"Chip-on-a-Transwell" Devices for User-Friendly Control of the Microenvironment of Cultured Cells.

We present microfabricated poly(dimethylsiloxane) templates assembled atop Transwell membranes ("Chip-on-a-Transwell") for focal delivery of soluble reagents to cells. This modified Transwell operates based on area-selective diffusion of soluble species to the basal side of cells. The device was capable of addressing small groups of cells, with a maximum spatial resolution of 20 µm, while sustaining focal signal for up to 48 hours. Furthermore, we developed a fluorosilane-based cell micropatterning method that complements the Transwell system, creating well-defined arrays of muscle cell cultures and enabling automated microscopy and image analysis protocols. The micropatterning method is based on plasma-etching hydrophilic cell-adhesive tracks against a hydrophobic fluorosilane cell-repellent background. We applied the platform as a model of the neuromuscular junction (NMJ), in which we effectively substituted motor neurons with apertures delivering agrin stimuli to subcellular regions of micropatterned myotubes. With the Transwell system, we demonstrated that focal agrin application to subcellular sections of myotubes induced localized acetylcholine receptor microclustering, mimicking the in vivo NMJ. We also demonstrated spatially selective transfection of a monolayer of cells using Lipofectamine carrying the plasmid DNA for pMax GFP, which is ubiquitously expressed in transfected cells. In summary, we present a user-friendly cell culture tool that provides spatiotemporal control of the fluidic and substrate microenvironments with potential applications to developmental biology and tissue engineering.

Analysis of the Myc-induced pancreatic ß cell islet tumor microenvironment using imaging ToF-SIMS.

Solid tumors are a structurally complex system, composed of many different cell types. The tumor microenvironment includes nonmalignant cell types that participate in complex interactions with tumor cells. The cross talk between tumor and normal cells is implicated in regulating cell growth, metastatic potential, and chemotherapeutic drug resistance. A new approach is required to interrogate and quantitatively characterize cell to cell interactions in this complex environment. Here, the authors have applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze Myc-induced pancreatic ß cell islet tumors. The high mass resolution and micron spatial resolution of ToF-SIMS allows detection of metabolic intermediates such as lipids and amino acids. Employing multivariate analysis, specifically, principal component analysis, the authors show that it is possible to chemically distinguish cancerous islets from normal tissue, in addition to intratumor heterogeneity. These heterogeneities can then be imaged and investigated using another modality such as sum harmonic generation microscopy. Using these techniques with a specialized mouse model, the authors found significant metabolic changes occurring within ß cell tumors and the surrounding tissues. Specific alterations of the lipid, amino acid, and nucleotide metabolism were observed, demonstrating that ToF-SIMS can be utilized to identify large-scale changes that occur in the tumor microenvironment and could thereby increase the understanding of tumor progression and the tumor microenvironment.

Effect of substrate storage conditions on the stability of "Smart" films used for mammalian cell applications.

When poly(N-isopropyl acrylamide) (pNIPAM) is tethered to a surface, it can induce the spontaneous release of a sheet of mammalian cells. The release of cells is a result of the reversible phase transition the polymer undergoes at its lower critical solution temperature (LCST). Many techniques are used for the deposition of pNIPAM onto cell culture substrates. Previously, we compared two methods of deposition (plasma polymerization, and co-deposition with a sol-gel). We proved that although both were technically appropriate for obtaining thermoresponsive pNIPAM films, the surfaces that were co-deposited with a sol-gel caused some disruption in cell activity. The variation of cell behavior could be due to the delamination of pNIPAM films leaching toxic chemicals into solution. In this work, we assessed the stability of these pNIPAM films by manipulating the storage conditions and analyzing the surface chemistry using X-ray photoelectron spectroscopy (XPS) and contact angle measurements over the amount of time required to obtain confluent cell sheets. From XPS, we demonstrated that ppNIPAM (plasma polymerized NIPAM) films remains stable across all storage conditions while sol-gel deposition show large deviations after 48 h of storage. Cell response of the deposited films was assessed by investigating the cytotoxicity and biocompatibility. The 37°C and high humidity storage affects sol-gel deposited films, inhibiting normal cell growth and proper thermoresponse of the film. Surface chemistry, thermoresponse and cell growth remained similar for all ppNIPAM surfaces, indicating these substrates are more appropriate for mammalian cell culture applications.

An unsupervised MVA method to compare specific regions in human breast tumor tissue samples using ToF-SIMS.

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA) were used to investigate two sets of pre- and post-chemotherapy human breast tumor tissue sections to characterize lipids associated with tumor metabolic flexibility and response to treatment. The micron spatial resolution imaging capability of ToF-SIMS provides a powerful approach to attain spatially-resolved molecular and cellular data from cancerous tissues not available with conventional imaging techniques. Three ca. 1 mm(2) areas per tissue section were analyzed by stitching together 200 µm × 200 µm raster area scans. A method to isolate and analyze specific tissue regions of interest by utilizing PCA of ToF-SIMS images is presented, which allowed separation of cellularized areas from stromal areas. These PCA-generated regions of interest were then used as masks to reconstruct representative spectra from specifically stromal or cellular regions. The advantage of this unsupervised selection method is a reduction in scatter in the spectral PCA results when compared to analyzing all tissue areas or analyzing areas highlighted by a pathologist. Utilizing this method, stromal and cellular regions of breast tissue biopsies taken pre- versus post-chemotherapy demonstrate chemical separation using negatively-charged ion species. In this sample set, the cellular regions were predominantly all cancer cells. Fatty acids (i.e. palmitic, oleic, and stearic), monoacylglycerols, diacylglycerols and vitamin E profiles were distinctively different between the pre- and post-therapy tissues. These results validate a new unsupervised method to isolate and interpret biochemically distinct regions in cancer tissues using imaging ToF-SIMS data. In addition, the method developed here can provide a framework to compare a variety of tissue samples using imaging ToF-SIMS, especially where there is section-to-section variability that makes it difficult to use a serial hematoxylin and eosin (H&E) stained section to direct the SIMS analysis.

Synthesis and optimization of fluorescent poly(N-isopropyl acrylamide)-coated surfaces by atom transfer radical polymerization for cell culture and detachment.

Although there are many stimulus-responsive polymers, poly(N-isopropyl acrylamide) (pNIPAM) is of special interest due to the phase change it undergoes in a physiologically relevant temperature range that leads to the release of cells and proteins. The nondestructive release of cells opens up a wide range of applications, including the use of pNIPAM for cell sheet and tissue engineering. In this work, pNIPAM surfaces were generated that can be distinguished from the extracellular matrix. A polymerization technique was adapted that was previously used by Mendez, and the existing protocol was optimized for the culture of mammalian cells. The resulting surfaces were characterized with X-ray photoelectron spectroscopy and goniometry. The developed pNIPAM surfaces were further adapted by incorporation of 5-acrylamidofluorescein to generate fluorescent pNIPAM-coated surfaces. Both types of surfaces (fluorescent and nonfluorescent) sustained cellular attachment and produced cellular detachment of ∼90%, and are therefore suitable for the generation of cell sheets for engineered tissues and other purposes. These surfaces will be useful tools for experiments investigating cellular detachment from pNIPAM and the pNIPAM/cell interface.