Name: isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin
Uploaded: 2017-07-14T14:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

这项工作得到了国家牙医学院和马萨诸塞州和皮肤疾病研究所（张（（R01AR069653）和美国国家卫生研究院（5T32AR062496-03至CA）的支持。

所有动物实验均得到UCSD机构动物保护和使用委员会的批准。 初级小鼠KC从新生儿皮肤中分离和培养<ol><li>使用剪刀将C57B / 6野生型小鼠品系的产后0-2新生儿斩首。切断手腕上方的四肢和踝关节，然后完全切断尾巴，留下一个小洞。 </li><li> 要剥离整个皮肤，首先将尖锐的剪刀穿过尾部的孔，将身体的背部中线的皮肤沿着颈部的开口切开。接下来，使用一个镊子抓住暴露的身体和其他镊子抓住皮肤，并用一个连续的运动轻轻地将整个皮肤从身体和腿部残余物上剥离。 <ol><li>剥离皮肤作为一个完整的部分，并小心不要将皮肤分解成碎片，因为这将导致细胞丢失步骤。 </li></ol></li> &lt;li&gt; 用10 cm培养皿中的15 mL无菌PBS冲洗去皮皮肤，然后将每只小鼠皮肤放入装有冰冷分散酶消化缓冲液的2 mL管中，该缓冲液在KC生长培养基中含有4 mg / mL的分泌酶（KC基础培养基具有0.06mM CaCl 2 ，dGS和抗生素/抗真菌剂）。 注意:可以将多个皮肤分离物合并并在含有12 mL分散液的一个15 mL管（每管最多5个皮肤）中孵育。在旋转器上的4℃冰箱中孵育皮肤管过夜。 <ol><li>确保皮肤没有折叠在管中，因为这将导致折叠区域暴露于分散液的不足。 </li></ol></li><li>在第二天（分泌12-18小时后），将皮肤与分散液一起转移到培养皿中。将每个组织块转移到具有15mL无菌PBS的新培养皿中，以洗涤多余的分散素。 </li><li>使用两对镊子，从PBS抓住皮肤，抬起皮肤，并转移到一个新的培养皿，表皮一面朝下，真皮面朝上。仔细拉伸皮肤褶皱，使其完全延伸到培养皿上。 </li><li> 在新的培养皿中放置500μL每种皮肤的基于胰蛋白酶的消化液。使用镊子，缓慢地将真皮提起并远离表皮，同时保持表皮下降。将真皮处理为生物危害材料。转移分离的表皮，并将基底层向下漂浮在每滴胰蛋白酶溶液上。 <ol><li>如果将表皮折叠在胰蛋白酶溶液上，则使用镊子去除任何褶皱，以确保从上皮细胞中有效消化基底KCs。 </li></ol></li><li>在室温下（盖上盖子）在胰蛋白酶溶液中培养培养皿上的皮肤，轻轻搅拌20分钟。 </li><li> 每个表皮添加2 mL补充的KC生长培养基（每平方英尺约1平方英寸）表皮）到培养皿。使用镊子抓住表皮，大力擦拭表皮，从表皮片上释放单细胞。 <ol><li>随着细胞与表皮分离，观察中等转变越来越浑浊。倾斜培养皿收集和转移细胞悬浮液到一个新的管，留下剩下的表皮片在盘子上。 </li></ol></li><li>在加入2 mL KC生长培养基后再重复两次摩擦表皮片，并将细胞悬液混合在同一根管中。 </li><li>将细胞溶液轻轻吸取数次，以使用适当的血清移液管破碎任何细胞团块，然后将其通过100μm过滤器，直至新的50 mL离心管。 </li><li>将过滤的细胞离心5分钟，180×g。 </li><li>吸出上清液，轻轻将细胞沉淀重悬于1 mL冷的KC生长培养基/表皮中。 </li><li>用血细胞计数器计数细胞。 <br/&gt;注意:KCs可以在悬浮液中自发分化，因此细胞应在分离后尽快铺板，以确保细胞的最佳性能。我们建议在细胞计数时将细胞置于冰上，因为这会降低KC自发分化的速度。 </li><li> 将细胞以5×10 4 / cm 2的密度接种在用促进细胞附着的细胞外基质（ECM）产品预培养的培养皿中的KC生长培养基中。 注意:我们使用商业用途（例如附着因子，它是一种明胶基涂层材料（详见材料表），细胞在37℃的5％CO 2的潮湿培养箱中培养，新鲜培养基每隔一天补充一次<ol><li>对于涂层，在细胞接种之前，将温热至室温的细胞附着涂层材料包被培养皿10分钟至2小时。删除附件因素加入细胞悬液。 </li></ol></li><li>在初次铺板后24小时更换培养基以除去未附着的细胞，每天更换培养基直到细胞在实验前达到所需的汇合度。 </li></ol> 2.成年尾部皮肤的主要小鼠KC隔离<ol><li>根据动物设施法规，牺牲成年人C57BL / 6小鼠（优选在6-15周龄之间;男性或女性）。从尾座上切下身体的尾巴，切断〜2 mm的尾尖，使尖端有一个可见的孔。 </li><li>要剥离尾巴皮肤，首先使用锋利的刀片将尾皮从基部切割到尾端。接下来，使用一个镊子抓住暴露的尾骨和其他镊子抓住皮肤，并用一个连续的运动轻轻地将尾皮从皮肤上剥下。从中线切下去皮的皮肤，使每片不到2-3厘米长。 </li><li> 去皮去皮皮肤在10cm培养皿中加入15mL无菌PBS，然后将每个尾部的皮肤放入装有冰冷分散酶消化缓冲液（KC生长培养基中的4mg / mL分散酶）的2mL管中。 注意:还可以将多个皮肤片合并，并在含有12 mL Dispase溶液的一个15 mL管（每管最多5尾）中孵育。 <ol><li>在旋转器上的4℃冰箱中孵育皮肤管过夜。 </li></ol></li><li>执行步骤1.4至1.13以从尾部表皮分离单细胞悬浮液。 </li><li>在用胶原蛋白预包被的培养皿中的KC生长培养基中以10×10 4 / cm 2的密度（通过血细胞计数器计数）种成虫细胞。 注意:我们使用市售的胶原基涂层套件（详见表格 ）。细胞在37℃的5％CO 2的潮湿培养箱中培养，每隔一天补充新鲜培养基。一般来说，c在细胞播种之前，将超声波培养皿涂上胶原蛋白30分钟至1小时。我们观察到，与作为明胶基涂层材料的附着因子相比，当涂覆胶原时，成人KC附着到培养皿上显着增强。 </li><li>在初次铺板后24小时更换培养基以除去未附着的细胞，每天更换培养基直到细胞在实验前达到所需的汇合度。 </li></ol> 3.高钙对KCs的体外分化<ol><li>为了诱导终末分化，在培养基中加入CaCl 2至0.2mM。 </li><li>或者，在添加高钙之前，将培养的KCs饿死过夜而不加入低钙KC基础培养基中的生长补充剂。 注意:生长因子饥饿将增强细胞对早期分化和早期分化标志物的诱导，如K10 5 </SUP&gt;。 </li></ol> UVB照射介导的细胞死亡和TNF释放从KCs <ol><li>在KC生长培养基（37℃，5％CO 2的加湿培养箱）中培养小鼠KCs，每天培养基更换直到细胞达到融合。在UVB照射之前，取出生长培养基，并用500μLPBS代替。然后使细胞达到25mJ / cm 2的 UVB或模拟对照（无UVB）。 注:UVB照射是使用手持UVB灯与如先前所述10 2个8瓦灯泡（312纳米）上进行。 UVB照射后，抽吸PBS，加入新鲜培养基。 </li><li>测量和在6,8量化由CCK-8细胞生存力试验中的细胞生存力，以及制造商的指令11以下24小时后UVB治疗。 </li><li>从所需时间点用UVB处理的KCs收集条件培养基，并测量释放的TNFα在由小鼠TNF（单/单）ELISA试剂盒制造商的指令7，12以下的条件培养基。 </li></ol>

<ol>
	<li>Mauro, T., 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=Acute+barrier+perturbation+abolishes+the+Ca2++and+K++gradients+in+murine+epidermis:+quantitative+measurement+using+PIXE.">Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.</a> J Invest Dermatol. 111 (6), 1198-1201 (1998).</li><li>Menon, G. K., Grayson, S., Elias, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+calcium+reservoirs+in+mammalian+epidermis:+ultrastructural+localization+by+ion-capture+cytochemistry.">Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry.</a> J Invest Dermatol. 84 (6), 508-512 (1985).</li><li>Elias, P. 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=Formation+of+the+epidermal+calcium+gradient+coincides+with+key+milestones+of+barrier+ontogenesis+in+the+rodent.">Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent.</a> J Invest Dermatol. 110 (4), 399-404 (1998).</li><li>Hennings, H., 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=Calcium+regulation+of+growth+and+differentiation+of+mouse+epidermal+cells+in+culture.">Calcium regulation of growth and differentiation of mouse epidermal cells in culture.</a> Cell. 19 (1), 245-254 (1980).</li><li>Zhang, L. J., Bhattacharya, S., Leid, M., Ganguli-Indra, G., Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip2+is+a+dynamic+regulator+of+epidermal+proliferation+and+differentiation+by+integrating+EGFR+and+Notch+signaling.">Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling.</a> J Cell Sci. 125 (Pt 23), 5733-5744 (2012).</li><li>Lai, Y. P., 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=Commensal+bacteria+regulate+Toll-like+receptor+3-dependent+inflammation+after+skin+injury.">Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury.</a> Nat Med. 15 (12), 1377-1382 (2009).</li><li>Bernard, J. 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=Ultraviolet+radiation+damages+self+noncoding+RNA+and+is+detected+by+TLR3.">Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.</a> Nat Med. 18 (8), (2012).</li><li>Zhang, L. 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=Antimicrobial+Peptide+LL37+and+MAVS+Signaling+Drive+Interferon-beta+Production+by+Epidermal+Keratinocytes+during+Skin+Injury.">Antimicrobial Peptide LL37 and MAVS Signaling Drive Interferon-beta Production by Epidermal Keratinocytes during Skin Injury.</a> Immunity. 45 (1), 119-130 (2016).</li><li>Borkowski, A. W., 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=Toll-Like+Receptor+3+Activation+Is+Required+for+Normal+Skin+Barrier+Repair+Following+UV+Damage.">Toll-Like Receptor 3 Activation Is Required for Normal Skin Barrier Repair Following UV Damage.</a> J Invest Dermatol. 135 (2), 569-578 (2015).</li><li>Borkowski, A. W., 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=Toll-like+receptor+3+activation+is+required+for+normal+skin+barrier+repair+following+UV+damage.">Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.</a> J Invest Dermatol. 135 (2), 569-578 (2015).</li><li>Zillessen, P., 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=Metabolic+role+of+dipeptidyl+peptidase+4+(DPP4)+in+primary+human+(pre)adipocytes.">Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes.</a> Sci Rep. 6, 23074 (2016).</li><li>Wells, C. 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=The+macrophage-inducible+C-type+lectin,+mincle,+is+an+essential+component+of+the+innate+immune+response+to+Candida+albicans.">The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.</a> J Immunol. 180 (11), 7404-7413 (2008).</li><li>Vasioukhin, V., Bauer, C., Yin, M., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+actin+polymerization+is+the+driving+force+for+epithelial+cell-cell+adhesion.">Directed actin polymerization is the driving force for epithelial cell-cell adhesion.</a> Cell. 100 (2), 209-219 (2000).</li><li>Zhuang, 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=TNF+receptor+p55+plays+a+pivotal+role+in+murine+keratinocyte+apoptosis+induced+by+ultraviolet+B+irradiation.">TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation.</a> J Immunol. 162 (3), 1440-1447 (1999).</li><li>Lichti, U., Anders, J., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+short-term+culture+of+primary+keratinocytes,+hair+follicle+populations+and+dermal+cells+from+newborn+mice+and+keratinocytes+from+adult+mice+for+in+vitro+analysis+and+for+grafting+to+immunodeficient+mice.">Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.</a> Nat Protoc. 3 (5), 799-810 (2008).</li><li>Dlugosz, A. A., Glick, A. B., Tennenbaum, T., Weinberg, W. C., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+utilization+of+epidermal+keratinocytes+for+oncogene+research.">Isolation and utilization of epidermal keratinocytes for oncogene research.</a> Methods Enzymol. 254, 3-20 (1995).</li><li>Jones, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+keratinocytes.">Isolation and culture of mouse keratinocytes.</a> CSH Protoc. 2008, (2008).</li><li>Pirrone, A., Hager, B., Fleckman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+keratinocyte+culture.">Primary mouse keratinocyte culture.</a> Methods Mol Biol. 289, 3-14 (2005).</li><li>Yuspa, S. H., Kilkenny, A. E., Steinert, P. M., Roop, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+murine+epidermal+differentiation+markers+is+tightly+regulated+by+restricted+extracellular+calcium+concentrations+in+vitro.">Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.</a> J Cell Biol. 109 (3), 1207-1217 (1989).</li><li>Yuspa, S. H., 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=Signal+transduction+for+proliferation+and+differentiation+in+keratinocytes.">Signal transduction for proliferation and differentiation in keratinocytes.</a> Ann N Y Acad Sci. 548, 191-196 (1988).</li><li>Fang, N. X., 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=Calcium+enhances+mouse+keratinocyte+differentiation+in+vitro+to+differentially+regulate+expression+of+papillomavirus+authentic+and+codon+modified+L1+genes.">Calcium enhances mouse keratinocyte differentiation in vitro to differentially regulate expression of papillomavirus authentic and codon modified L1 genes.</a> Virology. 365 (1), 187-197 (2007).</li><li>Kolly, C., Suter, M. M., Muller, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+cell+cycle+exit,+and+onset+of+terminal+differentiation+in+cultured+keratinocytes:+pre-programmed+pathways+in+control+of+C-Myc+and+Notch1+prevail+over+extracellular+calcium+signals.">Proliferation, cell cycle exit, and onset of terminal differentiation in cultured keratinocytes: pre-programmed pathways in control of C-Myc and Notch1 prevail over extracellular calcium signals.</a> J Invest Dermatol. 124 (5), 1014-1025 (2005).</li><li>Marcelo, C. L., Gold, R. C., Fairley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+1.2+mmol/l+calcium,+triamcinolone+acetonide,+and+retinoids+on+low-calcium+regulated+keratinocyte+differentiation.">Effect of 1.2 mmol/l calcium, triamcinolone acetonide, and retinoids on low-calcium regulated keratinocyte differentiation.</a> Br J Dermatol. 111, 64-72 (1984).</li></ol>

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皮肤表皮作为分离和保护身体免受外界环境的损害，水分流失，病原体，热量和紫外线照射的关键障碍。 KCs是表皮的主要细胞系，表皮KCs的原代培养是研究和了解屏障形成的生物学过程和KCs对体外环境侮辱的反应的有用工具。 在这里我们描述从新生儿和成年鼠皮肤分离和培养初级表皮KCs的方法。虽然原代小鼠KCs可以方便地从新生小鼠皮肤分离，但成年KCs的分离和成功?...

皮肤是身体最大的器官，表皮最外层。表皮在形成完整的表皮屏障以将身体与环境分离的过程中发挥关键作用，从而防止水分流失，并且防止环境污染，例如过敏原，病原体和UVB暴露。表皮从单层未分化基底角质形成细胞（KCs）发展成由基底层组成的多层分层上皮，其次是棘突层，颗粒层和角质层。由表皮干细胞和转运扩增细胞组成的基础KCs是增殖和未分化的。由于基底KCs离开细胞周期，细胞承诺分化，并逐渐迁移到表皮表面，伴随细胞细胞连接的成熟和表皮渗透屏障（EPB）的形成。棘突中的KCs表达早期差异离子标记如角蛋白10（K10）;随着KCs迁移到颗粒层，细胞表达晚期分化标志物，如丝胶蛋白（FLG），Loricrin（LOR）和Involucrin（INV）。在角质层，KCs成为终末分化的角质细胞，当新细胞取代它们时，它们最终通过脱屑而脱落。 钙被认为是表皮中最生理的药物，并以类似的方式在体外和体内触发分化。在正常皮肤表皮，钙离子形成的特性"浓度梯度"，在浓度朝向皮肤表面1，2，3增大。钙浓度从最低子层（基底和棘突层）的低水平上升到上部颗粒层中的峰值，然后在最表层（角质层）中降至可忽略的水平。<html>钙梯度也与成分渗透性屏障的出现同时发生，其支持钙信号传导起KC分化的关键作用。 体外 ，低钙（0.02-0.1mM）维持基底KCs作为单层的增殖，而高钙（&gt; 0.1mM）诱导细胞对终末分化的快速和不可逆的承诺，如紧密连接形成和诱导LOR和INV的在高钙处理至基础枯否4,5。 除了屏障形成之外，表皮KCs也是皮肤先天免疫系统的重要组成部分。针对在UVB照射或损伤时释放的病原体或损伤相关分子模式（DAMP），KCs可以产生大量的炎性细胞因子，如TNFα，IL6和IFNβ，导致免疫系统激活&gt; 6，7，8，9。虽然从枯否正确炎性信号所需的病原体清除，失控性炎症反应可以触发自动炎性皮肤疾病，如牛皮癣和酒渣鼻6,8的发展。 总体而言，KCs在维持完整的皮肤屏障和启动对病原体入侵或环境侮辱的免疫应答方面起着至关重要的作用。因此，表皮KCs的原代培养是研究上皮生物学，KC分化以及KC刺激的先天免疫应答的有用技术。由于KC对各种外部兴奋剂的敏感性和敏感性，原代小鼠表皮KCs的分离和培养可能是一个具有挑战性的过程。这里我们描述一种从新生小鼠皮肤中分离和培养KCs的方法成年老鼠尾皮。对于成年KC分离，我们不使用小鼠背部皮肤，因为从这个组织中分离出足够量的活KC可能是困难的，原因如下:首先，在头发周期的休眠阶段的成年背部皮肤（休止期）由薄的表皮只有1-2层细胞，导致低细胞产量和表皮与真皮的低效分离，这是成功进行KC分离的关键步骤。第二，存在于成年背皮上的高毛囊密度进一步有助于分离表皮与真皮的困难。相反，我们通常使用尾皮作为成年小鼠KCs的来源，因为上皮更厚，3-5层表皮KCs。它也具有较低的毛囊密度，其不会干扰表皮分离，从而允许KC从任何成年小鼠尾部皮肤分离，而不管小鼠的年龄和毛发循环阶段。孤立的霓虹灯将tal KCs接种到明胶包被的培养皿中，而使用胶原蛋白包被的培养皿来种植分离的成年KCs，这是因为与新生儿相比，成人细胞的粘附能力受损。为了培养小鼠KCs，补充含有表皮生长因子（EGF），牛转运蛋白，胰岛素样生长因子c1（IGF1），前列腺素E2（PGE2），牛血清白蛋白（BSA）和氢化可的松的dGS的低钙基础培养基。在初次铺板2-4天之后，大多数分化的KCs可以在日常培养基的变化过程中被洗去，剩下的贴壁细胞显示典型的鹅卵石形态4 ，正在增殖，并且不表达早期的分化标志物K10。

<table><tbody><tr><td>C57BL/6 neonates or adult wildtype mice</td><td>Jackson Laboratory</td><td>000664</td><td>Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td></tr><tr><td>KC basal medium (EpiLife)</td><td>Invitrogen, Carlsbad, CA</td><td>MEPICF500</td><td>basal medium for keratinocyte culture with 0.06 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Defined Growth Supplement (dGS)</td><td>Invitrogen, Carlsbad, CA</td><td>S0125</td><td>defined growth supplements for culture medium</td></tr><tr><td>Dispase powder</td><td>Invitrogen, Carlsbad, CA</td><td>17105041</td><td>enzyme to dissociate the epidermis from dermis</td></tr><tr><td>Attachment Factor</td><td>Invitrogen, Carlsbad, CA</td><td>S006100</td><td>gelatin-based coating material</td></tr><tr><td>Coating Matrix</td><td>Invitrogen, Carlsbad, CA</td><td>R011K</td><td>Collagen-based coating material</td></tr><tr><td>TrypLE</td><td>Invitrogen, Carlsbad, CA</td><td>12604-013</td><td>A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td></tr><tr><td>100 &amp;mu;m Cell Strainer Nylon mesh</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>CCK-8 cell viability Kit</td><td>Dojindo Molecular Technologies, Rockville, MD</td><td>CK04-11</td><td></td></tr><tr><td>Mouse TNF (Mono/Mono) ELISA Set II</td><td>BD Biosciences, San Jose, CA</td><td>555268</td><td></td></tr><tr><td>Corded Hand-Held UV Lamps</td><td>Spectronics, Westbury, NY</td><td>EB-280C</td><td></td></tr><tr><td>8-watt UV tubes</td><td>Spectronics, Westbury, NY</td><td>BLE-8T312</td><td></td></tr><tr><td>Light Inverted Microscope for cell culture</td><td>ZEISS, Jena, Germany</td><td>Axio Observer</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Olympus</td><td>BX41</td><td></td></tr></tbody></table>

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

角质形成细胞（KC）是表皮中最主要的细胞类型，即皮肤的最外层。表皮KCs通过形成完整的皮肤屏障来抵御环境污染，例如UVB照射或病原体，以及通过对这些侮辱发起炎症反应，在提供皮肤防御方面发挥关键作用。这里我们描述了从新生小鼠皮肤和成年小鼠尾皮分离KCs的方法。我们还描述了使用定义的生长补充剂（dGS）与穿梭胎牛血清（cFBS）相比的培养条件。在功能上，我们显示新生儿和成年KCs对高钙诱导的末端分化，紧密连接形成和分层具有高度反应性。此外，培养的成年KCs对UVB触发的细胞死亡敏感，并且可以在UVB照射时释放大量的TNF。一起，这里描述的方法对于体外模型设置的研究人员将是有用的以研究新生小鼠和/或成年小鼠中的表皮生物学。

高钙诱导的新生儿和成人KCs的终末分化。以0.06mM CaCl 2铺平并保持的主要小鼠表皮KCs作为单层生长，并且单个细胞具有不同细胞间隙的多边形形状，当汇合时显示出鹅卵石的外观（ 图1A和图2A ）。将CaCl 2升高至0.2mM导致细胞的快速形态变化。高钙开关后8 h内，细胞变平，细胞间隙明显不明显，24 h时?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

表皮角质形成细胞形成功能性皮肤屏障，位于主体防御的前线，防止外部环境污染。在这里，我们描述了从新生儿和成年小鼠皮肤分离和原代培养表皮角化细胞的方法，以及从角质形成细胞诱导终末分化和UVB引发的炎症反应。

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

isolation-culture-primary-mouse-keratinocytes-from-neonatal-adult

Research

JoVE Journal

Biology

30.7K 次观看.  UC San Diego. 表皮角质形成细胞形成功能性皮肤屏障，位于主体防御的前线，防止外部环境污染。在这里，我们描述了从新生儿和成年小鼠皮肤分离和原代培养表皮角化细胞的方法，以及从角质形成细胞诱导终末分化和UVB引发的炎症反应。 

视频: Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

科研

发育生物学

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

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

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

生物学

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

生成用于角质形成细胞活细胞成像的原代培养物

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

生物工程

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

人皮肤角质形成细胞分离:一种从皮肤样本中培养原代角质形成细胞的方法

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...