Periodic Reporting for period 1 - MultiStem (Multifunctional polymer scaffolds for stem cell differentiation)
Período documentado: 2020-09-01 hasta 2022-08-31
A variety of factors are known to direct cell growth and differentiation. Although, the role of biochemical and mechanical cues in cell growth and differentiation are now relatively well understood, the role of electrical cues in cell development remain poorly understood. Recently, with the advent of functional conducting materials with modular properties that are easy to fabricate, it has become practicable to interface live cells with conducting materials in a more biomimetic fashion. Three-dimensional (3D) bioelectronic devices proposed to bridge the dimensionality mismatch between 2D/static electronics and 3D/dynamic biology, comprising a versatile platform for hosting and monitoring cells. These highly biomimetic systems can more accurately represent native tissues thanks to mimicry of biochemical, mechanical, and electrical cues. Stem cells take their cues from their environment and surroundings. A reliable platform that mimics accurately the human body environment, would be an invaluable tool in stem cell research. In general, tissue engineering has benefitted enormously from different materials used to create scaffolds to grow cells in 3D, however, only few studies report the development of 3D tissue-like structures that exhibit multifunctional properties (e.g. electrical and optical).
Objectives
- Fabrication of 3D-polymer scaffolds, mimicking the human body environment, with electrical and optical properties.
- Development of healthy stem cell cultures within the scaffolds and being able to monitor their activity
- Control the proliferation and differentiation of stem cells by taking advantage of the scaffold’s properties, i.e. by using electrical or optical stimulating pulses
- Generation of a vast library of experimental data on how optical or electrical cues influence stem cells differentiation
Objectives
- Fabrication of 3D-polymer scaffolds, mimicking the human body environment, with electrical and optical properties.
- Development of healthy stem cell cultures within the scaffolds and being able to monitor their activity
- Control the proliferation and differentiation of stem cells by taking advantage of the scaffold’s properties, i.e. by using electrical or optical stimulating pulses
- Generation of a vast library of experimental data on how optical or electrical cues influence stem cells differentiation
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).
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).
The experimental results have revealed the potential of organic electronic materials for stem cell engineering. We showed for the first time the multifunctional organic electronic thin films can be produced – i.e. thin films that exhibit both electrical conductivity and photosensitivity. These materials can be used in the future for the development of novel bioelectronic devices such photosensitive organic electrochemical transistors. The biocompatible nature of these materials can also be utilised to produce multifunctional interfaces for electrogenic cells such as neurons. Further development of the proposed 3D platforms for monitoring stem cell proliferation can also have added features, such the ability to stimulate stem cell cultures aiming in controlled cell-fate direction. We believe the results presented here pave the way for further advancements in 3D stem cell research for both fundamental understanding as well as the development of personalized therapies. Finally, our experimental evidence on the positive effect of light stimulation on stem cell differentiation show a great potential for the development of more reliable and cost effective personalized treatments.