Micro-patterned porous substrates for cell-based assays

Abstract

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.

Application of Au cage/Ru(bpy)32+ nanostructures for the electrochemiluminescence detection of K562 cancer cells based on aptamer

“…Low-toxic calcein-AM, which could be incorporated into living cells through the membrane, was chosen to carry out staining experiment for the determination of cell viability. Under the fluorescent microscope, green fluorescence could be observed in cytoplasm of K562 cancer cells [34]. Fig.…”

Simple and non-toxic fabrication of poly(vinyl alcohol)-patterned polymer surface for the formation of cell patterns

“…Cell patterning on an artificial substrate has received a great amount of attention as an essential prerequisite for a variety of biological applications such as the fundamental study of cell biology, tissue engineering, cell-based bioassays, and cell-based drug screening [1][2][3][4][5][6][7]. To facilitate the formation of cell patterns, a variety of surface patterning techniques, including inkjet printing, photolithography, and micro-contact printing, has been extensively explored [8][9][10].…”

Micropatterned Porous Membranes for Combinatorial Cell-Based Assays

“…In particular, several microfluidics systems which allow the chemical environment of eukaryotic cells to be altered while simultaneously measuring the expression of key reporter genes by fluorescence microscopy have been proposed (Jovic, Howell, & Takayama, 2009;Taylor et al, 2009). A number of commercial systems exist; however, they are often limited in terms of operability and, without solid expertize, cannot be used to perform combinatorial and/or time-dependent cell-based assays (Castel, Pitaval, Debily, & Gidrol, 2006;Evenou, Di Meglio, Ladoux, & Hersen, 2012;Wu et al, 2010). A number of commercial systems exist; however, they are often limited in terms of operability and, without solid expertize, cannot be used to perform combinatorial and/or time-dependent cell-based assays (Castel, Pitaval, Debily, & Gidrol, 2006;Evenou, Di Meglio, Ladoux, & Hersen, 2012;Wu et al, 2010).…”

“…Our method is inspired by the fabrication method of "paper-based microfluidics" (Carrilho, Martinez, & Whitesides, 2009;Nery & Kubota, 2013), which aims to create cheap and "ready to use" diagnostic systems with the use of paper. In doing so, we modify the local porosity of the filter membrane (Evenou et al, 2012). This pattern defines hydrophobic and hydrophilic areas.…”