这项工作一直由奥地利研究基金会（FWF，授予N211南到DS）和奥地利研究促进署（FFG，授予N200到DS）的支持。作者感谢优秀的技术援助和莫妮卡法雷尔语言编辑克劳迪亚网址。

1。起始原料富含血小板血浆（PRP）由cytapheresis准备，或从Buffy大衣派生单位开始。 2。不育检查对于不育检查从每个PRP单位连接的小袋子（巴克斯特）传输量20毫升的样本。断开焊接袋。 3。覆检单位的冻结配制后，立即冻结覆检单位至少-20 ° C的原始存储袋，没有进一步的处理。 4。解冻的HPL单位当细菌测试呈阴性反应，解冻人体血小板裂解单位，现​​在要求在37℃（水浴），直到冰凝块消失的HPL单位。不暖的防火板。 5。汇集的HPL单位以至少十到十五的HPL单位之一血小板裂解液池解冻，准备标准化的产品。连续连接HPL袋池双袋（MacoPharma），并转移到这两种袋的防火板。断开焊接空的HPL袋。获取一个汇集了人类的血小板裂解液混合两袋的内容（pHPL）3至4大号的最终体积。一个小袋子（巴克斯特）连接，并采取了20毫升，汇集产品的无菌检查pHPL的样本。断开焊接袋。 6。饭的的pHPL 部分的pHPL得到适合作进一步处理等分。小包装袋（百特）连接池双包（Macopharma）和250毫升的小储物袋（百特）转让的卷。断开焊接填充袋。 7。重新冻结pHPL等分增加血小板破碎率和释放的生长因子，进一步冻结/解冻步骤。再次冻结分份pHPL的小袋子，至少-20 ° C。 8。再解冻pHPL等分饭解冻pHPL袋在37℃（水浴）。切割管使用无菌剪刀袋，倒入小瓶的pHPL的内容转移到50 mL样品瓶（猎鹰屋宇署）。执行此步骤，在层流台，以避免细菌或霉菌污染。 9。去除血小板片段由于血小板片段往往聚集，并可能诱发同种免疫，从pHPL删除。因此在4000克（15分钟，4℃）离心pHPL小瓶。在层流台移液器上清液进入决赛的存储小瓶（猎鹰屋宇署）的血浆及丢弃血小板颗粒，以避免在细胞培养中的片段。 10。存储的pHPL 再次冻结50毫升pHPL小瓶分装，在至少20℃，存放实验用。 11。在细胞培养中使用pHPL 最初添加防腐剂无肝素介质，以避免形成凝胶。在37 ° C解冻一个pHPL等份，并补充培养基除了8 - 10％。 介质 MSC介质： 使用500毫升的A - MEM，添加56毫升解冻pHPL（亦见进一步的细节参考1）和2 IU /毫升（= 224μL原液）（避免通过介质凝固结块的防腐剂，无肝素在血浆中的纤维蛋白原），达到了10％pHPL的终浓度。此外，添加青霉素（100U/mL）/链霉素（100μg/mL）解决方案的L - Glutamin 2MM（均为Sigma公司）。 过滤介质，通过一个20微米孔径真空过滤机（Millipore公司）。瓶适当的标签（内容，日期）。 ECFC，中等： 使用EBM的瓶（500毫升），添加细胞因子等分pHPL，10国际单位/毫升（原液1120μl），56毫升的不含防腐剂的肝素，青霉素（100U/mL）/链霉素（100克/毫升）溶液和培养基和20μL孔径真空过滤机（微孔）过滤器的L - 2MM Glutamin。瓶适当的标签（内容，日期）。 <img src="/files/ftp_upload/1523/1523fig1.jpg" alt="图1" /> 图1：从整个献血从局部血液银行或授权任何其他供应商的富血小板血浆的制备。离心后，血液可分为血浆，捉鬼外套和红细胞。四捉鬼外套单位，血型O和一个血AB型血浆可汇集前离心分离富含血小板血浆。一个定期的质量约300毫升的富含血小板血浆的单位，应包含1 × 10 9血小板每毫升或3X10 11 plateleTS总额。 <img src="/files/ftp_upload/1523/1523fig2.jpg" alt="图2" /><img src="/files/ftp_upload/1523/1523fig3.jpg" alt="图3" />

<ol>
	<li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front Biosci. 13, 3532-3548 (2008).</li><li>Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cytokines+in+wound+healing.">Growth factors and cytokines in wound healing.</a> Wound Repair Regen. 16, 585-601 (2008).</li><li>Giacco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin-activated+platelets+induce+proliferation+of+human+skin+fibroblasts+by+stimulating+autocrine+production+of+insulin-like+growth+factor-1.">Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1.</a> FASEB J. 20, 2402-2404 (2006).</li><li>Kocaoemer, A., Kern, S., Kluter, H., Bieback, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+AB+serum+and+thrombin-activated+platelet-rich+plasma+are+suitable+alternatives+to+fetal+calf+serum+for+the+expansion+of+mesenchymal+stem+cells+from+adipose+tissue.">Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.</a> Stem Cells. 25, 1270-1278 (2007).</li><li>Kakudo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation-promoting+effect+of+platelet-rich+plasma+on+human+adipose-derived+stem+cells+and+human+dermal+fibroblasts.">Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.</a> Plast Reconstr Surg. 122, 1352-1360 (2008).</li><li>Doucet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysates+promote+mesenchymal+stem+cell+expansion:+A+safety+substitute+for+animal+serum+in+cell-based+therapy+applications.">Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications.</a> J Cell Physiol. 205, 228-236 (2005).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum+Tissue+Eng+Part.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum Tissue Eng Part.</a> C Methods. 14, 185-196 (2008).</li><li>Lange, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+and+safe+expansion+of+human+mesenchymal+stromal+cells+in+animal+serum-free+medium+for+transplantation+and+regenerative+medicine.">Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.</a> J Cell Physiol. 213, 18-26 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Bernardo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+in+vitro+expansion+of+human+multipotent+mesenchymal+stromal+cells+for+cell-therapy+approaches:+further+insights+in+the+search+for+a+fetal+calf+serum+substitute.">Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.</a> J Cell Physiol. 211, 121-130 (2007).</li><li>Carrancio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+mesenchymal+stem+cell+expansion+procedures+by+cell+separation+and+culture+conditions+modification.">Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.</a> Exp Hematol. 36, 1014-1021 (2008).</li><li>Muller, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysate+as+a+serum+substitute+for+2D+static+and+3D+perfusion+culture+of+stromal+vascular+fraction+cells+from+human+adipose+tissue.">Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue.</a> Tissue Eng Part A. 15, 869-875 (2009).</li><li>Anitua, E., Sanchez, M., Orive, G., Andia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+impact+of+the+preparation+rich+in+growth+factors+(PRGF)+in+different+medical+fields.">The potential impact of the preparation rich in growth factors (PRGF) in different medical fields.</a> Biomaterials. 28, 4551-4560 (2007).</li><li>Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+and+novel+applications+for+platelet-rich+fibrin+therapies.">New insights into and novel applications for platelet-rich fibrin therapies.</a> Trends Biotechnol. 24, 227-234 (2006).</li><li>Martinez-Zapata, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+the+use+of+autologous+plasma+rich+in+platelets+for+tissue+regeneration:+a+systematic+review.">Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review.</a> Transfusion. 49, 44-56 (2009).</li><li>Weibrich, G., Kleis, W. K., Hafner, G., Hitzler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+levels+in+platelet-rich+plasma+and+correlations+with+donor+age,+sex,+and+platelet+count.">Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.</a> J Craniomaxillofac Surg. 30, 97-102 (2002).</li><li>Frechette, J. P., Martineau, I. &. a. m. p. ;. a. m. p., Gagnon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasmas:+growth+factor+content+and+roles+in+wound+healing.">Platelet-rich plasmas: growth factor content and roles in wound healing.</a> J Dent Res. 84, 434-439 (2005).</li><li>Seghatchian, J., Krailadsiri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+platelet+storage+lesion.">The platelet storage lesion.</a> Transfus Med Rev. 11, 130-144 (1997).</li><li>Selvaggi, T. A., Walker, R. E., Fleisher, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+antibodies+to+fetal+calf+serum+with+arthus-like+reactions+in+human+immunodeficiency+virus-infected+patients+given+syngeneic+lymphocyte+infusions.">Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions.</a> Blood. 89, 776-779 (1997).</li><li>Tonti, G. A., Mannello, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+bone+marrow+to+therapeutic+applications:+different+behaviour+and+genetic/epigenetic+stability+during+mesenchymal+stem+cell+expansion+in+autologous+and+foetal+bovine+sera.">From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera.</a> Int J Dev Biol. 52, 1023-1032 (2008).</li><li>Bieback, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Alternatives+to+Fetal+Bovine+Serum+for+the+Expansion+of+Mesenchymal+Stromal+Cells+from+Bone+Marrow.">Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow.</a> Stem Cells. , (2009).</li><li>Rohde, E., Schallmoser, K., Bartmann, C., Reinisch, A., Gad, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-Compliant+Propagation+of+Human+Multipotent+Mesenchymal+Stromal+Cells.">GMP-Compliant Propagation of Human Multipotent Mesenchymal Stromal Cells.</a> Pharmaceutical Manufacturing Handbook: Regulations and Quality. , 97-115 (2008).</li></ol>

在一些地区富含血小板血浆（PRP），棕黄色的大衣，否则被浪费的包装红细胞测试献血（图1）电池生产的产品可从。理想情况下，覆检委员会是立即使用做进一步的准备pHPL，集中在过时的血小板生长因子的可用性可能会减少由于血小板保存损伤和退化 20 。进一步建议匹配的O型血AB型血血浆的血小板，以避免可能影响ABH抗原和isoagglutinins生产的覆检。截至到现在，主要用于细胞培养胎牛血...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Double bag (2 x 3.5L)</td>
<td>&nbsp;</td>
<td>MacoPharma</td>
<td>VDL 8000XQ</td>
<td>originally for ascites puncture</td>
</tr>
<tr>
<td>50 mL Falcon tube</td>
<td>&nbsp;</td>
<td>Falcon BD</td>
<td>2098</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Stripettes</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>4501</td>
<td>&nbsp;</td>
<tr>
<td>Plasma bags (600mL)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>R4R2021</td>
<td>&nbsp;</td>
<tr>
<td>Sterile tubing welder</td>
<td>&nbsp;</td>
<td>Terumo</td>
<td>TSCD-II</td>
<td>&nbsp;</td>
<tr>
<td>Welding equipment </td>
<td>&nbsp;</td>
<td>Fresenius Kabi</td>
<td>Compo Seal</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of pooled human platelet lysate (phpl) as an efficient supplement for animal serum-free human stem cell cultures

血小板衍生生长因子已被证明有效刺激细胞增殖<em&gt;在体内</em<sup&gt; 1,2</sup&gt;<em&gt;在体外</em&gt;。凝血酶激活的血小板，这种效应已报告的间质基质细胞（MSCs），成纤维细胞和内皮细胞集落形成细胞<sup&gt; 3-5</sup&gt;或裂解冻结/解冻周期<sup&gt; 6-14</sup血小板releasate&gt;前加入细胞培养液中。血小板衍生生长因子的营养作用已在多次试验测试，组织工程和再生治疗。<sup&gt; 1,15-17</sup认为至少部分是由于个别发散生长因子浓度&gt;变效率<sup&gt; 18,19</sup&gt;和目前缺乏的血小板制剂的标准化协议。<sup&gt; 15,16</sup该协议提出了一个可行的步骤来生成一个从常规生产的富含血小板血浆（PRP）的四五十单献血的人血小板裂解液池（pHPL）。几个冻结/解冻循环血小板膜被破坏和生长因子，有效地释放入血浆。最后，血小板通过离心，以避免大量聚合的形成和消耗潜在抗原片段被删除。 pHPL成标准的文化协议执行代表一个有前途的工具，在动物蛋白系统传播的细胞疗法的进一步发展。

Watch this Scientific Journal Video about Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures at JoVE.com

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

人类血小板裂解物源丰富的生长因子和细胞培养的有力补充。该协议提出，准备从富含血小板血浆，执行几个冻融循环和消耗血小板片段大池的人血小板裂解过程。

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

preparation-pooled-human-platelet-lysate-phpl-as-an-efficient

Research

JoVE Journal

Biology

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

25.1K 次观看.  Medical University of Graz, Austria. 人类血小板裂解物源丰富的生长因子和细胞培养的有力补充。该协议提出，准备从富含血小板血浆，执行几个冻融循环和消耗血小板片段大池的人血小板裂解过程。

视频: Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

科研

生物学

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

制备海兔感觉运动神经元细胞培养

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...