RESUMO
The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.
Assuntos
Células Clonais/citologia , Ensaios de Triagem em Larga Escala/métodos , Análise de Célula Única/métodos , Software , Animais , Antígenos/metabolismo , Células CHO , Células Imobilizadas/citologia , Cricetulus , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Imagem Assistida por Computador , Imunoglobulina G/metabolismo , CamundongosRESUMO
We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 µm high and 200 µm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.
RESUMO
A novel microfabricated flow cytometer for simultaneous impedance and optical measurement of single cells and particles is described in this chapter. We discuss the sensitivity of the system with regard to the impedance sensor and describe the optical setup. The relevant parameters related to the experimental setup and sample preparation are discussed. The use of dielectrophoretic forces for particle manipulation is presented as a simple enabling technology, which allows the manipulation of particles within microfluidic devices. The fabrication processes required to produce the impedance sensor chips are described with relevance to the chip design and features. Finally, the system is used to discriminate between different marine algae populations.