CN113637631B - Extraction and culture method of rat peritoneal mesothelial cells - Google Patents
Extraction and culture method of rat peritoneal mesothelial cells Download PDFInfo
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Abstract
The invention discloses an extraction and culture method of rat peritoneal mesothelial cells, which comprises the following steps: (1) Preparing a culture solution, wherein the culture solution comprises a basic culture medium, fetal bovine serum and sodium bicarbonate; (2) mesenteric extraction: spreading the mesentery on the surface of rat liver, lifting the middle part of the mesentery, cutting off the middle part of small mesentery, and placing into buffer solution for cleaning; transferring the cleaned small mesentery to digestive juice for digestion to obtain primary cell suspension; (3) cell inoculation culture. The extraction and culture method of the invention not only can stably obtain the peritoneal mesothelial cells with uniform characters, high cell obtaining rate, less content of mixed cells and stronger activity at low cost, but also can see that the obtained peritoneal mesothelial cells are classical paving stones when observed by using a microscope in a bright field, and has stable morphology, high consistency and stable culture, thereby being beneficial to increasing the accuracy and repeatability of experiments when being used for subsequent related researches.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an extraction and culture method of rat peritoneal mesothelial cells.
Background
In the application aspect of basic experiments such as medicine, biology and the like, the cell line or the cell strain is subjected to multiple passages to possibly generate heterogeneity and limitation on gene level, while primary cells are directly derived from organism tissues, the biological properties are not changed greatly, and the in vivo state can be reflected to a certain extent.
The current studies involving peritoneal mesothelial cells have been very widespread, and many documents describe their process of extraction of peritoneal mesothelial cells in experimental methods or methods, partly from human beings and partly from other animals, but human tissues are generally difficult to obtain. Common primary mammalian cell culture steps include drawing materials, rinsing, shearing, digesting, preparing a digested cell suspension, and counting the number of inoculations.
The following two methods are used in the prior art (for example, non-patent documents 1 to 4):
(1) Trypsin method for intraperitoneal injection
(2) Macroreticular membrane or mesenteric membrane separation in vitro digestion culture method.
However, the disadvantages of these current methods are:
(1) At present, the studies cannot specifically explain the details of the operation of obtaining cells, but the subject group finds that the repeatability is not strong, and fibroblasts and mesothelial cells in peritoneum can be inevitably led to enter cell suspension together in the experimental process, especially when the pancreatin digestion time exceeds 15 minutes, so that the cells are mutually influenced in the subsequent culture to induce the peritoneal mesothelial cells to transdifferentiate, and the purity of the extracted cells is reduced in the process of adherence or growth, and the non-patent document 5 can be seen; (2) At present, commercial culture mediums (comprising DMEM/F12, M19 or RPMI 1640) are frequently used in China, but in practice, the components of the culture mediums sold and split-packed in China are slightly different from those of the same culture mediums sold in foreign countries, so that primary cells cannot grow or grow at a low speed, and the price of the culture mediums sold in foreign countries is too high.
Non-patent literature:
[1] effect of lipopolysaccharide on primary rat peritoneal mesothelial cell IL-1 beta and IL-6 expression, once li, phonon, li Wenlin, et al; effect of lipopolysaccharide on the expressions of IL-1β and IL-6 in rat peritoneal mesothelial cells[J J.A.of the elderly, 2016, 36:1560-1563.
[2] Once li, mao Chunqin, liu Tulin, et al, culture and identification of rat peritoneal mesothelial cells; culture and Identification of Peritoneal Mesothelium Cells in Rats [ J ]. Nanjing university of Chinese medicine, 2012, 28:334-336.
[3] Yan Libo, zhao Wenxiu, yin Zhenyu, et al, improved isolation and identification of human peritoneal mesothelial cells; isolation and identification of improved human peripheral peritoneum mesothelial cells Chinese tissue engineering research and clinical rehabilitation, 2011, 15:5007-5010.
[4] Zhu Yan the mechanism of action of ligustrazine nanometer spray against experimental abdominal adhesion was studied based on rat peritoneal mesothelial cell TGF-beta/Smad signaling pathway 2015, university of Nanjing traditional Chinese medicine.
[5]Stylianou, E, Jenner L A, Davies M，et al.， Isolation, culture and characterization of human peritoneal mesothelial cells[J].Kidney Int，1990，37:1563-70。
Disclosure of Invention
In order to solve the technical problems, the invention provides an extraction and culture method of rat peritoneal mesothelial cells.
The technical problems to be solved by the invention are realized by the following technical scheme:
the extraction and culture method of the rat peritoneal mesothelial cells comprises the following steps:
(1) Preparing a culture solution, wherein the culture solution comprises a basic culture medium, fetal bovine serum and sodium bicarbonate;
(2) Mesenteric extraction: spreading the mesentery on the surface of rat liver, lifting the middle part of the mesentery, cutting off the middle part of small mesentery, and placing into buffer solution for cleaning; transferring the cleaned small mesentery to digestive juice for digestion to obtain primary cell suspension;
(3) Stopping digestion, centrifuging the primary cell suspension, discarding the supernatant, and re-suspending the cells using the culture broth of step (1), and inoculating the cells for culture.
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the invention, in the step (1), each 100mL of culture solution comprises: 85-90 mL of basic culture medium and 10-15 mL of fetal bovine serum, and adding sodium bicarbonate to control the pH value of the culture solution to be 7.35-7.45.
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the invention, the process for preparing the culture solution is as follows: taking a centrifuge tube, and adding a basic culture medium; adding proper sodium bicarbonate solution to raise the pH value of the basic culture medium to 7.25; and (3) filtering by using a syringe and a filter, adding fetal bovine serum, uniformly mixing, and filtering to obtain a culture solution, wherein the pH value of the culture solution is 7.35-7.45.
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the invention, the sodium bicarbonate solution is 10wt% sodium bicarbonate solution.
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the present invention, the process of mesenteric extraction in step (2) is as follows: exposing the abdominal cavity of a rat, lifting the abdominal muscle of the rat upwards, finding the cecum of the rat, gently pulling out the ileum and the mesentery thereof from the ileocecum back, spreading the mesentery on the smooth liver surface, lifting the middle part of the mesentery to a proper height, cutting the middle part of a small piece of mesentery for multiple times, and putting the middle part of the small piece of mesentery into buffer solution for cleaning; and (3) transferring the cleaned small mesenteries into digestive juice for digestion to obtain primary cell suspension.
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the present invention, the large omentum is not separated and removed during the extraction of the mesentery of step (2).
As a preferred embodiment of the method for extracting and culturing rat peritoneal mesothelial cells provided by the invention, in the step (2), the rat peritoneal mesothelial cells are gently shaken 1 time during digestion.
As a preferred embodiment of the method for extracting and culturing the rat peritoneal mesothelial cells, provided by the invention, the specific conditions of the culture are as follows: 37 ℃ and 5% CO 2 The method comprises the steps of carrying out a first treatment on the surface of the Extracting, performing plate adherence culture for 24h, changing liquid, washing off residual undigested tissue blocks, and continuing culture; and (4) replacing the liquid once every 48 hours, and continuing culturing after replacing the liquid, wherein the cell coverage rate of the bottle bottom is more than or equal to 70% after culturing for 4-5 days.
The invention has the following beneficial effects:
according to the invention, the single source and high purity of cells are ensured by extracting the central part of the mesentery depending on jejunal tension through a specific extraction method, meanwhile, the cells do not tend to differentiate in the digestion and culture processes by matching with a specific culture solution, so that the peritoneal mesothelial cells which are uniform in character, high in cell obtaining rate, low in impurity cell content and high in activity can be obtained stably, and the obtained peritoneal mesothelial cells can be seen to be in a classical paving stone shape when observed by using a microscope in a bright field, are stable in shape, high in consistency and stable in culture, and are beneficial to increasing the accuracy and repeatability of experiments when used for subsequent related researches.
Drawings
FIGS. 1a-f are flowcharts of the operation of step (2) of example 1 of the present invention;
FIG. 2 is a schematic representation of the prior art of extracting whole mesenteries in a petri dish;
FIG. 3 is a graph showing the state of cells of example 1 of the present invention (wherein, (a) is 100-fold magnified and (b) is 200-fold magnified) after culturing for 7 days and observing the microscopic fields;
FIG. 4 is a graph showing the cell state of the cell line of comparative example 1 after the cell line had been grown on the wall and observed in the bright field of a microscope (wherein, (a) is at 100-fold magnification and (b) is at 200-fold magnification);
FIG. 5 is a graph showing the cell state of comparative example 1 after culturing for 7 days and observing the microscopic field (wherein, (a) is at 100-fold magnification and (b) is at 200-fold magnification);
FIG. 6 is a graph showing the cell state of the cell line of comparative example 2 after the cell line had been grown on the wall and observed in the bright field of a microscope (wherein, (a) is at 100-fold magnification and (b) is at 200-fold magnification);
FIG. 7 is a graph showing the cell state of comparative example 2 after culturing for 7 days and observing the microscopic field (wherein, (a) is at 100-fold magnification and (b) is at 200-fold magnification);
FIG. 8 is a schematic representation of the fluorescent immunoassay of example 1 of the present invention after 7 days of cell culture;
FIG. 9 is a schematic representation of the fluorescent immunoassay of comparative example 1 of the present invention after 7 days of cell culture;
FIG. 10 is a schematic representation of the fluorescent immunoassay of comparative example 2 of the present invention after 7 days of cell culture.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the examples described below, unless otherwise indicated, are conventional methods well known to those skilled in the art. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.
At present, a rat peritoneal mesothelial cell culture method is adopted for culturing pig peritoneal mesothelial cells, and most of cell sources are rat omentum or whole mesentery including mesentery blood vessels, but in the implementation process of extracting the omentum, the inventor finds that the rat omentum can retract into a group of soft tissues after exposing abdominal cavities, and the rat omentum is not large in practice, and part of tissues can be torn in the pulling and tearing process, so that the extraction amount is greatly reduced and the purity is influenced; in the implementation process of extracting the whole mesentery (shown in figure 2), the inventor finds that although the whole mesentery is convenient and quick to extract, cells not only contain peritoneal epithelial cells but also have a large number of fibroblasts and vascular epithelial cells when digestion is carried out, and the defects of unstable form, multiple mixed cells and the like of the peritoneal epithelial cells are easy to occur; it is also difficult to perform efficient isolation, i.e., the fibroblasts and mesothelial cells in the peritoneum are inevitably brought together into a cell suspension, so that they are induced to transdifferentiate by mutual influence in subsequent culture, resulting in a great decrease in the purity of the extracted cells. Moreover, commercial media (including DMEM/F12, M19, or RPMI 1640) are currently used more frequently in China, but in practice, these media sold separately in China and the same media sold abroad have different compositions, so that primary cells cannot grow or grow at a slower rate, and the price of purchasing the media imported abroad is too expensive.
In view of this, the invention provides a method for extracting and culturing rat peritoneal mesothelial cells, which extracts the central part of the mesentery depending on jejunal tension by a specific extraction method to ensure single source and high purity of the cells, and simultaneously, the cells do not tend to differentiate in the process of digestion and culture by matching with a specific culture solution, so that the peritoneal mesothelial cells with uniform properties, high cell yield, less impurity cell content and stronger activity can be obtained stably at low cost, and the obtained peritoneal mesothelial cells can be seen to be in classical paving stone shape when observed by using a microscope in bright field, and have stable morphology, high consistency and stable culture, thereby being beneficial to increasing the accuracy and repeatability of experiments in the following related research.
Specifically, the invention relates to a method for extracting and culturing rat peritoneal mesothelial cells, which comprises the following steps:
(1) Preparing a culture solution, wherein the culture solution comprises a basic culture medium, fetal bovine serum and sodium bicarbonate;
(2) Mesenteric extraction: spreading the mesentery on the surface of rat liver, lifting the middle part of the mesentery, cutting off the middle part of small mesentery, and placing into buffer solution for cleaning; transferring the cleaned small mesentery to digestive juice for digestion to obtain primary cell suspension;
(3) Stopping digestion, centrifuging the primary cell suspension, discarding the supernatant, and re-suspending the cells using the culture broth of step (1), and inoculating the cells for culture.
Wherein in step (1), each 100mL of the culture medium comprises: 85-90 mL of basic culture medium and 10-15 mL of fetal bovine serum, and adding sodium bicarbonate to control the pH value of the culture solution to be 7.35-7.45. The basal medium can be DMEM/F12, M19 or RPMI1640 containing various amino acids and glucose, and the DMEM/F12 basal medium is preferably used in the invention. The sodium bicarbonate solution is 10wt% sodium bicarbonate solution.
The process of mesenteric extraction of step (2) is as follows: exposing the abdominal cavity of a rat, lifting the abdominal muscle of the rat upwards, finding the cecum of the rat, gently poking the ileum and the mesentery thereof from the ileocecum back, spreading the mesentery on the smooth liver surface, lifting the middle part of the mesentery to a proper height, cutting the middle part of a small piece of mesentery for multiple times, putting the small piece of mesentery into buffer solution for cleaning, and cutting the small piece of mesentery into pieces no longer because the small piece is small enough; and (3) transferring the cleaned small mesenteries into digestive juice for digestion to obtain primary cell suspension. The macroreticular membrane is not separated during the extraction process because the macroreticular membrane itself is very soft, easily curls into clusters and contains many other types of cells. The digestion period is gently shaken for 1 time, and the cells may change in state due to impact caused by shaking or shaking, which is not beneficial to culture.
In the step (3), the specific conditions of the culture are: 37 ℃ and 5% CO 2 The method comprises the steps of carrying out a first treatment on the surface of the Extracting, performing plate adherence culture for 24h, changing liquid, washing off residual undigested tissue blocks, and continuing culture; and (4) replacing the liquid once every 48 hours, and continuing culturing after replacing the liquid, wherein the cell coverage rate of the bottle bottom is more than or equal to 70% after culturing for 4-5 days.
The technical scheme of the present invention will be described in further detail with reference to specific embodiments and drawings, it should be understood that,
the following examples are merely illustrative of the present invention and are not intended to limit the invention.
Example 1
The embodiment provides a method for extracting and culturing rat peritoneal mesothelial cells, which comprises the following steps:
(1) Preparation of culture solution (50 mL for example)
(1) The required equipment or consumable materials comprise a plurality of 50mL centrifuge tubes, 45mL DMEM/F12 basic culture medium, 5mL fetal calf serum, 1.5mL 10% sodium bicarbonate solution, 250 mL syringes, 2 0.22 mu m filters and an ultra clean bench, wherein the above materials are required to be sterilized in grade or subjected to sterilization treatment, and a pH meter;
(2) taking a 50mL centrifuge tube, and adding 45mL DMEM/F12 basal medium;
(3) the pH value of the basic culture medium is measured by a pH meter and is generally about 6.35, and at the moment, a proper amount of sodium bicarbonate solution is added to raise the pH value of the basic culture medium to about 7.25;
(4) filtering the basic culture medium with pH adjusted by using a syringe and a filter, adding 5mL of fetal bovine serum, uniformly mixing, reserving 5mL for detecting the final pH value, filtering the liquid by using a sterile filter with the aperture of 0.22 mu m, filtering out bacteria, sealing and preserving at the temperature of 4 ℃ for later use;
(5) and (3) detecting the pH value of 5mL obtained in the step 4, namely sealing and storing at 4 ℃ for standby, wherein the pH value is between 7.35 and 7.45.
(2) Mesenteric extraction (taking 1 experimental animal as an example)
(1) 180-250g of SPF-grade SD male rats of the required experimental animals; the equipment or consumable material needs 1L beaker, timer, 0.22 μm diameter 18cm filter paper two, diameter 60mm culture dish 2, 50mL sterile centrifuge tube 1, ophthalmic scissors 1 (sterilization), ophthalmic forceps straight bending one (sterilization), direct scissors 1 (sterilization), toothed forceps 1 (sterilization), centrifuge, ultra clean bench; 10mL of PBS is needed for the reagent, and 10mL of 0.25% pancreatin is needed for the culture solution prepared in the step (1);
(2) the rat is killed by neck breaking, soaked in 75% alcohol for 2 minutes, and transferred to a super clean bench double-layer filter paper;
(3) carefully expose the rat abdominal cavity, open the rat abdominal muscle upward, and not isolate the omentum (as shown in fig. 1 a);
(4) finding the cecum of the rat, gently pulling out the ileum and its mesentery from the ileocecum (as shown in fig. 1 b), taking the main extraction subject as a clear mesentery, spreading the mesentery on a smooth liver surface to maintain surface tension and wetness, carefully lifting the middle part of the mesentery with the minimum angle of opening with the forceps, and lifting the appropriate height (as shown in fig. 1 c); using an ophthalmic scissors to cut a small piece of mesenteric tissue, taking care to select a mesentery with a certain size and cut the central part (as shown in fig. 1d and 1 e), so that the purity of the cells can be ensured to the greatest extent, the cells can be quickly put into PBS, and the middle parts of the mesentery of a plurality of small pieces are repeatedly cut (as shown in fig. 1 f);
(5) after taking down all mesenteric small pieces, washing in PBS for 2 times, and directly transferring the small pieces into pancreatin digestive juice for digestion without cutting the small pieces into pieces; digestion at 37 ℃ for 10 minutes, during which time the cells are gently shaken for 1 time, do not shake vigorously or shake, and impact can change the state of the cells;
(3) Cell inoculation culture
Step (3) specifically means stopping digestion, centrifuging the primary cell suspension, discarding the supernatant, and resuspending the cells using the culture solution of step (1), and inoculating the cells for culture.
The method specifically comprises the following steps:
(1) adding the same amount of culture solution to stop digestion, and centrifuging at 1200rpm at room temperature for 10 minutes; discarding the supernatantAdding 5mL culture medium, gently stirring, adding into 10cm diameter culture dishes, adding 1.8 mL, shaking, standing at 37deg.C in 5% CO 2 Culturing in an incubator;
(2) after extraction, the culture is carried out for 24 hours by sticking the plates, the liquid can be changed, 1 ml PBS is used for washing gently to wash off the residual undigested tissue blocks, 1 mL culture liquid is added into each dish, then the liquid is changed once every 48 hours, the culture is continued after the liquid change, and the culture can be carried out for more than 70% after about 4-5 days. After 7 days of cell culture, the state of cells in the dishes was photographed using an inverted biomicroscope, as shown in FIG. 3. A portion of the cells were taken for immunofluorescent identification and were visualized under a fluorescent microscope as shown in fig. 8.
Example 2
This embodiment is based on embodiment 1, except that: (1) Preparation of culture solution (100 mL for example)
(1) The required equipment or consumable materials comprise a plurality of 100mL centrifuge tubes, 90mL of DMEM/F12 basic culture medium, 10mL of fetal calf serum, 3mL of 10% sodium bicarbonate solution, 2 100mL of injectors, 2 0.22 mu m filters and an ultra clean bench, and all the above materials need sterilization grade or are subjected to sterilization treatment and a pH meter;
(2) taking a 100mL centrifuge tube, and adding 90mL DMEM/F12 basal medium;
(3) the pH value of the basic culture medium is measured by a pH meter and is generally about 6.37, and at the moment, a proper amount of sodium bicarbonate solution is added to raise the pH value of the basic culture medium to about 7.3;
(4) filtering the basic culture medium with pH adjusted by using a syringe and a filter, adding 10mL of fetal bovine serum, uniformly mixing, reserving 5mL for detecting the final pH value, filtering the liquid by using a sterile filter with the aperture of 0.22 mu m, filtering out bacteria, sealing and preserving at the temperature of 4 ℃ for later use;
(5) and (3) detecting the pH value of the remaining 5mL in the step 4, namely sealing and storing at 4 ℃ for standby at 7.35-7.45.
Comparative example 1
The comparative example provides a method for extracting and culturing rat peritoneal mesothelial cells, which comprises the following steps:
(1) Preparation of culture solution (50 mL for example)
(1) The required equipment or consumable materials are provided with a plurality of 50mL centrifuge tubes, 45mL DMEM/F12 basic culture medium, 5mL fetal calf serum, 250 mL injectors, 2 0.22 mu m filters and an ultra clean bench, and all the above materials need sterilization grade or are subjected to sterilization treatment;
(2) taking a 50mL centrifuge tube, and adding 45mL DMEM/F12 basal medium;
(3) filtering the basic culture medium by using a syringe and a filter, adding 5mL of fetal bovine serum, uniformly mixing, sealing and preserving at 4 ℃ for later use.
(2) Mesenteric extraction (taking 1 experimental animal as an example)
(1) 180-250g of SPF-grade SD male rats of the required experimental animals;
(2) the rat is killed by neck breaking, soaked in 75% alcohol for 2 minutes, and transferred to a super clean bench double-layer filter paper;
(3) carefully expose the rat abdominal cavity and open the rat abdominal muscle upward;
(4) the rat cecum was found, and the entire mesentery was cut down the ileum and placed in PBS (as shown in fig. 2);
(5) washing in PBS for 2 times after shearing, and transferring into pancreatin digestion solution for digestion; digestion at 37℃for 10 minutes, during which time the cells are gently shaken 1 time, do not shake vigorously or shake, and the impact may change the state of the cells.
(3) Cell inoculation culture
The step (3) specifically comprises the following steps:
(1) adding the culture solution in the same amount in the step (1) to stop digestion, and centrifuging at 1200rpm at room temperature for 10 minutes; discarding supernatant, adding 5mL culture medium, gently stirring, mixing, adding into 10cm diameter culture dishes, adding 1.8 mL per dish, shaking, standing at 37deg.C under 5% CO 2 Culturing in an incubator;
(2) after extraction, the plates were subjected to adherent culture for 24 hours, the liquid was changed, the remaining undigested tissue pieces were washed away gently with 1 ml of PBS, 1 mL culture liquid was added to each dish, and after every 48 hours, the culture was continued after changing the liquid. After 24 hours of plate-attached culture, the state of cells in the culture dish was photographed using an inverted biomicroscope, as shown in fig. 4. After culturing the cells for 7 days, the state of the cells in the culture dish was photographed using an inverted biomicroscope, as shown in fig. 5. A portion of the cells were taken for immunofluorescent identification and were visualized under a fluorescent microscope as shown in fig. 9.
Comparative example 2
This comparative example differs from example 1 in that: the preparation of the culture solution in the step (1) is specifically changed into the following steps: (1) the required equipment or consumable materials are provided with a plurality of 50mL centrifuge tubes, 45mL DMEM/F12 basic culture medium, 5mL fetal calf serum, 250 mL injectors, 2 0.22 mu m filters and an ultra clean bench, and all the above materials need sterilization grade or are subjected to sterilization treatment; (2) taking a 50mL centrifuge tube, and adding 45mL DMEM/F12 basal medium; (3) filtering the basic culture medium by using a syringe and a filter, adding 5mL of fetal bovine serum, uniformly mixing, sealing and preserving at 4 ℃ for later use.
After 24 hours of the cell plating of this comparative example 2, the cell state in the petri dish was photographed using an inverted biomicroscope, as shown in fig. 6. After culturing the cells for 7 days, the state of the cells in the dishes was photographed using an inverted biomicroscope, as shown in fig. 7. A portion of the cells were taken for immunofluorescent identification and were visualized under a fluorescent microscope, as shown in fig. 10.
[ microscopic bright field contrast ]
From a morphological view, the peritoneal mesothelial cells should exhibit a uniform cobblestone-like growth, rather than long fusions or polygons (also known as fibroblast-like growth). From the comparison of fig. 3-7, the invention adopts a specific mesentery extraction method, can greatly reduce pollution of fibroblast-like cells to peritoneal mesothelial cells, namely few or no mixed cells appear, and when the cells are cultured for 7 days, namely the cells grow on a culture dish to form uniform paving cobblestones (shown in fig. 3), which shows that the invention adopts the specific extraction method, has short culture time and high cell yield, and is in the shape and relatively uniform in size and shape of typical peritoneal mesothelial cells. Comparing fig. 4 to 7, it can be seen that, under the same condition of the culture solution, the cell yield is lower when the existing whole mesentery extraction method is adopted for attaching the primary seed plates and culturing for 7 days, and the mixed cells are more, the growth state is poorer than that of the specific extraction method, and the consistency of the cell shape is also poorer.
In addition, as can be seen from comparing fig. 3 with fig. 7, the same culture time can be obtained by using the extraction culture method of example 1 of the present invention, so that the purity and survival of cells can be improved to some extent, while the interference of the miscellaneous cells can be effectively reduced by using the extraction method of the middle portion of the mesentery in the extraction culture method of comparative example 2, the growth state is worse than that of the specific culture solution of the present invention after using the conventional culture solution, and the uniformity of the cell shape and the activity of the cell are poor, and the bright white spot in the figure is dead cell corpse.
Immunofluorescence identification contrast of rat peritoneal mesothelium cells
In fig. 8 to 10, (a) in each figure is a blue fluorescence diagram of the nucleus, (b) is a red fluorescence diagram of Vimentin, and (c) is a green fluorescence diagram of keratin 18, and (d) is a graph of the overlapping effect of fig. (a), (b) and (c).
As can be seen from FIGS. 8 to 10, the nuclei of example 1, comparative example 1 and comparative example 2 all exhibited blue fluorescence. When whole-section mesenteric extraction mode was used (comparative example 1), the corresponding keratin 18 was not observed under green fluorescence, see fig. 9-c, which illustrates that the cell yield was lower when primary culture was performed for 7 days using the existing whole-section mesenteric extraction method. Furthermore, as can be seen from the red fluorescent diagrams (fig. 8-b and 9-b) of the vimentin in example 1 and comparative example 1, the fibroblasts expressed by the whole-section mesentery extraction method are obviously more and have different forms (fig. 9-b), but the specific extraction method of the invention basically does not observe the fibroblasts and the observed cell forms are uniform (fig. 8-b), which means that the whole-section mesentery extraction method has more heterogeneous cells and has a poorer growth state than the specific extraction method of the invention.
When the specific mesenteric extraction method of the invention is adopted, but no specific culture solution is adopted, most of the cytoplasms can be observed under a fluorescence microscope to show bright green fluorescence (as shown in fig. 10-c), but the mesothelial cells observed from fig. 10-c tend to differentiate, and the purity is not high; when the specific mesentery extraction method and the specific culture solution are combined, the specific mesentery extraction method and the specific culture solution act synergistically, and the mesothelial cells observed in the figure 8-c are not differentiated, and have higher purity and uniform morphology.
In summary, it can be understood that the technical effect of the present invention is the sum of the technical feature synergistic effects of the steps, and the steps have a certain inherent correlation, and are not simple superposition of the individual technical feature effects. The invention prepares specific culture solution by (1); (2) The specific mesentery extraction method not only avoids the trend differentiation of cells in the process of digestion and culture, but also can stably obtain the peritoneal mesothelial cells with uniform properties, high cell obtaining rate, less impurity cell content and stronger activity at low cost, and meanwhile, the obtained peritoneal mesothelial cells can be seen to be in a classical paving stone shape when observed by using a microscope in a bright field, and have stable shape and high consistency. The above effects of the present invention are obtained by mutual cooperation and are not separable.
The above examples only show embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the invention, but all technical solutions obtained by equivalent substitution or equivalent transformation shall fall within the scope of the invention.
Claims (6)
1. The extraction and culture method of the rat peritoneal mesothelial cells is characterized by comprising the following steps of:
(1) Preparing a culture solution, wherein the culture solution consists of a DMEM/F12 basic culture medium, fetal calf serum and sodium bicarbonate;
(2) Mesenteric extraction: spreading the mesentery on the surface of rat liver, lifting the middle part of the mesentery, cutting off the middle part of small mesentery, and placing into buffer solution for cleaning; transferring the cleaned small mesentery to digestive juice for digestion to obtain primary cell suspension;
(3) Terminating digestion, centrifuging the primary cell suspension, discarding the supernatant, and resuspending the cells using the culture broth of step (1), and inoculating the cells for culture;
in the step (1), each 100mL of the culture solution comprises: 85-90 mL of DMEM/F12 basal medium and 10-15 mL of fetal bovine serum, and adding sodium bicarbonate to control the pH value of the culture solution to be 7.35-7.45;
the process of mesenteric extraction of step (2) is as follows: exposing the abdominal cavity of a rat, lifting the abdominal muscle of the rat upwards, finding the cecum of the rat, gently pulling out the ileum and the mesentery thereof from the ileocecum back, spreading the mesentery on the smooth liver surface, lifting the middle part of the mesentery to a proper height, cutting the middle part of a small piece of mesentery for multiple times, and putting the middle part of the small piece of mesentery into buffer solution for cleaning; and (3) transferring the cleaned small mesenteries into digestive juice for digestion to obtain primary cell suspension.
2. The method for extracting and culturing rat peritoneal mesothelial cells according to claim 1, wherein the process of preparing the culture solution is as follows: taking a centrifuge tube, and adding a DMEM/F12 basal medium; adding proper sodium bicarbonate solution to raise the pH value of the basic culture medium to 7.25; and (3) filtering by using a syringe and a filter, adding fetal bovine serum, uniformly mixing, and filtering to obtain a culture solution, wherein the pH value of the culture solution is 7.35-7.45.
3. The method for extracting and culturing rat peritoneal mesothelial cells of claim 2, wherein said sodium bicarbonate solution is 10wt% sodium bicarbonate solution.
4. The method for extraction and culture of rat peritoneal mesothelial cells according to claim 1, wherein the extraction of mesentery of step (2) is performed without isolation of the omentum.
5. The method for extracting and culturing rat peritoneal mesothelial cells according to claim 1, wherein in step (2), the digestion period is gently shaken 1 time.
6. The method for extracting and culturing rat peritoneal mesothelial cells according to claim 1, wherein specific conditions of said culturing are: 37 ℃ and 5% CO 2 The method comprises the steps of carrying out a first treatment on the surface of the Extracting, performing plate adherence culture for 24h, changing liquid, washing off residual undigested tissue blocks, and continuing culture; and (5) replacing the liquid once every 48 hours, and continuing culturing after replacing the liquid.
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