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
Summary
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。
Abstract
血小板衍生生长因子已被证明有效刺激细胞增殖<em>在体内</em<sup> 1,2</sup><em>在体外</em>。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> 3-5</sup>或裂解冻结/解冻周期<sup> 6-14</sup血小板releasate>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> 1,15-17</sup认为至少部分是由于个别发散生长因子浓度>变效率<sup> 18,19</sup>和目前缺乏的血小板制剂的标准化协议。<sup> 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。
Protocol
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。 <p class="jove_t…
Discussion
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…
Acknowledgements
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。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Double bag (2 x 3.5L) | MacoPharma | VDL 8000XQ | originally for ascites puncture | |
| 50 mL Falcon tube | Falcon BD | 2098 | ||
| 50 mL Stripettes | Costar | 4501 | ||
| Plasma bags (600mL) | Baxter | R4R2021 | ||
| Sterile tubing welder | Terumo | TSCD-II | ||
| Welding equipment | Fresenius Kabi | Compo Seal | ||
| Water bath | ||||
| Sterile scissors | ||||
| Clamps | ||||
| Centrifuge |
References
- Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. Platelets and wound healing. Front Biosci. 13, 3532-3548 (2008).
- Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. Growth factors and cytokines in wound healing. Wound Repair Regen. 16, 585-601 (2008).
- Giacco, F. Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1. FASEB J. 20, 2402-2404 (2006).
- Kocaoemer, A., Kern, S., Kluter, H., Bieback, K. 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. Stem Cells. 25, 1270-1278 (2007).
- Kakudo, N. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 122, 1352-1360 (2008).
- Doucet, C. Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications. J Cell Physiol. 205, 228-236 (2005).
- Schallmoser, K. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion. 47, 1436-1446 (2007).
- Reinisch, A. Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application. Regen Med. 2, 371-382 (2007).
- Schallmoser, K. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum Tissue Eng Part. C Methods. 14, 185-196 (2008).
- Lange, C. Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol. 213, 18-26 (2007).
- Reinisch, A. Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood. 113, 6716-6725 (2009).
- Bernardo, M. E. 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. J Cell Physiol. 211, 121-130 (2007).
- Carrancio, S. Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Exp Hematol. 36, 1014-1021 (2008).
- Muller, A. M. Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue. Tissue Eng Part A. 15, 869-875 (2009).
- Anitua, E., Sanchez, M., Orive, G., Andia, I. The potential impact of the preparation rich in growth factors (PRGF) in different medical fields. Biomaterials. 28, 4551-4560 (2007).
- Anitua, E. New insights into and novel applications for platelet-rich fibrin therapies. Trends Biotechnol. 24, 227-234 (2006).
- Martinez-Zapata, M. J. Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review. Transfusion. 49, 44-56 (2009).
- Weibrich, G., Kleis, W. K., Hafner, G., Hitzler, W. E. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg. 30, 97-102 (2002).
- Frechette, J. P., Martineau, I. &. a. m. p. ;. a. m. p., Gagnon, G. Platelet-rich plasmas: growth factor content and roles in wound healing. J Dent Res. 84, 434-439 (2005).
- Seghatchian, J., Krailadsiri, P. The platelet storage lesion. Transfus Med Rev. 11, 130-144 (1997).
- Selvaggi, T. A., Walker, R. E., Fleisher, T. A. Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood. 89, 776-779 (1997).
- Tonti, G. A., Mannello, F. From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera. Int J Dev Biol. 52, 1023-1032 (2008).
- Bieback, K. Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow. Stem Cells. , (2009).
- Rohde, E., Schallmoser, K., Bartmann, C., Reinisch, A., Gad, S. C. GMP-Compliant Propagation of Human Multipotent Mesenchymal Stromal Cells. Pharmaceutical Manufacturing Handbook: Regulations and Quality. , 97-115 (2008).
Tags
Pooled Human Platelet LysatePHPLAnimal Serum-freeStem Cell CulturesPlatelet Derived Growth FactorsCell ProliferationMesenchymal Stromal CellsFibroblastsEndothelial Colony-forming CellsThrombin ActivationFreeze/thaw CyclesPlatelet ReleasateTrophic EffectRegenerative TherapyGrowth Factor ConcentrationsStandardized ProtocolsPlatelet PreparationHuman Platelet LysatePlatelet Rich PlasmaFreeze/thaw CyclesCentrifugationAggregate FormationPotential AntigensCulture ProtocolsCell Therapeutics
Cite This Article
Schallmoser, K., Strunk, D. Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures. J. Vis. Exp. (32), e1523, doi:10.3791/1523 (2009).