Prototyping in Polymethylpentene to Enable Oxygen-Permeable On-a-Chip Cell Culture and Organ-on-a-Chip Devices Suitable for Microscopy.

With the rapid development and commercial interest in the organ-on-a-chip (OoC) field, there is a need for materials addressing key experimental demands and enabling both prototyping and large-scale production. Here, we utilized the gas-permeable, thermoplastic material polymethylpentene (PMP). Three methods were tested to prototype transparent PMP films suitable for transmission light microscopy: hot-press molding, extrusion, and polishing of a commercial, hazy extruded film. The transparent films (thickness 20, 125, 133, 356, and 653 µm) were assembled as the cell-adhering layer in sealed culture chamber devices, to assess resulting oxygen concentration after 4 days of A549 cell culture (cancerous lung epithelial cells). Oxygen concentrations stabilized between 15.6% and 11.6%, where the thicker the film, the lower the oxygen concentration. Cell adherence, proliferation, and viability were comparable to glass for all PMP films (coated with poly-L-lysine), and transparency was adequate for transmission light microscopy of adherent cells. Hot-press molding was concluded as the preferred film prototyping method, due to excellent and reproducible film transparency, the possibility to easily vary film thickness, and the equipment being commonly available. The molecular orientation in the PMP films was characterized by IR dichroism. As expected, the extruded films showed clear orientation, but a novel result was that hot-press molding may also induce some orientation. It has been reported that orientation affects the permeability, but with the films in this study, we conclude that the orientation is not a critical factor. With the obtained results, we find it likely that OoC models with relevant in vivo oxygen concentrations may be facilitated by PMP. Combined with established large-scale production methods for thermoplastics, we foresee a useful role for PMP within the OoC field.

Application of Polymethylpentene, an Oxygen Permeable Thermoplastic, for Long-Term on-a-Chip Cell Culture and Organ-on-a-Chip Devices.

The applicability of a gas-permeable, thermoplastic material polymethylpentene (PMP) was investigated, experimentally and analytically, for organ-on-a-chip (OoC) and long-term on-a-chip cell cultivation applications. Using a sealed culture chamber device fitted with oxygen sensors, we tested and compared PMP to commonly used glass and polydimethylsiloxane (PDMS). We show that PMP and PDMS have comparable performance for oxygen supply during 4 days culture of epithelial (A549) cells with oxygen concentration stabilizing at 16%, compared with glass control where it decreases to 3%. For the first time, transmission light images of cells growing on PMP were obtained, demonstrating that the optical properties of PMP are suitable for non-fluorescent, live cell imaging. Following the combined transmission light imaging and calcein-AM staining, cell adherence, proliferation, morphology, and viability of A549 cells were shown to be similar on PMP and glass coated with poly-L-lysine. In contrast to PDMS, we demonstrate that a film of PMP as thin as 0.125 mm is compatible with high-resolution confocal microscopy due to its excellent optical properties and mechanical stiffness. PMP was also found to be fully compatible with device sterilization, cell fixation, cell permeabilization and fluorescent staining. We envision this material to extend the range of possible microfluidic applications beyond the current state-of-the-art, due to its beneficial physical properties and suitability for prototyping by different methods. The integrated device and measurement methodology demonstrated in this work are transferrable to other cell-based studies and life-sciences applications.

Markers of Mitochondrial Metabolism in Tumor Hypoxia, Systemic Inflammation, and Adverse Outcome of Rectal Cancer.

Tumor hypoxia contributes to therapy resistance and metastatic progression of locally advanced rectal cancer (LARC). We postulated that the tumor mitochondrial metabolism, manifested by reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage, reflects how hypoxic conditions connect to cancer-induced systemic inflammation and poor outcome. Levels of ROS and mtDNA damage were analyzed in three colorectal cancer (CRC) cell lines cultured for 24 hours under normoxia (21% O2) or hypoxia (0.2% O2) and serum sampled at the time of diagnosis from 35 LARC patients participating in a prospective therapy study. Compared with normoxia, ROS were significantly repressed and mtDNA damage was significantly enhanced in the hypoxic CRC cell lines; hence, a low ratio of ROS to mtDNA damage was an indicator of hypoxic conditions. In the LARC patients, low serum ROS were associated with elevated levels of circulating carcinoembryonic antigen and tumor choline concentration, both indicative of unfavorable biology, as well as adverse progression-free and overall survival. A low ratio of ROS to mtDNA damage in serum was associated with poor local tumor response to the neoadjuvant treatment and, of note, elevated systemic inflammation factors (C-reactive protein, the interleukin-1 receptor antagonist, and factors involved in tumor necrosis factor signaling), indicating that deficient treatment response locally and detrimental inflammation systemically link to a hypoxic mitochondrial metabolism. In conclusion, serum ROS and damaged mtDNA may be markers of the mitochondrial metabolism driven by the state of oxygenation of the primary tumor and possibly implicated in systemic inflammation and adverse outcome of LARC.

Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells.

BACKGROUND: The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)-collectively referred to as combo-Rx, in hypoxic CRC cell lines. METHODS: Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines. RESULTS: In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses. CONCLUSIONS: The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity.