CN113604419A - Extraction method of mouse primary liver Kupffer cells - Google Patents
Extraction method of mouse primary liver Kupffer cells Download PDFInfo
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Abstract
The invention relates to an extraction method of mouse primary liver Kupffer cells. The extraction method provided by the invention comprises the following steps: a disposable infusion device is adopted for buffer solution perfusion, and the collagenase perfusion is combined for liver digestion treatment; after the mouse liver is subjected to venous perfusion digestion, the liver envelope is torn up to filter liver tissues; and after filtration, centrifuging for many times to obtain 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 the mouse liver Kupffer cells, and the extraction amount of the mouse liver Kupffer cells is maintained at 5 multiplied by 106‑8×106And (4) respectively. The method provided by the invention has the advantages that the used equipment is simple and easy to obtain, great convenience is provided for a laboratory without a peristaltic pump, the cost of the used disposable transfusion device is low, and the sterile operation requirement in 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 feasible extraction method of mouse primary liver Kupffer cells with universality.
Background
The liver is an important metabolic organ and is composed of a plurality of cells, wherein hepatic parenchymal cells account for about 65% of the total number of the cells, and non-parenchymal cells such as liver endothelial cells, stellate cells, kupffer cells and liver-related lymphocytes account for 35%. Meanwhile, the liver is also an important immunocompetent organ which is also called as a lymphoid tissue-like organ, 90 percent of mononuclear-macrophages of the organism are in the liver, and Kupffer cells in the liver are the main component of the mononuclear-macrophages of the organism and account for 25 percent of the total number of the cells in the liver; most researches on extracting primary Kupffer cells adopt a non-perfusion method, namely a mechanical method to cut tissues into small pieces, and then the cells are separated from the tissues by mechanical means such as extrusion, shearing, vibration and the like so as to obtain the separated cells. Another non-perfusion method for extracting cells is to shear liver tissue and then use collagenase or pancreatin to digest and destroy the bridges or fibrous components among cells for cell separation. However, although this non-perfusion technique is simple and easy to operate, the problems of incomplete digestion during cell separation, multicellular aggregates in the separated cells, and the like often occur, and the demand of researchers cannot be met well.
In 1969, a perfusion method is introduced into Berry and Friend to extract primary cells, and a perfusion process can enable digestive juice to be in contact with liver tissues more fully, so that the separation efficiency is improved, and the activity and the number of the separated cells are greatly improved. Perfusion methods have been developed in an effort to improve by various researchers, such as in situ collagenase perfusion, semi-in situ collagenase perfusion, and ex vivo collagenase perfusion. In situ collagenase perfusion is commonly used, but it relies on peristaltic pumps, which is undoubtedly wasteful of laboratory equipment in laboratories that occasionally extract several primary cells for study, and often difficult to achieve the desired perfusion effect because of the thinness of the veins.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a simple and feasible method for extracting primary mouse liver histiocytes with universality.
In the prior art, the extraction method of rat primary liver cells is often referred to, and the extraction of the rat primary liver cells is performed by combining in-situ perfusion collagenase with a peristaltic pump. Because the portal vein of the mouse is very thin, the perfusion pressure is not easy to control when a peristaltic pump is adopted, and for a laboratory which extracts 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 easier to implement.
Therefore, the invention provides a method for extracting mouse primary liver Kupffer cells, which comprises the steps of using buffer solution to perfuse from the portal vein of a mouse in situ, discharging from the tail end of the inferior vena cava, and changing the color of the liver from dark red to yellow; and (3) performing perfusion by using collagenase instead of the buffer solution, wherein the collagenase perfusion time is kept for 6-10 min.
The extraction method provided by the invention is simple and feasible, does not depend on a peristaltic pump, has moderate perfusion pressure, and does not consider the survival condition of the mouse in the perfusion process, namely the heart of the mouse keeps beating or stops beating in the perfusion process, so that the red blood cells are not influenced to be discharged along with the buffer solution.
The invention also finds that in the experiment, the situation that the portal vein is punctured by an injector occasionally occurs when collagenase perfusion is carried out due to the small diameter of the portal vein of the mouse. In order to solve the problem, in the extraction method provided by the invention, the liver tissue is digested by adopting the forward in-situ perfusion of a buffer solution combined with the reverse in-situ perfusion of collagenase; i.e., when collagenase is used for perfusion, it can be perfused from the inferior vena cava and drained from the portal vein.
The invention finds that when collagenase is used for in-situ perfusion, in addition to forward in-situ perfusion (perfusion from the portal vein and drainage from the inferior vena cava) of collagenase, in order to overcome the defects that the portal vein of a mouse is too thin, the in-situ perfusion is not easy to operate and the like, reverse perfusion (perfusion from the inferior vena cava and drainage from the portal vein) of collagenase can be also used.
The invention realizes the cell extraction with high activity and high yield by combining the method of forward buffer solution in-situ perfusion and reverse collagenase in-situ perfusion, and provides a new way for extracting primary liver cells.
Based on the method, the invention provides an extraction method of mouse primary liver Kupffer cells, which uses Percoll separation liquid to carry out gradient centrifugation on liver tissues obtained after digestion by the extraction method; the Percoll separating medium is 30% of Percoll and 70% of Percoll.
Specifically, in the method for extracting liver kupffer cells provided by the invention, the gradient centrifugation comprises:
(1) centrifuging the digested liver tissue for 3-5 minutes at 35-70 g;
(2) collecting supernatant, and continuing to centrifuge for 650g for 5-7 minutes;
(3) abandoning the supernatant, resuspending the precipitate with HBSS, separating cells by using Percoll separating medium, centrifuging for 1800g for 15-20 minutes without braking; before starting the centrifugal machine, the speed of the centrifugal machine is increased and reduced to the lowest level, so that the damage of density gradient caused by too high lifting speed is avoided;
(4) the middle layer cells of two Percoll separating solutions with different concentrations are taken, resuspended by HBSS, and centrifuged for 5-7 minutes at 650 g.
The invention utilizes a closed passage formed among the liver, the portal vein and the inferior vena cava, utilizes buffer solution to perform forward perfusion combined with collagenase forward or reverse perfusion for digestion, and then obtains the high-purity high-yield liver primary Kupffer cells through multiple times of centrifugation.
In the method for extracting the kupffer cells of the liver, the cells obtained by centrifugation are inoculated to a six-well plate and then cultured for 12-16 minutes, and then the non-adherent cells are discarded. The invention discovers that because liver Kupffer cells are easy to adhere to the wall, after the liver Kupffer cells are cultured for 12-16 minutes, nonadherent cells are mostly non-Kupffer cells, and the purity of the extracted Kupffer cells can be increased by discarding the nonadherent cells.
In the method for extracting the kupffer cells of the liver, provided by the invention, the centrifugal temperature is 0-4 ℃; in step (1), step (2) or step (4), the centrifuge selects the maximum braking. Before starting the centrifuge, the speed of the centrifuge should be increased and decreased to the highest level.
In the method for extracting liver Kupffer cells provided by the invention, the 70% Percoll solution is prepared by 7 parts of 100% Percoll and 3 parts of 1 XPBS buffer solution; the 30% Percoll solution is prepared by 3 parts of 100% Percoll and 7 parts of 1 XPBS; the 100% Percoll solution is prepared from 9 parts Percoll stock solution and 1 part 10 XPBS buffer solution.
In the method for extracting the kupffer cells of the liver, the HBSS buffer solution and the collagenase solution are placed in a water bath at 35-38 ℃ for preheating for 4-7 minutes before perfusion.
According to the understanding of the skilled person, the invention also claims the application of the extraction method in the culture of the mouse liver tissue cells and in the improvement of the purity of the mouse primary liver tissue cells.
The invention has the beneficial effects that:
(1) the instruments and materials involved in the invention are common in laboratories, and are easy to obtain and low in cost; the survival condition of the mice is not considered in the treatment process of the invention.
(2) The invention can remove liver parenchymal cells as much as possible on the basis of ensuring the purity of the cells, and separates out the primary liver cells of the mice with high yield.
(3) The purity of the cells is identified by flow cytometry, the extracted, washed and purified cells are stained by F4/80 and CD11b flow antibodies, and the purity of the extracted mouse primary liver Kupffer cells is over 98 percent by flow cytometry detection.
Drawings
FIG. 1 is a microscopic image of Kupffer cells isolated from the liver in example 1 of the present invention.
FIG. 2 is a graph showing the results of purity measurement of liver Kupffer cells isolated in example 1 of the present invention.
FIG. 3 is a microscopic image of Kupffer cells isolated from liver in example 2 of the present invention.
FIG. 4 is a graph showing the results of purity measurement of liver Kupffer cells isolated in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, test 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 otherwise specified.
The mice used in the invention are all selected from healthy C57BL/6 male mice with 6-8 weeks old and 18-25 g of body weight in SPF-grade animal rooms, and are cleaned by 100 ml of 70% ethanol and then subjected to subsequent tests in a biological super clean bench.
Experimental materials, instruments used in the following examples:
main reagent consumables: healthy mouse, collagenase IV (Invit)rogen), Ca-containing2+1 × HBSS solution (Macgene), 1 × PBS (Macgene), 10 × PBS (Macgene), Percoll stock solution (GE Health), 0.4% (w/v) trypan blue solution (Sciencell), RPMI 1640 culture solution (Hyclone), fetal bovine serum (Gibco), diabody (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 (20mL), disposable 200 mesh cell screen (FALCON).
The main apparatus is as follows: cell culture incubator (Thermo Fisher); bench centrifuge (Thermo Fisher); a constant temperature incubator (Taicang scientific and education instrument factory); biological super clean bench (SW-CJ-1FD, Suzhou clarification facility Co., Ltd.); optical microscopy (olympus CKX 41); an electric heating constant temperature water bath (Jiangsu province JintanCity Ronghua apparatus manufacturing Co., Ltd.); different size micropipettes (Thermo Fisher).
Example 1
The embodiment provides a method for efficiently obtaining mouse liver Kupffer cells, which comprises the following steps:
(1) two 50mL sterile vials, one containing 40mL sterile HBSS and the other containing 40mL 0.04% (w/v) collagenase IV (prepared from Ca-containing solution) were prepared2+Prepared by 1 × HBSS), heating in a constant-temperature water bath at 37 ℃, and inversely hanging the two bottles at a high position beside a biological super clean bench for later use.
(2) The needle head at the liquid inlet end of the disposable infusion apparatus is opened and inserted into a bottle with HBSS, the control valve of the infusion apparatus is opened to fill liquid in the infusion apparatus pipeline to discharge air bubbles, and then the control valve is closed to fix the infusion apparatus for later use.
(3) 100% Percoll (9 parts Percoll stock solution +1 part 10 XPBS), 70% Percoll (prepared from 1 XPPBS), 30% Percoll solution (prepared from 1 XPPBS) were prepared.
(4) And (5) weighing.
(5) Mice were placed under deep anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg).
(6) Disinfecting a mouse with alcohol, fixing the mouse in a tray, cutting a small section of medical adhesive tape to fix four limbs of the mouse, cutting a gap at the middle position 1-2cm away from the hind legs, cutting the gap along the body edge of the mouse to the thorax, and lifting the skin to leak all liver tissues.
(7) The mouse organs were gently flicked to look for the inferior vena cava and portal vein.
(8) Slowly inserting the needle of the disposable infusion set into the portal vein of the mouse, and keeping the position by hands to avoid needle withdrawal.
(9) The control valve of the infusion set is opened to 1/2, and then the tail end of the vena cava is cut off, and then the control valve is opened to the maximum.
(10) The HBSS is perfused until the color of the liver changes from dark red to yellow, then collagenase is perfused, and the perfusion is continued for 8 minutes.
(11) After completion of perfusion digestion, liver tissue was removed and the outer cover blood was washed out with PBS and placed in sterile 10cm petri dishes containing 0.02% collagenase IV.
(12) The liver was gently agitated 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) Centrifuge at 35g, 4 ℃ for 3 minutes, brake max.
(15) The supernatant was collected into a new 50mL centrifuge tube, trimmed with HBSS, 650g, centrifuged at 4 ℃ for 7 minutes, and braked maximally.
(16) Discard the supernatant and gently resuspend the pellet by pipetting 10mL of HBSS with a micropipette.
(17) Gently (from top to bottom) spread 70% Percoll, 30% Percoll in a new 50mL centrifuge tube, and resuspend the pellet.
(18)1800g, centrifuging for 15 minutes at 4 ℃, adjusting the speed of the centrifuge to be low (ascending 2 and descending 1) and centrifuging.
(19) After centrifugation, the uppermost layer and the lowermost layer were discarded, and the cells at the junction of 30% Percoll and 70% Percoll were left and transferred to a new 50mL centrifuge tube, resuspended with HBSS, and then centrifuged at 650g at 4 ℃ for 7 minutes with maximum braking.
(20) After centrifugation, the supernatant was discarded, 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 and used for cell counting and viability determination.
(21) And (3) paving the cells in a six-hole cell culture plate at a proper density, culturing in a cell culture box for 15 minutes, then changing the culture solution to remove the non-adherent cells, adding a fresh culture medium to the cell culture box, culturing for more than 24 hours, and carrying out the next experiment.
When the kupffer cells are observed under an inverted microscope, the cells which are just separated are round, full and good in transparency (20 times of objective lenses), and contain fewer impurities. The growth state of the cells was still good after several days of culture, see FIG. 1; from the images obtained by analyzing the data determined by flow cytometry (fig. 2), it can be observed that the purity of the isolated kupffer cells can be as high as 91.4% double positive and as high as 99% single positive.
Example 2
This example is the same as example 1, and provides a method for obtaining mouse liver kupffer cells with high efficiency, except that in step (10), after the liver color changes from dark red to yellow, the disposable infusion needle is slowly inserted into the inferior vena cava, and collagenase is reversely perfused, and is discharged from the portal vein, and the perfusion lasts for 8 minutes.
The observation of the kupffer cells under an inverted microscope shows that the growth state of the cells is still good after the cells are cultured for several days, which is shown in figure 3; the resulting images were analyzed according to flow cytometry determined data (see fig. 4). The extraction amount of mouse liver Kupffer cells is maintained at 5 × 106-8×106In addition, the purity of the separated kupffer cells can be observed to be up to 89.7 percent in a double positive mode and up to 98 percent in a single positive mode.
Example 3
The embodiment provides a method for efficiently obtaining primary hepatocytes of a mouse, which comprises the following steps:
(1) two 50mL sterile vials, one containing 40mL sterile HBSS and the other containing 40mL 0.04% (w/v) collagenase IV (prepared from Ca-containing solution) were prepared2+Prepared by 1 × HBSS), heating in a constant-temperature water bath at 37 ℃, and inversely hanging the two bottles at a high position beside a biological super clean bench for later use.
(2) The needle head at the liquid inlet end of the disposable infusion apparatus is opened and inserted into a bottle with HBSS, the control valve of the infusion apparatus is opened to fill liquid in the infusion apparatus pipeline to discharge air bubbles, and then the control valve is closed to fix the infusion apparatus for later use.
(3) 100% Percoll (9 parts Percoll stock solution +1 part 10 XPBS), 70% Percoll (prepared from 1 XPPBS), 30% Percoll solution (prepared from 1 XPPBS) were prepared.
(4) And (5) weighing.
(5) Mice were placed under deep anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg).
(6) Disinfecting the 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 gap at the middle position 1-2cm away from the hind legs, cutting the gap along the body edge of the mouse to the thorax, and lifting the skin to leak all liver tissues.
(7) The mouse organs were gently flicked to look for the inferior vena cava and portal vein.
(8) Slowly inserting the needle of the disposable infusion set into the portal vein of the mouse, and keeping the position by hands to avoid needle withdrawal.
(9) The control valve of the infusion set is opened to 1/2, and then the tail end of the vena cava is cut off, and then the control valve is opened to the maximum.
(10) The HBSS is perfused until the color of the liver changes from dark red to yellow, then collagenase is perfused, and the perfusion is continued for 8 minutes.
(11) After completion of perfusion digestion, liver tissue was removed and the outer cover blood was washed out with PBS and placed in sterile 10cm petri dishes containing 0.02% collagenase IV.
(12) The liver was gently agitated 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) Centrifuge at 35g, 4 ℃ for 3 minutes, brake max.
(15) The pellet was collected, resuspended by adding HBSS, 35g, and centrifuged again at 4 ℃ for 3 minutes with maximum braking.
(16) After centrifugation, the supernatant was discarded, 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 and used for cell counting and viability determination.
(17) And (3) paving the cells in a six-hole cell culture plate at a proper density, culturing in a cell culture box overnight, then changing the culture solution to remove the non-adherent cells, adding a fresh culture medium into the cell culture box, culturing for more than 24 hours, and carrying out the next experiment.
When the hepatocytes are observed under an inverted microscope, the freshly isolated cells are round, full and have good transparency (20 times objective lens) and contain fewer impurities. Cells were round after several days of adherent culture and the growth state was still good.
Comparative example 1 different centrifugation conditions
Comparative example 1 is the same as example 1 except that the centrifugation conditions in this comparative example are:
(1)50g, 3 min, 4 ℃ and the supernatant was aspirated into a new 50mL centrifuge tube.
(2)550g, 5 min, 4 ℃, discard the supernatant and resuspend the pellet in HBSS.
(3) Gently (from top to bottom) spread 70% Percoll, 30% Percoll in a new 50mL centrifuge tube, and resuspend the pellet.
(4)800g, centrifuging for 15 minutes at 4 ℃, adjusting the speed of the centrifuge to be low (ascending 2 and descending 1) and then centrifuging.
(5) And (4) discarding the uppermost layer and the lowermost layer after the centrifugation is finished, leaving cells at the junction of 30% Percoll and 70% Percoll, transferring the cells to a new 50mL centrifuge tube, and performing counting and plating vitality identification by using RPMI (recombinant plasma amplification) heavy suspension.
After the centrifugation was completed, no distinct cell layer appeared at the delamination site.
Comparative example 2
This comparative example was the same as example 2 except that the collagenase was digested with 0.02% collagenase IV at 37 ℃ for 15 to 30 minutes in vitro, and then filtered and centrifuged. The result shows that the cell amount is obviously increased during cell counting, but adherent cells are reduced when the solution is changed after the culture for 15 minutes, which indicates that the increase of the enzyme digestion time causes obvious cell damage and the in vitro digestion after perfusion is not needed.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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| 严茂林;王耀东;田毅峰;赖智德;周松强;邱福南;: "小鼠肝Kupffer细胞分离方法探讨", 福建医科大学学报, vol. 42, no. 06, pages 526 - 1 * |
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