Developing ECM-based bioinks for stem cell and tissue-specific 3D bioprinting

• Main Author: Neo, Shao Ming
• Other Authors: Subbu S. Venkatraman
• Format: Final Year Project
• Language: English
• Published: 2018
• Subjects: DRNTU::Engineering::Materials
• Online Access: http://hdl.handle.net/10356/73752
• Institution: Nanyang Technological University

The overall goal of this project is to develop universal production methodologies for bioactive and printable bioinks for tissue-specific cell-extracellular matrix (ECM) bioprinting. This goal was achieved by pursuing the following 2 aims: Aim 1 – To culture and characterize possible cellular components for tissue printing: human mesenchymal stem cells (hMSC) and human umbilical vein endothelial cells (HUVEC). These two cell types are representative of two common cell-tissue interactions shown in many native tissues—interstitial stromal cells and basement membrane coating epithelium/endothelium, respectively. hMSC were characterised for their phenotypic surface markers and differentiation potentials. Three major cell characterisation techniques were used: immunofluorescence staining, fluorescence-activated cell sorting (FACS) analysis, and evaluation of cell multipotency by a biochemical differentiation assays. hMSC cells were positively expressing the common mesenchymal stem cell markers CD73, CD90 and CD105 and show no expression of CD31 (a common endothelial lineage marker used as a negative control) by both FACS and immunofluorescent staining. HUVEC were positively expressing the endothelial cell marker CD31. hMSC cells were also tested for their differentiation potential by differentiating into chondrocytes, osteocytes and adipocytes. These differentiated cells were successfully characterised by using the respective histological staining and dye indicators. Aim 2 – To study various ECM crosslinking protocols and characterize the gelation kinetics of promising formulations using a rheometer. Two ECM and crosslinkers respectively were investigated by varying the ECM/crosslinker combinations and concentrations. More specifically, we used Matrigel—a cell secreted ECM of commercial source containing a high amount of basement membrane components (e.g., collagen type IV and Laminin), and porcine cardiac ECM (pcECM) which was manufactured by our Israeli collaborating lab through a process of interstitial myocardial wall decellularization. The crosslinkers studied were genipin—a plant based biocompatible crosslinker, and transglutaminase (Tg)—a natural crosslinking enzyme in physiology which has been patented by our lab for application in gelatin cell-laden deposition and printing. The results indicated that genipin as a crosslinker produced generally weaker gelling as compared to Tg (preliminary study). For rheological study, frequency sweep and time sweep were used to characterize the gels’ resistance to shear and the time taken for gelation respectively. Whist preparing for a frequency sweep, pcECM gels were discovered to be too weak to handle. In the frequency sweep experiment, it was discovered that Matrigel had high shear resistance at higher frequencies, however, the actual maximum shear has yet to be tested. For the time sweep, Matrigel High/Tg High sample could gel within 10s at both 12oC and 37oC, albeit with a lower rheological strength for the 12oC sample as compared to the 37oC sample. This is in contrast with Matrigel High only sample, which took 24min to gel. The sample was unable to gel at 8oC. Future work should focus on increasing the frequency tested for up to 100Hz, and test wider Matrigel/Tg temperature (12-45oC) and concentration ranges towards development of a reliable printing test using our robotic dispensing bioprinter.