微血管碎片血管化产热脂肪组织的三维培养
Summary
在这里，我们提出了一个详细的方案，概述了使用从啮齿动物或人类脂肪组织中分离的微血管碎片作为设计功能性，血管化米色脂肪组织的直接方法。
Abstract
工程产热脂肪组织（例如，米色或棕色脂肪组织）已被研究为代谢疾病的潜在疗法或用于健康筛查和药物测试的个性化微组织设计。目前的策略通常相当复杂，无法准确完全描述产热脂肪组织的多细胞和功能特性。微血管碎片是由从脂肪组织中分离的小动脉、小静脉和毛细血管组成的完整小血管，作为细胞的单一自体来源，可实现血管化和脂肪组织形成。本文介绍了优化培养条件的方法，以便能够从微血管碎片中生成三维、血管化和功能性产热脂肪组织，包括从脂肪组织和培养条件中分离微血管碎片的方案。此外，还讨论了最佳实践，以及表征工程组织的技术，并提供了啮齿动物和人类微血管碎片的样品结果。这种方法有可能用于理解和开发肥胖和代谢疾病的治疗方法。
Introduction
该协议的目标是描述一种从单一的，潜在的自体来源微血管碎片（MVF）开发血管化米色脂肪组织的方法。棕色和米色脂肪组织已被证明具有与代谢调节相关的有益特性;然而，成人中这些脂肪组织库的小体积限制了对全身代谢的潜在影响，特别是在肥胖或2型糖尿病等疾病条件下1，2，3，4，5，6，7。棕色/米色脂肪作为预防与肥胖及其合并症相关的有害代谢作用的治疗靶点引起了浓厚的兴趣8，9，10，11，12。
MVF是可以直接从脂肪组织中分离，培养并以三维结构长时间保持的血管结构13，14，15。我们小组和其他小组以前的工作已经开始利用MVF的多细胞和多能能力，特别是因为它与脂肪组织形成有关16，17，18。作为这项工作的积累，我们最近证明，来自健康和2型糖尿病19的啮齿动物模型以及来自人类受试者（50岁以上的成年人）20的MVF含有能够被诱导形成产热或米色脂肪组织的细胞。
这是一种利用单一来源MVF的创新方法，不仅能够创建米色脂肪组织，而且还能够创建其相关和关键的血管成分21。该技术的使用对于寻找一种直接的组织工程方法来形成产热脂肪组织的研究可能具有重要价值。与有志于设计米色脂肪组织22，23，24，25，26，27，28的其他方法不同，本研究中描述的过程不需要使用多种细胞类型或复杂的诱导方案。可以使用源自啮齿动物和人类来源的MVF创建血管化的米色和白色脂肪模型，显示出巨大的翻译潜力。该方案的最终产品是一种工程米色产热脂肪组织，其结构和代谢功能与棕色脂肪组织相当。总体而言，该协议提出了这样一种想法，即易于访问且可能自体来源MVF可能是一种有价值的治疗干预和研究代谢紊乱的工具。
Protocol
本研究是根据《动物福利法》和《动物福利实施条例》根据《实验动物护理和使用指南》的原则进行的。所有动物程序均由德克萨斯大学圣安东尼奥分校的机构动物护理和使用委员会批准。 注意：对于下面描述的步骤，使用雄性刘易斯大鼠。必须对雌性以及小鼠微血管片段（MVF）收集进行轻微的方案调整29。对于使用人MVF（h-MVF）的方案，唯一需要的步骤是?…
Representative Results
米色/棕色脂肪组织有几个关键的表型形态特征：它是多房/含有小脂滴，具有大量的线粒体（这是其在 体内特征性“褐色”外观的原因），相应地具有高耗氧率/线粒体生物能量，高度血管化，脂肪分解/胰岛素刺激的葡萄糖摄取增加，以及最臭名昭著的， 表达高水平的解偶联蛋白 1 （UCP1），这是一种参与产热呼吸的线粒体蛋白19，30。 <p class="…
Discussion
棕色/米色脂肪组织工程领域在很大程度上尚未成熟22，23，24，25，26，27，28，大部分脂肪模型正在开发用于白色脂肪组织8，22，31.工程棕色/米色微组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
阿科斯塔博士得到了美国国立卫生研究院拨款CA148724和TL1TR002647的支持。Gonzalez Porras博士得到了美国国立卫生研究院国家糖尿病，消化和肾脏疾病研究所的支持，奖励号为F32-0DK122754。这项工作得到了美国国立卫生研究院（5SC1DK122578）和德克萨斯大学圣安东尼奥分校生物医学工程系的部分支持。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。数字部分是用 Biorender.com 创建的。
Materials
| Aminocaproic Acid | Sigma Aldrich | A2504-100G | Added in DMEM at the concentration of 1 mg/mL |
| Blunt-Tipped Scissors | Fisher scientific | 12-000-172 | Sterilize in autoclave |
| Bovin Serum Albumin (BSA) | Millipore | 126575-10GM | Diluted in PBS to 4 mg/mL and 1 mg/mL |
| Collagenase Type 1 | Fisher scientific | NC9633623 | Diluted to 6 mg/mL in BSA 4 mg/mL, Digestion of minced fat |
| Dexamethasone | Thermo Scientific | AC230302500 | Diluted in ethanol at a 2 mg/ml stock concentration |
| Disposable underpads | Fisher scientific | 23-666-062 | For fluid absorption during surgery |
| Dissecting Scissors | Fisher scientific | 08-951-5 | Sterilize in autoclave |
| Dulbecco′s Modified Eagle′s Medium (DMEM) | Fisher scientific | 11885092 | |
| Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) | Sigma Aldrich | D8062 | |
| Fetal Bovine Serum | Fisher scientific | 16140089 | Added in DMEM to 20% v/v. |
| Fibrinogen | Sigma Aldrich | F8630-25G | Solubilized in DMEM at the concentration of 20 mg/mL, Protein found in blood plasma and main component of hydrogel |
| Flask, 250 mL | Fisher scientific | FB500250 | Allows for digestion of fat using a large surface area |
| Forceps | Fisher scientific | 50-264-21 | Sterilize in autoclave, For handling of tissue and filters |
| Forskolin | Sigma Aldrich | F6886 | Diluted in ethanol at a 10 mM stock concentration |
| Human MVF | Advanced Solutions Life Scienes, LLC | https://www.advancedsolutions.com/microvessels | Human MVFs (hMVFs) isolated from three different patients (52-, 54-, and 56-year old females) were used in the current study. |
| Indomethacine | Sigma Aldrich | I7378 | Diluted in ethanol at a 12.5 mM stock concentration |
| Insulin from porcine pancreas | Sigma Aldrich | I5523 | Diluted in 0.01 N HCl at a 5 mg/ml stock concentration |
| MycoZap | Fisher scientific | NC9023832 | Added in DMEM to 0.2% w/v, Mycoplasma Prophylactic |
| Pennycilin/Streptomycin (10,000 U/mL) | Fisher scientific | 15140122 | Added in DMEM to 1% v/v. |
| Petri dishes, polystyrene (100 mm x 15 mm). | Fisher scientific | 351029 | 3 for removal of blood vessels and mincing, 8 (lid) for presoaking of screens & 8 (dish) for use when filtering with 500 or 37 µM screens |
| Petri dishes, polystyrene (35 mm x 10 mm). | Fisher scientific | 50-202-036 | For counting fragments |
| Phosphate Buffer Saline (PBS) | Fisher scientific | 14-190-250 | Diluted to 1x with sterile deionized water. |
| Rat Clippers (Andwin Mini Arco Pet Trimmer) | Fisher scientific | NC0854141 | |
| Rosiglitazone | Fisher scientific | R0106200MG | Diluted in DMSO at a 10 mM stock concentration |
| Scissors | Fine Science Tools | 14059-11 | 1 for initial incision, 1 for epididymal incision, 1 for tip clipping |
| Screen 37 µM | Carolina Biological Supply Company | 652222R | Cut into 3" rounded squares and sterilized in ethylene oxide, Fragment entrapment and removal of very small fragments/single cells and debris |
| Screen 500 µM | Carolina Biological Supply Company | 652222F | Cut into 3" rounded squares and sterilized in ethylene oxide, Removes larger fragments/debris |
| Serrated Hemostat | Fisher scientific | 12-000-171 | Sterilize in autoclave, For clamping of skin before incision |
| Steriflip Filter 0.22 μm | Millipore | SE1M179M6 | |
| Thrombin | Fisher scientific | 6051601KU | Diluted in deionzed water to 10 U/mL, Used as a clotting agent turning fibrinogen to fibrin |
| Thyroid hormone (T3) | Sigma Aldrich | T2877 | Diluted in 1N NaOH at a 0.02 mM stock concentration |
| Zucker diabetic fatty (ZDF) rats – obese (FA/FA) or lean (FA/+) male | Charles River | https://www.criver.com/products-services/find-model/zdf-rat-lean-fa?region=3611 https://www.criver.com/products-services/find-model/zdf-rat-obese?region=3611 |
Obtained from Charles River (Wilmington, MA). Rats were acquired at 4 weeks of age and fed Purina 5008 until euthanasia (15-19 weeks of age). Glucose levels (blood from the lateral saphenous vein) were greater than 300 mg/dL in all FA/FA rats used in the study. All animals were housed in a temperature-controlled environment with a 12-h light-dark cycle and fed ad libitum. |
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Cite This Article
Acosta, F. M., Gonzalez Porras, M. A., Stojkova, K., Pacelli, S., Rathbone, C. R., Brey, E. M. Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments. J. Vis. Exp. (192), e64650, doi:10.3791/64650 (2023).