Abstract
The need for in vitro models that mimic the human brain to replace animal testing and allow high-throughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput.
Highlights
The design and development of realistic experimental models of the human brain remain major constraints in studying nervous system functions in health and disease, developing new treatment strategies, and testing prosthetic devices[1]
Synaptic puncta per mm3t, and the vGAT/vGLUT1% ratio We quantified the number of vGAT and vGLUT1 synaptic puncta per mm[3] on three different confocal z-stacks acquired on each of the three distinct hydrogels (n = 3) analyzed for each group of DIVs reported; data are shown as mean values + standard errors; (e) Bar plot of the percent of viable cells of 3D cortical cultures over time
We analyzed three distinct z-stacks for 3 distinct hydrogels, in each reported condition; (f) Bar plot showing the mean firing rate (MFR), expressed as number of events per second, of 3D cortical cultures over time; data shown are the mean values + standard errors, (n = 6) for each DIVs interval; (g) Four frames of fluorescence calcium imaging acquired on a GCaMP6s-expressing 3D cortical culture at 26 DIVs
Summary
The design and development of realistic experimental models of the human brain remain major constraints in studying nervous system functions in health and disease, developing new treatment strategies, and testing prosthetic devices[1]. This, in turn, may cause the observed amplification of the activity of 2D neuronal networks on stiff substrates[7], which may lead to non-physiological states[8] In this regard, it is widely recognized that mechanical and topographical cues influence cell survival, proliferation and differentiation and the development of functional neural networks[9]. The temporal resolution of our system in monitoring neuronal activity was limited only by the sensitivity of the detector; with the CCD detector used in this study, we obtain a wide-volume scanning rate of 65 Hz. we achieved the current state of the art of 3D functional imaging[20, 33] in terms of scanned volume extension and neuronal sampling rate[15]: we are able to monitor 20000 ÷ 35000 cells per second in a volume of ~0.19 mm[3] using a simple and inertia-free optical architecture. The whole model is accessible by any laboratories and is a versatile and efficient model for high-throughput screening studies
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