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>
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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一些关键步骤, 通常可以导致产量下降或质量较低的产品。ELP 制剂的一些最常见的问题有以下几种: (1) 储存的细菌种群的质量, (2) 第一次冻融循环破坏细菌膜, (3) 通过热循环进行蛋白质纯化。
蛋白质表达与其他非生物生产方法的主要区别在于, 我们正在利用重组宿主的生物机械来合成聚合物。随后, 这种技术具有独特的局限性: 细胞死亡或损伤。细胞死亡最常见的表现是在裸板或异常小的菌落生长相对缓慢后, 细菌菌落数量减少。细菌类股如果保持谨慎, 可维持多年稳定;然而, 由于反复使用或冷冻衰竭而连续的冻融循环可以降低细胞的生存能力或导致 DNA 损伤。典型的 BL21 细菌种群使用10% 和40% 甘油之间的体积与悬浮细胞混合。甘油的目的是减少在冷冻过程中成核冰晶的膜损伤。因此, 使用低浓度 (< 10%) 可以导致一个受损的膜, 而较高浓度 (> 40%) 可以抑制冻结点足够到那里的股票从来没有冻结导致细胞死亡。然而, 即使在最佳甘油水平, 细菌库存不应该被允许完全解冻, 因为复合膜损伤从再结冰和细胞毒作用从甘油可能导致减少存货生存能力和脱氧核糖核酸损伤。因此, 如果观察到细菌储存会导致低菌落计数或细胞以一贯缓慢的速度分裂 (表现为在表达过程中缓慢 OD600坡道速率), 重新转换质粒并制作新的股票是一个简单的解决此问题的第一种方法。考虑到这一点, 为了确保细菌储存和 DNA 完整性的长期维持, 最好将你的质粒拷贝保存在水中, 而不是细胞内。以这种方式存储 dna 将确保在未预见的事件 (如失败的库存或冷藏库故障) 中, 原始 DNA 的可靠来源可以用于转化。
ELP 制造的另一个关键步骤是从表达宿主中纯化目标蛋白。蛋白质提取从大肠杆菌是通过打破细胞壁使用形成的成核冰晶在悬浮细胞裂解物在结冰以后, 与连续的冻融循环进一步复合。细胞壁破裂的替代方法可以使用, 如超声波或压力机。特别是, 连续冷冻解冻的裂解是有利的, 因为它只需要一个冷藏库, 没有其他专业设备。然而, 这个过程 nonspecifically 释放 DNA, RNA 和蛋白质污染物, 除了蛋白酶, 有可能降低目标蛋白。因此, 为了避免污染和降低产量, 脱氧核糖核酸酶 I (DNase) 和 phenylmethanesulfonyl 氟 (PMSF) 被添加到细胞裂解酶降解 DNA 和抑制蛋白酶, 分别。在加入 DNase 之前, DNA 的存在可以视作在第一次解冻后, 整个重新悬浮细胞裂解过程中的 "粘性" 出现。DNase 积极降解这种 DNA, 从而降低了细胞裂解液的粘度, 使其更容易通过离心提纯。DNA 的最佳分解可以通过确保细胞裂解液看起来是完全液态的, 并使粘着的外观不再可见而在视觉上得到证实。我们在实践中观察到, 添加0.1 毫克的 DNase 每毫升细胞裂解液足以达到必要的降解。然而, 如果仍然观察到 DNA 的存在, 可以增加更多的 DNase, 然后再增加三小时的躁动。如果 DNase 在任何裂解液有足够解冻的可能性之前过早添加, 也会出现类似的问题。在这种情况下, 较冷的温度可以限制 DNase 过早失活导致 DNA 降解的效率。为了避免这一问题, 通常最好的做法是允许重新悬浮颗粒解冻约8小时前与 DNase 治疗。此外, 如果报告的蛋白质产量低, 并且 DNA 的分解已经足够, 增加更多的 PMSF, 以帮助进一步减少可能的蛋白质降解蛋白酶可能需要。
确保 ELPs 最佳表达的其他考虑因素包括仔细了解所选择的抗生素的优点和局限性。在这里, 含有氨苄西林抗性基因的 pET15b 载体用于蛋白质表达。在功能上, pET 载体系列允许在一个成功的归纳32,33之后, 以50% 的细菌蛋白表达的方式对目标蛋白进行有效的蛋白质表达。然而, 氨苄西林作为一种选择抗生素具有一定的局限性, 可能干扰最佳表达。首先, 在出现大肠杆菌的情况下, 氨苄西林的降解可能会因酶的释放而迅速发生。如果氨苄西林的足够数量被降解, 氨苄西林编码质粒 (即ELP 编码质粒) 可能完全丧失。因此, 当表达 ELPs 的时间较长时, 应在连续的时间点仔细监测蛋白质的表达水平, 以确保足够数量的 ELP 编码基因保持适当的表达。可能的方法, 以解决 lactamases 的积累包括纺纱的起始培养和重新悬浮细胞在无抗生素培养基前接种的表达培养基。这一过程有效地限制了抗生素降解酶的转移, 并确保更大一部分的细胞含有靶蛋白编码载体。此外, 氨苄西林的保质期有限, 约三周。因此, 蛋白质表达的培养基应在使用前最多两周保存在4摄氏度。最后, 为了确保氨苄西林在起始和表达培养基中的有效性, 在使用前应立即产生新鲜的氨苄西林库存液, 因为长期贮存可能会导致一种不太有效的抗生素。
LCST 的存在允许通过热循环简单地净化 ELPs。具体地说, 在较高的温度和盐的存在下, 熵力导致 ELPs 变得不那么可溶性, 随后形成一个富含聚合物的凝聚相。另一方面, 在较低的温度下, ELPs 保持可溶性, 容易溶入溶液中。在这两个温度机制之间循环, 再加上离心步骤收集和丢弃不含 ELP 的相, 依次浓缩蛋白质, 同时减少非 ELP 污染物的存在。
然而, 在这个纯化过程中, 有许多阶段 ELPs 可以丢失。首先, 在每一个冷旋转之前, 含有蛋白质的溶液碱化9.0 的 pH 值。这种较高的 pH 值 deprotonate 某些氨基酸在蛋白质的主干上, 有效地使它们处于带电状态, 并进一步增强其溶解度。因此, 上述步骤或不允许足够的时间, 蛋白质溶解可能导致降低产量, 因为非可溶性的蛋白质将被颗粒在离心和丢弃。
同样, 当 ELP 为颗粒时, 靶蛋白在热旋转过程中可能会丢失。最初, 在富含蛋白质的上清液中加入氯化钠, 以减少 ELP 的溶解度。这种盐能保护蛋白质和水分子之间的静电相互作用, 导致蛋白质与水相分离。这种效应通过加热解决方案而得到放大, 由于熵的影响, 进一步分解了水中的 "笼" 周围的 ELPs, 并迫使蛋白质聚集。在较低的蛋白质浓度 (i. e, 第一个热循环), 单独添加盐往往不足以导致这种相分离。然而, 随着蛋白质浓度的增加 (i. e, 后来的热循环), 并有较少的次生污染物与盐相互作用, ELP 将更容易沉淀。因此, 如果添加的盐太快, 他们可能会成为物理陷阱的聚合蛋白, 从而有效地降低溶液的盐浓度, 并限制进一步的蛋白质沉淀。因此, 盐应添加三小批, 以确保他们有足够的时间 homogenously 分发通过解决方案。作为最后的说明, 变化的 ELP 骨干, 无论是通过进一步修改的客人残留的弹性蛋白样的区域或变化的生物活性区域可以显著影响 LCST 行为。因此, 为了确保蛋白质变异的最佳蛋白质产量, 最重要的是优化 pH 值, 盐浓度和盐类型 (e. g, 单价或价) 的冷和热旋转。
建议在协议完成时运行 SDS 页, 因为它可用于轻松确定在任何净化步骤中是否发生重大的 ELP 损失。简而言之, 如果 ELP 是在一个热旋转后的上清检测到的, 那么蛋白质就没有被有效地沉淀。同样, 如果 ELPs 是在一个冷旋后的可溶性颗粒样本中确定的, 那么蛋白质就不能有效地溶解。
ELP 水凝胶比合成或天然衍生材料具有许多优点。具体来说 , 胺反应联剂 THPC 的使用 , 提供了一种低成本、简单、可调谐的蛋白质去甲机制。但是, 在交联议定书中有明显的局限性, 应予注意。THPC 是氧敏感的, 如果储存在不适当的条件下, 反应效率会迅速恶化。另外, 由于它的反应性与主要胺, THPC 可能反应与周围的蛋白质在媒介或那些在细胞表面那些富含胺类。因此, 在形成 ELP 水凝胶时, 建议避免与细胞颗粒的介质污染, 以减少可能的外源蛋白的交叉反应性, 从而降低交联效率。最后, 这种交联机制将生物活性区域序列排除在不含赖氨酸残留物上, 从而限制了某些细胞粘附图案 (e. g, IKVAV34) 的潜在集成。为解决这些限制, 对 ELP 骨干的修改与叠氮化物和 bicyclononyne (BCN) 反应伙伴允许生物正交交联, 如前所述的27。
应注意的是, ELP LCST 行为在水凝胶显微结构的口述中起着重要的作用。在 LCST 之上的温度机制中, ELPs 沉淀出溶液, 导致蛋白质丰富和缺蛋白质的阶段的形成, 从而影响基质的孔隙率和交联效率的矩阵9。由于大多数细胞培养实验是在生理相关的温度 (约37°c) 以上的 ELP LCST, 这些影响应考虑。为了使水凝胶有效地交联并形成一个相互关联的蛋白质网络, 赖氨酸的主要胺必须在物理上可以 THPC 交联剂。如果 ELP 聚合发生在达到足够的交联之前, 被困在富含蛋白质的阶段的 ELPs 可能无法进入, 因而无法参与交联。为了解决这一限制, 我们的协议要求在室温下的初始15分钟交联期, 这使得水凝胶在 ELP 进行热相转变之前可以进行初步交联。此室温孵化后, 另外15分钟孵化37°c, 以完成水凝胶交联。这一过程对 ELP 材料的交联性和可靠、可再生的凝胶化至关重要。
总之, 使用 ELP 制备的重组蛋白水凝胶提供了特殊的调谐蛋白质序列, 因此3D 细胞微环境。ELP 聚合物已被证明是表述高产量, 容易纯化, 由于其 LCST 行为, 并在各种体外和体内系统中的生物相容性。使用大肠杆菌作为一个重组宿主提供了一个简单和廉价的程序, 使接近完美的控制聚合物分子量和功能。结合起来, 这种技术可以使水凝胶平台的强健调谐和重现性, 允许在3D 内多种细胞类型的培养。最后, 该 ELP 水凝胶平台是适合许多下游生化检测, 包括 qRT PCR, 西方印迹, DNA 提取和细胞染色9。

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。

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			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Petri Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">FB0875713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>RICCA Chemical</td>
			<td style="width:123px;">2546.70-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">A3920-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1760-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">9002-18-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21(DE3)pLysS Competent Cells</td>
			<td>Invitrogen</td>
			<td style="width:123px;">C606003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol</td>
			<td style="width:260px;">Amresco</td>
			<td style="width:123px;">0230-100G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA disodium salt, dihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">O2793-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">A451-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1755-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Broth</td>
			<td style="width:260px;">EMD Millipore</td>
			<td>1.10285.5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>VWR</td>
			<td style="width:123px;">52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>MP Biomedicals</td>
			<td style="width:123px;">195381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">S 8045-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Filter Unit (0.22 &#956;m)</td>
			<td>Millipore</td>
			<td style="width:123px;">SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terrific Broth</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">71754-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Encapsulation in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m syringe filters</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mm thick silicone sheet</td>
			<td>Electron Microscopy Science</td>
			<td style="width:123px;">70338-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well tissue culture plates&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (2 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (4 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-34</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (5 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">No. 1 12 mm glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">404861-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5% Tryspin/EDTA</td>
			<td>Thermo Fisher&#160;</td>
			<td style="width:123px;">15400054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Immunocytochemistry of Cells in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% (w/v) Paraformaldehyde (PFA)</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">15701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:260px;">Roche</td>
			<td style="width:123px;">3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes</td>
			<td style="width:123px;">D1306&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td style="width:260px;">Lampire Biological Labs</td>
			<td style="width:123px;">7332100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse Secondary Antibody (AF488)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit Secondary Antibody (AF546)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Gibco</td>
			<td style="width:123px;">16210-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Mouse Nestin Primary Antibody</td>
			<td style="width:260px;">BD Pharmingen</td>
			<td style="width:123px;">556309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Sox2 Primary Antibody</td>
			<td style="width:260px;">Cell Signaling Technology</td>
			<td style="width:123px;">23064S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Nail Polish</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">72180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">X100-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield Hardset Mounting Medium&#160;</td>
			<td style="width:260px;">Vector Labs</td>
			<td style="width:123px;">H-1400&#160;</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的中心重复包含了一个赖氨酸组在访客残渣站点, 使交联通过 THPC, 而侧翼重复含有异亮氨酸达到 LCST 32 °c31。
Post 表达式、SDS 页或西方印迹可用于可视化分子量并确认含有抗体标记的 ELPs 的标识, 如 T7 (MASMTGGQQMG) 或 His6 (HHHHHH) (图 2)。成功的表达在受控条件下产生一个高度均匀的产品代表的单一黑暗带在大约分子量 (约 37 kDa) 这些蛋白质使用 SDS 页 (图 2A) 和西方印迹 (图 2B)。
在不受控制的条件下, 在一个西方印迹存在较低的分子量带表明, 一些蛋白质的一部分没有完全翻译和/或在表达后退化 (图 2C)。具体来说, 这里的群众平均分布在 9 kDa, 大致相当于一个生物活性区域的重量和三个弹性蛋白样的区域 (一个 ' 重复 '), 或大约第四的目标蛋白。当表达式在更高的温度 (> 32 °c) 进行时, 如图 2C中所示, 这些较小的蛋白质片段通常会出现。这些低分子量蛋白质的存在会导致不可预知的机械特性。因此, 建议定期筛选后的表达式, 以确保高质量的最终产品。
ELP 水凝胶的力学刚度可以通过操纵 ELP 的浓度或 THPC 反应基团的比例来进行修正: ELP 主要胺。同时, 细胞粘附配体的浓度可以通过改变 ELP 的比例与细胞-胶粘剂 (RGDS) 到非胶粘剂 (RDGS) 序列在任何刚度制度。通过操纵这两个变量, 我们可以生产具有一系列机械性能和配体浓度的凝胶 (图 3)。
要封装 3D ELP 水凝胶中的单元格, 所需的单元数在介质和离心中悬浮, 以产生细胞颗粒 (图 4A)。培养基从管中吸气, 细胞在所需浓度的 ELP 溶液中均匀地重新悬浮。接下来, THPC 溶液被添加到细胞/ELP 悬浮体和 pipetted 彻底形成一个均匀的混合物。该解决方案被迅速转移到24井板内的无菌硅胶模具使用吸管和允许在室温交联和37°c 15 分钟 (图 4B)。最后, 将介质添加到井板中, 并在实验中孵化37摄氏度。
活/死染色可用于评估细胞活力和成功的细胞封装在 ELP 水凝胶。如图 5所示, 成年小鼠神经祖细胞 (npc) 在 3% (w/v) ELP 水凝胶中显示高细胞活力超过7天。
3D ELP 水凝胶以前已被证明支持通过表达的标准 npc 蛋白标记 SRY (性别决定区域 Y)-框 2 (Sox2) 和巢的9的 npc 干细胞维护。npc 封装在 3% (w/v) ELP 水凝胶低 THPC 交联显示核本地化 Sox2 和细胞质巢蛋白丝通过染色和共焦成像的高表达 (图 6)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57739/57739fig1.jpg"> 图 1: ELP 和相应氨基酸序列的示意图表示形式.本研究中使用的 ELP 包含一个 T7 和 His6 标记的抗体为基础的成像, 一个生物活性区域介绍细胞粘附领域, 和弹性体类似的区域, 赋予弹性力学性质, 并允许化学交联。<a href="/files/ftp_upload/57739/57739fig1large.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/57739/57739fig2.jpg"> 图 2: 目标蛋白表达式可以用 SDS 页和西方印迹验证, 以确认最终冻干产品的分子量和身份.使用 T7 (MASMTGGQQMG) 或组氨酸 6 (HHHHHH) 标记(B) , 由 SDS 页(a)和西方印迹报告的纯全长 ELP 以 37 kDa 的分子量运行。(C).由于 ELP 表达式协议中的偏差, ELP 的不纯批次可能导致低分子量的 ELPs 的表达。<a href="/files/ftp_upload/57739/57739fig2large.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/57739/57739fig3.jpg"> 图 3:RGDS 配体含量可以从 ELP 水凝胶内的力学性能中独立调节.5% (w/v) 和 3% (瓦特/v) ELP 水凝胶分别具有 800 pa 和 400 pa 的剪切模量。1:1 THPC 反应组的水凝胶: ELP 主要胺在室温下交联15分钟, 加热到37摄氏度, 在测量前允许平衡5分钟。数据的意思是 s.d., ***p < 0.001。<a href="/files/ftp_upload/57739/57739fig3large.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/57739/57739fig4.jpg"> 图 4:ELP 水凝胶中细胞封装的示意图.(A).单元格最初与介质中的单细胞悬浮液分离, 并使用离心机进行颗粒。介质从管中吸气, 细胞在 ELP 溶液中以所需浓度和混合井重新悬浮。最后, 加入 THPC 交联溶液, 混合良好。(B).在添加 THPC 后立即, 该解决方案被投进带有吸管的硅胶模具中。该溶液可以在室温下交联15分钟, 其次是15分钟孵化37摄氏度。在实验期间, 培养基被添加到培养井中。<a href="/files/ftp_upload/57739/57739fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57739/57739fig5.jpg"> 图 5:神经祖细胞在 ELP 水凝胶中维持高生存能力.3% (w/v) ELP 水凝胶与1:1 交联 (THPC 活性基团: ELP 主要胺) 在培养7天后的神经祖细胞的代表性图像。绿色: 活染色 (calcein);红: 死染色 (溴 homodimer)。刻度条 = 100 µm。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57739/57739fig6.jpg"> 图 6: 3D ELP 水凝胶支持神经祖细胞干细胞的表达.ELP 水凝胶中7天培养的神经祖细胞免疫荧光图像表达 Sox2 和巢蛋白。图像显示封装在 3% (w/v) ELP 凝胶的细胞与 0.5: 1 交联 (THPC 反应组: ELP 主要胺)。蓝色: DAPI (核);红色: Sox2;绿色: 巢蛋白。缩放条 = 25 µm.<a href="/files/ftp_upload/57739/57739fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

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12.2K 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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

科研

生物工程

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

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 ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

破碎散装水凝胶并加工成颗粒状水凝胶，用于生物医学应用

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

具有长距离纤维排列的微工程3D胶原蛋白水凝胶

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...