小鼠原代肝细胞分离提取技术 (经验帖)

孤苦伶仃，苦干五个月，终于成功了。实验期间各种坑都填过，该绕的弯路也都绕了，证明这套提取方法相当可靠，感谢论坛曾经的帮助，现在把core protocol共享，有问题欢迎楼下讨论。

如果已经做过一阵子原代肝细胞提取的话，对一些基本的实验操作和流程应该都比较熟悉了，论坛里也有一些经验贴。下面分享几点我认为成功分离原代肝细胞的关键:

1.动物我用的C57，同等周龄下，下腔静脉或者门静脉要比Balb/c粗壮很多，方便进针置管。

2.严格控制灌流速度，小鼠的最佳速度是5ml每分钟，推荐最好用蠕动泵代替手工注射器，灌流过程中保证灌流液的温度。

3.我认为最最最关键的一点就是胶原酶的浓度！！！(重点) 文献里提到胶原酶最佳浓度是100CDU/ML，推荐买sigma的胶原酶，根据批号可以查到具体的胶原酶活性值，非常重要！！！（后文有详细说明）

4.关于灌流时间，前灌和消化大约各5分钟即可，具体细节需要自己把控，但是按照前面的速度和浓度，绝对能分离出来活力较好的肝细胞！

5.每个肝约消化3-5千万肝细胞，用percoll纯化后可以极大提高细胞活率，且percoll浓度越高，得到的肝细胞活性越好，但与此同时，细胞产量会减少，二者需做一平衡。

6.小鼠插管的话建议下腔静脉逆行进针，用i.v. retractable cather 也就是套管针，配合使用一些鲁尔接头，插管的难度会降低很多，也不存在溜针或者刺破血管的问题。

7.重点还是第3点！！！

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20200716 更新 最近整理了一下之前的课题，把之前实验的步骤放上来，供有需要的人参考。从开始的一头雾水，到中间坎坷的独立摸索，最终用5个月的时间才把原代肝细胞成功搞定。可惜当时的课题夭折了，传统手艺也暂时搁置了。回想摸索过程不免感慨，也受到园子里很多前辈经验帖的帮助和启发！希望我分享的材料对大家有所帮助，能减少原代肝细胞分离提取实验的弯路！希望大家的实验都顺利！有错误也欢迎随时指正！~

(以下内容为原创)

Protocol on Isolation of Mouse Hepatocyte

Materials 液体配置表

1.PerfusateⅠ 前灌液

(小鼠一般25mL足够，各组分终浓度如下，可以等比例换算25ml需要的量)

• EGTA                     0.5mM
• HEPES                  25mM
• P/S (双抗)               1%
• D-HANKS              补至30mL

2.Perfusate Ⅱ 消化液

(小鼠一般25mL足够，各组分终浓度如下，可以等比例换算25ml需要的量；胶原酶预先用Hanks配成储备液分装)

• Collagenase Ⅳ (sigma)        100CDU/mL
• CaCl₂ (sigma)                3mM /14mg
• HEPES                      15mM
• P/S                         1%
• DMEM                      补至45mL

3.Dispersion 分散液

（普通DMEM可以添加地米和胰岛素，其他改良培养基本身已添加，不需要额外加）

• Dexamethasone         100nM
• Bovine insulin             0.5μg/ml
• HEPES                    15mM
• P/S                          1%
• FBS                         10%
• DMEM                      补至45ml

4.Culture medium (For first 4h, adding 10% FBS)

（普通培养基，前4h加FBS能够促进贴壁，4h后根据实验需求确定培养基是否添加FBS）

• Dexamethasone       100nM
• Bovine insulin           0.5μg/ml
• HEPES                     5mM
• P/S                          1%
• DMEM                     根据实际需求配置总体积

5.100%Percoll (9:1 v/v 体积比)

• Percoll                     90ml
• 1.5M NaCl or 10x PBS        10ml

6.其他浓度的Percoll, 例如40% Percoll

• 100% Percoll 40ml
• 0.15M NaCl or 1x PBS 60ml

Methods Perfusion 灌流方法

1.      Pre-warm the perfusate Ⅰ and Ⅱ in 42° water bath for 30min to ensure the temperature maintaining 37° after reaching liver. 预热，确保液体到肝脏还是37℃

2.      Turn on the peristaltic pump and adjust to the maximum flow rate to cycle the perfusate Ⅰ to warm the pipe and exhaust air bubble. 排空管道中的气泡

3.      Anaesthetize the mouse with isoflurane or chloral hydrate (4%, 1ml/kg). 小鼠麻醉

4.      Fix mouse feet with needle or sterilization indicator tape. 固定

5.      Sterilize the abdominal area with 75% ethanol. 腹部消毒

6.      Open the abdominal cavity and push intestines to the left side of mouse and expose the portal vein and inferior vena cava. 开腹，暴露下腔静脉

7.      Use cotton swabs or paper balls to bluntly separate the tissue around the inferior vena cava to make it completely exposed. 用棉签或卫生纸团钝性搓开血管周围的软组织

8.      Cannulate the inferior vena cava using I.V. retractable catheter (20G), withdraw the needle and then connect the pipe (no air bubble). 套管针插管，撤回针头，连接灌流管道

9.      Turn on the peristaltic pump at low flow rate of 2 ml/min, cut off the portal vein immediately. Now TIMER is on and gradually adjust the rate to 5ml/min. 打开蠕动泵，流速逐渐升高至5ml/min

10.   Open the thoracic cavity and close the upper segment of inferior vena cava with a venous hemostatic clip. 打开胸腔，把下腔静脉肝上段夹闭

11.   Add 25ml perfusate Ⅱ directly to the perfusate Ⅰ tube when it’s exhausted. 当前灌液快灌完的时候，直接把消化液倒进前灌液的管子，不用停泵，注意避免产生气泡

12.   Once the liver begins to swell and see liquid build-up under liver capsule, wait for 1-2min then turn off the pump, isolate liver and put into the rest part of the perfusate Ⅱ. 当肝包膜开始膨胀，出现透明状的裂纹时，考虑消化结束（消化5分钟足够）

13.   Step 9-12 generally take exactly 9.5-10 min From TIMER on. 整个灌流过程从打开蠕动泵后，持续大约10分钟

14.   Liver digestion is done with less than 25ml perfusate Ⅱ (20ml generally) and 5-min collagenase digestion time. That’s absolutely enough.

Note

a)      No bubble in pipe during perfusion. The I.V. retractable catheter should pre-filling a portion of perfusate Ⅰ to make it convenient to directly connect the pipe line after cannulation. 管道中预充液体，不要有气泡，防止组织肝内血管，影响灌流效果

b)     Collagenase Ⅳ(sigma) concentration. Refer to the certificate of analysis of different batch products. Optimal final concentration is 100CDU/ml. According to practice, perfusing the liver with perfuse Ⅱ (37°, 20-25ml, 100CDU/ml, flow rate 5ml/min) for about 5min is absolutely enough. Too harsh concentration and long-time perfusion doesn’t improve the yield but heavily impair the cell viability. Time is quality. One criterial to stop the digestion is that liver is absolutely swelled and in the upper segment of inferior vena cava near the thoracic cavity, transparent liquid accumulation can be identified. 胶原酶浓度很重要，过高过低都不行，严格按照介绍的来！

Methods Purification

1.      Isolated liver was put into the rest part of perfuse Ⅱ. Storage in ice box and execute the follow-up experiment on ice box and in sterile environment. 肝脏离体以后在冰上操作

2.      Pour the liver into a 10cm culture dish. Use two tweezers to gently tear open the liver capsule and shaken the liver to isolate the single liver cells.镊子撕碎肝包膜，很多细胞抖一抖，液体就浑浊了

3.      Filter the cell suspension with 70μm cell strainer above a new 50ml tube. 过筛

4.      Centrifugate at 4°, 50xg, 1min. Discard the supernatant and resuspend cell pellets with 15ml Dispersion. (Weight balance, no volume balance) 低速离心纯化

5.      Centrifugate at 4°, 50xg, 1min. Discard the supernatant and resuspend cell pellets with 10ml Dispersion. Repeat for two times.

6.      Resuspend the cell pellet in the remaining 10ml Dispersion.

7.      Add 15ml 60%/65%/70% Percoll to a new tube. Lean the tube and gently add cell suspension to the Percoll surface. Make sure that the dividing line of two layer is clearly visible. Percoll纯化，注意操作手法，细胞悬液要倾斜离心管，缓缓滴加，不然分层会被破坏

8.      Centrifugate at 4°, 400xg, 10min, no brake. Speed should rise and fall slowly.

9.      Discard the supernatant and resuspend the cell pellets in culture medium (containing FBS).

10.   Centrifugate at 4°, 50xg, 1min. Discard the supernatant and resuspend cell pellets with culture medium (containing FBS).

11.   Cell count and Trypan blue staining.

12.   Adjust the density of cell suspension to 3*10⁵/ml. It’s recommend to plate on 12-well or even larger well plate to make sure the most consistent plating result. The recommendatory volume is 800ul for 12-well plate, 1.7ml for 6-well plate, 360ul for 24-well plate. At this point, cell confluence under microscopy should be about 60-70%. 注意铺板密度，可以用鼠尾胶原蛋白先预铺孔板，有利于贴壁和细胞形态维持

13.   Leave the plate to rest for 5-10min. Then transfer it into cell culture box.

14.   After 3-4 hours, Change the medium into culture medium (No FBS). Binuclear of cell is clearly visible now even at 2h after plating.

15.   After overnight culture, cells are ready to be used for the following experiment.

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200810更新

sigma的4型胶原酶可以根据批号查到具体的胶原酶活性，一般说明书上标注的≥125U/mg的参考意义不大，实际的酶活或远大于125，切忌根据125u/mg来换算最终需要使用的100CDU/ml。这是我个人在实验中摸出来的关键一点，普通的文献大都按照mg/ml的方式标注，但不同批次不同厂家的胶原酶若仅按照重量换算，可能会导致实验的重复性变差，因此推荐使用sigma的胶原酶按照具体的酶活性值去换算需要的量（不是广告）。

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210530更新

再次强调胶原酶浓度的重要性，请参考200810更新的查询方法！

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220329 更新

肝细胞对流体剪切力很敏感，极易损伤，故全程操作小心。

肝细胞贴壁后，形态变化过程与去分化机制相关，可以检索文献查阅。

按照上面的步骤，肯定能提出来！

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220330更新

最近这段时间有很多人又来咨询原代肝细胞提取的问题，可以直接在下面留言，我会定期上来查看信息，并逐一解答。