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>
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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...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<td>Water bath</td>
<td>&nbsp;</td>
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<td>Sterile scissors</td>
<td>&nbsp;</td>
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<td>Clamps</td>
<td>&nbsp;</td>
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<td>Centrifuge</td>
<td>&nbsp;</td>
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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。

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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 ...

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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。