Name: production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d
Uploaded: 2018-05-19T13:30:00.000+00:00
Duration: 11 min 46 s
Description: Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

作者感谢 t. 帕尔默和 h. (斯坦福神经外科) 提供小鼠 npc. 矢量艺术在图 4被使用了和适应了从施维雅医学艺术在创造性的共同性归属 3.0 Unported 执照 (https://creativecommons.org/许可证/3.0/legalcode)。这项工作的一部分是在斯坦福纳米共享设施 (SNSF), 在国家科学基金会的支持下, ECCS-1542152 奖。N.A.S. 承认国立卫生研究院 (32GM008412) 国立医学科学院的支持。C.M.M. 承认 NIH NRSA 博士研究生奖学金 (F31 EB020502) 和 Siebel 学者计划的支持。S.C.H. 承认国家卫生研究院 (U19 AI116484 和 R21 EB018407)、国家科学基金会 (DMR 1508006) 和加利福尼亚再生医学研究所 (RT3-07948) 提供的支持。这项研究获得了再生康复研究 & 培训联盟 (AR3T) 的资助, 该协会得到了尤尼斯肯尼迪发育研究院国家儿童健康和人类发展研究所 (国家研究所) 的支持。神经系统疾病和中风 (NINDS), 和国家生物医学成像和生物工程研究所 (NIBIB) 的国家卫生研究院的奖励号 P2CHD086843。内容完全是作者的责任, 不一定代表国家卫生研究院的意见。

1. ELP 表达式协议
<ol><li>1天: 成长的起始群体
	<ol>
<li>用热处理25克 Luria 汤和15克琼脂每1升的超纯水制备氨苄西林和氯霉素琼脂板。一旦溶液冷却到摄氏60摄氏度, 加入1毫升氨苄西林 (100 毫克/毫升在超纯水) 和1毫升氯霉素库存 (34 毫克/毫升70% 乙醇) 至1升琼脂溶液的最终浓度100µg/毫升和34µg/毫升分别。用血清学吸管将20毫升最终溶液转移到10厘米培养皿中, 使琼脂凝固。包装培养皿与 parafilm, 并存储在4摄氏度。 注: 培养皿可储存在4摄氏度, 长达两周。</li>
		<li>从预先制作的细菌库存中条纹 BL21 (DE3) pLysS大肠杆菌的一个小样本, 其中含有 pET15b 的向量, 编码氨苄西林和氯霉素琼脂板上的 ELP。 注: 氨苄西林和氯霉素分别选择含有 pET15b 和 pLysS 载体的细菌。</li>
		<li>将条纹板倒在孵化器37摄氏度。让细菌菌落在一夜之间生长。 注: 不要孵育超过16小时的板材, 因为氨苄西林会降解, 不携带氨苄西林抵抗的菌落可以形成。</li>
	</ol></li>
	<li>2天: 起始文化和表达媒介的准备
	<ol>
<li>从孵化器中移除大肠杆菌区域性。Parafilm 在4摄氏度的情况下, 在4天的时间内储存。</li>
		<li>准备一个起始培养瓶 (250 毫升) 和表达培养烧瓶 (12x 1 升) 和高压釜。对于 1 l 的表达媒体, 添加47.6 克的了不起的汤和4毫升甘油到 1 l 的超纯水在 2 l 困惑的文化瓶, 并盖铝箔。 注: 典型产量为60-100 毫克/升蛋白表达培养基。</li>
		<li>将蒸压起动器装入预热前, 37 摄氏度摇动孵化箱, 无搅拌, 孵育。</li>
		<li>添加250µL 无菌过滤 (0.22 µm 过滤器) 氨苄西林股票 (100 毫克/毫升在超纯水) 到起始培养的最终浓度为100µg/毫升。立即开始搅拌 250 rpm 的起始文化。 注: 氯霉素仅用于琼脂板上的菌落选择, 不包括在液体培养中。</li>
		<li>通过在条纹板上添加一个含有大肠杆菌菌落的单 ELP 质粒, 并允许起始培养在37°c 时以16小时为起始培养基接种。</li>
		<li>将表达式培养基瓶放入预热前, 37 摄氏度摇动孵化器, 一夜无搅动地孵化, 使烧瓶在第二天早晨准备接种。</li>
	</ol></li>
	<li>3天: 诱导蛋白在大肠杆菌中的表达
	<ol>
<li>用新鲜的、无菌过滤的氨苄西林储存 (100 毫克/毫升的超纯水)。将1毫升氨苄西林储存到每瓶表达介质中, 最终浓度为100µg/毫升。</li>
		<li>开始搅动表达式媒体在 250 rpm。</li>
		<li>从任何表达式烧瓶中抽取2毫升的介质样本, 并在 600 nm (OD600) 的光学密度读数中添加试管作为空白。</li>
		<li>完成16小时的培养孵化后, 接种每一个表达瓶通过转移20毫升的起始培养到每个表达瓶通过血清吸管。</li>
		<li>完成1小时的搅拌后, 测量其中一个表达式烧瓶的 OD600 。执行此步骤后, 每20分钟测量 OD600 , 每次检查不同的烧瓶。</li>
		<li>在600的 OD 0.6 中, 将包含表达式烧瓶的振荡的温度降低到32摄氏度。</li>
		<li>每10分钟检查 OD600 。在600的 OD 0.8 中, 通过在超纯水中添加1毫升1米、无菌过滤β异丙基-β-d (IPTG) 来诱导表达式烧瓶。</li>
		<li>允许表达式烧瓶中的大肠杆菌表达为 7 h。</li>
		<li>20分钟前结束的表达, 预冷的大地板离心机到4°c。</li>
		<li>收集所有 12 L 的表达媒体到个别离心机容器和平衡。</li>
		<li>用地面离心机将表达式介质在 > 1.2万 x g 上离心15分钟, 4 摄氏度。</li>
		<li>从每个离心机容器中倒上清液。使用刮刀, 收集细胞颗粒到一个预先权衡的拉链袋。</li>
		<li>在无菌过滤的十缓冲器 (每25克颗粒中100毫升的缓冲器) 中重新悬浮颗粒, 并通过按摩颗粒去除多余的气泡。对于1升十缓冲器, 添加5.8 克氯化钠, 1.21 克的三基, 和0.37 克乙二胺四乙酸四酸 (EDTA) 钠盐二水合物到900毫升的超纯水。将 pH 值调整为 8.0, 并将其调至1升。对于这一步骤, pH 条是足够的。</li>
		<li>将含有重悬浮细胞颗粒的 zip 锁袋放入二次容器中, 并在一夜之间冻结-80 摄氏度。</li>
	</ol></li>
	<li>4-6 天: 通过冻融循环破裂细菌细胞壁
	<ol>
<li>从冰箱中取出冷冻颗粒, 并允许它在4摄氏度缓慢解冻, 用一个轨道振动筛轻轻搅拌。</li>
		<li>允许颗粒解冻, 直到有液体存在。添加〜30-40 毫克的脱氧核糖核酸酶 I (DNase) 解冻裂解。另外, 增加1毫升100毫米 phenylmethylsulphonyl 氟化物 (PMSF; 蛋白酶抑制剂) 在异丙醇每100毫升的细胞裂解物。让裂解液在一夜之间摇动。 注: 添加 DNase 和 PMSF 只对第一次冻融循环是必要的。 注意: PMSF 是有毒的, 如果吸入。处理 PMSF 粉时应使用口罩。</li>
		<li>一旦细胞裂解液完全解冻, 冻结裂解液在-80 摄氏度过夜, 或直到完全冻结。</li>
		<li>重复冻融过程共三个周期。在上一次冻融后, 把裂解液解冻4摄氏度。在纯化后, 贮存100µL 用于月桂酸钠-聚丙烯酰胺凝胶电泳 (SDS 页) 分析。</li>
		<li>最后解冻后, 用1米氢氧化钠将解冻的裂解液的 pH 值调整为9.0。对于这一步骤, pH 条是足够的。在一个轨道振动筛上孵育至少1小时 (过夜是适当的) 4 摄氏度。</li>
	</ol></li>
	<li>7-9 天: 冷热自旋热循环 ELP 净化
	<ol>
<li>将地面离心机冷却至离心前4摄氏度20分钟。</li>
		<li>整除和平衡解冻的裂解液成离心容器。</li>
		<li>离心样品在 > 1.5万 x g 为1小时在4°c。贮存100µL 的上清液, 用于 SDS 页分析纯化完成后。 注: 离心后, ELP 蛋白应留在上清, 由于其高溶解度在水中的温度低于其 LCST (< 32 摄氏度)。</li>
		<li>将上清液转移到新的离心容器中, 并适当平衡。</li>
		<li>在三部分中加入氯化钠 (nacl), 最终浓度为1米 (每上清液100毫升5.84 克氯化钠)。确保在三部分中加入氯化钠, 以使其有足够的溶解。</li>
		<li>鼓动3小时在37°c 在 250 rpm 在震动孵化器。</li>
		<li>1小时前搅拌结束, 预热一层离心机至37°c。</li>
		<li>离心机在 > 1.5万 x g 为1小时在37°c。贮存100µL 的上清液进行 SDS 页分析, 完成纯化后再丢弃剩余的上清。 注: 离心后, ELP 蛋白应为颗粒, 由于其在水中的溶解度低, 温度高于其 LCST (> 32 摄氏度)。</li>
		<li>通过添加10毫升的蒸压, 超纯水, 每1克颗粒, 重新悬浮颗粒。用金属铲把小球捣碎以帮助蛋白质的溶解。</li>
		<li>用1米氢氧化钠将解冻的裂解液的 pH 值调整为9.0。对于这一步骤, pH 条是足够的。</li>
		<li>在4摄氏度的一个轨道振动筛上一夜之间搅动。</li>
		<li>重复冷热旋转热循环过程共三个周期 (i. e, 重复步骤1.5.1 通过1.5.11 三总周期)。</li>
	</ol></li>
	<li>10-15 天: ELP 透析和冻干
	<ol>
<li>离心机在预冷离心机中, 在 > 1.5万 x g 1 小时, 在4摄氏度时再悬浮颗粒。贮存100µL 的上清液, 用于 SDS 页分析纯化完成后。</li>
		<li>将 3.5 kDa 透析膜中的剩余上清液透析为4摄氏度, 对预冷超纯水4升进行脱盐。每日两次更换透析水, 共3天6次。 注: 典型上清液量介于5至30毫升之间。</li>
		<li>离心机在预冷离心机的透析溶液在 > 1.5万 x g 1 小时在4摄氏度。储存100µL 的上清, 以 SDS 页分析在结束的纯化。</li>
		<li>将所产生的上清液冻结在预重锥形管的-80 摄氏度。</li>
		<li>Lyophilize 冷冻溶液3天, 质量最终产品, 以确定蛋白质产量。</li>
		<li>Parafilm 的管含有最终冻干 ELP 产品和存储在4摄氏度。</li>
		<li>运行 SDS 页, 以确定蛋白质的纯度。 注: SDS 的协议将根据特定的琼脂糖凝胶条件而异。在我们的实验中, 最终冻干蛋白被溶解到0.5 毫克/毫升在去离子水中, 在变性条件下, 在 12% (w/V) 丙烯酰胺凝胶中以140伏70-100 分钟的浓度运行。</li>
	</ol></li>
</ol>2. 3D 弹性蛋白类水凝胶中的细胞封装
<ol><li>硅胶模具的制备
	<ol>
<li>使用所需直径的活检冲床在0.5 毫米厚的硅胶板上创建孔, 并在每个孔周围切出一个正方形。重复所需的模具数量。 注: 模具的直径和厚度可根据特定的应用和细胞培养条件进行调整。在实践中, 对于 50 106细胞/mL 的细胞培养, 建议用4毫米和5毫米直径的0.5 毫米厚模具分别进行足够的 DNA/RNA 和蛋白质提取。对于染色, 一个2毫米的活检冲床可以用来代替每平方模创建三个相邻的孔, 以便三的复制可以被染色每井。</li>
		<li>用镊子去除单个模具两侧的塑料包装。 注: 避免接触暴露的硅胶表面, 因为污染会降低未来的等离子结合效率。</li>
		<li>使用镊子, 安排相同数量的裸硅胶模具和玻璃盖玻片 (1, 12 毫米直径) 的交替行上的倒盖48井板。一旦完成, 盖上48井板的盖子, 以避免污染。</li>
		<li>氧气等离子处理整个48井板盖子 (i. e, 模子和玻璃幻灯片)。紧接着, 用镊子将硅胶模具倒置到相邻的玻璃盖玻片上。压紧模具, 确保粘合。让模具在室温下孵化1小时。 注: 氧等离子体治疗的持续时间视使用的仪器而定。我们的仪器的典型条件是一个操作气体压力窗口在 0.3-4 毫巴之间, 氧气气体流动在 20 cm3/分钟, 并且样品暴露对血浆为 10-20 s。</li>
		<li>用热处理消毒霉菌。在室温下将模具贮存在无菌环境中, 直至使用。 注: 模具可以无限期储存在这个阶段。</li>
	</ol></li>
	<li>弹性蛋白类蛋白质库的制备方法研究
	<ol>
<li>从4摄氏度的储藏中取出冻干 ELP。在打开管子之前, 将蛋白质加热到室温, 以确保在重复使用的蛋白质上没有凝结。</li>
		<li>在 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 中溶解 ELP, 在4摄氏度时持续搅拌 (i. e, 旋转) 过夜。 注: ELP 库存溶液浓度将进一步稀释, 添加交联剂溶液在用户定义的体积比。例如, 对于 3% (瓦特/v) 最终 ELP 浓度, 准备一个 3.75% (瓦特/v) ELP 库存解决方案, 将稀释在4:1 容积率的 ELP 溶液: 交联剂溶液。根据需要调整所需应用的浓度。</li>
		<li>无菌过滤器 ELP 库存解决方案使用0.22 µm 注射器过滤器。不使用时, 将 ELP 储存在冰上。</li>
	</ol></li>
	<li>在生物安全柜中, 用无菌镊子将无菌的模具转移到24井组织培养板上。</li>
	<li>THPC 库存液的制备
	<ol>
<li>稀释 THPC 在 DPBS 前使用。根据最终 ELP 浓度和期望交联率调整 THPC 溶液的浓度。 注: 对于该协议, 最终的 ELP 浓度为 3% (瓦特/v) 将通过混合 ELP 股票溶液与 THPC 溶液的体积比为4:1。必要时进行调整。</li>
		<li>添加2.6 µL THPC 溶液 (80% 在水中) 到997.4 µL DPBS。 注: 这种浓度的 THPC 相当于1:1 的羟基甲基基团的化学计量比的 THPC 和主要胺的 ELP 蛋白时, 混合溶液在描述的4:1 容量比为最后 3% (w/v) ELP 水凝胶。THPC 溶液是粘性的, 液滴可以粘在吸管尖端的一侧。为了准确的浓度, 避免这样的水滴稀释成 DPBS。用氮气清除 THPC 储存容器, 防止磷化氢的氧化和交联剂的失活。</li>
		<li>漩涡的解决方案, 以混合和保持冰。</li>
		<li>无菌过滤器 THPC 库存解决方案使用0.22 µm 注射器过滤器。用无菌 DPBS 进一步稀释 THPC 的溶液, 以达到较低的化学计量交联率 (e. g, 0.5: 1 或 0.75:1)。 注: THPC 是氧敏感的, 稀释溶液应在准备后几个小时内使用。</li>
	</ol></li>
	<li>分离细胞通过孵化与胰蛋白酶-EDTA 进入单细胞悬浮, 颗粒细胞, 并计数细胞重新悬浮在培养基中使用 hemocytometer。 注意: 此步骤的确切协议将严重依赖于所需的单元格类型和应用程序。对于整个协议中使用的神经祖细胞, 进行了室温下0.025% 胰蛋白酶-EDTA 孵育, 1.5 分钟。细胞以 200 x g 的颗粒为2分钟。为了提高细胞的生存能力, 应在正常培养基条件下悬浮细胞。用于细胞封装的典型细胞密度范围从 1-50 106细胞/最终水凝胶体积的 mL。必要时进行调整。</li>
	<li>将所需的细胞数整除成无菌的1.5 毫升离心管。</li>
	<li>将细胞在 200 x g 的3分钟内离心, 吸入上清, 并将细胞颗粒放在冰上。 注: 要完全吸入所有上清液, 以减轻 ELP 和 THPC 交联剂的进一步稀释是至关重要的。特定的离心速度会因细胞类型而异。</li>
	<li>在 ELP 库存溶液中重新悬浮细胞颗粒, 使体积为最终体积的 80% (假设 4:1 ELP 溶液的比值: THPC 溶液)。吸管混合20-25 倍, 产生细胞和 ELP 的均匀混合物。 注意: 避免夹紧管子底部, 以减轻 ELP 的温度升高和随后的相变。每 2 mm 模具与三复制需要7.5 µL 的最终卷 (i. e, 6 µL 的 ELP 库存解决方案和1.5 µL THPC 库存解决方案) 平均分成3个孔 (i. e, 2.5 µL 最终体积每孔)。对于4和 5 mm 的模具, 分别需要7.5 µL 和15.5 µL 的最终体积。</li>
	<li>添加 THPC 库存解决方案的细胞/ELP 暂停剩余的20% 最终体积。吸管混合20-25 倍, 产生一个均匀的混合物。</li>
	<li>立即用圆形运动将细胞/ELP/THPC 混合物的相应最终体积注入到每个模具中。对所有模具重复。</li>
	<li>在室温下孵化15分钟的样品, 其次是37摄氏度的额外孵化15分钟。 注: 第一潜伏期将有助于促进水凝胶的初始交联, 然后将温度升高到 ELP 的 LCST 以上, 并诱导其热相分离。</li>
	<li>慢慢添加750µL 的暖细胞培养基, 每井24井板, 避免扰乱凝胶。</li>
	<li>将水凝胶孵化37摄氏度, 7 天。 注: 根据单元格类型, 建议每1-2 天进行全介质更改。要限制凝胶的应力, 请使用200µL 吸管尖端的玻璃巴斯德吸管, 当从井中吸气时。</li>
</ol>3. 3D ELP 水凝胶细胞免疫化学分析
<ol><li>在30毫升 DPBS 中混合10毫升 16% (瓦特/v) 多聚甲醛 (粉煤灰), 制备固定溶液。加热溶液到37摄氏度。</li>
	<li>从24井板中吸取培养基, 用1毫升的 DPBS 轻轻冲洗。</li>
	<li>添加750µL 的固定液, 每一个井和孵化在37°c 30 分钟。不要使用组织培养孵化器, 以避免污染其他文化与粉煤灰蒸气。</li>
	<li>小心地从每个井中抽出固定液, 将粉煤灰丢弃到适当的危险废物容器中。 注意: 接触到粉煤灰会引起皮肤和眼睛发炎。戴手套/安全眼镜, 在化学油烟机工作。</li>
	<li>在每个样品中加入1毫升的 DPBS。立即吸入 DPBS 并丢弃在粉煤灰废料容器中。</li>
	<li>每两次用1毫升的 DPBS 清洗样品10分钟。 注: 样品可以储存在 DPBS 4 摄氏度, 长达一周后, 密封板与 Parafilm。</li>
	<li>Permeabilize 每个样品与750µL 的通透溶液 (100 毫升 DPBS 和0.25 毫升的海卫一 X-100;PBST) 为1小时在室温上的摇杆在 15 rpm。</li>
	<li>吸入 PBST 并添加750µL 的阻断溶液 (PBS 95 毫升, 5 克牛血清白蛋白 (BSA), 5 毫升血清, 和0.5 毫升的海卫 X-100) 给每个样本。在室温下孵育3小时, 在 15 rpm 的摇杆上。 注: 阻断溶液应包含在使用前通过0.22 µm 过滤器引发的宿主物种的血清 (e. g, 山羊, 驴) 和无菌过滤。</li>
	<li>准备抗体稀释液 (97 毫升的 DPBS, 2.5 克 BSA, 2.5 毫升血清 (从同一寄主物种, 在步骤 3.8) 和0.5 毫升的海卫 X-100)。使用抗体稀释液稀释原抗体, 并在每个样品中添加500µL 溶液。用 Parafilm 封住盘子, 在4摄氏度的时候在摇杆上孵化一夜。 注: 巢蛋白和 Sox2 的抗体稀释液中的原发抗体稀释 1:400, 从制造商的原始浓度。</li>
	<li>从每个样品中吸取抗体溶液, 用 PBST 在15转每分钟的摇杆上以60分钟的温度清洗样品。重复洗涤步骤3次。</li>
	<li>使用抗体稀释液稀释二次抗体和5毫克/毫升 DAPI (1:2, 000), 并将溶液的500µL 添加到每个样品中。用铝箔盖住24井板, 在4摄氏度的摇杆上孵化过夜。 注意: 由于次要抗体是轻敏感的, 必须保护样品免受漂白的所有后续步骤。山羊抗小鼠和山羊抗兔二次抗体在抗体稀释溶液中稀释 1:500, 从制造商的原始浓度。</li>
	<li>从每个样品中抽取抗体溶液, 在摇杆的室温下用 PBST 清洗样品30分钟。重复洗涤步骤3次。</li>
	<li>将硬集安装介质的一滴放置到玻璃滑轨的表面上。使用镊子, 从模具中取出多余的溶液, 轻轻地将模具的边缘沾在纸巾上。小心地将模具倒置到安装介质上, 避免引入气泡。</li>
	<li>允许安装介质在室温下硬化48小时。将样品存放在室温或4摄氏度。在成像前, 允许安装介质完全设置为48小时, 因为介质的折射率会在硬化过程中发生变化。用透明指甲油将样品密封在玻璃盖板上, 以减少污染或试样运动。</li>
	<li>使用共焦显微镜对样品进行图像处理。</li>
</ol>

<ol>
	<li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+Cell-Matrix+Adhesions+to+the+Third+Dimension.">Taking Cell-Matrix Adhesions to the Third Dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Birgersdotter, A., Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+perturbation+in+vitro-A+growing+case+for+three-dimensional+(3D)+culture+systems.">Gene expression perturbation in vitro-A growing case for three-dimensional (3D) culture systems.</a> Seminars in Cancer Biology. 15 (5), 405-412 (2005).</li><li>G&#243;mez-Lech&#243;n, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expression+of+differentiated+functions+in+hepatocytes+cultured+in+three-dimensional+collagen+matrix.">Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix.</a> Journal of Cellular Physiology. 177 (4), 553-562 (1998).</li><li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension+-+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension - how 3D culture microenvironments alter cellular cues.</a> Journal of Cell Science. 125 (13), 3015-3024 (2012).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature Reviews Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Justice, B. A., Badr, N. A., Felder, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture+opens+new+dimensions+in+cell-based+assays.">3D cell culture opens new dimensions in cell-based assays.</a> Drug Discovery Today. 14 (1-2), 102-107 (2009).</li><li>Caliari, S. R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+hydrogels+for+cell+culture.">A practical guide to hydrogels for cell culture.</a> Nature Methods. 13 (5), 405-414 (2016).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnology and Bioengineering. 103 (4), 655-663 (2009).</li><li>Madl, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+neural+progenitor+cell+stemness+in+3D+hydrogels+requires+matrix+remodelling.">Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.</a> Nature Materials. 16 (12), 1233-1242 (2017).</li><li>Discher, D. E., Janmey, P., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Cells+Feel+and+Respond+to+the+Stiffness+of+Their+Substrate.">Tissue Cells Feel and Respond to the Stiffness of Their Substrate.</a> Science. 310 (5751), 1139-1143 (2005).</li><li>Sun, Y., Villa-Diaz, L. G., Lam, R. H. W., Chen, W., Krebsbach, P. H., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+Regulates+Fate+Decisions+of+Human+Embryonic+Stem+Cells.">Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells.</a> PLoS ONE. 7 (5), e37178 (2012).</li><li>Ehrbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+Role+of+Matrix+Stiffness+in+3D+Cell+Migration+and+Remodeling.">Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling.</a> Biophysical Journal. 100 (2), 284-293 (2011).</li><li>Rowlands, A. S., George, P. A., Cooper-White, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+osteogenic+and+myogenic+differentiation+of+MSCs:+interplay+of+stiffness+and+adhesive+ligand+presentation.">Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation.</a> American Journal of Physiology - Cell Physiology. 295 (4), 1037-1044 (2008).</li><li>Lampe, K. J., Antaris, A. L., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+three-dimensional+engineered+protein+hydrogels+for+tailored+control+of+neurite+growth.">Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth.</a> Acta Biomaterialia. 9 (3), 5590-5599 (2013).</li><li>Kilian, K. A., Mrksich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+Stem+Cell+Fate+by+Controlling+the+Affinity+and+Density+of+Ligand-Receptor+Interactions+at+the+Biomaterials+Interface.">Directing Stem Cell Fate by Controlling the Affinity and Density of Ligand-Receptor Interactions at the Biomaterials Interface.</a> Angewandte Chemie International Edition. 51 (20), 4891-4895 (2012).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Hydrogel+Substrates+with+Tunable+Mechanical+Properties.">Preparation of Hydrogel Substrates with Tunable Mechanical Properties.</a> Current Protocols in Cell Biology. , 10.16.1-10.16.16 (2010).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+A+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: A complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>DiMarco, R. L., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+Materials+through+Modular+Protein+Engineering.">Multifunctional Materials through Modular Protein Engineering.</a> Advanced Materials. 24 (29), 3923-3940 (2012).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+proteins+by+fusion+with+thermally-responsive+polypeptides.">Purification of recombinant proteins by fusion with thermally-responsive polypeptides.</a> Nature Biotechnology. 17 (11), 1112-1115 (1999).</li><li>Aladini, F., Araman, C., Becker, C. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+characterization+of+elastin-like+polypeptides+(ELPs)+with+variable+guest+residues.">Chemical synthesis and characterization of elastin-like polypeptides (ELPs) with variable guest residues.</a> Journal of Peptide Science. 22 (5), 334-342 (2016).</li><li>McMillan, R. A., Caran, K. L., Apkarian, R. P., Conticello, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Topographic+Imaging+of+Environmentally+Responsive,+Elastin-Mimetic+Hydrogels.">High-Resolution Topographic Imaging of Environmentally Responsive, Elastin-Mimetic Hydrogels.</a> Macromolecules. 32 (26), 9067-9070 (1999).</li><li>McMillan, R. A., Conticello, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+Elastin-Mimetic+Protein+Gels+Derived+from+a+Well-Defined+Polypeptide+Precursor.">Synthesis and Characterization of Elastin-Mimetic Protein Gels Derived from a Well-Defined Polypeptide Precursor.</a> Macromolecules. 33 (13), 4809-4821 (2000).</li><li>Chung, C., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrakis(hydroxymethyl)+Phosphonium+Chloride+as+a+Covalent+Cross-Linking+Agent+for+Cell+Encapsulation+within+Protein-Based+Hydrogels.">Tetrakis(hydroxymethyl) Phosphonium Chloride as a Covalent Cross-Linking Agent for Cell Encapsulation within Protein-Based Hydrogels.</a> Biomacromolecules. 13 (12), 3912-3916 (2012).</li><li>Romano, N. H., Madl, C. M., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+RGD+ligand+density+and+L1CAM-mediated+Schwann+cell+interactions+synergistically+enhance+neurite+outgrowth.">Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth.</a> Acta Biomaterialia. 11, 48-57 (2015).</li><li>Shah, M., Hsueh, P. Y., Sun, G., Chang, H. Y., Janib, S. M., MacKay, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+elastin-like+polypeptide+nanoparticles.">Biodegradation of elastin-like polypeptide nanoparticles.</a> Protein Science. 21 (6), 743-750 (2012).</li><li>Nettles, D. L., Chilkoti, A., Setton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+elastin-like+polypeptides+in+tissue+engineering.">Applications of elastin-like polypeptides in tissue engineering.</a> Advanced Drug Delivery Reviews. 62 (15), 1479-1485 (2010).</li><li>Madl, C. M., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine-Selective+Functionalization+for+Bio-Orthogonal+Cross-Linking+of+Engineered+Protein+Hydrogels.">Tyrosine-Selective Functionalization for Bio-Orthogonal Cross-Linking of Engineered Protein Hydrogels.</a> Bioconjugate Chemistry. 28 (3), 724-730 (2017).</li><li>Zhu, D., Wang, H., Trinh, P., Heilshorn, S. C., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+protein-hyaluronic+acid+(ELP-HA)+hydrogels+with+decoupled+mechanical+and+biochemical+cues+for+cartilage+regeneration.">Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.</a> Biomaterials. , 132-140 (2017).</li><li>Madl, C. M., Katz, L. M., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-Orthogonally+Crosslinked,+Engineered+Protein+Hydrogels+with+Tunable+Mechanics+and+Biochemistry+for+Cell+Encapsulation.">Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation.</a> Advanced Functional Materials. 26 (21), 3612-3620 (2016).</li><li>Cai, L., Dinh, C. B., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-pot+synthesis+of+elastin-like+polypeptide+hydrogels+with+grafted+VEGF-mimetic+peptides.">One-pot synthesis of elastin-like polypeptide hydrogels with grafted VEGF-mimetic peptides.</a> Biomater Sci. 2 (5), 757-765 (2014).</li><li>Straley, K. S., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+tuning+of+multiple+biomaterial+properties+using+protein+engineering.">Independent tuning of multiple biomaterial properties using protein engineering.</a> Soft Matter. 5 (1), 114-124 (2009).</li><li>Baneyx, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Escherichia+coli.">Recombinant protein expression in Escherichia coli.</a> Current Opinion in Biotechnology. 10 (5), 411-421 (1999).</li><li>Graumann, K., Premstaller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacturing+of+recombinant+therapeutic+proteins+in+microbial+systems.">Manufacturing of recombinant therapeutic proteins in microbial systems.</a> Biotechnology Journal. 1 (2), 164-186 (2006).</li><li>Tashiro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Synthetic+Peptide+Containing+the+IKVAV+Sequence+from+the+A+Chain+of+Laminin+Mediates+Cell+Attachment,+Migration,+and+Neurite+Outgrowth.">A Synthetic Peptide Containing the IKVAV Sequence from the A Chain of Laminin Mediates Cell Attachment, Migration, and Neurite Outgrowth.</a> Journal of Biological Chemistry. 264 (27), 16174-16182 (1989).</li></ol>

作者没有什么可透露的。

重组蛋白的表达和纯化是制备高重现性生物材料的有力工具。由于商业化分子克隆的出现, 自定义的重组质粒可以从几个供应商购买, 这大大减少了使用 ELPs 等材料的时间。同样, 当原始的工作得到联邦合同的支持, 未来的工作将用于非营利用途时, 可以直接从原始实验室请求质粒。以前已为几个 ELP 变体31发布了完整的 ELP 氨基酸序列。然而, 从表达到最终纯化重组蛋白的过程涉及?...

在过去的一个世纪中, 二维 (2D) 组织培养已发展成为一个完整的工具集, 研究基本细胞生物学在体外。此外, 相对低成本和简单的2D 细胞培养协议导致了它在许多生物和医学学科的应用。然而, 过去的研究表明, 传统的2D 平台可能导致结果与收集的在体内明显偏离, 导致宝贵的时间和资金浪费为临床研究1,2, 3。我们和其他人推测, 这种差异可能是由于缺乏对2D 表面培养的细胞所提供的本土生物化学和生物物理线索, 这可能是对各种细胞类型的最佳增殖和成熟所必需的。
为了解决这些限制并帮助弥合 2D体外和体内研究之间的差距, 研究人员开发了三维 (3D) 水凝胶平台, 用于细胞封装14,5 ,6。水凝胶是一种理想的材料, 用于重述细胞外基质 (ECM)在体内的内源性微环境, 因为它们的组织样的机械特性和水肿胀结构, 使养分迅速运输和信号因子7,8。此外, 3D 水凝胶可设计为对脚手架的机械和生化性能有独立的控制。矩阵力学9,10,11,12和细胞胶粘剂配体13,14,15是众所周知的影响细胞行为体外和体内.因此, 3D 水凝胶具有可调谐性质, 为研究细胞与微环境的因果关系提供了平台。理想的3D 水凝胶基质的标准包括简单的, 无细胞毒性的细胞封装, 以及独立调谐生理相关的力学特性和模仿本机细胞粘附图案。
合成 (e. g, 聚乙二醇, 聚乳酸, 聚 (乙醇酸)) 和自然地 (e. g, 海藻酸盐, 胶原, 胶) 水凝胶具有超过 2D体外培养平台的优势;但是, 它们也有很大的缺点, 限制了它们的适用性。首先, 许多合成和自然衍生的平台需要苛刻的交联条件, 可能对哺乳动物细胞有潜在的毒性, 导致细胞活力降低7。此外, 许多合成平台缺乏本土生物活性, 需要通过二次化学反应进行功能化, 这可以增加成本和复杂性16。最后, 虽然自然派生的材料通常包含固有的生物活动域, 但它们往往被高批对批次的可变性所困扰, 并且通常仅限于形成相对较弱的凝胶7,17。
重组蛋白工程为材料设计提供了一个独特的工具集, 允许对蛋白质序列进行显式控制, 并通过扩展, 得到最终的水凝胶支架的潜在机械和生化特性18。此外, 通过利用众所周知的生物机械的大肠杆菌 ( 大肠杆菌) 来表达蛋白质, 材料可以生产成本效益和一贯的有限的内部和批内的变化。这里提出的弹性蛋白样蛋白质 (ELP) 有三个工程领域: (1) T7 和 His6 标签, 允许标签通过荧光标记抗体, (2) 一个 "弹性体样" 区域, 赋予弹性力学性能, 并允许化学交联, (3) 一个 ' 生物活性 ' 区域, 编码的细胞粘接图案。
我们的弹性蛋白样区域基于规范 (Val-甘-Xaa-甘)5弹性蛋白序列, 其中四 ' Xaa ' 氨基酸站点是异亮氨酸 (微笑), 但可以被设计为任何氨基酸, 除了脯氨酸。这一序列赋予了具有较低临界溶液温度 (LCST) 行为的重组 ELPs, 通过热循环19、20, 可以利用它进行简单的纯化后表达式。通过修改来宾 "Xaa" 残滓21、22, 可以将此 LCST 属性调整为在不同温度下进行热聚合。
在这里, "Xaa" 的位置上的五弹性蛋白样重复已取代了胺呈现赖氨酸 (赖氨酸) 氨基酸, 这是用于水凝胶交联。我们以前的工作表明, 与胺反应交联剂四 (羟甲基) 磷氯 (THPC)23的反应, 非细胞毒性和可靠的结合。通过改变总的蛋白质含量和交联剂浓度, 我们能够产生水凝胶, 可以调谐到跨生理相关的刚度范围 (~ 0.5-50 帕)9,23,24。除了调整机械性能外, 水凝胶内的细胞黏附力是由 ELP 蛋白的主干内的规范细胞粘附域整合而来的结果。例如, 采用扩展的纤维连接蛋白衍生的 ' RGDS ' 氨基酸序列允许细胞黏附和构象的灵活性, 而炒, 无约束力的 ' RDGS ' 变体限制细胞基质黏附力24。通过调节细胞胶粘剂与非粘附蛋白的比值以及总蛋白浓度, 我们能够有效地生产出跨越多种配体浓度的水凝胶。Resultantly, 我们开发了一个水凝胶平台, 具有分离的生物化学和生物物理性质, 可以独立调整, 以最佳的3D 文化的各种细胞类型。
除了基体刚度和胶粘剂配体调谐, 重组水凝胶提供了设计特定材料退化剖面的能力, 这对于细胞在3D 上下文中的扩散、增殖和迁移是必要的4,9. 这种退化是由蛋白酶的细胞分泌所提供的, 具体目标是扩展的 "RGDS"9或弹性蛋白样序列25。ELP 水凝胶也被证明支持后续的生化化验, 这是必要的研究细胞的生存能力和功能, 包括免疫化学和 DNA/RNA/蛋白质提取的定量逆转转录-聚合酶链反应 (qRT PCR) 和西方印迹9。ELP 变体也已在许多体内模型中使用, 并且已知免疫系统26可以很好地耐受。
结合起来, ELP 作为细胞封装研究的物质平台, 与合成或自然衍生的材料平台相比, 具有广泛的优势, 它们往往缺乏相同程度的生物化学和生物物理调谐和重复。另外, ELP 的简单和非细胞毒性使用与各种各样的细胞类型 (e. g, 小鸡背根神经节14,24, 小鼠神经祖细胞9, 人骨髓间充质干细胞27, 牛新生儿软骨细胞28, 人内皮细胞29,30) 允许与2D 细胞培养相比, 更具生理学意义的内生 3D ECM 模型。在此, 我们提出了一个协议, 以表达 recombinantly 衍生, ELPs 作为一个可调谐的水凝胶平台, 3D 细胞封装。进一步提出了包覆细胞的荧光标记和共焦显微术的方法。

<table><tbody><tr><td>&lt;strong&gt;Elastin-Like Protein Expression and Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>70% Ethanol</td><td>RICCA Chemical</td><td>2546.70-1</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich</td><td>A3920-500G</td><td></td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25G</td><td></td></tr><tr><td>Bacto Agar</td><td>Thermo Fisher Scientific</td><td>9002-18-0&amp;nbsp;</td><td></td></tr><tr><td>BL21(DE3)pLysS Competent Cells</td><td>Invitrogen</td><td>C606003</td><td></td></tr><tr><td>Chloramphenicol</td><td>Amresco</td><td>0230-100G&amp;nbsp;</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>EDTA disodium salt, dihydrate</td><td>Thermo Fisher Scientific</td><td>O2793-500</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific</td><td>A451-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP1755-10G</td><td></td></tr><tr><td>Luria Broth</td><td>EMD Millipore</td><td>1.10285.5007</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals</td><td>195381</td><td></td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S 8045-1KG</td><td></td></tr><tr><td>Syringe Filter Unit (0.22 &amp;mu;m)</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>Terrific Broth</td><td>Millipore</td><td>71754-4</td><td></td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>&lt;strong&gt;Cell Encapsulation in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>0.5 mm thick silicone sheet</td><td>Electron Microscopy Science</td><td>70338-05</td><td></td></tr><tr><td>24-well tissue culture plates&amp;nbsp;</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Disposable Biopsy Punch (2 mm)</td><td>Integra Miltex</td><td>33-31</td><td></td></tr><tr><td>Disposable Biopsy Punch (4 mm)</td><td>Integra Miltex</td><td>33-34</td><td></td></tr><tr><td>Disposable Biopsy Punch (5 mm)</td><td>Integra Miltex</td><td>33-35</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>No. 1 12 mm glass coverslips</td><td>Thermo Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td><td>Sigma-Aldrich</td><td>404861-100ML</td><td></td></tr><tr><td>0.5% Tryspin/EDTA</td><td>Thermo Fisher&amp;nbsp;</td><td>15400054</td><td></td></tr><tr><td>&lt;strong&gt;Immunocytochemistry of Cells in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16% (w/v) Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15701</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306&amp;nbsp;</td><td></td></tr><tr><td>Donkey Serum</td><td>Lampire Biological Labs</td><td>7332100</td><td></td></tr><tr><td>Goat anti-mouse Secondary Antibody (AF488)</td><td>Molecular Probes</td><td>A-11017</td><td></td></tr><tr><td>Goat anti-rabbit Secondary Antibody (AF546)</td><td>Molecular Probes</td><td>A-11071</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td></td></tr><tr><td>Mouse Nestin Primary Antibody</td><td>BD Pharmingen</td><td>556309</td><td></td></tr><tr><td>Mouse Sox2 Primary Antibody</td><td>Cell Signaling Technology</td><td>23064S</td><td></td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Vectashield Hardset Mounting Medium&amp;nbsp;</td><td>Vector Labs</td><td>H-1400&amp;nbsp;</td><td></td></tr></tbody></table>

production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d

二维 (2D) 组织培养技术对于我们理解基本细胞生物学是必不可少的。然而, 传统的2D 组织培养系统缺乏三维 (3D) 矩阵, 导致在收集的体外和体内中的结果之间存在显著的脱节。为了解决这一限制, 研究人员设计了3D 水凝胶组织培养平台, 可以模拟体内细胞微环境的生物化学和生物物理特性。这项研究促使需要开发支持3D 细胞封装和下游生化检测的材料平台。重组蛋白工程为3D 水凝胶材料的设计和开发提供了独特的工具集, 允许对蛋白质序列进行特定的控制, 因此, 通过推广, 其潜在的机械和生化性质的结果矩阵。在这里, 我们提出了一个协议, 以表达 recombinantly 衍生弹性体样蛋白 (ELP), 它可以用来形成水凝胶具有独立可调谐的机械性能和细胞粘附物的浓度。我们进一步提出了 ELP 水凝胶内的细胞封装方法, 以及随后的嵌入细胞的免疫荧光染色, 用于下游分析和定量。

此协议中使用的 ELPs 包括五区域: T7 标记、His6 标记、肠激酶 (克朗) 解切站点、生物活动区域和弹性蛋白样区域 (图1)。T7 和 His6 标签允许通过标准的西方印迹技术容易识别。如果需要的话, 引入克朗解理的地方允许酶去除标记区域。生物活性区域编码的扩展, 纤维连接蛋白衍生细胞胶粘剂 (' RGDS ') 或非胶粘剂 (' RDGS ') 序列。最后, 类似弹性蛋白的区域?...

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

重组蛋白工程水凝胶有利于3D 细胞培养, 因为它们允许完整的调谐的聚合物骨干, 因此, 细胞微环境。在这里, 我们描述了重组弹力蛋白样蛋白质纯化的过程及其在3D 水凝胶细胞封装中的应用。

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

production-elastin-like-protein-hydrogels-for-encapsulation

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Stanford University. 重组蛋白工程水凝胶有利于3D 细胞培养, 因为它们允许完整的调谐的聚合物骨干, 因此, 细胞微环境。在这里, 我们描述了重组弹力蛋白样蛋白质纯化的过程及其在3D 水凝胶细胞封装中的应用。

视频: Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

科研

生物化学

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

生物工程

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

糖注射水凝胶的制备及其在3D 细胞培养中的应用

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

化学

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

生物学

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

琼脂糖流体凝胶在凝胶过程中通过剪切处理形成，用于悬浮3D生物打印

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

光调谐生物打印水凝胶，用于探测细胞对 ECM 硬化的反应

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

作者聚焦：使用 3D 打印光可调谐水凝胶了解慢性肺病 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

3D生物打印光可调水凝胶研究成纤维细胞活化

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...