我们要感谢奥尔布里希花园的约翰 Wirth 慷慨地提供了这个项目使用的标本。这项工作由国家心脏、肺和血液学会部分支持 (R01HL115282 到 G.R.G.)国家科学基金会 (DGE1144804 和 G.R.G.), 威斯康星大学外科和校友基金 (H.D.L.)。这项工作也部分由环境保护局 (83573701 号星赠款)、国立卫生研究院 (R01HL093282-01A1 和 UH3TR000506) 和国家科学基金会 (IGERT DGE1144804) 支持。

1. 用洗涤剂为基础的方法去细胞植物组织
<ol><li>
使用新鲜或冻结的F. 刚毛, 叶样本。冻结未使用的新鲜样品在一个-20 °c 冷藏库和存储, 以供将来使用 (最多一年)。 注: 使用几乎任何所需植物的茎或叶组织。延长贮存时间会对组织造成损害。<ol>
<li>根据样品的预期用途确定要处理的样品的尺寸和形状 (即切成条的样品非常适合于机械测试应用, 同时8毫米圆盘样品在多井培养应用中很有用。).在室温下 (20-25 °c) 去离子 H2O, 用锋利的、干净的活检冲床将叶子切成8毫米的圆盘。 注: 本协议可用于全叶和茎。然而, 较小的样本将 decellularize 更快。</li>
		<li>将样品在室温 (20-25 °c) 去离子 H2O 上孵育5-10 分钟, 设置为低速设置以清洗和/或解冻。使用足够的去离子 H2O 确保所有样品都被彻底润湿。</li>
	</ol>
</li>
	<li>
在去离子 H2中制备 10% (w/v) 月桂酸钠 (SDS) 的溶液在合适的容器中放置样品 (玻璃或塑料碟是理想的), 并添加 SDS 解决方案, 完全覆盖样品。在室温 (20-25 °c) 上孵育5天的样品, 设置为低速以防止样品损坏。 注意: 不要过度拥挤容器, 因为这会减慢去细胞过程, 并导致 SDS 不均匀的处理。在这一步骤中, 样品应获得棕色色调。<ol>
<li>5天后, 用去离子 H2取代 SDS 溶液, 在摇板上孵化样品, 以彻底冲洗任何残留的 sds 溶液。</li>
	</ol>
</li>
	<li>在 10% (v/v) 漂白剂溶液中制备 1% (v/v) 非离子表面活性剂。为使500毫升溶液, 混合5毫升的非离子表面活性剂与50毫升漂白剂, 然后添加445毫升的去离子 H2o 淹没样品在新鲜准备的解决方案。 注: 非离子表面活性剂/漂白剂溶液不具有较长的保质期, 因此, 应在48小时内使用准备。</li>
	<li>
将非离子表面活性剂/漂白剂溶液每24小时更换一次, 直到完全清除样品 (参照图 1A进行可视比较)。然后在2分钟的摇动板上用去离子水孵化样品, 冲洗掉多余的非离子表面活性剂/漂白剂溶液。将摇板设置为低设置以防止损坏样品。 注: 清除所用样品所需的时间因植物的种类和种类而异。完全褪色的样品表明完全去细胞 (执行 DNA 量化, 以确保彻底清除)。<ol>
<li>Lyophilize 样品保存一年 (使用液氮的闪光冷冻是优选的, 冷冻样品在-80 °c 也是可接受的) 并且存放他们在室温 (20-25 °c) 在低湿气。</li>
		<li>重建样品在三 HCl (10 毫米, pH 值 8.5) 使用足够的涂层样品。在使用前用微在无血清介质中2-3 次冲洗样品 (即使用150-300 µL 每次冲洗, 在标准24井板)。 注: 三盐酸缓冲液和无血清培养基 (即DMEM, 但任何基本细胞培养基均可替代) 更有效地降低对 SDS 治疗样品的细胞活力的影响, 而不是仅以去离子 H2O 治疗。</li>
	</ol>
</li>
</ol>2. 使用无洗涤剂去细胞方法制备样品
注意: 此过程的初始步骤与步骤 1.1-1.1. 2 (见上文) 一致。
<ol><li>准备 5% (v/v) 漂白剂 (次氯酸钠) 和 3% (瓦特/v) 碳酸氢钠 (NaHCO 3) 解决方案.将溶液在通风罩中加热至60-70 摄氏度, 用于F. 刚毛叶片样品在热板上搅拌时切成8毫米的圆盘。 注: 碳酸氢钠可替代碳酸钠 (Na2CO3), 或氢氧化钠 (氢氧化钠)。所需的温度变化很大 (从室温到90°c), 应根据所用样品的性能进行调整。</li>
	<li>一旦溶液达到所需的温度范围, 淹没样品并降低搅拌速度, 以避免损坏它们。在明显清除示例后 (参阅图 1B进行可视比较), 请仔细地从浴缸中移除。在去离子 H2O 上孵化一次样品, 1-2 分钟, 以去除多余的漂白剂溶液。 注: 清除样品所需的时间可能会有很大差异。例如, 切香菜可以清除10-15 分钟, 而厚和/或较大的样品, 如整个叶子或茎可能需要数小时的高温浴完全清除。</li>
	<li>Lyophilize 样品并存放在室温下 (20-25 摄氏度) 的低湿度。</li>
</ol>

<ol><li>Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+engineering+and+regenerative+medicine%3A+from+first+principles+to+state+of+the+art%5BTitle%5D)+AND+%22Journal+of+Pediatric+Surgery%22%5BJournal%5D)">Tissue engineering and regenerative medicine: from first principles to state of the art.</a> Journal of Pediatric Surgery. 45 (2), 291-294 (2010).</li>
<li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Survival+and+function+of+hepatocytes+on+a+novel+three-dimensional+synthetic+biodegradable+polymer+scaffold+with+an+intrinsic+network+of+channels%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels.</a> Annals of Surgery. 228 (1), 8-13 (1998).</li>
<li>Park, A., Wu, B., Griffith, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+surface+modification+and+3D+fabrication+techniques+to+prepare+patterned+poly%28L-lactide%29+substrates+allowing+regionally+selective+cell+adhesion%5BTitle%5D)+AND+%22Journal+of+Biomaterial+Science%2C+Polymer+Edition%22%5BJournal%5D)">Integration of surface modification and 3D fabrication techniques to prepare patterned poly(L-lactide) substrates allowing regionally selective cell adhesion.</a> Journal of Biomaterial Science, Polymer Edition. 9 (2), 89-110 (1998).</li>
<li>Steinhoff, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+engineering+of+pulmonary+heart+valves+on+allogenic+acellular+matrix+conduits%3A+in+vivo+restoration+of+valve+tissue%5BTitle%5D)+AND+%22Circulation%3A+JAMA.+102+%28Suppl+3%29%2C+III-50-+III+-55%22%5BJournal%5D)">Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue.</a> Circulation: JAMA. 102 (Suppl 3), III-50- III -55. 102 (Suppl 3), III-50-III-55 (2000).</li>
<li>Stock, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+valved+conduits+in+the+pulmonary+circulation%5BTitle%5D)+AND+%22Journal+of+Thoracic+Cardiovascular+Surgery%22%5BJournal%5D)">Tissue-engineered valved conduits in the pulmonary circulation.</a> Journal of Thoracic Cardiovascular Surgery. 119 (4 Pt 1), 732-740 (2000).</li>
<li>Ott, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfusion-decellularized+matrix%3A+using+nature%27s+platform+to+engineer+a+bioartificial+heart%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nature Medicine. 14 (2), 213-221 (2008).</li>
<li>Guyette, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioengineering+Human+Myocardium+on+Native+Extracellular+Matrix%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Bioengineering Human Myocardium on Native Extracellular Matrix.</a> Circulation Research. 118 (1), 56-72 (2016).</li>
<li>Huerta, S., Varshney, A., Patel, P., Mayo, H., Livingston, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+Mesh+Implants+for+Abdominal+Hernia+Repair%3A+US+Food+and+Drug+Administration+Approval+Process+and+Systematic+Review+of+Its+Efficacy%5BTitle%5D)+AND+%22JAMA+Surgery%22%5BJournal%5D)">Biological Mesh Implants for Abdominal Hernia Repair: US Food and Drug Administration Approval Process and Systematic Review of Its Efficacy.</a> JAMA Surgery. 151 (4), 374-381 (2016).</li>
<li>Catalano, E., Cochis, A., Varoni, E., Rimondini, L., Azzimonti, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+skin+substitutes%3A+an+overview%5BTitle%5D)+AND+%22Journal+of+Artificial+Organs%22%5BJournal%5D)">Tissue-engineered skin substitutes: an overview.</a> Journal of Artificial Organs. 16 (4), 397-403 (2013).</li>
<li>Fang, B., Wan, Y., Tang, T., Gao, C., Dai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proliferation+and+osteoblastic+differentiation+of+human+bone+marrow+stromal+cells+on+hydroxyapatite%2Fbacterial+cellulose+nanocomposite+scaffolds%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds.</a> Tissue Engineering. 15 (5), 1091-1098 (2009).</li>
<li>Wan, Y., et al. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/S0928493106003523">Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications.</a> Materials Science and Engineering. 27 (4), 855-864 (2007).</li>
<li>Vinatier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+injectable+cellulose-based+hydrogel+for+the+transfer+of+autologous+nasal+chondrocytes+in+articular+cartilage+defects%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects.</a> Biotechnology and Bioengineering. 102 (4), 1259-1267 (2009).</li>
<li>Vinatier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+silanized+hydroxypropyl+methylcellulose+hydrogel+for+the+three-dimensional+culture+of+chondrocytes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.</a> Biomaterials. 26 (33), 6643-6651 (2005).</li>
<li>Vinatier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+cartilage+with+human+nasal+chondrocytes+and+a+silanized+hydroxypropyl+methylcellulose+hydrogel%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel.</a> Journal of Biomedical Materials Research Part A. 80 (1), 66-74 (2007).</li>
<li>Helenius, G., Bäckdahl, H., Bodin, A., Nannmark, U., Gatenholm, P., Risberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+biocompatibility+of+bacterial+cellulose%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">In vivo biocompatibility of bacterial cellulose.</a> Journal of Biomedical Materials Research Part A. 76 (2), 431-438 (2006).</li>
<li>Zhong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+aligned+nanofibrous+collagen+scaffold+by+electrospinning+and+its+effects+on+in+vitro+fibroblast+culture%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture.</a> Journal of Biomedical Materials Research Part A. 79 (3), 456-463 (2006).</li>
<li>Thomas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+shape-controlled+tuneable+microgel+platform+to+modulate+angiogenic+paracrine+responses+in+stem+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.</a> Biomaterials. 35 (31), 8757-8766 (2014).</li>
<li>Lai, C., Zhang, S., Wang, L., Sheng, L., Zhou, Q., Xi, T. <a target="_blank" href="http://pubs.rsc.org/en/content/articlelanding/2015/tb/c5tb01640a">The relationship between microstructure and in vivo degradation of modified bacterial cellulose sponges.</a> Journal of Materials Chemistry B. 3 (46), 9001-9010 (2015).</li>
<li>Märtsonad, M., Viljantoa, J., Hurmea, T., Laippalac, P., Saukkob, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+cellulose+sponge+degradable+or+stable+as+implantation+material%3F+An+in+vivo+subcutaneous+study+in+the+rat%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.</a> Biomaterials. 20 (21), 1989-1995 (1999).</li>
<li>Miyamoto, T., Takahashi, S., Ito, H., Inagaki, H., Noishiki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+biocompatibility+of+cellulose+and+its+derivatives%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research%22%5BJournal%5D)">Tissue biocompatibility of cellulose and its derivatives.</a> Journal of Biomedical Materials Research. 23 (1), 125-133 (1989).</li>
<li>Entcheva, E., Bien, H., Yin, L., Chung, C., Farrell, M., Kostov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+cardiac+cell+constructs+on+cellulose-based+scaffolding%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Functional cardiac cell constructs on cellulose-based scaffolding.</a> Biomaterials. 25 (26), 5753-5762 (2004).</li>
<li>Modulevsky, D., Cuerrier, C., Pelling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biocompatibility+of+Subcutaneously+Implanted+Plant-Derived+Cellulose+Biomaterials%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials.</a> PLoS One. 11 (6), e0157894(2016).</li>
<li>McCulloh, K., Sperry, J., Adler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Water+transport+in+plants+obeys+Murray%27s+law%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Water transport in plants obeys Murray's law.</a> Nature. 421 (6926), 939-942 (2003).</li>
<li>Gershlak, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crossing+kingdoms%3A+Using+decellularized+plants+as+perfusable+tissue+engineering+scaffolds%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.</a> Biomaterials. 125, 13-22 (2017).</li>
<li>Fontana, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofunctionalized+Plants+as+Diverse+Biomaterials+for+Human+Cell+Culture%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">Biofunctionalized Plants as Diverse Biomaterials for Human Cell Culture.</a> Advanced Healthcare Materials. 6 (8), (2017).</li>
<li>Wegst, U., Bai, H., Saiz, E., Tomsia, A., Ritchie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioinspired+structural+materials%5BTitle%5D)+AND+%22Nature+Materials%22%5BJournal%5D)">Bioinspired structural materials.</a> Nature Materials. 14 (1), 23-36 (2015).</li>
<li>Gibson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+hierarchical+structure+and+mechanics+of+plant+materials%5BTitle%5D)+AND+%22Journal+of+the+Royal+Society+Interface%22%5BJournal%5D)">The hierarchical structure and mechanics of plant materials.</a> Journal of the Royal Society Interface. 9 (76), 2749-2766 (2012).</li>
<li>Hoshiba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularized+Extracellular+Matrix+as+an+In+vitro+Model+to+Study+the+Comprehensive+Roles+of+the+ECM+in+Stem+Cell+Differentiation%5BTitle%5D)+AND+%22Stem+Cells+International%22%5BJournal%5D)">Decellularized Extracellular Matrix as an In vitro Model to Study the Comprehensive Roles of the ECM in Stem Cell Differentiation.</a> Stem Cells International. 2016, (2016).</li>
<li>Guyette, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfusion+decellularization+of+whole+organs%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li>
<li>Modulevsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apple+derived+cellulose+scaffolds+for+3D+mammalian+cell+culture%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Apple derived cellulose scaffolds for 3D mammalian cell culture.</a> PLoS ONE. 9 (5), e97835(2014).</li>
<li>Blonder, B. How to make leaf skeletons. , Available from:
 <a target="_blank" href="http://benjaminblonder.org/The_secrets_of_leaves/Making_skeletons.html">http://benjaminblonder.org/The_secrets_of_leaves/Making_skeletons.html</a> (2017).</li>
<li>Seba, A., Sloane, H. <a target="_blank" href="https://www-jstor-org.remotexs.ntu.edu.sg/stable/103557?seq=1">The Anatomical Preparation of Vegetables, by Albertus Seba, F. R. S. Communicated to the Royal Society by Sir Hans Sloane, Bart. Pr. R. S. and Col. Med. Lond. Translated from the German, by Mr. Zolman, F. R. S.</a> Philosophical Transactions. 36 (407), 441-444 (1775).</li>
<li>Parrish, E. <a target="_blank" href="https://archive.org/details/phantombouquetpo00parriala">The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste.</a> The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste. , (1865).</li>
<li>Coffin, S., Gaudette, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aprotinin+extends+mechanical+integrity+time+of+cell-seeded+fibrin+sutures%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">Aprotinin extends mechanical integrity time of cell-seeded fibrin sutures.</a> Journal of Biomedical Materials Research Part A. 104 (9), 2271-2279 (2016).</li>
<li>Zangala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+Genomic+DNA+from+Mouse+Tails%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Isolation of Genomic DNA from Mouse Tails.</a> Journal of Visualized Experiments: JoVE. (6), e246(2007).</li>
<li>Borselli, C., Cezar, C., Shvartsman, D., Vandenburgh, H., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+multifunctional+delivery+scaffold+in+the+ability+of+cultured+myoblasts+to+promote+muscle+regeneration%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration.</a> Biomaterials. 32 (34), 8905-8914 (2011).</li>
<li>Hill, E., Boontheekul, T., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designing+Scaffolds+to+Enhance+Transplanted+Myoblast+Survival+and+Migration%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">Designing Scaffolds to Enhance Transplanted Myoblast Survival and Migration.</a> Tissue Engineering. 12 (5), 1295-1304 (2006).</li>
<li>Hill, E., Boontheekul, T., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulating+activation+of+transplanted+cells+controls+tissue+regeneration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Regulating activation of transplanted cells controls tissue regeneration.</a> Proceedings of the National Academy of Sciences. 103 (8), 2494-2499 (2006).</li>
<li>Ma, J., Holden, K., Zhu, J., Pan, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Application+of+Three-Dimensional+Collagen-Scaffolds+Seeded+with+Myoblasts+to+Repair+Skeletal+Muscle+Defects%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">The Application of Three-Dimensional Collagen-Scaffolds Seeded with Myoblasts to Repair Skeletal Muscle Defects.</a> Journal of Biomedicine and Biotechnology. 2011, 1-9 (2011).</li>
<li>Tyree, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+hydraulics%3A+the+ascent+of+water%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Plant hydraulics: the ascent of water.</a> Nature. 424 (6943), 923(2003).</li>
<li>Raven, P., Evert, R., Eichhorn, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRaven%2C+P+%22Biology+of+Plants%22">Biology of Plants</a>. , 7th edition, (2005).</li>
<li>Turrell, F. <a target="_blank" href="https://www-jstor-org.remotexs.ntu.edu.sg/stable/2435976">The area of the internal exposed surface of dicotyledon leaves.</a> American Journal of Botany. 23 (4), 255-264 (1936).</li>
</ol>

作者没有什么可透露的。

本文介绍了两种 decellularize 植物组织的方法。这里提出的结果, 加上先前研究的结果25, 表明所提出的协议可能适用于广泛的植物种类, 可以在茎和叶上进行。这些程序是简单的, 不需要专门的设备, 所以植物去细胞可以在大多数实验室进行。值得注意的是, 去细胞后, 支架必须具有功能性, 以促进哺乳动物细胞黏附。不同的功能化技术, 使哺乳动物细胞黏附和生长的植物组织已被描述在其他地方25 , 并不是当前手稿的主题。
这里提出的两个去细胞协议的最后一步 (图1) 对它们的成功至关重要。当执行任一方法时, 不过度拥挤浴缸, 因为这会导致不均匀的清除。这些方法可应用于全叶和茎;但是, 这将增加清除它们所需的时间长度。确保样品完全浸没在整个样品中保持清洁一致。理想情况下, 去细胞应该清除大多数细胞物质的植物组织, 同时尽量减少结构损伤。这是可能的无洗涤剂的方法可以根据需要修改, 以配合所需的标本。较高的浴缸温度可能会导致较快的清除时间, 但也可能会导致更多的损害, 如果这些样品是过度孵化。较低的温度可能需要更长的孵化时间, 并适合更脆弱的样品。溶液中漂白剂的浓度也可以改变以加速或减速去细胞过程。当测试与无洗涤剂方法使用的新样品时, 建议先以50-60 摄氏度的浴温开始, 并经常检查去细胞的进展情况 (理想情况下每15-20 分钟), 直到它们充分清除为止。这种温度范围是很好的耐受性的大多数植物物种, 在优化的技术。在完成后, 样品可以目视和手动检查, 以确保他们没有过度孵化和破坏不可挽回的, 因为将表明撕裂或解体后, 从浴缸中删除。使用此方法制备的样品对细胞生长的影响 (参考图 3A) 比以洗涤剂为基础的方法, 只有在去离子 H2O 的洗涤。然而, 建议使用三 HCl (10 毫米, pH 值 8.5) 和无血清的培养基, 特别是在敏感的应用。
以洗涤剂为基础的 (SDS) 方法很可能适用于大多数植物物种, 对于清除整个叶子和微妙的样品特别有用, 因为它需要最小的物理搅拌。如上所述, 最后一步对样品的去细胞至关重要。通过调整非离子表面活性剂浴的孵化时间, 可以清除具有明显物理特性的样品。这个过程的一个重要缺点是, 瓣膜组织可能含有洗涤剂的痕迹, 这会随后影响哺乳动物细胞的细胞代谢活动。因此, 在使用前建议彻底清洗步骤。在这里发现, 用三 HCl 缓冲液 (10 毫米, pH 值 8.5) 冲洗的样品在使用(图 3B)之前, 与去离子 H2O 所洗的细胞相比有更高的生存能力。
当储存未经处理的样品供将来使用的任何一种方法, 重要的是监测他们的损害, 以确保所产生的脚手架不会受到影响。当处理时, 损伤可能会出现在植物组织的变色以及开裂或过度的脆性。建议每周对样品进行监测, 以便在它们不再有用并需要处理之前利用它们。损伤的发生在标本类型之间变化, 较厚的更结实的样品持久。
总的来说, 植物作为生物材料的潜在来源有着很大的希望。这是由它们自然发展起来的错综复杂的结构和特征所支撑的, 它们有助于它们在当地环境中生长。成功利用植物组织作为组织工程支架提出了一个潜在的便宜的替代品昂贵, 往往难以设计, 生物材料。植物组织的表面形貌, 从一个物种到另一种自然变化, 也可以用来指导空间定向的细胞生长25。例如, 先前的一项研究表明, 人类细胞对瓣膜植物上存在的地形线索作出反应25, 因此这些支架可用于培养高度组织模式25中的细胞。组织工程中需要的另一项功能是从头perfusability。植物组织已经进化了数以亿计的时间, 在流体运输中非常有效, 它们的血管网络可以有效地灌注24。此属性可用于指导细胞迁移和血管生成, 最终目的是为临床应用创建替代组织。此前的研究结果表明, 高表面积和连通孔隙率的生物材料有利于细胞增殖, 当植入体内时, 可以使宿主细胞向内迁移。进入植入36,37,38,39。高表面积和连通孔隙度也是植物组织的标志40,41,42, 因此植物派生的脚手架有潜力与周围的组织集成在体内中。此外, 最近的研究发现, 瓣膜叶可以成功地灌注细胞24 , 当人类细胞被播种在瓣膜植物组织, 他们符合他们的地形提示25。植物源性支架在全部结合后, 具有一定的性能, 可成功应用于组织工程应用。

组织工程出现在二十世纪八十年代, 以创建活体组织替代品, 并有可能解决重大器官和组织短缺1。一种策略是使用支架刺激和引导身体再生缺失的组织或器官。尽管先进的制造方法, 如3维印刷生产的脚手架具有独特的物理性能, 但能够制造具有多种可实现的物理和生物特性的脚手架仍然是一个挑战2,3. 此外, 由于缺乏功能性血管网络, 这些技术在再生3维组织方面受到限制。使用瓣膜动物和人体组织作为支架帮助规避此问题4,5,6,7。但是, 高成本、批处理可变性和有限的可用性可能限制了瓣膜动物支架的广泛使用8。还有人担心潜在的疾病传播给病人和免疫反应的一些瓣膜哺乳动物组织9。
纤维素, 来源于植物和细菌来源, 已广泛用于产生生物材料的广泛应用于再生医学。一些示例包括: 骨骼10、11、软骨12、13、14和伤口愈合15。由纤维素组成的脚手架有一个额外的好处, 因为它们是耐用的, 并耐被哺乳动物细胞分解。这是由于哺乳动物细胞不产生分解纤维素分子所必需的酶。相比之下, 使用大分子从胞外基质 (如胶原蛋白) 生产的脚手架容易被分解为16 , 可能不适合于长期应用。胶原支架可以通过化学交联来稳定。然而, 由于影响到支架的生物相容性的交叉连接器的固有毒性, 有一个权衡 17.反之, 纤维素有可能长期留在植入地点, 因为它不受哺乳动物细胞的酶降解18,19,20。通过水解预处理和与多种纤维素酶21 的协同提供, 可以改变降解速率。在小鼠22的研究中也证明了瓣膜植物衍生纤维素支架的生物相容性在体内。
经过数以百万年的进化, 植物改良了它们的结构和组成, 以提高流体输送和保持的效率。植物血管容器通过分枝成较小的血管来最小化水力阻力, 类似于根据默里定律23的哺乳动物血管。去细胞后, 该植物的复杂网络的船只和互联毛孔保持。考虑到大量不同种类的植物, 植物衍生的支架有可能克服目前影响组织工程中支架的设计限制24,25。例如, Modulevsky et表明, 当瓣膜苹果组织植入在鼠标22的背面时, 血管生成和细胞迁移发生。同样, Gershlak et表明, 内皮细胞可以在瓣膜叶的血管内生长24。在一个单独的实验中, Gershlak et 等人也能够表明心肌细胞可以生长在叶子表面, 并且能够收缩24。
植物还包括从细胞到宏观尺度的复杂组织, 即使用迄今为止开发的最先进的制造技术, 也很难实现。植物组织的复杂层次设计使它们比其成分的总和26强。植物拥有大量不同的机械性能, 从刚性和坚韧的成分, 如茎, 更灵活和柔韧的, 如叶27。叶片的大小、形状、断裂强度、血管化程度等不同种类的不同, 并可进行多种亲水性。总的来说, 这些植物的特性表明, 瓣膜植物可以作为独特和功能强大的医疗设备, 包括组织工程支架。
本议定书的重点是两种方法, decellularize 植物组织, 如叶和茎, 用作支架在组织工程。第一种方法是以洗涤剂为基础的技术, 使用一系列的浴池来去除 DNA 和细胞物质, 这已经从广泛使用的技术 decellularize 哺乳动物和植物组织中进行了调整6,22,25 ,28,29,30。第二种方法是无洗涤剂的, 它适应于通常用于去除叶子的软组织的 "骨架" 协议31。先前的工作表明, 在漂白和碳酸氢钠溶液中煨的叶子促进了从周围的软组织分离的血管31。该技术可引回到 17th和 18th世纪进行的实验, 例如 Albertus 塞巴32和爱德华帕里什33的工作。这些实验围绕着离开植物物质, 如叶子和水果, 在水中浸泡了很长一段时间 (几周到几个月), 让柔软的组织自然腐烂。这里的 "骨架" 方法适合使用较温和的条件, 如较长的潜伏期在较低的温度, 以消除细胞残留, 并避免严重扰乱软组织结构。在此详细的实验中, 使用了三种植物类型:榕刚毛、 Pachira aquatica和一个藤黄的种类。对这两种方法的 DNA 定量、力学测试和对细胞代谢活动的影响进行了描述。

<table><tbody><tr><td>Sodium dodecyl sulfate</td><td>Sigma Life Science</td><td>75746-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>MP Biomedicals, LLC</td><td>807426</td><td>Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td></tr><tr><td>Concentrated bleach (8.25% sodium hypochlorite)</td><td>Clorox</td><td>Item #: 31009</td><td>Standard concentrated bleach.</td></tr><tr><td>Sodium bicarbonate</td><td>Acros Organics</td><td>217120010</td><td>Can be substituted with sodium hydroxide or sodium carbonate.</td></tr><tr><td>8 mm Biopunch</td><td>HealthLink</td><td>15111-80</td><td>Cuts samples that fit well in 24 well plate</td></tr><tr><td>Belly Dancer-Shake table</td><td>Stovall Life Sciences</td><td>BDRAA115S</td><td>Use low speeds to not damage tissues. Can use any model/brand of shake table.</td></tr><tr><td>Isotemp hot/stir plate</td><td>Fisher Scientific</td><td></td><td>Can use any style/brand of hot/stir plate.</td></tr><tr><td>Beaker</td><td>Any</td><td></td><td>Can use any size beaker as long as it will fit your samples and not overcrowd them.</td></tr><tr><td>Tris Hydrochloride</td><td>Fisher Scientific</td><td>BP153-500</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT50003PC</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA assay</td><td>Life Technologies</td><td>P11496</td><td>Can use any dsDNA quantification mehtod on hand.</td></tr></tbody></table>

two methods for decellularization of plant tissues for tissue engineering applications

目前用作组织置换支架的自体、人工合成和动物衍生的移植物由于可用性低、生物兼容性差和成本高而受到限制。植物组织具有良好的特性, 使其独特的适合作为脚手架, 如高表面积, 优良的水传输和保留, 相互连通的孔隙率, 现有的血管网络, 和广泛的机械性能。本文介绍了两种用于组织工程应用的植物去细胞的成功方法。第一种方法是基于洗涤剂浴去除细胞物质, 这与以前建立的用于清除哺乳动物组织的方法相似。第二种是一种无洗涤剂的方法, 它适应于一种隔离叶脉的协议, 包括使用加热漂白剂和盐浴清除叶子和茎。这两种方法都能产生类似机械性能和低细胞代谢影响的脚手架, 从而使用户能够选择更适合其预期应用的协议。

这两种方法都产生了适合细胞培养和组织工程应用的支架。图 1显示了去细胞过程的一般工作流, 该流程使用完整的叶为洗涤剂的方法和切割样品 (8 毫米直径) 为无洗涤剂的方法。成功去细胞的榕刚毛组织后, 这两种方法产生了清晰和完整的样本 (图1A 和 1B). 可以 decellularize 整个植物组织 (图 1A);然而, 较小的样品似乎能够更快、更有效地清除。以洗涤剂为基础的方法所观察到的一个潜在问题是由于非离子表面活性剂浴的过早去除 (图 1C) 而导致异构去细胞。无洗涤剂方法的缺点是, 由于在加热的浴缸中长时间孵化 (图 1D), 损坏可能会发生。
用单轴拉伸试验研究了两种方法制备的瓣膜支架的力学性能, 如其他研究24,34所做。此测试产生了最大切线模数 (母语) (图 2A)、故障应变 (SAF) (图 2B) 以及F. 刚毛和Pachira aquatica示例的最终抗拉强度 (众信) (图 2C)。使用两个去细胞协议编写的 aquatica 示例在测量的所有参数中显示了类似的机械性能. 除了众信测试之外,刚毛示例在所有情况下都显示了类似的趋势。特别是, 使用以洗涤剂为基础的 (SDS) 协议制备的样品比用无洗涤剂 (漂白剂) 方法制备的产品的平均结果更高。这些结果表明, 从不同的植物物种收集的样本可能产生独特的瓣膜脚手架的性质, 当准备通过两个去细胞协议。
为了测量 DNA 的去除, 样本被清除使用任何一种方法和他们的基因组 DNA 被隔离使用以前建立的协议35。两种去细胞方法 (洗涤剂为基础和无洗涤剂) 显著降低了样本的基因组 dna 含量为 2.47 ng 的 dna/毫克 (n = 3, s = 0.18) 和 2.71 ng 的 DNA/镁的组织 (n = 3, s = 1.60) 分别 (图 2D)。非瓣膜样本有 104.67 ng 的 DNA/毫克的组织 (n = 3, s = 26.21) (图 2D)。去细胞后 DNA 含量的大幅下降表明植物细胞物质的有效去除。
通过测定在瓣膜aquatica或藤黄支架的存在下培养2天的人真皮成纤维细胞 (高密度纤维板) 的代谢活动, 评估了两种去细胞方法对细胞活力的影响。hDFs 有较高的新陈代谢活动时, 在存在的脚手架瓣膜通过无洗涤剂的方法与那些通过洗涤剂的方法 (图 3A)。为了确保彻底去除去细胞中使用的试剂, 在细胞培养之前, 进行了一系列的实验, 其中制备的支架被广泛洗涤。用洗涤剂为基础的方法对样品进行了两种不同的洗涤方法: 在三 HCl 缓冲液 (10 毫米, pH 8.5) 的洗涤, 其次是在血清自由介质中的洗涤vs 2 洗涤在去离子 H2O。这两种方法都使用了 1x PBS 的最终洗涤步骤。对随后暴露在瓣膜植物支架上的哺乳动物细胞的影响使用与上述相同的代谢活性测定进行测量。三盐酸缓冲器 (10 毫米, pH 值 8.5), 其次是血清游离培养基有助于减少对细胞代谢活动的影响, 与去离子 H2O 相比, 使样品处理的洗涤剂为基础的方法可媲美的样品从无洗涤剂方法 (图 3B)。
以前的研究表明, 哺乳动物细胞可以生长在使用基于洗涤剂的方法25制备的支架上;因此, 有必要对以前未经测试的无洗涤剂方法所制备的样品进行演示。间充质干细胞 (MSCs) 在瓣膜F. 刚毛叶样本 (8 毫米圆盘) 上播种, 每支架2万个细胞, 并允许孵育24小时。这些支架是功能化之前, 细胞介绍与一个多巴胺共轭, 以促进黏附。图 4阐释从典型示例中收集的图像。收集了一个明亮的字段图像, 以显示所用示例的一节的总体结构 (图 4A)。MSCs 用 calcein 染色, 用荧光显微镜进行成像 (图 4B)。然后, 图像被叠加 (图 4C) 以显示叶子表面上生长的细胞的位置。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57586/57586fig1.jpg"> 图 1.植物组织去细胞的一般工作流程。植物组织去细胞的典型工作流程 (洗涤和准备步骤是普遍的);(A) 顶部路径为基于洗涤剂的方法 (B) 底部的无洗涤剂方法。(C) 由于从非离子表面活性剂/漂白剂浴 (D) 中过早去除的不完全/失败的P. aquatica叶的去细胞在 F 刚毛叶的损坏的去细胞上使用基于洗涤剂的方法。由于长期孵化在加热漂白剂溶液中无洗涤剂的方法。<a href="/files/ftp_upload/57586/57586fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57586/57586fig2.jpg"> 图 2.方法比较去细胞的力学性能和有效性。意思是 * SD;统计分析是使用两个样本的 t 检验和p值列出的, 在统计上显著 (小于或等于 0.05)。比较F. 刚毛和aquatica的示例之间的机械测试结果 (用于基于洗涤剂的方法的蓝色条, 红色用于无洗涤剂, 绿色用于未经处理的样品) (F. 刚毛洗涤剂基于 n = 4,无洗涤剂 n = 2;aquatica洗涤剂-基 n = 3, 无洗涤剂 n = 4) (A) 最大切线模数 (母语) (B) 应变在故障 (SAF) (C) 和极限抗拉强度 (众信)。(D) 在每组三个样本中以三份 dsDNA 量化分析: 未经处理 (新鲜, n = 3), 无洗涤剂方法 (漂白剂, n = 3) 和基于洗涤剂的方法 (SDS, n = 3), p值为去细胞方法组与未经处理的样本组比较。<a href="/files/ftp_upload/57586/57586fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57586/57586fig3.jpg"> 图 3.代谢活性测定结果比较两种去细胞方法之间的影响。意思是统计分析是使用两个样本的 t 检验和 p值列出的, 在统计上有显著性 (小于或等于 0.05)。在aquatica的组织样本和藤黄的种类 (规范化以控制未添加样本的水井) 的情况下测量细胞代谢活动, 以显示 (a) 两种代谢影响的比较去细胞方法 (每个组的 n = 3) 和 (B) 使用基于洗涤剂的协议中的两种洗涤方法对代谢活动的影响比较: 三 HCl 缓冲 (10 毫米, pH 8.5) 与血清游离介质 (n = 4) vs 去离子 H 2O (n = 4)。<a href="/files/ftp_upload/57586/57586fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57586/57586fig4.jpg"> 图 4.骨髓间充质干细胞 (MSCs) 生长在瓣膜的表面上榕刚毛叶, 功能性与多巴胺共轭.MSCs 生长在F. 刚毛叶上, 瓣膜由无洗涤剂的方法和功能性与一个 RGD-多巴胺共轭 (500 µm 标尺被增加用于参考) (A) 瓣膜叶部分的亮场图像 (B) 在叶表面上生长的 calcein 染色的 MSCs 的荧光图像 (C) 合并了明亮的场和荧光图像。<a href="/files/ftp_upload/57586/57586fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

组织工程应用中植物组织去细胞的两种方法

在这里, 我们介绍, 并对比两个协议用于 decellularize 植物组织: 洗涤剂为基础的方法和无洗涤剂的方法。这两种方法都留下了植物组织的细胞外基质, 然后可以用作组织工程应用的支架。

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

two-methods-for-decellularization-plant-tissues-for-tissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.5K 次观看.  University of Wisconsin-Madison. 在这里, 我们介绍, 并对比两个协议用于 decellularize 植物组织: 洗涤剂为基础的方法和无洗涤剂的方法。这两种方法都留下了植物组织的细胞外基质, 然后可以用作组织工程应用的支架。

视频: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

整个肝脏的脱细胞和Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

科研

生物工程

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

脱细胞猪心逆行灌注的程序

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

一脱细胞方法对天然脱细胞基质肠的生产

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

胎儿和成人心脏组织外植体作为三维体外研究平台的可比较去角质

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

胎儿小鼠骨骼肌3D脱细胞基质的制备用于细胞培养

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

发育生物学

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

人隐静脉的去细胞和 Recellularization 方法学研究

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

倒置方向下整个人的心脏在加压袋内的去细胞化

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

<meta charset="utf8"></head><body>用于制备高度保存的人类脱细胞皮肤基质的脱细胞化，用于再生医学

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

用于制备高度保存的人类脱细胞皮肤基质的脱细胞化，用于再生医学

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

组织工程应用中植物组织去细胞的两种方法

摘要

探索更多视频

整个肝脏的脱细胞和Recellularization

脱细胞猪心逆行灌注的程序

一脱细胞方法对天然脱细胞基质肠的生产

胎儿和成人心脏组织外植体作为三维体外研究平台的可比较去角质

胎儿小鼠骨骼肌3D脱细胞基质的制备用于细胞培养

人隐静脉的去细胞和 Recellularization 方法学研究

倒置方向下整个人的心脏在加压袋内的去细胞化

用于制备高度保存的人类脱细胞皮肤基质的脱细胞化，用于再生医学

用于制备高度保存的人类脱细胞皮肤基质的脱细胞化，用于再生医学

在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣