CN113604419A - Extraction method of mouse primary liver Kupffer cells - Google Patents
Extraction method of mouse primary liver Kupffer cells Download PDFInfo
- Publication number
- CN113604419A CN113604419A CN202110706246.0A CN202110706246A CN113604419A CN 113604419 A CN113604419 A CN 113604419A CN 202110706246 A CN202110706246 A CN 202110706246A CN 113604419 A CN113604419 A CN 113604419A
- Authority
- CN
- China
- Prior art keywords
- cells
- percoll
- liver
- perfusion
- extraction method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000004185 liver Anatomy 0.000 title claims abstract description 44
- 210000001865 kupffer cell Anatomy 0.000 title claims abstract description 34
- 238000000605 extraction Methods 0.000 title claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 54
- 230000010412 perfusion Effects 0.000 claims abstract description 50
- 102000029816 Collagenase Human genes 0.000 claims abstract description 33
- 108060005980 Collagenase Proteins 0.000 claims abstract description 33
- 229960002424 collagenase Drugs 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 27
- 210000005228 liver tissue Anatomy 0.000 claims abstract description 18
- 239000007853 buffer solution Substances 0.000 claims abstract description 13
- 230000029087 digestion Effects 0.000 claims abstract description 9
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 22
- 210000003240 portal vein Anatomy 0.000 claims description 16
- 238000005119 centrifugation Methods 0.000 claims description 15
- 238000011065 in-situ storage Methods 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 10
- 210000001631 vena cava inferior Anatomy 0.000 claims description 10
- 238000004113 cell culture Methods 0.000 claims description 8
- 230000001464 adherent effect Effects 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims 3
- 238000001802 infusion Methods 0.000 abstract description 15
- 230000002572 peristaltic effect Effects 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 238000001914 filtration Methods 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 32
- 239000008188 pellet Substances 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 210000005229 liver cell Anatomy 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 210000004738 parenchymal cell Anatomy 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 239000002390 adhesive tape Substances 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000001174 ascending effect Effects 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000038 chest Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229960001412 pentobarbital Drugs 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 210000004500 stellate cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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.
Claims (10)
1. A method for extracting mouse primary liver Kupffer cells is characterized in that buffer solution is used for in-situ perfusion from a portal vein of a mouse and is discharged from the tail end of a inferior vena cava, and the color of the liver changes 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.
2. The extraction method according to claim 1, wherein the liver tissue is digested by forward in situ perfusion of the buffer combined with reverse in situ perfusion of collagenase; when collagenase is used for retrograde perfusion, perfusion is from the inferior vena cava and drainage is from the portal vein.
3. The extraction method according to claim 2, wherein the liver tissue obtained after digestion is subjected to gradient centrifugation using Percoll separating medium; the Percoll separating medium is 30% of Percoll and 70% of Percoll.
4. The extraction method according to claim 3, characterized in that said 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;
(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.
5. The extraction method according to claim 4, wherein the non-adherent cells are discarded after the cells obtained by centrifugation are inoculated and cultured for 12 to 16 minutes.
6. The extraction process according to claim 5, wherein the centrifugation temperature is 0 to 4 ℃; in step (1), step (2) or step (4), the centrifuge selects the maximum braking.
7. The extraction method according to claim 6, wherein the 70% Percoll solution is prepared from 7 parts of 100% Percoll and 3 parts of 1 XPBS buffer; 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.
8. The method of claim 7, wherein the HBSS buffer and the collagenase solution are preheated in a water bath at 35-38 ℃ for 4-7 minutes before perfusion.
9. Use of the extraction method of any one of claims 1-8 in mouse liver tissue cell culture.
10. Use of the extraction method of any one of claims 1 to 8 for increasing the purity of mouse primary liver tissue cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110706246.0A CN113604419B (en) | 2021-06-24 | 2021-06-24 | Method for extracting mouse primary liver cumic cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110706246.0A CN113604419B (en) | 2021-06-24 | 2021-06-24 | Method for extracting mouse primary liver cumic cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604419A true CN113604419A (en) | 2021-11-05 |
| CN113604419B CN113604419B (en) | 2024-06-21 |
Family
ID=78303694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110706246.0A Active CN113604419B (en) | 2021-06-24 | 2021-06-24 | Method for extracting mouse primary liver cumic cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604419B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114908040A (en) * | 2022-06-21 | 2022-08-16 | 贵州医科大学 | SD rat liver kupffer cell and liver stellate cell primary separation and culture method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109385394A (en) * | 2018-11-02 | 2019-02-26 | 大连理工大学 | A kind of method of extracting and developing and the androgynous xenogenesis liver primary cell of culture |
-
2021
- 2021-06-24 CN CN202110706246.0A patent/CN113604419B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109385394A (en) * | 2018-11-02 | 2019-02-26 | 大连理工大学 | A kind of method of extracting and developing and the androgynous xenogenesis liver primary cell of culture |
Non-Patent Citations (4)
| Title |
|---|
| 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 * |
| 姚一楠;卢珊;李和权;周建英;: "小鼠肝脏原位持续灌流方法的建立及其在肝脏非实质细胞分离中的应用", 中国药理学通报, vol. 28, no. 10, pages 1475 - 1477 * |
| 李亮;彭琼;戴夫;: "SD大鼠肝脏枯否细胞分离方法改进研究", 安徽医学, vol. 36, no. 03, pages 1 * |
| 潘君风;姜颖;贺福初;段敏;郭蔼光;: "改进原位循环灌流法分离小鼠肝细胞研究", 西北农林科技大学学报(自然科学版), vol. 39, no. 11, pages 184 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114908040A (en) * | 2022-06-21 | 2022-08-16 | 贵州医科大学 | SD rat liver kupffer cell and liver stellate cell primary separation and culture method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604419B (en) | 2024-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10537596B2 (en) | Bone marrow adipose portion isolation device and methods | |
| TWI836505B (en) | A method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord | |
| CN114317443B (en) | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN107475190B (en) | Method for clinical-level efficient preparation and cryopreservation of fat SVF cells and application thereof | |
| CN108300688B (en) | Primary hepatocyte separation and culture method | |
| CN114292816B (en) | Lung cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| US20180155691A1 (en) | Mechanical apparatus and method for isolating stromal vascular fraction | |
| US20220267712A1 (en) | Cell isolation for use in automated bioreactors | |
| CN112029717B (en) | Serum-free in vitro domestication culture of human mesenchymal stem cells | |
| CN113604419B (en) | Method for extracting mouse primary liver cumic cells | |
| CN107354130B (en) | Human placenta chorion mesenchymal stem cell separation method | |
| CN111925985B (en) | Domestication culture method of mesenchymal stem cells | |
| CN111235100B (en) | Culture method of human umbilical cord blood mesenchymal stem cells | |
| CN110117570B (en) | Primary culture method of rheumatoid arthritis synovial fibroblasts | |
| CN108660108A (en) | A kind of method enhancing umbilical cord mesenchymal stem cells immunoregulation capability | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN108774630A (en) | A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell | |
| CN112342182B (en) | Method for efficiently separating mouse liver bile duct cells | |
| CN117660325B (en) | Culture medium for preparing umbilical cord blood MSC and method thereof | |
| CN114134122B (en) | Macrophage-mediated mesenchymal stem cell directional differentiation culture method | |
| CN111548991B (en) | Technical method for extracting mouse skull osteoclast precursor cells | |
| CN113957035B (en) | Duck embryo primary liver cell separation culture method | |
| TWI782492B (en) | A method for obtaining the small p stem cells | |
| CN108130307B (en) | Improved method for enriching islet cells of mice | |
| US20240301354A1 (en) | System and method for converting adipose derived mesenchymal stems cells to hematopoietic stem/progenitor cells and differentiating into blood cells and applications of same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |