孤苦伶仃，苦干五個月，終于成功了。實驗期間各種坑都填過，該繞的彎路也都繞了，證明這套提取方法相當可靠，感謝論壇曾經(jīng)的幫助，現(xiàn)在把core protocol共享，有問題歡迎樓下討論。

如果已經(jīng)做過一陣子原代肝細胞提取的話，對一些基本的實驗操作和流程應該都比較熟悉了，論壇里也有一些經(jīng)驗貼。下面分享幾點我認為成功分離原代肝細胞的關鍵:

1.動物我用的C57，同等周齡下，下腔靜脈或者門靜脈要比Balb/c粗壯很多，方便進針置管。

2.嚴格控制灌流速度，小鼠的最佳速度是5ml每分鐘，推薦最好用蠕動泵代替手工注射器，灌流過程中保證灌流液的溫度。

3.我認為最最最關鍵的一點就是膠原酶的濃度！??！(重點) 文獻里提到膠原酶最佳濃度是100CDU/ML，推薦買sigma的膠原酶，根據(jù)批號可以查到具體的膠原酶活性值，非常重要?。。。ê笪挠性敿氄f明）

4.關于灌流時間，前灌和消化大約各5分鐘即可，具體細節(jié)需要自己把控，但是按照前面的速度和濃度，絕對能分離出來活力較好的肝細胞！

5.每個肝約消化3-5千萬肝細胞，用percoll純化后可以極大提高細胞活率，且percoll濃度越高，得到的肝細胞活性越好，但與此同時，細胞產(chǎn)量會減少，二者需做一平衡。

6.小鼠插管的話建議下腔靜脈逆行進針，用i.v. retractable cather 也就是套管針，配合使用一些魯爾接頭，插管的難度會降低很多，也不存在溜針或者刺破血管的問題。

7.重點還是第3點！??！

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20200716 更新?最近整理了一下之前的課題，把之前實驗的步驟放上來，供有需要的人參考。從開始的一頭霧水，到中間坎坷的獨立摸索，最終用5個月的時間才把原代肝細胞成功搞定?？上М敃r的課題夭折了，傳統(tǒng)手藝也暫時擱置了?；叵朊鬟^程不免感慨，也受到園子里很多前輩經(jīng)驗帖的幫助和啟發(fā)！希望我分享的材料對大家有所幫助，能減少原代肝細胞分離提取實驗的彎路！希望大家的實驗都順利！有錯誤也歡迎隨時指正！~

(以下內(nèi)容為原創(chuàng))

Protocol on Isolation of Mouse Hepatocyte

Materials 液體配置表

1.PerfusateⅠ 前灌液

(小鼠一般25mL足夠，各組分終濃度如下，可以等比例換算25ml需要的量)

• EGTA?????????????????????0.5mM

• HEPES??????????????????25mM

• P/S (雙抗)???????????????1%

• D-HANKS??????????????補至30mL

2.Perfusate Ⅱ 消化液

(小鼠一般25mL足夠，各組分終濃度如下，可以等比例換算25ml需要的量；膠原酶預先用Hanks配成儲備液分裝)

• Collagenase Ⅳ (sigma)????????100CDU/mL

• CaCl2?(sigma)????????? ????? 3mM /14mg

• HEPES????????????????????? 15mM

• P/S???????????????????????? 1%

• DMEM???????????????????? ?補至45mL

3.Dispersion 分散液

（普通DMEM可以添加地米和胰島素，其他改良培養(yǎng)基本身已添加，不需要額外加）

• Dexamethasone?????????100nM

• Bovine insulin???????????? 0.5μg/ml

• HEPES????????????????? ??15mM

• P/S??????????????????????? ??1%

• FBS???????????????????? ????10%

• DMEM??????????????? ??????補至45ml

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

（普通培養(yǎng)基，前4h加FBS能夠促進貼壁，4h后根據(jù)實驗需求確定培養(yǎng)基是否添加FBS）

• Dexamethasone???????100nM

• Bovine insulin???????????0.5μg/ml

• HEPES?????????????????????5mM

• P/S??????????????????????? ??1%

• DMEM?????????????????????根據(jù)實際需求配置總體積

5.100%Percoll (9:1 v/v 體積比)

• Percoll????????????????? ???90ml

• 1.5M NaCl or 10x PBS????????10ml

6.其他濃度的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. 預熱，確保液體到肝臟還是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. 排空管道中的氣泡

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. 開腹，暴露下腔靜脈

7.??????Use cotton swabs or paper balls to bluntly separate the tissue around the inferior vena cava to make it completely exposed. 用棉簽或衛(wèi)生紙團鈍性搓開血管周圍的軟組織

8.??????Cannulate the inferior vena cava using I.V. retractable catheter (20G), withdraw the needle and then connect the pipe (no air bubble). 套管針插管，撤回針頭，連接灌流管道

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. 打開蠕動泵，流速逐漸升高至5ml/min

10.???Open the thoracic cavity and close the upper segment of inferior vena cava with a venous hemostatic clip. 打開胸腔，把下腔靜脈肝上段夾閉

11.???Add 25ml perfusate Ⅱ directly to the perfusate Ⅰ tube when it’s exhausted. 當前灌液快灌完的時候，直接把消化液倒進前灌液的管子，不用停泵，注意避免產(chǎn)生氣泡

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. 當肝包膜開始膨脹，出現(xiàn)透明狀的裂紋時，考慮消化結束（消化5分鐘足夠）

13.???Step 9-12 generally take exactly 9.5-10 min From TIMER on. 整個灌流過程從打開蠕動泵后，持續(xù)大約10分鐘

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. 管道中預充液體，不要有氣泡，防止組織肝內(nèi)血管，影響灌流效果

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. 膠原酶濃度很重要，過高過低都不行，嚴格按照介紹的來！

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. 肝臟離體以后在冰上操作

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.鑷子撕碎肝包膜，很多細胞抖一抖，液體就渾濁了

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

4.??????Centrifugate at 4°, 50xg, 1min. Discard the supernatant and resuspend cell pellets with 15ml Dispersion. (Weight balance, no volume balance) 低速離心純化

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純化，注意操作手法，細胞懸液要傾斜離心管，緩緩滴加，不然分層會被破壞

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%. 注意鋪板密度，可以用鼠尾膠原蛋白先預鋪孔板，有利于貼壁和細胞形態(tài)維持

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型膠原酶可以根據(jù)批號查到具體的膠原酶活性，一般說明書上標注的≥125U/mg的參考意義不大，實際的酶活或遠大于125，切忌根據(jù)125u/mg來換算最終需要使用的100CDU/ml。這是我個人在實驗中摸出來的關鍵一點，普通的文獻大都按照mg/ml的方式標注，但不同批次不同廠家的膠原酶若僅按照重量換算，可能會導致實驗的重復性變差，因此推薦使用sigma的膠原酶按照具體的酶活性值去換算需要的量（不是廣告）。

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210530更新

再次強調(diào)膠原酶濃度的重要性，請參考200810更新的查詢方法！

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220329 更新

肝細胞對流體剪切力很敏感，極易損傷，故全程操作小心。

肝細胞貼壁后，形態(tài)變化過程與去分化機制相關，可以檢索文獻查閱。

按照上面的步驟，肯定能提出來！

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最近這段時間有很多人又來咨詢原代肝細胞提取的問題，可以直接在下面留言，我會定期上來查看信息，并逐一解答。