Cell Culture
pancreatic cancer cell models in 3D matrices. The device consists of an array of microfluidic chan- nels specifically designed for the culture of cells in 3D format. For the pancreatic tumour cell model, BxPC3 cells were added to the channel suspended in neutralised Collagen Type I which traps the cells in 3D upon gelling. The channels are open at each end and allow for a droplet placed at one end to flow through the porous fibrillar collagen matrix to equilibrate the pressure across the chan- nel. As opposed to well-based 3D culture, this sim- ple addition and removal of droplets of growth media and assay reagents to the channels is done without disruption of the matrix and cells. After an overnight compound treatment, the cells were fixed, permeabilised and stained for EdU incorpo- ration during DNA replication using Invitrogen’s ClickIT Alexa 594 EdU Imaging Kit. Compounds that inhibit cell proliferation or are overtly cyto- toxic have a lower incorporation of EdU. The channels are 140µm tall, which allows for imaging of the full depth of the 3D matrix with low power objectives. Here, (see Figure 9) we captured the channel region in one shot for each label (Hoescht and Alexa 594 EdU). Image analysis was per- formed with Metamorph’s Multi-wavelength Cell Scoring application in which cells are first seg- mented by their nuclear Hoescht stain and then scored for EdU incorporation.
BioTek (
www.biotek.com) is interested in the automation of cell-based assays, particularly in Global Cell Solutions GEM 3-D matrix as it sim- plifies the work flow of cell-based high throughput screening assays. Current cell culture techniques are two-dimensional (2-D) where cells attach to the microplate surface in a single monolayer. Trypsinisation is required to split cell cultures, and prepare them for downstream applications, which becomes increasingly difficult as scale-up is required for primary and secondary screens. 3-D cell culture using GEM, where cells grow on micro- carriers suspended within the culture, eliminates the need for trypsinisation and enables the growth of high-density cultures in a small volume. Cells on the optically-transparent microcarrier can be dis- pensed directly from culture or frozen in situ to be thawed when needed without further culturing. Figure 10 demonstrates the comparison of Histamine 1 antagonist pharmacology using GeneBLAzer® H1-NFAT-bla HEK 293T cells freshly cultured on GEM; frozen on GEM, then thawed; and using standard 2-D cell culture proto- cols. Receptor pharmacology was found to be equivalent across the three formats.
Drug Discovery World Summer 2010
Figure 10: Comparison of Histamine 1 antagonist pharmacology using GeneBLAzer® H1- NFAT-bla HEK 293T cells. All assay steps were automated: cells and LiveBLAzer substrate were dispensed using the BioTek MicroFlo™ peristaltic pump dispenser; antagonist dose responses were serially diluted using the Precision™ automated pipetting station; and GeneBLAzer FRET signals measured using the BioTek Synergy™ 4 MultiMode Microplate Reader
CellASIC (
www.cellasic.com/3D) has developed the 3D:M microfluidic plate, enabling long term perfu- sion culture of cells in a 3D environment. The microfluidics are integrated with a standard 96-well frame and do not require any external pumps, max- imising compatibility with existing assays. Each perfusion unit consists of four well positions (inlet, culture chamber, cell/gel inlet, outlet) resulting in 24 independent units per plate. The user loads their selected cell/gel combination via capillary flow to fill the 150 nanolitre culture chamber. Different protocols allow for embedding cells in gel, overlay of gel on a layer of cells, or 2D culture without gel. The micro-chamber floor is a #1.5 thickness glass cover slide to facilitate high magnification microscopy. The cells are fed by gravity driven per- fusion of medium, set to a rate of 40
Figure 11 (a) The 24-unit CellASIC 3D:M microfluidic 3D perfusion plate. (b) MCF-10A breast epithelial cells cultured in Matrigel (BD Biosciences) for four days in the micro- chamber, and (c) actin staining highlights the acinar organisation of cells in 3D culture
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