Novel Method using PEMF Cell Culture Reproducibility

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Bioengineering 2022, 9, 595
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position of the samples and in real time (except for constant background fields) throughout the course of the experiment. To ensure a valid biological readout, the experiment uses a homogeneous, immortalized and genetically defined human reporter gene cell line (HEK-BlueTM hTLR4). This model cell line is used for studies of inflammation and is engineered to secrete alkaline phosphatase into the cell culture medium upon triggering the TLR4-dependent inflammatory response. As a consequence, using this experimental system, the biological readout is a simple colorimetric substrate assay, which can be readily standardized and replicated under laboratory conditions.
2. Materials and Methods
2.1. The Magnetic-Field Exposure System
The exposure system allows the application of controlled magnetic-field parameters with respect to signal shapes g(t), frequency f, intensity/amplitude B0, duty cycle D as well as controlled environmental conditions characterized by temperature T, relative humidity rH and CO2 for the incubation of the cell culture. For each experiment, there are 2 parallel running systems: an ’exposure’ cell-culture incubator (called incubator E; IC E) to which the controlled magnetic field is applied and a ’control’ cell-culture incubator (called incubator C; IC C) running in parallel with no exposure condition. Both systems are controlled by an in-house developed software application.
2.1.1. Incubator Design
The incubator housing is made out of black acrylic glass (PMMA) with an inner vol- ume of 200 mm × 200 mm × 150 mm (x × y × z) enclosed by a water jacket to regulate the temperature via an external thermostat. A water bath provides passive humidity regula- tion. For faster temperature regulation, a Pt100-sensor (special design, Innovative Sensor Technology IST AG, 2.8 mm, 4-wire, quality class AA or F 0.1) connected to the thermostat is embedded directly in the water jacket. In addition, the incubator lid is temperature- controlled by the water circuit. An external CO2 controller provides a premixed CO2–air mixture. The gas mixture passes a humidifier (bubble water bottle) before entering the incubator. Since CO2 has a higher density than air, the incubator was constructed as a top loader. During an experiment, the internal incubator conditions regarding temperature, rel- ative humidity and CO2 are logged by sensors. For temperature, an NTC sensor (negative temperature coefficient thermistor) is used and for humidity measurements a multisensor module (Ahlborn GmbH GmbH, Holzkirchen, Germany). The NTC sensor (TS-NTC-103A, B & B Sensors, Donaueschingen, Germany) was calibrated with a calibration bath (FK31- SL, Julabo GmbH, Seelbach, Germany) versus a DAkkS-calibrated Ahlborn Mess- und Regelungstechnik GmbH, Eichenfeldstraße 1, 83607 Holzkirchen, Germany (supplement, Calibration Certificate 2019-06)) Pt100 (PT100 1/10th DIN liquid probe, 4.8 mm × 40 mm long, Electronic Temperature Instruments Ltd. (ETI), Easting Close, Worthing, Sussex, BN14 8HQ, UK) with an absolute error of ∆T = ±0.1 K (Supplement, Figure S7). The NTC sensor and the multisensor module were placed within the coil in vicinity of the sample and close to the inner wall of the coil. The precision of the atmospheric sensors in the incubator are ∆T = 0.1 K, ∆rH = 0.1% and ∆[CO2] = 0.1%. For CO2 measurement, we use a heated flow through IR-CO2-sensor (SPRINTIR-WF-20, Gas Sensing Solutions Ltd., Cumbernauld, UK) logging the incubator gas. During the experiments, the setpoints for temperature and CO2 in the incubators are: 37 ◦ C, 5 % CO2 , while rH is passively regulated by evaporation.
2.1.2. Coil Specifications
The biological sample is placed into a plexiglass holder at a fixed position inside the coil, which is then placed in the incubator. The coil was constructed to provide a homogenous field distribution regarding field intensity across the entire microplate (Supplement, Figure S1). The homogeneity is within ≤1% over a space of 3 stacked mi- croplates. Furthermore, the coil reduces the far field (via a counter winding) to avoid the