WP1:The first work package aimed to guarantee the fellow’s technical training for the successful realization of the action, as well as the fellow’s professional development. Supervised research management activities such as, writing research and supervision of graduate students, occurred continuously throughout the fellowship. Technical trainings were conducted intensively in the first six months for cell culture, freeze drying and scaffold engineering and for stem cell culture and differentiation protocols.
WP2: This WP aimed at identifying polymer composite thin films that exhibit high electrical conductivity as well as photo-sensitivity when operated in aqueous, biological media. First, we evaluated 4 different semi-conducing polythiophenes purchased from Rieke Metals. We found that two of them combined high solubility in water – a requirement for good mixing with water soluble PEDOT:PSS – and therefore we continued working with them, namely P3C4PT and P3C6T. Based on a series of experiments we selected the 50 vol.% mixture of P3C6PT – PEDOT:PSS as the one that combines the highest electrical conductivity and photosensitivity. We then used these mixtures to construct polymer scaffolds as described in WP3.
WP3:This WP targeted the realization of multifunctional 3D porous polymer scaffolds based on the best combinations revealed in WP2. We found that the properties of the scaffolds can be tuned by using different crosslinkers – used to render these scaffolds durable in electrolytes. We showed that by using PEGDE crosslinker instead of the established GOPS, the pore size of the scaffolds is enlarged (more suitable for human adipose derived stem cells), the scaffolds are becoming more elastic and the electrical conductivity is enhanced. We then 3D bioelectronic devices with a characteristic impedance response. The increase of the stem cell population within the porous network impedes increase the device resistance and enables an electrical figure of merit to monitor the proliferation of stem cells. We then moved on the fabrication of polymer scaffolds, combining electrical and optical properties. The best mixtures were used, i.e. P3CP6PT-PEDOT:PSS 50 vol.%. Homogeneous porous scaffold slices were obtained with pore size between 50 -200 μm. The scaffolds exhibited multifunctional properties such as high elasticity, high electrical conductivity, and high photo-conductivity.
WP4:This WP aimed at the successful integration of adipose derived stem cells into the scaffolds/bioreactors. A seven-day long protocol was established where cells were able to fully colonize the scaffolds. Materials and device designs were kept constant and both electrical pulses as well as light pulses were applied to the cultures. For the electrical stimulation the devices described in WP3 were used. Electrical stimulation protocols were applied for more than 5 experimental batches. The results did not show any clear effect on the influence of electrical stimulation on these cultures. Therefore, we moved on optical stimulation. For these experiments, free floating multifunctional scaffolds were used. Immunofluorescence imaging showed that light cues can alter the morphology of cells. Importantly, we have developed a neurogenic differentiation protocol of these cultures growing within the 3D scaffolds. Immunofluorescence imaging with specific early neuron markers showed that the cells differentiated to neuron-like cells and exposed to light stimulation showed enhanced expression of neurofilament and b-tubulin, compared to the cells that have not been exposed to light stimulation.
Publications
1) 3-D Light sensitive Scaffolds for controlling differentiation of human adipose derived stem cells into neuron like cells. In preparation
2) 3-Dimensional Organic Bioelectronics for Electrical Monitoring of In Vitro Stem Cell Cultures, A. Savva, J. Saez, C. Barberio, C.-M. Moysidou, Z. Lu, K. Kallitsis, C. Pitsalidis, R.M. Owens, biorxiv: 486455v1 (2022).
3) Photo-Electrochemical Stimulation of Neurons with Organic Donor-Acceptor Heterojunctions, A. Savva, A. Hama, G. Herrera, N. Gasparini, L. Migliaccio, P. Magistretti, I. McCulloch, E. Glowacki, D. Baran, S. Inal, biorxiv: 480608 (2022).
4) Organic Bioelectronics for In Vitro Systems, C. Pitsalidis, A.-M. Pappa, A.J. Boys, Y. Fu, C.-M. Moysidou, D. van Niekerk, J. Saez, A. Savva, D. Iandolo, R. M. Owens, Chemical Reviews,(2021).
