该的作者感谢的M. Dietel教授和德克·舒马​​赫为支持这项工作，并批判性阅读的手稿。 导致这些结果的研究创新药物倡议联合承诺根据赠款协议Ñ°115234，资源的财政贡献，这是由欧盟第七框架计划（FP7/2007-2013）和EFPIA公司中所获得的支持实物捐助。也支持这项工作的：柏林Krebsgesellschaft（BKG）。

实验的总体方案包括制备初级单细胞从肿瘤组织中，表征初级细胞培养，成纤维细胞耗竭和磁性细胞分选的CD133 +和CD133 -黑色素瘤细胞的是，在图1中所示。 1。样品采集<ol><li>伦理审查的检查，然后再考虑下一个步骤。 </li><li>准备一个50毫升的试管，用无菌PBS与1％青霉素 - 链霉素的最终浓度和交出手术或团队补充。 </li><li>组织快速运输手术的肿瘤组织细胞培养实验室，在4°C。 </li></ol> 2。准备原发性黑色素瘤单细胞肿瘤组织<ol><li>在无菌条件下执行所有步骤。 </li><li>在切除肿瘤之前，需要准备的分解组合。 10毫升的分解组合，每2-4克组织是必需的。可以使用的混合immediately或存储在10毫升的等分试样在-20℃下，购买使用。 <ol><li>准备含有150mM氯化钠的溶液。混合使用的涡流，直到溶液澄清。如果有必要的无菌滤液的氯化钠溶液，用0.22μm的过滤器。 </li><li>称取DNA酶I，并含有10毫克/毫升DNA酶I在150MM氯化钠制备的溶液。通过翻转混合，直到溶液澄清。 </li><li>在PBS中含有100mg/ml胶原酶IV准备的解决方案。通过翻转混合，直到溶液澄清。胶原酶溶液的等分试样在-20℃下，可以存储数个月。 </li><li>准备与最终浓度为1％青霉素 - 链霉素，1毫克/毫升胶原酶IV和0.1毫克/毫升DNA酶I量子263介质中离解混合。混合使用的旋涡。可选：当使用来自从皮肤添加两性霉素B溶液至终浓度为1.5微克/毫升两性霉素B的解离混合的黑色素瘤组织</li></ol></li><li>温暖的REQuired量的PBS和量子分解组合，263中等至37°C </li><li>将肿瘤组织在无菌培养皿中。组织和吸入过量的溶液，加入500μl分解组合。百果2-4毫米，采用新鲜，无菌手术刀切成小块肿瘤。 </li><li>传输2-4克肿瘤组织绞碎成gentleMACS C管含5毫升的分解组合。冲洗培养皿中，用4.5毫升分离混合，并加入剩余的组织碎片的gentleMACS C管。 </li><li>关闭gentleMACS C管，并将它附加倒挂在套筒上的gentleMACS离解。运行的gentleMACS计划“h_tumor_01。 </li><li>分离的gentleMACS C管的离解器和样品孵育30分钟，在37℃下连续旋转使用的MACSmix管肩的最高运行速度（12转）或通过手动转动管，每5分钟，以重新悬浮结算组织碎片。 </li><li>颠倒gentleMACS C管安装在套筒上的t他gentleMACS离解和运行的gentleMACS的节目“h_tumor_02”的。 </li><li>分离的gentleMACS C管的离解器和样品孵育30分钟，在37℃下连续旋转使用的MACSmix管肩的最高运行速度（12转）或通过手动转动管，每5分钟，以重新悬浮结算组织碎片。 </li><li>将gentleMACS C管，倒挂在套筒上的gentleMACS离解，运行gentleMACS计划“h_tumor_03”。 </li><li>分离的gentleMACS的C管的离解，重悬样品，应用细胞悬液50毫升的试管放在一个70微米的细胞过滤器。如果过滤器的堵塞时，搅拌细胞悬浮液用无菌的过滤嘴，直到完整的悬浮液穿过。 </li><li>用5毫升的量子263培养基冲洗细胞过滤网。 </li><li>可选：如果你还有组织碎片留在过滤器，重悬片段在263昆腾的介质和种子，他们在适当的细胞培养显示小时。孵育片段，在37℃和5％CO 2的 。 </li><li>弃细胞过滤器，并关闭50毫升管与消化的细胞悬液。 5分钟离心细胞悬液300×g离心，弃去上清液。 </li><li>可选：如果细胞沉淀出现红色的，由于高量的红细胞准备1个红血细胞裂解液500μLPBS稀释10倍的解决方案，双蒸水重悬细胞沉淀在1:10，加入5毫升1个红细胞裂解解决方案。涡5秒后，在室温下孵育10分钟的温度。在300 XG离心5分钟，弃去上清液。 </li><li>重悬与Quantum 263和种子细胞在适当的细胞培养瓶中的肿瘤细胞。文化的肿瘤细胞，在37℃和5％CO 2和常规传代细胞，当他们达到80％汇合。 </li></ol> 3。原代细胞培养的表征和成纤维细胞耗竭<ol><li>分析原代细胞培养的表型使用microscoPE。 </li><li>收获的原代细胞培养中的一个小的量（0.5×10 6个细胞），并与肿瘤，黑色素瘤，干细胞和基质细胞的标记基因的引物，进行RT-PCR。最重要的基因分析的是CD90（成纤维细胞标记），TYR（黑色素瘤/黑素细胞标记物），CD133（癌干细胞标志物），CD83（标记为成熟树突状细胞）和管家基因（ 例如 HPRT或GAPDH）。 </li><li>如果微观分析，在RT-PCR显示高污染的原发性黑色素瘤文化与成纤维细胞高表达CD90，你需要消耗使用反成纤维细胞微珠和D列美天旎的成纤维细胞。 </li></ol> 4。磁性细胞分选的CD133 +和CD133 -黑色素瘤细胞LS列<ol><li>准备MACS缓冲液含有2mM EDTA和5％FCS的PBS。混合使用0.22微米Steritop的GP过滤器的反相和无菌滤液缓冲。 4-8°C的冷藏缓冲并对其进行脱气后方可使用。 </li><li>前的温暖Accutase，PBS和Quantum 263中的37°C。 </li><li>用PBS洗净80％汇合的细胞。加入适量的Accutase和孵化，直到细胞的培养表面脱离。在一个50毫升的试管使用量子263培养基，收集细胞。 </li><li>确定的细胞数和离心的所需数目的细胞（根据Miltenyi公司的协议的一个最大数的2×10 9细胞可以被加载每LS柱。细胞行具体的最优数目应被确定，并可以相当低的。）5分钟，在300 xg和4-8°C。完全吸出上清液。 </li><li>重悬最大为1×10 8个细胞，350μLMACS缓冲（更高的手机号码规模以下所有MACS缓冲，FcR阻断的试剂，CD133/1-Biotin和抗生物素微珠体积相应）。 </li><li>加入100μlFcR阻断剂。 </li><li>加入50微升CD133/1-Biotin。混合使用的旋涡，并培育4-8°C F或10分钟。 </li><li>在300 xg和4-8℃，加入10ml MACS缓冲液和5分钟离心洗涤细胞完全吸出上清液。 </li><li>重复洗涤步骤（4.8） </li><li>重悬细胞沉淀用400μl的MACS缓冲。 </li><li>加入100μl抗生物素微球。混合使用涡，并在4-8°C孵育15分钟。 </li><li>同时，准备MACS分离器，磁分离。附加QuadroMACS MACS分离器多架。插入所需数量的LS列与列翼的前部中的磁场的QuadroMACS。预分离过滤器（30μm的尼龙网）放置到每个LS柱和一个适当的集合管（15毫升或50毫升），根据每列。准备过滤器和列的通过用3ml MACS缓冲液漂洗。丢弃流过。 </li><li>在300 xg和4-8℃，加入10ml MACS缓冲液和5分钟离心洗涤细胞完全吸出上清液。 </li><li>重悬细胞用500μlMACS缓冲和应用细胞悬液的LS栏上与前分离滤芯。 </li><li>洗涤三次，用3ml MACS每列的缓冲区。列水库是空的，只有添加新的缓冲区。 </li><li>将收集的总流出物含有未标记的CD133 -细胞组分。 </li><li>丢弃前分离过滤器，删除列从分离器，并把它放在一个合适的收集管中。 </li><li>加5毫升MACS缓冲。立即冲洗出来的标记CD133 +细胞，用力推将柱塞压入列。 </li><li>若要增加纯度负馏分，细胞悬浮液应用到一个新的平衡LS柱插入在QuadroMACS。污水中含有的CD133 -分数。 </li><li>丢弃列。 </li></ol>

<ol>
	<li>Garibyan, L., Fisher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+sunlight+causes+melanoma.">How sunlight causes melanoma.</a> Curr. Oncol. Rep. 12, 319-326 (2010).</li><li>Radovic-Kovacevic, V., 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=Survival+analysis+in+patients+with+cutaneous+malignant+melanoma.">Survival analysis in patients with cutaneous malignant melanoma.</a> Srp. Arh. Celok. Lek. 125, 132-137 (1997).</li><li>Lens, M. B., Eisen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+chemotherapy+in+the+treatment+of+malignant+melanoma.">Systemic chemotherapy in the treatment of malignant melanoma.</a> Expert Opin. Pharmacother. 4, 2205-2211 (2003).</li><li>Nashan, D., Muller, M. L., Grabbe, S., Wustlich, S., Enk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+therapy+of+disseminated+malignant+melanoma:+an+evidence-based+overview+of+the+state-of-the-art+in+daily+routine.">Systemic therapy of disseminated malignant melanoma: an evidence-based overview of the state-of-the-art in daily routine.</a> J. Eur. Acad. Dermatol. Venereol. 21, 1305-1318 (2007).</li><li>Eigentler, T. K., Meier, F., Keilholz, U., Hauschild, A., Garbe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Der Onkologe. 16, 1160-1166 (2010).</li><li>Keilholz, U., Tilgen, W., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemische+Therapie+des+metastasierten+Melanoms:+Ergebnisse+randomisierter+Studien+der+letzten+zehn+Jahre.">Systemische Therapie des metastasierten Melanoms: Ergebnisse randomisierter Studien der letzten zehn Jahre.</a> Deutsches &#196;rzteblatt. 100, (2003).</li><li>Chapman, P. B., 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=Improved+survival+with+vemurafenib+in+melanoma+with+BRAF+V600E+mutation.">Improved survival with vemurafenib in melanoma with BRAF V600E mutation.</a> N. Engl. J. Med. 364, 2507-2516 (2011).</li><li>Burdall, S. E., Hanby, A. M., Lansdown, M. R., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines:+friend+or+foe.">Breast cancer cell lines: friend or foe.</a> Breast Cancer Research : BCR. 5, 89-95 (2003).</li><li>Osborne, C. K., Hobbs, K., Trent, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+differences+among+MCF-7+human+breast+cancer+cell+lines+from+different+laboratories.">Biological differences among MCF-7 human breast cancer cell lines from different laboratories.</a> Breast Cancer Research and Treatment. 9, 111-121 (1987).</li><li>Hiorns, L. R., 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=Variation+in+RNA+expression+and+genomic+DNA+content+acquired+during+cell+culture.">Variation in RNA expression and genomic DNA content acquired during cell culture.</a> Br. J. Cancer. 90, 476-482 (2004).</li><li>Nelson-Rees, W. A., Daniels, D. W., Flandermeyer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-contamination+of+cells+in+culture.">Cross-contamination of cells in culture.</a> Science. 212, 446-452 (1981).</li><li>MacLeod, R. 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=Widespread+intraspecies+cross-contamination+of+human+tumor+cell+lines+arising+at+source.">Widespread intraspecies cross-contamination of human tumor cell lines arising at source.</a> Int. J. Cancer. 83, 555-563 (1999).</li><li>Masters, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cancer+cell+lines:+fact+and+fantasy.">Human cancer cell lines: fact and fantasy.</a> Nature reviews. Molecular Cell Biology. 1, 233-236 (2000).</li><li>Pennartz, S., Reiss, S., Biloune, R., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Single-Cell+Suspensions+from+Mouse+Neural+Tissue.">Generation of Single-Cell Suspensions from Mouse Neural Tissue.</a> J. Vis. Exp. (29), e1267 (2009).</li><li>Zabierowski, S. E., Fukunaga-Kalabis, M., Li, L., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermis-derived+stem+cells:+a+source+of+epidermal+melanocytes+and+melanoma.">Dermis-derived stem cells: a source of epidermal melanocytes and melanoma.</a> Pigment Cell Melanoma Res. 24, 422-429 (2011).</li><li>Singh, S. K., 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Ieta, K., 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=Biological+and+genetic+characteristics+of+tumor-initiating+cells+in+colon+cancer.">Biological and genetic characteristics of tumor-initiating cells in colon cancer.</a> Ann. Surg. Oncol. 15, 638-648 (2008).</li><li>Bertolini, G., 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=Highly+tumorigenic+lung+cancer+CD133++cells+display+stem-like+features+and+are+spared+by+cisplatin+treatment.">Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 16281-16286 (2009).</li><li>Monzani, E., 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=Melanoma+contains+CD133+and+ABCG2+positive+cells+with+enhanced+tumourigenic+potential.">Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential.</a> Eur. J. Cancer. 43, 935-946 (2007).</li><li>Gedye, 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=Cancer/testis+antigens+can+be+immunological+targets+in+clonogenic+CD133++melanoma+cells.">Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells.</a> Cancer Immunology, Immunotherapy : CII. 58, 1635-1646 (2009).</li><li>Kobari, 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=CD133++cell+selection+is+an+alternative+to+CD34++cell+selection+for+ex+vivo+expansion+of+hematopoietic+stem+cells.">CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells.</a> J. Hematother. Stem Cell Res. 10, 273-281 (2001).</li><li>Quirici, N., 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=Differentiation+and+expansion+of+endothelial+cells+from+human+bone+marrow+CD133(+)+cells.">Differentiation and expansion of endothelial cells from human bone marrow CD133(+) cells.</a> Br. J. Haematol. 115, 186-194 (2001).</li><li>Pfenninger, C. V., 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=CD133+is+not+present+on+neurogenic+astrocytes+in+the+adult+subventricular+zone,+but+on+embryonic+neural+stem+cells,+ependymal+cells,+and+glioblastoma+cells.">CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells.</a> Cancer Res. 67, 5727-5736 (2007).</li><li>Dubreuil, V., Marzesco, A. M., Corbeil, D., Huttner, W. B., Wilsch-Brauninger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbody+and+primary+cilium+of+neural+progenitors+release+extracellular+membrane+particles+enriched+in+the+stem+cell+marker+prominin-1.">Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1.</a> J. Cell Biol. 176, 483-495 (2007).</li><li>Florek, 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=a+neural+and+hematopoietic+stem+cell+marker,+is+expressed+in+adult+human+differentiated+cells+and+certain+types+of+kidney+cancer.">a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer.</a> Cell Tissue Res. 319, 15-26 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+Mouse+Lymph+Nodes+Using+Miltenyi+MACS+Purification.">Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification.</a> J. Vis. Exp. (9), e409 (2007).</li><li>Watkins, S. K., Zhu, Z., Watkins, K. E., Hurwitz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Immune+Cells+from+Primary+Tumors.">Isolation of Immune Cells from Primary Tumors.</a> J. Vis. Exp. (64), e3952 (2012).</li><li>Mollamohammadi, S., 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=A+simple+and+efficient+cryopreservation+method+for+feeder-free+dissociated+human+induced+pluripotent+stem+cells+and+human+embryonic+stem+cells.">A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells.</a> Hum. Reprod. 24, 2468-2476 (2009).</li></ol>

作者宣称，他们没有竞争的金融利益。

为了去除红细胞污染的肿瘤细胞沉淀，我们强烈建议您使用红细胞裂解液从美天旎。比传统的Ficoll密度梯度离心的红细胞裂解的优点是，它是更快，更简单。此外，利用Ficoll或红血细胞和肿瘤细胞的损失，避免污染。当你观察到的原代细胞培养成纤维细胞生长，应立即删除，因为它们通常生长肿瘤细胞时等待时间过长。 排序时细胞与美天旎MACS技术，我们建议收集细胞酶。使?...

皮肤恶性黑色素瘤是最常见的，也是最致命的皮肤癌类型。由于发病率越来越高，一个高品位的恶性和快速的传播，恶性黑色素瘤占75％的恶性皮肤肿瘤相关死亡1,2。除了 ​​手术切除原发肿瘤，化疗，放疗，免疫治疗和化疗和免疫治疗转移性黑色素瘤是国家的最先进的黑色素瘤的治疗策略，3,4。然而，恶性黑色素瘤的特点是对化疗药物的反应不佳（5-12％）5,6，和只有50％-70％的恶性黑色素瘤患者携带BRAF V600E基因突变的好处有前途的新的靶向治疗药物的治疗vemurafenib 7提高总生存率，更好地了解黑色素瘤肿瘤的机制是必要的。 为了研究这些机制在过去，完善的市售单独购买可用的细胞株。利用最近的方法来研究癌症的发生和发展的分子事件的主要直接来源于肿瘤组织的文化 - 一种策略轴承多方面的好处：研究者具有完全控制的患者和组织的选择。采样的细胞获得的专业选择肿瘤组织的肿瘤，像所有的异质性，对病人的随访数据和详细的病理是允许的文化进行比较的特点与原发肿瘤8。 与此相反，在癌症研究中作为相关的模型系统的建立的细胞系的使用有争议的讨论。早在1987年，奥斯本和他的同事们描述了来自不同实验室9人乳腺癌MCF-7细胞株显着的生物差异。这种广泛的基因组不稳定性，在传代过程中RNA的表达变化合作nfirmed由Hiorns 等，并提供证据表明，细胞系在培养演变，从而削弱了这种既定文化作为人类癌症10的模型直接相关的数据支持。 多年来，这在很大程度上被忽视了，是另一个重要的考虑无关的&#39;假&#39;的细胞，这是第一次在20年前通过展示突出文化与污染或过度生长的风险，大量被污染的细胞系HeLa细胞细胞8,11。误解&#39;假&#39;细胞株的数据，最近在文献中再次暴露出来。使用DNA分析和分子细胞遗传学的结合，MacLeod 等人发现，252个新的人类肿瘤源性细胞株存放在德国收集的微生物和细胞培养（DSMZ），近18％被认为是种内或种间杂种污染物12,13。为了避免这些问题，并利用上述的优点，我们决定建立低传代新鲜切除转移的黑色素瘤细胞系。 同时限制细胞的死亡和破坏特征的表面蛋白单细胞的准备工作，以产生强大的细胞分离和分析技术。台式仪器的自动分离成单细胞悬液组织是由美天旎制造gentleMACS离解。当使用结合gentleMACSÇ管和优化的解离的解决方案，在一个封闭的系统中的肿瘤组织的一个有效和温和解离来实现，同时保留抗原表位，并减少信元丢失。该仪器提供了优化，预先设定的程序的各种特定的应用程序和担保标准化制备单细胞悬浮液从黑色素瘤组织14。 &lt;P类=的“jove_content”&gt;最近的报告表明，多数肿瘤怀有小亚群所谓的肿瘤干细胞（CSCs），只表现出肿瘤的启动和自我更新的能力。的皮肤的真皮层中的黑素细胞产生的干细胞的识别导致的假设，即这些细胞可能会对在黑素瘤中的肿瘤干细胞（CSCs）的起源，因为暴露于紫外线辐射可以灌注，这些细胞的恶性转化15。这种遗传病变的结果将是怀有相结合的肿瘤干细胞特性的细胞。 建议在黑色素瘤干细胞亚群的代表的关键标志物之 ​​一是CD133的16-20。 CD133（也称为作为Prominin 1），一个部件的pentaspan跨膜糖蛋白，是表示，在造血干细胞，血管内皮祖细胞，神经元和神经胶质干细胞21-23。 CD133表达与不对称细胞分裂24，以及被证明是糖基化的抗原表位的CD133下调细胞分化时25。最近，CD133 +黑色素瘤细胞具有自我更新和肿瘤起始容量19,20。 为了研究CD133 +公认的黑色素瘤干细胞的功能，我们修改和优化的磁性活化细胞分选系统（MACS）从美天旎生物技术获得的丰富和可存活种群的CD133 +和CD133 -细胞。 基于磁珠细胞分离可以是负选择Matheu 等 26或积极的选择，我们在这里展示。阳性选择的过程的特定的靶细胞类型， 例如 CD133的表达细胞，微珠，50-nm的超顺磁性粒子，是共轭高度特异性抗体针对磁标记特定的细胞表面抗原。分离过程中，磁标记的细胞被保留在柱中，在磁场中的隔板，而标记的细胞流经。洗涤步骤之后，该列被删除从磁场的分离器，和靶细胞，从柱中洗脱。 LS或使用MS列的分数高纯度的标记和未标记的细胞阳性选择的过程。另一方面，LD列，用于负选择，导致稍微低的纯度的标记的部分。由于它们的矩阵变浓的包装在这些列中的流速是低级所得在被困在列中的未标记细胞的风险较高。 LD列，因此，应仅用于不需要的细胞亚群的严格耗尽。可以通过直接（耦合到微珠的抗体）或间接的磁性标记（孵化后incubatio与微珠的阳性筛选n与第一抗体）。具体MACS微珠可用于许多类型的细胞的阳性筛选原发性肿瘤细胞，如前列腺癌或黑素瘤27。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> Accutase </td><td> PAA实验室公司</td><td> L11-007 </td><td></td></tr><tr><td>两性霉素B溶液250微克/毫升的去离子水</td><td> Sigma-Aldrich公司</td><td> A2942-50ML </td><td></td></tr><tr><td>抗成纤维细胞微</td><td>美天旎生物技术有限公司</td><td> 130-050-601 </td><td></td></tr><tr><td> CD133 / 1（W6B3C1） </td><td>美天旎生物技术有限公司</td><td> 130-092-395 </td><td></td></tr><tr><td>胶原酶IV </td><td> Sigma-Aldrich公司</td><td> C5138 </td><td></td></tr><tr><td> DNA酶I </td><td> Applichem有限公司</td><td> A3778.0050 </td><td></td></tr><tr><td>无钙，镁的D-PBS </td><td> PAA实验室公司</td><td> H15-002 </td><td></td></tR&gt; <tr><td>乙二胺四乙酸二钠二水（EDTA）的</td><td> Sigma-Aldrich公司</td><td> E-7889 </td><td></td></tr><tr><td> FBS高级</td><td> Biochrom </td><td> S0615 </td><td></td></tr><tr><td>间接CD133微珠试剂盒人</td><td>美天旎生物技术有限公司</td><td> 130-091-895 </td><td>包括：CD133 / 1（AC133）-生物素，抗生物素微球和Fc受体阻断剂</td></tr><tr><td>青霉素 - 链霉素（100倍），液体</td><td> Invitrogen公司有限公司</td><td> 15140-122 </td><td></td></tr><tr><td>量子263 </td><td> PAA实验室公司</td><td> U15-815 </td><td></td></tr><tr><td>红血细胞裂解液（10倍） </td><td>美天旎生物技术有限公司</td><td> 130-094-183 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr> &lt;TD&gt;细胞过滤器（70微米） </td><td> BD Biosciences公司</td><td> 352350 </td><td></td></tr><tr><td> gentleMACSÇ管</td><td>美天旎生物技术有限公司</td><td> 130-093-237 </td><td></td></tr><tr><td> gentleMACS离解</td><td>美天旎生物技术有限公司</td><td> 130-093-235 </td><td></td></tr><tr><td> MACS分离多架</td><td>美天旎生物技术有限公司</td><td> 130-042-303 </td><td></td></tr><tr><td> MACS分选列25 LS的列， </td><td>美天旎生物技术有限公司</td><td> 130-042-401 </td><td></td></tr><tr><td> MACSmix管肩</td><td>美天旎生物技术有限公司</td><td> 130-090-753 </td><td></td></tr><tr><td> Natriumchlo​​ride </td><td>默克</td><td> 567440-1KG </td><td></td></tr><tr><td>预分离过滤器</td><td>美天旎生物技术有限公司</td><td> 130-041-407 </td><td></TD&gt; </tr><tr><td> QuadroMACS分离</td><td>美天旎生物技术有限公司</td><td> 130-090-976 </td><td></td></tr><tr><td> Rotilabo针头式过滤器（0.22微米，PES） </td><td>卡尔·罗斯GMBH＆CO KG </td><td> P668.1 </td><td></td></tr><tr><td> Steritop-GP过滤器单元500毫升</td><td>美天旎生物技术有限公司</td><td> SCGPS05RE </td><td></td></tr></tbody></table>

patient derived cell culture and isolation of cd133+ putative cancer stem cells from melanoma

尽管改进治疗方法的选择黑色素瘤的今天，晚期恶性黑色素瘤患者的无进展生存和总体生存的预后较差。因此，翻译研究的需要，以改善恶性黑色素瘤的靶向治疗提供进一步的分子生物学证据。在过去，致癌黑色素瘤有关的机制在已建立的细胞系中进行了广泛的研究。在个人基因图谱的基础上，以更加个性化的治疗方案，我们建议使用患者来源的细胞系，而不是普通的细胞株。再加上高品质的临床数据，尤其是在患者随访，这些细胞将有助于更好地理解背后的分子机制黑色素瘤的进展。 在这里，我们报告的原发性黑色素瘤的解剖新鲜肿瘤组织文化的建立。此过程包括切碎的组织分离成单个细胞，再moval与红细胞和成纤维细胞的原代培养细胞的黑色素瘤起源和可靠的核查以及污染。 最近的报告表明，多数肿瘤，黑色素瘤，如港口小亚群的肿瘤干细胞（CSCs），这似乎是专门推动肿瘤的发生和发展对转移状态。 CSC在黑色素瘤的识别和隔离的关键指标之一是CD133。隔离CD133 +干细胞从原发黑色素瘤的文化，我们修改和优化，磁激活细胞分选（MACS）从美天旎程序导致排序纯度高和活力的CD133 +干细胞，CD133 -散装，它可以培育和功能分析此后。

从肿瘤组织中的单细胞的制备 图2例示的晚期黑色素瘤患者切除的淋巴结转移前（A）和（B）机械分离后为2-4毫米。酶解后的组织并通过70微米尼龙网过滤，将细胞沉淀，并弃去上清。在这一步骤中，我们观察到高污染的红细胞的细胞沉淀，在图2C中所表示的颜色偏红。接着，我们耗尽的红血细胞，这导致在细胞沉淀的浅棕色（ 图2D）。这些?...

Watch this Scientific Journal Video about Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma at JoVE.com

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

本文介绍了准备的新鲜获得的原代细胞培养，以及如何去除污染的红细胞和成纤维细胞的肿瘤细胞的黑色素瘤组织到。最后，我们介绍了如何CD133<sup&gt; +</sup&gt;假定黑色素瘤干细胞的CD133排序<sup-</sup&gt;批量使用磁性细胞分选（MACS）。

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

patient-derived-cell-culture-isolation-cd133-putative-cancer-stem

Research

JoVE Journal

Medicine

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

21.1K 次观看.  Charité - Universitätsmedizin Berlin. 本文介绍了准备的新鲜获得的原代细胞培养，以及如何去除污染的红细胞和成纤维细胞的肿瘤细胞的黑色素瘤组织到。最后，我们介绍了如何CD133 +假定黑色素瘤干细胞的CD133排序

视频: Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

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 ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...