Method for extracting primary hepatocyte lipid of ICR mouse

Abstract

The invention discloses a method for extracting primary hepatocyte lipid of an ICR mouse. The method comprises the following steps: digesting and extracting hepatocytes by using a buffer working solution 2 containing type IV collagenase by using a two-step collagenase perfusion method, then laying rat tail collagen in a pore plate, inoculating the extracted hepatocytes on the pore plate, culturing the cells, and finally extracting lipid from the cultured cells. The method is oriented to the ICR mouse, the problem that a primary hepatocyte extraction and culture system is unstable is solved, the cell extraction time is greatly shortened, the survival rate and the cell activity of the cells are improved, and the ICR mouse primary hepatocyte model is more effectively applied to lipidomics research.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture and relates to a method for extracting lipids from primary hepatocytes of ICR mice. Background Art

[0002] The liver is the main organ for drug metabolism. General in vitro metabolic models are based on the liver. Common in vitro models include liver microsomes, liver S9, liver cytosol, primary hepatocytes, liver tissue slices, recombinant human metabolic enzymes, etc. Among them, primary hepatocytes have good in vitro test reproducibility, basically maintain the metabolic function of the liver, and especially better retain the enzyme level consistent with the body, and are widely used in drug metabolism and toxicology research.

[0003] The key to in vitro culture of hepatocytes is how to isolate and culture hepatocytes with complete morphology and high in vitro metabolic activity. Since the method of hepatocyte isolation was first created, various laboratories have been continuously improving the method of stem cell isolation. At present, the main method for extracting primary hepatocytes is perfusion. The common perfusion solution (working solution 1) formula is Ca-free 2+ Mg 2+ and phenol red HBSS buffer, 0.5 mMol EDTA and 25 mMol HEPES; the digestion solution formula (working solution 2) is 97.5% Ca 2+ Mg 2+ and phenol red in HBSS buffer and 25mMol HEPES (Protocol for Primary Mouse Hepatocyte Isolation, Meital Charni-Natan, Ido Goldstein, STAR Protocols). However, the perfusion working solution with different components and the details of the different operation steps will affect the stability of primary hepatocyte extraction and the activity of the extracted hepatocytes. At the same time, the conditions for culturing hepatocytes also determine the metabolic activity of the cells later. At present, the laboratory extraction process of primary hepatocytes of ICR mice has great instability, and the experimental process is time-consuming. In summary, it is necessary to conduct a detailed exploration of the isolation and culture conditions of primary hepatocytes of ICR mice.

[0004] Lipids can be broadly defined as fatty acids and their derivatives, as well as substances related to the biosynthesis or function of these compounds. Lipids have the following functions in organisms: (1) major structural components of biological membranes, as well as functional and regulatory components of membrane protein signaling; (2) biologically active intracellular and intercellular signaling molecules; and (3) energy storage molecules used to maintain energy homeostasis. Numerous studies have shown that lipid metabolites play a functional role in the dynamic regulation of cellular homeostasis and that dysregulation of lipid metabolism plays a role in the onset and progression of a range of human diseases, including diabetes, neurodegeneration, and cancer.

[0005] The main method of lipid extraction is liquid-liquid extraction, which uses low-polarity organic reagents to extract low-polarity lipids. The more commonly used methods include: (1) Bligh and Dyer; (2) Folch; (3) MTBE; (4) BUME. The first two methods both use chloroform, which is highly toxic. The third method uses methyl tert-butyl ether to extract lipids, which is relatively less toxic. At the same time, MTBE extracts lipids in the upper layer, which is simpler to operate than the first two methods. MTBE has a higher extraction efficiency for phosphoglycerides, ceramides, and unsaturated fatty acid lipids, while the first two methods MeOH:CHCl3 has a higher extraction efficiency for saturated fatty acids and acetal phospholipids. In these methods, different reagent ratios also have different differences, and the extracted lipids also have differences. In the process of using the Floch method and the Bligh-Dyer method, taking the lower phase may cause contamination; while the MTBE method takes the upper phase, and there is no contamination. Regarding the lipid extraction method, the overall process is roughly adding a mixture, vortexing and centrifuging, then adding a mixture of other proportions to extract again, and collecting and nitrogen blowing to dissolve again. The improvement of the method mainly involves changing the proportion and type of the mixed solution. Currently, there are methods for obtaining various lipids that are not efficient. In summary, a specific and feasible experimental treatment method is needed for lipid extraction in primary hepatocytes to ensure that a considerable amount and type of lipids can be extracted. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting lipids from primary hepatocytes of ICR mice.

[0007] The technical solution for achieving the purpose of the present invention is as follows:

[0008] The method for extracting lipids from primary hepatocytes of ICR mice comprises the following steps:

[0009] S1. Isolation of primary hepatocytes: Using a two-step collagenase perfusion method, the superior and inferior vena cava of ICR mice were first perfused with buffer working solution 1 until no blood flowed out of the superior vena cava, and then the liver was perfused with buffer working solution 2 to digest and extract hepatocytes. The concentration of type IV collagenase in the buffer working solution 2 was 0.56 mg / mL.

[0010] S2. Culture of primary hepatocytes: Rat tail collagen was laid in the well plate at a density of 1.2×10 5 / mL cell density, the hepatocytes extracted from S1 were seeded on the well plate and cultured for 7 to 15 days;

[0011] S3. Extraction of lipids in primary hepatocytes: Remove the culture medium from the well plate, rinse the well plate with pre-cooled PBS solution, add 0.25% Trypsin-EDTA trypsin solution to each well, place in a cell culture incubator for 4-5 minutes to digest the cells, then add methanol to the well plate, transfer the mixed solution to a centrifuge tube, rinse the well with methanol, transfer the mixed solution in the well, then add chloroform to the centrifuge tube, vortex for 10-20 seconds, centrifuge, and combine the supernatant so that the cell concentration in the supernatant is 3.6×10 5 / sample to obtain intracellular lipid molecules, and the volume ratio of chloroform:methanol:water is 15:14:2.

[0012] Furthermore, in S1, the ICR mice were 6-8 week old ICR male mice.

[0013] In S1, collagenase type IV can be used to digest a variety of tissues.

[0014] Furthermore, in S1, during the digestion process, the liver was filled once every one minute, and this was repeated three times, which greatly shortened the cell digestion time.

[0015] Further, in S1, the formula of buffer working solution 1 is: 8g / L NaCl, 0.4g / L KCl, 0.078g / L NaH2PO4·2H2O, 0.151g / L Na2HPO4·12H2O, 0.35g / L NaHCO3, 0.19g / L EGTA, 0.9g / L glucose, 0.006g / L phenol red, 0.5% penicillin-streptomycin mixture; the formula of buffer working solution 2 is: 8g / L NaCl, 0.4g / L KCl, 0.078g / L NaH2PO4·2H2O, 0.151g / L Na2HPO4·12H2O, 0.35g / L NaHCO3, 0.24g / L HEPES, 0.006g / L phenol red, 0.5% penicillin-streptomycin mixture, 0.56mg / mL type IV collagenase.

[0016] Furthermore, in S2, the rat tail collagen is rat tail collagen type I, and the coating concentration is 2 μg / cm 2 By laying rat tail collagen on the well plate and controlling the cell seeding density to 1.2×10 5 / mL, which greatly improved the cell adhesion efficiency and viability in the well plate.

[0017] In S3, the adherent cells were digested with trypsin, the proteins were denatured and precipitated with methanol twice, and the non-polar substances therein were extracted with chloroform.

[0018] Compared with the existing methods, the present invention has the following advantages:

[0019] The present invention utilizes a two-step collagenase perfusion method to digest and extract liver cells, and achieves better separation and extraction of living cells by adjusting the concentration and perfusion speed of collagenase in the buffer working solution 2; obtains the best cell growth state by controlling the cell inoculation density and the well plate laying conditions; and achieves considerable quantity and type of lipid extraction by controlling the number of cells in the sample and the lipid extraction step. The present invention solves the problem of instability of the primary mouse hepatocyte extraction and culture system in the laboratory, greatly shortens the time of cell extraction, and improves the survival rate and cell viability of cells, and enables the primary mouse hepatocyte model to be more effectively applied to lipidomics. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is a flow chart of the lipid extraction method of primary hepatocytes from ICR mice.

[0021] Figure 2 These are cells obtained under different collagenase concentrations during the extraction process. The upper layer of the liquid is dead cells, and the lower sediment is living cells. (A) is the cell obtained under a collagenase concentration of 0.28 mg / mL, and (B) is the cell obtained under a collagenase concentration of 0.56 mg / mL.

[0022] Figure 3 The figures are the growth status of mouse primary hepatocytes at different times under different culture conditions, including (A) the growth status of mouse primary hepatocytes at different laying densities in a well plate without rat tail collagen, and (B) the growth status of mouse primary hepatocytes at different laying densities in a well plate with rat tail collagen.

[0023] Figure 4 Schematic diagram of the process of lipid extraction from primary hepatocytes.

[0024] Figure 5 It is the number of lipids extracted from samples containing different numbers of cells. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0026] The formula of the working solution used in the following examples is as follows:

[0027] (1) Buffer working solution 1: 8 g / L NaCl, 0.4 g / L KCl, 0.078 g / L NaH2PO4·2H2O, 0.151 g / L Na2HPO4·12H2O, 0.35 g / L NaHCO3, 0.19 g / L EGTA, 0.9 g / L glucose, 0.006 g / L phenol red, 0.5% penicillin-streptomycin mixture.

[0028] (2) Buffer working solution 2: 8 g / L NaCl, 0.4 g / L KCl, 0.078 g / L NaH2PO4·2H2O, 0.151 g / L Na2HPO4·12H2O, 0.35 g / L NaHCO3, 0.24 g / L HEPES, 0.006 g / L phenol red, 0.5% penicillin-streptomycin mixture, 0.56 mg / mL type IV collagenase.

[0029] Example 1

[0030] (1) Isolation of primary hepatocytes from ICR mice: A two-step collagenase perfusion method was used. In the first step, the needle was inserted into the inferior vena cava of the ICR mouse, and at the same time, perfusion buffer working solution 1 was injected and the superior vena cava was cut off, and the superior vena cava was flushed until no blood flowed out of the superior vena cava. In the second step, the liver was perfused with buffer working solution 2. During this process, the superior vena cava was clamped every 1 minute to allow the liver to fill and swell and be fully digested. After filling 3-4 times, the liver was found to be completely swollen and transparent fluid accumulated at the bottom, and the digestion was stopped. In the preparation of buffer working solution 2, in addition to diluting the original solution 10 times, type IV collagenase was also added. While the other conditions remained unchanged, the concentration of type IV collagenase was changed (0.28 mg / mL and 0.56 mg / mL, respectively), and the number and activity of live cells extracted were compared. The results are as follows Figure 2 As shown in the figure, most of the cells obtained after digestion and separation with working solution 2 having a collagenase concentration of 0.28 mg / mL were suspended above the separation solution and were dead cells; while there were a considerable number of live cells in the cells obtained after digestion and separation with working solution 2 having a collagenase concentration of 0.56 mg / mL, and the live cells showed good metabolic activity in subsequent culture. Therefore, the final collagenase concentration in working solution 2 was determined to be 0.56 mg / mL.

[0031] (2) Cultivation of ICR mouse primary hepatocytes: The mouse primary hepatocytes with good metabolic activity extracted above were cultured at different densities (0.8×10 5 / mL, 1.2×10 5 / mL, 1.6×10 5 / mL, 2×10 5 / mL) were inoculated into two well plates with different well plate conditions (whether rat tail collagen type I was laid on the bottom of the well plate in advance). More than three parallel wells were set up in each group. The other culture conditions remained unchanged, and all were placed in a cell culture incubator at 37°C and a carbon dioxide concentration of 5%. The cell growth status was observed under a microscope. The results are as follows Figure 3 As shown, it can be seen that the cell seeding density is 2×10 5 When the number of cells in the field of view was large and most of them were clustered together. The cells were mostly spherical and had basically no obvious binuclear structure. The cell seeding density was 1.6×10 5 When the number of cells in the field of view was large, some binuclear hepatocytes were expanded, but most of them were still spherical. The cell seeding density was 1.2×10 5 At 4 s, the cell density in the field of view was normal, the cell distribution was reasonable, the number of binuclear hepatocytes was large, and a small number of them were spherical; the cell inoculation density was 0.8×10 5 When the cells were plated with rat tail collagen, the cells were arranged slightly sparsely in the field of view, and almost all of them showed an unfolded binucleate structure. Regardless of whether rat tail collagen was used for plating, the cells showed enlarged nuclei and unclear edges over time, and the number of dead cells increased over time. Comparing the cells of the two, the cells plated with rat tail collagen had better adhesion to the wall than those not plated, and the cell growth morphology was better, which was particularly obvious in the comparison of high seeding density.

[0032] (3) Method for extracting lipids from primary hepatocytes of ICR mice: aspirate the culture medium in a clean well plate; rinse the wells of the well plate with pre-cooled PBS solution; add 60uL of 0.25% Trypsin-EDTA trypsin solution to each well and place it in a cell culture incubator for 4-5 minutes to digest the cells; add 220uL of methanol to the well plate; transfer the mixed solution to a 2.0mL centrifuge tube, and then rinse the wells again with 200uL of methanol, and transfer the mixed solution in the wells in the same way; add 450uL of chloroform to each 2.0mL centrifuge tube and vortex for 10-20s. Follow the above steps to obtain intracellular lipid molecules. Thereafter, by combining the sample supernatants and changing the number of cells therein, the cell numbers obtained were 0.6×10 5 (take half of the normal sample supernatant), 1.8×10 5 (combined supernatant of one sample and half sample), 3.6×10 5 The samples (the supernatants of the three samples were combined) were tested for lipid molecules to select the appropriate number of cells for subsequent experiments.

[0033] In summary, the growth status of mouse primary hepatocytes over time can be seen Figure 2The obvious binuclear structure proves that the cells have strong activity. It can be seen that the collagenase concentration (0.56 mg / mL) can well separate and extract liver cells while retaining good activity. By comparison, according to 1.2×10 5 The cells grown best when inoculated at a density of 1.2 × 10 / mL on a plate with rat tail collagen on the bottom were selected. 5 / mL and rat tail collagen should be laid on the bottom of the well plate. The number of lipids obtained according to the above lipid extraction method is as follows Figure 3 As shown in the figure, it can be seen that this method has a good effect on lipid extraction, and the number of cells is 3.6×10 5 When the number of lipids per sample is increased, better lipid extraction can be achieved.

Claims

1. A method for extracting lipids from primary hepatocytes of ICR mice, characterized in that: The following steps are involved: S1. Isolation of primary hepatocytes: Using a two-step collagenase perfusion method, the superior and inferior vena cava of ICR mice were first perfused with buffer working solution 1 until no blood flowed out of the superior vena cava, and then the liver was perfused with buffer working solution 2 to digest and extract hepatocytes. The concentration of type IV collagenase in the buffer working solution 2 was 0.56 mg / mL. S2. Culture of primary hepatocytes: Rat tail collagen was laid in the well plate at a density of 1.2×10 5 / mL cell density, the hepatocytes extracted from S1 were seeded on the well plate and cultured for 7 to 15 days; S3. Extraction of lipids in primary hepatocytes: Remove the culture medium from the well plate, rinse the well plate with pre-cooled PBS solution, add 0.25% Trypsin-EDTA trypsin solution to each well, place in a cell culture incubator for 4-5 minutes to digest the cells, then add methanol to the well plate, transfer the mixed solution to a centrifuge tube, rinse the well with methanol, transfer the mixed solution in the well, then add chloroform to the centrifuge tube, vortex for 10-20 seconds, centrifuge, and combine the supernatant to make the cell concentration in the supernatant 3.6×10 5 / sample to obtain intracellular lipid molecules, and the volume ratio of chloroform:methanol:water is 15:14:

2.

2. The extraction method according to claim 1, characterized in that In S1, ICR mice were 6-8 weeks old ICR male mice.

3. The extraction method according to claim 1, characterized in that In S1, during the digestion process, the liver was filled once every one minute, and this was repeated three times.

4. The extraction method according to claim 1, characterized in that In S1, the formula of buffer working solution 1 is: 8g / LNaCl, 0.4g / L KCl, 0.078g / L NaH2PO4•2H2O, 0.151g / L Na2HPO4•12H2O, 0.35g / L NaHCO3, 0.19g / L EGTA, 0.9g / L glucose, 0.006g / L phenol red, 0.5% penicillin-streptomycin mixture; the formula of buffer working solution 2 is: 8g / L NaCl, 0.4g / L KCl, 0.078g / L NaH2PO4•2H2O, 0.151g / L Na2HPO4•12H2O, 0.35g / L NaHCO3, 0.24g / L HEPES, 0.006g / L phenol red, 0.5% Penicillin-streptomycin mixture, 0.56 mg / mL type IV collagenase.

5. The extraction method according to claim 1, characterized in that In S2, the rat tail collagen was rat tail collagen type I, and the coating concentration was 2 μg / cm 2 .

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