Patterned substrates for cell culture
Keywords: Patterned substrates, cell culture, large scale imaging
Reference: InnoStamp 40™ and InnoScan 1100AL™: a complete automated platform for microstructured cell arrays, Adriana Lagraulet, J. Foncy, Benjamin Berteloite, Aurore Esteve, Marie-Charline Blatche, Laurent Malaquin andChristophe Vieu, A. Nature Methods12
ECM-microarrays are then proved as promising tools in cell biology. However, two main limitations have to be overcome to transfer this technology as a high throughput approach easy to use in cell biology laboratories. First of all the construction of ECM-microarrays, ECM should be homogenously distributed in the pattern to control as much as possible cell spread. Also, patter size and position in the substrate should be controlled. Microcontact printing (µCP) is a cost-effective solution to create micro-patterned ECM arrays. In µCP a polydimethylsiloxane (PDMS) stamp is used to transfer ECM molecules with a desired micro-features. However, manual µCP have shown limitations in the quality of protein transfer since contact forces are not controlled, making manual µCP difficult of use as routine technique.
The second limitation to overcome concerns the image acquisition. ECM-arrays and, in general all cell arrays, needs of whole-slide imaging to be able of comparing each of the conditions presents in the same slide under the same imaging parameters. Also, high throughput approaches need of automated processes with reduced human intervention. Even when motorized microscope and other digital microscopes can be used to get whole-slide images, most of vendors use a tiling system. To obtain a whole-slide digital image, the tiling systems acquire multiple individual images which are then stitched together. This process can be tedious and long, and very often require the supervision of the user, making the process poorly automated.
To overcome both ECM-patterning and cell array image acquisition, Biosoft has developed a complete platform consisting of µCP automate, the InnoStamp 40, and of a three color fluorescence scanner, the InnoScan 100AL, capable of whole-slide high resolution scanning.
Fig 1: Micro-structured cell array automatic process. Panel 1: (a) A magnetic PDMS stamp containing the features is used as transfer stamp. (b) The magnetic PDMS stamp is inked with ECM components such as collagen at a concentration of 100µg/ml. (c) The stamp is air-dried to avoid liquid excess. (d) Once dried, the magnetic stamp is put in contact with the substrate. (e) ECM components are transferred to the substrate following the stamp patterns. (f) The slide is blocked with a non-adhesive molecule such as polylysine-grafted polyethylenglycol (PLL-g-PEG). (g) The non-adhesive molecule will cover those sites without ECM patterns. (h) The InnoStamp 40 micro-contact printinting automate, some examples of ECM patterned slides. Spot diameter goes from 50 to 150µm; lines height 50µm. Panel 2: Cells are then cultured in micro-patterned slides, after 24h cells will selectively attach to those sites containing ECM components. Panel 3: After fluorescence staining cells are detected with the InnoScan 1100AL scanner. Whole-slide imaging at 0.5µm/pixel resolution is shown.
Fig 2: ECM cell spread patterning. PC3 prostate cancer cells expressing green fluorescence protein (GFP) were cultured on an ECM-line-patterned slide. Nuclei were labeled using Draq5 dye detected in the red (635nm) channel. GFP was detected within the blue (488nm) channel. (a) A whole-slide image, the slide was scanned at 0.5µm/pixel resolution. (b) Image zoom-in. Cells were attached to those sites containing collagen, while the sites with PLL-g-PEG are mostly free of cells.