Subprojeto 18 – Microfluidics as a tool for investigation of biological agents in dynamic conditions

Participantes: Aline Furtado Oliveira1, Franciele Flores Vit1, Marcelo Lancellotti2, Reinaldo Gaspar Bastos3 and Lucimara Gaziola de la Torre1
(1) School of Chemical Engineering, University of Campinas, (2) Institute of Biology, University of Campinas, (3) Centro de CiênciasAgrárias, Center for Agricultural Sciences, Federal University of São Carlos

Microfluidics has the technological potential for biological agents (cell and enzymes) investigation under dynamic conditions, mimicking cellular micro environment. Microfluidic systems use small volumes that flow inside microchannels in laminar flow, with precise control of molecules in space and time. Considering these characteristics, microfluidics allows the evaluation of the culture conditions, microbial behavior and chemotaxis under different substrate concentration. Microfluidic systems can also be used for biological agent immobilization and cell culture using microbioreactors, allowing the investigation of kinetic parameter contributing to the improvement of industrial bioprocesses. Microfluidics coupled the techniques of biophotonics, such as timelapse confocal microscopy, FluoresenceCorrelaction Spectroscopy (FCS), Fluorescence Lifetime Image Microscopy (FLIM), Raman and Spinning Disk expands the research possibilities. Previous experience from our group identified the ideal limiting substrate concentration for microbial grown by using timelapse confocal microscopy. We designed diffusion-based concentration gradient to investigate the ideal culture conditions, allowing the study of free cells, in anaerobic conditions. As concept proof, Saccharomycecerevisiae ATCC7754 were cultured under glucose concentration gradient, in anaerobic condition at 30 ºC. The cellular behavior was observed over time through confocal microscopy Zeiss LSM 780-NLO (Carl Zeiss AG, Germany) and the images were acquired automatically every 30 min. The cellular growth was evaluated in different substrate concentration in only one device, allowing the determination of kinetic data. The next research steps involve the study of bacteria behavior in these microdevices. Other approach will be the development new microfluidics devices to study biological agent (as cell and enzyme) free or immobilized, contributing to the development of industrial bioprocesses that operate continuously.

Microfluidic droplets device to transfect non-adherent mammalian cells in vitro
Micaela Tamara Vitor, Caroline Casagrande Sipoli, Lucimara Gaziola de La Torre
Micaela Tamara Vitor1, Caroline Casagrande Sipoli1, Charles Baroud2and Lucimara Gaziola de la Torre1
(1) Faculdade de Engenharia Química, Universidade Estadual de Campinas, (2) ÉcolePolytechnique

This project aims to develop a microfluidic droplets system to transfect mammalian cells using nanoparticles as nucleic acid nanocarriers. For this, nucleic acids encoding the green fluorescent protein will be incorporated into cationic liposomes, and then inserted in the microfluidic droplets system with mammalian cells, to induce the transfection within the droplets. Traditional case of stagnant flow transfection in wells allows only diffusive transport of nanoparticles/nucleic acid to the cell surface; in contrast, the convective contribution in micro-droplets facilitates and enhances the control of the transfection. Additionally, droplet-based microfluidics demonstrated as a potentially more sensitive method for biomarker discovery than conventional microfluidic systems, since the encapsulation permits amplified detection of extremely low levels of biomarker molecules. Furthermore, based on the “rails and anchors” channel design, the motion of droplets can be control, optimizing the number of cells that can be observed and the flow within the stationary droplets to control the shear stress that is felt by the cells while they remain in the field of view of the microscope.Microfluidic droplet systems coupled the techniques of biophotonics, such as timelapse confocal microscopy, FluoresenceCorrelaction Spectroscopy (FCS), Fluorescence Lifetime Image Microscopy (FLIM), Raman and Spinning Disk, allows the quantification of fluorescent protein production and characterize the transfection process.Thus, this system will enable us to study the optimum molar ratio between the nanoparticles and nucleic acids, the rate between nanoparticle/cell and the effect of shear stress inside the droplets on transfection. Then, the transfection occurred in the microfluidic system will be compared to the traditional transfection, performed in wells (bulk method). This project will contribute in nanobiotechnology, microfluidics and gene delivery areas.