从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞
Summary
研究与肌肉骨骼损伤相关的伤口愈合通常需要评估施万细胞（SCs）、角质形成细胞和成纤维细胞之间的 体外 相互作用。该协议描述了从人包皮中分离，培养和表征这些原代细胞。
Abstract
该协议描述了分离方法，培养条件以及使用皮肤的快速酶解离具有高产量和活力的人类原代细胞的表征。原代角质形成细胞、成纤维细胞和施万细胞都是从人类新生儿包皮中采集的，可按照标准护理程序获得。去除的皮肤被消毒，皮下脂肪和肌肉被用手术刀去除。该方法包括表皮和真皮层的酶促和机械分离，然后进行额外的酶消化，以从这些皮肤层中的每一层获得单细胞悬浮液。最后，按照标准的细胞培养方案在适当的细胞培养基中培养单细胞，以在几周内保持生长和活力。总之，这种简单的方案允许从一块皮肤中分离，培养和表征所有三种细胞类型，以进行皮肤 – 神经模型的 体外 评估。此外，这些细胞可以在共培养物中一起使用，以测量它们对彼此的影响以及它们对 体外 创伤的反应，其形式是在与伤口愈合相关的培养物中机器人进行的划痕。
Introduction
衍生自活组织并在 体外 条件下培养的原代细胞与生理状态1非常相似，使其成为研究生理和病理生理过程的理想模型。皮肤含有多种细胞类型，包括角质形成细胞，成纤维细胞，皮脂细胞，黑素细胞和施万细胞（SC），可以分离和培养用于 体外 实验。尚未描述从单块皮肤中分离和培养角质形成细胞、成纤维细胞和 SC 的方法。该协议的目标是双重的：1）建立一种可靠且可重复的真皮SC分离和培养的方法，以及2）使用一种有效，稳健的方法从单个人包皮中分离角质形成细胞，成纤维细胞和SC。
目前，已有分离皮肤角质形成细胞2，3，4和成纤维细胞5，6的既定方案。这些研究描述了从皮肤中分离角质形成细胞，成纤维细胞或两者，但没有方案解决如何从人类皮肤建立原代SC的培养物。最近的研究表明，神经元SCs调节角质形成细胞和成纤维细胞过程并调节正常的皮肤生理功能7。因此，SC对皮肤稳态至关重要，并且对影响相邻皮肤细胞类型行为的调节生理学有重大贡献8。因此，允许分离每种细胞类型的方案非常适合涉及细胞 – 细胞间通信或细胞类型之间串扰的体外实验。
该协议描述了从单个皮肤建立原代细胞的单个细胞培养物。当可用组织量有限时，该方案特别有用。此外，从单个供体中分离所有三种细胞类型可以在细胞类型或共培养实验之间进行强有力的比较，同时减轻所需实验期间遗传学的影响。
Protocol
出于研究目的的去识别化人类包皮组织的获取和使用进行了审查，并获得了宾夕法尼亚州立大学医学院机构审查委员会（IRB #17574）确定的“非人类研究”。 注意：通过遵循以下方案，从单个包皮中获得2.4 x 106 角质形成细胞，4.4 x 106 成纤维细胞和1.1 x 106 SC。通常，这些原代细胞可用于3次传代，具体取决于实验条件。 1. 原代?…
Representative Results
正常新生儿包皮用于原发性表皮角质形成细胞和真皮SC和成纤维细胞的分离。将分离的原代细胞在含有生长因子的相应细胞培养基中培养。在培养瓶中接种SCs和成纤维细胞后，大多数细胞在2小时内粘附在烧瓶底部。在角质形成细胞的情况下，大多数角质形成细胞粘附24小时。分离的表皮原代角质形成细胞在第7天达到85%汇合，并表现出特征性细胞形态（鹅卵石形状）（图1A）?…
Discussion
该协议描述了一种从包皮的单个片段中分离三个不同细胞群的方法，即角质形成细胞，成纤维细胞和施旺细胞。有一些分离方案可用于分离角质形成细胞和成纤维细胞2，3，5，6，但没有一种描述SC分离。除了皮肤中的关键结构细胞、角质形成细胞和成纤维细胞外，皮肤还受到感觉传入结构和自主神经?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Fadia Kamal博士和Reyad Elbarbary博士允许我们使用实验室仪器和技术支持。这项工作得到了NIH（K08 AR060164-01A）和国防部（W81XWH-16-1-0725）向J.C.E.的资助，以及宾夕法尼亚州立大学好时医学中心的机构支持。
Materials
| 0.22 µM sterile filters (Millex-GP Syringe Filter Unit,polyethersulfone) | MilliporeSigma | SLGPR33RS | |
| 70 µM cell strainers | CELLTREAT | 229483 | |
| 100 µM cell strainers | CELLTREAT | 229485 | |
| 1 mL disposable syringes | BD Luer-Lok | BD-309659 | |
| 5 mL disposable syringes (Syringe sterile, single use) | BD Luer-Lok | BD309646 | |
| 10 mL disposable syringes | BD Luer-Lok | BD305462 | |
| 1% TritonX-100 | Sigma | X100-1L | Prepared at the time of use |
| 4% paraformaldehyde solution | ThermoFisher Scientific | J19943.K2 | Ready to use and store at 4 °C |
| 5% BSA | Sigma | A7906-100G | Prepared at the time of use |
| 70% ethanol | Pharmco | 111000200 | |
| Antibiotic | ScienCell Research | 503 | |
| Chemometec Vial1-Cassette | Fisher Scientific | NC1420193 | |
| Collagenase | Gibco | 17018-029 | |
| Coverslip | Fisherbrand | 12544D 22*50-1.5 | |
| Dispase I | Sigma-Aldrich | D46693 | |
| DMEM basal medium | ScienCell Research | 9221 | |
| Dulbecco's phosphate-buffered saline free from Ca2+ and Mg2+ (DPBS) | Corning | 21-031-CV) | |
| Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes | ThermoFisher Scientific | 339651 | |
| Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes | ThermoFisher Scientific | 339653 | |
| 1.5 mL micro-centrifuge tubes | Fisherbrand | 02-681-5 | |
| Fetal bovine serum (FBS) | ThermoFisher Scientific | 10082147 | |
| Fibroblast complete medium | ScienCell Research | 2331 | |
| Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 | Invitrogen | A11032 | Dilution (1:500) |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A11008 | Dilution (1:500) |
| Hanks' Buffered Saline Solution (HBSS buffer) | Lonza | CC-5022 | |
| Human foreskin | De-identified human foreskin tissue for research purposes (Institutional Review Board- IRB #17574). | ||
| KGM-GOLD keratinocyte medium (KGM gold and supplements) | Lonza | 00192151 and 00192152 | |
| Mouse alpha-smooth muscle actin antibody | ThermoFisher Scientific | 14-9760-82 | Dilution (1:200) |
| Mouse Cytokeratin14 antibody | Abcam | ab7800 | Dilution (1:100) |
| Mouse S100 antibody | ThermoFisher Scientific | MA5-12969 | Dilution (1:200) |
| Multi chambered (4 well glass slide) | Tab-Tek | 154526 | |
| NucleoCounter -Via1-Cassette | Chemometec | 941-0012 | |
| Poly-L-Lysisne (PLL) | ScienCell Research | 32503 | |
| ProLong Gold Anti-fade Mountant with DAPI | Invitrogen | P36935 | |
| Rabbit K10 antibody | Sigma-Aldrich | SAB4501656 | Dilution (1:100) |
| Rabbit p75-NTR antibody | Millipore | AB1554 | Dilution (1:500) |
| Rabbit vimentin | ProteinTech | 10366-1-AP | Dilution (1:200) |
| Schwann cell culture medium | ScienCell Research | 1701 | |
| Precision tweezers DUMONT straight with extra fine tips Dumostar, 5 | ROTH | LH75.1 | Sterilize with 70% alcohol before use |
| IRIS Scissors, sharp/sharp. Length 4–3/8"(111mm) | Codman | 54-6500 | Sterilize with 70% alcohol before use |
| Sterilized surgical – sharp blade (Duro Edge Economy Single Edge Blades) | Razor blade company | 94-0120 | Sterilize with 70% alcohol before use |
| T25 culture flask | Corning | 353109 | |
| Trypsin neutralization buffer (TNS) | Lonza | CC-5002 | |
| Trypsin/EDTA | Lonza | CC-5012 | |
| Inverted microscope | ZEISS | Axio Observer 7- Axiocam 506 mono – Apotome.2 microscope | For immunofluorescence of chamber slide containing stained cells |
| Inverted microscope | ZEISS | Primovert | For visulaizing/observing cell attachment or detachment |
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Cite This Article
Jagadeeshaprasad, M. G., Govindappa, P. K., Nelson, A. M., Elfar, J. C. Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin. J. Vis. Exp. (181), e63776, doi:10.3791/63776 (2022).