Development of cell sheet based bioink for 3D bioprinting applications
Bakırcı, Ezgi (2016) Development of cell sheet based bioink for 3D bioprinting applications. [Thesis]
Fabrication of functional tissue/organ constructs is becoming more feasible considering the recent developments in bioprinting technologies. There are two major approaches for the 3D bioprinting of tissue and organs. The scaffold based bioprinting approaches are studied to develop biocompatible, and at the same time, printable biomaterials with mechanical properties similar to the native tissue. The scaffold-free approaches have recently been taking attention due to its ability to fabricate tissue constructs without any exogenous materials and mimicking the natural development process of the targeted tissue. This approach provides reproducible native tissue in a shorter period of time compare to the scaffold-based approaches. Cell sheet technology is one of the scaffold-free tissue engineering techniques which forms a sheet of interconnected cells to establish cell-extracellular matrix (ECM) and cell-cell interactions. Furthermore, it provides a mechanically stronger structure and better mimicking of natural tissue. Poly (N-isopropylacrylamide) (pNIPAAm) coated thermoresponsive culture dishes are commonly used as one of the advanced cell sheet technology methods. pNIPAAm coated dishes are used to produce cell sheets and to detach them from the surface by using the switchable amphiphilic properties of the surface at two different temperatures. Although cell sheet technology has been clinically successful, there exist some limitations when fabricating 3D constructs, because the approach allows for only a simple and flat shaped tissue constructs. The implementation of cell sheet technology within the scaffold free bioprinting approach was investigated in this study. A novel method was developed to prepare bioink from cell sheets for 3D bioprinting applications. pNIPAAm coated surfaces were prepared by using initiated Chemical Vapor Deposition (iCVD) techniques for growing cell-sheets and also removing them from the pNIPAAm coated surfaces easily. The results showed that the prepared human skin fibroblast cell sheet aggregates increase the structural integrity of the bioink compared to the bioink composed of single cell aggregates, suggesting that cell sheet aggregates preserved interconnected ECM proteins and cells by harvesting with thermoresponsive dishes. The viability, fusion and ECM deposition of cell sheet based aggregates were compared to single cell aggregates three times in a seven-day period after printing. The results showed that using cell sheet aggregates as a novel bioink in 3D bioprinting applications has several advantages in terms of shape fidelity, reproducibility and shorter preparation time. The results also demonstrated that the constructs secreted collagen type I which is a strong indication of starting ECM deposition by bioprinted cell sheet aggregates. Moreover, the immunostaining results showed that the fibronectin in cell sheets was preserved during the preparation process of bioink.
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