cytometry and immunofluorescence.
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| CN115521901A (en) * | 2022-10-12 | 2022-12-27 | 中国医学科学院医学生物学研究所 | Immortalized tree shrew retina microvascular endothelial cell strain and construction method and application thereof |
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