Name: production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion
Uploaded: 2021-02-24T12:30:00
Description: Watch this Scientific Journal Video about Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion at JoVE.com

我们感谢Grégory Schneiter先生在流式细胞术数据方面的技术援助;Muriel Cuendet教授（药学院和日内瓦大学生药学实验室）允许使用Atune流式细胞仪和Cytation 3高通量显微镜;Brigitte Pittet教授提供科学建议。

研究方案符合赫尔辛基宣言，所有患者在参与研究前都提供了书面知情同意书。皮肤样本来自在日内瓦大学医院（瑞士日内瓦）整形、重建和美容外科接受腹部整形术的健康女性。该程序符合《赫尔辛基宣言》的原则，并得到了当地机构伦理委员会的批准（协议#3126）。
1. 准备PRP
注意：CuteCell-PRP 管（材料表）设计用于在闭路系统中从少量患者...

<ol>
	<li>Kumar, S., Mahajan, B. B., Kaur, S., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+therapies+in+dermatology.">Autologous therapies in dermatology.</a> The Journal of Clinical and Aesthetic Dermatology. 7 (12), 38-45 (2014).</li><li>Martin, I., 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+survey+on+cellular+and+engineered+tissue+therapies+in+Europe+in+2009.">The survey on cellular and engineered tissue therapies in Europe in 2009.</a> Tissue Engineering. Part A. 17 (17-18), 2221-2230 (2011).</li><li>Stunova, A., Vistejnova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+fibroblasts-A+heterogeneous+population+with+regulatory+function+in+wound+healing.">Dermal fibroblasts-A heterogeneous population with regulatory function in wound healing.</a> Cytokine &amp; Growth Factor Reviews. 39, 137-150 (2018).</li><li>Thangapazham, R. L., Darling, T. N., Meyerle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+skin+properties+with+autologous+dermal+fibroblasts.">Alteration of skin properties with autologous dermal fibroblasts.</a> International Journal of Biological Sciences. 15 (5), 8407-8427 (2014).</li><li>Costa-Almeida, R., Soares, R., Granja, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+as+maestros+orchestrating+tissue+regeneration.">Fibroblasts as maestros orchestrating tissue regeneration.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (1), 240-251 (2018).</li><li>Weiss, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+cell+therapy:+will+it+replace+dermal+fillers.">Autologous cell therapy: will it replace dermal fillers.</a> Facial Plastic Surgery Clinics of North America. 21 (2), 299-304 (2013).</li><li>Ichim, T. E., O'Heeron, P., Kesari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+as+a+practical+alternative+to+mesenchymal+stem+cells.">Fibroblasts as a practical alternative to mesenchymal stem cells.</a> Journal of Translational Medicine. 16 (1), 212 (2018).</li><li>Gstraunthaler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+the+use+of+fetal+bovine+serum:+serum-free+cell+culture.">Alternatives to the use of fetal bovine serum: serum-free cell culture.</a> ALTEX. 20 (4), 275-281 (2003).</li><li>Karnieli, O., 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+consensus+introduction+to+serum+replacements+and+serum-free+media+for+cellular+therapies.">A consensus introduction to serum replacements and serum-free media for cellular therapies.</a> Cytotherapy. 19 (2), 155-169 (2017).</li><li>van der Valk, 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=Fetal+Bovine+Serum+(FBS):+Past+-+Present+-+Future.">Fetal Bovine Serum (FBS): Past - Present - Future.</a> ALTEX. 35 (1), 99-118 (2018).</li><li>Cavallo, 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=Platelet-Rich+Plasma:+The+Choice+of+Activation+Method+Affects+the+Release+of+Bioactive+Molecules.">Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules.</a> BioMed Research International. 2016, 6591717 (2016).</li><li>Peng, G. L. <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+Plasma+for+Skin+Rejuvenation:+Facts,+Fiction,+and+Pearls+for+Practice.">Platelet-Rich Plasma for Skin Rejuvenation: Facts, Fiction, and Pearls for Practice.</a> Facial Plastic Surgery Clinics of North America. 27 (3), 405-411 (2019).</li><li>Fadadu, P. P., Mazzola, A. J., Hunter, C. W., Davis, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+concentration+yields+in+commercially+available+platelet-rich+plasma+(PRP)+systems:+a+call+for+PRP+standardization.">Review of concentration yields in commercially available platelet-rich plasma (PRP) systems: a call for PRP standardization.</a> Regional Anesthesia and Pain Medicine. 44, 652-659 (2019).</li><li>Berndt, S., Turzi, A., Pittet-Cuenod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+Platelet-Rich+Plasma+(CuteCell+PRP)+Safely+Boosts+In+Vitro+Human+Fibroblast+Expansion.">Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion.</a> Tissue Engineering. Part A. 25 (21-22), 1550-1563 (2019).</li><li>Zeng, W., 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=Preclinical+safety+studies+on+autologous+cultured+human+skin+fibroblast+transplantation.">Preclinical safety studies on autologous cultured human skin fibroblast transplantation.</a> Cell Transplantation. 23 (1), 39-49 (2014).</li><li>Lee, E. C. 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=Efficacy+of+Autologous+Cultured+Fibroblast+Cells+as+a+Treatment+for+Patients+with+Facial+Contour+Defects:+A+Clinical+Replication+Study.">Efficacy of Autologous Cultured Fibroblast Cells as a Treatment for Patients with Facial Contour Defects: A Clinical Replication Study.</a> Journal of Cosmetics, Dermatological Sciences and Applications. 7, 306-317 (2017).</li><li>Eca, L. P., Pinto, D. G., de Pinho, A. M., Mazzetti, M. P., Odo, 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=Autologous+fibroblast+culture+in+the+repair+of+aging+skin.">Autologous fibroblast culture in the repair of aging skin.</a> Dermatologic Surgery. 38 (2), 180-184 (2012).</li><li>Nilforoushzadeh, M. 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=Autologous+fibroblast+suspension+for+the+treatment+of+refractory+diabetic+foot+ulcer.">Autologous fibroblast suspension for the treatment of refractory diabetic foot ulcer.</a> Indian Journal of Dermatology, Venereology and Leprology. 82 (1), 105-106 (2016).</li><li>Cowper, 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=Human+Platelet+Lysate+as+a+Functional+Substitute+for+Fetal+Bovine+Serum+in+the+Culture+of+Human+Adipose+Derived+Stromal/Stem+Cells.">Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells.</a> Cells. 8 (7), 724 (2019).</li><li>Atashi, F., Jaconi, M. E., Pittet-Cuenod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+platelet-rich+plasma:+a+biological+supplement+to+enhance+adipose-derived+mesenchymal+stem+cell+expansion.">Autologous platelet-rich plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expansion.</a> Tissue Engineering Part C: Methods. 21 (3), 253-262 (2015).</li><li>Martinez-Zapata, M. 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=Autologous+platelet-rich+plasma+for+treating+chronic+wounds.">Autologous platelet-rich plasma for treating chronic wounds.</a> The Cochrane Database of Systematic Reviews. (5), (2016).</li><li>Cervelli, 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=Use+of+platelet-rich+plasma+and+hyaluronic+acid+in+the+loss+of+substance+with+bone+exposure.">Use of platelet-rich plasma and hyaluronic acid in the loss of substance with bone exposure.</a> Advances in Skin &amp; Wound Care. 24 (4), 176-181 (2011).</li><li>Nicoli, F., 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=Severe+hidradenitis+suppurativa+treatment+using+platelet-rich+plasma+gel+and+Hyalomatrix.">Severe hidradenitis suppurativa treatment using platelet-rich plasma gel and Hyalomatrix.</a> International Wound Journal. 12 (3), 338-343 (2015).</li><li>Gentile, P., Bottini, D. J., Spallone, D., Curcio, B. C., Cervelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+platelet-rich+plasma+in+maxillofacial+surgery:+clinical+evaluation.">Application of platelet-rich plasma in maxillofacial surgery: clinical evaluation.</a> The Journal of Craniofacial Surgery. 21 (3), 900-904 (2010).</li><li>Saleem, 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=Adjunctive+Platelet-Rich+Plasma+(PRP)+in+Infrabony+Regenerative+Treatment:+A+Systematic+Review+and+RCT's+Meta-Analysis.">Adjunctive Platelet-Rich Plasma (PRP) in Infrabony Regenerative Treatment: A Systematic Review and RCT's Meta-Analysis.</a> Stem Cells International. 2018, 9594235 (2018).</li><li>Everts, P. A., Pinto, P. C., Girao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+pure+platelet-rich+plasma+injections+for+facial+skin+rejuvenation:+Biometric+instrumental+evaluations+and+patient-reported+outcomes+to+support+antiaging+effects.">Autologous pure platelet-rich plasma injections for facial skin rejuvenation: Biometric instrumental evaluations and patient-reported outcomes to support antiaging effects.</a> Journal of Cosmetic Dermatology. 18 (4), 985-995 (2019).</li><li>Fiaschetti, 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=Magnetic+resonance+imaging+and+ultrasound+evaluation+after+breast+autologous+fat+grafting+combined+with+platelet-rich+plasma.">Magnetic resonance imaging and ultrasound evaluation after breast autologous fat grafting combined with platelet-rich plasma.</a> Plastic and Reconstructive Surgery. 132 (4), 498-509 (2013).</li><li>Gentile, P., Scioli, M. G., Orlandi, A., Cervelli, 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+Reconstruction+with+Enhanced+Stromal+Vascular+Fraction+Fat+Grafting:+What+Is+the+Best+Method.">Breast Reconstruction with Enhanced Stromal Vascular Fraction Fat Grafting: What Is the Best Method.</a> Plastic and Reconstructive Surgery. 3 (6), 406 (2015).</li><li>Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platlet+Rich+Plasma+(PRP)+Improves+Fat+Grafting+Outcomes.">Platlet Rich Plasma (PRP) Improves Fat Grafting Outcomes.</a> World Journal of Plastic Surgery. 2 (1), 6-13 (2013).</li><li>Muraglia, 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=Culture+Medium+Supplements+Derived+from+Human+Platelet+and+Plasma:+Cell+Commitment+and+Proliferation+Support.">Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.</a> Frontiers in Bioengineering and Biotechnology. 5, 66 (2017).</li><li>Ferrao, A. V., Mason, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+heparin+on+cell+proliferation+and+type-I+collagen+synthesis+by+adult+human+dermal+fibroblasts.">The effect of heparin on cell proliferation and type-I collagen synthesis by adult human dermal fibroblasts.</a> Biochimica et Biophysica Acta. 1180 (3), 225-230 (1993).</li><li>Gonzalez-Delgado, P., Fernandez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypersensitivity+reactions+to+heparins.">Hypersensitivity reactions to heparins.</a> Current Opinion in Allergy and Clinical Immunology. 16 (4), 315-322 (2016).</li><li>Atashi, F. S. B., Nayernia, Z., Pittet-Cu&#233;nod, B., Modarressi, A. <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+Plasma+Promotes+Proliferation+of+Adipose+Derived+Mesenchymal+Stem+Cells+via+Activation+of+AKT+and+Smad2+Signaling+Pathways.">Platelet Rich Plasma Promotes Proliferation of Adipose Derived Mesenchymal Stem Cells via Activation of AKT and Smad2 Signaling Pathways.</a> Stem Cell Research &amp; Therapy. 5 (8), (2015).</li></ol>

与伤口细胞治疗中的其他填充材料相比，使用自体成纤维细胞作为天然替代品的优点包括良好的生物相容性、最小的副作用以及易于收获和使用。然而，在日常临床环境中使用这些疗法之前，有必要进行适当的临床前研究，以确定移植前后分离成纤维细胞的生长特征并评估其生物学功能和安全性。因此，在分离过程之后，必须快速进行细胞的体外扩增，以限制细胞分裂的次数。必须严格控制培养?...

再生医学旨在治愈或替换因年龄、疾病或创伤而受损的组织和器官，并纠正先天性缺陷。在自体治疗中，从患者身上取出细胞或组织，扩增或修饰，然后重新引入供体。这种形式的疗法在皮肤病学领域具有广泛的潜力1。在自体成纤维细胞治疗中，对患者的成纤维细胞进行培养并重新注射以治疗皱纹、鼻痘或痤疮疤痕。由于成纤维细胞是真皮中的主要功能细胞，因此注射自体成纤维细胞可能比其他疗法更有益于面部年轻化2。
在皮肤中，成纤维细胞负责细胞外蛋白（即胶原蛋白、弹性蛋白、透明质酸和糖胺聚糖）的合成和分泌。它们还释放生长因子，调节正常皮肤稳态和伤口愈合中的细胞功能、迁移和细胞-基质/细胞-细胞相互作用3。皮肤成纤维细胞已被引入为一种潜在的临床细胞疗法，用于皮肤伤口愈合4，组织再生5或美容和整形外科手术中的皮肤填充剂6。一些研究甚至表明，在再生医学的背景下，成纤维细胞可能比间充质干细胞更实用和有效的细胞疗法7。
为了获得足够数量的成纤维细胞用于临床应用，细胞扩增通常是强制性的。体外/体外细胞培养需要补充生长因子、蛋白质和酶的基础培养基来支持细胞粘附和增殖。胎牛血清（FBS）是细胞培养基的常用补充剂，因为胎儿血液1）与成人血液相比富含生长因子，2）抗体含量低8。随着细胞治疗的进展，人们担心将FBS添加到培养基中的经典细胞培养条件的安全性。此外，现在有一种用替代品9取代FBS的趋势。几种FBS替代品已显示出有希望的结果10。
这里选择了富血小板血浆（PRP）替代品，我们开发了一种医疗设备来生产PRP的标准化制剂，名为CuteCell-PRP。该装置的预期用途是制备自体PRP，用作GMP条件下自体细胞体外扩增的培养基补充剂。
PRP被定义为血浆中的浓缩血小板悬浮液。由于有许多制备方案，它们在1）所需的血液量，2）使用的装置类型和3）离心方案方面有所不同，因此所得血小板浓度从略高于血液基线值的10倍到超过10倍不等。此外，PRP制剂含有不同水平的红细胞和白细胞污染。因此，术语“PRP”用于描述其生物成分和潜在治疗效果差异很大的产品。
在大多数研究中，FBS替代是使用不同浓度的PRP激活（通过凝血酶或钙）实现的。这种人工激活引起血小板生长因子从15分钟到24小时11的立即和重要的释放。因此，据信血小板活化对于细胞培养中的应用是不希望的，其中需要从逐渐的血小板脱颗粒中缓慢释放生长因子。
PRP治疗涉及在浓缩血浆中制备自体血小板12。最佳血小板浓度尚不清楚，有多种商业设备可用于制备PRP13。这种缺乏标准化是由于研究之间的不一致造成的，并导致了关于注射剂量和时间的黑匣子。该协议描述了使用这种专用PRP装置获得自体PRP的程序，以在100%自体离体培养模型中扩增皮肤成纤维细胞。

<table>
	<tbody>
		<tr>
			<td>96 well black clear flat bottom</td>
			<td>BD Falcon</td>
			<td>353219</td>
			<td>32/case</td>
		</tr>
		<tr>
			<td>Cell trace Violet Dye</td>
			<td>Thermo Fischer Scientific</td>
			<td>C34557</td>
			<td>180 assays</td>
		</tr>
		<tr>
			<td>CuteCell PRP</td>
			<td>Regen Lab SA</td>
			<td>CC-PRP-3T</td>
			<td>3 tubes per package</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>52400-025</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Glutamine 200 mM</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hematology Counter</td>
			<td>Sysmex</td>
			<td>KK-21N</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 5000E Liquemine</td>
			<td>Drossapharm AG</td>
			<td></td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution 1M</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Liberase DH</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td>2x 5 mg per package</td>
		</tr>
		<tr>
			<td>MEM NEAA 100x</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Na Pyruvate 1mg/mL</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Phalloidin alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A12379</td>
			<td>300 units</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin 1x 0.25%</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25%</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
	</tbody>
</table>

production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion

目前，临床上对使用自体成纤维细胞进行皮肤修复有很大的兴趣。在大多数情况下，需要在体外培养皮肤细胞。然而，使用异种或同种异体培养基的细胞培养有一些缺点（即感染因子传播或细胞扩增缓慢的风险）。在这里，开发了一种自体培养系统，用于使用患者自身的富血小板血浆（PRP）在体外扩增人类皮肤成纤维细胞。在进行腹部成形术时，从患者身上分离出人真皮成纤维细胞。使用补充有胎牛血清 （FBS） 或 PRP 的培养基随访培养物长达 7 天。评估PRP制剂中的血细胞含量、增殖和成纤维细胞分化。该协议描述了使用专用医疗设备获得标准化，非活化PRP制剂的方法。该制剂只需要一个医疗设备（CuteCell-PRP）和离心机。该装置适用于足够的医疗实践条件，是一种一步法、无热原和无菌封闭系统，需要 1，500 x g 的单次软旋转离心 5 分钟。离心后，血液成分被分离，富血小板血浆很容易收集。该装置可实现快速、一致和标准化的PRP制备，可用作人体细胞体外扩增的细胞培养补充剂。这里获得的PRP含有1.5倍的血小板浓度，与全血相比，优先去除红细胞和白细胞。结果表明，与FBS（7.7x）相比，PRP在细胞增殖中表现出促进作用，并且在PRP处理后成纤维细胞被激活。

这项专利技术是一种简单、快速且可重复的医疗设备，用于生产标准化的 PRP 制剂。这是一个一步法，全封闭系统，允许在以1，500 x g 离心5分钟后从静脉全血制备PRP（由于分离凝胶技术）。离心后获得的PRP从位于凝胶下方的红细胞和白细胞中清除。经过几次试管倒置后，凝胶顶部的血小板重新悬浮在血浆中，PRP即可使用（图1）。
为了评估制剂中?...

Watch this Scientific Journal Video about Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion at JoVE.com

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

该协议提出了一种产生PRP以促进100%自体成纤维细胞培养系统中细胞体外扩增的装置。

生产自体富血小板血浆以促进体外人成纤维细胞扩增

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

该方案是用于人成纤维细胞培养的自体系统，使用患者自己的富血小板血浆和五分钟的标准化制剂。PRP促进培养中成纤维细胞的大量细胞扩增。我建议您使用这种方法使用PRP作为细胞补充剂，当您需要大量的小溪和安全的细胞扩增进行自体细胞治疗时。当您尝试此协议时，我建议您检查PRP完整性。您的血浆必须非常干净，没有红细胞。当你转移它时，你必须非常小心地做。关于组织的消化，在继续之前必须非常干净。首先，小心地将充满血液的富血小板血浆（PRP）管倒置三次，以将血液与抗凝剂混合。在固定的 45 度角转子中以 1500g 离心血液样品五分钟并平衡试管。离心后，分离的血液在分离凝胶下捕获红细胞和白细胞，凝胶表面有血小板。轻轻倒置PRP管20次，将血小板沉积物重悬于血浆上清液中。确保血小板与凝胶完全分离，血浆应从透明变为浑浊。使用连接到10毫升注射器的转移装置从管中收集血浆溶液。将皮肤样品放入装有PBS的50毫升聚丙烯管中，轻轻摇动试管。如果皮肤样本相对较大，请将其放在 150 平方厘米的组织培养皿的盖子上，表皮面朝下。用一把镊子和剪刀取出皮下脂肪组织。为防止纸巾变干，请每隔几分钟在PBS中冲洗一次纸巾。脂肪去除完成后，使用手术刀将皮肤样本切成大约 0.5 x 1.5 厘米的条状。接下来，将 5 毫升胶原酶/分散酶混合物添加到无菌 15 毫升管中。将切割的组织转移到管中。确保将组织碎片浸没在溶液中并牢固地盖住试管。将试管放入37摄氏度的培养箱中，轨道振荡150分钟。孵育后，将装有组织的管移动到生物安全柜中。将无菌100毫米培养皿的盖子倒置在生物安全柜中。将组织悬浮液转移到100毫米培养皿的底部，不要飞溅。如果试管中残留任何组织碎片，请使用无菌的一毫升移液管或无菌镊子将碎片转移到培养皿底部。表皮朝上，用一对镊子握住真皮。用另一对镊子抓住表皮的边缘，将真皮和表皮分开。将分离的碎片放在同一个盖子上，将真皮碎片放入含有PBS的新100毫米培养皿中。接下来，使用实验室镊子将真皮片转移到含有三毫升0.3%胰蛋白酶PBS的15毫升聚丙烯管中。将试管在 37 摄氏度的水浴中孵育，并通过倒置试管几次每两到三分钟混合一次内容物。10至20分钟后，加入三至五毫升冰冷的完全生长培养基停止反应。用力涡旋管子几次。将含有成纤维细胞的悬浮液通过85微米尼龙网过滤到50毫升管中。将成纤维细胞溶液以150g离心10分钟。吸出上清液并将细胞沉淀重悬于100至200微升完全生长培养基中以进行细胞计数。将细胞接种在25平方厘米的组织培养瓶中。然后，将烧瓶在 37 摄氏度下孵育。第二天更换含有非贴壁细胞的培养基，每三到四天更换一次。当成纤维细胞达到70%至80%汇合度时，用PBS洗涤并加入胰蛋白酶-EDTA溶液。将烧瓶在37摄氏度下孵育三分钟后，加入三至五毫升温热的完全生长培养基停止反应。然后，将细胞悬液以200g离心五分钟。抽吸除去上清液。在PRP培养基中培养成纤维细胞并在37摄氏度下孵育。推荐的最小细胞密度为每平方厘米4， 000个活细胞。专用医疗设备用于通过处理来自10名不同患者的全血来产生PRP。该装置去除了大部分红细胞和白细胞。PRP中的平均血小板浓度比全血中的血小板浓度高约50%。自体成纤维细胞在含有10%FBS的培养基或PRP浓度范围为1至50%的培养基中培养7天后，在不改变培养基的情况下，用20%PRP引发的培养物表现出更多数量的活成纤维细胞。与FBS相比，PRP作为增殖助推器具有高达7.7倍的有效效果。鬼笔环肽染色显示，PRP活化时观察到的形态变化与F-肌动蛋白重组有关，F-肌动蛋白重组是成纤维细胞活化的标志。快速细胞扩增的一个主要问题是基因组修饰的风险。为了评估这一点，您可以进行比较基因组杂交测定以评估细胞的基因组稳定性。该技术的主要优点是用PRP代替FBS，PRP是一种更有效的自体生物补充剂。有了这个，研究人员可以在不添加任何异种物质的情况下更快地生长更多的细胞。我们可以将这种技术用于临床应用前需要离体扩增的其他细胞类型，例如角质形成细胞或间充质干细胞。

Research

JoVE Journal

Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

科研

生物工程

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

间质干细胞培养和交付自体条件：用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...