导致这些结果的研究得到了欧洲联盟第七框架方案 FP7/2007-2013 根据赠款协议编号 607051 (生物启发) 的资助。我们感谢 Carolien van Spreuwel-Goossens 从富士制造欧洲 bv 公司, 为技术援助在 RCP 制造, 和沃纳 Stracke 从弗劳恩霍夫研究所的硅酸盐研究 ISC, 以协助与 SEM 分析。

hBMSCs 是骨关节炎患者股骨头置换术后股骨头的分离。该程序是在 Wuerzburg 大学地方伦理委员会的批准和病人的知情同意下进行的。原发微血管内皮细胞从幼年捐献者的包皮活检中分离出来。他们的法律代表以书面形式提供了充分的知情同意。这项研究得到了 Wuerzburg 大学地方伦理委员会的批准 (投票 182/10)。
1. hBMSCs 和 HDMECs 的隔离
<ol><li>人骨髓间充质细胞 (hBMSCs)<ol>
<li>用无菌勺从股骨头上取出海绵骨。 注: 海绵状骨在皮质骨内呈松散和粒状物质, 坚硬而坚硬。可能需要用手术刀从皮质骨中划出海绵骨的部分。</li>
		<li>将取出的海绵骨插入两个50毫升的管子中。在髋关节置换手术中, 海绵状骨的体积可以从5到10毫升不等。</li>
		<li>用 DMEM-F12 (1:1 混合 DMEM 和火腿 F12 培养基) 冲洗, 用大约30毫升培养基填充管。</li>
		<li>用一种纵向的方式, 手动摇动试管, 5 秒让脂肪细胞脱离海绵状骨。离心机在 300 x g 和22° c 为5分钟。</li>
		<li>吸用一个毕业的塑料吸管, 脂肪细胞层和大约20毫升的剩余上清液。让足够的培养基覆盖海绵骨。</li>
		<li>用10毫升的 DMEM-F12 填满管子, 用手用力摇动, 10-十五年代, 让细胞出来。</li>
		<li>转移 10-15 毫升的细胞悬浮与一个毕业的塑料吸管从管到一个新的50毫升管。</li>
		<li>重复 2-3 次从步骤1.1.6 池一起获得的细胞悬浮, 直到海绵状骨收集在50毫升管是白色的。</li>
		<li>转移的细胞悬浮与一个毕业的塑料吸管, 以两个新的管, 避免了在50毫升管的底部沉积的胶原颗粒。</li>
		<li>离心机悬浮在 300 x g 和22° c 为 5 min. 通过用一只毕业的塑料吸管抽吸它来丢弃上清。</li>
		<li>添加40毫升的 DMEM-F12 10% FCS 1% 笔/链球菌50µg/毫升 Ascorbate-2-phosphate 在第三管。将这种培养基的5毫升转移到每个含有细胞颗粒的管子上。通过将管子的底部用力闪烁, 将细胞悬浮液转移到只含有培养基的管子上, 从而使细胞颗粒重新悬浮。</li>
		<li>可选: 稀释100µL 细胞悬浮液进行细胞计数, 稀释前1:100 与 PBS, 然后1:10 在台盼蓝, 通过增加15µL 的第一稀释15µL 的 DPBS 和30µL 锥盼蓝获得 DPBS: 台盼蓝比1:1。用标准的 Neubauer 室计数细胞。</li>
		<li>种子的细胞在 109 cells/175 厘米2 T 烧瓶在25毫升的培养基中所描述的1.1.11。或者, 将整个电池悬浮液除以十 175 cm2细胞培养烧瓶中, 不加计数。在这个阶段, hBMSCs 是不能区分的红细胞, 这大大超过他们。hBMSCs 将出现作为稀疏殖民地 (1-2/瓶) 在第一7天的过程中。</li>
		<li>改变培养基4天后播种, 使血液细胞与 hBMSCs 的相互作用。在第一次媒介交换以后, 每 2-3 天改变媒介。执行培养基交换, 完全去除培养基与一个毕业的塑料吸管, 洗涤二次与 DPBS (通过增加10毫升 DPBS 到黏附的细胞和完全地去除 DPBS 与一个毕业的塑料吸管) 然后增加20毫升新鲜培养基, 如1.1.11 所述。</li>
	</ol>
</li>
	<li>人真皮微血管内皮细胞 (HDMECs)<ol>
<li>根据以前发布的协议21, 治疗皮肤活检并隔离 HDMECs。简要地, 在真皮从表皮分离后, 在胰蛋白酶中孵育40分钟在37° c 和 5% CO2的真皮。孵育时间后, 将真皮转移到含有培养基的培养皿中, 用手术刀划伤每一块。将所获得的细胞悬浮液转移到50毫升塑料管和离心机5分钟, 在 300 x g 和22° c。使用 Neubauer 室和种子在 1.2 x 104单元格/cm2的密度下计算单元悬浮。每隔一天更换一次培养基。</li>
	</ol>
</li>
</ol>2. 旋转瓶、载体和生物反应器的设置和灭菌
<ol><li>微调瓶和 RCP 载体<ol>
<li>在实验前的一天, 设置微调烧瓶, 并准备他们灭菌的高压釜。 注意: 将微调瓶的瓶盖松开, 并在可能的情况下, 将其放在直立位置进行消毒。<ol>
<li>包装的微调瓶与铝箔周围的侧盖分开, 以避免在展开过程中的污染。将烧瓶用 1-2 层铝箔灭菌, 以减少灭菌后的污染风险。</li>
		</ol>
</li>
		<li>在20毫升的 DPBS 中称载体大孔的需要量。 注: 每锭瓶需30毫克。让他们水合物至少1小时前热灭菌的高压釜 (121 ° c 15 分钟)。</li>
	</ol>
</li>
	<li>灌注生物反应器<ol>
<li>使用描述的生物反应器系统产生带血管的组织等效物20。这个生物反应器包括容器, 博美被安置, 中等水库和压力瓶。<ol>
<li>通过硅管将容器与介质库和压力瓶连接 (请参见图 3C)。把瓶盖松开, 放在高压釜塑料袋里, 关好, 放在第二个高压釜塑料袋里。灭菌釜。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. hBMSCs 和 HDMECs 对大孔载体的培养
注: HDMECs 用于种子的 RCP 载体被标记与 RFP 的慢转导。此协议在以前发布的协议5之后被修改。
<ol><li>让 RCP 大孔载体沉淀到底部, 并用10毫升培养基洗涤。重复3次。</li>
	<li>用10毫升培养基洗涤微调烧瓶。</li>
	<li>在8毫升培养基中加入30毫克的 RCP 大孔载体。平衡的微调瓶包含 RCP 载体在37° c 和 5% CO2为30分钟, 然后播种细胞。 注: 这是基于载体的近似干重在灭菌前。</li>
	<li>种子 5 x 105 hBMSCs 或 HDMECs (通道 3) 每个微调瓶在2毫升培养基中。</li>
	<li>将微调烧瓶放在搅拌器板上, 开始搅拌 90 rpm, 5 分钟, 休息55分钟, 在37° c 和 5% CO2, 总共 1 h 静态/动态孵化。重复4次。</li>
	<li>添加10毫升的细胞培养基每个微调瓶, 然后开始连续搅拌 95 rpm。孵育在37° c 和 5% CO2。</li>
	<li>与用途微采取样品在天 1, 4 和 7: 333 µL 为脱氧核糖核酸内容 (~ 0.5 毫克), 1000 µL 为 SEM 分析 (~ 1.5 毫克) 和333µL 为活或死的染色 (~ 0.5 毫克)。 注: 这是基于载体的近似干重在灭菌前。</li>
	<li>每隔一天改变10毫升培养基。为此, 等待, 直到 RCP 载体落户到底部, 非常小心地从微调瓶10毫升, 使用10毫升吸管, 然后添加10毫升的新鲜培养基。</li>
	<li>保持在搅拌器板上的文化设置在 95 rpm, 在37° c 和 5% CO2为7天。</li>
</ol>4. DNA 含量、SEM 分析、活死人染色和发芽试验
<ol><li>DNA 含量<ol>
<li>使用微, 收集约0.5 毫克的载体从每个纺纱瓶 (包含在333µL 的悬浮) 和转移到2毫升塑料管。 注: 这是基于载体的近似干重在灭菌前。</li>
		<li>用1毫升的 DPBS 洗一次, 然后吸上清液。</li>
		<li>删除剩余的 DPBS 在一个冻和重量冻干载体后正常化。</li>
		<li>将样品在-80 ° c 冻结至少6小时。</li>
		<li>在60° c 的夜间孵育样品, 300 µL 木瓜蛋白酶 5 U/毫升。</li>
		<li>建立适当的滴定曲线后, manufacturer´s 指示的定量测定 (例如, PicoGreen dsDNA 化验) 试剂盒和执行荧光信号的测量, 在 520 nm 与发光探测器。</li>
	</ol>
</li>
	<li>扫描电子显微镜 (SEM) 分析<ol>
<li>从每个纺纱瓶收集约1毫克的载体, 并转移到2毫升塑料管。</li>
		<li>用1毫升的 DPBS 洗一次载体。</li>
		<li>去除 DPBS 和孵育在1毫升70% 乙醇为1小时。</li>
		<li>去除上清液, 在1毫升的80% 乙醇中孵育1小时。</li>
		<li>去除上清液, 在1毫升的90% 乙醇中孵育1小时。</li>
		<li>去除上清液, 在1毫升的100% 乙醇中孵育1小时。</li>
		<li>去除上清液, 在1毫升的烷中孵育10分钟。</li>
		<li>打开瓶盖, 让样品在化学罩下过夜。</li>
		<li>在适当的支持下修复样品, 并根据既定的协议进行 SEM 分析。</li>
	</ol>
</li>
	<li>DAPI 和活/死染色<ol>
<li>在2、4和7天, 在载体上的细胞播种后, 从每个纺纱瓶收集0.5 毫克的载体, 并转移到2毫升塑料管。用1毫升的 DPBS 洗一次。</li>
		<li>dapi<ol>
<li>在足够的4% 甲醛中修复10分钟的样品以覆盖它们。</li>
			<li>用1毫升的 DPBS 洗一次。</li>
			<li>在室温下孵育45分钟的摇摆振动筛的染色液。</li>
			<li>在荧光显微镜下, 用 420 nm 发射滤波器检测 DAPI 荧光信号, 获取图像。</li>
		</ol>
</li>
		<li>活/死染色<ol>
<li>在500µL 的活/死染料溶液中孵育样品。保持样品在37° c 为20分钟, 保护免受光。</li>
			<li>用1毫升的 DPBS 洗一次。</li>
			<li>检测活细胞荧光信号的 490 nm 发射过滤器和死细胞与 545 nm 发射过滤器的荧光显微镜。获取图像。</li>
		</ol>
</li>
		<li>HDMECs 发芽试验<ol>
<li>混合330µL 的胶原蛋白 R 溶液 0.4%, 170 µL 0.1% 乙酸, 和50µL 中 M199 在15毫升塑料管。继续冰</li>
			<li>使用微的载体 (250 µL) 的0.375 毫克。转移到一个1.5 毫升塑料管, 等待 RCP 载体解决到底部, 并完全删除介质。 注: 这是基于载体的近似干重在灭菌前。</li>
			<li>添加氢氧化钠 0.2 N 的体积, 以中和胶原蛋白混合物, 并立即与 RCP 载体混合。 注: 预先确定所需的氢氧化钠 0.2 N, 将其加入到混合物中, 直到 pH 值改变 (从黄色变成粉红色), 然后将混合物放在孵化器中1分钟, 之后必须形成凝胶。</li>
			<li>将胶原凝胶-RCP 载体混合物放在一个24井板的井中。孵育在37° c 和 5% CO2为 30 min。</li>
			<li>增加200µL 血管内皮生长因子 (如, VascuLife VEGF-Mv 培养基)。孵育在37° c 和 5% CO2为 24 h。</li>
			<li>在反相对比显微镜下观察, 使用10X 放大物体, 获取图像。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. HDMECs 生物反应器系统的博美播种和启动
<ol><li>在启动生物反应器培养的前一天, 将 BioVaSc (博美) 纵向切割在 antimesenteric 一侧, 并将其固定在先前经过消毒的聚碳酸酯框架中 (参见图 3A, B)。 注: BioVaSc 是从细胞猪空肠段获得的博美, 其制备方法与以前发布的21相同。 注: 由于聚碳酸酯框架不能通过加热灭菌, 在音速浴 (40 赫) 中消毒20分钟, 然后在乙醇70% 中进行3次孵育, 25 分钟。然后, 用 DPBS 消毒3次。<ol>
<li>将博美框架系统放入一个 100 mm 的培养皿中, 用血管内皮生长因子 + 1% 青霉素链霉素 (笔/链球菌) 填充。</li>
		<li>平衡博美在37° c 和 5% CO2的夜间孵化。</li>
	</ol>
</li>
	<li>分离和计数的细胞后, 注入 10 x 107 HDMECs 在1000年µL 的培养基通过博美保存的血管, 使用2毫升注射器。 注: 注入〜700µL 通过动脉入口和〜300µL 通过静脉出口。</li>
	<li>孵育在37° c 和 5% CO2为 3 h, 允许细胞附着。</li>
	<li>用350毫升血管内皮生长因子 + 1% 笔/链球菌填充生物反应器系统, 在37° c 和 5% CO2孵育。</li>
	<li>将框架置于生物反应器的容器内。将动脉和静脉蒂连接到血管。</li>
	<li>通过将生物反应器系统连接到蠕动泵来开始灌注。设置压力在10柱和振幅±。</li>
	<li>每小时增加压力, 直到达到100柱的压力, 振幅±20。</li>
	<li>在这些条件下, 在37° c 和 5% CO2下保持生物反应器系统7天。</li>
</ol>6. 将 RFP-HDMECs 添加到博美
<ol><li>在50毫升猎鹰管中混合330µL 0.4% 胶原蛋白 R 溶液, 170 µL 0.1% 乙酸, 15 µL 中 M199。继续冰</li>
	<li>从旋转烧瓶中取载体。完全删除区域性媒体。</li>
	<li>添加氢氧化钠 0.2 N 的体积, 以中和胶原蛋白混合物, 并立即与 RCP 载体混合。</li>
	<li>在博美的流明上加入胶原凝胶-RCP 载体混合物。允许凝胶30分钟。</li>
	<li>同时, 改变生物反应器的培养基, 完全去除培养基, 然后加入350毫升新鲜, 预热, 血管内皮生长因子 + 1% 笔/链球菌。</li>
	<li>将生物反应器连接到蠕动泵, 设置100柱和振幅±20的压力。</li>
	<li>每7天更换一次培养基。</li>
	<li>将生物反应器培养21天。</li>
</ol>7. 生物反应器培养的分析与读取
<ol><li>样品的准备<ol>
<li>断开博美与生物反应器的连接, 并将其从聚碳酸酯框架中取出。</li>
		<li>将获得的部分切成6段: 2 用于 MTT, 2 用于石蜡包埋/染色, 2 用于扫描电镜分析。</li>
	</ol>
</li>
	<li>石蜡嵌入<ol>
<li>将切片放置在培养皿中, 并用足够的 DPBS 来覆盖它们。丢弃 DPBS。</li>
		<li>在一夜之间修复4% 甲醛的部分。</li>
		<li>准备盒式石蜡嵌入, 并按照标准协议进行。</li>
		<li>使用切片, 在石蜡切片中准备切片, 并将3µm 的样品切割为 H & E 染色。</li>
	</ol>
</li>
	<li>H & E 染色<ol>
<li>根据标准协议32着色水化部分。</li>
	</ol>
</li>
	<li>扫描电子显微镜 (SEM) 分析<ol>
<li>将切片放置在培养皿中, 并用足够的 DPBS 来覆盖它们。丢弃 DPBS。</li>
		<li>一夜之间用4.75% 戊二醛固定切片。</li>
		<li>执行脱水链随着乙醇浓度的增加 50%-100%。</li>
		<li>用烷 (HMDS) 覆盖部分, 10 分钟丢弃使用的 HMDS, 并添加新鲜的 HMDS。</li>
		<li>允许部分在油烟罩下过夜, 然后准备样品进行成像。</li>
	</ol>
</li>
	<li>3-(45-Dimethylthiazol-2-基)-25-diphenyltetrazolium 溴化铵 (MTT)<ol>
<li>将 MTT 试剂与血管内皮生长因子混合, 浓度为1毫克/毫升。</li>
		<li>在 MTT 溶液中孵育90分钟, 在37° c 和 5% CO2。</li>
		<li>从宏观上或在明亮场显微镜下观察血管结构和细胞种子载体, 以检测新陈代谢活性细胞。获取图像。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

载体的一个主要目标是细胞的扩展, 同时保持它们的分化, 以便将细胞送到需要的地方。所代表的方法引入 RCP 载体, 细胞能够附着, 增殖, 并殖民载体高细胞密度。这是观察活/死染色, 其中超过90% 的存活细胞被检测, 而只有少数死细胞获得后, 7 天的动态文化。同样, SEM 图像证实, 细胞覆盖整个表面的载体后, 7 天的文化。
为了确保3D 模型中的细胞存活, 重要的是要保持细胞的氧?...

组织工程应用中的一个普遍问题是在需要的位置上产生一个具有正确的分化表型的高细胞质量。在1967年开始应用载体来解决这一问题, 在诸如骨科组织工程等领域, 对于大规模的皮肤、骨骼、软骨和肌腱的产生具有越来越重要的意义1。它们允许通过在微尺度的三维 (3D) 基板上扩展细胞, 以类似于悬浮培养物的方式来处理附着的区域性2 。从而细胞体验均匀的营养素供应和细胞矩阵互作用导致更好的维护在体内3,4差异, 这通常在2D 方法5中随时间而丢失。一个更高的表面体积比-最终导致更高的细胞产量6,7, 与静态系统相比, 更高的气体和养分交换率8, 有可能调节并将文化置于物理刺激9, 并且扩展进程7的扩展潜力是进一步的优势。直径、密度、孔隙度、表面电荷和附着力等几个特性10,11区分了不同的商业可用的微型和宏载体。然而, 其中一个主要优势是他们的交付潜力, 作为 microtissues 的网站缺陷或需求。
对于载体技术在骨组织工程中的应用, 我们在以前的报告12中说明了一种新的载体类型的生产, 它是由重组的 i 型胶原肽 (RCP, 在商业上可作为 Cellnest)。这一新的载体允许符合 GMP 的支架和细胞生产, 根据需要的细胞分娩在临床情况。在这种情况下, 通过适当选择合适的交联策略来调整支架的稳定性、降解率和表面特性, 可以使技术适应选定的应用、细胞类型的兴趣或目标组织13。特别是, 这个载体作为一个可注射的治疗应用的细胞递送系统的潜在就业14使他们特别有趣的临床设置。
因此, 本文对人骨髓间充质基质细胞 (hBMSCs) 和人真皮微血管内皮细胞 (HDMECs) 的分离和扩展的培养程序进行了阐述, 对 i. 型胶原重组多肽基载体, 及其在临床设置分娩的准备。此外, 我们还描述了用于在移植后维持细胞活力的附加协议。
植入后的细胞活力实际上强烈依赖于血管化的15,16,17, 这保证了氧气和营养素的交换, 并促进了废物的去除。生物反应器是克服组织工程中血管化挑战和维持细胞活力的一种方法, 通过对培养基进行灌注, 从而提供氧气和营养素18。在这里, 我们演示了一个体外方法, 以评估从 RCP 载体微血管内皮细胞的迁移能力, 以博美和他们的能力, 以促进de 从头血管和血管生成。这博美是猪空肠的细胞段, 称为 BioVaSc (生物血管支架), 富含胶原蛋白和弹性蛋白, 并与保存血管结构, 其中包括喂养动脉和引流静脉19已已应用于植入问题20。

<table>
	<tbody>
		<tr>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>20395.01</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-phenylindoldihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid 100%</td>
			<td>Sigma-Aldrich</td>
			<td>533,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance Kern EG 2200-2NM</td>
			<td>Kern &#38; Sohn GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope Axiovert 40C</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellnest</td>
			<td>Fujifilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL, 50 mL)</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen R solution 0,4%</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>47254.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Modified, without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth GmbH</td>
			<td>T171.4</td>
			<td>Denatured</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Bio&#38;SELL</td>
			<td>FCS.ADD.0500</td>
			<td>not heat-inactivated</td>
		</tr>
		<tr>
			<td>Fluorescence microscope BZ-9000</td>
			<td>Keyence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Morphisto</td>
			<td>10231.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma-Aldrich</td>
			<td>440191</td>
			<td>Reagent grade, &#8805;99%</td>
		</tr>
		<tr>
			<td>Incubator for bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Double Staining Kit</td>
			<td>Fluka</td>
			<td>04511KT-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer plate</td>
			<td>2Mag</td>
			<td>80002</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M0650</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>Microplate reader 
			Tecan Infinite M200</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 21G 16mm</td>
			<td>VWR</td>
			<td>613-5389</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma-Aldrich</td>
			<td>P4762</td>
			<td>lyophilized powder, &#8805; 10 units/mg protein</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Carl Roth GmbH</td>
			<td>6642.6</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quanti-iT PicoGreen dsDNA assay kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>Histofix 4%</td>
			<td>Carl Roth GmbH</td>
			<td>P087</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope Supra 25</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flasks (25 mL)</td>
			<td>Wheaton</td>
			<td>356879</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>VWR</td>
			<td>720-2561</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks (25 cm2, 75 cm2, 150 cm2)</td>
			<td>TPP Techno Plastik Products AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF-Mv</td>
			<td>Lifeline cell technology</td>
			<td>LL-0005</td>
			<td></td>
		</tr>
	</tbody>
</table>

recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model

组织工程学是一个有前途的领域, 重点是开发解决方案, 以增加对组织和器官的移植目的的需求。生成这种组织的过程是复杂的, 包括特定细胞类型、支架和物理或生物化学刺激的适当组合, 以指导细胞生长和分化。载体代表一个吸引人的工具来扩展三维 (3D) 微环境中的细胞, 因为它们提供了更高的表面体积比, 并且与传统的二维方法相比更接近于在体内的情况。血管系统, 提供氧气和养分的细胞和确保废物清除, 构成一个重要的积木时, 生成工程组织。事实上, 大多数的结构在植入后由于缺乏血管支持而失败。在这项研究中, 我们提出了一个协议的内皮细胞扩展的重组胶原基载体在动态条件下, 在微调瓶和生物反应器, 我们解释如何确定在这个设置细胞的可行性和功能。此外, 我们提出了一种细胞分娩的方法, 不需要额外的剥离步骤。此外, 我们还提供了一种在细胞生物基质上评价灌注生物反应器中细胞的血管潜能的策略。我们认为, 使用所提出的方法可能会导致开发新的细胞为基础的治疗组织工程的广泛应用在临床实践。

如图 1A所示, 在经过7天的培养后, 我们在 RCP 载体上获得了大量可行的细胞, 由活/死染色确定。这些结果通过 SEM 分析证实, 在毛孔周围观察完全地被殖民的载体, 部分 overgrowing 他们 (图 1B)。另一方面, 实验中的细胞不均匀播种导致了几个空载体。失败的实验的特征是异常高的死细胞数量不应超过总细胞数量的 10% (...

Watch this Scientific Journal Video about Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model at JoVE.com

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

提出了大孔载体的细胞扩展协议及其在灌注生物反应器中的传递系统, 以细胞组织基质为种子。我们还包括不同的技术, 以确定细胞增殖和细胞在载体培养的生存能力。此外, 我们还展示了生物反应器培养后细胞的功能。

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

recombinant-collagen-i-peptide-microcarriers-for-cell-expansion-their

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.5K 次观看.  University Hospital Wuerzburg. 提出了大孔载体的细胞扩展协议及其在灌注生物反应器中的传递系统, 以细胞组织基质为种子。我们还包括不同的技术, 以确定细胞增殖和细胞在载体培养的生存能力。此外, 我们还展示了生物反应器培养后细胞的功能。

视频: Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统:作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...