No abstract

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

Chemical patterns have attracted substantial interest for applications in the field of biosensors, fundamental cell–surface interaction studies, tissue engineering, and biomaterials. A novel micropatterning technique is proposed here that combines a top–down approach based on photolithography and a bottom–up strategy through self‐organization of multifunctional molecules. The development of the molecular‐assembly patterning by lift‐off (MAPL) has been driven by the need to economically produce patches incorporating a controlled surface density of bioligands while inhibiting non‐specific adsorption. In the MAPL process, a photoresist pattern is transferred into the desired biochemical pattern by means of spontaneous adsorption of biologically relevant species and photoresist lift‐off. The surface between the interactive patches is subsequently rendered non‐fouling through immobilization of a polycationic poly(ethylene glycol) (PEG)‐graft polymer. We demonstrate that surface density of biotin molecules inside adhesive islands can be tailored quantitatively and that cells grow selectively on cell‐adhesive peptide patterns. MAPL is considered to be a valuable addition to the toolbox of soft‐lithography techniques for life‐science applications combining simplicity (no clean‐room equipment needed), cost‐effectiveness, reproducibility on the scale of whole wafer surfaces, and flexibility in terms of pattern geometry, chemistry, and substrate choice.

We describe a novel parallel method for the patterning of proteins with nanoscale resolution. Combining nanoimprint lithography (NIL) and molecular assembly patterning by lift-off (MAPL), we produced streptavidin patterns with feature sizes in the order of 100 nm. A stamp is imprinted into a heated PMMA film followed by a dry etching step that converts the topography into a PMMA/Nb 2 O 5 contrast. A biotin functionalized copolymer, poly(L-lysine)-graft-poly(ethylene glycol)-biotin (PLL-g-PEG/PEG-biotin), spontaneously adsorbs on the oxide surfaces. After PMMA lift-off, the background is backfilled with protein-resistant PLL-g-PEG. We show that streptavidin selectively adsorbs on the biotin areas and thus can be used as a universal platform for immobilization of biotin-tagged molecules. This novel process is a parallel patterning method that is fast, reproducible, and economic. The PEG-copolymer can be functionalized with a variety of bioactive groups and thus allows a great flexibility in terms of surface chemistry.