CN113604419B - Method for extracting mouse primary liver cumic cells - Google Patents
Method for extracting mouse primary liver cumic cells Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
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Abstract
The invention relates to a method for extracting mouse primary liver cumic cells. The extraction method provided by the invention comprises the following steps: adopting a disposable infusion device to perform buffer solution infusion and performing liver digestion treatment by combining collagenase infusion; after the mice are digested by intravenous infusion, the liver envelopes are shredded for liver tissue filtration; and (5) performing centrifugal treatment for multiple times after filtering to obtain the high-purity liver tissue cells. The method provided by the invention can ensure that the cells keep better activity on the basis of ensuring the purity of the cells. The extraction method provided by the invention has higher efficiency of separating out the mouse liver cells, and the extraction amount of the mouse liver cells is maintained to be 5 multiplied by 10 6-8×106. The method provided by the invention has the advantages that the use equipment is simple and easy to obtain, great convenience is provided for a laboratory without a peristaltic pump, the cost of the disposable transfusion device is low, and the aseptic operation requirement of the cell extraction process can be better met.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a simple and easy extraction method of primary liver cells of mice with universality.
Background
The liver is an important metabolic organ and is composed of a plurality of cells, wherein liver parenchymal cells account for about 65% of the total number of cells, and liver endothelial cells, astrocytes, cumic cells, liver-related lymphocytes and other non-parenchymal cells account for 35%. Meanwhile, the liver is also an important immunocompetent organ, which is also called as a lymphoid tissue-like organ, 90% of organism mononuclear-macrophages are in the liver, and the dead cells in the liver are the main components of the organism mononuclear-macrophages and account for 25% of the total number of cells in the liver; most researches are conducted to extract primary cumic cells, namely, a non-perfusion method, namely, a mechanical method is adopted to cut tissues into small blocks, and then the cells are separated from the tissues through mechanical means such as extrusion, shearing, vibration and the like, so that the separated cells are obtained. Another non-perfusion method is to cut liver tissue and then separate cells by using collagenase or pancreatin digestion to break the bridge between cells or fiber components. However, although the non-perfusion technology is simple and easy to operate, the problem that the cells are not completely digested and the separated cells have multicellular aggregates often exists in the process of separating the cells, and the method cannot well meet the needs of researchers.
In 1969, berry and Friend introduced a perfusion method to extract primary cells, and the perfusion process can make the digestive juice more fully contact with liver tissue, so that not only separation efficiency is improved, but also activity and quantity of cells obtained by separation are greatly improved. Perfusion methods have been developed under the improvement of efforts of many researchers, such as in situ collagenase perfusion, semi-in situ collagenase perfusion, ex vivo collagenase perfusion, and the like. In situ collagenase infusion is commonly used, but relies on peristaltic pumps, which certainly results in wasted laboratory equipment for laboratory study of occasional extraction of several primary cells, and often makes it difficult to achieve the desired infusion effect because of the excessive fineness of the veins.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a simple and easy extraction method of the primary liver tissue cells of the mice with universality.
In the prior art, the method for extracting the primary liver cells of the rat is often referred to, and the primary liver cells of the mouse are extracted by combining in-situ perfusion collagenase and a peristaltic pump. Because the portal vein of the mouse is very thin, when a peristaltic pump is adopted, the perfusion pressure is not easy to control, and for a laboratory for extracting a small amount of primary liver cells of the mouse, the extraction method which does not depend on the peristaltic pump is more convenient and feasible.
Thus, in a first aspect, the present invention provides a method for extracting primary liver cells from a mouse, the method comprising the steps of perfusing the primary liver cells from the portal vein of the mouse with a buffer solution in situ, draining the primary liver cells from the tail end of the inferior vena cava, and changing the liver color from dark red to yellow; the collagenase is used for replacing buffer solution for perfusion, and the collagenase perfusion time is maintained for 6-10min.
The extraction method provided by the invention is simple and easy to implement, does not depend on peristaltic pumps, has moderate filling pressure, and does not need to consider the survival condition of the mice in the filling process, namely, the discharge of erythrocytes along with the buffer solution is not influenced by the heart of the mice in the filling process or stopping beating.
The invention also discovers that in the experiment, due to the small diameter of the portal vein of the mouse, the situation that the portal vein is pricked by a syringe occasionally occurs when collagenase is infused. In order to solve the problem, in the extraction method provided by the invention, buffer forward in-situ perfusion is adopted to combine with collagenase reverse in-situ perfusion to digest liver tissue; i.e., when using collagenase infusion, it may be infused from the inferior vena cava and expelled from the portal vein.
The invention discovers that when the collagenase is in-situ perfused, collagenase forward in-situ perfused (perfused from the portal vein and discharged from the inferior vena cava) can be adopted, and in order to solve the defects of the mice such as ultra-thin portal vein, poor in-situ perfused operation and the like, collagenase reverse perfused (perfused from the inferior vena cava and discharged from the portal vein) can also be adopted.
The invention realizes high-activity and high-yield cell extraction by combining the method of forward buffer solution in-situ perfusion with reverse collagenase in-situ perfusion, and provides a new way of a new idea for primary liver cell extraction.
Based on the above, the invention provides a method for extracting the primary liver cells of mice, which uses Percoll separating liquid to carry out gradient centrifugation on liver tissues obtained after digestion by adopting the extracting method; the Percoll separating liquid is 30% Percoll and 70% Percoll.
Specifically, in the method for extracting liver cumic cells provided by the invention, the gradient centrifugation comprises:
(1) Centrifuging the digested liver tissue for 35-70g and 3-5 min;
(2) Collecting supernatant, and centrifuging 650g for 5-7 min;
(3) Discarding the supernatant, re-suspending the sediment with HBSS, separating cells by using Percoll separating liquid, centrifuging 1800g for 15-20 min, and keeping the sediment brake free; before starting the centrifuge, the speed of the centrifuge is firstly increased and decreased to the lowest gear, so that density gradient damage caused by the excessively high speed of the centrifuge is avoided;
(4) The middle layer cells of two different concentrations of Percoll isolates were resuspended in HBSS and centrifuged 650g for 5-7 min.
The invention utilizes a closed passage formed among the liver, the portal vein and the inferior vena cava, utilizes buffer solution forward perfusion and collagenase forward or reverse perfusion to digest, and then extracts and obtains the high-purity and high-yield liver primary cumic cell through multiple times of centrifugation.
In the method for extracting liver cumic cells provided by the invention, cells obtained by centrifugation are inoculated into a six-hole plate and then cultured for 12-16 minutes, and then non-adherent cells are discarded. According to the invention, since the liver cells are easy to adhere, most of the non-adherent cells are non-adherent cells after being cultured for 12-16 minutes, and the purity of the extracted non-adherent cells can be increased by discarding the non-adherent cells.
In the method for extracting liver cumic cells provided by the invention, the centrifugation temperature is 0-4 ℃; in step (1), step (2) or step (4), the centrifuge selects maximum braking. The speed up and down of the centrifugal machine should be adjusted to the highest grade before the centrifugal machine is started.
In the method for extracting liver cumic cells provided by the invention, the 70% Percoll solution is prepared from 7 parts of 100% Percoll and 3 parts of 1 XPBS buffer solution; the 30% Percoll solution was prepared from 3 parts of 100% Percoll and 7 parts of 1 XPBS; the 100% percoll solution was prepared from 9 parts of percoll stock solution and 1 part of 10×pbs buffer.
In the method for extracting liver cumic cells provided by the invention, HBSS buffer solution and collagenase solution are placed in a water bath at 35-38 ℃ for preheating for 4-7 minutes before perfusion.
The invention also claims the application of the extraction method in the culture of mouse liver tissue cells and in improving the purity of the primary liver tissue cells of the mouse according to the understanding of the person skilled in the art.
The invention has the advantages that:
(1) The instrument and the equipment and the consumable related by the invention are all commonly used in laboratories, and the materials are easy to obtain and the cost is lower; in addition, the treatment process of the invention does not need to avoid the survival condition of mice.
(2) The invention can remove as many liver parenchymal cells as possible on the basis of ensuring the purity of the cells, and separate the primary liver cells of the mice with high yield.
(3) The invention identifies the purity of the cells by flow cytometry, the cells after extraction, washing and purification are stained by F4/80 and CD11b flow antibody, and the purity single positive of the primary liver cells of the mice obtained by extraction reaches more than 98% by detecting by a flow cytometer.
Drawings
FIG. 1 is a microscopic view of liver cells isolated in example 1 of the present invention.
FIG. 2 is a graph showing the results of purity detection of liver cells isolated in example 1 of the present invention.
FIG. 3 is a microscopic view of liver cells isolated in example 2 of the present invention.
FIG. 4 is a graph showing the results of purity detection of liver cells isolated in example 2 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention may be made without departing from the spirit and nature of the invention and are intended to be within the scope of the present invention.
Unless otherwise indicated, all experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art unless specifically indicated.
The mice used in the invention are all taken from C57BL/6 male mice which are healthy in SPF-class animal houses, are 6-8 weeks old and have a weight of 18-25 g, and are washed by 100 ml of 70% ethanol and then subjected to subsequent tests in a biological clean bench.
Experimental materials, instrumentation used in the following examples:
Main reagent consumable: healthy mice, collagenase IV (Invitrogen), ca 2+ in 1 XHBSS solution (Macgene), 1 XPBS (Macgene), 10 XPBS (Macgene), percoll stock (GE Health), 0.4% (w/v) trypan blue solution (scientific), RPMI 1640 medium (Hyclone), fetal bovine serum (Gibco), double antibody (Macgene), isoflurane (RWD LIFE SCIENCE Co.), scissors, forceps, medical tape, disposable infusion set, 50mL/15mL tip centrifuge tube (Corning), six well plate (Corning), hemocytometer, 0.22 μm filter, disposable syringe (20 mL), disposable 200 mesh cell screen (FAN).
The main instrument is as follows: cell incubator (Thermo Fisher); bench top centrifuge (Thermo Fisher); constant temperature incubator at 37 ℃ (Taicang scientific and teaching instruments factory); biological clean bench (SW-CJ-1 FD, suzhou purification plant Co., ltd.); optical microscope (olympus CKX 41); electric constant temperature water bath (Nanguo instruments manufacturing Co., ltd., jiangsu province, gold altar); micropipettes (Thermo Fisher) of different gauges.
Example 1
The embodiment provides a method for efficiently obtaining mouse liver cumic cells, which comprises the following steps:
(1) Two 50mL sterilized small-mouth glass bottles were prepared, one containing 40mL of sterile HBSS and the other containing 40mL of 0.04% (w/v) collagenase IV (prepared from 1 XHBSS containing Ca 2+), and after heating in a 37℃thermostat water bath, the two bottles were hung upside down at the height beside the biological super clean bench for use.
(2) The needle of the liquid inlet end of the disposable infusion set is opened and inserted into a bottle provided with the HBSS, the control valve of the infusion set is opened to enable liquid to be filled in the pipeline of the infusion set to discharge bubbles, and then the control valve is closed to fix the infusion set for standby.
(3) 100% Percoll (9 parts Percoll stock+1 part 10 XPBS), 70% Percoll (1 XPBS), 30% Percoll solution (1 XPBS) were prepared.
(4) And (5) weighing.
(5) Abdominal injection of pentobarbital sodium (50 mg/kg) places the mice under deep anesthesia.
(6) The method comprises the steps of sterilizing a mouse with alcohol, fixing the mouse in a tray, cutting a small section of medical adhesive tape to fix the limbs of the mouse, cutting a notch at the middle position 1-2cm away from the rear leg, cutting the notch along the edge of the body of the mouse until the chest is opened, lifting the skin, and leaking all liver tissues.
(7) The mouse viscera were gently stirred for searching the inferior vena cava and portal vein.
(8) The disposable infusion set needle is slowly inserted into the portal vein of the mouse, and the position is kept by hand to avoid needle discharge.
(9) Opening the control valve of the infusion apparatus to 1/2 of the position, then cutting off the tail end of the inferior vena cava, and then opening the control valve to the maximum.
(10) The HBSS is infused until the color of the liver changes from dark red to yellow, and then collagenase is infused for 8 minutes.
(11) After the end of perfusion digestion, liver tissue was removed and the covered blood was washed off with PBS and placed in a sterile 10cm dish containing 0.02% collagenase IV.
(12) The liver was gently stirred with forceps until it became a solution.
(13) The cell suspension was then filtered through a 200 mesh screen, and the filtered suspension was placed in two 50mL centrifuge tubes and trimmed with HBSS.
(14) 35G, centrifuged at 4℃for 3min, maximum braking.
(15) The supernatant was collected into a new 50mL centrifuge tube, trimmed with HBSS, 650g and centrifuged at 4℃for 7 min with maximum braking.
(16) The supernatant was discarded and 10mL of HBSS was pipetted into a micropipette to gently resuspend the pellet.
(17) Sequentially lightly spreading (from top to bottom) 70% percoll, 30% percoll and re-suspending the pellet in a new 50mL centrifuge tube.
(18) 1800G, centrifuging at 4deg.C for 15 min, and adjusting the speed of the centrifuge to low level (1/2).
(19) After centrifugation, the uppermost and lowermost layers were discarded, cells were left at the interface of 30% and 70% percoll and transferred to a new 50mL centrifuge tube, resuspended in HBSS, and centrifuged at 650g for 7min at 4 ℃ for maximum braking.
(20) After centrifugation, the supernatant was discarded, and the pellet was resuspended in 1mL of RPMI-1640 cell culture medium, and 10. Mu.L of the suspension and 10. Mu.L of 0.4% trypan blue were mixed for cell counting and viability identification.
(21) Cells were plated at appropriate densities in six well cell culture plates, cultured in a cell incubator for 15 minutes, the adherent cells were removed by pipetting, and fresh culture was added to the incubator for greater than 24 hours based on culturing in the cell incubator for the next experiment.
The observation of the cumulated cells under an inverted microscope shows that the freshly isolated cells are round, full and bright (20-fold objective) and contain few impurities. The cell growth state remained good after several days of culture, see fig. 1; from the images obtained by analysis of the data from flow cytometry assays (fig. 2), it was observed that isolated cumic cells could be double positive up to 91.4% pure, single positive up to 99%.
Example 2
This example is the same as example 1, and provides a method for efficiently obtaining mouse liver cells, except that in step (10), after the liver color changes from dark red to yellow, the disposable infusion set needle is slowly inserted into the inferior vena cava, collagenase is reversely infused instead, and collagenase is discharged from the portal vein for 8 minutes.
The observation of the cumic cells under an inverted microscope shows that the growth state of the cells is still good after a few days of culture, and the result is shown in figure 3; the resulting image was analyzed from the data measured by flow cytometry (see fig. 4). The extraction amount of the mouse liver cells is maintained at 5 multiplied by 10 6-8×106, and the purity of the isolated cells can be observed to be as high as 89.7% in double positive and as high as 98% in single positive.
Example 3
The embodiment provides a method for efficiently obtaining primary hepatocytes of a mouse, comprising the following steps:
(1) Two 50mL sterilized small-mouth glass bottles were prepared, one containing 40mL of sterile HBSS and the other containing 40mL of 0.04% (w/v) collagenase IV (prepared from 1 XHBSS containing Ca 2+), and after heating in a 37℃thermostat water bath, the two bottles were hung upside down at the height beside the biological super clean bench for use.
(2) The needle of the liquid inlet end of the disposable infusion set is opened and inserted into a bottle provided with the HBSS, the control valve of the infusion set is opened to enable liquid to be filled in the pipeline of the infusion set to discharge bubbles, and then the control valve is closed to fix the infusion set for standby.
(3) 100% Percoll (9 parts Percoll stock+1 part 10 XPBS), 70% Percoll (1 XPBS), 30% Percoll solution (1 XPBS) were prepared.
(4) And (5) weighing.
(5) Abdominal injection of pentobarbital sodium (50 mg/kg) places the mice under deep anesthesia.
(6) The method comprises the steps of sterilizing the mouse with alcohol, fixing the sterilized mouse in a tray, cutting a small section of medical adhesive tape to fix the limbs of the mouse, cutting a notch at the middle position 1-2cm away from the rear leg, cutting the notch along the edge of the body of the mouse until the chest is opened, lifting the skin, and leaking all liver tissues.
(7) The mouse viscera were gently stirred for searching the inferior vena cava and portal vein.
(8) The disposable infusion set needle is slowly inserted into the portal vein of the mouse, and the position is kept by hand to avoid needle discharge.
(9) Opening the control valve of the infusion apparatus to 1/2 of the position, then cutting off the tail end of the inferior vena cava, and then opening the control valve to the maximum.
(10) The HBSS is infused until the color of the liver changes from dark red to yellow, and then collagenase is infused for 8 minutes.
(11) After the end of perfusion digestion, liver tissue was removed and the covered blood was washed off with PBS and placed in a sterile 10cm dish containing 0.02% collagenase IV.
(12) The liver was gently stirred with forceps until it became a solution.
(13) The cell suspension was then filtered through a 200 mesh screen, and the filtered suspension was placed in two 50mL centrifuge tubes and trimmed with HBSS.
(14) 35G, centrifuged at 4℃for 3min, maximum braking.
(15) The pellet was collected, resuspended in 35g of HBSS and centrifuged again at4℃for 3 min with maximum braking.
(16) After centrifugation, the supernatant was discarded, and the pellet was resuspended in 1mL of RPMI-1640 cell culture medium, and 10. Mu.L of the suspension and 10. Mu.L of 0.4% trypan blue were mixed for cell counting and viability identification.
(17) Cells were plated at appropriate densities in six well cell culture plates, cultured overnight in a cell incubator, the adherent cells removed by pipetting, and fresh culture was added for greater than 24 hours based on culturing in the cell incubator for further experiments.
The observation of hepatocytes under an inverted microscope revealed that the freshly isolated cells appeared round, full and bright (20-fold objective) and contained fewer impurities. After the cells are subjected to the adherence culture for a few days, the cells are round, and the growth state is still good.
Comparative example 1 different centrifugation conditions
This comparative example 1 is identical to example 1, except that the centrifugation conditions in this comparative example are:
(1) 50g,3 min, 4℃and the supernatant was pipetted into a new 50mL centrifuge tube.
(2) 550G,5 min, 4 ℃, discard supernatant, HBSS resuspend pellet.
(3) Sequentially lightly spreading (from top to bottom) 70% percoll, 30% percoll and re-suspending the pellet in a new 50mL centrifuge tube.
(4) 80 G, centrifugation for 15 minutes at 4℃and the speed of the centrifuge was adjusted to low level (1/2) and then centrifugation was performed.
(5) After centrifugation, the uppermost layer and the lowermost layer were discarded, cells at the interface of 30% and 70% percoll were left and transferred to a new 50mL centrifuge tube, and counted and plated for viability assay by RPMI resuspension.
After centrifugation, no apparent cell layer appeared at the delamination.
Comparative example 2
This comparative example was conducted by performing in vitro digestion at 37℃for 15 to 30 minutes with 0.02% collagenase IV only after completion of collagenase perfusion digestion, and then performing filtration centrifugation. As a result, the cell count was found to be significantly increased, but the adherent cells were found to be decreased by changing the liquid after 15 minutes of culture, indicating that the increase in the enzyme digestion time resulted in significant cell damage and no in vitro digestion after perfusion was necessary.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. The extraction method of the primary liver cumic cells of the mice is characterized in that for the dead mice, HBSS buffer solution is used for in-situ perfusion from the portal vein of the mice, the mice are discharged from the tail end of the inferior vena cava, and the liver color is changed from dark red to yellow; using collagenase to replace buffer solution for perfusion, and maintaining the collagenase perfusion time for 6-10min; when reverse perfusion is performed by collagenase, the solution is perfused from the inferior vena cava and discharged from the portal vein; before pouring, placing the HBSS buffer solution and the collagenase solution in a water bath at the temperature of 35-38 ℃ for preheating for 4-7 minutes;
gradient centrifugation is carried out on the liver tissue obtained after digestion by using a Percoll separating liquid; the Percoll separating liquid is 30% Percoll and 70% Percoll;
the gradient centrifugation includes:
(1) Centrifuging the digested liver tissue for 35-70 g for 3-5 min;
(2) Collecting supernatant, and centrifuging for 650 g for 5-7 min;
(3) Discarding the supernatant, re-suspending the sediment by HBSS, separating cells by using Percoll separating liquid, centrifuging 1800 g for 15-20 minutes without braking;
(4) Taking two middle layer cells of Percoll separating liquid with different concentrations, re-suspending with HBSS, centrifuging 650g for 5-7 min;
The centrifugal temperature is 0-4 ℃; in step (1), step (2) or step (4), the centrifuge selects maximum braking;
Inoculating and culturing cells obtained by centrifugation for 12-16 minutes, and discarding non-adherent cells to obtain primary liver cumic cells of the mice;
The collagenase is collagenase IV with the mass volume ratio of 0.04%.
2. The method of claim 1, wherein the 70% Percoll solution is prepared from 7 parts 100% Percoll and 3 parts 1 x PBS buffer; the 30% Percoll solution was prepared from 3 parts 100% Percoll and 7 parts 1 XPBS; the 100% Percoll solution was prepared from 9 parts of Percoll stock solution and 1 part of 10×pbs buffer.
3. Use of the extraction method of claim 1 or 2 for the preparation of mouse primary liver cells.
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| 小鼠肝Kupffer细胞分离方法探讨;严茂林;王耀东;田毅峰;赖智德;周松强;邱福南;;福建医科大学学报;第42卷(第06期);第1.1.3，1.2.1-1.2.3节，表1 * |
| 改进原位循环灌流法分离小鼠肝细胞研究;潘君风;姜颖;贺福初;段敏;郭蔼光;;西北农林科技大学学报(自然科学版);第39卷(第11期);第184页左栏第3段，第2.1节 * |
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