建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性
1Department of Pathology, Microbiology and Immunology,University of South Carolina School of Medicine, 2Department of Dermatology,Brigham and Women’s Hospital, Harvard Medical School, 3Department of Biochemistry and Molecular Biology,University of Maryland School of Medicine Baltimore, 4Department of Pathology,Albert Einstein College of Medicine, 5Department of Pediatrics,Vanderbilt University Medical Center, 6Department of Drug Discovery and Biomedical Sciences, College of Pharmacy,University of South Carolina
Summary
在这里，我们描述了在3D悬架培养中系统地培养表皮球体的协议。本协议具有广泛的应用，用于各种上皮组织类型和几个人类疾病和条件的建模。
Abstract
表皮调节障碍是各种人类疾病和疾病的节点，包括慢性伤害、炎症和超过 80% 的人类癌症。作为衬里组织，皮肤上皮经常受到损伤，并通过获得修复受损组织所需的细胞可塑性进行进化适应。多年来，我们已作出若干努力，利用体外和体外细胞模型研究上皮可塑性。然而，这些努力在回顾上皮细胞可塑性各个阶段的能力方面受到限制。我们在这里描述一个从原发性新生儿角膜细胞中生成3D表皮球体和表皮球体衍生细胞的协议。本协议概述了表皮球体培养物在功能上模拟角膜细胞生成可塑性不同阶段的能力，并表明表皮球体重新镀层可以丰富异质正常人类角膜细胞 （NHKc） 培养物，用于具有增强茎状特征的特异性基因细胞 6hi/EGFRlo 角细胞亚聚变。我们的报告描述了用于研究皮肤角膜可塑性和表皮再生的高吞吐量系统的开发和维护。
Introduction
哺乳动物分层上皮是所有生物系统中最复杂的上皮结构，经常受到损伤和伤害。作为一种保护性组织，分层上皮已经进化产生复杂而有效的组织损伤反应。受伤后，这些细胞必须激活血统可塑性程序，使它们能够迁移到受伤地点，并进行修复1，2，3。这种多方面反应发生在几个连续步骤中，这些步骤仍然鲜为人知。
研究上皮再生复杂过程的一个主要障碍在于缺乏高吞吐量模型系统，这些模型系统可以在细胞再生的指定阶段捕获动态细胞活动。虽然在体内小鼠模型提供了有关伤口愈合的见解，并最密切地概括了人类再生过程，但它们的发展需要付出艰苦的努力和巨大的成本，限制了它们的吞吐能力。因此，迫切需要建立系统，以便能够在高吞吐量尺度上对人类上皮组织再生进行功能性研究。
近年来，已作出若干努力，以应付可扩展性的挑战。这通过大量创新的体外和前体内细胞模型的扩展来观察，这些模型与体内再生环境紧密结合。这包括芯片上的器官4、球体5、器官6和有机培养7方面的进步。这些基于3D的基于细胞的系统各有优势，并具有独特的实验局限性。迄今为止，球形培养仍然是最具成本效益和广泛使用的3D细胞培养模型。虽然一些报告表明，球形培养物可用于研究皮肤干细胞特征，但这些研究主要用动物组织8、9或皮肤成纤维细胞10进行，几乎没有报告能彻底描述人类表皮球体培养物的再生特性。在此协议中，我们详细介绍了正常人类角膜细胞 （NHKc） 表皮球体培养物的功能发展、文化和维护。我们同样描述了该系统在体外模拟表皮再生和角膜细胞干细胞可塑性的顺序阶段的效用。
Protocol
南卡罗来纳大学（UofSC）IRB审查了收集和处理皮肤标本和隔离人类角膜细胞的规程，并将其归类为”不涉及人类受试者的研究”，因为包皮标本是在常规外科手术（新生儿男孩的割礼）期间产生的手术丢弃物，完全缺乏识别信息。UofSC生物安全委员会还定期审查和批准该议定书，所有实验室人员都接受了实验室生物安全培训。所有程序均按照UofSC的安全和道德标准进行。 1. 新生儿?…
Representative Results
在皮肤表皮球测定过程中，NHKc培养物被播种在96井板的玫瑰涂层井中（图1A）。球形形成细胞应在48小时内自我聚集。使用标准倒置相对比显微镜，可尽早在 24 小时评估自体球体形成。皮肤表皮球的形成和重新电镀检测模型表皮组织再生的各个阶段（图1B）。图2显示了在3D培养中对表皮球体形成能力进行测定的各种NHKc菌株的高分?…
Discussion
3D球形培养系统的使用在评估细胞干细胞方面具有广泛的效用。这些系统已被证明可以增强组织干细胞的富集性，然而它们对人类表皮干细胞研究的效用却被有限的探索。在这里，我们描述了一种利用3D培养技术丰富人类角膜细胞干细胞的策略。在这个系统中，NHKc 被培养为自组装多细胞球体悬浮物，由几个角膜细胞亚型组成，这些亚型悬浮在含有 KSFM-scm 的玫瑰床上。此协议的设…
Disclosures
The authors have nothing to disclose.
Acknowledgements
UofSC医学院仪器资源设施（IRF）提供了成像和细胞分拣设备以及技术援助。这项工作部分得到了赠款1R21CA201853的支持。MCF 和 IRF 从 NIH 赠款 P20GM103499（SC INBRE）获得部分支持。MCF 还获得 NIH 赠款 P20GM109091 的支持。Yvon Woappi 部分得到了 NIH 赠款 2R25GM06652626-06A1 （PREP） 和 R25GM076277 （IMSD） 的支持，以及 UofSC 格蕾丝·乔丹·麦克法登教授项目奖学金的支持。杰拉尔丁·埃泽卡和贾斯汀·韦尔塞利诺在UofSC获得NIH赠款2R25GM066526-10A1（PREP）的支持。肖恩·布洛斯在UofSC获得2016年麦哲伦学者奖。
Materials
| Affymetrix platform | Affymetrix | For microarray experiments | |
| Affymetrix’s HuGene-2_0-st library file | Affymetrix | Process | |
| Agilent 2100 Bioanalyzer | Agilent | For microarray experiments | |
| All Prep DNA/RNA Mini Kit | Qiagen | 80204 | Used for RNA isolation |
| Analysis Console Software version 3.0.0.466 | analyze cell type specific transcriptional responses using one-way between-subject analysis of variance | ||
| BD FACSAria II flow cytometer | Beckman | For flow cytometry | |
| Console Software version 3.0.0.466/Expression console Software | Affymetrix/Thermo Fisher Scientific | For confirming data quality | |
| Cytokeratin 14 | Santa Cruz Biotechnology | sc-53253 | 1:200 dilution |
| Dispase | Sigma-Aldrich | D4818 | For cell media |
| FITC-conjugated anti-integrinα6 | Abcam | ab30496 | For FACS analysis |
| GeneChip Command Console 4.0 software | Affymetrix/Thermo Fisher Scientific | For confirming data quality | |
| GeneChip Fluidics Stations 450 (Affymetrix/Thermo Fisher Scientific) | Affymetrix/Thermo Fisher Scientific | For washing and staining of hybridized arrays | |
| GeneChip HuGene 2.0 ST Arrays | Affymetrix/Thermo Fisher Scientific | For hybridization and amplifycation of total RNA | |
| GeneChip Hybridization Oven 640 | Thermo Fisher Scientific | For hybridization and amplifycation of total RNA | Amplify labeled samples | |
| GeneChip Hybridization Wash, and Stain Kit (Affymetrix/Thermo Fisher Scientific). | Affymetrix/Thermo Fisher Scientific | For washing and staining of hybridized arrays | |
| GeneChip Scanner 3000 7G system | Affymetrix/Thermo Fisher Scientific | Scanning hybridized arrays | |
| GeneChip WT PLUS Reagent Kit | Affymetrix/Thermo Fisher Scientific | For amplifycation of biotinylating total RNA | |
| Human Basic Fibroblast Growth Factor (hFGF basic/FGF2) | Cell Signaling Technology | 8910 | For cell media |
| Human Epidermal Growth Factor (hEGF) | Cell Signaling Technology | 8916 | For cell media |
| Human Insulin | Millipore Sigma | 9011-M | For cell media |
| iQ SYBR Green Supermix (Bio-Rad) | Bio-Rad | 1708880 | Used for RT-qPCR |
| iScript cDNA Synthesis Kit | Bio-Rad | 1708890 | Used for RT-qPCR |
| KSFM | ThermoFisher Scientific | 17005041 | Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 µg/ml insulin |
| KSFM-scm | ThermoFisher Scientific | 17005042 | Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 µg/ml insulin |
| MCDB 153-LB basal medium | Sigma-Aldrich | M7403 | MCDB 153-LB basal media w/ HEPES buffer |
| NEST Scientific 1-Well Cell Culture Chamber Slide, BLACK Walls on Glass Slide, 6/PK, 12/CS | Stellar Scientific | NST230111 | For immunostaining |
| P63 | Thermo Scientific | 703809 | 1:200 dilution |
| PE-conjugated anti-EGFR ( San Jose, CA; catalog number ) | BD Pharmingen | 555997 | For FACS analysis |
| pMSCV-IRES-EGFP plasmid vector | Addgene | 20672 | For transfection |
| Promega TransFast kit | Promega | E2431 | For transfection |
| Qiagen RNeasy Plus Micro Kit | Qiagen | For microarray experiments | |
| Thermo Scientific™ Sterile Single Use Vacuum Filter Units | Thermo Scientific | 09-740-63D | For cell media |
| Zeiss Axionvert 135 fluorescence microscope | Zeiss | Use with Axiovision Rel. 4.5 software |
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Cite This Article
Woappi, Y., Ezeka, G., Vercellino, J., Bloos, S. M., Creek, K. E., Pirisi, L. Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity. J. Vis. Exp. (167), e62182, doi:10.3791/62182 (2021).