CN102634480B - Method for isolating and culturing liver primary cells - Google Patents

Abstract

Translated fromChinese

本发明提供一种分离培养肝脏原代细胞的方法，包括以下步骤：1)从肝脏的肝门静脉输入灌流液；2)输入胶原酶灌流液；3)将消化成熟的肝脏浸泡于胶原酶灌流液中；4)加清洗液终止消化，过滤，收集肝细胞悬液；5)离心，弃上清，用完全培养基重悬，培养。本发明方法操作简单，成本低，稳定高效，肝细胞得率高，活力高，易贴壁。此法可在实验室推广，成为科学研究的常规方法。The invention provides a method for isolating and culturing primary liver cells, comprising the following steps: 1) inputting perfusate from the hepatic portal vein of the liver; 2) inputting collagenase perfusate; 3) immersing the digested mature liver in the collagenase perfusate Middle; 4) Add cleaning solution to terminate digestion, filter, and collect liver cell suspension; 5) Centrifuge, discard supernatant, resuspend with complete medium, and culture. The method of the invention is simple in operation, low in cost, stable and efficient, high in liver cell yield, high in vigor and easy to adhere to the wall. This method can be promoted in the laboratory and become a routine method of scientific research.

Description

Translated fromChinese

一种分离培养肝脏原代细胞的方法A method for isolating and culturing primary liver cells

技术领域technical field

本发明涉及细胞领域，具体地，涉及一种分离培养肝脏原代细胞的方法。The invention relates to the field of cells, in particular to a method for isolating and culturing primary liver cells.

背景技术Background technique

原代细胞作为一种体外模型，在基因功能研究方面有其突出的优点：可同时获得大量性状较均一的样本；与生物体内情况基本保持一致，可以在接近生理状态的情况下研究基因的表达；排除许多复杂因素干扰，如神经、激素的干扰；原代肝细胞具有较好的体外实验重现性，基本维持了肝脏的代谢功能，特别是很好的保留了与体内一致的细胞色素P450酶(cytochrome P450)的水平，基本保留肝脏原有的代谢功能和细胞分化状态。As an in vitro model, primary cells have outstanding advantages in the study of gene function: a large number of samples with relatively uniform characteristics can be obtained at the same time; it is basically consistent with the situation in vivo, and gene expression can be studied under conditions close to physiological conditions ; Eliminate the interference of many complex factors, such as the interference of nerves and hormones; primary hepatocytes have good reproducibility in vitro experiments, basically maintain the metabolic function of the liver, especially well retain the cytochrome P450 consistent with the body Enzyme (cytochrome P450) level basically retains the original metabolic function and cell differentiation state of the liver.

肝细胞(Hepatocytes)是肝脏最主要的实质细胞类型，生理状况下占60％，属于高度分化的多功能、实质性脏器细胞。肝细胞既能高度分化，又能很快进入细胞周期而增殖，在体外培养体系中，根据研究目的的不同，既有支持其增殖的培养体系，也有支持其分化的培养体系。肝细胞离体后，在不适宜的环境中很快出现肝细胞衰老和失功能，pH值不适合、无激素支持导致细胞死亡，基因的激活造成细胞凋亡等。原代肝细胞在一般培养条件下，几小时内便丧失其缝隙连接，几天内丧失其组织特异性代谢活动。现有技术中常用的肝细胞分离方法是灌流法，传统灌流法操作环节多，时间长，肝细胞容易损伤，活力低、效率低，纯度不高。因此，迫切需要制备体外培养活力旺盛、功能良好的猪肝脏原代细胞以满足细胞生物学、组织培养技术等研究的要求。Hepatocytes are the most important parenchymal cell type in the liver, accounting for 60% under physiological conditions, and are highly differentiated multifunctional and substantial organ cells. Hepatocytes can not only be highly differentiated, but also quickly enter the cell cycle and proliferate. In vitro culture systems, according to different research purposes, there are culture systems that support their proliferation and differentiation. After hepatocytes are isolated from the body, hepatic cell aging and dysfunction will soon appear in an unsuitable environment. The pH value is not suitable, and there is no hormone support leading to cell death, and the activation of genes leads to cell apoptosis. Under normal culture conditions, primary hepatocytes lose their gap junctions within a few hours and their tissue-specific metabolic activities within a few days. The commonly used hepatocyte separation method in the prior art is the perfusion method. The traditional perfusion method has many operation steps, takes a long time, easily damages the hepatocytes, has low vitality, low efficiency, and low purity. Therefore, it is urgent to prepare porcine liver primary cells with strong vigor and good function in vitro to meet the research requirements of cell biology and tissue culture technology.

发明内容Contents of the invention

本发明的目的在于提供一种分离培养肝脏原代细胞的方法，以克服上述现有技术中存在的缺陷。The object of the present invention is to provide a method for isolating and culturing primary liver cells, so as to overcome the defects in the above-mentioned prior art.

本发明提供的分离培养肝脏原代细胞的方法，包含以下步骤：The method for isolating and culturing primary liver cells provided by the present invention comprises the following steps:

(1)从肝脏的肝门静脉输入灌流液；所述的灌流液，其1L配方为：NaCl 8-9.4g，KCl 0.2-0.4，HEPES 2.4-4.8g，双蒸水定容至1L，pH值7.2-7.4；(1) Input the perfusate from the hepatic portal vein of the liver; the 1L formula of the perfusate is: NaCl 8-9.4g, KCl 0.2-0.4, HEPES 2.4-4.8g, distilled water to 1L, pH 7.2-7.4;

(2)输入胶原酶灌流溶液，肝组织呈龟背状裂开后，停止灌流；(2) Input the collagenase perfusion solution, after the liver tissue splits in a turtle-like shape, stop the perfusion;

(3)将消化成熟的肝脏浸泡于胶原酶溶液中；(3) Soak the digested mature liver in the collagenase solution;

(4)加清洗液终止消化，过滤，收集肝细胞悬液；(4) Add cleaning solution to stop digestion, filter, and collect liver cell suspension;

(5)离心，弃上清，用完全培养基重悬培养。(5) Centrifuge, discard the supernatant, and resuspend culture with complete medium.

所述步骤1)的肝脏为新鲜采集的离体肝脏，或新鲜离体肝脏经肝门静脉以80-100ml/min速度注射3倍以上肝体积的0-4℃预冷UW液，肝温降至10℃以下，保持1-4℃离体3h之内的肝脏。当肝温降至10℃以下后，可将肝脏置于冰盒或其他能够保持1-4℃的容器中保存肝脏不超过3h。The liver in step 1) is a freshly collected isolated liver, or a fresh isolated liver is injected with 0-4°C pre-cooled UW solution more than three times the liver volume through the hepatic portal vein at a rate of 80-100ml/min, and the liver temperature drops to Below 10°C, keep the liver at 1-4°C within 3 hours in vitro. When the liver temperature drops below 10°C, the liver can be stored in an ice box or other containers capable of maintaining 1-4°C for no more than 3 hours.

本发明方法采用蠕动泵进行灌流，用无菌PBS排空蠕动泵管道内气泡。其中所述步骤(1)是以恒温37℃灌流液开放灌流10-15min，流速50-60ml/min。The method of the invention adopts a peristaltic pump for perfusion, and uses sterile PBS to empty the air bubbles in the pipeline of the peristaltic pump. Wherein the step (1) is to open the perfusion with the perfusate at a constant temperature of 37° C. for 10-15 minutes, with a flow rate of 50-60 ml/min.

其中所述步骤(2)胶原酶灌流溶液灌流流速50-60ml/min。Wherein said step (2) the perfusion flow rate of the collagenase perfusion solution is 50-60ml/min.

其中所述步骤(2)的胶原酶灌流液，其1L配方为NaCl 8-9.4g，KCl 0.2-0.4g，HEPES 2.4-4.8g，Na₂HPO₄0.5-0.6g，CaCl₂0.10-0.15g，II型或IV型胶原酶0.5g，双蒸水定容至1L。Wherein the collagenase perfusate of step (2), its 1L formula is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na₂ HPO₄ 0.5-0.6g, CaCl₂ 0.10-0.15g , type II or type IV collagenase 0.5g, distilled water to 1L.

其中所述步骤(3)浸泡时间为5-10min。Wherein said step (3) soaking time is 5-10min.

其中所述步骤(4)清洗液配方为NaCl 8-9.4g，KCl 0.2-0.4g，HEPES 2.4-4.8g，Na₂HPO₄0.5-0.6g，CaCl₂0.10-0.15g，双蒸水定容至1L。Wherein the formula of step (4) cleaning liquid is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na₂ HPO₄ 0.5-0.6g, CaCl₂ 0.10-0.15g, distilled water to volume to 1L.

其中所述步骤(4)过滤是将肝脏细胞悬液通过两层纱布和两层细胞筛，纱布和细胞筛均用清洗液浸润，所述细胞筛上层100目，下层200目。Wherein said step (4) filtering is to pass the liver cell suspension through two layers of gauze and two layers of cell sieves, both gauze and cell sieves are infiltrated with cleaning solution, the upper layer of the cell sieve is 100 mesh, and the lower layer is 200 mesh.

本发明分离培养肝脏原代细胞所用几种溶液的配方见表1。The formulations of several solutions used for the isolation and culture of liver primary cells in the present invention are shown in Table 1.

表1灌流液，胶原酶灌流液，清洗液配方表Table 1 perfusate, collagenase perfusate, cleaning solution formula table

注：用盐酸把pH调至7.2-7.4，ddH₂O定容至1000ml，过滤除菌。Note: Adjust the pH to 7.2-7.4 with hydrochloric acid, adjust the volume to 1000ml with ddH₂ O, and filter to sterilize.

其中，所述步骤(5)离心是将肝细胞滤液分别加入离心管中，400rpm，3min，弃上清，再次离心500rpm，3min，弃上清。在本发明的实施例中，所用离心管规格为50ml。Wherein, the centrifugation in the step (5) is to add the liver cell filtrate into the centrifuge tube respectively, 400 rpm, 3 min, discard the supernatant, and centrifuge again at 500 rpm, 3 min, discard the supernatant. In the embodiment of the present invention, the specification of the centrifuge tube used is 50ml.

其中所述步骤(5)中完全培养基其配方为：含20％胎牛血清(FBS)的DMEM培养基添加终浓度为200-600U/ml青霉素，20-60μg/ml链霉素，1％-2％二甲基亚砜，1-10mg/L胰岛素，1-10mmol/L尼克酰胺，0.1-1mmol/L抗坏血酸，用1M的NaHCO₃调节pH至7.2-7.4。Wherein said step (5) its prescription of complete culture medium is: the DMEM culture medium that contains 20% fetal bovine serum (FBS) adds final concentration and is 200-600U/ml penicillin, 20-60 μ g/ml streptomycin, 1% -2% dimethyl sulfoxide, 1-10mg/L insulin, 1-10mmol/L nicotinamide, 0.1-1mmol/L ascorbic acid, adjust the pH to 7.2-7.4 with 1M NaHCO₃ .

其中所述步骤(5)的重悬培养条件为制成5×10⁵/ml的细胞悬液，在37℃、5％二氧化碳培养箱内培养。Wherein the resuspension culture condition of the step (5) is to make a cell suspension of 5×10⁵ /ml and culture it in a 5% carbon dioxide incubator at 37°C.

DMSO能让肝细胞稳定复制，保持其特异性功能，抑制非实质细胞过度生长；胰岛素可促进细胞增殖分化；尼克酰胺促使肝细胞快速克隆增生分化；抗坏血酸可延长肝细胞生命期，长期保持肝细胞活性和功能。本发明中的培养基采用上述添加物和加入较高含量的FBS来替代昂贵的生长因子(如EGF，HGF等)，同样能保持肝细胞的活力和功能，同时也降低了成本。DMSO can make hepatocytes replicate stably, maintain their specific functions, and inhibit the excessive growth of non-parenchymal cells; insulin can promote cell proliferation and differentiation; nicotinamide can promote rapid clonal proliferation and differentiation of hepatocytes; activity and function. The culture medium of the present invention adopts the above-mentioned additives and a higher content of FBS to replace expensive growth factors (such as EGF, HGF, etc.), which can also maintain the viability and function of liver cells and reduce costs.

用10ml完全培养基重悬一个离心管中的细胞，取0.05ml细胞悬液稀释100倍取出0.5ml加入1.5mlEP管，再加入0.5ml0.4％台盼蓝染液，染色2min。将血球计数板及盖片用擦试干净，并将盖片盖在计数板上。将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间。静置2min后镜下观察，可见活细胞呈饱满圆形，透光性好，胞核清晰。取任意几个视野对拒染的活细胞和染成深蓝色的死细胞计数，计算活率，结果显示，细胞活率大于95％。计算计数板四大格细胞总数，按照下式计数：细胞数/ml＝4大格细胞数×10⁴×稀释倍数/4。细胞计数后制成5×10⁵/ml的细胞悬液，在37℃、5％二氧化碳培养箱内培养。4h后换液除去死细胞和未贴壁细胞，20h后细胞状态良好，获得可用于后续实验研究的猪肝脏原代细胞。Resuspend the cells in a centrifuge tube with 10ml of complete medium, take 0.05ml of the cell suspension and dilute it 100 times, take out 0.5ml and add it to a 1.5ml EP tube, then add 0.5ml of 0.4% trypan blue staining solution, and stain for 2min. Clean the hemocytometer and cover slip with a wipe, and cover the cover slip on the counting plate. Aspirate a little of the cell suspension and add it dropwise to the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate. Observe under the microscope after standing for 2 minutes, it can be seen that the living cells are plump and round, with good light transmission and clear nuclei. Take any number of fields of view to count the living cells that refuse to be dyed and the dead cells that are stained dark blue, and calculate the viability. The results show that the cell viability is greater than 95%. Calculate the total number of cells in the four major grids of the counting plate, and count according to the following formula: cell number/ml=number of cells in 4 large grids×10⁴ ×dilution factor/4. After cell counting, a cell suspension of 5×10⁵ /ml was made, and cultured in a 5% carbon dioxide incubator at 37°C. After 4 hours, the medium was changed to remove dead cells and non-adherent cells. After 20 hours, the cells were in good condition, and primary pig liver cells that could be used for subsequent experimental research were obtained.

本发明分离培养肝脏原代细胞镜下观察肝细胞呈圆形，形状饱满，透亮，胞核清晰，非实质细胞污染率低，细胞碎片和其他非实质细胞污染少。本发明的方法分离培养肝脏原代细胞的得率为6.5×10⁷，肝细胞活率为95％-99％，培养贴壁率为95％-99％，纯度为90％-95％，较传统的灌流法获得的肝细胞各项均有明显的提高。The invention isolates and cultures primary liver cells and observes under a microscope that the hepatocytes are round, full in shape, translucent, clear in nuclei, low in non-parenchymal cell pollution rate, and less in cell fragments and other non-parenchymal cell pollution. The yield of primary liver cells isolated and cultured by the method of the present invention is 6.5×10⁷ , the viability of liver cells is 95%-99%, the culture adherence rate is 95%-99%, and the purity is 90%-95%. The hepatocytes obtained by the traditional perfusion method were significantly improved.

本发明提供的分离培养肝脏原代细胞的方法与传统的四步灌流法相比，省去两次更换灌流液的操作，省去梯度离心过程除杂细胞，采用分离时用灌流液彻底的除去血细胞，消化肝细胞时彻底过滤除去消化较慢的结缔组织，低速短时离心减少成纤维细胞即得到纯度较高的肝脏原代细胞，本发明方法操作简单，且降低污染几率。本发明提供的灌流液和清洗液配方简单，配制原材料易获得，清洗液中不使用DNase，而是采用加大清洗液用量来冲开细胞间的粘连，节约成本；无需使用肝素钠等抗凝剂处理；无需使用胶原或其他基质包被培养皿，使用普通细胞培养皿即可实现高贴壁率。本发明方法操作简单易行，成本低，稳定性好，可广泛应用于生物领域大规模分离肝细胞的研究，为细胞移植和药物筛选等研究提供高质量的肝细胞。Compared with the traditional four-step perfusion method, the method for isolating and culturing primary liver cells provided by the present invention saves the operation of replacing the perfusate twice, eliminates the gradient centrifugation process for removing impurities, and completely removes blood cells with the perfusate during separation. When digesting liver cells, thoroughly filter to remove slow-digesting connective tissue, reduce fibroblasts by low-speed short-time centrifugation, and obtain primary liver cells with high purity. The method of the invention is simple to operate and reduces the probability of contamination. The formula of the perfusate and cleaning solution provided by the present invention is simple, and the preparation raw materials are easy to obtain. Instead of using DNase in the cleaning solution, the amount of cleaning solution is increased to break away the adhesion between cells, saving costs; no need to use heparin sodium and other anticoagulants Reagent treatment; no need to use collagen or other substrates to coat the culture dish, and a high adherence rate can be achieved using ordinary cell culture dishes. The method of the invention is simple and easy to operate, low in cost and good in stability, can be widely used in the research of large-scale separation of hepatocytes in the biological field, and provides high-quality hepatocytes for researches such as cell transplantation and drug screening.

附图说明Description of drawings

图1为4×镜下新鲜分离乳猪肝细胞。Figure 1 shows freshly isolated suckling pig hepatocytes under a 4× microscope.

图2为10×镜下新鲜分离乳猪肝细胞。Fig. 2 is freshly isolated suckling pig liver cells under a 10× microscope.

图3为20×镜下新鲜分离乳猪肝细胞。Fig. 3 is freshly isolated suckling pig hepatocytes under a 20× microscope.

图4为4×镜下培养24h乳猪肝细胞。Fig. 4 shows suckling pig hepatocytes cultured for 24 hours under a 4× microscope.

图5为10×镜下培养24h乳猪肝细胞。Fig. 5 shows suckling pig hepatocytes cultured for 24 hours under a 10× microscope.

图6为20×镜下培养24h乳猪肝细胞。Fig. 6 shows suckling pig hepatocytes cultured for 24 hours under a 20× microscope.

图7为4×镜下培养12h乳猪肝细胞。Figure 7 shows suckling pig hepatocytes cultured for 12 hours under a 4× microscope.

图8为10×镜下培养12h乳猪肝细胞。Fig. 8 shows suckling pig hepatocytes cultured for 12 hours under a 10× microscope.

图9为20×镜下培养12h乳猪肝细胞。Fig. 9 shows suckling pig hepatocytes cultured for 12 hours under a 20× microscope.

具体实施方式Detailed ways

以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，实施例中化学试剂除特别说明外，均为市售。Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the chemical reagents in the examples are commercially available unless otherwise specified.

实施例1原代肝细胞的分离Example 1 Isolation of Primary Hepatocytes

本实施例所用的肝脏为从屠宰场采集的新鲜猪肝脏，离体后立即用注射器经肝门静脉把3倍于肝体积的预冷4℃UW液(购自美国Dupont Critical Care公司)以80ml/min速度灌入肝脏，使肝脏迅速均匀降温至10℃以下，再将肝脏放入冰盒(保持2℃左右)3小时之内运输至实验室进行后续操作。The liver used in this example is a fresh pig liver collected from a slaughterhouse. Immediately after isolation, pre-cooled 4°C UW solution (purchased from Dupont Critical Care, USA) 3 times the volume of the liver is injected with a syringe through the hepatic portal vein at 80ml/ The liver was poured into the liver at a speed of 1 min, so that the temperature of the liver was rapidly and evenly lowered to below 10°C, and then the liver was placed in an ice box (maintained at about 2°C) and transported to the laboratory for subsequent operations within 3 hours.

取100g左右的肝脏，启动蠕动泵从肝门静脉先以恒温37℃灌流液(1L配方为：NaCl 9.397g，KCl 0.235g，HEPES 2.383g，双蒸水定容至1L，pH值7.3)流速60ml/min开放灌流12min，然后以相同速度灌流0.05％IV型胶原酶(购自sigma公司)溶液，该胶原酶溶液其1L配方为NaCl 9.397g，KCl 0.235g，HEPES 2.383g，Na₂HPO₄0.504g，CaCl₂0.1g，IV型胶原酶0.5g，双蒸水定容至1L，约3min，包膜下的肝组织呈龟背状裂开后结束灌流。迅速取下消化成熟至肝脏表面呈龟背状裂开的肝脏置于另一无菌平皿中，浸泡于0.05％IV型胶原酶溶液中。由肝脏中心向周围剪几下肝脏，钝性撕开肝包膜，让肝脏在100ml 0.05％IV型胶原酶中继续消化约7min。Take about 100g of liver, start the peristaltic pump, and start the perfusate from the hepatic portal vein at a constant temperature of 37°C (1L formula: NaCl 9.397g, KCl 0.235g, HEPES 2.383g, distilled water to 1L, pH 7.3) at a flow rate of 60ml /min open perfusion_for 12min,_and then perfuse 0.05% type IV collagenase (purchased from sigma company) solution at the same speed. g, CaCl₂ 0.1g, type IV collagenase 0.5g, double-distilled water to 1L, about 3min, the liver tissue under the capsule cracked in a turtle-like shape, and then the perfusion was terminated. Quickly remove the liver that has been digested and matured until the surface of the liver is cracked like a turtle, and placed in another sterile plate, soaked in 0.05% type IV collagenase solution. Cut the liver several times from the center of the liver to the periphery, tear the liver capsule bluntly, and let the liver continue to digest for about 7 minutes in 100ml 0.05% type IV collagenase.

待肝脏消化到“烂泥状”，加入500ml清洗液，本发明清洗液的配方为NaCl 9.397g，KCl 0.235g，HEPES 2.383g，Na₂HPO₄0.504g，CaCl₂0.1g，双蒸水定容至1L。终止消化，让消化好的肝脏细胞悬液通过两层清洗液浸润过的纱布和两层清洗液湿润的细胞筛(上层100目，下层200目)。再把过滤后的悬液分别转入若干50ml离心管中，400rpm，3min，弃上清，加入与沉淀体积比为8∶1的清洗液，再次离心500rpm，3min，弃上清，得到分离的肝脏原代细胞。When the liver is digested to a "slushy state", add 500ml of cleaning solution. The formula of the cleaning solution of the present invention is NaCl 9.397g, KCl 0.235g, HEPES 2.383g, Na₂ HPO₄ 0.504g, CaCl₂ 0.1g, distilled water to volume to 1L. Stop the digestion, let the digested liver cell suspension pass through two layers of gauze soaked in the cleaning solution and two layers of cell sieves wetted with the cleaning solution (the upper layer is 100 mesh, and the lower layer is 200 mesh). Then transfer the filtered suspension into several 50ml centrifuge tubes, 400rpm, 3min, discard the supernatant, add a cleaning solution with a volume ratio of 8:1 to the precipitate, centrifuge again at 500rpm, 3min, discard the supernatant, and obtain the separated Liver primary cells.

实施例2原代肝细胞的分离Example 2 Isolation of Primary Hepatocytes

本实施例所用的肝脏为从屠宰场采集的新鲜动物肝脏，离体后立即用注射器经肝门静脉把4倍于肝体积的预冷4℃UW液(购自美国Dupont Critical Care公司)以100ml/min速度灌入肝脏，使肝脏迅速均匀降温至10℃以下，再将肝脏放入冰盒(保持1℃左右)3小时之内运输至实验室进行后续操作。The liver used in this example is a fresh animal liver collected from a slaughterhouse. Immediately after isolation, pre-cooled 4°C UW solution (purchased from Dupont Critical Care, U.S.) that is 4 times the volume of the liver is injected with a syringe through the hepatic portal vein at 100ml/ The liver was poured into the liver at a speed of 1 min, so that the temperature of the liver was rapidly and evenly lowered to below 10°C, and then the liver was placed in an ice box (maintained at about 1°C) and transported to the laboratory for follow-up operations within 3 hours.

取100g左右的肝脏，启动蠕动泵从肝门静脉先以恒温37℃灌流液(1L配方为：NaCl 9g，KCl 0.2g，HEPES 4.8g，双蒸水定容至1L，pH值7.2)流速55ml/min开放灌流15min，然后以相同速度灌流0.05％II型胶原酶(购自sigma公司)溶液，该胶原酶溶液其1L配方为NaCl9g，KCl 0.2g，HEPES 4.8g，Na₂HPO₄0.6g，CaCl₂0.14g，II型胶原酶0.05g，双蒸水定容至1L，约3min，包膜下的肝组织呈龟背状裂开后结束灌流。迅速取下消化成熟至肝脏表面呈龟背状裂开的肝脏置于另一无菌平皿中，浸泡于0.05％II型胶原酶溶液中。由肝脏中心向周围剪几下肝脏，钝性撕开肝包膜，让肝脏在100ml 0.05％II型胶原酶中继续消化约10min。Take about 100g of liver, start the peristaltic pump, and start the perfusion solution (1L formula: NaCl 9g, KCl 0.2g, HEPES 4.8g, distilled water to 1L, pH value 7.2) from the hepatic portal vein at a flow rate of 55ml/ open perfusion for 15 min, and then perfuse 0.05% type II collagenase (purchased from sigma company) solution at the same speed. The 1L formula of the collagenase solution is NaCl 9g, KCl 0.2g, HEPES 4.8g, Na₂ HPO₄ 0.6g, CaCl₂ 0.14g, 0.05g type II collagenase, distilled water to 1L, about 3min, the liver tissue under the capsule cracks in a turtle-like shape, and then the perfusion ends. Quickly remove the liver that has been digested and matured until the surface of the liver is cracked like a turtle, and placed in another sterile plate, soaked in 0.05% type II collagenase solution. Cut the liver several times from the center of the liver to the periphery, tear the liver capsule bluntly, and let the liver continue to digest for about 10 minutes in 100ml 0.05% type II collagenase.

待肝脏消化到“烂泥状”，加入600ml清洗液，本发明清洗液的配方为NaCl 9g，KCl 0.2g，HEPES 4.8g，Na₂HPO₄0.6g，CaCl₂0.14g，双蒸水定容至1L。终止消化，让消化好的肝脏细胞悬液通过两层清洗液浸润过的纱布和两层清洗液湿润的细胞筛(上层100目，下层200目)。再把过滤后的悬液分别转入若干50ml离心管中，400rpm，3min，弃上清，加入与沉淀体积比为7∶1的清洗液，再次离心500rpm，3min，弃上清，得到分离的肝脏原代细胞。After the liver is digested to "slushy", add 600ml of cleaning solution. The formula of the cleaning solution of the present invention is 9g of NaCl, 0.2g of KCl, 4.8g of HEPES, 0.6g of Na₂ HPO₄ , 0.14g of CaCl₂ . 1L. Stop the digestion, let the digested liver cell suspension pass through two layers of gauze soaked in the cleaning solution and two layers of cell sieves wetted with the cleaning solution (the upper layer is 100 mesh, and the lower layer is 200 mesh). Then transfer the filtered suspension into several 50ml centrifuge tubes, 400rpm, 3min, discard the supernatant, add a cleaning solution with a volume ratio of 7:1 to the precipitate, centrifuge again at 500rpm, 3min, discard the supernatant, and obtain the separated Liver primary cells.

实施例3原代肝细胞的分离Example 3 Isolation of Primary Hepatocytes

本实施例实验乳猪为纯种大白猪，来自北京顺新隆种猪场。预先准备好37℃水浴锅和蠕动泵(ALC-B6型恒流泵，上海奥尔科特生物科技有限公司)，用无菌PBS排空管道气泡。通过以下方法采集乳猪的肝脏，操作方法为：5日龄实验乳猪禁食12h后，采用3％戊巴比妥钠(灭菌生理盐水配制)腹腔注射麻醉(1.6ml/kg)，用温水清洗乳猪皮肤，并浸泡75％酒精5min。保定乳猪后，T型口开腹，暴露并分离肝门静脉和下腔静脉，穿线备用。在门静脉上剪一小口，插入16号灌胃针，动脉夹固定。结扎肝动脉，胃左右静脉，脾静脉，肠系膜静脉。剪断肝脏韧带，其他血管及系膜并将肝脏转移至无菌平皿中。The experimental suckling pigs in this example are purebred large white pigs from Beijing Shunxinlong Breeding Pig Farm. A 37°C water bath and a peristaltic pump (ALC-B6 constant flow pump, Shanghai Alcott Biotechnology Co., Ltd.) were prepared in advance, and the air bubbles in the pipeline were evacuated with sterile PBS. Collect the liver of suckling pig by the following method, the operating method is: after 5 days old experimental suckling pig fasted 12h, adopt 3% sodium pentobarbital (sterilized normal saline preparation) intraperitoneal injection anesthesia (1.6ml/kg), use Wash the suckling pig skin with warm water, and soak in 75% alcohol for 5 minutes. After Baoding the suckling pigs, the T-shaped port was opened to expose and separate the hepatic portal vein and inferior vena cava, and thread them for later use. Cut a small opening on the portal vein, insert a 16-gauge gavage needle, and fix the arterial clip. Hepatic artery, left and right gastric vein, splenic vein, and mesenteric vein were ligated. Hepatic ligaments, other blood vessels and mesentery were cut and the liver was transferred to a sterile dish.

启动蠕动泵后立即剪开下腔静脉。先以恒温37℃灌流液(1L配方为：NaCl 8g，KCl 0.4g，HEPES2.4g，双蒸水定容至1L，pH值7.4)流速50ml/min开放灌流10min，然后以相同速度灌流0.05％IV型胶原酶溶液购自sigma公司，胶原酶溶液(1L配方为：NaCl 8g，KCl 0.4g，HEPES 2.4g，Na₂HPO₄0.6g，CaCl₂0.1g，IV型胶原酶0.5g，双蒸水定容至1L)，约3min，包膜下的肝组织呈龟背状裂开后结束灌流。迅速取下消化成熟至肝脏表面呈龟背状裂开的肝脏置于另一无菌平皿中，浸泡于0.05％IV型胶原酶溶液中。由肝脏中心向周围剪几下肝脏，钝性撕开肝包膜，让肝脏在100ml 0.05％IV型胶原酶中继续消化约5-10min。Cut the inferior vena cava immediately after starting the peristaltic pump. Firstly, perfusate at a constant temperature of 37°C (1L formula: NaCl 8g, KCl 0.4g, HEPES 2.4g, distilled water to 1L, pH value 7.4) at a flow rate of 50ml/min for 10min, and then perfuse at the same speed of 0.05% Type IV collagenase solution was purchased from sigma company, collagenase solution (1L formula: NaCl 8g, KCl 0.4g, HEPES 2.4g,_Na2HPO4 0.6g,_CaCl2 0.1g, type IV collagenase_0.5g , double distilled The volume of water was adjusted to 1L), and the perfusion was terminated after about 3 minutes. Quickly remove the liver that has been digested and matured until the surface of the liver is cracked like a turtle, and placed in another sterile plate, soaked in 0.05% type IV collagenase solution. Cut the liver several times from the center of the liver to the periphery, tear the liver capsule bluntly, and let the liver continue to digest for about 5-10 minutes in 100ml 0.05% type IV collagenase.

待肝脏消化到“烂泥状”，加入400ml清洗液(1L配方为：NaCl 8g，KCl 0.4g，HEPES 2.4g，Na₂HPO₄0.6g，CaCl₂0.1g，双蒸水定容至1L)，终止消化，让消化好的肝脏细胞悬液通过两层清洗液浸润过的纱布和两层清洗液湿润的细胞筛(上层100目，下层200目)。再把过滤后的悬液分别转入若干50ml离心管中，400rpm，3min，弃上清，加入与沉淀体积比为5∶1的清洗液，再次离心500rpm，3min，弃上清，沉淀即为肝脏原代细胞。After the liver is digested to "slushy", add 400ml of cleaning solution (1L formula: NaCl 8g, KCl 0.4g, HEPES 2.4g, Na₂ HPO₄ 0.6g, CaCl₂ 0.1g, distilled water to 1L), Stop the digestion, let the digested liver cell suspension pass through two layers of gauze soaked in the cleaning solution and two layers of cell sieves wetted with the cleaning solution (the upper layer is 100 mesh, and the lower layer is 200 mesh). Then transfer the filtered suspension into several 50ml centrifuge tubes, 400rpm, 3min, discard the supernatant, add a cleaning solution with a volume ratio of 5:1 to the precipitate, centrifuge again at 500rpm, 3min, discard the supernatant, and the precipitate is Liver primary cells.

实施例4原代肝细胞的培养The culture of embodiment 4 primary hepatocytes

用完全培养基重悬。本发明中完全培养基为20％FBS的DMEM培养基添加了600U/ml青霉素，20μg/ml链霉素，2％二甲基亚砜，10mg/L胰岛素，10mmol/L尼克酰胺，1mmol/L抗坏血酸，另用1M的NaHCO₃调节pH至7.2。Resuspend with complete medium. In the present invention, the complete medium is 20% FBS DMEM medium with 600U/ml penicillin, 20 μg/ml streptomycin, 2% dimethyl sulfoxide, 10mg/L insulin, 10mmol/L nicotinamide, 1mmol/L Ascorbic acid and additionally 1 M NaHCO₃ to adjust the pH to 7.2.

分别用10ml完全培养基重悬实施例1、2、3分离得到的肝脏原代细胞，取0.05ml细胞悬液稀释100倍取出0.5ml加入1.5mlEP管，再加入0.5ml 0.4％台盼蓝染液，染色2min。将血球计数板及盖片用擦试干净，并将盖片盖在计数板上。将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间。静置2min后镜下观察，可见活细胞呈饱满圆形，透光性好，胞核清晰。取任意几个视野对拒染的活细胞和染成深蓝色的死细胞计数，计算活率，结果显示，细胞活率均大于95％。计算计数板四大格细胞总数，按照下式计数：细胞数/ml＝4大格细胞数×10⁴×稀释倍数/4。细胞计数后按照培养基与沉淀体积比为250∶1制成5×10⁵/ml的细胞悬液，在37℃、5％二氧化碳培养箱内培养。4h后换液除去死细胞和未贴壁细胞，20h后细胞状态良好，双核细胞呈岛状连接，可用于实验。Resuspend the primary liver cells isolated in Examples 1, 2, and 3 with 10ml of complete medium, take 0.05ml of the cell suspension and dilute it 100 times, take out 0.5ml and add it to a 1.5ml EP tube, then add 0.5ml of 0.4% trypan blue to stain solution, stained for 2min. Clean the hemocytometer and cover slip with a wipe, and cover the cover slip on the counting plate. Aspirate a little of the cell suspension and add it dropwise to the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate. Observe under the microscope after standing for 2 minutes, it can be seen that the living cells are plump and round, with good light transmission and clear nuclei. Take any number of fields of view to count the living cells that refuse to be dyed and the dead cells that are dyed dark blue, and calculate the viability. The results show that the cell viability is greater than 95%. Calculate the total number of cells in the four major grids of the counting plate, and count according to the following formula: cell number/ml=number of cells in 4 large grids×10⁴ ×dilution factor/4. After cell counting, a cell suspension of 5×10⁵ /ml was prepared according to the volume ratio of the medium to the pellet at 250:1, and cultured in a 5% carbon dioxide incubator at 37°C. After 4 hours, the medium was changed to remove dead cells and non-adherent cells. After 20 hours, the cells were in good condition, and the binuclear cells were connected in an island shape, which could be used for experiments.

本发明与传统的灌流法(见后附的参考文献1-3)获得的肝细胞相比较如下表2所示：The hepatocytes obtained by the present invention and the traditional perfusion method (see attached references 1-3) are compared as shown in the following table 2:

表2Table 2

  本发明灌流法Perfusion method of the present invention  传统灌流法^[1-3]Traditional perfusion method^[1-3]  得率(个/克肝组织)Yield (one/gram liver tissue)  6.5×10⁷6.5×10⁷  1.25×10⁷1.25×10⁷  活率(％)Live rate (%)  9999  80-9080-90  贴壁率(％)Adhesion rate (%)  9898  80-9080-90  纯度(％)Purity (%)  9393  80-9080-90

培养后0小时，24小时4倍镜，10倍镜和20倍镜下细胞生长状态见图1-6。See Figures 1-6 for the cell growth status under 0 hour, 24 hour 4X, 10X and 20X microscopes after culture.

镜下观察肝细胞呈圆形，形状饱满，透亮，胞核清晰，非实质细胞污染率低，细胞碎片和其他非实质细胞污染少。分离培养的肝脏原代细胞的各项指标均显著高于传统灌流法。本发明方法操作简单易行，成本低，稳定性好，可广泛应用于生物领域大规模分离肝细胞的研究，为细胞移植和药物筛选等研究提供高质量的肝细胞。(参考文献：[1]P.O.Seglen.Preparation of rat liver cells：I.Effect of Ca²⁺onenzymatic dispersion of isolated，perfused liver[J]Experimental Cell Research，1972，74(2)，450-454.Under the microscope, the hepatocytes were round, full in shape, translucent, with clear nuclei, low rate of non-parenchymal cell contamination, less cell debris and other non-parenchymal cell contamination. The indicators of isolated and cultured primary liver cells were significantly higher than those of the traditional perfusion method. The method of the invention is simple and easy to operate, low in cost and good in stability, can be widely used in the research of large-scale separation of hepatocytes in the biological field, and provides high-quality hepatocytes for researches such as cell transplantation and drug screening. (References: [1] POSeglen. Preparation of rat liver cells: I. Effect of Ca²⁺ onenzymatic dispersion of isolated, perfused liver [J] Experimental Cell Research, 1972, 74 (2), 450-454.

[2]P.O.Seglen.Preparation of rat liver cells：II.Effects of ions and chelators on tissuedispersion.Experimental Cell Research，1973，76(1)，25-30.[2] P.O. Seglen. Preparation of rat liver cells: II. Effects of ions and chelators on tissue dispersion. Experimental Cell Research, 1973, 76(1), 25-30.

[3]P.O.Seglen.Preparation of rat liver cells：III.Enzymatic requirements for tissuedispersion.Experimental Cell Research，1973，82(2)，391-398.)[3] P.O. Seglen. Preparation of rat liver cells: III. Enzymatic requirements for tissue dispersion. Experimental Cell Research, 1973, 82(2), 391-398.)

实施例5原代肝细胞的培养The cultivation of embodiment 5 primary hepatocytes

用完全培养基重悬。本发明中完全培养基为20％FBS的DMEM培养基添加了200U/ml青霉素，60μg/ml链霉素，1％二甲基亚砜，1mg/L胰岛素，1mmol/L尼克酰胺，0.1mmol/L抗坏血酸，另用1M的NaHCO₃调节pH至7.4。Resuspend with complete medium. In the present invention, the DMEM culture medium of 20% FBS has added 200U/ml penicillin, 60 μg/ml streptomycin, 1% dimethyl sulfoxide, 1mg/L insulin, 1mmol/L nicotinamide, 0.1mmol/ L ascorbic acid and additionally adjust the pH to 7.4 with 1 M NaHCO₃ .

分别用10ml完全培养基重悬实施例1、2、3分离得到的肝脏原代细胞，取0.05ml细胞悬液稀释100倍取出0.5ml加入1.5mlEP管，再加入0.5ml 0.4％台盼蓝染液，染色2min。将血球计数板及盖片用擦试干净，并将盖片盖在计数板上。将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间。静置2min后镜下观察，可见活细胞呈饱满圆形，透光性好，胞核清晰。取任意几个视野对拒染的活细胞和染成深蓝色的死细胞计数，计算活率，结果显示，细胞活率均大于95％。计算计数板四大格细胞总数，按照下式计数：细胞数/ml＝4大格细胞数×10⁴×稀释倍数/4。细胞计数后按照培养基与沉淀体积比为300∶1制成5×10⁵/ml的细胞悬液，在37℃、5％二氧化碳培养箱内培养。4h后换液除去死细胞和未贴壁细胞，20h后细胞状态良好，双核细胞呈岛状连接，可用于实验。Resuspend the primary liver cells isolated in Examples 1, 2, and 3 with 10ml of complete medium, take 0.05ml of the cell suspension and dilute it 100 times, take out 0.5ml and add it to a 1.5ml EP tube, then add 0.5ml of 0.4% trypan blue to stain solution, stained for 2min. Clean the hemocytometer and cover slip with a wipe, and cover the cover slip on the counting plate. Aspirate a little of the cell suspension and add it dropwise to the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate. Observe under the microscope after standing for 2 minutes, it can be seen that the living cells are plump and round, with good light transmission and clear nuclei. Take any number of fields of view to count the living cells that refuse to be dyed and the dead cells that are dyed dark blue, and calculate the viability. The results show that the cell viability is greater than 95%. Calculate the total number of cells in the four major grids of the counting plate, and count according to the following formula: cell number/ml=number of cells in 4 large grids×10⁴ ×dilution factor/4. After counting the cells, a cell suspension of 5×10⁵ /ml was prepared according to the volume ratio of the medium to the pellet at 300:1, and cultured in a 5% carbon dioxide incubator at 37°C. After 4 hours, the medium was changed to remove dead cells and non-adherent cells. After 20 hours, the cells were in good condition, and the binuclear cells were connected in an island shape, which could be used for experiments.

本发明与传统的灌流法获得的肝细胞相比较如下表3所示：Compared with the hepatocytes obtained by the traditional perfusion method, the present invention is shown in Table 3 below:

表3table 3

  本发明灌流法Perfusion method of the present invention  传统灌流法^[1-3]Traditional perfusion method^[1-3]  得率(个/克肝组织)Yield (one/gram liver tissue)  6.5×10⁷6.5×10⁷  1.25×10⁷1.25×10⁷  活率(％)Live rate (%)  9999  80-9080-90  贴壁率(％)Adhesion rate (%)  9595  80-9080-90  纯度(％)Purity (%)  9595  80-9080-90

培养后12小时4倍镜，10倍镜和20倍镜下细胞生长状态见图7-9。12 hours after culture, see Figure 7-9 for the cell growth status under 4X, 10X and 20X microscopes.

镜下观察肝细胞呈圆形，形状饱满，透亮，胞核清晰，非实质细胞污染率低，细胞碎片和其他非实质细胞污染少。肝细胞的得率、活率、贴壁率和纯度均显著高于传统灌流法。本发明方法操作简单易行，成本低，稳定性好，可广泛应用于生物领域大规模分离肝细胞的研究，为细胞移植和药物筛选等研究提供高质量的肝细胞。Under the microscope, the hepatocytes were round, full in shape, translucent, with clear nuclei, low rate of non-parenchymal cell contamination, less cell debris and other non-parenchymal cell contamination. The yield, viability, adherence rate and purity of hepatocytes were significantly higher than those of the traditional perfusion method. The method of the invention is simple and easy to operate, low in cost and good in stability, can be widely used in the research of large-scale separation of hepatocytes in the biological field, and provides high-quality hepatocytes for researches such as cell transplantation and drug screening.

实施例6传代效果试验Embodiment 6 subculture effect test

当肝脏原代细胞汇合度达到80-90％即可传代。以10cm细胞培养皿为例，吸走培养基，用1ml PBS(购自北京钮因华信)清洗，反复3遍，加入2ml 0.05％胰酶，37℃消化1min，加入2ml完全培养基(配方同实施例4)终止消化，用移液器轻轻吹打，使培养皿壁上的细胞脱落，形成细胞悬液的转入离心管，800rpm，离心5min。倒去上清，加入2ml完全培养基重悬后往每个盛有9ml预热完全培养基的10cm细胞培养皿加1ml。4h后，镜下观察传代细胞贴壁率达99.5％以上，60-72h后传代细胞汇合度达到80-90％又可传代。对传代10代内的观察，肝细胞无衰老死亡现象，形态正常，生长速度和状态均正常。When the primary liver cells reach 80-90% confluence, they can be passaged. Take a 10cm cell culture dish as an example, suck away the culture medium, wash it with 1ml PBS (purchased from Beijing Niuyin Huaxin),repeat 3 times, add 2ml 0.05% trypsin, digest at 37°C for 1min, add 2ml complete medium (the formula is the same as the implementation Example 4) Terminate the digestion, blow gently with a pipette to make the cells on the wall of the culture dish fall off, and transfer the cell suspension into a centrifuge tube, centrifuge at 800rpm for 5min. Pour off the supernatant, add 2ml of complete medium to resuspend, then add 1ml to each 10cm cell culture dish filled with 9ml of preheated complete medium. After 4 hours, the adhesion rate of the passaged cells was observed under a microscope to reach over 99.5%, and after 60-72 hours the confluence of the passaged cells reached 80-90% and could be passaged again. Observation within 10 passages showed that the hepatocytes had no senescence and death phenomenon, and the morphology was normal, and the growth speed and state were normal.

实施例7冻存及复苏效果试验Embodiment 7 cryopreservation and recovery effect test

冻存：计划冻存前12h换成新鲜完全培养基，细胞长到汇合度80％即可冻存。配制冻存液：50％DMEM+40％FBS+双抗+1mg/L胰岛素+10％DMSO，4℃预冷备用。细胞消化收集后用预冷的冻存液缓慢的重悬细胞至浓度约为5×10⁶/ml，放入冻存管后放置在异丙醇细胞冻存盒(购自美国Nalgene公司)中置于-80℃冰箱，16-18h后转移至液氮罐永久保存。Cryopreservation: Replace with fresh complete medium 12 hours before the planned freezing, and the cells can be frozen when they grow to a confluence of 80%. Prepare cryopreservation solution: 50% DMEM + 40% FBS + double antibody + 1 mg/L insulin + 10% DMSO, pre-cool at 4°C for later use. After cell digestion and collection, slowly resuspend the cells with pre-cooled freezing solution to a concentration of about 5×10⁶ /ml, put them into cryopreservation tubes and place them in isopropanol cell freezing boxes (purchased from Nalgene, USA) Store in a -80°C refrigerator, and transfer to a liquid nitrogen tank for permanent storage after 16-18 hours.

复苏：预热水浴锅至40℃。从液氮罐中取出保存肝原代细胞的冻存管，迅速置于温水中并不断搅动，使之在1分钟之内融化。超净盒中打开冻存管，将细胞悬液缓慢滴入盛有5ml完全培养基的离心管中。800rpm离心5分钟，弃上清。每管沉淀加10ml完全培养基，吹打均匀，将细胞转移至10cm培养皿中，37℃培养，4h后观察贴壁率95％以上，肝细胞形状饱满，生长状态好。Recovery: Preheat the hot water bath to 40°C. Take out the cryopreservation tube that preserves the primary liver cells from the liquid nitrogen tank, place it in warm water quickly and keep stirring, so that it melts within 1 minute. Open the freezing tube in the ultra-clean box, and slowly drop the cell suspension into the centrifuge tube containing 5ml of complete medium. Centrifuge at 800 rpm for 5 minutes and discard the supernatant. Add 10ml of complete medium to each tube of precipitation, pipette evenly, transfer the cells to a 10cm culture dish, and culture at 37°C. After 4 hours, observe that the adherence rate is over 95%, and the shape of the liver cells is full and the growth state is good.

实验结果说明本发明分离培养的肝脏原代细胞经冻存、复苏后，其肝细胞形状饱满，生长状态良好，稳定性好，可以继续应用于生物学领域的各项实验中。The experimental results show that the hepatic primary cells isolated and cultured in the present invention have plump shape, good growth state and good stability after cryopreservation and resuscitation, and can be continuously applied in various experiments in the field of biology.

Claims (1)

Translated fromChinese

1.一种分离培养肝脏原代细胞的方法，包含以下步骤：1. A method for isolating and culturing liver primary cells, comprising the following steps:1）从离体肝脏的肝门静脉输入灌流液，所述的灌流液，其1L配方为：NaCl 8-9.4g，KCl 0.2-0.4g，HEPES 2.4-4.8g，双蒸水定容至1L，pH值7.2-7.4；1) Input the perfusate from the hepatic portal vein of the isolated liver. The 1L formula of the perfusate is: NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, distilled water to 1L, pH 7.2-7.4;其中步骤1）中，所述离体肝脏为：新鲜离体肝脏经肝门静脉以80-100ml/min速度注射肝体积3倍以上的0-4℃预冷UW液，肝温降至10℃以下后，将肝脏置于冰盒保持1-4℃，保存时间不超过3h；In step 1), the isolated liver is as follows: the fresh isolated liver is injected with 0-4°C pre-cooled UW solution more than three times the liver volume through the hepatic portal vein at a rate of 80-100ml/min, and the liver temperature drops below 10°C Afterwards, put the liver in an ice box and keep it at 1-4°C for no more than 3 hours;其中步骤1）中所述灌流是以恒温37℃灌流液开放灌流10-15min，流速50-60ml/min；The perfusion described in step 1) is to open the perfusion with the perfusate at a constant temperature of 37°C for 10-15 minutes, and the flow rate is 50-60ml/min;2）输入胶原酶灌流液，肝组织呈龟背状裂开后，停止灌流；所述步骤2）胶原酶灌流液灌流流速50-60ml/min；2) Input the collagenase perfusate, and stop the perfusion after the liver tissue cracks in a turtle-like shape; the step 2) the perfusion flow rate of the collagenase perfusate is 50-60ml/min;其中步骤2）中的胶原酶灌流液，其1L配方为NaCl 8-9.4g，KCl 0.2-0.4g，HEPES 2.4-4.8g，Na₂HPO₄ 0.5-0.6g，CaCl₂ 0.10-0.15g，II型或IV型胶原酶0.5g，双蒸水定容至1L；Among them, the collagenase perfusate in step 2), its 1L formula is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na₂ HPO₄ 0.5-0.6g, CaCl₂ 0.10-0.15g, II Type or IV collagenase 0.5g, distilled water to 1L;3）将消化成熟的肝脏浸泡于胶原酶灌流液中；所述步骤3）浸泡时间为5-10min；3) Soak the digested mature liver in the collagenase perfusate; the step 3) soaking time is 5-10min;4）加清洗液终止消化，过滤，收集肝细胞悬液；其中所述步骤4）清洗液配方为NaCl 8-9.4g，KCl 0.2-0.4g，HEPES 2.4-4.8g，Na₂HPO₄0.5-0.6g，CaCl₂ 0.10-0.15g，双蒸水定容至1L；4) Add cleaning solution to stop digestion, filter, and collect liver cell suspension; the step 4) cleaning solution formula is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na₂ HPO₄ 0.5- 0.6g, CaCl₂ 0.10-0.15g, distilled water to 1L;所述步骤4）过滤是将肝脏细胞悬液通过两层纱布和两层细胞筛，纱布和细胞筛均用清洗液浸润，所述细胞筛上层100目，下层200目；The step 4) filtering is to pass the liver cell suspension through two layers of gauze and two layers of cell sieves, both gauze and cell sieves are infiltrated with cleaning solution, the upper layer of the cell sieve is 100 mesh, and the lower layer is 200 mesh;5）离心，弃上清，用完全培养基重悬培养；5) Centrifuge, discard the supernatant, and resuspend culture with complete medium;其中，所述步骤5）离心是将肝细胞滤液加入离心管中，400rpm，3min，弃上清，按与沉淀的体积比5-8：1加入清洗液，再次离心500rpm，3min，弃上清；Wherein, the step 5) centrifugation is to add the liver cell filtrate into the centrifuge tube, 400rpm, 3min, discard the supernatant, add the cleaning solution according to the volume ratio of the precipitate to 5-8:1, centrifuge again at 500rpm, 3min, discard the supernatant ;其中，所述步骤5）中完全培养基其配方为：含20%FBS的DMEM培养基添加终浓度为200-600U/ml青霉素，20-60μg/ml链霉素，1%-2%二甲基亚砜，1-10mg/L胰岛素，1-10mmol/L尼克酰胺，0.1-1mmol/L抗坏血酸，用1M的NaHCO₃调节pH至7.2-7.4。Wherein, the formula of the complete medium in step 5) is: DMEM medium containing 20% FBS is added with a final concentration of 200-600U/ml penicillin, 20-60 μg/ml streptomycin, 1%-2% dimethyl Base sulfoxide, 1-10mg/L insulin, 1-10mmol/L nicotinamide, 0.1-1mmol/L ascorbic acid, adjust the pH to 7.2-7.4 with 1M NaHCO₃ .

Images