使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分
Summary
该协议描述了从小鼠脂肪组织中半自动分离基质血管部分（SVF）以获得前脂肪细胞并在 体外实现脂肪细胞分化。使用组织解离剂进行胶原酶消化可减少实验变异并提高可重复性。
Abstract
白色、棕色和米色脂肪细胞分化的 体外 研究能够研究脂肪细胞的细胞自主功能及其机制。永生化的白色前脂肪细胞系是公开的，并被广泛使用。然而，使用公开的白色脂肪细胞系很难充分概括白色脂肪细胞响应外部线索在白色脂肪组织中出现米色脂肪细胞。通常从小鼠脂肪组织中分离基质血管部分（SVF）以获得原代前脂肪细胞并进行脂肪细胞分化。然而，用手切碎和胶原酶消化脂肪组织会导致实验变化，并且容易受到污染。在这里，我们提出了一种改进的半自动方案，该方案利用组织解离剂进行胶原酶消化，以实现更容易的SVF分离，目的是减少实验变异，减少污染并提高可重复性。获得的前脂肪细胞和分化的脂肪细胞可用于功能和机理分析。
Introduction
由于全球肥胖和2型糖尿病的患病率日益增加，脂肪组织生物学一直受到越来越多的关注1。脂肪细胞以脂滴的形式储存多余的能量，这些脂滴在饥饿时释放。此外，脂肪组织通过充当内分泌器官并与其他组织交流来维持全身能量稳态2，3。有趣的是，过多的脂肪组织（肥胖）和脂肪损失（脂肪营养不良）都与胰岛素抵抗和糖尿病有关1。脂肪细胞分为三种类型：白色、棕色和米色1.白色脂肪细胞主要以脂质的形式储存多余的能量，而棕色和米色的脂肪细胞通过线粒体解偶联蛋白-1（Ucp1）以热量的形式耗散能量1，4。值得注意的是，米色脂肪细胞（也称为“诱导”棕色脂肪细胞）响应寒冷或交感神经刺激出现在白色脂肪组织中，并表现出与“经典”棕色脂肪细胞重叠但不同的基因表达模式5。最近，棕色和米色脂肪细胞有望成为抗肥胖和抗糖尿病治疗的潜在靶标，旨在“增强能量耗散”而不是“抑制能量摄入”4。支持性地，FTO肥胖变体rs1421085在人类中的风险等位基因，在常见变异6，7中表现出与较高体重指数（BMI）的最强关联，并表现出各种基因 – 环境相互作用8，9，据报道对米色脂肪细胞分化和功能产生负调节10.过氧化物酶体增殖物激活受体γ（PPARγ）被称为脂肪生成的主要转录调节因子，对于脂肪细胞分化是必要且充分的11。转录调节因子，例如含有 16 （PRDM16）、早期 B 细胞因子 2 （EBF2） 和核因子 I-A （NFIA） 的 PRD1-BF1-RIZ1 同源结构域，对于棕色和米色脂肪细胞分化和功能至关重要 12，13，14，15，16，17，18.另一方面，白色脂肪细胞基因编程需要转录调节因子，例如转导蛋白样增强蛋白3（TLE3）和锌指蛋白423（ZFP423）19，20，21。
体外模型系统能够进行分子研究，旨在提高对脂肪细胞功能和功能障碍机制的理解。尽管存在公开可用且永生化的前脂肪细胞系，例如3T3-L1和3T3-F442A222222，但原代前脂肪细胞的培养和分化为脂肪细胞将是更适合研究体内脂肪生成的模型。从小鼠脂肪组织中分离基质血管部分（SVF）是获得原代前脂肪细胞25，26的公知方法。然而，脂肪组织的胶原酶消化通常使用带有试管架的细菌摇床进行，可导致实验变化，并且容易受到污染27，28。在这里，我们描述了一种替代方案，该方案使用温和的磁激活细胞分选（MACS）组织解离剂进行胶原酶消化，以实现更容易的SVF分离。
Protocol
Representative Results
Discussion
在这里，我们描述了一种从小鼠脂肪组织中分离SVF以获得前脂肪细胞并在 体外进行脂肪细胞分化的方案。使用组织解离剂进行胶原酶消化减少了实验变异，降低了污染风险，并提高了可重复性。虽然此过程是所提出的协议中的关键步骤，但该过程是高度自动化的，不需要优化。但是，根据小鼠年龄和脂肪组织库，可能需要优化切碎，例如大小碎片或切割时间。
SVF 被称…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢Takahito Wada和Saiko Yoshida（东京大学，东京，日本）的实验帮助。这项工作由以下对Y.H.的赠款资助：东京大学优秀青年研究员计划的研究资助;日本科学促进会（JSPS）KAKENHI早期职业科学家补助金，资助号19K17976;日本应用酶学基金会未来糖尿病研究领跑者（FFDR）资助，资助号17F005;药理学研究基金会的资助;Mochida纪念医学和药物研究基金会的资助;默沙东生命科学基金会的资助;大和证券健康财团的赠款;东京生化研究财团资助;武田科学基金会生命科学研究基金;以及SENSSHIN医学研究基金会的资助。
Materials
| 100 mm dish | Corning | 430167 | |
| 12 well plate | Corning | 3513 | |
| 60 mm dish | IWAKI | 3010-060 | |
| Adipose Tissue Dissociation Kit, mouse and rat | Miltenyi Biotec | 130-105-808 | contents: Enzyme D, Enzyme R, Enzyme A and Buffer A |
| Cell strainer 70 µm | BD falcon | #352350 | |
| Collagen coated dishes, 100 mm | BD | #356450 | |
| Collagen coated dishes, 60 mm | BD | #354401 | |
| Collagen I Coat Microplate 6 well | IWAKI | 4810-010 | |
| Dexamethasone | Wako | 041-18861 | |
| Dissecting Forceps | N/A | N/A | autoclave before use |
| Dissecting Scissors, blunt/sharp | N/A | N/A | autoclave before use |
| Dissecting Scissors, sharp/sharp | N/A | N/A | autoclave before use |
| DMEM/F-12, GlutaMAX supplement | Gibco | 10565-042 | |
| Fetal Bovine Serum (FBS) | N/A | N/A | |
| gentleMACS C Tubes | Milteny Biotec | 130-093-237 | |
| gentleMACSOcto Dissociator with Heaters | Miltenyi Biotec | 130-096-427 | |
| Humulin R Injection U-100 | Eli Lilly | 872492 | |
| Indomethacin | Sigma | I7378-5G | |
| Isobutylmethylxanthine (IBMX) | Sigma | 17018-1G | |
| Lipofectamine 2000 | Life Technologies | 11668-019 | |
| Neomycin Sulfate | Fujifilm | 146-08871 | |
| Opti-MEM | Invitrogen | 31985-062 | |
| pBABE-neo largeTcDNA (SV40) | Add gene | #1780 | |
| PBS tablets | Takara | T900 | |
| Platinum-E (Plat-E) Retroviral Packaging Cell Line | cell biolab | RV-101 | |
| Polybrene | Nacalai Tesque | 12996-81 | |
| Power Sybr Green Master Mix | Applied Biosystems | 4367659 | |
| ReverTra Ace qPCR RT Master Mix | TOYOBO | #FSQ-201 | |
| RNeasy Mini Kit (250) | QIAGEN | 74106 | |
| Rosiglitazone | Wako | 180-02653 | |
| T3 | Sigma | T2877-100mg | |
| Trypsin-EDTA (0.05%) | Gibco | 25200-056 |
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