Name: isolation, culture, and characterization of primary dermal fibroblasts from human keloid tissue
Uploaded: 2023-07-28T14:10:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue at JoVE.com

这项工作得到了中国国家自然科学基金（批准号81903189和82073418）和广州市科学技术基金（批准号202102020025）的资助。

这项研究得到了南方医科大学皮肤病医院机构审查委员会的批准（2020081）。在从个体收集组织之前，已获得知情的患者同意。
1. 准备
注意：以下程序应在生物安全柜下的无菌环境中进行。
<ol><li>通过将 10% 胎牛血清 （FBS） 和 1% 青霉素-链霉素-两性霉素 B 溶液 （PSA） 添加到高葡萄糖 Dulbecco 的改良 Eagle 培养基 （DMEM） 中来制备完整的培养?...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nintedanib+inhibits+keloid+fibroblast+functions+by+blocking+the+phosphorylation+of+multiple+kinases+and+enhancing+receptor+internalization.">Nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization.</a> Acta Pharmacologica Sinica. 41 (9), 1234-1245 (2020).</li><li>Wang, X. M., Liu, X. M., Wang, Y., Chen, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+transcription+factor+3+(ATF3)+regulates+cell+growth,+apoptosis,+invasion+and+collagen+synthesis+in+keloid+fibroblast+through+transforming+growth+factor+beta+(TGF-beta)/SMAD+signaling+pathway.">Activating transcription factor 3 (ATF3) regulates cell growth, apoptosis, invasion and collagen synthesis in keloid fibroblast through transforming growth factor beta (TGF-beta)/SMAD signaling pathway.</a> Bioengineered. 12 (1), 117-126 (2021).</li><li>Sato, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+obtained+from+amnion-derived+mesenchymal+stem+cell+culture+prevents+activation+of+keloid+fibroblasts.">Conditioned medium obtained from amnion-derived mesenchymal stem cell culture prevents activation of keloid fibroblasts.</a> Plastic and Reconstructive Surgery. 141 (2), 390-398 (2018).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+explant+culture:+An+improved+method+for+consistently+harvesting+homogeneous+populations+of+keloid+fibroblasts.">Long-term explant culture: An improved method for consistently harvesting homogeneous populations of keloid fibroblasts.</a> Bioengineered. 13 (1), 1565-1574 (2022).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+glucose+metabolism+and+cell+function+in+keloid+fibroblasts+under+hypoxia.">Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia.</a> Redox Biology. 38, 101815 (2021).</li><li>Fan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptome+integration+analysis+reveals+the+correlation+between+mesenchymal+stromal+cells+and+fibroblasts.">Single-cell transcriptome integration analysis reveals the correlation between mesenchymal stromal cells and fibroblasts.</a> Frontiers in Genetics. 13, 798331 (2022).</li><li>Yao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+control+of+PDGFR&#945;+regulates+the+fibroblast-to-myofibroblast+transition+in+wound+healing.">Temporal control of PDGFR&#945; regulates the fibroblast-to-myofibroblast transition in wound healing.</a> Cell Reports. 40 (7), 111192 (2022).</li><li>Domdey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consecutive+dosing+of+UVB+irradiation+induces+loss+of+ABCB5+expression+and+activation+of+EMT+and+fibrosis+proteins+in+limbal+epithelial+cells+similar+to+pterygium+epithelium.">Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.</a> Stem Cell Research. 40, 102936 (2022).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+tubular+epithelial+cell+necroptosis+promotes+tubulointerstitial+fibrosis+in+patients+with+chronic+kidney+disease.">Renal tubular epithelial cell necroptosis promotes tubulointerstitial fibrosis in patients with chronic kidney disease.</a> FASEB Journal. 36 (12), e22625 (2022).</li><li>Korosec, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+identity+and+location+within+the+dermis+determine+the+function+of+papillary+and+reticular+fibroblasts+in+human+skin.">Lineage identity and location within the dermis determine the function of papillary and reticular fibroblasts in human skin.</a> Journal of Investigative Dermatology. 139 (2), 342-351 (2019).</li></ol>

从瘢痕疙瘩组织获得原代成纤维细胞是进一步研究的关键基础。到目前为止，有两种方法可以获得原代成纤维细胞：酶消化和外植体培养11,12,13,14。然而，两种传统方法都有局限性，例如易受污染，与其他类型的细胞混合，培养期长，成功率低15,16。这项研?...

瘢痕疙瘩是一种纤维增殖性疾病，表现为斑块的持续生长，这些斑块经常侵入周围的正常皮肤而没有自限性，并引起患者不同程度的瘙痒、疼痛以及美容和心理负担1.成纤维细胞是参与瘢痕疙瘩的原代细胞，通过过度增殖、多余的细胞外基质产生和无序的胶原蛋白在这种疾病的形成和发展中起着至关重要的作用2,3。然而，潜在的发病机制尚不清楚，仍然缺乏有效的瘢痕疙瘩治疗方法;因此，迫切需要进一步研究4,5。
由于体内瘢痕疙瘩研究没有理想的动物模型6,7，通过从瘢痕疙瘩组织中获取原代成纤维细胞来构建体外模型可以为瘢痕疙瘩研究提供可行性和可靠性2,6。原代细胞是直接来源于活组织的细胞，人们普遍认为，与细胞系8,9相比，这些细胞可以更接近多个个体的生理状态和遗传背景。原代细胞培养为研究细胞的生长和代谢以及其他细胞表型提供了一种强有力的手段。
目前，获得原代成纤维细胞的方法有两种：酶消化和外植体培养。然而，已经确定了获得原代成纤维细胞的几个障碍，例如被各种细菌或真菌污染的风险，与不易去除的其他类型的细胞混合，培养周期的长周期，与原始细胞相比细胞特性的后续变化，等等9.因此，开发一种可行且有效的方法来获得原代成纤维细胞是进一步研究和应用的基础。本研究描述了一种优化的方案，用于从瘢痕疙瘩组织中提取原代成纤维细胞，可以有效和稳定地提供纯净和可行的成纤维细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL sterile centrifuge tube</td>
			<td>JETBIOFIL</td>
			<td>CFT002015</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tube</td>
			<td>JETBIOFIL</td>
			<td>8076</td>
			<td></td>
		</tr>
		<tr>
			<td>4% polyformaldehyde</td>
			<td>Beyotime Biotechnology</td>
			<td>P0099</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tube</td>
			<td>JETBIOFIL</td>
			<td>8081</td>
			<td>Put keloid tissue</td>
		</tr>
		<tr>
			<td>Alexa Fluor-555 goat anti-rabbit IgG&#160;</td>
			<td>Abcam</td>
			<td>Alexa&#160;Fluor&#160;555&#160;</td>
			<td>second antibody for immunofluorescence staining assay</td>
		</tr>
		<tr>
			<td>Anti human CD90</td>
			<td>BioLegend</td>
			<td>B301002</td>
			<td>Identify the purity of fibroblasts</td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Beyotime Biotechnology</td>
			<td>P0262</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet&#160;</td>
			<td>Thermo Scientific</td>
			<td>1300 series A2</td>
			<td>Isolation and culture cells</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>aladdin</td>
			<td>B265993</td>
			<td>Blocking for immunofluorescence staining assay</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator</td>
			<td>ESCO</td>
			<td>CCL-170B-8</td>
			<td>Using for culturing cells</td>
		</tr>
		<tr>
			<td>Cell cryotubes</td>
			<td>Corning</td>
			<td>43513</td>
			<td>Store the cells in low temperature</td>
		</tr>
		<tr>
			<td>centrifugal machine</td>
			<td>Thermo Fisher</td>
			<td>ST 16R</td>
			<td>Discard supernatant&#160;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime Biotechnology</td>
			<td>C1006</td>
			<td>Stain the cellular nucleus</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>MP Biomedicals</td>
			<td>196055</td>
			<td>Using for preserving cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td>Culture medium solution</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BI</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>BD FACSCelesta</td>
			<td>Observing the identity of cells</td>
		</tr>
		<tr>
			<td>frozen box</td>
			<td>Thermo Scientific</td>
			<td>&#160;5100-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser confocal microscope</td>
			<td>Nikon</td>
			<td>AIR-HD25</td>
			<td>Observing the immunofluorescence staining assay</td>
		</tr>
		<tr>
			<td>PDGFR-&#945; antibody</td>
			<td>CST</td>
			<td>3174T</td>
			<td>First antibody for immunofluorescence staining assay</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin-Am solution</td>
			<td>Solarbio</td>
			<td>P1410</td>
			<td>Add in culture medium solution to avoid contamination</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>JETBIOFIL</td>
			<td>7556</td>
			<td>Culture fibroblasts</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline solution</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Culture medium solution</td>
		</tr>
		<tr>
			<td>Rabbit (DAIE) mAB IgG XR (R) Isotuge Control (PE)</td>
			<td>Cell Signaling Technology</td>
			<td>5742S</td>
			<td>As a control for flow cytometry</td>
		</tr>
		<tr>
			<td>Round coverslip</td>
			<td>Biosharp</td>
			<td>801007</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td>Punch holes in the cell membrane</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Used for passaging cells</td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Abcam</td>
			<td>ab8978</td>
			<td>First antibody for immunofluorescence staining assay</td>
		</tr>
	</tbody>
</table>

isolation, culture, and characterization of primary dermal fibroblasts from human keloid tissue

成纤维细胞是瘢痕疙瘩组织中的主要细胞类型，在瘢痕疙瘩的形成和发展中起着至关重要的作用。来自瘢痕疙瘩组织的原代成纤维细胞的分离和培养是进一步研究瘢痕疙瘩的生物学功能和分子机制以及治疗瘢痕疙瘩的新治疗策略的基础。获得原代成纤维细胞的传统方法具有局限性，例如细胞状态差，与其他类型的细胞混合以及易受污染。本文描述了一种优化且易于重现的方案，可以减少获取成纤维细胞时可能出现问题的发生。在该方案中，可以在分离后5天观察到成纤维细胞，并在培养10天后达到近80%的汇合度。然后，使用用于免疫荧光测定的PDGFRα和vimentin抗体以及用于流式细胞术的CD90抗体传代和验证成纤维细胞。总之，通过该方案可以轻松获得来自瘢痕疙瘩组织的成纤维细胞，这可以为瘢痕疙瘩研究提供实验室中丰富而稳定的细胞来源。

该协议的时间线总结在 图1A中。分离过程的一些代表性图像如图 2所示;小心地去除表皮和脂肪层，并将真皮层分离成3-4mm2的小碎片，接种到培养皿中。
如图3A所示，在处理后5天在显微镜下观察到组织碎片的几个成纤维细胞生长。如图3B所示，成纤维细胞显示出高增殖速率，并...

Watch this Scientific Journal Video about Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue at JoVE.com

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

本研究描述了一种优化的方案，用于从瘢痕疙瘩组织中建立原代成纤维细胞，可以有效和稳定地提供纯净和可行的成纤维细胞。

人瘢痕疙瘩组织中原代真皮成纤维细胞的分离、培养和表征

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

来自瘢痕疙瘩组织的原代成纤维细胞的分离和培养是进一步研究瘢痕疙瘩的基础。通过该协议可以轻松获得来自瘢痕疙瘩组织的成纤维细胞，这可以为瘢痕疙瘩研究提供实验室中丰富而稳定的细胞来源。首先使用无菌镊子将瘢痕疙瘩组织放入含有10至25毫升补充有1%PSA的PBS的50毫升无菌离心管中。同时，向六孔板的每个孔中加入四毫升PBS PSA溶液。现在将纸巾从管中取出，并用PBS PSA溶液洗涤两次。使用一对无菌镊子，将组织从一个孔依次转移到下一个孔。然后用手术剪刀去除脂肪和表皮层，保持真皮不变。用一把剪刀将修剪过的真皮解剖成三到五平方毫米的碎片，然后用一对无菌镊子将碎片转移到下一个孔中。在PBS PSA溶液中清洗碎片。使用无菌镊子，将10至30个相距小于5毫米的真皮组织碎片放置在培养皿中。将盘子倒置放入培养箱中，直到块变干并粘在盘子上。接下来，加入7毫升补充有10%FBS和1%PSA的DMEM，并像以前一样孵育培养皿。三天后，用完全培养基替换一半的上清液。每天在40X显微镜下观察成纤维细胞。当成纤维细胞达到90%汇合度时，去除培养基中的组织碎片。在PBS中洗涤成纤维细胞，并加入两毫升无菌胰蛋白酶EDTA溶液。在加湿的培养箱中孵育细胞后，轻轻敲击培养皿并在显微镜下观察。加入两毫升完全培养基，一旦大多数细胞分离，即可结束消化。现在将细胞悬液转移到15毫升无菌离心管中，并在室温下以300G离心管三分钟。小心丢弃上清液并将细胞沉淀重悬于完全培养基中。将成纤维细胞接种到9厘米的细胞培养皿中，并在加湿的培养箱中孵育。要在圆形盖玻片上培养成纤维细胞并将其在一抗和二抗中孵育后进行免疫荧光分析，向载玻片中加入 50 微升 DAPI 溶液以染色细胞核。用镊子将洗好的盖玻片放在载玻片上。最后，将样品转移到湿暗盒中进行荧光显微镜检查。在处理后五天观察到组织的成纤维细胞生长。成纤维细胞表现出高增殖速率，并在10天后达到汇合。成纤维细胞具有细长的纺锤状细胞体，它们在高汇合度下排列成束。免疫荧光染色显示成纤维细胞的红色免疫荧光呈阳性，细胞核的蓝色免疫荧光呈阳性。流式细胞术测定显示几乎所有成纤维细胞的CD90阳性。这项研究描述了一种优化的方法，并提供了明确的说明，以解决现有的挑战，并增加瘢痕疙瘩成纤维细胞分离和培养的成功机会。

Research

JoVE Journal

Medicine

Isolation of Human Islets from Partially Pancreatectomized Patients

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Primary Culture of Human Vestibular Schwannomas

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...