CN116694554A - Liver cell separation, culture and cryopreservation method for littoral-like red-rooted salvia - Google Patents
Liver cell separation, culture and cryopreservation method for littoral-like red-rooted salvia Download PDFInfo
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- 235000017276 Salvia Nutrition 0.000 title description 2
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for separating, culturing and freezing preservation of liver cells of climbing stone, which is suitable for the technical field of bioengineering, healthy climbing stone is selected for dissection, the liver is taken out in an HM culture medium under the aseptic condition, the liver is cut into blocks after being washed for three times, cell suspension is obtained by filtering with a cell sieve after trypsin digestion, after counting under a microscope, the liver is inoculated into a 24-hole cell culture plate with a certain concentration, and DM culture medium is added for culture; and then, adopting a cryopreservation agent to carry out liquid nitrogen cryopreservation on the cultured liver cells to obtain an optimal preservation system for the liver cells of the herba lycopodii. The liver cell culture method of the macula littoralis adopted by the invention has strong repeatability and good effect; the invention establishes a stable primary culture system of liver cells, uses a special culture medium, improves the activity of the extracted primary culture cells of the liver, provides a cell model for further developing the research of the liver functions of the climbing stone, and has important practical significance for the conservation of the climbing stone.
Description
Technical Field
The invention discloses a method for separating, culturing and cryopreserving liver cells of a littoralis, which is applicable to the technical field of bioengineering.
Background
The liver is the largest digestive gland in fish body, and has bile synthesis, lipid metabolism, sugar metabolism, hormone metabolism and detoxification functions. In recent years, the intensive aquaculture industry in China develops rapidly, and meanwhile, a serious problem is also exposed: in production, the problems of intensified feeding, drug abuse, unbalanced nutritional composition of feed, addition of spoilage or forbidden substances and the like often occur, so that aquatic animal diseases frequently occur, wherein the diseases characterized by liver injury and liver lesion induction are the greatest. Since the liver is the most main metabolic organ in the fish body, the damage or the lesion of the liver often leads to metabolic dysfunction and disease resistance reduction of the aquatic animal body, the outbreak of secondary infectious diseases and the surprise of syndromes are extremely easy to cause, and the continuous healthy development of the intensive aquaculture industry is seriously threatened. The most common manifestation of fish liver lesions is fatty liver, especially the most prominent nutritional fatty liver hazard, has attracted widespread attention by aquaculture workers and breeders.
Disclosure of Invention
The invention aims to provide a method for separating, culturing and freezing preservation of liver cells of gliocladium littoralis, which is simple, and can obtain liver cultured cells with high quantity, high purity and stable activity by utilizing the culture solution suitable for primary culture of the liver cells of the gliocladium littoralis and the method for separating the liver cells.
According to the physiological characteristics of the gliptin, the invention specially prepares the culture solution suitable for the culture of the gliptin liver cells, improves the separation process of the liver cells, and can obtain a large number of liver primary cultured cells with stable activity.
In order to achieve the above object, the present invention adopts the following technical measures:
a method for separating, culturing and cryopreserving liver cells of a glyptophana huashanensis, comprising the following steps:
(1) Cleaning tissue blocks of 0.1-1.0mm of fresh liver of healthy stone-climbing with HM culture medium;
in the above scheme, preferably, the tissue block is a tissue block which is obtained by cleaning fresh liver tissue of healthy stone-like green-leaf silkworms with HM culture medium and then cutting the cleaned liver tissue into 0.1-1.0 mm;
(2) Adding HM culture medium containing 0.1-1mM EDTA into the tissue block, gently shaking and mixing, standing until the tissue block is settled at the bottom, and sucking out the culture medium;
(3) Adding HM culture medium containing 100-1000unit/ml Collagenase IV and 0.01-0.1mg/ml DNase II into tissue block, and digesting in water bath at 28deg.C for 15-60 min;
(4) Removing HM culture medium containing Collagenase IV and DNase II, adding HM culture medium, and blowing off hepatocytes with pipette;
(5) Filtering and separating and removing the un-blown cells by using a 22-100 mu M pore size filter screen, and collecting the scattered and separated cells;
(6) Centrifuging at 200-1000rpm and 4deg.C for 5-10min to collect liver cells, removing supernatant medium with a pipette, and adding 10-50ml DM medium to resuspend cells;
(7) Sucking 10-50. Mu.l of the cell suspension, adding 10-100. Mu.l of the phenol blue-stained cells, and counting the cells under a microscope;
(8) Diluting cells to 1.0-5.0X10 with DM Medium 6 Inoculating cells/ml into culture dish with diameter of 35mm 1.0-5.0X10 6 Cells, 28 ℃,5% CO 2 Culturing the cells in an incubator for 6-36 hours;
(9) Density is 2.0X10 with cryopreservative 6 Gradient cooling is carried out on cells/ml of the gliocladium littoralis, and the cells/ml of the gliocladium littoralis are frozen and stored in liquid nitrogen.
Further, the formula of the HM culture medium comprises:
KCl:1.0-1.0mg/ml,K 2 HPO 4 :0.01-0.1mg/ml；NaCl:1.0-10.0mg/ml；NaH 2 PO 4 :0.01-0.1mg/ml；Glucose:1.0-10.0mg/ml；HEPES:1.0-10.0mg/ml；NaHCO 3 0.1-1.0mg/ml; penicillin 50-100U/ml, streptomycin: 50-100 mug/ml.
Still further, the formulation of the DM medium and the cryopreservation agent comprises:
DMEM/F-12:1.0-10.0mg/ml; naHCO3 1.0-10.0mg/ml; BSA 1.0-10.0mg/ml; penicillin 50-100U/ml, streptomycin: 50-100 mug/ml.
Cryopreservation agent: DMSO concentration was 10% and serum concentration was 90%.
Compared with the prior art, the invention has the following advantages:
1. the invention explores a method suitable for separating, culturing and freezing preservation of the liver cells of the coralloides, and obtains the liver cells of the coralloides with a large quantity and high purity; the culture medium prepared according to the characteristics of the liver cells of the glabrous greenbrier rhizome is used for improving the activity of the extracted liver culture cells, providing a cell model for further developing liver function research of the glabrous greenbrier rhizome, and having important practical significance for the conservation of the glabrous greenbrier rhizome. .
2. The culture medium used in the invention is prepared by adopting the own formula, so that not only can the vitality of liver cells of the glabrous sarcandra herb be improved, but also the cost is well controlled, and the culture medium can meet the requirement of mass culture.
Drawings
FIG. 1 shows liver cell pattern of Gekko Swinhonis
FIG. 2 is a graph showing the effect of NKB (1. Mu.M), IGF-1 (0.1. Mu.M), EGF (0.1. Mu.M) and SLα (0.1. Mu.M) on the expression level of beta-actin and GHR mRNA in cultured cells of stone-climbing liver.
Detailed Description
The technical scheme of the invention is conventional in the field unless specifically stated otherwise, and the reagents or materials are commercially available unless specifically stated otherwise.
Example 1
A method for separating, culturing and cryopreserving liver cells of a glyptophana huashanensis, comprising the following steps:
(1) Cleaning fresh liver tissue of healthy littermate with HM culture medium;
(2) 3g of cleaned liver tissue is cut into tissue blocks of 0.5 mm;
(3) The minced tissue pieces were transferred to 50ml centrifuge tubes and washed three times with HM media;
(4) 30ml of HM culture medium containing 1mM EDTA is added into the tissue block, the tissue block is settled at the bottom after being gently mixed by shaking, and the culture medium is sucked by a suction pipe;
(5) 30ml HM medium (500 unit/ml Collagenase IV and 0.01mg/ml DNase I) was added to the tissue mass and digested in a water bath at 28℃for 30 min;
(6) Removing HM culture medium containing Collagenase IV and DNase II by pipette, adding 20ml new HM culture medium, and blowing off hepatocytes by using 2ml pipette for 30 times;
(7) Filtering and separating and removing the un-blown cells by using a 40 mu M pore size filter screen, and collecting the scattered and separated cells;
(8) Centrifugation at 480rpm at 4℃for 5min was performed to collect liver cells, the supernatant medium was removed using a pipette, and 30ml of DM medium was added to resuspend the cells;
(9) Mu.l of the cell suspension was aspirated, 90. Mu.l of the cells were stained with phenol blue and then counted under a microscope;
(10) Cells were diluted to 3.5X10 using DM medium 6 cells/ml, and inoculating 3.5X10 s to a culture dish with a diameter of 35mm 6 Cells, 28 ℃,5% CO 2 Culturing the cells in an incubator for 24 hours;
the formula of the HM culture medium comprises the following components:
KCl:0.4mg/ml,K 2 HPO 4 :0.06mg/ml；NaCl:8.0mg/ml；NaH 2 PO 4 :0.04788mg/ml；Glucose:1.0mg/ml；HEPES:2.383mg/ml；NaHCO 3 0.35mg/ml; penicillin 100U/ml, streptomycin: 100. Mu.g/ml;
the formula of the DM culture medium comprises:
DMEM/F-12:9.6mg/ml; naHCO3, 1.2mg/ml; BSA 2.0mg/ml; penicillin 100U/ml, streptomycin: 100. Mu.g/ml.
Example 2
Growth of liver cells of the glabrous greenbrier:
the liver cells of the glabrous sarcandra herb are in a one-to-one separation state on the first day of being inoculated to a cell culture plate, the cells are round and single in shape (A in figure 1), and the number and the shape of the cells are not obviously changed after 24 hours of culture; the cells still exhibited a circular separation state (B in fig. 1); the results show that the liver cells of the gliptin are maintained in a relatively stable state in a culture system.
Example 3
Liver cells of stone climbing obtained in the method of example 1 were treated with NKB (1. Mu.M), IGF-1 (0.1. Mu.M), EGF (0.1. Mu.M) and SLα (0.1. Mu.M), respectively, for 24 hours; removing the culture medium, adding 0.5ml Trizol into each hole to collect cells for extracting RNA, carrying out reverse transcription to obtain cDNA, and detecting the regulation and control effects of the four polypeptides on related functional genes in the liver cells of the Uygorskia littoralis by quantitative PCR. The detection analysis shows that the expression quantity of the reference gene beta-actin among the groups is not significantly different (the left graph of fig. 2), which shows that the cell culture system is relatively stable and can meet the experiment of drug treatment. Furthermore, NKB has no effect on Growth Hormone Receptor (GHR) mRNA expression in hepatocytes, EGF and slα can inhibit GHRmRNA expression in hepatocytes, while IGF-I can significantly promote GHR mRNA expression in hepatocytes (right panel of fig. 2); in addition, the standard deviation between four replicates in each group is controlled within 10%, and the results show that the cultured liver cells keep good activity, and the cells in each hole are relatively uniform in growth, so that the method is suitable for being used as a cell model for researching the liver of the glabrous.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (5)
1. A method for separating, culturing and freezing and preserving liver cells of a gliotope glabra is characterized in that: comprising the following steps:
(1) Cleaning tissue blocks of 0.1-1.0mm of fresh liver of healthy stone-climbing with HM culture medium;
(2) Adding HM culture medium containing 0.1-1mM EDTA into the tissue block, gently shaking and mixing, standing until the tissue block is settled at the bottom, and sucking out the culture medium;
(3) Adding HM culture medium containing 100-1000unit/ml Collagenase IV and 0.01-0.1mg/ml DNase II into tissue block, and digesting in water bath at 28deg.C for 15-60 min;
(4) Removing HM culture medium containing Collagenase IV and DNase II, adding HM culture medium, and blowing off hepatocytes with pipette;
(5) Filtering and separating and removing the un-blown cells by using a 22-100 mu M pore size filter screen, and collecting the scattered and separated cells;
(6) Centrifuging at 200-1000rpm and 4deg.C for 5-10min to collect liver cells, removing supernatant medium with a pipette, and adding 10-50ml DM medium to resuspend cells;
(7) Sucking 10-50. Mu.l of the cell suspension, adding 10-100. Mu.l of the phenol blue-stained cells, and counting the cells under a microscope;
(8) Diluting cells to 1.0-5.0X10 with DM Medium 6 Inoculating cells/ml into culture dish with diameter of 35mm 1.0-5.0X10 6 Cells, 28 ℃,5% co 2 Culturing the cells in an incubator for 6-36 hours;
(9) Density is 2.0X10 with cryopreservative 6 Gradient cooling is carried out on cells/ml of the gliocladium littoralis, and the cells/ml of the gliocladium littoralis are frozen and stored in liquid nitrogen.
2. The method for separating, culturing and cryopreserving liver cells of littoral in accordance with claim 1, wherein: in the step (1), the tissue blocks are obtained by cleaning fresh liver tissue of healthy stone climbing by using an HM culture medium and then cutting the cleaned liver tissue into tissue blocks with the diameter of 0.1-1.0 mm.
3. The method for separating, culturing and cryopreserving liver cells of littoral in accordance with claim 1, wherein: the formula of the HM culture medium comprises the following components:
KCl:1.0-1.0mg/ml,K 2 HPO 4 :0.01-0.1mg/ml；NaCl:1.0-10.0mg/ml；NaH 2 PO 4 :0.01-0.1mg/ml；Glucose:1.0-10.0mg/ml；HEPES:1.0-10.0mg/ml；NaHCO 3 0.1-1.0mg/ml; penicillin 50-100U/ml, streptomycin: 50-100 mug/ml.
4. The method for separating, culturing and cryopreserving liver cells of littoral in accordance with claim 1, wherein: the DM medium comprises the following components in percentage by weight:
DMEM/F-12:1.0-10.0mg/ml; naHCO3 1.0-10.0mg/ml; BSA 1.0-10.0mg/ml; penicillin 50-100U/ml, streptomycin: 50-100 mug/ml.
5. The method for separating, culturing and cryopreserving liver cells of littoral in accordance with claim 1, wherein: the formula of the cryopreservative comprises the following components:
cryopreservation agent: DMSO concentration was 10% and serum concentration was 90%.
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