School of Biomedical Engineering, McMaster University, Ontario L8S4K1, Canada.
Department of Mechanical Engineering, McMaster University, Ontario L8S4L7, Canada.
ACS Biomater Sci Eng. 2020 Sep 14;6(9):5346-5356. doi: 10.1021/acsbiomaterials.0c01073. Epub 2020 Aug 27.
The ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration. Newly emerging bioprinting methods sometimes lack the high cellular density needed to provide biophysical cues to orchestrate cellular behavior to recreate tissue architecture and function. Alternate methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of spheroids, organoids, or cell sheets. Cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues. Until now, the preparation of these sheets has involved culturing on specialized substrates that can be triggered by temperature or phase change (hydrophobic to hydrophilic) to release cells growing on them and form sheets. Here a new technique is proposed that allows delamination of cells and secreted ECM and rapid self-assembly into a cell sheet using a simple pH trigger and without the need to use responsive surfaces or applying external stimuli such as electrical and magnetic fields, only with routine tissue culture plates. This technique can be used with cells that are capable of syncytialization and fusion such as skeletal muscle cells and placenta cells. Using C2C12 myoblast cells we show that the pH trigger induces a rapid delamination of the cells as a continuous layer that self-assembles into a thick dense sheet. The delamination process has little effect on cell viability and maturation and preserves the ECM components that allow sheets to adhere to each other within a short incubation time enabling formation of thicker constructs when multiple sheets are stacked (double- and quadruple-layer constructs are formed here). These thick grafts can be used for regeneration purposes or as in vitro models.
形成具有适当细胞间和细胞-细胞外基质相互作用的高细胞密度组织样结构的能力对于许多应用至关重要，包括用于药物发现和组织再生的组织模型。新出现的生物打印方法有时缺乏提供生物物理线索以协调细胞行为以重现组织结构和功能所需的高细胞密度。可使用替代的自组装方法来创建具有高细胞密度和明确定义的微结构的组织样结构，例如球体、类器官或细胞片。在组织再生和修复的背景下，细胞片具有特别有趣的结构，因为它们可以作为补丁应用以与周围组织整合。到目前为止，这些片的制备涉及在特殊基质上培养，这些基质可以通过温度或相变化（从疏水性到亲水性）触发，以释放在其上生长的细胞并形成片。这里提出了一种新技术，该技术允许使用简单的 pH 触发从细胞和分泌的细胞外基质中分层，并快速自组装成细胞片，而无需使用响应表面或施加外部刺激，例如电和磁场，仅使用常规的组织培养板。该技术可用于能够发生合胞作用和融合的细胞，例如骨骼肌细胞和胎盘细胞。使用 C2C12 成肌细胞，我们表明 pH 触发会迅速将细胞分层为连续层，该层会自组装成厚而致密的片。分层过程对细胞活力和成熟的影响很小，并保留了细胞外基质成分，从而使片在短孵育时间内彼此粘附，从而在堆叠多个片时形成更厚的结构（这里形成双层和四层结构）。这些厚移植物可用于再生目的或作为体外模型。
