Periodic Reporting for period 1 - COSMOS (Curved Optoelectronic System to Monitor Ocular Signals)
Periodo di rendicontazione: 2022-09-01 al 2024-08-31
The COSMOS project vision targeted the broad field of bioelectricity beyond cellular excitability, advancing the path toward bioelectricity-based theranostics. This field, also known as bioelectronic medicine, aims at unravelling the fundamental role of bioelectricity in homeostatic regulation of key life processes happening at the cellular and tissue scales, and develop translatable technologies for critical areas of medical research, including wound healing, cancer, ageing, and morphogenesis.
Endogenous electric fields (EFs) play a crucial role both in cell development and pathology. The directional migration of cells in an EF is known as galvanotaxis and represents a dominant mechanism in guiding the behaviour of multiple cell populations in mammals, fishes, amphibians, and plants. EF perturbations induce localized bioelectrical changes which trigger different cell responses, such as mitosis, migration, and mutation. Naturally occurring EFs can be altered by pharmacological administration to foster different cellular responses. For example, upon manipulating the EF in wounds, epithelial cells follow the direction of the signal, promoting or inhibiting corneal regeneration based on EF polarity. Another key example in the field of cancer biology regards the employment of tumor treating fields (TTFs) and electroceuticals acting on dysregulated ion channels in cellular membranes.
Regenerative medicine and pharmaceutical strategies aim at restoring diseased or damaged tissues resulting from a range of conditions. However, their clinical translation remains a major challenge, due to the lack of knowledge on the mechanisms underlying these processes. This technological and knowledge gap has a high price for our healthcare systems. For example, medicare cost estimates for acute wound treatments are $28-$96 billion per year.
In this scenario, technology can play two important roles:
1) Scientific Advancement: Knowledge as the Main Goal. Exploit existing microsystems to investigating bioelectricity beyond cellular excitability, and access unknown biological information by targeting unexplored applications.
2) Technological Advancement: Translation as the Main Goal. In parallel, existing technologies can be refined and improved to develop nanosystems that can interface living matter either non-invasively and with higher throughput, or probing biological signals unaccessible with current methods.
In the context of COSMOS, preliminary steps were conducted in both directions in the fields of non-excitable cells bioelectricity, with a focus on epithelial cells and cancer cells.
Endogenous electric fields (EFs) play a crucial role both in cell development and pathology. The directional migration of cells in an EF is known as galvanotaxis and represents a dominant mechanism in guiding the behaviour of multiple cell populations in mammals, fishes, amphibians, and plants. EF perturbations induce localized bioelectrical changes which trigger different cell responses, such as mitosis, migration, and mutation. Naturally occurring EFs can be altered by pharmacological administration to foster different cellular responses. For example, upon manipulating the EF in wounds, epithelial cells follow the direction of the signal, promoting or inhibiting corneal regeneration based on EF polarity. Another key example in the field of cancer biology regards the employment of tumor treating fields (TTFs) and electroceuticals acting on dysregulated ion channels in cellular membranes.
Regenerative medicine and pharmaceutical strategies aim at restoring diseased or damaged tissues resulting from a range of conditions. However, their clinical translation remains a major challenge, due to the lack of knowledge on the mechanisms underlying these processes. This technological and knowledge gap has a high price for our healthcare systems. For example, medicare cost estimates for acute wound treatments are $28-$96 billion per year.
In this scenario, technology can play two important roles:
1) Scientific Advancement: Knowledge as the Main Goal. Exploit existing microsystems to investigating bioelectricity beyond cellular excitability, and access unknown biological information by targeting unexplored applications.
2) Technological Advancement: Translation as the Main Goal. In parallel, existing technologies can be refined and improved to develop nanosystems that can interface living matter either non-invasively and with higher throughput, or probing biological signals unaccessible with current methods.
In the context of COSMOS, preliminary steps were conducted in both directions in the fields of non-excitable cells bioelectricity, with a focus on epithelial cells and cancer cells.
Technical activities included:
• Optical mirroring of bioelectric signals. Cleanroom fabrication of suspended pass-through nanoelectrodes across PMMA and silicon nitride membranes. Two independent processes were developed for the two materials. Silicon nitride was then chosen as the main material for subsequent steps, implementations and experiments due to its higher reproducibility in manufacturing methods, a better handling, and results obtained. The system was further interfaced with cells and with a chanrged fluorophores dispersion in the two compartments defined by the suspended membranes. The platform was enclosed in a rigid microfluidic chamber to preserve the liquid and to favour refillment during usage. The functionality of the pass-through electrodes in acting as an electrochemical cell was validated by optically mirroring the electric charge of non-excitable cells. These results advanced the state of the art in the charge mirroring technology, previously focused on cardiomyocytes action potential recording. Collectively, these results highlight the possibility of interfacing cells in dye contact-free non-invasive manner while recording their electrical activity via a proportional fluorescence signal.
• Cancer bioelectricity. In parallel, the existing microelectrode array technology was implemented toward the recording of cancer signaling, associated to migration and metastasis. In this context, epithelial cells, breast cancer cells and glioblastoma cells were cultured on microelectrodes, both cleanroom fabricated (Ti-Au) and commercial (TiN). Both configurations revealed the possibility to record electrical signaling in cancer, advancing the field of in vitro electrophysiology.
• Curved biointerfaces. Ultra-thin microelectrodes were fabricated on PVF membranes and transferred onto curved substrates to create conformable devices for ex-vivo biointerfaces. Cellular migration was monitored in wounded epithelial cell monolayers using IncuCyte assays and electrical recording via current amplifiers across wound sites.
• Imaging the cell-material interface. The cell-material interface was imaged in the context of two side activities revolving around cardiomyocyte electrical recording and brain organoid maturations. Here, the work performed involved using a combination of scanning electron microscopy (SEM), focused ion beam (FIB), and transmission electron microscopy (TEM) to evaluate cellular adhesion and tissue constructs maturation stage/biological development.
• Optical mirroring of bioelectric signals. Cleanroom fabrication of suspended pass-through nanoelectrodes across PMMA and silicon nitride membranes. Two independent processes were developed for the two materials. Silicon nitride was then chosen as the main material for subsequent steps, implementations and experiments due to its higher reproducibility in manufacturing methods, a better handling, and results obtained. The system was further interfaced with cells and with a chanrged fluorophores dispersion in the two compartments defined by the suspended membranes. The platform was enclosed in a rigid microfluidic chamber to preserve the liquid and to favour refillment during usage. The functionality of the pass-through electrodes in acting as an electrochemical cell was validated by optically mirroring the electric charge of non-excitable cells. These results advanced the state of the art in the charge mirroring technology, previously focused on cardiomyocytes action potential recording. Collectively, these results highlight the possibility of interfacing cells in dye contact-free non-invasive manner while recording their electrical activity via a proportional fluorescence signal.
• Cancer bioelectricity. In parallel, the existing microelectrode array technology was implemented toward the recording of cancer signaling, associated to migration and metastasis. In this context, epithelial cells, breast cancer cells and glioblastoma cells were cultured on microelectrodes, both cleanroom fabricated (Ti-Au) and commercial (TiN). Both configurations revealed the possibility to record electrical signaling in cancer, advancing the field of in vitro electrophysiology.
• Curved biointerfaces. Ultra-thin microelectrodes were fabricated on PVF membranes and transferred onto curved substrates to create conformable devices for ex-vivo biointerfaces. Cellular migration was monitored in wounded epithelial cell monolayers using IncuCyte assays and electrical recording via current amplifiers across wound sites.
• Imaging the cell-material interface. The cell-material interface was imaged in the context of two side activities revolving around cardiomyocyte electrical recording and brain organoid maturations. Here, the work performed involved using a combination of scanning electron microscopy (SEM), focused ion beam (FIB), and transmission electron microscopy (TEM) to evaluate cellular adhesion and tissue constructs maturation stage/biological development.
COSMOS delivered preliminary results on both directions: scientific advancement and technology advancement, with a focus on in epithelial cells and cancer cells. The latter share a common denominator in the context of bioelectricity beyond cellular excitability. For example, endogenously driven cellular migration phenomena have key implications both in wound healing and cancer metastasis.
Scientific advancement. We have found that cancer cells exhibit spontaneous electrical activity both at high (> 100 Hz) and low (< 100 Hz) frequencies, in the form of voltage spikes and bursts, that can be recorded using both commercial (TiN) and cleanroom-fabricated (Ti-Au) microelectrode systems. In addition, we have demonstrated that the firing rate correlates to the metastatic ability of the cancer cell, resulting in higher signaling in hihgly metastatic cancer cell cohorts. Studies were performed on epithelial cells, breast cancer cells, and glioblastoma cells. These results open a new path of exploration in the field of ion channels in cancer, bioelectronic medicine, and in vitro electrophysiology.
Technology advancement. The project has delivered the first method for dye contact-free optical recording of electrical signals from non-electrogenic cells in a non-invasive and high-throughput manner. These results have expanded existing knowledge of this method employed in excitable cells. Findings will be further exploited to individually target different contributions of this cumulative electrical signal. Examples include the optical monitoring of cell proliferation in wound healing, non-invasive evaluation of cell-material adhesion, surface charge- based recognition of cancer cells.
Scientific advancement. We have found that cancer cells exhibit spontaneous electrical activity both at high (> 100 Hz) and low (< 100 Hz) frequencies, in the form of voltage spikes and bursts, that can be recorded using both commercial (TiN) and cleanroom-fabricated (Ti-Au) microelectrode systems. In addition, we have demonstrated that the firing rate correlates to the metastatic ability of the cancer cell, resulting in higher signaling in hihgly metastatic cancer cell cohorts. Studies were performed on epithelial cells, breast cancer cells, and glioblastoma cells. These results open a new path of exploration in the field of ion channels in cancer, bioelectronic medicine, and in vitro electrophysiology.
Technology advancement. The project has delivered the first method for dye contact-free optical recording of electrical signals from non-electrogenic cells in a non-invasive and high-throughput manner. These results have expanded existing knowledge of this method employed in excitable cells. Findings will be further exploited to individually target different contributions of this cumulative electrical signal. Examples include the optical monitoring of cell proliferation in wound healing, non-invasive evaluation of cell-material adhesion, surface charge- based recognition of cancer cells.