Stand-alone MEA device for medium-throughput testing of 3D engineered neural tissue
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1
Hepia HES-SO, Laboratory of Tissue Engineering, Switzerland
Motivation:
Testing of 3D engineered neural tissue (ENT) made of neurons derived from human embryonic stem cells (hESC) or from human induced pluripotent stem cells (hiPSC) are among the most promising tools for the next decade in drug discovery and neurotoxicology. It represents a cheaper, faster and more ethical alternative to in vivo animal testing that will likely close the bridge between in vitro animal models and human clinical trials. MEA recordings of spontaneous activity in ENTs offer a non-invasive, rapid as well as possibly real-time and long-term assessment of compounds' effects. However the use of ENTs on MEAs involves a series of constraints. Notably, the lack of vascularization drastically limits diffusion of oxygen and nutrients. As a consequence, ENTs are extremely sensitive to experimental conditions and thus are not resistant to frequent manipulations out of the incubator. Moreover, traditional methods using immersion of the cells in a culture medium don't provide sufficient short-term stability nor allow long-term survival of ENTs. To address these constraints we developed a novel stand-alone device integrating porous MEA chips, which allows the maintenance of an air-liquid interface around ENTs during recording. This device is fully operational inside an incubator. It provides 8 wells with independent recording capabilities and microfluidic systems for pharmacological testing. A large number of these devices can be used in parallel to achieve medium-throughput screening of compounds.
Material and Methods:
We have developed a small-volume in vitro device in the frame size of the standard 24 wells format wherein ENTs can be cultivated or transferred onto eight independent porous membrane MEAs. These are connected to eight independent microfluidic systems - with a volume of 800 µl - which allow the maintenance of an air-liquid interface around the ENTs and the application of pharmacological or neurotoxic compounds. Spontaneous electrophysiological activities recorded from the eight integrated MEAs are transmitted using a Wireless 2100 system (MCS), through two W2100 HS32 digital headstages. These headstages are directly linked to the chip and integrated to the main body of the device. MEA are made of porous polymide and contain 8 circular platinum electrodes (40 µm diameter, covered with black platinum) separated by 200 µm.
To assess whether our device could provide reliable, stable and reproducible recordings we tested it with ENTs originating from three different types of neuroprecursor cells (NPCs). One type obtained from hESCs: cell line CHES6, BAG-hES-GEW-002 (de Geyter's lab, Switzerland) and two from commercial hiPSCs: HIP from GlobalStem (USA) and DOPA4U from Axiogenesis (Germany).
A standardized tissue preparation was used for the three types of ENTs. Briefly, after amplification NPCs were transferred in 24-wells plates (20'000 cells/well) on an orbital shaker in an incubator. After 4-6 days, self-aggregated neurospheres were collected and dropped onto circular hydrophilic membranes, which were immediately transferred onto an insert in a 6-well plate containing the differentiation medium. This led to the flattening of the tissue and to the creation of an air-liquid interface. Typical ENTs have the shape of a 3D gaussian function with a diameter of ~1 mm and a maximum thickness of ~250 µm. Tissue were kept in an incubator until the day of the recording two months after NPC differentiation, with medium change every five day. The day of recording, simultaneous recordings of 5 ENTs were made for each type of tissue. Signals were digitized at 20 kHz and high-pass filtered at 0.1 Hz using MC_Rack (MCS). Each ENT was recorded three times 40 sec, 90, 120 and 150 min after transfer onto MEAs.
Based on their firing pattern (cv, ISI and mean frequency) neurons could be classified into four types: bursting, tonic, slow or irregular. Bursting neurons: cv > 1 AND at least 2 ISI < 50 ms; tonic neurons: cv < 0.25 AND frequency > 0.2 Hz; slow neurons: frequency < 0.2 Hz; irregular neurons: neurons that could not be classified as bursting, tonic or slow.
Results:
For ENTs obtained from hESCs, three different culture series separated by one week were tested. 90 min after transfer (t1), all ENTs tested showed spontaneous activity under in average 85.3 +/-3.6 % of the functional electrodes in a MEA. After PCA-based spike sorting (custom-written routine in Igor Pro, Wavemetrics) we could discriminate an average of 11.93 +/-0.82 firing neurons with a mean firing frequency of 0.65 +/-0.07 Hz. According to our classification of firing patterns 5.3 +/-1.0 % of the neurons were bursting, 1.6 +/-0.8% tonic, 38.9 +/-3.5 % slow and 54.2 +/-5.1 % irregular.
To assess whether these parameters were stable over time (1 hour) we recorded ENTs at two additional time points, 120 (t2) and 150 (t3) min after transfer onto MEAs. There were no significant changes (Wilcoxon Signed Rank test for paired data) between t1 and t2 as well as t1 and t3, neither in the number of units (p = 0.63 and 0.85 respectively), the proportion of active electrodes (p = 0.08 and 0.19) and the mean firing frequency (p = 0.59 and 0.40).
Conclusion:
We have built and validated a novel stand-alone MEA device specifically designed for medium-throughput testing of 3D engineered neural tissue (ENTs). Overall our data indicate that our ENTs are composed of a widespread population of functional neurons, since they show stable spontaneous firing under more than 4/5 of the electrodes. Furthermore the series production of such tissues leads to a tolerable variability in intra- and inter- series firing parameters and thus supports their use for medium-throughput testing. We show that the use of our ENTs with our device allows reliable and stable recordings for a period of time encompassing the duration of an acute pharmacological experiment. As a next step, we plan to assess the use of our ENTs and device for long-term recordings (longer than one day).
Acknowledgements
Laetitia Nikles for excellent technical assistance.
Keywords:
Drug Discovery,
Neurotoxicology,
hiPSC,
hESC,
3D engineered neural tissue,
porous MEA,
spontaneous firing activity,
standalone MEA device
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Eggermann
E,
Heuschkel
M,
Fischler
G and
Stoppini
L
(2016). Stand-alone MEA device for medium-throughput testing of 3D engineered neural tissue.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00009
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Emmanuel Eggermann, Hepia HES-SO, Laboratory of Tissue Engineering, Geneva, Switzerland, emmanuel.eggermann@hesge.ch