Name: leukodepletion filters-derived cd34+ cells as a cell source to study megakaryocyte differentiation and platelet formation
Uploaded: 2021-05-20T14:00:00
Description: Watch this Scientific Journal Video about Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation at JoVE.com

这项工作得到了ANR（国家研究机构）Grant ANR-17-CE14-0001-1的支持。

对照组的人体样本是从自愿献血者那里获得的，他们给予了由进行研究的输血中心（Etablissement Français du Sang-Grand Est）招募的书面知情同意书。所有程序均由法国高等教育和研究部注册和批准，并以AC_2015_2371号注册，捐赠者在CODHECO编号AC-2008 - 562同意书中给予批准，以便将样品用于研究目的。根据赫尔辛基宣言进行了人体研究。
1. 从LRF中提取和选择CD34 +细胞（HP）
<ol><li>
<stro...

<ol>
	<li>Deutsch, V. R., Tomer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+development+and+platelet+production.">Megakaryocyte development and platelet production.</a> British Journal of Haematology. 134 (5), 453-466 (2006).</li><li>Lefrancais, 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=The+lung+is+a+site+of+platelet+biogenesis+and+a+reservoir+for+haematopoietic+progenitors.">The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.</a> Nature. 544 (7648), 105-109 (2017).</li><li>de Sauvage, F. 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=Stimulation+of+megakaryocytopoiesis+and+thrombopoiesis+by+the+c-Mpl+ligand.">Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.</a> Nature. 369 (6481), 533-538 (1994).</li><li>Almomani, M. H., Mangla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2020).</li><li>Strassel, C., Hechler, B., Bull, A., Gachet, C., Lanza, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+mice+lacking+the+GPIb-V-IX+complex+question+the+role+of+this+receptor+in+atherosclerosis.">Studies of mice lacking the GPIb-V-IX complex question the role of this receptor in atherosclerosis.</a> Journal of Thrombosis and Haemostasis. 7 (11), 1935-1938 (2009).</li><li>Delalat, 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=Isolation+and+ex+vivo+expansion+of+human+umbilical+cord+blood-derived+CD34++stem+cells+and+their+cotransplantation+with+or+without+mesenchymal+stem+cells.">Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells.</a> Hematology. 14 (3), 125-132 (2009).</li><li>Yin, T., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem+cell+niches+in+bone.">The stem cell niches in bone.</a> The Journal of Clinical Investigation. 116 (5), 1195-1201 (2006).</li><li>Salunkhe, V., Papadopoulos, P., Guti&#233;rrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+megakaryocytes+from+human+peripheral+blood+mononuclear+cells.">Culture of megakaryocytes from human peripheral blood mononuclear cells.</a> Bio-protocol. 5 (21), 1639 (2015).</li><li>Peytour, Y., Villacreces, A., Chevaleyre, J., Ivanovic, Z., Praloran, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discarded+leukoreduction+filters:+a+new+source+of+stem+cells+for+research,+cell+engineering+and+therapy.">Discarded leukoreduction filters: a new source of stem cells for research, cell engineering and therapy.</a> Stem Cell Research. 11 (2), 736-742 (2013).</li><li>Lapostolle, 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=Repopulating+hematopoietic+stem+cells+from+steady-state+blood+before+and+after+ex+vivo+culture+are+enriched+in+the+CD34(+)CD133(+)CXCR4(low)+fraction.">Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34(+)CD133(+)CXCR4(low) fraction.</a> Haematologica. 103 (10), 1604-1615 (2018).</li><li>Ivanovic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-blood+leuko-depletion+filters+as+a+source+of+CD+34++progenitors+potentially+usable+in+cell+therapy.">Whole-blood leuko-depletion filters as a source of CD 34+ progenitors potentially usable in cell therapy.</a> Transfusion. 46 (1), 118-125 (2006).</li><li>Strassel, 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=Aryl+hydrocarbon+receptor-dependent+enrichment+of+a+megakaryocytic+precursor+with+a+high+potential+to+produce+proplatelets.">Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets.</a> Blood. 127 (18), 2231-2240 (2016).</li><li>Do Sacramento, 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=Functional+properties+of+human+platelets+derived+in+vitro+from+CD34(+)+cells.">Functional properties of human platelets derived in vitro from CD34(+) cells.</a> Scientific Reports. 10 (1), 914 (2020).</li><li>Blin, 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=Microfluidic+model+of+the+platelet-generating+organ:+beyond+bone+marrow+biomimetics.">Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.</a> Scientific Reports. 6, 21700 (2016).</li><li>Ito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbulence+activates+platelet+biogenesis+to+enable+clinical+scale+ex+vivo+production.">Turbulence activates platelet biogenesis to enable clinical scale ex vivo production.</a> Cell. 174 (3), 636-648 (2018).</li><li>Pallotta, I., Lovett, M., Kaplan, D. L., Balduini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+system+for+the+in+vitro+study+of+megakaryocytes+and+functional+platelet+production+using+silk-based+vascular+tubes.">Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes.</a> Tissue Engineering. Part C, Methods. 17 (12), 1223-1232 (2011).</li></ol>

该协议描述了一种生产MK的方法，该MK能够从血液衍生的HP中释放丙酸盐并从培养基中释放血小板。HP从LRF（血库的副产品）中获得，用于从细胞血液制品中去除污染的白细胞并避免不良反应。虽然这种方法相对简单，但有几点值得特别注意。
必须轻柔地在密度梯度培养基上沉积细胞悬浮液（步骤1.3.1）以避免混合（红色含量）。如果未仔细执行此步骤，则协议应在此点停止。?...

血小板来自专门的大多倍体细胞，即巨核细胞（MK），其起源于称为巨核细胞（MKP）的恒定和微调的生产过程。该过程的顶点是造血干细胞，它们与骨髓环境（细胞因子，转录因子，造血龛）接触，将能够增殖并分化成能够致力于巨核细胞通路的造血祖细胞（HP），从而产生未成熟的MKs1。在各种细胞因子的影响下，特别是血小板生成素（TPO），这是MKP的主要细胞因子;然后，MK将经历两个主要的成熟阶段：内膜分裂和分界膜（DMS）的发展。然后，这种完全成熟的MK出现在正弦血管附近，在该血管中它可以发射细胞质延伸，即丙酸盐，其将在血流下释放并随后重塑为功能性血小板2。1994年TPO的克隆3 通过加速体 外 培养技术的发展，促进了HP分化和MK成熟的MKP研究。
有许多影响血小板的病理，无论是在血小板数量（增加或减少）和功能方面4，5。能够从人类HP体外概括MKP可以提高对这一过程背后的分子和细胞机制的理解，并最终改善患者的治疗管理。
各种来源的人HP是合适的：脐带血，骨髓和外周血6，7，8。与从脐带血或骨髓中恢复相比，从外周血中采集HP引起的后勤和伦理问题更少。HP可以从白细胞分离术或黄褐色外套中恢复，但这些来源很昂贵，并且在血液中心并不总是可用的。其他方案，更便宜，更容易执行，允许直接回收人外周血单核细胞（PBMC），而无需事先CD34驱动的分离4，8。然而，这种方法对巨核细胞的纯度并不令人满意，建议从PBMC中选择CD34 +细胞以最佳分化为MK。这导致我们从白细胞还原过滤器（LRF）中实施了HP纯化，该过滤器通常用于血库中以去除白细胞，从而避免不良免疫反应9。事实上，自1998年以来，法国的血小板浓缩物已被自动白细胞化。在此过程结束时，LRF被丢弃，所有保留在LRF中的细胞都被破坏。因此，LRF中的细胞很容易获得，无需额外费用。LRF具有接近白细胞分离术或黄褐色外套获得的细胞含量，特别是其CD34 + HP的组成使它们成为非常有吸引力的来源10。LRF作为人类HP来源已被证明可以为细胞提供完整的功能容量11。这种来源的优点是丰富且价格合理，可用于实验室研究。在此背景下，本文依次描述了：i）从LRF中提取和选择CD34+ HP;ii）两阶段优化培养，其概括了HP对巨核细胞途径的承诺以及能够释放丙酸盐的MK的成熟;iii）从这些MK中有效释放血小板的方法;和iv）表型MK和培养血小板的程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >7-AAD</td>
			<td >Biolegend</td>
			<td >558819</td>
			<td ></td>
		</tr>
		<tr >
			<td >ACD</td>
			<td>EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PE&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-081-002</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PECy7</td>
			<td >eBioscience</td>
			<td >25-0349-42</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD41-Alexa Fluor 488</td>
			<td >Biolegend</td>
			<td >303724</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD42a-PE</td>
			<td >BD Bioscience</td>
			<td >559919</td>
			<td></td>
		</tr>
		<tr >
			<td >Apyrase</td>
			<td >EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Trucount Tubes</td>
			<td >BD Bioscience</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr >
			<td >CD34 MicroBead Kit UltraPure, human&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-100-453</td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td>Heraeus</td>
			<td>Megafuge 1.OR</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Compteur ADAM&#160;</td>
			<td>DiagitalBio</td>
			<td >NA</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Cryotubes</td>
			<td>Dutscher</td>
			<td>55002</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Dextran from leuconostoc spp&#160;</td>
			<td>Sigma</td>
			<td>31392-50g</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >DMSO Hybri-max&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA 0.5 M&#160;</td>
			<td>Gibco</td>
			<td>15575-039</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf 1,5 mL&#160;</td>
			<td>Dutscher</td>
			<td>616201</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Filtration unit Steriflip PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow Cytometer</td>
			<td >BD Bioscience</td>
			<td >Fortessa</td>
			<td></td>
		</tr>
		<tr >
			<td >Human LDL</td>
			<td>Stemcell technologies</td>
			<td>#02698</td>
			<td></td>
		</tr>
		<tr >
			<td >ILOMEDINE 0,1 mg/1 mL</td>
			<td >Bayer</td>
			<td>MA038EX</td>
			<td></td>
		</tr>
		<tr >
			<td >Inserts</td>
			<td>Fenwal</td>
			<td>R4R1401</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Laminar flow hood&#160;</td>
			<td>Holten</td>
			<td >NA</td>
			<td>Archived product</td>
		</tr>
		<tr >
			<td >LS Columms&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td>Stemcell</td>
			<td>7861</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen Strep Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS (-)</td>
			<td>Life Technologies</td>
			<td>14190-169&#160;</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >PGi2</td>
			<td >Sigma</td>
			<td >P6188</td>
			<td></td>
		</tr>
		<tr >
			<td >Poches de transferts 600ml&#160;</td>
			<td>Macopharma</td>
			<td>VSE4001XA</td>
			<td></td>
		</tr>
		<tr >
			<td >Pre-Separation Filters (30&#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >StemRegenin 1 (SR1)</td>
			<td>Stemcell technologies</td>
			<td>#72344</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan Expansion Supplement (100x)</td>
			<td>Stemcell technologies</td>
			<td>#02696</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan-SFEM&#160;</td>
			<td>Stemcell technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr >
			<td >Stericup Durapore 0,22&#181;m PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SCGVU05RE</td>
			<td></td>
		</tr>
		<tr >
			<td >SVF Hyclone&#160;</td>
			<td>Thermos scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringues 30 mL&#160;</td>
			<td>Terumo</td>
			<td>SS*30ESE1</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Syringe filters Millex 0,22&#181;M PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr >
			<td >TPO</td>
			<td>Stemcell technologies</td>
			<td>#02822</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubes 50 mL</td>
			<td>Sarstedt</td>
			<td>62.548.004 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubes 15 mL&#160;</td>
			<td>Sarstedt</td>
			<td>62.554.001 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubulures</td>
			<td>B Braun</td>
			<td>4055137</td>
			<td>Or equivalent material</td>
		</tr>
	</tbody>
</table>

leukodepletion filters-derived cd34+ cells as a cell source to study megakaryocyte differentiation and platelet formation

人造血祖细胞 的体外 扩增和分化为能够伸长丙酸盐和释放血小板的巨核细胞，可以深入研究血小板生物发生的机制。现有的培养方案主要基于来自骨髓或脐带血的造血祖细胞，这引发了许多伦理、技术和经济问题。如果已经有可用的方案可以从外周血中获取CD34细胞，那么本手稿提出了一种简单而优化的方案，用于从血液中心现成的白细胞消除过滤器中获取CD34 +细胞。这些细胞是从白细胞破灭过滤器中分离出来的，用于制备输血产品，相当于八次献血。这些过滤器应该被丢弃。描述了从这些过滤器中收集鉴定为CD34 +细胞的造血祖细胞的详细程序。还详细介绍了在讨论其表型进化的同时获得延伸丙酸盐的成熟巨核细胞的方法。最后，该协议提出了一种经过校准的移液方法，以有效地释放在形态学和功能上与天然血小板相似的血小板。该协议可以作为评估在过程的各个步骤中起作用的药理学化合物的基础，以剖析潜在的机制并接近 体内 血小板产量。

从LRF中提取和选择CD34 +细胞 在这里，该方法源自Peytour等人9，描述了从白细胞去除后血库中可用的废弃LRF中提取和选择CD34+细胞。在反冲洗程序之后，通常恢复1.03 x 10 9±2.45 x10 8个细胞/ LRF（平均值±SEM; n = 155），存活率为94.88±0.10%（图2A i）。在CD34阳性选择后，获得平均615.54×10 3±12.28个细胞/LRF（n = 155）?...

Watch this Scientific Journal Video about Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation at JoVE.com

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

该协议详细描述了获得白细胞衍生的CD34 +造血祖细胞及其 体外 分化和成熟为能够释放培养基中血小板的含丙酸盐的巨核细胞所涉及的所有步骤。该程序可用于深入分析控制巨核细胞生成的细胞和分子机制。

白细胞消除过滤器衍生的CD34 +细胞作为研究巨核细胞分化和血小板形成的细胞来源

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

该协议提出了一种获得人类造血祖细胞并诱导其分化为巨核细胞的简单方法。它可以作为更好地了解巨核细胞生成的基础 该技术使用来源，但为造血的实验室研究提供了负担得起和丰富的优势。这种方法用于更好地了解巨核生成，用于治疗的细胞需要在GMP条件下制备，目前情况并非如此。了解包括PBMC收集在内的步骤只能通过视觉演示才能完全完成。在开始实验之前，将白质还原过滤器连接到空的600毫升转运袋和管路组。在生物安全柜中，将每个白质还原过滤器将25毫升过滤的洗脱缓冲液注入空袋中，并使用30毫升注射器轻轻地通过过滤器吸入袋内物品。将收集的红细胞转移到新的50毫升管中，并用2%葡聚糖将细胞悬浮液稀释一半。充分混合以聚集血细胞，并使细胞在室温下沉淀30分钟。当红细胞沉降时，将上清液转移到50毫升的管中，并用两毫摩尔PBS EDTA溶液填充管中。将一半的上清液轻轻覆盖到两个50毫升管中每个25毫升的密度梯度培养基上，而不会干扰梯度表面，并通过密度梯度离心分离细胞。使用一次性移液器将细胞从每个界面转移到新的50毫升管中。每次洗涤2毫升的50毫升PBS EDTA洗涤细胞两次。第二次洗涤后，将颗粒重悬于单个50毫升体积的新鲜PBS EDTA中以进行计数。在这里，可以通过在夜间将收集的细胞保持在四摄氏度的搅拌下来停止该过程。对于CD34 +细胞选择，确定细胞数量并通过离心收集细胞，并将细胞沉淀重悬于每10个细胞2毫摩尔PBS EDTA的300微升至第八个细胞中。向第八个细胞加入适当体积的FC受体阻断试剂和每10个细胞50微升CD34微珠。在4摄氏度下30分钟后，用两毫摩尔PBS EDTA洗涤细胞，并将沉淀重悬于500微升新鲜2毫摩尔PBS EDTA中，每10个细胞至8个细胞。用PBS EDTA加湿并适当尺寸的磁柱，然后将样品添加到色谱柱中。每次洗涤用三毫升两毫摩尔PBS EDTA洗涤色谱柱两次。在逃避CD34 +细胞之前，每次洗脱用两个五毫升体积的两毫摩尔PBS EDTA。为了评估磁珠所选细胞的纯度，将两微升适当的人抗CD34抗体加入100微升等分试样中分离的细胞中，并在4摄氏度下孵育15分钟之前充分混合。孵育后，在PBS中洗涤细胞并将沉淀重悬于200微升PBS中，以通过流式细胞术分析细胞样品的纯度。计数后，通过离心收集CD34 +细胞，并立即将沉淀重悬于冷溶液一至密度为10至每毫升6个细胞，然后快速将细胞悬浮液加入冷溶液二中。然后立即将冷冻管置于零下80摄氏度的冰箱中24小时，然后将冷冻管转移到液氮储存中。在这项具有代表性的研究中，细胞通过白质还原过滤回收，其存活率约为95%。经磁珠选择后，获得纯度大于90%的CD34+细胞群。细胞增殖通常在培养一周后减少，但细胞活力没有显着变化。到第七天，CD34 +细胞应该开始表达CD41，CD41是一种特异性的早期标志物，或用于巨核细胞和血小板发育。到第10天，培养物中的大多数细胞通常发育成表达巨核细胞的成熟CD41。到第13天，巨核细胞pro血小板的延伸和血小板的释放可以通过光学显微镜观察。然后可以使用校准数量的荧光珠来定量释放到培养物中的CD41，CD42，8阳性血小板的总数。该方法为增强巨核生成的潜在机制和提高视觉中的血小板产量铺平了道路。即使这种方法看起来很乏味，也很简单，你只需要耐心等待并提前准备所有区域。虽然我们严重关注巨核生成，但我们获得的CD42细胞可用于造血途径。

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

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

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

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 ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

在3D甲基纤维素基水凝胶中培养巨核细胞，以改善细胞成熟并研究僵硬和禁闭的影响

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D ...