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

<ol>
	<li>Schneider, M., Angele, P., J&#228;rvinen, T. A. H., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+plan+for+Achilles:+Therapeutics+steering+the+fate+and+functions+of+stem+cells+in+tendon+wound+healing.">Rescue plan for Achilles: Therapeutics steering the fate and functions of stem cells in tendon wound healing.</a> Adv Drug Deliv Rev. 129, 352-375 (2018).</li><li>Steinmann, S., Pfeifer, C. G., Brochhausen, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+tendon+pathologies:+Triggers,+trails+and+end-state.">Spectrum of tendon pathologies: Triggers, trails and end-state.</a> Int J Mol Sci. 21 (3), 844 (2020).</li><li>Snedeker, J. G., Foolen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+injury+and+repair+-+A+perspective+on+the+basic+mechanisms+of+tendon+disease+and+future+clinical+therapy.">Tendon injury and repair - A perspective on the basic mechanisms of tendon disease and future clinical therapy.</a> Acta Biomater. 63, 18-36 (2017).</li><li>Lomas, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+in+scaffold-based+tendon+treatments.">The past, present and future in scaffold-based tendon treatments.</a> Adv Drug Deliv Rev. 84, 257-277 (2015).</li><li>Gomez-Florit, M., Labrador-Rached, C. J., Domingues, R. M. A., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tendon+microenvironment:+Engineered+in+vitro+models+to+study+cellular+crosstalk.">The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.</a> Adv Drug Deliv Rev. 185, 114299 (2022).</li><li>Docheva, D., M&#252;ller, S. A., Majewski, M., Evans, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologics+for+tendon+repair.">Biologics for tendon repair.</a> Adv Drug Deliv Rev. 84, 222-239 (2015).</li><li>Kroner-Weigl, N., Chu, J., Rudert, M., Alt, V., Shukunami, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+Is+not+sufficient+to+facilitate+tenogenic+differentiation+of+dermal+fibroblasts+in+a+3D+organoid+model.">Dexamethasone Is not sufficient to facilitate tenogenic differentiation of dermal fibroblasts in a 3D organoid model.</a> Biomedicines. 11 (3), 772 (2023).</li><li>Chu, J., Pieles, O., Pfeifer, C. G., Alt, V., Morsczeck, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+follicle+cell+differentiation+towards+periodontal+ligament-like+tissue+in+a+self-assembly+three-dimensional+organoid+model.">Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.</a> Eur Cell Mater. 42, 20-33 (2021).</li><li>Yan, Z., Yin, H., Brochhausen, C., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aged+tendon+stem/progenitor+cells+are+less+competent+to+form+3D+tendon+organoids+due+to+cell+autonomous+and+matrix+production+deficits.">Aged tendon stem/progenitor cells are less competent to form 3D tendon organoids due to cell autonomous and matrix production deficits.</a> Front Bioeng Biotechnol. 8, 406 (2020).</li><li>Larkin, L. M., Calve, S., Kostrominova, T. Y., Arruda, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functional+evaluation+of+tendon-skeletal+muscle+constructs+engineered+in+vitro.">Structure and functional evaluation of tendon-skeletal muscle constructs engineered in vitro.</a> Tissue Eng. 12 (11), 3149-3158 (2006).</li><li>Schiele, N. R., Koppes, R. A., Chrisey, D. B., Corr, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cellular+fibers+for+musculoskeletal+soft+tissues+using+directed+self-assembly.">Engineering cellular fibers for musculoskeletal soft tissues using directed self-assembly.</a> Tissue Eng Part A. 19 (9-10), 1223-1232 (2013).</li><li>Florida, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structural+and+cellular+remodeling+of+engineered+bone-ligament-bone+constructs+used+for+anterior+cruciate+ligament+reconstruction+in+sheep.">In vivo structural and cellular remodeling of engineered bone-ligament-bone constructs used for anterior cruciate ligament reconstruction in sheep.</a> Connect Tissue Res. 57 (6), 526-538 (2016).</li><li>Mubyana, K., Corr, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+uniaxial+tensile+strain+enhances+the+mechanical+properties+of+engineered,+scaffold-free+tendon+fibers.">Cyclic uniaxial tensile strain enhances the mechanical properties of engineered, scaffold-free tendon fibers.</a> Tissue Eng Part A. 24 (23-24), 1808-1817 (2018).</li><li>Brassard, J. A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+stem+cell+self-organization+to+build+better+organoids.">Engineering stem cell self-organization to build better organoids.</a> Cell Stem Cell. 24 (6), 860-876 (2019).</li><li>Kim, J., Koo, B. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+model+systems+for+human+biology+and+medicine.">Human organoids: model systems for human biology and medicine.</a> Nat Rev Mol Cell Biol. 21 (10), 571-584 (2020).</li><li>Hsieh, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comparison+of+2D-cell+culture+and+3D-cell+sheets+of+scleraxis-programmed+bone+marrow+derived+mesenchymal+stem+cells+to+primary+tendon+stem/progenitor+cells+for+tendon+repair.">In vitro comparison of 2D-cell culture and 3D-cell sheets of scleraxis-programmed bone marrow derived mesenchymal stem cells to primary tendon stem/progenitor cells for tendon repair.</a> Int J Mol Sci. 19 (8), 2272 (2018).</li><li>Chu, J., Lu, M., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+tendons:+A+concise+review+on+the+potential+of+dermal+fibroblasts.">Rebuilding tendons: A concise review on the potential of dermal fibroblasts.</a> Cells. 9 (9), 2047 (2020).</li><li>Gaspar, D., Spanoudes, K., Holladay, C., Pandit, A., Zeugolis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+cell-based+therapies+for+tendon+repair.">Progress in cell-based therapies for tendon repair.</a> Adv Drug Deliv Rev. 84, 240-256 (2015).</li><li>Sacco, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> J Cell Mol Med. 23 (6), 4256-4268 (2019).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+predominant+tenogenic+phenotype+in+human+dermal+fibroblasts+via+synergistic+effect+of+TGF-&#946;+and+elongated+cell+shape.">Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-&#946; and elongated cell shape.</a> Am J Physiol Cell Physiol. 310 (5), C357-C372 (2016).</li><li>Pryce, B. A., Watson, S. S., Murchison, N. D., Staverosky, J. A., D&#252;nker, N., Schweitzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+and+maintenance+of+tendon+progenitors+by+TGFbeta+signaling+are+essential+for+tendon+formation.">Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation.</a> Development. 136 (8), 1351-1361 (2009).</li><li>Pattappa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physioxia+has+a+beneficial+effect+on+cartilage+matrix+production+in+interleukin-1+beta-inhibited+mesenchymal+stem+cell+chondrogenesis.">Physioxia has a beneficial effect on cartilage matrix production in interleukin-1 beta-inhibited mesenchymal stem cell chondrogenesis.</a> Cells. 8 (8), 936 (2019).</li><li>Shafiee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+physiologically+relevant+skin+organoids+from+human+induced+pluripotent+stem+cells.">Development of physiologically relevant skin organoids from human induced pluripotent stem cells.</a> Small. 2304879, (2023).</li><li>Zeiger, A. S., Hinton, B., Van Vliet, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+the+dish+makes+a+difference:+quantitative+comparison+of+polystyrene+culture+surfaces.">Why the dish makes a difference: quantitative comparison of polystyrene culture surfaces.</a> Acta Biomater. 9 (7), 7354-7361 (2013).</li><li>Koh, T. M., Feih, S., Mouritz, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengthening+mechanics+of+thin+and+thick+composite+T-joints+reinforced+with+z-pins.">Strengthening mechanics of thin and thick composite T-joints reinforced with z-pins.</a> Compos Part A Appl Sci. 43 (8), 1308-1317 (2012).</li><li>Gelberman, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+administration+of+ASCs+and+BMP-12+promotes+an+M2+macrophage+phenotype+and+enhances+tendon+healing.">Combined administration of ASCs and BMP-12 promotes an M2 macrophage phenotype and enhances tendon healing.</a> Clin Orthop Relat Res. 475 (9), 2318-2331 (2017).</li><li>Hofer, M., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+organoids.">Engineering organoids.</a> Nat Rev Mater. 6 (5), 402-420 (2021).</li><li>Driehuis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+organoids+recapitulate+disease+and+allow+personalized+drug+screening.">Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.</a> Proc Natl Acad Sci U S A. 116 (52), 26580-26590 (2019).</li><li>Yan, H. H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+human+gastric+cancer+organoid+biobank+captures+tumor+subtype+heterogeneity+and+enables+therapeutic+screening.">A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.</a> Cell Stem Cell. 23 (6), 882-897 (2018).</li></ol>

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Ascorbic acid&#160;&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;&#160;</td>
			<td>A8960</td>
			<td></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>
		</tr>
		<tr>
			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-medium</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&#160;&#160;</td>
			<td>4583</td>
			<td></td>
		</tr>
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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>
			<td></td>
		</tr>
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			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
			<td></td>
		</tr>
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			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
			<td></td>
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			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
			<td></td>
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			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>&#160;AppliChem, Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>A3813</td>
			<td></td>
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			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15140122</td>
			<td></td>
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			<td>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</td>
			<td></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>
			<td></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>
			<td></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。

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 ...

作者聚焦：使用 3D 类器官模型推进肌腱组织工程

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。

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

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

1.2K 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?...

视频: 作者聚焦：使用 3D 类器官模型推进肌腱组织工程

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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 ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...