Towards a 3D-Printed and Autonomous Culture Platform Integrated with Commercial Microelectrode Arrays

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by IEEE in Proceedings of the 2021 13th Spanish Conference on Electron Devices on June 2021, available at: https://doi.org/10.1109/CDE52135.2021.9455748 © 2021 IEEE. En idUS Licencia Creative Commons CC BY-NC-ND Towards a 3D-Printed and Autonomous Culture Platform Integrated with Commercial Microelectrode Arrays Jesús David Urbano-Gámez, Carmen Aracil, Francisco Perdigones, José Antonio Fontanilla, José Manuel Quero Universidad de Sevilla, [email protected], [email protected], perd[email protected], jfonta[email protected], [email protected] 1. Abstract Biologists and physicians need tools that may study the biological mechanisms involved in the degeneration of tissues to understand and slow down the effects of the diseases. In this way, systems able to maintain a culture in its appropriate conditions, even long term, and compatible with electro-stimulation and acquisition, open a wide field of applications. The proposed design is a low-cost portable system that accomplishes all these requirements. It is based on the integration of an MEA with electronic systems, and a culture platform, offering the possibility of electro-stimulation of a cell or tissue culture and studying its electrical activity. 2. Introduction Focusing on the long-term cultures, most applications involve the use of incubators to maintain the culture in good conditions [1], and the use of electronic systems like Multi Channel Systems MCS GmbH where the electro-stimulation and acquisition are performed. However, there is a lack of integrated systems where both functions are combined. The development of integrated platforms with the benefits of having the supply of nutrients, and even drugs to study their effects, while the acquisition and electro-stimulation can be carried out is fundamental [2], and would allow obtaining all the relevant information in the understanding of biological processes underlying pathologies and the possible effect of new therapeutic treatments. Regarding the culture platform, strict conditions are required for the correct development of cell and tissue cultures. This implies some parameters of the physiochemical environment must be controlled such as nutrients and oxygen supply, pH, and temperature [3]. In addition, the design should be compatible with the addition of other liquids, to add drugs if required. On the side of electronics, an electrical interface is required to be in direct contact with the culture, and an electronic system has to be designed in a bidirectional manner, so the culture could be stimulated for studying its effect and being able to capture the evolution of the culture along the time of the experiment. This particular requirement should be highlighted, since monitoring the activity of the culture would allow feedback in realtime, achieving interesting results before the end of the experiment and generating a continuous report of cell state with minimal invasiveness. The integration of electronics and microfluidics is the key to the challenge, using a low-cost technology easy to customize. In this way, it could be applied to many different cultures, even long-term ones, which are the more stringent ones. 3. Proposed design The proposed design is shown in Fig. 1. On one hand, it is composed of a microfluidic platform printed with biocompatible resin, where the ports are located. It is the interface with the impulsion system if required. Through these ports, nutrients are supplied, and the waste of the culture is discarded. On the other, the electronic elements are taken into account: the MEA and the electronic system. Both elements will be connected between them thanks to spring-loaded pins and will be also connected to a PC to monitor the culture during the experiments. Regarding the MEA, it is the substrate where the culture is in direct contact. It is a commercial MEA of Multi Channel Systems. The electronic system, on the other, is in charge of the generation and acquisition of signals, designed ad-hoc to the specific biomedical application. It can also include any signal conditioning circuit, amplifiers, etc. In the following sections, MEA and the microfluidic platform will be explained in detail. Fig.1. Proposed design. 4. Microelectrode Array Although multielectrode arrays (MEAs) appeared over the 70s [4], in recent years these devices have become a powerful tool in the field of electrophysiology. MEAs allow the simultaneous recording and stimulation of large areas using bidirectional electrodes distributed across the preparation. The complexity of these devices has grown with technology, giving today the possibility of recording and stimulating with more than 65000 electrodes in an area of 42,6 mm 2 [5]. Considering the relevance of these devices in the current context of electrophysiology and the need of automated systems for culturing, the proposed device provides a low-cost portable solution with a potential application in biomedical research. The proposed design is based on the use of 60MEA500/30iR-Ti of Multi Channel Systems MCS GmbH [6], although it is compatible with other models. This specific model is composed of 60 microelectrodes (Fig. 2), distributed in an array of 6x10 lines, with a separation of 500 µm. This MEA can be used for both organotypic or cell cultures. The culture would be located directly over the electrodes. Fig.2. 60MEA500/30iR-Ti of Multi Channel Systems. 5. Culture platform The following requirements have to be considered in the design of the culture platform. First of all, it has to contain the nutrients, encircling the culture, so one or more wells have to be included, where the culture is deposited. Moreover, it has to be connected to the pump system in charge of controlling its delivery along with the experiment. In long-term cultures the flow used to be very slow, so a controlled syringe pump, connected to this culture platform, can be used. The culture platform must therefore have two ports, an inlet for nutrients and an outlet of waste. Another important requirement is to design the platform for avoiding the creation of bubbles since they would result in the culture lacking nutrients and could deteriorate. In this way, a tilted surface has been designed between the inlet and outlet ports, so that in the purging process, the bubbles that are created move towards the outlet port. The choice of the material for the culture platform has to consider many aspects. First of all, the material has to be biocompatible, low cost, and easy to manufacture. The material chosen was a biocompatible Uniz zSG amber resin. The manufacture has been performed with 3D Anycubic Photon printer. The fluidic platform was designed using Fusion 360 software from Autodesk. The outer limits of the design were fitted to the space between electrodes and external pads of the MEA device. An internal space of 21x21 mm was defined for the base where the culture (cell or tissue) would be seeded. The top part was designed according to the electrode matrix to guarantee a correct visualization. Two circular Luer connectors were added to the design at opposite sites to connect the perfusion and waste system. The perfusion entry hole was open lower than the waste one, to use the waste height as the filling limit. Both ports were surrounded by a frame with a 10º slope designed to attach a glass coverslip, for the isolation of the culture chamber. Both, the base and the top part have 4 vertical cylindrical holes aligned at each corner to couple both bodies using a system of screw and nut (Fig. 3). Fig.3. Microfluidic platform 3D design B. Transversal section showing perfusion and waste channels. For printing, the model was rotated 45º in the X and Y axes and cone supports (diameter 1,30 mm, circular base) were added automatically using the LycheeSlicer software. In order to ensure accurate printing, additional supports were manually added to the lowest corner of the body. The inner space was fully filled to generate a robust piece. The model was printed using the Anycubic Photon 3D printer with the biocompatible zSG amber resin from Uniz. The printing settings were customized, A B 8 bottom layers were defined with an exposure time of 60 seconds, the normal exposure time was set to 20 s, and the layer thickness was fixed to 0,05 mm. After printing, the pieces were washed with isopropanol, followed by an additional wash with distilled water and a post-cure period of 45 minutes with UV light. The optimization of the culture area was a key point when designing the fluidic platform. Due to space limits, alignment of both MEA and fluidic platform become a critical step to keep electrodes and pads accessible. For this reason, alignment support was designed and printed. The alignment support was composed of two pieces: a base where the MEA device is placed and a cap where the base of the fluidic platform fits. For adhesion of the fluidic platform and the MEA, the epoxy resin EPO-TEK 301 was used. Biocompatibility of EPO-TEK 301 adhesive was previously evaluated in a cell line [7]. Additionally, as previously mentioned, a glass coverslip was attached to the top part of the platform using the same adhesive, EPO-TEK 301. Isolation of the culture chamber has been ensured using EVA (ethylene-vinyl acetate) polymer in the gap between the base and top part of the fluidic platform. Fig. 4. Setup of the integration between the culture platform and MEA. 6. Results and discussion As previously described, a critical point is the assembly of the fluidic platform and the MEA device. The results are successful using EPO-TEK 301 epoxy. Alignment of both the MEA and the platform was assured using a customized two-piece aligner. Fig. 4 shows the resulting assembly. Microfluidic tests have been carried out. Water resistance and correct adhesion were ensured by keeping the inner chamber filled with distilled water for more than 24 hours. No leakages were observed and the bubbles were removed during the filling thank to the tilted configuration. Regarding the functionality, a continuity test was performed on all the electrodes in the MEA using a saline buffer (PBS, Phosphate-Buffered Saline; Fisher BioReagents Ref. BP2944-100). No disruption or alteration of the electrodes was observed after the coupling process. 7. Conclusion A portable culture platform has been designed and fabricated. The proposed design consists of the integration of microfluidic and electronic elements. The final structure counts with a transparent window to follow the evolution of the culture along with the experiments, and a 3D biocompatible platform with inlet and outlet ports to manage the medium. This part is assembled to a commercial microelectrodes array to electro-stimulate and acquire biological signals. It is compatible with the connection to an electronic system that monitors the culture. Acknowledgements This work was funded by the European Regional Development Fund (ERDF) under the responsibility of Andalusian Government (Consejería de Economía y Conocimiento). The reference of the project is US- 1265983, Retina-on-a-chip. References [1] Danisovic, L., et al. "Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem cells." J Physiol Pharmacol 68.1 (2017): 149-158. [2] Langhans, Sigrid A. "Three-dimensional in vitro cell culture models in drug discovery and drug repositioning." Frontiers in pharmacology 9 (2018): 6. [3] Charis-P. Segeritz, Ludovic Vallier, Chapter 9 - Cell Culture: Growing Cells as Model Systems In Vitro, Editor(s): Morteza Jalali, Francesca Y.L. Saldanha, Mehdi Jalali, Basic Science Methods for Clinical Researchers. [4] C.A. Thomas, P.A. Springer, G.E. Loeb, Y. Berwald- Netter, L.M. Okun, A miniature microelectrode array to monitor the bioelectric activity of cultured cells, Experimental Cell Research, Volume 74, Issue 1, 1972, Pages 61-66, ISSN 0014-4827. [5] Tsai, D., Sawyer, D., Bradd, A. et al. A very large-scale microelectrode array for cellular-resolution electrophysiology. Nat Commun 8, 1802 (2017). [6] https://www.multichannelsystems.com/ (accessed on 01/02/2021). [7] Cabello, M., Domínguez, I., Macías, C. et al. Low cost SU-8 lift-off process to fabricate a gold/glass microelectrodes array for culturing applications. Microsyst Technol (2021).