First, the researchers scaled down the size of the fluid-filled chambers used to hold the cells. Chemistry graduate student Matthew Stewart made the small chambers out of a molded gel of polydimethylsiloxane (PDMS). The reduced chamber size also reduced by several orders of magnitude the amount of fluid around the cells, said Biotechnology Center director Jonathan Sweedler, an author on the study. This miniaturization of experimental architectures will make it easier to identify and measure the substances released by the cells, because these releasates are less dilute.
If you bring the walls in and you make an environment thats cell-sized, the channels now are such that youre constraining the releasates to physiological concentrations, even at the level of a single cell, Sweedler said.
Second, the researchers increased the purity of the material used to form the chambers. Cell and developmental biology graduate student Larry Millet exposed the PDMS to a series of chemical baths to extract impurities that were killing the cells.
Millet also developed a method for gradually perfusing the neurons with serum-free media, a technique that resupplies depleted nutrients and removes cellular waste products. The perfusion technique also allows the researchers to collect and analyze other cellular secretions a key to identifying the biochemical contributions of individual cells.
We know there are factors that are communicated in the media between the cells, Millet said. The question is what are they, and how can we get at those"
This combination of techniques enabled the research team to grow postnatal primary hippocampal neurons from rats for up to 11 days at extremely low densities. Prior to this work, cultured neurons in closed-channel devices made of untreated, native PDMS remained viable for two days at best.
The cultured neurons also developed more axons and dendrites, the neural te
|Contact: Diana Yates|
University of Illinois at Urbana-Champaign