Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion.
Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion.
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DOI：
10.3791/56993
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发表时间：
2018-02-12
期刊：
影响因子：
--
通讯作者：
Harris EN
中科院分区：
文献类型：
--
作者：
Cabral F;Miller CM;Kudrna KM;Hass BE;Daubendiek JG;Kellar BM;Harris EN
该方案证明了一种适合于培养或获得细胞裂解物的小鼠肝细胞和肝窦内皮细胞(SECs)获得高产率和活性的方法。在这个方案中，门静脉被用作插管的部位，而不是腔静脉，因为这限制了在最终的肝脏准备中对其他可能的细胞类型的污染。在整个过程中不需要特殊的仪器。水浴被用作热源，以保持所有缓冲液和溶液的温度。使用标准的蠕动泵驱动液体，离心机需要冷藏的台式离心机。这项技术的唯一限制是将导管放置在门静脉内，这对一些18-25g大小的小鼠来说是一种挑战。这项技术的一个优点是只利用一条静脉进行灌流，而且进入静脉的速度很快，从而最大限度地减少了肝脏的缺血和再灌注，从而降低了肝细胞的活力。该方案的另一个优点是，由于离心步骤中细胞密度的不同，很容易用肉眼区分活的和死的肝细胞。来自该方案的细胞可用于任何下游应用的细胞培养，以及用于任何生化评估的处理。
This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.
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影响因子：
1.6
作者：
EDSTROM, S;EKMAN, L;LUNDHOLM, K
通讯作者：
LUNDHOLM, K
影响因子：
1.2
作者：
Gopalakrishnan, Sandhya;Harris, Edward N.
通讯作者：
Harris, Edward N.
影响因子：
15.3
作者：
MILLER, LL;BLY, CG;BALE, WF
通讯作者：
BALE, WF
影响因子：
3.7
作者：
Meyer, Jeremy;Lacotte, Stephanie;Buhler, Leo
通讯作者：
Buhler, Leo
DOI：
10.1007/978-1-4939-6700-1_21
发表时间：
2017-01-01
期刊：
HEPATITIS B VIRUS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Liu, Jia;Huang, Xuan;Lu, Mengji
通讯作者：
Lu, Mengji