## 4 Discussion  
This study describes the assembly of a hybrid brain model, combining a neocortical biopsy from a patient with epilepsy with a layer of hiPSC-derived cortical neurons cultured on a MEA. This setup allowed recordings of spontaneous electrophysiological activity for 6 days, with the main advantage being the possibility to study patient-derived biopsy postoperatively with commercially available consumables and a standardized readout system. During the first week of co-culturing of the hybrid model, we confirmed that the network activity of the neuronal cell layer can serve as an interface between the patient-derived 3D tissue plug and the MEA electrodes. Gradually, the co-culture became less responsive as of day 6, which suggests its degradation. Various read-outs (fire rate, burst rate, inter-spike interval, etc. ; see Supplementary Table S1) were extracted from the electrophysiological signal. Based on literature [26; 27; 28], eight read-outs (endpoints) were selected to characterize the network's behaviour of our hybrid model and revealed that it was reminiscent of pro-convulsive and epileptiform electrophysiological signatures [30; 31; 32; 33; 34]. The hereby obtained epileptiform phenotype was not observed in setups where either the biopsy alone or the hiPSC-derived cortical neuron layer alone were cultured on a MEA. Here we will discuss the simplicity of our approach and the results obtained with our hybrid system, but also the challenges remaining. Recordings from the stand-alone biopsy in 37 \({}^{\circ}\)C maintenance medium hardly surpassed the signal-to-noise ratio, i.e., single electrodes showed some local field potentials in the first few minutes. There was no network activity observed and all signals were lost within the hour (Supplementary Figure S1). Technically, without a dedicated chip (i.e., cortical neurons grown onto a MEA) and suction ports [35] to keep the biopsy in place, an alternative is to place the MEA in the recording apparatus and load the biopsy atop the electrodes directly and measure for as long as possible (up to a few hours) before discarding it. Moving the MEA chip back and forth between the recorder and the incubator can displace the biopsy, thereby increasing the risk that electrical activity is not always obtained from the same location. Therefore, we added a PDMS ring that kept the biopsy in place. However, for the stand-alone biopsy, adding a PDMS ring to fix the biopsy did not improve the electrophysiological signal, as the neurons of the biopsy plugs were not in close enough contact with the electrodes of the MEA chip. However, loading the 3D biopsy plug onto a PDMS ring containing a layer of cortical neurons at the bottom resulted in a good signal-to-noise ratio of spontaneous electrophysiological activity on all electrodes for up to 6 days. When we measured activity from the layer of hiPSC-derived cortical neurons prior to assembly of the biopsy plug, we retrieved an expected spike activity and bursting behaviour (Figure 2D), which is commonly seen for such cultures [25], but no seizure-like discharge patterns. DIV16 was chosen to assemble the biopsy plugs with the neuronal culture because morphological (i.e., immunohistochemical positivity of neuronal markers beta tubulin III, MAP2 and synaptophysin) and electrophysiological (i.e., a firing rate of \(\sim\)3 Hz and the presence of network bursts) read-outs indicated that the hiPSC-derived neuronal cell layer reached a stage that permitted their functioning as a transducer between the biopsy and the MEA. When comparing the electrophysiological recordings of the three conditions (i.e., the hiPSC-derived neuronal layer alone (Figure 1D), the hybrid setup (Figure 1E) and the biopsy alone (Figure S1)), in the hybrid model we identified not only an increase in firing and bursting rates, but also an increase in burst duration for both single-channel and network bursts, as well as an increase in inter-spike interval coefficient of variation.