斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定
1Doctorado en Ciencias Biológicas y de la Salud,Universidad Autónoma Metropolitana, 2Medical Research Unit in Biochemistry, Specialties Hospital, National Medical Center SXXI,Instituto Mexicano de Seguro Social, 3Department of Agricultural and Animal Production, Division of Biological and Health Science,Universidad Autónoma Metropolitana-Xochimilco, 4Molecular Biology Research Laboratory, Center for Congenital Malformations,Children Hospital of Mexico Federico Gomez (HIMFG)
Summary
该协议描述了从Sprague Dawley大鼠中分离和鉴定脂肪组织来源的间充质干细胞（MSCs）的方法。
Abstract
近几十年来，成人间充质细胞彻底改变了分子和细胞生物学。它们可以分化成不同的特化细胞类型，此外还有强大的自我更新、迁移和增殖能力。脂肪组织是间充质细胞侵入性最小、最容易获得的来源之一。据报道，与其他来源相比，它具有更高的产量，以及优越的免疫调节特性。最近，已经发表了从不同组织来源和动物物种获得成年间充质细胞的不同程序。在评估了一些作者的标准后，我们标准化了一种适用于不同目的且易于复制的方法。来自肾周和附睾脂肪组织的基质血管分数（SVF）池使我们能够开发具有最佳形态和功能的原代培养物。观察到细胞粘附在塑料表面上24小时，并表现出成纤维细胞样形态，具有延长和形成集落的趋势。流式细胞术（FC）和免疫荧光（IF）技术用于评估膜标志物CD105，CD9，CD63，CD31和CD34的表达。脂肪来源的干细胞（ASCs）分化成成脂肪谱系的能力也使用多种因素（4μM胰岛素，0.5mM 3-甲基异丁基黄嘌呤和1μM地塞米松）进行评估。48小时后，观察到成纤维细胞样形态逐渐丧失，并且在12天时，确认存在对油红色染色阳性的脂滴。总之，提出了一种程序来获得最佳和功能性ASC培养物以应用于再生医学。
Introduction
间充质干细胞（MSCs）因其自我更新，增殖，迁移和分化为不同细胞谱系的高能力而强烈影响再生医学1，2。目前，大量的研究集中在它们治疗和诊断各种疾病的潜力上。
间充质细胞有不同的来源：骨髓、骨骼肌、羊水、毛囊、胎盘和脂肪组织等。它们是从不同的物种获得的，包括人类、小鼠、大鼠、狗和马3.骨髓来源的间充质干细胞（BMSC）多年来一直被用作再生医学中干细胞的主要来源，并作为胚胎干细胞使用的替代品4。然而，脂肪来源的MSCs或脂肪来源的干细胞（ASCs）是一种重要的替代品，由于其易于收集和分离，以及每克脂肪组织获得的细胞产量5，6，具有很大的优势。据报道，ASC的收获率普遍高于BMSCs7。最初提出ASC的修复/再生能力是由于它们分化成其他细胞谱系的能力8。然而，近年来的研究加强了ASC释放的旁分泌因子在其修复潜力中的主要作用9，10。
脂肪组织（AT）除了作为能量储备外，还与内分泌，神经和心血管系统相互作用。它还参与出生后的生长发育、维持组织稳态、组织修复和再生。AT 由脂肪细胞、血管平滑肌细胞、内皮细胞、成纤维细胞、单核细胞、巨噬细胞、淋巴细胞、前脂肪细胞和 ASC 组成。后者由于其低免疫原性而在再生医学中具有重要作用11，12。ASC可以通过酶消化和机械加工或通过脂肪组织外植体获得。ASC的原代培养物易于维持、生长和扩增。通过使用免疫荧光和流式细胞术等方法评估特定膜标志物的表达，ASC 的表型表征对于验证细胞的身份至关重要13.国际脂肪治疗与科学联合会（IFATS）和国际细胞治疗学会（ISCT）已经定义ASC表达CD73，CD90和CD105，而缺乏CD11b，CD14，CD19，CD45和HLA-DR14的表达。因此，这些阳性和阴性标志物被认为对ASC的表征是可靠的。
该项目的重点是描述从大鼠AT中提取的成年间充质细胞的分离和鉴定程序，因为与胚胎干细胞不同，这种细胞来源不存在伦理挑战。这巩固了该程序作为可行选择的地位，因为与骨髓来源的干细胞相比，该方法易于访问和微创方法。
来自这种组织来源的间充质细胞因其免疫调节能力和低免疫排斥反应而在再生医学中起着重要作用。因此，本研究是未来研究其分泌组及其在不同疾病（包括糖尿病等代谢性疾病）中作为再生疗法应用的基础部分。
Protocol
所有实验程序均按照墨西哥动物护理指南进行，该指南基于国际实验动物护理评估和认证协会（墨西哥官方规范NOM-062-200-1999，墨西哥）的建议。该协议由墨西哥社会研究所健康研究伦理委员会审查、批准和注册 （R-2021-785-092）。 1.通过手术切除从大鼠身上去除脂肪组织 用 20 mL 无菌 1x 磷酸盐缓冲盐水-抗生素抗真菌剂 （PBS-AA） 溶液（1 x PBS、庆大霉素 [20 μ…
Representative Results
脂肪组织取自3-4个月大的成年Sprague Dawley大鼠，体重为401±41g（几何平均值±SD）。附睾和肾周脂肪组织的平均值为3.8g对应于15次实验提取的分析。培养24小时后，细胞群仍然粘附在塑料表面上并表现出异质形态。第一次传代在8±2天实现，在总共8个实验中±0.6 x 106 个细胞的产量为1.4。直接明场观察（图1）和苏木精-伊红染色（图2）有助于用光学?…
Discussion
自发现间充质干细胞以来的过去四十年中，几组研究人员已经描述了从不同组织和物种获得间充质干细胞的程序。使用大鼠作为动物模型的优点之一是它们易于维护和快速发育，以及易于从脂肪组织获得MSC。已经描述了用于获得ASC的不同组织来源，例如内脏，肾周，附睾和皮下脂肪12，13，14，15，16。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢墨西哥社会保障研究所（IMSS）和墨西哥儿童医院，Federico Gomez（HIMFG）和IMSS研究协调的Bioterio工作人员为开展该项目提供的支持。我们感谢国家科学技术委员会提供的AOC（815290）奖学金和安东尼奥·杜阿尔特·雷耶斯在视听材料方面的技术支持。
Materials
| Amphotericin B | HyClone | SV30078.01 | |
| Analytical balance | Sartorius | AX224 | |
| Antibody anti- CD9 (C-4) | Santa Cruz | Sc-13118 | |
| Antibody anti-CD34 (C-18) | Santa Cruz | Sc-7045 | |
| Antibody anti-C63 | Santa Cruz | Sc-5275 | |
| Antibody anti-Endoglin/CD105 (P3D1) Alexa Fluor 594 | Santa Cruz | Sc-18838A594 | |
| Antibody anti-CD31/PECM-1 Alexa Fluor 680 | Santa Cruz | Sc-18916AF680 | |
| Antibody Goat anti-rabitt IgG (H+L) Cy3 | Novus | NB 120-6939 | |
| Antibody Donkey anti-goat IgG (H+L) DyLight 550 | Invitrogen | SA5-10087 | |
| Antibody anti-mouse IgG FITC conjugated goat F (ab´) | RD Systems. | No. F103B | |
| Bottle Top Filter Sterile | CORNING | 10718003 | |
| Cell and Tissue Culture Flasks | BIOFIL | 170718-312B | |
| Cell Counter Bright-Line Hemacytometer with cell counting chamber slides | SIGMA Aldrich | Z359629 | |
| Cell wells: 6 well with Lid | CORNING | 25810 | |
| Centrifuge conical tubes | HeTTICH | ROTANA460R | |
| Centrifuge eppendorf tubes | Fischer Scientific | M0018242_44797 | |
| Collagen IV | Worthington | LS004186 | |
| Cryovial | SPL Life Science | 43112 | |
| Culture tubes | Greiner Bio-One | 191180 | |
| CytExpert 2.0 | Beckman Coulter | Free version | |
| CytoFlex LX cytometer | Beckman Coulter | FLOW-2463VID03.17 | |
| DMEM | GIBCO | 31600-034 | |
| DMSO | SIGMA Aldrich | 67-68-5 | |
| DraQ7 Dye | Thermo Sc. | D15106 | |
| EDTA | SIGMA Aldrich | 60-00-4 | |
| Eosin yellowish | Hycel | 300 | |
| Ethanol 96% | Baker | 64-17-5 | |
| Falcon tubes 15 mL | Greiner Bio-One | 188271 | |
| Falcon tubes 50 mL | Greiner Bio-One | 227261 | |
| Fetal Bovine Serum | CORNING | 35-010-CV | |
| Gelatin | SIGMA Aldrich | 128111163 | |
| Gentamicin | GIBCO | 15750045 | |
| Glycerin-High Purity | Herschi Trading | 56-81-5 | |
| Hematoxylin | AMRESCO | 0701-25G | |
| Heracell 240i CO2 Incubator | Thermo Sc. | 50116047 | |
| Ketamin Pet (Ketamine clorhidrate) | Aranda | SV057430 | |
| L-Glutamine | GIBCO/ Thermo Sc. | 25030-081 | |
| LSM software Zen 2009 V5.5 | Free version | ||
| Biological Safety Cabinet Class II | NuAire | 12082100801 | |
| Epifluorescent microscope | Zeiss Axiovert 100M | 21.0028.001 | |
| Inverted microscope | Olympus CK40 | CK40-G100 | |
| Non-essential amino acids 100X | GIBCO | 11140050 | |
| Micro tubes 2 mL | Sarstedt | 72695400 | |
| Micro tubes 1,5 mL | Sarstedt | 72706400 | |
| Micropipettes 0.2-2 μL | Finnpipette | E97743 | |
| Micropipettes 2-20 μL | Finnpipette | F54167 | |
| Micropipettes 20-200 μL | Finnpipette | G32419 | |
| Micropipettes 100-1000 μL | Finnpipette | FJ39895 | |
| Nitrogen tank liquid | Taylor-Wharton | 681-021-06 | |
| Paraformaldehyde | SIGMA Aldrich | SLBC3029V | |
| Penicillin / Streptomycin | GIBCO/ Thermo Sc. | 15140122 | |
| Petri dish Cell culture | CORNING Inc | 480167 | |
| Pipet Tips | Axygen Scientific | 301-03-201 | |
| Pisabental (pentobarbital sodium) | PISA Agropecuaria | Q-7833-215 | |
| Potassium chloride | J.T.Baker | 7447-40-7 | |
| Potassium Phosphate Dibasic | J.T Baker | 2139900 | |
| S1 Pipette Fillers | Thermo Sc | 9531 | |
| Serological pipette 5 mL | PYREX | L010005 | |
| Serological pipette 10 mL | PYREX | L010010 | |
| Sodium bicarbonate | J.T Baker | 144-55-8 | |
| Sodium chloride | J.T.Baker | 15368426 | |
| Sodium Phosphate Dibasic Anhydrous | J.T Baker | 7558-79-4 | |
| Sodium pyruvate | GIBCO BRL | 11840-048 | |
| Syringe Filter Sterile | CORNING | 431222 | |
| Spectrophotometer | PerkinElmer Lambda 25 | L6020060 | |
| Titer plate shaker | LAB-LINE | 1250 | |
| Transfer pipets | Samco/Thermo Sc | 728NL | |
| Trypan Blue stain | GIBCO | 1198566 | |
| Trypsin From Porcine Pancreas | SIGMA Aldrich | 102H0234 | |
| Tween 20 | SIGMA Aldrich | 9005-64-5 | |
| Universal Blocking Reagent 10x | BioGenex | HK085-GP | |
| Xilapet 2% (xylazine hydrochloride) | Pet's Pharma | Q-7972-025 |
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Tags
Keywords:
Mesenchymal Stem CellsAdipose-derived Mesenchymal Stem CellsSprague Dawley RatsCellular CommunicationParacrine FunctionPancreatic RegenerationDiabetesCell TherapiesSecretomeMiRNAsOxidative StressCell DifferentiationSelf-renewalMigrationProliferationStromal Vascular FractionFlow CytometryImmunofluorescence
Cite This Article
Oliva Cárdenas, A., Zamora-Rodríguez, B. C., Batalla-García, K. A., Ávalos-Rodríguez, A., Contreras-Ramos, A., Ortega-Camarillo, C. Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats. J. Vis. Exp. (194), e65172, doi:10.3791/65172 (2023).