A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers.

Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.We show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators

Abstract 3309: Tumor-treating fields decrease proliferation and clonogenicity of patient-derived WHO grade IV glioma cell lines

Abstract Despite decades of research, efficacious treatments for malignant glioma tumors are limited. Tumor-treating fields (TTFields) are FDA-approved for the treatment of newly-diagnosed and recurrent glioblastoma. In this study, in vitro experiments comparing TTFields to untreated controls were performed on patient-derived grade IV glioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity. Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Patient tumor specimens (glioblastoma and gliosarcoma) were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Equal numbers of cells were plated on plastic coverslips in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at 200 kHz to half of the coverslips. Culture media was replaced every day. At the conclusion of the 2 week treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (10,000 cells/well). The resulting colonies were fixed and stained with crystal violet and counted with an automated colony counter. Control vs. TTFields treated groups were compared by two-tailed t-tests. Results: The two-week TTFields treatment significantly reduced cell proliferation in both the glioblastoma (41.6 ± 11.1 % control; n=4; p&amp;lt;0.001) and in the gliosarcoma (41.6 ± 16.6 % control; n=4; p&amp;lt;0.002) as measured by XTT assay. The clonogenic assay revealed that the number of colonies generated from both cell lines was reduced by TTFields treatment. For the glioblastoma cell line, control cells yielded 847 colonies with an average diameter of 462.8 ± 5.6 µM while TTFields-treated cells yielded 561 colonies with an average diameter of 435.1 ± 7.3 µM, which was a statistically significant decrease (p=0.0022). For the gliosarcoma cell line, control cells yielded 809 colonies vs. 144 colonies from the TTFields treated cells, although the average diameter of the colonies was unchanged between groups (control 375.1 ± 176.1 µM; TTFields 362.9 ± 196.0 µM). Conclusions: In vitro application of TTFields markedly reduced cell proliferation and clonogenicity in both patient-derived WHO grade IV glioblastoma and gliosarcoma cell lines. This is the first report on the in vitro effects of TTFields on gliosarcoma. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Adrianne Wallace-Povirk, Sandeep Mittal. Tumor-treating fields decrease proliferation and clonogenicity of patient-derived WHO grade IV glioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3309. doi:10.1158/1538-7445.AM2017-3309</jats:p

Outline of the wiring diagram.

<p>A Fritzing (fritzing.org) sketch is presented outlining the wiring connections between the microcontroller, the logic converter, and the oxygen sensor.</p

Schematic of the logic converter and connections.

<p>Pin designations of the 4-channel bi-directional logic converter (low-voltage pins A1, A2 and high voltage pins B1, B2 were used. A3, A4 and B3, B4 pins are extra, and not needed in this application). The logic converter is supplied as a mini printed circuit board (PCB) with a pair of 6-pin headers to facilitate mounting on a breadboard. We used a 30W soldering iron (RadioShack) fitted with a 15-watt soldering iron tip (cat. no. 64–2052, RadioShack) and 0.787 mm (0.032") flux-cored solder wire (cat. no. 64–005) to solder the PCB to the 6-pin headers. The soldered PCB was fitted on the half-sized breadboard mounted alongside the Arduino board.</p

Screen-capture images of CoolTerm terminal window.

<p><b>Upper panel</b>: CoolTerm quick access bar and ribbon; <b>middle panel</b>: format of the initial data output from CoolTerm; <b>lower panel</b>: format of the final data saved by CoolTerm with time-stamps (saved and opened as a Microsoft Notepad file).</p