CN113957035B - Duck embryo primary liver cell separation culture method - Google Patents
Duck embryo primary liver cell separation culture method Download PDFInfo
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Abstract
The invention relates to the field of cell culture, in particular to a duck embryo primary liver cell separation culture method. The method comprises the steps of: sterilizing the outer shell of the duck embryo, and taking thirteen-day-old to fifteen-day-old duck embryos under the aseptic condition; separating duck embryo livers, removing gall bladder, soaking and washing the obtained livers in a D-Hank's buffer solution; removing loose connective tissue and liver capsule adhered around liver, soaking the obtained small liver tissue in D-Hank's buffer solution drop, and washing; multi-step digestion; the obtained cells were resuspended, sieved through a 70 μm cell sieve, and the cells were inoculated into a flask for culturing. The invention establishes a simple, economical and rapid method for separating primary hepatocytes of duck embryo from the methods of selecting the day-old age of the duck embryo, combining a mechanical method with an enzyme digestion method on tissues and the like.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a duck embryo primary liver cell separation culture method.
Background
The liver is an important internal organ of animal body metabolism, is a main place of carbohydrate, fat and protein metabolism, and has important functions of regulating and controlling body metabolism and maintaining body metabolism balance. Unlike mammal, the ability of fat cell to synthesize fat is limited, and fat is mainly derived from liver synthesis and intestinal absorption, so that the liver of meat duck has extremely important function in lipid metabolism regulation, and separation and culture of liver cells of meat duck provide good foundation for exploring lipid metabolism and regulation mechanism of meat duck.
Liver cells are classified into hepatic parenchymal cells, which are the main cell group that performs liver function, and hepatic non-parenchymal cells, such as hepatic stellate cells, cumic cells, etc., which mainly assist the hepatic parenchymal cells to complete normal liver function. In vitro culture of liver parenchymal cells, it has been shown that liver parenchymal cells are beneficial to the growth of liver parenchymal cells, and therefore in liver cell culture, in order to maintain morphological characteristics and physiological functions of primary liver cells to the maximum extent, a mode of co-culturing liver cells and liver parenchymal cells is often selected. In addition, the liver primary cells can well simulate the liver state of a host organism, and are ideal models for researching the liver metabolic mechanism of animals. The duck embryo liver cells are separated, primary culture is carried out on the duck embryo liver cells, and the research on the metabolism mechanism and the regulation and control effect of the liver of the meat duck can be realized under the state closest to the in vivo state.
In 1953, anderson first separated hepatocytes from liver by shearing method, and then, the separation method is modified for many times in practical application, and is mainly divided into mechanical method and enzyme digestion method, and the two-step collagenase perfusion method is always a classical method for separating liver cells due to the great damage caused by mechanical method, so far, it is still adopted by most laboratories. The method has the advantages of good effect, high cell yield, high purity and good activity, but complicated operation, high perfusion technical requirement, easy failure of intubation, large consumption of collagenase and the like because of the need of special devices such as constant flow pumps and the like, and difficult development in a common laboratory. In recent years, many laboratories are optimizing and improving this process. In addition, no matter what method is adopted to separate the liver cells, the problem of liver cell purification exists, and the main purpose is to remove the fibroblast, the method comprises a differential adherence method, a differential centrifugation technology and a Percoll separation technology, the cost of the latter separation technology is relatively high, and the separation and purification are carried out by adopting the method of the former two in application.
Disclosure of Invention
The invention is proposed and completed for solving the problems of complex operation, high cost, low survival rate and purity of extracted cells and the like in the prior art.
The method for separating and culturing the duck embryo primary liver cells comprises the following steps of
S1: sterilizing the outer shell of the duck embryo, and taking thirteen-day-old to fifteen-day-old duck embryos under the aseptic condition;
s2: separating duck embryo livers, removing gall bladder, soaking and washing the obtained livers in buffer solution;
s3: removing loose connective tissue and liver capsule adhered around liver, and soaking the obtained small liver tissue in buffer solution;
s4: multi-step digestion:
(4-1) immersing the liver tissue block in pancreatin-EDTA with concentration of 0.25% for 15 s-30 s, transferring to cell culture solution for washing after the tissue is softened and whitened,
(4-2) aseptically cutting the tissue into 1mm pieces 3 The pellet is washed with cell culture medium, the supernatant is discarded by self-sedimentation of cells and tissues,
(4-3) using 10mL of 0.25% pancreatin-EDTA, the tissue pieces were blown off to a cell suspension of the tissue pieces invisible to the naked eye within 30s, left at room temperature for 30s, then pancreatin digestion was stopped with fetal bovine serum,
(4-4) centrifuging to remove the supernatant, and washing the cells with the cell culture medium;
s5: the cells obtained were resuspended in complete medium, sieved through a 70 μm cell sieve, and inoculated into a flask for culture.
The method for separating and culturing the duck embryo primary hepatocytes by specific implementation of water drainage comprises the following steps of
S1: sterilizing the outer shell of the duck embryo, and taking thirteen-day-old to fifteen-day-old duck embryos under the aseptic condition;
s2: separating duck embryo livers, removing gall bladder, soaking and washing the obtained livers in a D-Hank's buffer solution;
s3: removing loose connective tissue and liver capsule adhered around liver, soaking the obtained small liver tissue in D-Hank's buffer solution drop, and repeating the soaking and washing process for three times;
s4: multi-step digestion:
(4-1) immersing the liver tissue block in pancreatin-EDTA (0.25%) for 15-30 s, transferring to cell culture solution after softening and whitening the tissue, washing for three times,
(4-2) aseptically cutting the tissue into 1mm pieces 3 The pellet was washed twice with DMEM, the supernatant was discarded by self-sedimentation of cells and tissues,
(4-3) Using 10mL of pancreatin-EDTA (0.25%), the tissue pieces were blown off to a cell suspension of the tissue pieces invisible to the naked eye within 30 seconds, left at room temperature for 30 seconds, and then the fetal bovine serum was terminated with pancreatin digestion,
(4-4) centrifuging to discard the supernatant, washing the cells with DMEM, and repeating the process twice;
s5: the cells obtained were resuspended in complete medium, sieved through a 70 μm cell sieve, and inoculated into a flask for culture.
According to the duck embryo primary hepatocyte separation culture method, the cell culture solution is DMEM.
The invention relates to a duck embryo primary hepatocyte separation culture method, wherein the complete culture medium comprises high sugar culture medium DMEM rich in 10% fetal calf serum and penicillin 10 5 U·L －1 Streptomycin 100 mg.L －1 。
The invention establishes a simple, economical and rapid method for separating primary hepatocytes of duck embryo from the methods of selecting the day-old age of the duck embryo, combining a mechanical method with an enzyme digestion method on tissues and the like.
The invention has the advantages that:
1. the conventional duck embryo primary liver cell separation and culture method is a perfusion method, the method is only suitable for adult ducks, a large amount of collagenase, peristaltic pumps and other complex devices are required to be used for treatment, meanwhile, the challenges of intubation failure and external environment pollution are also required, the technical and environmental requirements are strict, the method disclosed by the invention can be used for selecting duck embryo to separate duck liver primary cells, the problem of operation complexity can be avoided, the method can be easily realized on an ultra-clean workbench, the pollution risk is reduced, and meanwhile, sufficient digestion of tissue samples can be realized by a small amount of pancreatin, and the separation cost is reduced.
2. The existing pancreatin enzymolysis method for separating liver cells often causes cell damage due to overdigestion because the digestion process is not easy to control, so that the cell survival rate is reduced.
3. Because primary hepatocytes obtained by the conventional perfusion method and enzymolysis method are poor in purity, a differential adherence method, a differential centrifugation technology and a Percoll separation technology are often required for cell purification, the cost of the latter purification technology is relatively high, and the former two methods are adopted for separation and purification in application, so that the separation cost and the operation complexity are obviously increased.
4. After primary hepatocytes are separated, hormone drugs such as insulin, hydrocortisone sodium succinate and the like are often added into a culture medium to promote growth such as cell adhesion and the like.
5. The multi-step pancreatin digestion method of the invention has the characteristic of less damage, and the survival rate of the obtained cells is up to more than 95%.
Drawings
FIG. 1 shows a multi-step digestion process for the isolation of duck embryo primary hepatocytes;
FIG. 2 shows hepatocytes prepared from duck embryos of different ages of days, wherein A is 11 years old B is 14 days old C is 20 days old, and the scale is 50 μm;
FIG. 3 shows cell viability determination by trypan blue exclusion;
FIG. 4 shows duck embryo primary hepatocytes at different culture times;
FIG. 5 shows the results of identifying alb and afp at the mRNA level, wherein A: alb and afp nucleic acids are electrophoresed, B: sequence alignment of amplified alb and afp;
fig. 6 shows the results of glycogen staining of duck primary hepatocytes, wherein a: normal glycogen staining, B: negative control (no oxidant in) with a scale of 100 μm shown in the figure;
FIG. 7 shows indirect immunofluorescence results;
FIG. 8 is a graph of cell growth curve.
Detailed Description
EXAMPLE 1 Duck embryo Primary liver cell isolation and culture
1. Isolation and culture
1. Wiping and sterilizing the duck embryo shell by using iodophor, and spraying and sterilizing the duck embryo shell by using 75% alcohol;
2. under the aseptic condition, knocking the shell from the air chamber end, opening the egg shell membrane of the duck embryo by using aseptic forceps, and taking out the duck embryo;
3. the operation scissors and the tweezers cooperate, duck embryo is dissected to separate duck embryo livers and remove gall bladder, and the obtained liver D-Hank's buffer solution is soaked and washed;
4. the scalpel and the forceps cooperate to remove loose connective tissue and liver capsule adhered around the liver, the obtained small liver tissue is soaked in D-Hank's buffer solution drops, and the soaking and washing process is repeated for three times;
5. the method comprises the steps of multi-step digestion, namely firstly soaking liver tissue blocks in pancreatin-EDTA (0.25%) for 15-30 s, transferring the liver tissue blocks into DMEM liquid drops after softening and whitening the tissues, and washing for three times;
6. the tissue was transferred to a 15mL sterile centrifuge tube and cut into 1mm pieces with a scalpel 3 The pellet was washed twice with 5mL DMEM, which process discarded the supernatant by self-sedimentation of cells and tissues;
7. 10mL of pancreatin-EDTA (0.25%) was added to a 15mL sterile centrifuge tube, and the tissue mass was blown off with a 5mL pipette to a cell suspension of the tissue mass invisible to the naked eye within 30s, and left at room temperature for 30s, whereupon 1/10 volume of fetal bovine serum was added to terminate pancreatin digestion;
8. centrifugal force 500g, time 5min, centrifugal at 4 ℃, discard the upper liquid, 10mL DMEM wash the cells, the process is repeated twice;
9. the cells obtained were resuspended in medium (complete medium: high sugar medium DMEM enriched with 10% fetal bovine serum, penicillin 10) 5 U·L －1 Streptomycin 100 mg.L －1 ) Through a 70 μm cell sieve (Corning) and counted with a blood cell counting plate, whereupon the cells were inoculated into a culture flask.
As shown in figure 1, the result of separating and culturing the duck embryo primary liver cells by a multi-step digestion method is shown, and the result shows that the invention establishes a simple, economical and rapid duck embryo primary liver cell separation method.
2. Day-age selection
With the gradual increase of the day-age of the duck embryo, the liver of the duck embryo gradually increases, however, the activity and the adaptability of the duck embryo in an in-vitro environment are different. In addition, as the age of the day increases, the attachments on the liver surface of duck embryo are increased, and the purity of the prepared cells is directly affected. As shown in figure 2, the duck embryos of eleven, fourteen and twenty days are selected in sequence, the states of the obtained cells are observed morphologically, the 14-day-old duck embryos are found to have obvious advantages compared with the other two groups, the cells are in an epithelial cell shape, the cell nucleus is positioned in the center of the cells, the binuclear liver cells are visible, and the duck embryos accord with typical characteristics of the liver cell morphology. As shown in FIG. 4, thirteen, fourteen, fifteen and sixteen-day-old duck embryos are selected and respectively cultured for 24 hours, 48 hours, 72 hours and 96 hours, and observation results show that in the preparation of primary hepatocytes of the duck embryos, excessive-day-old duck embryos are not suitable to be selected, and thirteen-day-old to fifteen-day-old duck embryos are preferentially selected.
Cell viability was identified by trypan blue exclusion (Soxhaust: C0040). Wherein,,
1. mixing the cell suspension with 0.4% trypan blue solution at a ratio of 9:1 (final concentration 0.04%), and staining for 3min;
2. a small amount of stained cells was aspirated and counted with a hemocytometer. Dead cells are blue and enlarged, and are matt; living cells are not colored and remain in normal form, and are shiny;
3. cell viability (%) =total number of living cells/(total number of living cells+total number of dead cells) ×100%.
As shown in FIG. 3, the cell viability of the duck embryo isolated by the method according to the present invention is >95% (upper panel in FIG. 3), and the duck embryo primary hepatocytes isolated by enzymatic methods such as high Yu Tan brilliance (lower panel in FIG. 3).
3. Preliminary identification of isolated cells from mRNA levels
Albumin (ALB) is secreted by hepatocytes, alpha fetoprotein (Alpha fetoprotein, AFP) is normally mainly derived from embryonic hepatocytes, cellular RNA is extracted according to the characteristics of hepatocytes, cDNA is obtained by reverse transcription, the two genes are amplified by PCR, and then the amplified gene sequences are sequenced to determine ALB and AFP obtained by reverse transcription, and the isolated cells are initially identified. ALB is located on the fourth chromosome, the genome accession number NC_040049.1 is located at 46869333-46879914, the gene ID is NM_001310394.1, the CDs sequence is 1848bp long, and the primer design ALB-F is ATGAAGTGGGTAACATTAATTTC, ALB-R is TTAAGCACCAATTCCTAATGT. AFP is located on the fourth chromosome, genome accession NC_040049.1, at 46836428-46864918, gene ID XM_021267672.2, CDs sequence length 1809bp, primer design AFP-F ACTGTAGTCAAAGCCCTGC, AFP-R TTGGAATCAATCCTCTTTCACAAA. As shown in FIG. 5, the sequencing results showed that the method of the present invention resulted in hepatocytes.
4. Cell purity and functional identification
The morphology of the liver cells is observed, and the functions of the liver cells are detected. Liver glycogen is a stock polysaccharide in humans and animals, also known as liver starch. The energy required for survival of liver cells is derived from anaerobic glycolysis in cells, glycogen in liver cells can be decomposed into glucose, and a substrate is provided for anaerobic glycolysis. The lack of hepatocyte bioenergy is an initiating factor in a series of pathological changes in the liver, and thus, the method of periodic acid-schiff staining was chosen to evaluate the biological function of primary hepatocytes by the condition of cell glycogen content. As shown in FIG. 6, the microscopic examination result shows that the cytoplasm of the liver cell contains rich glycogen, and the glycogen is dyed into purplish red and distributed in a homogeneous or granular form, which indicates that the liver cell separated by the invention has normal functional activity.
The primary cultured liver cells have the function of secreting AFP, but with the extension of the culture time, the concentration of the AFP secreted by the liver cells gradually decreases, and the liver cells after multiple passages basically lose the function of secreting AFP. In addition, since albumin is expressed and secreted by hepatic parenchymal cells, the secretion of albumin and alpha fetoprotein is also a common detection index of the functional state of liver cells, and the functional activity of liver cells of the isolated cells is evaluated by adopting an indirect immunofluorescence method aiming at albumin. In addition, DAPI staining was performed on nuclei at the same time, and cell purity was identified. The observation result of a fluorescence microscope shows that the separated cells have the capacity of secreting albumin, and the combined result shows that more than 90% of the cells are liver parenchymal cells capable of secreting albumin, and the primary liver cells separated from duck embryo have higher purity.
5. Cell growth curve
The cell growth curve is a common method for determining the absolute growth number of cells, is also an important index for determining the activity of cells, and is one of basic parameters for the biological characteristics of cultured cells. The cell growth curve drawn by MTT method as shown in FIG. 7 shows that the isolated cells have typical cell growth characteristics including slow-growing latency period, large-slope exponential growth period, plateau-like plateau period and 4 parts of degenerated decay.
The prior art is faced with problems such as cell purity, cell viability, etc. during the isolation of primary cells using duck embryos. In order to improve the purity of cells, those skilled in the art often perform further purification after separating the cells, such as differential adherence method, differential centrifugation technology, percoll separation technology, etc., and depending on the purification technology, there are methods for early treatment of fresh tissues in the prior literature materials. Furthermore, the person skilled in the art generally selects ducks with a relatively large age in days, and the adhesion around the liver and the increase of non-liver cells in the liver increase the difficulty of removal. According to the technical scheme, the duck embryo with thirteen-day-old to fifteen-day-old is selected, and the duck embryo with the embryo age is used, so that liver non-parenchymal cells of the liver are fewer, and meanwhile, the development condition of the liver is considered.
From the standpoint of the prior art, in order to improve the cell viability, a person skilled in the art generally grows to determine a suitable isolated culture method from cumbersome conditions such as the kind of medium, the addition of hormones, the kind and concentration of digestive enzymes used, and the like. The multi-step digestion method disclosed by the invention indirectly reduces the damage of pancreatin to cells while utilizing the advantage of high digestion efficiency of pancreatin, so that the cell survival rate is improved.
Sequence listing
<110> institute of feed for national academy of agricultural sciences
<120> duck embryo primary liver cell separation culture method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
actgtagtca aagccctgc 19
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttggaatcaa tcctctttca caaa 24
Claims (3)
1. A method for separating and culturing duck embryo primary liver cells is characterized by comprising the following steps of
S1: sterilizing the outer shell of the duck embryo, and taking thirteen-day-old to fifteen-day-old duck embryos under the aseptic condition;
s2: separating duck embryo livers, removing gall bladder, soaking and washing the obtained livers in buffer solution;
s3: removing loose connective tissue and liver capsule adhered around liver, soaking the obtained small liver tissue in buffer solution, and washing;
s4: multi-step digestion:
(4-1) immersing the liver tissue block in pancreatin-EDTA with concentration of 0.25% for 15 s-30 s, transferring to cell culture solution for washing after the tissue is softened and whitened,
(4-2) aseptically cutting the tissue into 1mm pieces 3 The pellet is washed with cell culture medium, the supernatant is discarded by self-sedimentation of cells and tissues,
(4-3) using 10ml of 0.25% pancreatin-EDTA, the tissue pieces were blown off to a cell suspension of the tissue pieces invisible to the naked eye within 30s, left at room temperature for 30s, then pancreatin digestion was stopped with fetal bovine serum,
(4-4) centrifuging to remove the supernatant, and washing the cells with the cell culture medium;
s5: resuspension the obtained cells with the complete culture medium, sieving with a 70 μm cell sieve, and inoculating the cells into a culture flask for culture;
wherein,,
the cell culture solution is DMEM culture solution,
the buffer solution is D-Hank's buffer solution,
the complete culture medium is prepared from high sugar culture medium DMEM rich in 10% fetal bovine serum and contains penicillin 10 5 U·L －1 Streptomycin 100 mg.L －1 。
2. The method for isolated culture of duck embryo primary hepatocytes according to claim 1, wherein in step S3, the soaking washing is repeated three times.
3. The method of claim 1, wherein in step (4-4), the supernatant is centrifuged off and the cells are washed with DMEM, and the process is repeated twice.
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