D.D. 和 S.M.-D.承认 BMBF 资助“CellWiTaL：用于药物研究的可重复细胞系统 - 三维细胞结构中高度特异性单细胞的转移层无激光打印”提案编号 13N15874。D.D. 和 V.R.A. 承认欧盟 MSCA-COFUND 授予 OSTASKILLS，“下一代骨关节炎研究的整体培训”，GA Nr. 101034412。所有作者都感谢Beate Geyer夫人提供的技术援助。

注意：所有步骤都必须使用无菌技术进行。
1. NHDF的培养和预扩增
<ol><li>在37°C下快速解冻含有成体冷冻保存的正常人真皮成纤维细胞（NHDF，1 x 106 个细胞）的冻存瓶，直到它们几乎解冻。</li>
	<li>缓慢加入1mL预热的成纤维细胞生长培养基2（即用型试剂盒，包括基础培养基，2%胎牛血清（FCS），碱性成纤维细胞生长因子（bFGF）和胰岛素），补充有1%青霉素/链霉素（pen/strep）（NHDF培养基），在37°C下预热到细胞中。</li>
	<li>将细胞转移到 15 mL 离心管中。在室温（RT）下以300× g 离心细胞5分钟。</li>
	<li>取出上清液。将细胞沉淀重悬于 12 mL 预热的 NHDF 培养基中。</li>
	<li>将细胞转移到 T-75 烧瓶中，并以交叉方式短暂摇动。将T-75烧瓶在37°C下置于5%CO2 加湿培养箱中。</li>
	<li>每 2 天更换一次培养基。在显微镜下观察NHDF，直到它们达到70%-80%汇合度。</li>
</ol>2. 2D扩展
<ol><li>取出培养箱的 T-75 烧瓶并取出 NHDF 培养基。用预热的磷酸盐缓冲盐水（PBS）洗涤细胞。</li>
	<li>向细胞中加入 3 mL 0.05% 胰蛋白酶-EDTA。将细胞在37°C下在5%CO2 湿化培养箱中孵育，直到细胞从烧瓶中分离（约3分钟）。</li>
	<li>加入 6 mL 预热的 NHDF 培养基以中和胰蛋白酶作用。将细胞转移到 15 mL 离心管中。</li>
	<li>在室温下以300× g 离心细胞5分钟，并除去上清液。</li>
	<li>将细胞沉淀重悬于补充有10%胎牛血清（FBS），1x MEM氨基酸，1%笔/链球菌的Dulbecco改良Eagles培养基（DMEM）低葡萄糖中，在37°C预热。</li>
	<li>将NHDFs铺在10cm贴壁细胞培养皿中，密度为8×103NHDF /cm 2 （总共，每10cm培养皿4.4×105NHDF ）。以交叉方式轻轻摇晃。</li>
	<li>将10cm细胞培养皿置于37°C的5%CO2 加湿培养箱中。每 2 天更换一次培养基</li>
	<li>在显微镜下监测细胞，直至达到100%汇合度（约5天）。</li>
</ol>3. 2D刺激
<ol><li>从培养箱中取出含有NHDF（来自步骤2.6和2.7）的10cm细胞培养皿。取出培养基，用预热的PBS洗涤细胞。</li>
	<li>加入补充有10%FBS，50μg/ mL抗坏血酸和1%pen / strep的DMEM高葡萄糖培养基，在37°C下预热。</li>
	<li>将培养皿置于37°C的5%CO2 加湿气氛中。每 2 天更换一次培养基。</li>
	<li>在显微镜下监测NHDFs14天。</li>
</ol>4. 3D成熟
<ol><li>从培养箱中取出 10 cm 细胞培养皿，取出 DMEM 高葡萄糖培养基。</li>
	<li>用细胞刮刀轻轻快速地将形成的细胞片从培养皿中分离出来。在分离时，同时将细胞片卷成 3D 棒状类器官。</li>
	<li>拾取NHDF类器官并将其置于10cm非粘附（用于细胞）培养皿中。这种培养皿类型用于避免细胞从类器官迁移到塑料。</li>
	<li>在此时间点或之后的一天（3D 成熟的第 0 天或第 1 天），收集一些类器官进行进一步分析，例如湿重、组织学评估（第 1 天类器官更可取，因为它们变得更加紧凑）、RNA 和蛋白质分离。</li>
	<li>用金属销固定类器官的边缘，如下所示：<ol><li>用镊子小心地握住类器官的一侧，然后用另一个镊子轻轻地手动拉伸大约 10% 的轴向伸长率。要估计 10% 的轴向伸长率，请使用毫米纸或尺子。</li>
		<li>接下来，用镊子拿起一根金属针，然后手动将类器官边缘向下压入塑料盘以固定它。重复此过程，将第二个引脚钉在类器官的第二个边缘上。</li>
	</ol></li>
	<li>加入10mL补充有10%FBS，1x MEM氨基酸，50μg/ mL抗坏血酸，10ng / mL TGFß3和1%pen / strep的DMEM高葡萄糖培养基，在37°C下预热。</li>
	<li>将10厘米的培养皿置于37°C的5%CO2 加湿气氛中。每 2 天更换一次培养基。</li>
	<li>定期监测类器官 14 天。 注意： 销钉可能会松动，因此请使用步骤 4.5 中所述的相同技术重新修复。</li>
	<li>14 天后，从类器官中取出 DMEM 高葡萄糖培养基。用预热的PBS洗涤类器官。</li>
	<li>使用精密秤在第 0 天和第 14 天测量类器官的湿重。<ol><li>将一个 10 厘米的空培养皿放在秤上，将重量去皮至零，以确保仅测量类器官的重量。从10cm培养皿中完全取出培养基，并逐个测量类器官湿重。 注：在这项研究中，在第 0 天，每个供体称重三个类器官，在第 14 天，称量每个供体两个类器官。</li>
	</ol></li>
	<li>根据研究目的，在进一步的组织学分析中实施一些 NHDF 类器官，或立即使用无菌 DNA/RNA 管将它们冷冻在液氮中，用于后续的 DNA、RNA 和蛋白质分离，然后将它们储存在 -80 °C 下。</li>
	<li>对于组织学研究，将每个类器官固定在5mL预冷（4°C）4%多聚甲醛的PBS（调节至中性pH）冰上45分钟，然后在室温下洗涤PBS（每次2×5分钟）。</li>
	<li>通过将类器官放入玻璃罐中进行组织学检查，使用蔗糖梯度对固定的类器官进行冷冻保护。将类器官在4°C的PBS溶液中的10%蔗糖中孵育2小时，然后在4°C下孵育20%蔗糖/ PBS2小时，最后在4°C下将30%蔗糖/ PBS孵育过夜。 通过首先移出，然后用新溶液填充来改变蔗糖溶液。</li>
	<li>将类器官嵌入冷冻培养基中。<ol><li>将铜板放在聚苯乙烯泡沫塑料盒中的干冰上，冷却10分钟。</li>
		<li>接下来，将预装冷冻培养基的塑料冷冻模具放在铜板上，然后使用镊子将类器官轻轻按压到冷冻模具的底部。等到冷冻介质完全冷冻。</li>
		<li>对于长类器官，首先用手术刀切成两半，然后将两半彼此平行放入冷冻模具中。对打算进行组织学分析的每种类器官重复该过程。</li>
	</ol></li>
	<li>将样品储存在-20°C直至冷冻切片。</li>
	<li>使用冷冻组，将类器官纵向切割成 10 μm 厚的切片，并使用标准方案8 进行组织学染色，例如苏木精和伊红 （H&E）。</li>
</ol>

用于肌腱研究和工程的三步类器官模型

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提交人无需声明任何利益冲突。

本研究结果为NHDF 3D类器官模型的建立和表征提供了宝贵的见解，用于研究T/L组织。3 步方案导致 3D 杆状类器官的形成，这些类器官表现出 T/L 生态位的典型特征。该模型之前在 Kroner-Weigl 等人 20237 中报道，并在此处进行了非常详细的演示。
图 2 中显示的相差图像显示了在扩增和刺激步骤期间 NHDF 汇合和片状形成的进展。3D成熟过...

肌腱和韧带 （T/L） 是肌肉骨骼系统的重要组成部分，为身体提供必要的支撑和稳定性。尽管这些结缔组织起着关键作用，但它们容易退化和受伤，导致疼痛和行动障碍1。此外，他们的血液供应有限和愈合能力缓慢会导致慢性损伤，而衰老、重复运动和康复不当等因素则进一步增加了退化和受伤的风险2.常规治疗，如休息、物理治疗和手术干预，无法完全恢复 T/L 结构和功能。在过去的几年中，研究人员一直在努力更好地了解 T/L 的复杂性，以便寻求 T/L 疾病的有效治疗方法 3,4,5。T/L 的特点是层次结构合理、以细胞外基质 （ECM） 为主的结构，主要由 I 型胶原纤维和蛋白聚糖组成，这一特征在体外难以复制 6。传统的二维（2D）细胞培养模型无法捕捉T/L组织的特征性三维（3D）组织，限制了其转化潜力，并阻碍了T/L再生领域的创新进展。
最近，3D类器官模型的发展为推进各种组织类型的基础研究和无支架组织工程提供了新的可能性7,8,9,10,11,12,13。例如，为了研究肌腱连接处，Larkin等人，2006年开发了3D骨骼肌结构以及源自大鼠尾部肌腱10的自组织肌腱段。此外，Schiele 等人，2013 年，通过使用微机械纤连蛋白包被的生长通道，指导人真皮成纤维细胞的自组装形成细胞纤维，而无需 3D 支架的帮助，这种方法可以捕捉胚胎肌腱发育的关键特征11。在 Florida 等人 2016 年的研究中，骨髓基质细胞首先被扩解为骨骼和韧带谱系，然后用于生成自组装的单层细胞片，然后实施这些细胞片以创建模仿天然前交叉韧带的多相骨-韧带-骨结构，该模型旨在提高对韧带再生的理解12.为了阐明肌腱机械转导过程，Mubyana 等人，2018 年使用了一种无支架方法，通过该方法，创建单肌腱纤维并经受机械加载协议13。类器官是自组织的 3D 结构，模仿组织的天然结构、微环境和功能。3D 类器官培养为研究组织和器官生物学以及病理生理学提供了更具生理相关性的模型。此类模型还可用于诱导不同干细胞/祖细胞类型的组织特异性分化14,15。因此，在 T/L 生物学和组织工程领域实施 3D 类器官模型成为一种非常有吸引力的方法 9,16。可以为类器官组装实施替代细胞来源，并刺激其实现硬原分化。本研究中用于演示的一种相关细胞类型是真皮成纤维细胞 7,17,18。这些细胞很容易通过皮肤活检程序获得，与骨髓穿刺或吸脂术相比，皮肤活检程序的侵入性较小，并且由于其良好的增殖能力，可以相当快地繁殖到大量细胞。相比之下，更特化的细胞类型，如T/L驻留成纤维细胞，在分离和扩增方面更具挑战性。因此，真皮成纤维细胞也被用作细胞重编程技术的起点，以达到诱导多能胚胎干细胞19。将真皮成纤维细胞置于特定的 3D 培养条件和信号信号线索下，例如转化生长因子-β 3 （TGFß3），据报道，TGFß3 是各种细胞过程的关键调节因子，包括 T/L 的形成和维持，可以增强其体外致肌腱分化，导致肌腱特异性基因的表达和 T/L 典型 ECM20 的沉积，21.
在这里，我们描述并演示了先前建立和实施的 3 步（2D 扩增、2D 刺激和 3D 成熟）类器官方案，使用市售的正常成人真皮成纤维细胞 （NHDF） 作为细胞源，为研究 体外 肌张力生成提供了有价值的模型7。尽管该模型并不等同于 体内 T/L组织，但它仍然提供了一个生理学上更相关的系统，可用于研究细胞分化机制，在 体外模拟T/L病理生理学，以及建立T/L个性化医疗和药物筛选平台。此外，在未来，研究可以通过进一步优化来评估 3D 类器官是否适合于无支架 T/L 工程，以及可用于开发与天然 T/L 组织的尺寸、结构和生物物理特性非常相似的放大机械鲁棒结构。

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			<td>Ascorbic acid&#160;&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;&#160;</td>
			<td>A8960</td>
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			<td>10 cm adherent cell culture dish</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430167</td>
			<td></td>
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			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</td>
			<td></td>
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			<td>Cryo-medium</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&#160;&#160;</td>
			<td>4583</td>
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			<td>Cryomold standard&#160;</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands</td>
			<td>4557</td>
			<td></td>
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			<td>D(+)-Sucrose&#160;</td>
			<td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td>
			<td>A2211</td>
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			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
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			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
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			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
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			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</td>
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			<td>Inverted microscope with high resolution camera</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Zeiss Axio Observer with&#160; Axiocam 506</td>
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			<td>MEM amino acids&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>NEAA-B</td>
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			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
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			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</td>
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			<td>Paraformaldehyde</td>
			<td>&#160;AppliChem, Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>A3813</td>
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			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15140122</td>
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			<td>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</td>
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			<td>TGF&#223;3&#160;</td>
			<td>R&#38;D Systems, Wiesbaden, Germany&#160;</td>
			<td>&#160;8420-B3</td>
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			<td>Trypsin-EDTA 0,05% DPBS&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>&#160;TRY-1B</td>
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demonstration of self-assembled cell sheet culture and manual generation of a 3d tendon/ligament-like organoid by using human dermal fibroblasts

肌腱和韧带 （T/L） 是连接肌肉骨骼系统的坚固、层次分明的结构。这些组织具有严格排列的富含 I 型胶原的细胞外基质 （ECM） 和 T/L 谱系细胞，主要分布在平行行中。损伤后，T/L需要很长时间进行康复，故障风险高，修复结果往往不尽如人意。尽管 T/L 生物学研究最近取得了进展，但仍然存在的挑战之一是 T/L 领域仍然缺乏能够概括体 外 T/L 形成过程的标准化分化方案。例如，间充质前体细胞的骨和脂肪分化只需要标准的二维 （2D） 细胞培养和添加特定的刺激培养基。为了分化到软骨，需要三维 （3D） 颗粒培养和补充 TGFß。然而，细胞分化为肌腱需要一个非常有序的 3D 培养模型，理想情况下，该模型也应该能够受到动态机械刺激。我们已经建立了一个 3 步（扩增、刺激和成熟）类器官模型，以从自组装细胞片中形成 3D 棒状结构，该细胞片提供具有自身 ECM、自分泌和旁分泌因子的自然微环境。这些棒状类器官在丰富的 ECM 中具有多层细胞结构，并且可以很容易地处理暴露于静态机械应变。在这里，我们通过使用市售的真皮成纤维细胞演示了 3 步方案。我们可以证明，这种细胞类型形成了健壮且富含ECM的类器官。所描述的程序可以在培养基方面进一步优化，并针对动态轴向机械刺激进行优化。同样，可以测试替代细胞来源形成 T/L 类器官的潜力，从而进行 T/L 分化。综上所述，建立的3D T/L类器官方法可作为肌腱基础研究的模型，甚至可以作为无支架T/L工程的模型。

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

作者聚焦：使用 3D 类器官模型推进肌腱组织工程

使用人真皮成纤维细胞自组装细胞片培养和手动生成 3D 肌腱/韧带样类器官的演示

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

3D T/L 类器官模型之前是通过实施商业购买的 NHDF 建立并演示的（n=3，每个供体 3 个类器官，在第 5-8 次传代使用 NHDF）。 图 1 总结了模型工作流。 图2 显示了在T-75烧瓶中预扩增期间NHDF培养物的代表性相差图像（图2A），以及在10cm细胞培养皿中的2D扩增步骤中培养5天开始和5天后（图2B）。 图 ...

在此，我们展示了一种三步类器官模型（二维[2D]扩展、二维刺激、三维[3D]成熟），为肌腱基础研究提供了一种有前途的工具，并为肌腱组织工程提供了一种潜在的无支架方法。

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

我们展示了先前建立并实施的三步类器官方案，使用市售的正常人真皮成纤维细胞作为细胞来源。3D类器官模型在组织工程和再生医学应用中具有多项优势。它密切模拟肌腱组织的细胞组成和组织以及细胞间和细胞间基质的相互作用。建立的三维肌腱-韧带类器官方法可作为以下领域的肌腱基础和应用研究的模型。体外肌腱发生、疾病机制、药物检测、无支架肌腱组织工程。

demonstration-self-assembled-cell-sheet-culture-manual-generation-3d

Research

JoVE Journal

Bioengineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

722 次观看.  University of Würzburg. 我们展示了先前建立并实施的三步类器官方案，使用市售的正常人真皮成纤维细胞作为细胞来源。3D类器官模型在组织工程和再生医学应用中具有多项优势。它密切模拟肌腱组织的细胞组成和组织以及细胞间和细胞间基质的相互作用。建立的三维肌腱-韧带类器官方法可作为以下领域的肌腱基础和应用研究的模型。体外肌腱发生、疾病机制、药物检测、无支架肌腱组织工?...