我们感谢中国国家自然科学基金 (31770968) 和天津应用基金会和先进技术研究项目 (17JCZDJC33400) 的财政支持。

1. 肝素 Starpeg Nanocoating 的制备
<ol><li>
肝素琥珀酰亚胺琥珀酸 (肝素-NHS) 的合成
	<ol>
<li>重出0.69 克肝素, 溶于2.0 毫升冰冷的去离子水。</li>
		<li>重出0.23 克 NHS 和溶解在0.5 毫升冰冷的去离子水在一个圆底烧瓶。</li>
		<li>在一个圆底烧瓶中, 重出0.77 克的 EDC, 溶解在0.5 毫升的冰冷的去离子水中。</li>
		<li>将 NHS 和 EDC 溶液混合在一个圆底烧瓶中。在室温下将混合溶液放在室温下30分钟的工作台上。</li>
		<li>用 5031 x g的10分钟离心机, 去除过量的 edc 和 nhs。</li>
		<li>将30毫升的冷乙醇添加到混合物溶液中, 离心机在 5031 x g处为10分钟。<ol>
<li>重复两次。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
starPEG 肝素 nanocoating 的制备
	<ol>
<li>加入1.0 毫升 10% (瓦特/v) starPEG-(NH2)8到一个圆底烧瓶。</li>
		<li>添加0.67 毫升 10% (w/v) 肝素-NHS 到相同的圆底烧瓶。</li>
		<li>将 starPEG-(NH2)8和肝素-NHS 的混合溶液放置在25摄氏度的孵化器中, 以获得一个具有粘度的清晰溶液。 注: 对于需要荧光标记肝素的实验, 制作程序是相同的, 除了星钉-(NH2)8被溶于去离子水补充 5 (6)-羧基n-琥珀酰亚胺酯 0.1% (摩尔比) 的 starPEG-(NH2)8。)</li>
	</ol>
</li>
	<li>
傅里叶变换红外光谱法表征肝素 PEG nanocoating
	<ol>
<li>开始前, 用丙酮和去离子水冲洗玛瑙砂浆、杵和制粒装置 (直径为13毫米的小圆盘)。使用前将玻璃器皿烘干。</li>
		<li>将大约0.1–1% 肝素 PEG 样品与200–250镁 KBr 粉混合。</li>
		<li>将肝素 PEG 和 KBr 混合物放入玛瑙砂浆中, 细粉碎成直径为2µm 的小颗粒。</li>
		<li>将混合物转化成造粒装置。在真空中应用高压缩力 (8 T/厘米2) 形成透明颗粒。</li>
		<li>轻轻地将样品丸从造粒装置中取出。小心不要拉得太硬, 以免招致裂痕。</li>
		<li>将样品小球非常小心地转移到傅里叶变换红外光谱仪中, 以确定样品的化学结构。</li>
	</ol>
</li>
	<li>
用扫描电镜检查肝素 PEG nanocoating 内孔结构 (SEM)
	<ol>
<li>为下列步骤, 准备标准的48井细胞培养板和吸管。</li>
		<li>将肝素 PEG 共聚物溶液注入48井培养板的水井中。</li>
		<li>把盘子冻在-80 摄氏度, 24 小时。</li>
		<li>Lypophilize 样品在真空环境 (0.1 Pa)-50 °c 为 24 h。</li>
		<li>在铝存根的粘性表面上散布样品。</li>
		<li>用金涂上样品, 观察扫描电镜下观察内孔结构。</li>
	</ol>
</li>
	<li>
原子力显微镜 (AFM) 检测肝素 PEG nanocoating 表面
	<ol>
<li>清洁硅玻璃幻灯片与食人鱼溶液 (70% 小时2,4, 30% H2O2) 在80°c 为40分钟。</li>
		<li>油脂实验在甲醇中的10分钟, 然后在甲苯10分钟的幻灯片。</li>
		<li>用真空干燥干燥幻灯片。</li>
		<li>用大量的 3-氨基丙基 triethoxysilane 溶液 (2% 甲苯) 冲洗幻灯片, 轻轻摇动。</li>
		<li>油脂实验在甲醇中的10分钟的幻灯片, 然后在甲苯10分钟。</li>
		<li>使用标准吸管将100µL 3% 肝素 PEG 溶液放在幻灯片表面。</li>
		<li>在原子力显微镜下检查肝素 PEG 的表面 (50–80赫的共振频率, 0.350 N m 21 的力常数, 尖端半径为 600 nm)。</li>
	</ol>
</li>
</ol>2. 用肝素 PEG Nanocoating 的小鼠胰岛表面工程
<ol><li>
肝素 PEG nanocoating 小鼠胰岛表面涂层
	<ol>
<li>用胶原酶消化和 histopaque 梯度纯化法提取小鼠胰岛, 如前所述的朱庇特19。</li>
		<li>精选200胰岛解剖显微镜下的1.5 毫升管。</li>
		<li>增加500µL 的 PBS 到胰岛和离心机在 503 x g 1 分钟。</li>
		<li>脱上清液, 添加250µL 的肝素 PEG 溶液, 并保持在冰上的混合物10分钟左右, 使胰岛与肝素 peg 溶液混合良好。</li>
		<li>离心机在 503 x g 1 分钟。</li>
		<li>取出上清, 并添加500µL 的 PBS。吸管上下混合好。</li>
		<li>离心机在 503 x g 1 分钟, 取出上清, 并保留胰岛进一步使用。对用肝素 peg 包覆的小鼠胰岛进行检查后, 用肝素 peg 对胰岛涂层进行了这些步骤。</li>
	</ol>
</li>
	<li>
nanocoating 肝素 PEG 对小鼠胰岛的检测
	<ol>
<li>添加 RPMI 1640 补充10% 胎牛血清的包覆胰岛和转移这些小岛成一个非带电的无菌细菌培养皿。</li>
		<li>在倒置荧光显微镜下观察 nanocoating-肝素 PEG 包膜胰岛的形态和荧光信号。</li>
	</ol>
</li>
</ol>3. 肝素 PEG 包膜小鼠胰岛与非包衣胰岛的功能比较
<ol><li>
肝素 PEG 包膜小鼠胰岛的可行性研究
	<ol>
<li>精选20的肝素 PEG 包覆小鼠胰岛, 并将其放置在一个96井细胞培养板的一个井。</li>
		<li>用20个肝素 PEG 包覆的小鼠胰岛填充10口井。</li>
		<li>精选20的 noncoated 控制胰岛, 并将它们放入同一96井细胞培养板的一个井中。</li>
		<li>填充10口水井, 每个井都有 20 noncoated 控制胰岛。</li>
		<li>将200µL 的 RPMI 1640 补充10% 胎牛血清纳入96井板, 其中包含胰岛和保持在养殖14天。</li>
		<li>每2天更换胰岛培养基。</li>
		<li>在14天结束时用活/死染色试剂盒治疗胰岛, 并在倒置荧光显微镜下观察。</li>
		<li>计数 PI+ (红色) 细胞在每个胰岛下倒置荧光显微镜。</li>
	</ol>
</li>
	<li>
肝素 PEG 包膜小鼠胰岛的体外重建
	<ol>
<li>前冷却所有实验塑料包括培养板和吸管提示至少12小时前使用。</li>
		<li>把一个24井的地方放在一个冷却垫上。在24井细胞培养板中添加250µL 的冰冷生长因子-减少胶到每一个井。将涂敷的24井板放在4摄氏度, 至少30分钟。</li>
		<li>当基质溶液在冰箱中设置时, 胰蛋白酶处理老鼠胰岛内皮细胞 1 (MS1)。<ol>
<li>从组织培养瓶中取出所有培养基。用5毫升的 PBS 冲洗烧瓶。</li>
			<li>取出 PBS, 加入3毫升的胰蛋白酶-EDTA。在标准孵化器中孵化37摄氏度, 3 分钟。</li>
			<li>轻敲烧瓶底部, 分离细胞, 加入5毫升的血清补充培养基。</li>
		</ol>
</li>
		<li>将细胞收集成15毫升离心管, 离心机在 1000 x g处, 5 分钟。</li>
		<li>脱去上清, 加入5毫升的 PBS。吸管上下混合好。</li>
		<li>离心机在 1000 x g 5 分钟, 脱去上清。</li>
		<li>将血清补充的 DMEM 培养基加入管中, 并将种子5万细胞注入到基质溶液涂敷的24井板中的每个井中。</li>
		<li>添加5肝素 PEG 涂层胰岛或 noncoated 控制胰岛每井。</li>
		<li>在每个井中添加血清补充 DMEM 培养基, 每µL 500 个。</li>
		<li>观察4h 和24小时在光镜下孵化后的管形成。</li>
	</ol>
</li>
	<li>
肝素-PEG 包膜胰岛葡萄糖刺激胰岛素分泌的研究 注: 评估 nanocoating 是否会影响小鼠胰岛的功能, 评估葡萄糖刺激的胰岛素分泌肝素-PEG 涂层和 noncoated 胰岛。<ol>
<li>精选30肝素-PEG 涂层胰岛或 noncoated 控制胰岛成1.5 毫升管。准备一共有10管的涂层胰岛和 noncoated 胰岛每个。</li>
		<li>添加1毫升的生理盐溶液补充2毫摩尔葡萄糖20和孵化在37°c 为 2 h。 注: 有关生理盐溶液的配方, 请参阅表 1。在孵化过程中保持管帽松动。</li>
		<li>尽可能多地删除解决方案。</li>
		<li>通过添加600µL 的生理盐溶液刺激胰岛, 补充20毫摩尔/升葡萄糖37摄氏度30分钟。</li>
		<li>用商用胰岛素 elisa 试剂盒从每管中收集200µL, 用于胰岛素 elisa 定量。</li>
	</ol>
</li>
</ol>

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Clin. Pract. 100, 162-169 (2013).</li></ol>

作者没有什么可透露的。

在本文中, 我们展示了一种 "易于采用" 的方法, 为活细胞表面工程的肝素结合 starPEG nanocoating 通过伪 bioorthogonal 化学之间的 nanocoating 琥珀组和胰岛表面膜胺组。事实上, 细胞膜内的氨基酸是非常有反应性的, 因此早期的研究报告说, 在生理条件下, 与活化的n-hydroxysuccinimidyl (NHS) 酯的主要氨基组之间的相互作用14 ,16,21。此外, 广泛的研究报告说, 肝素, 一个高度硫酸盐多糖和重要组成部分的细胞外基质, 在胰岛封装, 可导致增强后移植重建和减少 IBMIR22,23。考虑到 peg 的生物相容性和肝素的多价蜂胶性质, 在 nanocoating 制备过程中, 我们使用8臂 peg 进行最大肝素负荷。肝素的改变与-NHS, 随后将与 NH2组在胰岛细胞膜上的反应。通过使 nanocoating 之间的共价键形成-NH2 (细胞膜) 和-NHS 的丙肝, 胰岛将容易 "涂层" 的肝素结合 PEG, 从而形成一个纳米薄层 () 在胰腺的外表面胰 岛。
目前的方法不同于以前发表的方法, 也选择 PEG 作为胰岛微囊化的主要聚合物, 在这种伪 bioorthogonal 化学之间的-NHS (nanocoating) 和-NH2的胰岛细胞膜使用。考虑到胰岛/细胞涂层的稳定性, 特别是在诸如血浆等复杂的环境中, 对于移植后的血管重建和生存至关重要, 而 NHS 和-NH2之间的形成与PEG 与细胞膜间的疏水作用24、静电相互作用9、15、24、25、26或生物素之间的生物学联系链亲和素14。
此外, 与胰岛涂层方法相比, 依赖于 LBL 方法与扩展胰岛处理期的多层沉积14,16,25, 目前的技术也需要最小处理和非常短的涂层期间孤立的胰岛。这两个因素都是移植后胰岛生存的必要条件, 因为在酶消化过程中, 由于破坏了 ECM, 胰岛的存活率往往已经受到破坏。然而, 目前方法的一个局限性是, 与 LBL 不同的是, 通过增加或减少层沉积的数量, 可以控制外层涂层的厚度, 目前尚不能对 nanocoating 的厚度进行定制。
此外, 由于温和的条件, 即 NHS 和-NH2之间的化学反应发生, 目前的方法适用于活细胞表面工程不限于胰岛, 但大多数细胞治疗。此外, 考虑到肝素是已知的与一系列的细胞因子和生物活性分子, 肝炎 nanocoating 也提出了一个开放的平台, 有潜力纳入无限的生物调解人, 以及接口为更加复杂的细胞表面工程学。

细胞治疗的疗效受低细胞滞留和生存率1、2的限制。为了改善细胞治疗的结果, 细胞表面工程通过酶操作, 肽共轭, bioorthogonal 化学和物理封装与生物材料已被利用3,4, 5,6,7,8,9,10。本议定书旨在通过将超薄肝素结合的 starPEG (肝素 PEG) nanocoating 细胞表面, 实现活细胞的表面工程, 采用 "易于采用" 的方法。本文以胰岛表面工程为例, 以胰岛的异质性和目前临床胰岛移植的贬低结果为实例。
事实上, 临床胰岛移植目前是通过直接注射孤立的胰岛进入肝门静脉, 这一程序只适用于选择性的病人, 因为缺乏捐助材料和低治疗效果11. 传统上, 海藻酸盐一直是胰岛封装和表面改性最常用的生物材料, 尽管它不太理想, 因为海藻酸盐和炎症相关纤维化的化学不稳定性12, 13。此外, 与100至200µm 的胰岛的自然大小相比, 海藻酸钠-胰岛微囊更大, 介于400和800µm 之间, 超过了氧气的生理扩散距离。在不显著改变胰岛体积的情况下, 建立了保形胰岛包封, 并进行了封装。因此, nanomembranes 的沉积, 由 PEG, 四氟乙烯, 硅膜或多层 nanocoating (也称为 "逐层" [LBL] 技术) 已报告, 从而改善离体胰岛生存率14 ,15,16,17,18, 虽然 LBL 方法往往需要广泛的胰岛处理期, 以沉积多层, 这可能会损害胰岛的生存能力.此外, 依赖于生物膜层之间的静电或共价相互作用的 nanomembranes 的不稳定性, 或 nanomembranes 与胰岛表面的疏水性相互作用也引起了有关9、14,15,16,22,23,24,25,26。
另一个限制因素, 押账治疗结果门静脉胰岛移植是即时血液介导的炎症反应 (IBMIR) 引起的直接接触的植入胰岛与血液, 导致血小板聚集,凝血和不良免疫效应或不想要的细胞活化9。为解决这些问题, 制备了一种由星形聚乙二醇 (starPEG) 组成的超薄 nanocoating, 其建立的生物相容性和多功能性作为胰岛壳体材料。肝素, 一个高度硫酸盐多糖, 也被纳入 starPEG nanocoating 其抗炎, 抗凝血性能和能力, 以促进血管化通过招募促血管生成生长因子22, 23。

<table border="0" cellpadding="0" cellspacing="0" style="width:531px;" width="531">
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			<td height="38" style="height:38px;width:131px;">Sodium chloride, 99.5%</td>
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			<td style="width:120px;">105864</td>
			<td style="width:120px;"></td>
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			<td height="38" style="height:38px;width:131px;">Potassium chloride, 99%, extra pure</td>
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			<td style="width:120px;">991468</td>
			<td style="width:120px;"></td>
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			<td height="38" style="height:38px;width:131px;">Sodium bicarbonate, 99.7%, ACS reagent</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">988639</td>
			<td style="width:120px;"></td>
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			<td height="57" style="height:57px;width:131px;">Magnesium chloride hexahydrate, 99%, ACS reagent</td>
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			<td style="width:120px;">182158</td>
			<td style="width:120px;"></td>
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			<td height="76" style="height:76px;width:131px;">Potassium dihydrogen phosphate, 99%, extra pure</td>
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			<td style="width:120px;">128839</td>
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		<tr height="57">
			<td height="57" style="height:57px;width:131px;">Magnesium sulfate heptahydrate, 99%, for analysis</td>
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			<td style="width:120px;">119370</td>
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			<td height="57" style="height:57px;width:131px;">Calcium chloride solution volumetric, 1.0 M CaCl2</td>
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			<td style="width:120px;">EZRMI-13K</td>
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		</tr>
	</tbody>
</table>

surface engineering of pancreatic islets with a heparinized starpeg nanocoating

细胞表面工程可以保护植入细胞免受宿主免疫攻击。它还可以重塑细胞的景观, 以改善移植功能和生存后移栽。本协议的目的是实现胰岛的表面工程使用一个超薄肝素结合 starPEG (肝炎) nanocoating。以 n-(3-二甲氨基丙基) n-'-乙基 carbodiimide 为原料, 对胰岛表面工程中的 nanocoating, 制备肝素琥珀酰亚胺琥珀酸 (肝素-NHS)。盐酸和n-琥珀 (NHS)。然后由氨基端功能性的八武装 starPEG (starPEG-(NH2)8) 和肝素-NHS 的交联, 形成了 PEG 混合物。对于胰岛表面涂层, 通过 Histopaque 胶原酶消化和梯度纯化分离小鼠胰岛。然后用冰冷型的 PEG 溶液对孤立的胰岛进行10分钟的治疗, 使 NHS 与胰岛细胞膜胺组之间的共价键结合。Nanocoating 与肝炎, 对胰岛大小和体积和肝素化胰岛的数量和容量的最小改变, 也可能减少即时血液介导的炎症反应在胰岛移植。这种 "易于采用" 的方法对于活细胞的表面工程来说是足够温和的, 而不影响细胞的生存能力。考虑到肝素对多种细胞因子有约束力, nanocoating 也提供了一个开放的平台, 能够将无限的功能性生物介质和多层表面用于活细胞表面。生物工程。

用 starPEG-(NH2)8和肝素结合 nanocoating 和 NHS 作为偶联剂, 合成了肝素 PEG (图 1)。采用红外光谱法检测肝素 PEG nanocoating 的化学结构, 如图 2所示, 肝素的特征峰可观察到3,300–3,600 厘米-1, 对应于肝素的羟基 (图 2 红色)。峰值在3,300–3,600 厘米-1 (图 2 蓝色) 的振幅下降表示 starPEG-(NH2)8酰胺组和肝素羰基基团之间的共轭。峰值1650厘米-1对应的酰胺羰基拉伸振动也减少, 表明有充分的反应与琥珀酰亚胺的肝素组和 starPEG 氨基甲酸酯-(NH2)8. 用原子力显微镜检查了肝素 PEG nanocoating 的表面结构, 并从娄等方面进行了研究, nanocoating 的高度约为 30 nm, 2 µm 宽, 有小孔特征 (暗斑) 范围在100到200毫微米之间直径10。从扫描电子显微镜获得的数据也证实了肝素 PEG 的高度互联多孔结构 (图 3), 这表明它可以适合在体内分娩期间的细胞存活。
对离体小鼠胰岛的表面涂层进行了检查, 如图 4所示, nanocoating 薄层, 绿色荧光显示, 均匀地沉积在包覆的胰岛表面, 不引起胰岛体积/大小的明显变化。在涂层过程中, 建议将胰岛保持在冰上以保持其生存能力。同样, 在这种情况下, 10 分钟的涂层周期也被优化, 以允许维持胰岛的生存能力。值得注意的是, 为了更好地观察 nanocoating, 检查被涂覆的小岛的横断面的电子显微术, 如前所报告的9,16将是更加适当的, 虽然数据将产生从固定的和嵌入的胰岛而不是在文化中的活胰岛。
对包膜胰岛的存活和功能进行了体外评价。考虑到肝素的有益性质, 肝素在胰岛表面的功能化可以促进胰岛的细胞重建, 从而使其存活。我们观察到, 肝素 PEG 包覆的小鼠胰岛在培养中表现出强健的胰岛生存能力 (图 5)。明显更先进的血管形成也明显从胰岛内皮细胞 (MS1), 与肝素 PEG 包膜胰岛共同培养, 由细长的微血管样结构和网络状血管结构表示 (图6).
nanocoating 过程不影响葡萄糖刺激的胰岛素分泌能力的肝素 PEG 涂层胰岛。所有治疗组均观察到, 当胰岛以生理盐液灌注, 辅以葡萄糖亚刺激水平 (2 毫摩尔/升) 时, 胰岛素分泌水平较低;图 7)。当胰岛以超生理水平的葡萄糖 (20 毫摩尔葡萄糖) 刺激时, 所有治疗组均观察到胰岛素分泌量的增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56879/56879fig1v2.jpg"> 图 1:胰岛表面工程 nanocoating 的化学结构.胰岛涂层是通过共价交联的肝素-NHS 和主要胺在细胞膜和星 PEG-(NH2)8。每个肝素分子都拥有多个羧基组, 它们被改成有一个 NHS 组。NHS 激活的羧基组将与蛋白质的主要胺反应形成酰胺的联系, 通过该 nanocoating 的 PEG 和胰岛的稳定。<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig1v2large.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/56879/56879fig2v2.jpg"> 图 2:星 peg 的红外光谱 (NH2)8, 肝素和肝炎 nanocoating 在干燥状态.<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig2v2large.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/56879/56879fig3.jpg"> 图 3:扫描电子显微图像的 PEG 在干燥状态.<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig3large.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/56879/56879fig4.jpg"> 图 4:荧光显微镜下肝素 PEG 包膜胰岛的代表性图像. 肝素被预先贴上了家族的标签 (绿色显示)。图像代表100个小岛。刻度条 = 100 µm.<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56879/56879fig5.jpg"> 图 5:肝素 PEG 涂层胰岛表现出强健的胰岛活力.(A)以 mean±标准误差表示的数据, 每组n = 50 个小岛。(B)活细胞以红色的绿色和死细胞显示。缩放条 = 100 µm. 图象代表50个小岛。<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56879/56879fig6.jpg"> 图 6:肝素 PEG nanocoating 促进胰岛内 revascularisation.胶管形成的 MS1 细胞共培养与肝素 PEG 涂层胰岛和 noncoated 控制胰岛。图像被采取了在4和 24 h. 刻度条 = 100 µm.<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56879/56879fig7.jpg"> 图 7:肝素 PEG nanocoating 对胰岛胰岛素分泌功能无改变.肝素-PEG 涂层胰岛和 noncoated 控制胰岛 (30) 被暴露到2毫摩尔/升 (白条) 或20毫摩尔/升 (黑条) 葡萄糖30分钟. 胰岛素分泌反应20毫摩尔/升葡萄糖是可比的涂层和控制胰岛。数据显示为平均值, 即标准误差, n = 10。<a href="https://cloudflare.jove.com/files/ftp_upload/56879/56879fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating at JoVE.com

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

胰岛血气 StarPEG Nanocoating 的表面工程

该协议的目的是实现胰岛的表面工程使用肝素结合 starPEG nanocoating 通过伪 bioorthogonal 化学之间的 nanocoating 琥珀组和胺组的胰岛细胞膜。

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

surface-engineering-pancreatic-islets-with-heparinized-starpeg

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.3K 次观看.  Chinese Academy of Medical Science & Peking Union Medical College. 该协议的目的是实现胰岛的表面工程使用肝素结合 starPEG nanocoating 通过伪 bioorthogonal 化学之间的 nanocoating 琥珀组和胺组的胰岛细胞膜。

视频: Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构:转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...