The use of substrate materials and topography to modify growth patterns and rates of differentiation of muscle cells.

Cells are cultured on platforms made of a variety of materials with selected topographies during studies of cell response and behavior. Understanding the effects of substrates is essential for such applications as developing effective interfaces between body cells and implanted materials and devices. In this study, the effects of substrate surface properties on cell differentiation and alignment on C2C12 myoblasts cultured on conventional or fabricated polymeric cell culture substrates were investigated. Comparisons were made between cells cultured on tissue culture grade polystyrene (TCPS), glass, Permanox, and cured polydimethylsiloxane (PDMS) substrates. Fluorescent immunohistochemistry of cell markers was used to analyse the extent of differentiation. Alignment and guidance of cell growth and spread were studied using patterned platforms. Gratings were made on polystyrene (PS) and PDMS and differentiation was facilitated after 5 days by media exchange. Differences in cell morphology were observed between cells cultured on TCPS and PDMS substrates. Fully differentiated myotubes were observed in highest numbers on TCPS substrates and were non-detectable on PDMS substrates in the time frame of 144 h. Muscle cell alignment and their differentiation followed along the grating patterns on PS and elongated along the pattern length. On the other hand, on PDMS cells formed sheets of tissue and peeled from the substrate. We have revealed the potential for the combinations of surface materials and topography on cell behavior to induce accelerated differentiation and coordinated alignment. The results demonstrate that culture environment can be designed or engineered to modify or regulate muscle cell functions. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1638-1645, 2016.

Positive and negative bioimprinted polymeric substrates: new platforms for cell culture.

Bioimprinting, which involves capturing cell morphological details into a polymer matrix, provides a new class of patterned surfaces which opens an opportunity to investigate how cells respond to their own signatures and may introduce possibilities for regulating their behaviour. In this study, phenotypic details of human nasal chondrocytes (HNCs) were replicated in soft polydimethylsiloxane (PDMS) mould resulting in inverse replicas of cells, which have been termed here as 'negative bioimprint'. For the first time, the information from this negative bioimprint was then transferred into another PDMS layer resulting in surfaces which resemble cell morphology and were called 'positive bioimprints'. Soft lithography was used to transfer these details from PDMS into different polymers like polystyrene, tissue culture polystyrene and clinically used block co-polymer poly (ethylene glycol) terephthalate-poly (butylene terephthalate) (PEGT-PBT). Results obtained from surface characterization confirmed that fine details of cells were successfully replicated from cells to different polymer matrices without any significant loss of information during the different steps of pattern transfer. HNCs seeded on different polymer surfaces with positive and negative bioimprints exhibited distinct behaviour. Cells cultured on positive bioimprints were more spread out and displayed high levels of proliferation compared to those on negative bioimprints, where cells were more compact with lower proliferation.

Investigation of a nanofabrication process to achieve high aspect-ratio nanostructures on a quartz substrate.

This work investigates the development of a nanofabrication process to achieve high aspect-ratio nanostructures on quartz substrates using electron beam lithography (EBL) patterning and fluorinated plasma etching processes. An imaging layer of a poly(methyl methacrylate) bi-layer resist was spun coated on quartz substrate and exposed by an e-beam with the designed patterns of sub-100 nm feature sizes using a Raith-150 EBL patterning tool. Additive pattern transfer was employed by depositing a 40 nm thick Nichrome layer on the resist pattern using a metal evaporator which was later lifted off by soaking in acetone. Nichrome was employed as an etch mask and an Oxford Plasmalab 80Plus reactive ion etcher was used for the etching process. The etching process was carried out in a gas mixture of CHF(3)/Ar with a flow rate ratio of 50/30 sccm, pressure of 20 mTorr, radiofrequency power of 200 W and at room temperature. These etching process parameters were found to achieve a 10 nm min(-1) etch rate and tall vertical side walls profile. An aspect-ratio of 10:1 was achieved on 60 nm feature size structures.

Cellular transfer and AFM imaging of cancer cells using Bioimprint.

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.

Cellular replication and atomic force microscope imaging using a UV-Bioimprint technique.

Replication and fixation techniques have been of considerable interest for imaging and analysis of biological cells since the introduction of electron and scanning probe microscopy. Although such tools as the atomic force microscope (AFM) permit in situ morphological studies at a magnitude of resolution beyond traditional optical microscopy, they are difficult to operate and their resolution capabilities are rarely realized. We used a UV-Bioimprint replication technique to imprint a polymer layer onto cells attached to a substrate and rapidly cure to create an impression of cell topography. Replicas of chemically fixed and untreated cells analyzed by atomic force microscopy demonstrate nanometer resolution in the transfer of replicated features. UV-Bioimprint presents an improvement over techniques using heat-curable polymers as well as an alternative technique to the direct imaging of cells. The motivation for UV-Bioimprint is to effectively integrate scanning probe microscopy tools for imaging of cellular ultrastructure.