小鼠肝部分切除术后再生肝细胞的分离
Summary
富含脂质的肝细胞是肝脏再生所固有的，但通常在密度梯度离心时丢失。在这里，我们提出了一种优化的细胞分离方案，该方案保留了脂肪变性肝细胞，在小鼠部分肝切除术后产生具有代表性的再生肝细胞群。
Abstract
部分肝切除术已被广泛用于研究小鼠的肝脏再生，但分离高产量的活肝细胞用于下游单细胞应用具有挑战性。在小鼠正常肝脏再生的前2天观察到再生肝细胞内脂质的显着积累。这种所谓的短暂再生相关脂肪变性（TRAS）是暂时的，但部分与主要增殖期重叠。密度梯度纯化是大多数现有原代肝细胞分离方案的支柱。由于梯度纯化依赖于细胞的密度和大小，因此它将非脂肪性肝细胞群与脂肪性肝细胞群分开。因此，脂肪肝细胞经常丢失，产生不具代表性的肝细胞分数。
所提出的方案描述了一种简单可靠的方法，用于 体内 分离再生肝细胞，无论其脂质含量如何。通过经典的两步胶原酶灌注方法在肝切除术后24-48小时分离来自雄性C57BL / 6小鼠的肝细胞。标准蠕动泵使用逆行灌注技术将加热的溶液 通过 导管下腔静脉驱动到残余静脉中，并通过门静脉流出。肝细胞被胶原酶解离，从Glisson的胶囊中释放出来。洗涤和仔细离心后，肝细胞可用于任何下游分析。总之，本文描述了一种直接且可重复的技术，用于分离小鼠部分肝切除术后代表性的再生肝细胞群体。该方法也可能有助于脂肪肝疾病的研究。
Introduction
即使在组织大量流失后，肝脏也可以自我再生。这种独特的再生能力在部分（70%）肝切除术的实验模型中得到了明确说明，希金斯和安德森于1931年首次在大鼠中描述了1。在这个模型中，通过手术从动物身上切除70%的肝脏，方法是通过剪掉较大的肝叶。然后，剩余的叶通过代偿性肥大生长，在手术后约1周内恢复原始肝脏肿块，尽管没有恢复原始肝脏结构2，3。已经开发了具有不同组织切除量的其他肝切除术，例如86%扩展的肝切除术，其中肝脏残留物太小而无法恢复，最终导致肝切除术后肝衰竭（PHLF）和随后30%-50%的动物死亡4，5，6。这些模型能够研究正常和失败的肝脏再生，具体取决于切除组织的数量（图1）。
尽管肝脏切除术的小鼠模型已经成功使用多年，但直到最近才有更先进的分析方法允许在单细胞水平上更深入地了解。然而，对于大多数这些方法，单个肝细胞的存在是一个基本的先决条件。大多数分离原代肝细胞的方案基于两步胶原酶灌注技术和随后的密度梯度纯化，以从碎片和非实质以及死细胞中分离活肝细胞7，8，9。这种方法首先由Berry和Friend在196910中描述，并由Seglen及其同事在197211，12中进行了改编。然而，由于梯度离心依赖于细胞的密度和大小，因此在标准纯化过程中通常会丢失富含脂质的肝细胞。虽然对于许多研究问题来说，这种损失可以忽略不计，但它是早期肝脏再生的一个关键方面。在前2天，再生小鼠肝脏内的肝细胞积累脂质，从而增大大小并降低密度。这种短暂的再生相关脂肪变性（TRAS）用于提供再生燃料，并且是暂时的，但部分重叠了主要的增殖阶段，并且在肝小叶（肝脏的功能单位）内分布不均匀13，14。然而，在延长86%肝切除术后，TRAS也发生但持续存在，因为再生停滞并且脂质没有被氧化14。因此，在70%或86%肝切除术后对肝细胞进行梯度纯化将产生不具代表性的组分，因为大多数含脂质的肝细胞由于其低密度而丢失15。
在这种改良的分离方案中，通过经典的两步胶原酶灌注方法在肝切除术后24-48小时分离来自C57BL / 6小鼠的肝细胞。通常，用于细胞分离的残余物的插管和灌注是通过门 静脉完成的 。然而，大切除后留下的小残留物的门血管阻力高达16，因此灌注很微妙。由于腔静脉不受肝切除术的影响，因此可以通过插管 腔静脉轻松地 在逆行方向进行灌注。标准蠕动泵通过导管插入的下腔静脉 将 加热的溶液驱动到肝脏残余物中，使用逆行灌注并通过门静脉流出（补充图S1）。肝细胞被胶原酶解离并从Glisson的胶囊中释放出来。在使用低速离心方法逐步分离和仔细处理活肝细胞后，肝细胞可用于任何下游分析。
Protocol
所有动物实验均符合瑞士联邦动物法规，并经苏黎世兽医局批准（n° 007/2017，156/2019），确保人类护理。将10-12周龄的雄性C57BL / 6小鼠保持在12小时的昼夜循环中，并自由获取食物和水。每个实验组由六到八只动物组成。有关本协议中使用的所有材料、设备和试剂的详细信息，请参阅 材料表 。 1.小鼠肝部分切除术 对于标准肝切除术（70%），结?…
Representative Results
TRAS在肝切除术后16小时达到峰值，并在标准肝切除术后32-48小时逐渐消失，但在延长肝切除术后持续超过48小时。在肉眼上，TRAS很容易看到肝脏残留物的苍白肤色（图1F），并且可以在手术后16小时至48小时之间在肝切除小鼠中观察到。 估计的最终产量为70%肝切除术后10-15×106 肝细胞和延长86%肝切除术后4-9×106 肝细胞，平均最终生存能力分…
Discussion
已发布的方案提供了一种可靠且直接的方法来分离高产量的正常和脂肪变性小鼠肝细胞，用于单细胞下游分析或FACS分选后的细胞批量分析。与密度梯度纯化相比，其明显优势在于细胞脂质含量对肝细胞的有效产量基本上没有影响。因此，脂肪变性肝细胞的比例将被保留并包含在下游分析中。这不仅对于脂肪源性肝病的研究至关重要，而且对于主要肝切除术后的任何过程分析也至关重要，其中肝细…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了瑞士国家基金（项目赠款310030_189262）的支持。
Materials
| Reagents | |||
| Alexa Fluor 488 Zombie green | BioLegend | 423111 | Amine-reactive viability dye |
| Attane Isoflurane ad us. vet. 99.9% | Provet AG | QN01AB06 | CAUTION: needs ventilation |
| EDTA solution | Sigma-Aldrich | E8008-100ML | – |
| Ethanol | Sigma-Aldrich | V001229 | Dilute with water to 70% |
| Fetal bovine serum (FCS) | Gibco | A5256701 | – |
| Hanks' Balanced Salt Solution (HBSS), Ca2+, Mg2+, phenol red | Sigma-Aldrich | H9269-6x600ML | For digestion/preservation |
| Hanks' Balanced Salt solution (HBSS), w/o Ca2+, w/o Mg2+, no phenol red | Sigma-Aldrich | H6648-6x500ML | For perfusion buffer |
| HEPES solution, 1 M | Sigma-Aldrich | 83264-100ML-F | – |
| Histoacryl tissue adhesive (butyl-2-cyanoacrylate) | B. Braun | 1050052 | For stabilization of cannulation site |
| Hoechst 33258 Staining Dye Solution | Abcam | ab228550 | – |
| Liberase Research Grade | Roche | 5401119001 | Lyophilized collagenases I/II |
| NaCl 0.9% 500 mL Ecotainer | B. Braun | 123 | – |
| Paralube Vet Ointment | Dechra | 17033-211-38 | – |
| Phosphate buffered saline (PBS) | Gibco | A1286301 | – |
| Sudan IV – Lipid staining | Sigma-Aldrich | V001423 | – |
| Temgesic (Buprenorphine hydrochloride), Solution for Injection 0.3 mg/mL | Indivior Europe Ltd. | 345928 | Narcotics. Store securely. |
| Trypan blue, 0.4%, sterile-filtered | Sigma-Aldrich | T8154 | For cell counting |
| Williams’ Medium E | Sigma-Aldrich | W4128-500ML | – |
| Materials | |||
| 25 mL serological pipette, Greiner Cellstar | Merck | P7865 | – |
| 50 mL Falcon tubes | TPP | – | – |
| BD Neoflon, Pro IV Catheter 26 G | BD Falcon | 391349 | – |
| Cell scraper, rotating blade width 25 mm | TPP | 99004 | – |
| Falcon Cell Strainer 100 µm Nylon | BD Falcon | 352360 | – |
| Fenestrated sterile surgical drape | – | – | Reusable cloth material |
| Filling nozzle for size 16# tubing (ID 3.1 mm) | Drifton | FILLINGNOZZLE#16 | To go into the tubes |
| Flow cytometry tubes, 5 mL | BD Falcon | 352008 | – |
| Male Luer to Barb, Tubing ID 3.2 mm | Drifton | LM41 | Connection tube to syringe |
| Petri dishes, 96 x 21 mm | TPP | 93100 | – |
| Prolene 5-0 | Ethicon | 8614H | To retract the sternum |
| Prolene 6-0 | Ethicon | 8695H | For skin suture |
| Prolene 8-0 | Ethicon | EH7470E | Ligature gall bladder |
| Tube 16#, WT 1.6 mm, ID 3.2 mm, OD 6.4 mm | Drifton | SC0374T | Perfusion tube |
| Equipment | |||
| BD LSRFortessa Cell Analyzer Flow Cytometer | BD | – | – |
| Isis rodent shaver | Aesculap | GT421 | – |
| Isofluran station | Provet | – | – |
| Low-speed centrifuge – Scanspeed 416 | Labogene | – | – |
| Neubauer-improved counting chamber | Marienfeld | – | – |
| Oxygen concentrator – EverFlo | Philips | 1020007 | 0 – 5 L/min |
| Pipetboy – Pipettor Turbo-Fix | TPP | 94700 | – |
| Shenchen perfusion pump – YZ1515x | Shenchen | YZ1515x | – |
| Surgical microscope – SZX9 | Olympus | – | – |
| ThermoLux warming mat | Thermo Lux | – | – |
| Vortex Genie 2, 2700 UpM | NeoLab | 7-0092 | – |
| Water bath – Precision GP 02 | Thermo scientific | – | Adjust to 42 °C |
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Citar este artigo
Breuer, E., Humar, B. Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice. J. Vis. Exp. (190), e64493, doi:10.3791/64493 (2022).