A method for high-content functional imaging of intracellular calcium responses in gelatin-immobilized non-adherent cells.

Functional imaging of the intracellular calcium concentration [Ca2+]i using fluorescent indicators is a powerful and frequently applied method for assessing various biological questions in vitro, including ion channel function and intracellular signaling in homeostasis and disease. In functional [Ca2+]i imaging experiments, the fluorescence intensity of single cells is typically recorded during application of a chemical stimulus, i.e. by exchange of modified extracellular media, exposure to drugs and/or ligands. The concomitant mechanical perturbation caused by the perfusion of different solution during experimentation severely hinders calcium imaging in non-adherent cells, including peripheral immune cells, as cells in suspension are dislocated by turbulent flow during chemical stimulation. The quantitative analysis, involving time-courses of intracellular fluorescence signal changes, necessitates cells to remain at the same position throughout the experiment. To prevent dislocation of cells during solution exchange, and to enable imaging as well as analysis of Ca2+ responses in immune cells, a gelatin-based method for immobilization of non-adherent cells was developed. Gelatin has been a long-serving material for cell immobilization, e.g. in 3D bio-printing of cells and has thus, also been employed in the context of this study. To demonstrate the applicability of the established method for functional Ca2+ imaging in gelatin-immobilized suspension cells, a proof-of-concept study was conducted using human peripheral blood model cell lines (Jurkat/T-lymphocytes and THP-1/monocytes), Ca2+ indicators (Fluo-4 and Fura-2) and two different fluorescence microscopy rigs. The data presented that the established methodology is applicable for studying Ca2+ signaling by in vitro high-content functional imaging of [Ca2+]i in suspension cells, including but not restricted to human immune cells.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation.

GABAA receptors are ligand-gated anion channels that form pentameric arrangements of various subunits. Positive allosteric modulators of GABAA receptors have been reported as being isolated either from plants or synthesized analogs of known GABAA receptor targeting drugs. Recently, we identified monoterpenes, e.g. myrtenol as a positive allosteric modulator at α1ß2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in the loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis and electrophysiological recordings in whole-cell configurations were used for analysis of transfected cells. C-atoms 1 and 2 of the myrtenol backbone were identified as crucial to preserve positive allosteric potential. A modification at C-atom 2 and lack of the hydroxyl group at C-atom 1 exhibited significantly reduced GABAergic currents at α1ß2, α1ß2γ, α2ß3, α2ß3γ and α4ß3δ receptors. This effect was independent of the γ2 subunit. A sub-screen with side chain length and volume differences at the C-atom 1 identified two compounds that inhibited GABAergic responses but without receptor subtype specificity. Our combined approach of pharmacophore-based virtual screening and functional readouts reveals that side chain modifications of the bridged six-membered ring structure of myrtenol are crucial for its modulatory potential at GABAA receptors.

Optimizing neuronal differentiation of human pluripotent NT2 stem cells in monolayer cultures.

Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity testing at different stages of maturation. However, differentiation of NT2 cells is not straightforward. There are different protocols available in the literature reporting varying results with regard to differentiation efficiency, expression of neuronal markers and morphological characteristics of differentiated cells. Yet, the efficiency of available protocols has not been systematically compared. To address this question, we quantified the number and size of cell cluster formed during differentiation using published and modified protocols and analyzed the abundance of neuronal and non-neuronal expression markers using immunocytochemistry. In the course of the experiments we observed that differentiation results strongly depend on the cell density at differentiation-initiation as well as on the type of used cell culture plastic ware. Based on those observations and the results from our comparative analysis, we created our own optimized and robust protocol that reproducibly reveals differentiated cells with high yield. We conclude that our method may be superior to differentiation of NT2 cells for systematic in vitro-based primary screening for developmental toxicants and neurotoxicants at different stages of maturation over previous protocols used. Our approach will also contribute to reduce animal testing in the context of the 3Rs.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines.

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Sesterterpene glycinyl-lactams: a new class of glycine receptor modulator from Australian marine sponges of the genus Psammocinia.

Bioassay guided fractionation of three southern Australian marine sponges of the genus Psammocinia, selected for their ability to modulate glycine-gated chloride channel receptors (GlyRs), yielded the rare marine sesterterpenes (-)-ircinianin (1) and (-)-ircinianin sulfate (2), along with the new biosynthetically related metabolites (-)-ircinianin lactam A (3), (-)-ircinianin lactam A sulfate (4), (-)-oxoircinianin (5), (-)-oxoircinianin lactam A (6) and (-)-ircinianin lactone A (7). Acetylation of 1 returned (-)-ircinianin acetate (8). Whole cell patch-clamp electrophysiology on 1-8 established 3 as an exceptionally potent and selective α3 GlyR potentiator, and 6 as a selective α1 GlyR potentiator. The discovery and characterization of sesterterpenes 1-8, and in particular the glycinyl-lactams 3 and 6, provide valuable new insights into GlyR pharmacology. These insights have the potential to inform and inspire the development of new molecular tools to probe GlyR distribution and function, and therapeutics to treat a wide array of GlyR mediated diseases and disorders.

Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator.

Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five known sponge indole alkaloids, aplysinopsin (5), 8E-3'-deimino-3'-oxoaplysinopsin (6), 8Z-3'-deimino-3'-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist activity with a bias towards α3 over α1 GlyR, while tubastrindole B (9) exhibited a bias towards α1 over α3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator of α1 GlyR, with no effect on α3 GlyR-a pharmacology that could prove useful in the treatment of movement disorders such as spasticity and hyperekplexia. Our investigations into the GlyR modulatory properties of 1-9 were further supported by the synthesis of a number of structurally related indole alkaloids.

Assaying Proliferation Characteristics of Cells Cultured Under Static Versus Periodic Conditions.

Two-dimensional in vitro culture models are widely being employed for assessing a vast variety of biological questions in different scientific fields. Common in vitro culture models are typically maintained under static conditions, where the surrounding culture medium is replaced every few days-typically every 48 to 72 h-with the aim to remove metabolites and to replenish nutrients. Although this approach is sufficient for supporting cellular survival and proliferation, static culture conditions do mostly not reflect the in vivo situation where cells are continuously being perfused by extracellular fluid, and thus, create a less-physiological environment. In order to evaluate whether the proliferation characteristics of cells in 2D culture maintained under static conditions differ from cells kept in a dynamic environment, in this chapter, we provide a protocol for differential analysis of cellular growth under static versus pulsed-perfused conditions, mimicking continuous replacement of extracellular fluid in the physiological environment. The protocol involves long-term life-cell high-content time-lapse imaging of fluorescent cells at 37 °C and ambient CO2 concentration using multi-parametric biochips applicable for microphysiological analysis of cellular vitality. We provide instructions and useful information for (i) the culturing of cells in biochips, (ii) setup of cell-laden biochips for culturing cells under static and pulsed-perfused conditions, (iii) long-term life-cell high-content time-lapse imaging of fluorescent cells in biochips, and (iv) quantification of cellular proliferation from image series generated from imaging of differentially cultured cells.

Pathophysiological mechanisms of dominant and recessive GLRA1 mutations in hyperekplexia.

Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) alpha1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric alpha1 or heteromeric alpha1beta GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2.

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exon-skipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC(50) and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity.

Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae.

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure-activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against alpha1 and alpha3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

Synthesis of Tamoxifen-Artemisinin and Estrogen-Artemisinin Hybrids Highly Potent Against Breast and Prostate Cancer.

In the search for new and effective treatments of breast and prostate cancer, a series of hybrid compounds based on tamoxifen, estrogens, and artemisinin were successfully synthesized and analyzed for their in vitro activities against human prostate (PC-3) and breast cancer (MCF-7) cell lines. Most of the hybrid compounds exhibit a strong anticancer activity against both cancer cell lines - for example, EC50 (PC-3) down to 1.07 µM, and EC50 (MCF-7) down to 2.08 µM - thus showing higher activities than their parent compounds 4-hydroxytamoxifen (afimoxifene, 7; EC50 =75.1 (PC-3) and 19.3 µM (MCF-7)), dihydroartemisinin (2; EC50 =263.6 (PC-3) and 49.3 µM (MCF-7)), and artesunic acid (3; EC50 =195.1 (PC-3) and 32.0 µM (MCF-7)). The most potent compounds were the estrogen-artemisinin hybrids 27 and 28 (EC50 =1.18 and 1.07 µM, respectively) against prostate cancer, and hybrid 23 (EC50 =2.08 µM) against breast cancer. These findings demonstrate the high potential of hybridization of artemisinin and estrogens to further improve their anticancer activities and to produce synergistic effects between linked pharmacophores.

Improved stability of polyclonal antibodies: A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite Plasmodium falciparum.

Antibodies can be produced as polyclonal (pAb) or monoclonal (mAb) liquid formulations with limited shelf-life. For pAbs, unlike mAbs, only little is known about excipients and lyophilization affecting antibody stability upon reconstitution. We used a model pAb directed against Plasmodium falciparum (Pf) pyridoxal 5'-phosphate synthase 2 (Pdx2) to systemically study effects of bulking agents (amino acids, phosphate buffers, salt solutions), sugar(alcohols), surfactants and protein additions (bovine serum albumin, BSA) in liquid pAb formulations (isolated or in combinations) on the activity to detect the antigen in Pf extracts by Western blots. Repeated freeze-thaw cycles (20x) and extended room temperature storage markedly compromised pAb stability, the former being ameliorated by addition of cryoprotectants (glycerol, sucrose). Lyophilization of pure liquid pAb formulation markedly decreased antibody reactivity upon reconstitution which was not preserved by most bulking agents tested (e.g., histidine, arginine, acetate). Among the tested salt solutions (NaCl, Ringer, PBS), phosphate buffered saline had the largest lyoprotective potential, alongside sucrose, but not trehalose or maltitol. Among combinations of excipients, PBS, sucrose, low concentration BSA and Tween potently preserved PfPdx2 stability. Results for PBS were transferable to PfEnolase pAb, indicating that some of the formulations investigated here might be a low-cost solution for more general applicability to pAbs.

New Oleoyl Hybrids of Natural Antioxidants: Synthesis and In Vitro Evaluation as Inducers of Apoptosis in Colorectal Cancer Cells.

Nowadays, the beneficial role of a healthy lifestyle, particularly emphasizing the quality of foods and cancer management, is accepted worldwide. Polyphenols and oleic acid play a key role in this context, but are still scarcely used as anti-cancer agents due to their bio-accessibility limits. Therefore, we aimed to synthesize a set of new oleoyl-hybrids of quercetin, morin, pinocembrin, and catechin to overcome the low bioavailability of polyphenols, throughout a bio-catalytic approach using pancreatic porcine lipase as a catalyst. The in vitro assays, using a wide panel of human cancer cell lines showed, mainly for two novel regioisomer oleoyl-hybrids of quercetin, a remarkable increase in apoptotic cell populations. We suggested that the DNA damage shown as ɣH2AX signals might be the major cause of apoptotic cell death. Finally, we demonstrated convincing data about two novel polyphenol-based hybrids displaying a highly selective anti-cancer cytotoxicity and being superior compared to their reference/parental compounds.

Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy.

3D Printed Lab-on-a-Chip Platform for Chemical Stimulation and Parallel Analysis of Ion Channel Function.

Functional imaging has been a widely established method for the assessment of ion channel function in vitro. Conventional infrastructure used for in vitro functional analysis of ion channels is typically proprietary, non-customizable, expensive, and requires a high level of skill to use and maintain. 3D desktop printing, which is employed in the rapid prototyping field, allows for quick engineering of alternatives to conventional imaging infrastructure that are customizable, low cost, and user friendly. Here, we describe an ultra-low-cost microfluidic lab-on-a-chip (LOC) device manufactured using acrylonitrile butadiene styrene (ABS) for in vitro functional imaging of ion channels that can quickly and easily be reconstructed using three-dimensional (3D) desktop printing. The device is light weight (<5 g), small (20 mm × 49 mm), and extremely low cost (

Portoporator©: A portable low-cost electroporation device for gene transfer to cultured cells in biotechnology, biomedical research and education.

Electroporation has been a widely established method for delivering DNA and other material into cells in vitro. Conventional electroporation infrastructure is typically immobile, non-customizable, non-transparent regarding the characteristics of output pulses, and expensive. Here, we describe a portable electroporator for DNA delivery into bacterial cells that can quickly be reconstructed using 3D desktop printing and off-the-shelf components. The device is light weight (700 g), small (70 × 180 × 210 mm) and extremely low-cost (

Proliferation characteristics of cells cultured under periodic versus static conditions.

In vitro culture models have become an indispensable tool for assessing a vast variety of biological questions in many scientific fields. However, common in vitro cultures are maintained under static conditions, which do not reflect the in vivo situation and create a non-physiological environment. To assess whether the growth characteristics of cells cultured at pulsed-perfused versus static conditions differ, we observed the growth of differentially cultured cells in vitro by life-cell time-lapse imaging of recombinant HEK293YFPI152L cells, stably expressing yellow fluorescent protein. Cells were grown for ~ 30 h at 37 °C and ambient CO2 concentration in biochips mounted into a custom-designed 3D printed carrier and were imaged at a rate of ten images per hour using a fluorescence microscope with environment control infrastructure. Cells in one chip were maintained under static conditions whereas cells in another chip were recurrently perfused with fresh media. Generated image series were quantitatively analyzed using a custom-modified cell detection software. Imaging data averaged from four biological replicates per culturing condition demonstrate that cells cultured under conventional conditions exhibit an exponential growth rate. In contrast, cells cultured in periodic mode exhibited a non-exponential growth rate. Our data clearly indicate differential growth characteristics of cells cultured under periodic versus static conditions highlighting the impact of the culture conditions on the physiology of cells in vitro.

Ultra-Low-Cost 3D Bioprinting: Modification and Application of an Off-the-Shelf Desktop 3D-Printer for Biofabrication.

3D bioprinting has become a versatile and powerful method in tissue engineering and regenerative medicine and is increasingly adapted by other disciplines due to its tremendous potential beyond its typical applications. However, commercially available 3D bioprinting systems are typically expensive circumventing the broad implementation, including laboratories in low-resource settings. To address the limitations of conventional and commercially available technology, we developed a 3D bioprinter by modification of an off-the-shelf 3D desktop printer, that can be installed within a single day, is of handy size to fit into a standard laminar flow hood, customizable, ultra-low cost and thus, affordable to a broad range of research labs, or educational institutions. We evaluate accuracy and reproducibility of printing results using alginate and alginate/gelatin-hydrogels and demonstrate its potential for biomedical use by printing of various two-and three-dimensional cell-free and mammalian cell-laden objects using recombinant HEKYFP cells, stably expressing yellow fluorescent protein (YFP) as a model system and high-content imaging. We further provide a parts list and 3D design files in STL and STEP format for reconstructing the device. A time-lapse video of the custom-built device during operation is available at https://vimeo.com/274482794.

Nano- and Micro-Patterned S-, H-, and X-PDMS for Cell-Based Applications: Comparison of Wettability, Roughness, and Cell-Derived Parameters.

Polydimethylsiloxane (PDMS) is a promising biomaterial for generating artificial extracellular matrix (ECM) like patterned topographies, yet its hydrophobic nature limits its applicability to cell-based approaches. Although plasma treatment can enhance the wettability of PDMS, the surface is known to recover its hydrophobicity within a few hours after exposure to air. To investigate the capability of a novel PDMS-type (X-PDMS) for in vitro based assessment of physiological cell properties, we designed and fabricated plane as well as nano- and micrometer-scaled pillar-patterned growth substrates using the elastomer types S-, H- and X-PDMS, which were fabricated from commercially available components. Most importantly, we compared X-PDMS based growth substrates which have not yet been investigated in this context with H- as well as well-known S-PDMS based substrates. Due to its applicability to fabricating nanometer-sized topographic features with high accuracy and pattern fidelity, this material may be of high relevance for specific biomedical applications. To assess their applicability to cell-based approaches, we characterized the generated surfaces using water contact angle (WCA) measurement and atomic force microscopy (AFM) as indicators of wettability and roughness, respectively. We further assessed cell number, cell area and cellular elongation as indirect measures of cellular viability and adhesion by image cytometry and phenotypic profiling, respectively, using Calcein and Hoechst 33342 stained human foreskin fibroblasts as a model system. We show for the first time that different PDMS types are differently sensitive to plasma treatment. We further demonstrate that surface hydrophobicity changes along with changing height of the pillar-structures. Our data indicate that plane and structured X-PDMS shows cytocompatibility and adhesive properties comparable to the previously described elastomer types S- and H-PDMS. We conclude that nanometer-sized structuring of X-PDMS may serve as a powerful method for altering surface properties toward production of biomedical devices for cell-based applications.