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. 2018 Feb;70(1):375-386.
doi: 10.1007/s10616-017-0152-x. Epub 2017 Oct 14.

A novel lab-on-a-chip platform for spheroid metabolism monitoring

Frank Alexander Jr  1 , Sebastian Eggert  1   2 , Joachim Wiest  3
Affiliations

A novel lab-on-a-chip platform for spheroid metabolism monitoring

Frank Alexander Jr et al. Cytotechnology. 2018 Feb.

Abstract

Sensor-based cellular microphysiometry is a technique that allows non-invasive, label-free, real-time monitoring of living cells that can greatly improve the predictability of toxicology testing by removing the influence of biochemical labels. In this work, the Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) was utilized to perform cellular microphysiometry on 3D multicellular spheroids. Using a commercial 3D printer, 3 × 3 microwell arrays were fabricated to maintain nine previously cultured HepG2 spheroids on a single BioChip. Integrated layers above and under the spheroids allowed fluidic contact between spheroids in microwells and BioChip sensors while preventing wash out from medium perfusion. Spheroid culturing protocols were optimized to grow spheroids to a diameter of around 620 μm prior to transfer onto BioChips. An ON/OFF pump cycling protocol was developed to optimize spheroid culture within the designed microwells, intermittently perfuse spheroids with fresh culture medium, and measure the extracellular acidification rate (EAR) and oxygen uptake rate (OUR) with the BioChips of the IMOLA-IVD platform. In a proof-of-concept experiment, spheroids were perfused for 36 h with cell culture medium before being exposed to medium with 1% sodium dodecyl sulphate (SDS) to lyse cells as a positive control. These microphysiometry studies revealed a repeatable pattern of extracellular acidification throughout the experiment, indicating the ability to monitor real-time metabolic activity of spheroids embedded in the newly designed tissue encapsulation. After perfusion for 36 h with medium, SDS exposure resulted in an instant decrease in EAR and OUR signals from 37 mV/h (± 5) to 8 mV/h (± 8) and from 308 mV/h (± 21) to -2 mV/h (± 13), respectively. The presented spheroid monitoring system holds great potential as a method to automate screening and analysis of pharmaceutical agents using 3D multicellular spheroid models.

Keywords: Cellular respiration; Extracellular acidification; Label-free sensing; Microphysiometry; Organ-on-chip; Spheroid-on-chip.

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Figures

Fig. 1
Fig. 1
a Six Intelligent Mobile Labs for in vitro diagnostics are installed in an incubator and connected to a personal computer for temporary data storage and control of experimental settings. b The illustration presents the modular structure of the technology with the sensor chip (BioChip), the analog module for the operation of the microsensors, the fluidic system for the application of cell culture medium and test substances, the power supply, and the digital module which communicates with a personal computer
Fig. 2
Fig. 2
a The IMOLA BioChips allows label-free and continuous measurements of cellular cultures on the chip. b The BioChip-D is presented with a detailed view of the sensor chip with the two pH sensors (pH1, pH2), two electric impedance sensors (Imp1, Imp2), one amperometric sensor (pO2), and one temperature sensor (T)
Fig. 3
Fig. 3
a To maintain spheroid cultures on the IMOLA system a new spheroid encapsulation was designed. The exploded view shows that the encapsulation consisting of a standard IMOLA fluidic head, a separation ring, a net filter membrane, a scaffold disc which houses nine spheroids, another net filter membrane and a support disc. b The assembly is stacked onto a standard IMOLA BioChip. c Medium is perfused through the fluidic head and the porous membrane shields cells from direct sheer forces, while allowing access to fresh medium
Fig. 4
Fig. 4
Fluid flow conditions were tested for the new spheroid encapsulation design without cell. As the pH solution is switched between values of 6.4 and 7.4, the measured voltage fluctuates as well. Steady state values are reached for pH values of 6.4 whereas a dedicated drop in signal is seen when switching to pH values of 7.4
Fig. 5
Fig. 5
The extracellular pH of HepG2 spheroids was monitored with the developed spheroid encapsulation design with an applied pump cycle of a 1 h on and 1 h off and b 30 min on and 30 min off. Changes in pH signal are presented in mV (black), and the pump phase is presented with on or off (blue). An increase in pH signal during the pump off phase was observed for both pump set-ups. During the pump on phase, the pH signal decreased to a basal line. (Color figure online)
Fig. 6
Fig. 6
Exposure to sodium dodecyl sulphate (SDS) was performed to evaluate the ability for the system to perform real-time recording of changes due to toxic exposure. The applied pump cycle was 1 h on and 1 h off, and 1% SDS was added after 36 h. a Original pH signals and b the normalized extracellular acidification rate (EAR) indicate spheroid metabolism before SDS exposure. During pump off cycles, spheroids metabolism showed an increase in measured pH that corresponds with the acidification of the medium. Subsequent pump on cycles resulted in a return to basal pH levels. After the addition of 1% SDS solution this trend disappeared as cells no longer acidify the medium. EAR decreased over 10 h after SDS exposure and reached a base line afterwards
Fig. 7
Fig. 7
Oxygen consumption was monitored for nine HepG2 spheroids encapsulated on a single BioChip. The applied pump cycle was 1 h on and 1 h off, and 1% SDS was added after 36 h. Both a original oxygen signals and b the normalized oxygen uptake rate (OUR) indicated a base line before the SDS exposure and then an instant drop. Oxygen consumption increased during pump off phase and decreased instantly to a basal line in the pump on phase before SDS exposure. The addition of 1% SDS resulted in an instant toxic response, indicated by the decrease of the signal values and OUR. IMOLA oxygen sensors showed consistent consumption of oxygen by cells in spheroid morphologies. At the addition of SDS the consumption curves halted and OUR reached a base line after 2 h of SDS exposure
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