CONICET | Buscador de Institutos y Recursos Humanos

The production of monoclonal antibodies for therapeuticuse is one of the fastest growing areas in the biopharmaceutical industry. In2011, 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 monoclonalantibodies and other biotherapeutics is based on the synthesis in bioreactors withsuspended mammalian cells with agitation operated in fed-batch or perfusionmode [1-2]. The production of monoclonal antibodies in these stirred tanksfaced challenges related to product quality and process such as demand forhigher productivity, control of glycosylation, reproducibility, and otherprocess controls. Most of these challenges are related to the large spatial andtemporal variability of the intrinsic conditions of the fermenters. One way toimprove the control is to reduce the scale of the system by miniaturization inthe form of micro devices [3]. A micro device offers several advantages, includingshorter time response, a higher surface / volume ratio and a more homogeneousand controllable microenvironment.In this work wasstudy 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 andlengths between 12 mm and 80 mm was used. Feed by a central channel of 40x1.9mm (length x width), and spacers channels of 12 x1,2mm (length x width) betweenserpentines. The height of all channels was 40 μm.Several microfabrication processes have beenimplemented for the microfluidicdevice. Amold of the design in high relief was made by photolithography in a silicon(100) wafer 700 µm thick (Virginia Semiconductor, Inc.), by usingthe negative resin SU-8 (MicroChem). PDMS was mixed with curing agent in a 15:1ratio. The mixture was placed under vacuum to remove air bubbles. After this,the mixture was poured into the master; placed under vacuum once again, andcured in an oven at 70° C during 70 min. Finally the sample waspeeled off. Oncethe processes were finished (PDMS microchannels and glass), both parts werejoined 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 substantiallyimproves cell attachment. Before cell seeding, the chip was treated with 0.1mg/ml Poly-D-lisine Hydrobromide (Sigma) sterile solution in water in order toimprove the attachment of the cells. The microfluidic devices were incubatedwith Poly-D-lisine solution for one hour at 37°C. The solution was removed andlet 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 serumheat-inactivated (SBF) (Internegocios SA, Mendoza, Argentina), 2 mM L-glutamine(Gibco, Grand Island, NY, USA), and Antibiotic-Antimycotic 100 units/mlpenicillin, 100 ug/ml streptomycin and 0.25 g fungizone ml (Gibco, GrandIsland, NY, USA) at 37°C in an incubator with 5% CO2. The cells wereresuspended with trypsin solution 250 mg/ml, EDTA 100 mg/ml (Gibco, GrandIsland, NY, USA) and incubated at 37°C for 3 minutes. Trypsin was inactivatedwith 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 aconcentration of 107 cells/ml. Before seeding the cells, the microchannels were washedwith complete DMEM medium and were kept filled with medium for HEK-293T cellseeding. Next, cells were seeded in a 20 μl volume in the loading well of themicrofluidic device. Once the cell suspension reached the outlet well, 20 μl ofDMEM complete medium without cells was applied to equilibrate the flow in the microchannels. The cells were allowed to settle and the microfluidic device wasincubated at 37°C in incubator with 5% CO2. The medium was changed every 24hours with fresh DMEM complete medium. The microchannels of the device werevisualized using an inverted Olympus microscope CKX41. Brightfield images weretaken with Olympus objectives LUCPlan FL N 40X/0.60; LCAch N 20X/0.40; PlanC N10X/0.25; and PlanC N 4X/0.10 with an Olympus QColor 5; and they were processedwith QCapture Pro 6.0 software.At the time of seeding, HEK-293T cells were distributedin aggregates or individually along the microchannels (Figure 1). At day two,the larger aggregates were washed and the individual cells were adhering to thebottom of the wide channels (canal central y canales espaciadores) with theDMEM complete medium change. Cells then began to form clusters and to extendprocesses around the clusters (Figure 2). At day five the cells consolidatetheir growth in clusters on the floor of the wide microchannels, with almost nocells in the serpentine channels. At day eight, cells began to show signs ofstress, with granulations in the cytoplasm. In these early tests, it has been found that the widechannels showed better growth of cells in comparison with serpentine channels.This is because wide microchannels exhibit slow fluid speed respect the narrowchannels, allowing the cells to settle and grown. The high fluid velocity ofthe narrow microchannels may result in constant washing of the factors that thecell secretes to attach to the solid substrate, favoring their carrying anddeposition to quieter areas. This paper introduces a new concept for the grownof cells to produce monoclonal antibodies, using microfluidics chips aspotential bioreactors. An extensive characterization of the cell grown as wellas the study of antibody production, will be the subject of future works. [1] A. Gilbert., et al.Biotechnol Prog. 29 (2013) 1519-1527.[2] R. Kshirsagar., et al.Biotechnol Bioeng. 109 (2012) 2523-2532.[3] H.E. Abaci, et al. Biomed Microdevices. 14 (2012)145-152. [4] D. Lightwood et al. mAbs.(2014) 143?159