Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding
Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding
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DOI：
10.3791/53642
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发表时间：
2016-08-01
影响因子：
1.2
通讯作者：
Raghunath, Michael
中科院分区：
文献类型：
--
作者：
Benny, Paula;Badowski, Cedric;Raghunath, Michael
胶原蛋白糖蛋白家族是人体的主要结构蛋白，是现代组织工程中生物材料的重要组成部分。胶原蛋白在体外的沉积是一个技术瓶颈，因为它是出了名的缓慢，导致结缔组织的形成和随后的组织凝聚力不理想，特别是在皮肤模型中。在这里，我们描述了一种方法，该方法涉及到在皮肤培养物中添加不同大小的蔗糖共聚物以产生大分子拥挤（MMC），这导致胶原沉积的显着增强。特别是，与对照组相比，MMC下的真皮成纤维细胞沉积了大量的胶原I/IV/VII和纤维连接蛋白。该方案还描述了一种方法来脱细胞拥挤的细胞层，暴露了大量的细胞外基质（ECM），其保留在培养表面，如免疫细胞化学所证明的。用干涉反射显微镜研究了基质的总质量和分布规律。有趣的是，成纤维细胞、角化细胞和共培养物产生了不同成分和形态的细胞衍生基质（CDM）。CDM可以用作二次细胞播种的“生物支架”，在这种情况下，可以避免目前使用的涂层或支架（通常来自异种动物），从而转向更临床相关的应用。此外，该方案描述了在3d器官型皮肤共培养模型的浸没阶段MMC的应用，这足以增强真皮-表皮连接处（DEJ）的ECM沉积，特别是胶原蛋白VII，锚定原纤维的主要成分。与对照组相比，电子显微镜证实了MMC培养物中锚定原纤维的存在。这一点很重要，因为锚定原纤维将真皮层与表皮连接在一起，因此，在移植物稳定性和整体伤口愈合方面，预先形成成熟的DEJ可能有利于皮肤移植物受体。此外，当使用MMC时，培养时间从5周缩短到3周，以获得成熟的构建物，从而降低了成本。
The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.