CONICET | Buscador de Institutos y Recursos Humanos

The production of monoclonal antibodies for therapeutic use is one of the fastest growing areas in the biopharmaceutical industry. In 2011, the production of therapeutic recombinant proteins was estimated at $56 billion, with an estimated projection of more than $ 80 billion by 2015.Currently, the commercial production of monoclonal antibodies and other biotherapeutics is based on the synthesis in bioreactors with suspended mammalian cells with agitation operated in fed-batch or perfusion mode [1-2]. The production of monoclonal antibodies in these stirred tanks faced challenges related to product quality and process such as demand for higher productivity, control of glycosylation, reproducibility, and other process controls. Most of these challenges are related to the large spatial and temporal variability of the intrinsic conditions of the fermenters. One way to improve the control is to reduce the scale of the system by miniaturization in the form of micro devices [3]. A micro device offers several advantages, including shorter time response, a higher surface / volume ratio and a more homogeneous and controllable microenvironment.In this work was study the effect of the microchip geometry for the growth of HEK-293T cells, which are antibody production cells [4]. A Chip design with different serpentine shapes of 100 μm wide and lengths between 12 mm and 80 mm was used. Feed by a central channel of 40x1.9 mm (length x width), and spacers channels of 12 x1,2mm (length x width) between serpentines. The height of all channels was 40 μm.Several microfabrication processes have been implemented for the microfluidic device. A mold of the design in high relief was made by photolithography in a silicon (100) wafer 700 µm thick (Virginia Semiconductor, Inc.), by using the negative resin SU-8 (MicroChem). PDMS was mixed with curing agent in a 15:1 ratio. The mixture was placed under vacuum to remove air bubbles. After this, the mixture was poured into the master; placed under vacuum once again, and cured in an oven at 70 C during 70 min. Finally the sample was peeled off. Once the processes were finished (PDMS microchannels and glass), both parts were joined by exposure to oxygen plasma in PECVD.Cells were grown on surfaces of polymethyl siloxane (PDMS) microchannels, the effect of adding Poly-D-Lisine was evaluated which substantially improves cell attachment. Before cell seeding, the chip was treated with 0.1 mg/ml Poly-D-lisine Hydrobromide (Sigma) sterile solution in water in order to improve the attachment of the cells. The microfluidic devices were incubated with Poly-D-lisine solution for one hour at 37°C. The solution was removed and let dry 24h at 4°C. HEK-293T cells were cultured in complete DMEM medium (Gibco, Grand Island, NY, USA), that was supplemented with 10% fetal calf serum heat-inactivated (SBF) (Internegocios SA, Mendoza, Argentina), 2 mM L-glutamine (Gibco, Grand Island, NY, USA), and Antibiotic-Antimycotic 100 units/ml penicillin, 100 ug/ml streptomycin and 0.25 g fungizone ml (Gibco, Grand Island, NY, USA) at 37°C in an incubator with 5% CO2. The cells were resuspended with trypsin solution 250 mg/ml, EDTA 100 mg/ml (Gibco, Grand Island, NY, USA) and incubated at 37°C for 3 minutes. Trypsin was inactivated with FBS and the cells were washed with PBS (50 mM NaH2PO4, 300 mM NaCl, pH = 7.6). They were then resuspended in DMEM supplemented with 20% FBS at a concentration of 107 cells/ml. Before seeding the cells, the microchannels were washed with complete DMEM medium and were kept filled with medium for HEK-293T cell seeding. Next, cells were seeded in a 20 μl volume in the loading well of the microfluidic device. Once the cell suspension reached the outlet well, 20 μl of DMEM complete medium without cells was applied to equilibrate the flow in the micro channels. The cells were allowed to settle and the microfluidic device was incubated at 37°C in incubator with 5% CO2. The medium was changed every 24 hours with fresh DMEM complete medium. The microchannels of the device were visualized using an inverted Olympus microscope CKX41. Brightfield images were taken with Olympus objectives LUCPlan FL N 40X/0.60; LCAch N 20X/0.40; PlanC N 10X/0.25; and PlanC N 4X/0.10 with an Olympus QColor 5; and they were processed with QCapture Pro 6.0 software.At the time of seeding, HEK-293T cells were distributed in aggregates or individually along the microchannels (Figure 1). At day two, the larger aggregates were washed and the individual cells were adhering to the bottom of the wide channels (canal central y canales espaciadores) with the DMEM complete medium change. Cells then began to form clusters and to extend processes around the clusters (Figure 2). At day five the cells consolidate their growth in clusters on the floor of the wide microchannels, with almost no cells in the serpentine channels. At day eight, cells began to show signs of stress, with granulations in the cytoplasm. In these early tests, it has been found that the wide channels showed better growth of cells in comparison with serpentine channels. This is because wide microchannels exhibit slow fluid speed respect the narrow channels, allowing the cells to settle and grown. The high fluid velocity of the narrow microchannels may result in constant washing of the factors that the cell secretes to attach to the solid substrate, favoring their carrying and deposition to quieter areas. This paper introduces a new concept for the grown of cells to produce monoclonal antibodies, using microfluidics chips as potential bioreactors. An extensive characterization of the cell grown as well as the study of antibody production, will be the subject of future works.

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