Effects of Nanotextured PDMS on Cell Culture and Growth

Cell adhesion, morphology and growth are known to be influenced by microscale topography of the surfaces. Taking this a step further, nanotextured polydimethylsiloxane (PDMS) substrates were fabricated by micro reactive ion etching (Micro-RIE).  This simple process gave good control on surface texture to improve the affinity of PDMS to enhance cell growth. The effects of various nanoscale roughnesses on cell growth were also quantified. Three different recipes were used to prepare nanotextured PDMS by micro-RIE using oxygen (O₂) and carbon tetrafluoride (CF₄). First of all etching was performed for 12 min and flow rate of O₂ and CF₄ was kept at a ratio of 1:1 to fabricate surface 1. Surface 2 was fabricated just by changing the ratio of O₂ and CF₄ to 1:2. Then the flow rates of gases were kept same as recipe 1 but the etching time was increased to 25 min to prepare surface 3. Next, surface topography of these PDMS substrates was evaluated quantitatively by measuring root mean square surface roughness using an atomic force microscope (AFM). The measured average roughness of three nanotextured PDMS surfaces were found 242.92±87.709 nm, 515±102.28 nm and 629.17±109.67 nm for surface 1, surface 2 and surface 3 respectively. The roughnesses of three types of nanotextured surfaces showed a correlation on cell growth. The cell growth rate on flat PDMS surfaces is known to be very low but nanotextured PDMS surfaces allowed exciting cell growth for three different types of cells (human glioblastoma (hGBM), astrocytesand fibroblast cells).The cells were seeded on various surfaces coated with laminin and incubated for 3 days at 37^oC. The cell densities of experimental surfaces were calculated from their images using ImageJ software. For all types of cells, growth was higher on nantoextured PDMS surfaces compare to flat PDMS surface and surface 3 exhibited highest cell density among three nanotextured surfaces. However density of astrocytes cells was 114 ± 16.15 cells per mm² in surface 3 where as flat PDMS had only 28 ± 3.58 cells per mm². But standard well plate had higher cell density compare to surface 3 and it was 140 ± 18.07 cells per mm². Again density of fibroblasts cells in surface 3 was 272.80 ± 13.68 cells per mm² which was higher compare to flat PDMS but lower than standard well plate. On the other hand surface 3 exhibited very similar cell density as standard well plate for hGBM cells and cell densities were 346 ± 70.64 cells per mm² and 334.67 ± 63.23 cells per mm² respectively. Consequently it can be deduced that cell growth strongly depends on surface nanotexturing. By comparing three nanotextured surfaces it was found that surface 1 exhibited lowest roughness compare to surface 2 and surface 3. Therefore available surface area for laminin attachment was lowest in this surface. But surface 2 and surface 3 had higher roughness and therefore provided larger surface area to attach more laminin. As cell growth is a direct function to available laminin attached in the surface therefore cell density was more in later surfaces. In addition to that circulating tumor cells (CTCs) are known to have very high affinity towards nanotextured surfaces. Therefore density of only hGBM cells in nanotextured surface was very similar to cell density in standard well plate. As a result nanotextured PDMS may be thus useful for cancer cytology devices, especially to culture isolated CTCs for further investigation in microfluidic platforms.