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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>AAJ207798</td><td></td></tr><tr><td>Streptozototin</td><td>Sigma</td><td>S0130</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>10831</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>31800022</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO, by Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Cell Dissociation Solution</td><td>GIBCO, by Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>12800017</td><td></td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma</td><td>G8644</td><td></td></tr><tr><td>488 phalloidin</td><td>Sigma</td><td>A12379</td><td></td></tr><tr><td>CFSE</td><td>Sigma</td><td>21888-25mg-F</td><td></td></tr><tr><td>Annexin V/PI apoptosis kit</td><td>Dojindo</td><td>AD10</td><td></td></tr><tr><td>DAPI Fluoromount-G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Collagenase from Clostridium, Type XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>NHS</td><td>Sigma-Aldrich</td><td>56480</td><td></td></tr><tr><td>EDC</td><td>Sigma-Aldrich</td><td>3449</td><td></td></tr><tr><td>8-armed PEG</td><td>J&amp;amp;K Scientific Ltd</td><td>1685176</td><td></td></tr><tr><td>FAM</td><td>Sigma-Aldrich</td><td>M041100</td><td></td></tr><tr><td>5(6)-carboxyfluorescein N-succinimidyl ester</td><td>Sigma-Aldrich</td><td>21888</td><td></td></tr><tr><td>KBr</td><td>J&amp;amp;K Scientific Ltd</td><td>32036</td><td></td></tr><tr><td>3-aminopropyl-triethoxysilane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>toluene</td><td>J&amp;amp;K Scientific Ltd</td><td>S-15497-20X</td><td></td></tr><tr><td>Live/dead staining kit</td><td>Biovision, US</td><td>K501</td><td></td></tr><tr><td>BD Matrigel&lt;sup&gt;TM&lt;/sup&gt;, basement membrane matrix, growth factor reduced</td><td>BD Bioscience</td><td>354230</td><td></td></tr><tr><td>Sodium chloride, 99.5%</td><td>J&amp;amp;K Scientific Ltd</td><td>105864</td><td></td></tr><tr><td>Potassium chloride, 99%, extra pure</td><td>J&amp;amp;K Scientific Ltd</td><td>991468</td><td></td></tr><tr><td>Sodium bicarbonate, 99.7%, ACS reagent</td><td>J&amp;amp;K Scientific Ltd</td><td>988639</td><td></td></tr><tr><td>Magnesium chloride hexahydrate, 99%, ACS reagent</td><td>J&amp;amp;K Scientific Ltd</td><td>182158</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, 99%, extra pure</td><td>J&amp;amp;K Scientific Ltd</td><td>128839</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, 99%, for analysis</td><td>J&amp;amp;K Scientific Ltd</td><td>119370</td><td></td></tr><tr><td>Calcium chloride solution volumetric, 1.0 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>J&amp;amp;K Scientific Ltd</td><td>21114</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>V900933</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA kit</td><td>Millipore-linco</td><td>EZRMI-13K</td><td></td></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

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

生物工程

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 ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

免疫与感染

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 ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

生物学

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰岛血气 StarPEG Nanocoating 的表面工程

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