我们要感谢安德斯 Jeppsson 教授提供实验用血管的帮助。这项研究是由 LUA 赠款资助的 SSH。

使用的组织, 本文的协议遵循了哥德堡大学的伦理准则。
1. 组织准备和贮存
<ol><li>用手术钳和剪刀切开所提取的隐静脉的周围组织。 注: 隐静脉由血管外科医生从人类下肢解剖。本论文所用的隐静脉是旁路术后未使用的部分。</li>
	<li>为防止在灌注过程中出现渗漏, 结扎所有侧支在其末端用缝线和镊子。</li>
	<li>将静脉放在含有40毫升磷酸盐缓冲盐水 (PBS) 的50毫升管中。通过改变 PBS 2-3 倍来清洗静脉, 以清除多余的血液。将静脉冷冻保存在摄氏-80 摄氏度。</li>
</ol>2. 去细胞设置和 Recellularization 生物反应器的制备
注: 使用剪刀, 切割硅管如表 1所示。
<ol><li>
去细胞设置 1 (用于海卫 X-100 灌注和洗涤)
	<ol>
<li>到2升室 (塑料浴缸), 胶带所有三件管 a, 如图 1A所示。 注: 胶带一管到一壁, 边缘触及腔底 (洗涤剂的入口) 和其他两个管到对面墙上的距离为 5-10 厘米之间的长度取决于静脉。至少5厘米的这些管子也应贴在房间的地板 (静脉的入口和出口)。</li>
		<li>使用男女鲁尔连接器, 串联洗涤剂的入口管到管 B, 然后管 F, 管 C, 最后到静脉入口。把管子 F 放进蠕动泵的卡带里。</li>
		<li>再拿一根管子, 把一端连接到静脉的出口。将另一端放入玻璃罐中, 底部软管插座放置45厘米以上的房间 (洗涤剂出口) 和磁带, 以确保安全。取管 G 并将一端推入玻璃罐的软管出口, 然后将另一端置于静脉腔内。 注: 完整设置 (红色箭头)、腔室和流方向 (白色箭头) 的图片如图 1A所示。</li>
	</ol>
</li>
	<li>
去细胞设置 2 (用于 TnBP 灌注)
	<ol>
<li>拿一个1升的玻璃罐子, 把管子 C (洗涤剂入口) 的一端放到罐子里, 直到管子碰到罐子的底部。</li>
		<li>将另一端与管 F 连接, 然后管 c 将管 c 的另一端放入罐中, 直到半深度 (静脉入口)。将管 F 放入蠕动泵盒中。</li>
		<li>取管 D, 将一端放入罐子 (静脉的出口), 直到半深度, 另一端进入玻璃罐, 底部软管放置在45厘米高的静脉入口 (洗涤剂出口)。为了确保, 胶带的洗涤剂的入口和出口管。</li>
		<li>将1升玻璃罐放在磁力搅拌器上, 将磁铁放在瓶子内。</li>
		<li>取管 G, 将一端推到玻璃瓶的软管出口上, 将另一端置于1升玻璃罐中。 注:图 1B显示了完整设置 (红色箭头)、腔室和流方向 (白色箭头) 的图片。</li>
	</ol>
</li>
	<li>
去细胞设置 3 (用于 DNase 灌注)
	<ol>
<li>取一个250毫升的玻璃瓶, 将管的两端放置在软管的中间区域进入蠕动泵的卡带中。 注: 管的一端是溶液入口, 管的另一端连接到静脉的入口。静脉的出口在溶液中是无保留的。</li>
		<li>将整个安装, 包括泵在摄氏37摄氏度。图 1C显示了完整的设置和流方向的图片。</li>
	</ol>
</li>
	<li>
Recellularization 生物反应器
	<ol>
<li>准备好所有的零件, 如图 2A所示。</li>
		<li>如图 2B所示, 取4端口盖并插入到每个端口, 管 H (静脉的出口), 管 I (介质入口) 和管 J (静脉的入口)。将管 H 的另一端插入4端口 (介质插座)。 注意: 从顶部的4端口盖的视图显示在图 2C中。为了便于插入管子, 用剪刀将边缘剪成尖点。</li>
		<li>将管 J 的另一端弯曲成 U 形, 并将其与缝合线栓在一起。将还原连接器连接到管子的内端 & J。</li>
		<li>将60毫升管与一个扁平底座放入250毫升玻璃瓶中, 然后将上述设置安装到管中, 如图 2D所示。如图 2E所示, 连接管 k、E 和 k 串联。连接一个管 k 到静脉的入口和另一个管 k 到介质入口。 注意: 安装的示意图和流量方向见图 2F。</li>
		<li>用热处理消毒生物反应器。</li>
	</ol>
</li>
</ol>3. 解决方案的准备工作
<ol><li>解决方案 1 (10x 水): 对1升的超纯水, 添加2克叠氮化钠和18.6 克乙二胺乙酸 (EDTA)。搅拌直到盐溶解。</li>
	<li>解决方案 2 (1x 水): 采取100毫升的溶液1和添加900毫升的超纯水。</li>
	<li>解决方案 3 (5x 海卫 X-100): 950 毫升的超纯水, 添加50毫升的海卫 X-100, 1 克叠氮化钠和9.3 克 EDTA。搅拌直至溶解。</li>
	<li>解决方案 4 (1x 海卫 X-100): 采取200毫升的溶液3和添加800毫升的超纯水。</li>
	<li>解决方案 5 (1x TnBP): 添加10毫升的 TnBP 与10毫升吸管到990毫升的解决方案2。</li>
	<li>解决方案 6 (40 Kunitz 单位/毫升 DNase): 添加 2000 Kunitz 单位 DNaseI 50 毫升 Dulbecco 的磷酸盐缓冲盐水含有 CaCl2和氯化镁2。准备新鲜, 立即使用。</li>
	<li>解决方案 7 (PBS): 加入24克氯化钠, 0.6 克氯化钾, 4.32 克磷酸磷酸钠, 0.72 克磷酸钾为3升的超纯水。搅拌直至溶解。将 pH 值调整为7.4。在高压釜内消毒1升 PBS, 并分别贮存。</li>
	<li>溶液 8 (0.1% 过氧乙酸): 对50毫升的无菌溶液 7, 添加50µL 的过氧乙酸。准备新鲜, 立即使用。</li>
</ol>4. 内皮介质的制备
<ol><li>添加5毫升的 l-谷氨酰胺, 5 毫升抗反, 50 毫升的人 AB 血清和所有成分的 EGM-2 生长因子套件, 除胎牛血清到500毫升的 MCDB131 培养基下的无菌罩。</li>
</ol>5. 隐静脉去细胞
<ol><li>在室温下将人隐静脉从-80 摄氏度解冻。再次冻结在-80 摄氏度和解冻再次在室温下。</li>
	<li>用剪刀切开2毫米长的活检, 在室温下固定甲醛 24-48 小时。将静脉的每一端与一条缝合的雄性和雌性鲁尔连接器的倒钩系在一起。</li>
	<li>将静脉连接至去细胞设置 1, 填充溶液2的1升。灌注为15分钟, 在35毫升/分钟. 清空安装程序的内容。</li>
	<li>添加 1 L 的溶液4和灌注为4小时在35毫升/分钟. 清空安装程序的内容。</li>
	<li>添加500毫升溶液2和灌注5分钟. 清空安装程序的内容。重复此步骤。从灌注设置1断开静脉, 并连接到灌注设置2。</li>
	<li>添加 1 L 的解决方案 5, 打开搅拌器和灌注4小时在35毫升/分钟。 注意: 在打开搅拌器和整个灌注过程中, 解决方案5看起来多云。</li>
	<li>断开静脉从灌注设置2和洗涤静脉的外部和内部与注射器或10毫升吸管在4变化的超纯水 5-10 分钟。</li>
	<li>将静脉连接至灌注设置 3, 在37°c 室中添加50毫升溶液6和灌注, 4 摄氏度. 清空安装程序的内容, 并断开静脉与安装程序的连接。</li>
	<li>将静脉连接至灌注设置 1, 将溶液2和灌注的1升填入35毫升/分钟. 重复步骤5.4 至步骤 5.9 4 次 (总共5个循环)。如步骤5.2 所述, 再次进行活检。 注: 跳过步骤5.8 第二部分在周期1和3。</li>
	<li>清空安装程序的内容。1 L 的解决方案2和灌注为24小时在35毫升/分钟. 更改解决方案2后 12 h. 清空安装程序的内容。在35毫升/分钟内, 1L 的超纯水和灌注24小时. 在12小时后改变超纯水. 清空安装程序的内容。1 L 的解决方案7和灌注24小时在35毫升/分钟. 更改解决方案7后 12 h。</li>
</ol>6. 去细胞的核查
<ol><li>用超纯水清洗福尔马林固定活检15分钟, 在组织处理器中进行标准协议, 并嵌入石蜡22。</li>
	<li>5µm 部分使用切片和染色与迈耶的苏木精和0.2% 酒精的伊红 (H & E) 遵循标准协议22。</li>
	<li>在显微镜下查看与正常组织相比, 瓣膜组织中核的损失。</li>
</ol>7. Recellularization
<ol><li>将静脉放在含有50毫升溶液8的50毫升管中杀菌。搅拌在80旋转每分钟 (rpm) 在振动筛为1小时37摄氏度。</li>
	<li>从现在起, 处理层流 (黎巴嫩) 内阁下的静脉以维持不孕。用无菌钳将静脉转移到新的50毫升管中, 含有50毫升的无菌溶液7和500µL 抗反。为12小时以上的搅动, 改变解决方案7至少两次之间。</li>
	<li>使用无菌钳, 将静脉转移到新的50毫升管, 并添加50毫升的内皮介质。11小时以上的鼓动。</li>
	<li>使用无菌手套在黎巴嫩武装部队内阁中组装生物反应器。用无菌缝合和镊子将静脉栓在生物反应器的静脉入口和出口处。</li>
	<li>将静脉连接到生物反应器, 并用相同的培养基填充。添加肝素在50毫升/毫升和灌注1小时在 2 mL/分钟37摄氏度。</li>
	<li>收集 15-25 毫升的新鲜血液 (取决于静脉的长度) 从捐赠者在肝素涂层 vacutainer 管和稀释1:1 与斯蒂恩溶液。随后, 添加内分泌腺体衍生血管内皮生长因子 (如 VEGF, 80 ng/毫升), 碱性成纤维细胞生长因子 (b 蛋白, 4 µL/毫升) 和乙酰水杨酸 (5 µg/毫升)。</li>
	<li>将生物反应器移动到一个37°c 孵化器与 5% CO2和灌注为 48 h 在2毫升/分钟。</li>
	<li>大约在12小时之后, 将生物反应器移动到一个黎巴嫩武装部队的柜子里, 用血糖监测装置取一滴血, 用葡萄糖来测量血糖。如果水平小于3毫摩尔, 添加葡萄糖, 使 7-9 毫摩尔/l 的范围重复此步骤每 8-12 h。</li>
	<li>48小时后, 将生物反应器移到黎巴嫩武装部队的柜子中, 将血液从生物反应器中排出。添加30毫升的无菌溶液7和灌注5分钟排出溶液。添加30毫升的无菌溶液7和灌注5分钟. 重复两次或直到血红色消失。 注: 活检可用于验证细胞附件。拔下静脉, 用剪刀将静脉切开5毫米边缘, 然后丢弃。采取步骤5.2 所述的活检, 再次将静脉连接到生物反应器 (可选步骤)。</li>
	<li>增加30-45 毫升内皮培养基和灌注96小时的孵化器在2毫升/分钟。 注: 添加内皮介质, 直到静脉被淹没。</li>
	<li>将生物反应器移动到黎巴嫩武装部队的内阁, 断开 recellularized 静脉, 用剪刀剪断静脉的5毫米边缘, 然后丢弃。按照步骤5.2 中的说明进行活检。</li>
</ol>8. Recellularization 的核查
<ol><li>按照6节中的说明操作, 执行 H & E 染色。检查显微镜下的幻灯片是否存在细胞。</li>
</ol>

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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+effect+of+exercise+and+body+position+on+the+venous+pressure+at+the+ankle+in+patients+having+venous+valvular+defects.">The effect of exercise and body position on the venous pressure at the ankle in patients having venous valvular defects.</a> Journal of Clinical Investigation. 28 (3), 559-563 (1949).</li><li>Li, 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=Low-molecular-weight+peptides+derived+from+extracellular+matrix+as+chemoattractants+for+primary+endothelial+cells.">Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells.</a> Endothelium. 11 (3-4), 199-206 (2004).</li><li>van Ark, 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=Type+2+diabetes+mellitus+is+associated+with+an+imbalance+in+circulating+endothelial+and+smooth+muscle+progenitor+cell+numbers.">Type 2 diabetes mellitus is associated with an imbalance in circulating endothelial and smooth muscle progenitor cell numbers.</a> Diabetologia. 55 (9), 2501-2512 (2012).</li><li>Steen, S., 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=Transplantation+of+lungs+from+a+non-heart-beating+donor.">Transplantation of lungs from a non-heart-beating donor.</a> Lancet. 357 (9259), 825-829 (2001).</li><li>Van Raemdonck, D., Neyrinck, A., Cypel, M., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-vivo+lung+perfusion.">Ex-vivo lung perfusion.</a> Transplant International. 28 (6), 643-656 (2015).</li><li>Cross, M. 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SSH 持有 Verigraft 的股份, 该公司拥有组织工程血管技术许可。其他作者没有什么可透露的。

去细胞隐静脉的技术是一种简单、简便、经济有效的方法, 也可应用于所有小直径静脉, 如脐和乳腺静脉。该方法中使用的去细胞解决方案及其浓度是从我们以前的结果6,7。虽然我们建议5周期的去细胞, 在某些静脉, 我们也注意到去细胞在3周期内完成。然而, 利用5周期获得了可重现的结果。应用此协议, 我们成功地瓣膜了30厘米的不同长度的静脉 (未公布的结果)。将去细胞溶液的出口提升45厘米, 会在静脉内产生 33 mmHg 的压力。在我们的经验中, 我们注意到这是一个关键步骤 decellularizing 整个静脉均匀和重现性在5周期。所选择的压力比正常的隐静脉压力高3倍 (5-10 mmHg), 但与不称职静脉 (静脉曲张)23相同。此外, 我们推测, 这种高压将造成静脉壁的一个巨大的力量, 因此, 可以帮助有效和更快的细胞去除。
由于 TnBP 是一种有机溶剂, 不溶于水, 搅拌洗涤剂, 直到它变成多云是重要的;否则, 洗涤剂将浮动。出于同样的原因, 为了保持 TnBP 混合在溶液中, 洗涤剂的出口管被放置在玻璃罐顶部与软管出口。在冲洗水中没有漂浮的 TnBP 液滴, 可以看出在每一个循环中使用 TnBP 从静脉中有效去除。我们还注意到, 跳过 DNase 的步骤也产生了一个瓣膜组织, 但在少数情况下, 一个相对较高的 DNA 含量在瓣膜组织被注意到。由于高效 DNase 活动不需要高压和高流速, 所以可以使用低灌注率 (10 毫升/分钟)。使用不同的蠕动泵作为其较小的尺寸有助于方便地处理安装。由于我们注意到大多数细胞在去细胞期间的损伤导致周期 2, 我们建议跳过 DNase 治疗在周期1和 3 (未发表的结果)。尽管本手稿没有对细胞外基质蛋白进行定性和定量化, 但我们以往类似去细胞协议的经验表明, 生物力学性能的保存,细胞外基质结构和蛋白质7。虽然我们的初步量化实验与这些静脉产生了类似的结果 (未发表), 我们已经公布的结果将加强这种信心。
Recellularization 使用血液是一个方便和容易的过程, 在骨髓扩张细胞, 因为你可以避免长时间的细胞扩张时间, 自发突变的扩张细胞, 外科入侵和不适的病人。由于众所周知, 内皮化也可能发生在循环 EPCs, 我们假设, 血液灌注后, 内皮介质灌注将足以 recellularization。采用类似方法工程的静脉组织的安全性, 从移植的成功结果看, 当两个这样的静脉植入到额外的肝门静脉梗阻7的儿童。我们推测瓣膜细胞外基质中的生长因子将允许血液循环 EPCs 的附着24。Recellularization 的静脉通过类似的协议显示细胞阳性 VEGF receptor-2 和分化 (CD) 14 在流明, 而 CD45 表达细胞的膜7。我们还认为, 在所有情况下, 都不能观察到持续的内皮层, 特别是在使用老年和患病患者的血液时, 因为众所周知, 这些人的循环祖细胞数量减少了25。然而, 我们假设, 用接受者自己的血液灌注可能会掩盖许多由于去细胞而暴露的抗原, 从而减少移植时的不良炎症反应的几率, 而不是仅移植瓣膜血管。此外, 血液灌注可以沉积增加的生长因子水平的流明和膜, 反过来可能会招募更多的循环祖细胞, 导致一个快速的 recellularization 过程中的体内。
本研究所用的生物反应器设计的优点是热处理完整、组装方便、成本效益高、处理方便、破坏可能性最小。根据我们的经验, 使用目前的设计, 10 厘米长的脉 recellularized。即使25厘米长的血管也可以通过保持在生物反应器内的 "U" 形的静脉 recellularized, 这应该得到验证。生物反应器的设计表明, 静脉内的流动方向与重力有关, 因此设计是因为它是人类这些静脉的正常流向。12 h 灌注的内皮介质步骤是先决条件的静脉和增加亲和力的依恋传入 EPCs。增加额外的肝素和灌注1小时将降低在血液灌注过程中形成血栓的风险。
所需的血量取决于静脉的长度。我们遵循的基本原则是血液中的静脉应该被水淹没。在处理大于45毫升的血容量时, 可能需要偶尔进行混合以防止生物反应器底部的细胞积聚。我们增加了斯蒂恩的血液的解决方案, 因为它含有大量的蛋白质和成分需要保持组织健康的器官移植26,27。增加 VEGF 和 b 细胞因子是有益的, 因为它们是有效的血管生成因子28和它们的存在诱导迁移, 增殖和分化的 EPCs29,30,31,32.增加 VEGF 的数量是根据我们以前未发表的结果, 在那里 EPCs 的增殖被看见在 80 ng/毫升。阿司匹林的加入抑制了血小板33的活化, 从而减少了它们附着到内皮层的几率。持续监测和添加葡萄糖也有利于细胞增殖和防止红细胞溶血。
由于接受方只需要简单的血样样本, 所以可以认为这是一种简单可行的程序, 需要较少的技术专长。虽然, 从开始到结束的整个过程需要20天, 储存瓣膜静脉作为一个现成的产品将缩短程序8天的病人。虽然瓣膜静脉的贮存技术不应影响 recellularization 效率, 但必须对其进行评估。本程序产生的组织工程静脉可用于临床旁路手术, 取代受阻静脉, 静脉不足导致静脉曲张不需要免疫抑制, 从而提供了更好的质量病人的生活。

血管导管是需要的几个临床条件, 如动脉瘤, 颈动脉狭窄和动脉粥样硬化导致严重的血管问题。外科医生使用自体, 异基因或合成血管导管来恢复功能性血液供应。虽然自体血管的使用仍然被认为是理想的方法, 但患者的可用性是有限的。同种异体或合成移植等替代品在免疫抑制剂治疗和通畅的畅通方面存在着严重的问题, 导致再手术12, 给各国带来重大的健康经济负担。血管组织工程旨在为移植物提供自然同源性和自体细胞。因此, 受体免疫系统承认移植的移植物为自体, 因为这样的移植物包含了原始构型中的自然蛋白质和细胞, 因此与目前的替代物相比, 它可能更好地发挥作用。组织工程器官, 如膀胱3, 尿道4, 气管5, 静脉6,7, 已成功地用于临床。
组织工程生产个性化移植需要一个移植的捐赠者后, 去细胞和 recellularization。去细胞是一种有前景的技术, 从组织和器官中去除细胞8,9,10。去细胞可以通过特定的物理, 化学和酶方法11或结合它们来执行。在最佳使用这些方法, 瓣膜组织可以有类似的结构和功能蛋白的细胞外基质类似于本地组织。这些器官具有增强进入干细胞的附着、迁移、增殖和分化的内在能力。
Recellularization 是种子细胞进入移植的动态过程, 受体干细胞可用于临床移植。目前用于此类用途的干细胞包括骨髓、间充质和器官居民3、5、6。以动物和研究为导向的研究已经使用了胚胎和诱导多能性12,13,14的骨髓间质来源的干细胞。这个过程需要一个生物反应器 (一个容纳静脉的腔室, 并提供必要的条件, 如温度, 气体, pH 值和压力), 细胞和培养基。recellularization 的挑战是获得特定类型细胞所需的数量, 以及细胞能够到达整个组织或器官的播种策略。尽管直到目前为止, 在结构和功能上没有完整的组织或器官被产生和评估, 但在这一领域的一些进展和初步结果显示了未来的可能性15。静脉的关键功能在于控制炎症细胞渗入组织中的血管内皮, 以及有助于收缩的中层平滑肌层, 同时也提供了保持血压16的力量。研究表明, 在损伤期间, 内皮化发生于吻合或血液循环内皮祖细胞 (EPCs)17,18,19。我们的 recellularization 的策略依赖于循环血液中存在的 EPCs。
组织工程的静脉和动脉是由几个组遵循不同的去细胞和 recellularization 策略20,21。我们的小组还执行和发展了髂和乳腺静脉6,7去细胞和 recellularization 战略。去细胞是由静脉在海卫 X-100, 三正丁基磷酸酯 (TnBP) 和酶脱氧核糖核酸酶 (DNase) 的搅拌。recellularization 用骨髓源性内皮和平滑肌细胞6或外周血7进行。任一协议的静脉 recellularized 显示在提供功能性血液供应的儿科患者肝门静脉梗阻6,7的临床承诺。
我们目前已经开发了一个改进版本的相同的协议, 以改善和容易的性能去细胞, recellularization 和生物反应器处理小直径静脉。目前的去细胞协议要求用压力来灌注洗涤剂, 而不是用洗涤剂搅动。recellularization 协议包括额外的预处理步骤, 以改善细胞黏附力和增加循环血液中的生长因子, 以改善细胞黏附力, 生存和增殖。我们还改进了使用商用产品的生物反应器的设计。本文详细介绍了对人隐静脉执行去细胞和 recellularization 的改进协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:1159px;" width="1158">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:333px;">4-Port Cap</td>
			<td style="width:208px;">CPLabSafety</td>
			<td style="width:119px;">WF-GL45-4Kit</td>
			<td style="width:499px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Acetyl salicylic acid</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">A5376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Anti-Anti</td>
			<td style="width:208px;">Life Technologies</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">B-FGF</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:119px;">cc-4113B</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:333px;">Blood Glucose monitoring system - Free style Lite</td>
			<td style="width:208px;">Abbott</td>
			<td style="width:119px;">70808-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">D-Glucose</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">G8769</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">DNase-I</td>
			<td style="width:208px;">Worthington</td>
			<td style="width:119px;">LS0020007</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:333px;">Dulbecco&#39;s PBS with CaCl2 and MgCl2</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">EDTA disodium salt dihydrate</td>
			<td style="width:208px;">AlfaAesar</td>
			<td style="width:119px;">A15161.OB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">EGM-2 Growth Factors Kit</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:119px;">CC-4176</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:333px;">EG-VEGF</td>
			<td style="width:208px;">Peprotech</td>
			<td style="width:119px;">100-44</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:333px;">Glass bottle 250ml</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2151593</td>
			<td>Any bottle with GL45 cap can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Glass bottle 1L</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2151595</td>
			<td>Any bottle with GL45 cap can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Glass jar 500ml with bottom hose outlet</td>
			<td style="width:208px;">Kimble Chase Life Science</td>
			<td style="width:119px;">14607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Heparin</td>
			<td style="width:208px;">Leo</td>
			<td style="width:119px;">387107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Heparin Coated Vacutainer Tubes</td>
			<td style="width:208px;">Becton Dickinson</td>
			<td style="width:119px;">368480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Human AB Serum</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">H3667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">L-Glutamine</td>
			<td style="width:208px;">Life Technologies</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Luer Female with 1/8&#34; ID Barb</td>
			<td style="width:208px;">Oina</td>
			<td style="width:119px;">LF-2PP-QC</td>
			<td>For 3X5mm silicon tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Luer Female with 3/32&#34; ID Barb</td>
			<td style="width:208px;">Oina</td>
			<td style="width:119px;">LF-1.5PP-QC</td>
			<td>For 2X4mm silicon tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Luer Male with 3/32&#34; ID Barb</td>
			<td style="width:208px;">Oina</td>
			<td style="width:119px;">LM-1.5PP-QC</td>
			<td>For 2X4mm silicon tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Luer Male with 1/8&#34; ID Barb</td>
			<td style="width:208px;">Oina</td>
			<td style="width:119px;">LM-2PP-QC</td>
			<td>For 3X5mm silicon tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Luer Male with 5/32&#34; ID Barb</td>
			<td style="width:208px;">Cole Parmer</td>
			<td style="width:119px;">EW-45518-06</td>
			<td>For 5X8mm silicon tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">MCDB 131</td>
			<td style="width:208px;">Life Technologies</td>
			<td style="width:119px;">10372-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Per acetic acid</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">433241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Peristaltic pump I</td>
			<td style="width:208px;">Masterflex</td>
			<td style="width:119px;">7524-45</td>
			<td>For Decellularization setup 1 and 2. The cassette used is 7519-75</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Peristaltic pump II</td>
			<td style="width:208px;">Ismatec</td>
			<td style="width:119px;">ISM941</td>
			<td>For Decellularization setup 3 and Recellularization bioreactor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Potassium chloride</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">P5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Potassium hydrogen phosphate</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Reducing Connector 1.5mmX2.5mm</td>
			<td style="width:208px;">Biotech</td>
			<td style="width:119px;">ISM569A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Shaker</td>
			<td style="width:208px;">Ika</td>
			<td style="width:119px;">KS4000 i&#160; control</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sodium Azide</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">71290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sodium chloride</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:119px;">13423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sodium hydrogen phosphate</td>
			<td style="width:208px;">Merck</td>
			<td style="width:119px;">71640-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Steen solution</td>
			<td style="width:208px;">Xvivo</td>
			<td style="width:119px;">19004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Suture</td>
			<td style="width:208px;">Agnthos</td>
			<td style="width:119px;">14817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Tri-n-butyl Phosphate</td>
			<td style="width:208px;">AlfaAesar</td>
			<td style="width:119px;">A16084.AU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Triton-X-100</td>
			<td style="width:208px;">AlfaAesar</td>
			<td style="width:119px;">A16046.OF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Tube 60ml with flat base</td>
			<td style="width:208px;">Sarstedt</td>
			<td style="width:119px;">60596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube A</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280706</td>
			<td>Cut 3 pieces, each of 25 cm length for decellularization perfusion steup 1 and 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube B</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280706</td>
			<td>Cut 1 piece of 35 cm length for decellularization perfusion steup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube C</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280706</td>
			<td>Cut 5 pieces, each of 75 cm length for decellularization perfusion steups 1-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube D</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280706</td>
			<td>Cut 1 piece of 90 cm length for decellularization perfusion steup 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube E</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280706</td>
			<td>Cut 1 piece of 20 cm length for recellularization perfusion steup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube F</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280713</td>
			<td>Cut 2 pieces, each of 15 cm length for decellularization perfusion steup 1 and 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube G</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280713</td>
			<td>Cut 2 pieces, each of 15 cm length for decellularization perfusion steup 1 and 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube H</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280703</td>
			<td>Cut 1 piece of 15 cm length for recellularization perfusion steup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube I</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280703</td>
			<td>Cut 1 piece of 20 cm length for recellularization perfusion steup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube J</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280703</td>
			<td>Cut 1 piece of 25 cm length for recellularization perfusion steup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube K</td>
			<td style="width:208px;">VWR</td>
			<td style="width:119px;">2280703</td>
			<td>Cut 2 pieces, each of 35 cm length for recellularization perfusion steup</td>
		</tr>
	</tbody>
</table>

decellularization and recellularization methodology for human saphenous veins

血管导管在大多数血管手术中使用的是同种异体或合成移植, 通常会导致并发症的免疫抑制和不良的通畅。组织工程提供了一个新的解决方案, 以生成个性化移植的自然细胞外基质含有受体的细胞使用去细胞和 recellularization 的方法。我们展示了一个详细的方法来执行去细胞的人隐静脉和 recellularization 的外周血灌注。静脉由灌注1% 瓣膜 X-100, 1% 三正丁基磷酸酯 (TnBP) 和 2000 Kunitz 单位脱氧核糖核酸酶 (DNase)。海卫 X-100 和 TnBP 在35毫升/分钟内灌注4小时, DNase 以10毫升/分钟的时间在37摄氏度下灌注4小时。该静脉在超纯水和 PBS 洗涤, 然后在0.1% 过氧乙酸消毒。在 PBS 中再次被洗涤, 在内皮介质中进行预处理。静脉连接到一个生物反应器, 并灌流的内皮介质含有50毫升/mL 肝素 1 h. Recellularization 是用新鲜血液填充生物反应器, 稀释1:1 在斯蒂恩溶液中, 并添加内分泌腺衍生血管内皮生长因子 (80 ng/毫升), 碱性成纤维细胞生长因子 (4 µL/毫升), 乙酰水杨酸 (5 µg/毫升)。然后将该生物反应器移入孵化器中, 以2毫升/分钟的时间灌注48小时, 同时维持 3-9 毫摩尔/升之间的葡萄糖。随后, 该静脉用 PBS 冲洗, 填充内皮介质, 并在孵化器中灌注96小时。用海卫 X-100、TnBP 和 DNase 在5个周期内瓣膜隐静脉进行治疗。瓣膜静脉与正常和 recellularized 静脉呈白色对照 (浅红色)。苏木精 & 瓣膜 (H & E) 染色显示细胞核的存在只在正常的, 但不是在静脉。在 recellularized 静脉中, H & E 染色显示血管腔表面存在细胞。

正常静脉的毛形态为浅红色 (图 3A)。红色在渐进的去细胞周期中丢失 (周期 2,图 3B; 周期 3,图 3C), 5 次周期,它看起来苍白和白色 (图 3D)。血流灌注后的 recellularized 静脉 (图 3E) 和内皮介质灌注 (图 3F) 的颜色呈鲜艳的红色。5周期的去细胞治疗成功地移除从静脉的细胞, 因为没有蓝色的细胞核在 H & E 染色 (图 4B) 看到。相比之下, 在正常的静脉中发现了几个细胞核 (图 4A)。在 recellularized 静脉的 h & E 染色中, 在腔侧存在附着细胞, 其血为48小时 (图 4C、黑色箭头), 并在内皮介质中灌注 96 h (图 3D、黑色箭头)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57803/57803fig1.jpg"> 图 1: 去细胞灌注装置的装配. A)图片显示组装去细胞安装1为海卫 X-100 和洗涤。白色箭头显示解决方案的流路径, 红色箭头表示静脉和解决方案的入口和插座。B)显示组装的去细胞设置2用于 TnBP 灌注的图片。类似地, 如在去细胞安装1中, 白色箭头表示解决方案的流路径, 红色箭头表示静脉和解决方案的入口和插座。C)显示组装的去细胞设置3用于脱氧核糖核酸酶灌注的图片。白色箭头显示解决方案的流路径, 红色箭头表示静脉和解决方案的入口。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57803/57803fig1large.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/57803/57803fig2.jpg"> 图 2: recellularization 生物反应器的制备与组装.A)显示组装生物反应器所需材料的图片。B) 4 端口帽内侧的图片, 显示减少连接器 (红色箭头) 的位置, 指向连接静脉入口和出口 (白色箭头) 和排列的 H、I 和 J。管 H 的自由端被放置到生物反应器中以返回介质。C)从4端口盖的顶部可以看到进出的各个管。D)显示组装的生物反应器的图片。E)用蠕动泵显示整个生物反应器设置的图片。管 K 从生物反应器延伸到蠕动泵的连接。F)整个生物反应器灌注系统的示意图表示。橙色箭头指示流的方向。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57803/57803fig2large.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/57803/57803fig3.jpg"> 图 3: 去细胞和 recellularization 期间的静脉毛形态.A)正常静脉的毛形态呈鲜艳的红色。随着去细胞周期B)周期2和C)周期3的增加, 颜色丢失。D)由5个周期, 静脉看起来苍白和白色。静脉后灌注与 E) 血液48小时和 F) 与内皮介质为 96 h 看起来再次明亮的红色的颜色。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57803/57803fig3large.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/57803/57803fig4.jpg"> 图 4:瓣膜和 recellularized 脉的表征.正常静脉的苏木精和伊红染色图像含有许多蓝核, 但在B)瓣膜静脉中不存在。在静脉 recellularized 与C)血液为 48 h 和与D)内皮介质为 96 h, 细胞 (黑箭头) 的附件在流明被注意了。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57803/57803fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Decellularization and Recellularization Methodology for Human Saphenous Veins at JoVE.com

Decellularization and Recellularization Methodology for Human Saphenous Veins

人隐静脉的去细胞和 Recellularization 方法学研究

在这里, 我们描述了一个协议的隐静脉去细胞使用洗涤剂和 recellularization 的血液灌注的外周血和内皮介质。

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

decellularization-recellularization-methodology-for-human-saphenous

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.1K 次观看.  University of Gothenburg. 在这里, 我们描述了一个协议的隐静脉去细胞使用洗涤剂和 recellularization 的血液灌注的外周血和内皮介质。

视频: Decellularization and Recellularization Methodology for Human Saphenous Veins

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

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术:协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

在振荡压力灌流器程序大鼠肝脏的脱细胞

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

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 ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

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