Spheroid Trapping and Calcium Spike Estimation Techniques toward Automation of 3D Culture.

We present a spheroid trapping device, compatible with traditional tissue culture plates, to confine microtissues in a small area and allow suspension cultures to be treated like adherent cultures with minimal loss of spheroids due to aspiration. We also illustrate an automated morphology-independent procedure for cell recognition, segmentation, and a calcium spike detection technique for high-throughput analysis in 3D cultured tissue. Our cell recognition technique uses a maximum intensity projection of spatial-temporal data to create a binary mask, which delineates individual cell boundaries and extracts mean fluorescent data for each cell through a series of intensity thresholding and cluster labeling operations. The temporal data are subject to sorting for imaging artifacts, baseline correction, smoothing, and spike detection algorithms. We validated this procedure through analysis of calcium data from 2D and 3D SHSY-5Y cell cultures. Using this approach, we rapidly created regions of interest (ROIs) and extracted fluorescent intensity data from hundreds of cells in the field of view with superior data fidelity over hand-drawn ROIs even in dense (3D tissue) cell populations. We sorted data from cells with imaging artifacts (such as photo bleaching and dye saturation), classified nonfiring and firing cells, estimated the number of spikes in each cell, and documented the results, facilitating large-scale calcium imaging analysis in both 2D and 3D cultures. Since our recognition and segmentation technique is independent of morphology, our protocol provides a versatile platform for the analysis of large confocal calcium imaging data from neuronal cells, glial cells, and other cell types.

Brain-on-a-Chip Device for Modeling Multiregional Networks.

Animal models are frequently used in drug discovery because they represent a mammalian in vivo model system, they are the closest approximation to the human brain, and experimentation in humans is not ethical. Working with postmortem human brain samples is challenging and developing human in vitro systems, which mimic the in vivo human brain, has been challenging. However, the use of animal models in drug discovery for human neurological diseases is currently under scrutiny because data from animal models has come with variations due to genetic differences. Evidence from the literature suggests that techniques to reconstruct multiple neurotransmission projections, which characterize neurological disease circuits in humans, in vitro, have not been demonstrated. This paper presents a multicompartment microdevice for patterning neurospheres and specification of neural stem cell fate toward networks of multiple neuronal phenotypes. We validated our design by specification of human neural stem cells to dopaminergic and GABAergic neurons in different compartments of the device, simultaneously. The neurospheres formed unrestricted robust neuronal circuits between arrays of neurospheres in all compartments of the device. Such a device design may provide a basis for formation of multineurotransmission circuits to model functional connectivity between specific human brain regions, in vitro, using human-derived neural stem cells. This work finds relevance in neurological disease modeling and drug screening using human cell-based assays and may provide the impetus for shifting from animal-based models.

Secretome-Based Prediction of Three-Dimensional Hepatic Microtissue Physiological Relevance.

Early biomarkers for indication of the complex physiological relevance (CPR) of a three-dimensional (3D) tissue model are needed. CPR is detected late in culture and requires different analytical techniques. Albumin production, CYP3A4 expression, and formation of bile canaliculi structures are commonly used to compare in vitro hepatic cells to their in vivo counterpart. A universal biomarker independent of the cell type would bring this to a common detection platform. We make the case that these hepatic characteristics are not sufficient to differentiate traditional (2D) cell culture from the more complex 3D culture. We explored the cytokine secretion profile (secretome) for its potential as a 3D early culture biomarker. PDGF-AB/BB and vascular endothelial growth factor (VEGF) were found to be upregulated in 3D compared to 2D cultures at early time points (days 3 and 4). These observations provide a foundation upon which in vivo validation of cytokines can lead to physiologically relevant 3D in vitro cell culture.

Calcium Oscillation Frequency Is a Potential Functional Complex Physiological Relevance Indicator for a Neuroblastoma-Based 3D Culture Model.

In vitro screening for drugs that affect neural function in vivo is still primitive. It primarily relies on single cellular responses from 2D monolayer cultures that have been shown to be exaggerations of the in vivo response. For the 3D model to be physiologically relevant, it should express characteristics that not only differentiate it from 2D but also closely emulate those seen in vivo. These complex physiologically relevant (CPR) outcomes can serve as a standard for determining how close a 3D culture is to its native tissue or which out of a given number of 3D platforms is better suited for a given application. In this study, Fluo-4-based calcium fluorescence imaging was performed followed by automated image data processing to quantify the calcium oscillation frequency of SHSY5Y cells cultured in 2D and 3D formats. It was found that the calcium oscillation frequency is upregulated in traditional 2D cultures while it was comparable to in vivo in spheroid and microporous polymer scaffold-based 3D models, suggesting calcium oscillation frequency as a potential functional CPR indicator for neural cultures.

Engineering microsystems to recapitulate brain physiology on a chip.

The structural and functional organization of the human brain consists of 52 regions with distinct cellular organization. In vitro models for normal and pathological states using isolated brain-region-specific 3D engineered tissues fail to recapitulate information integration and/or transfer that arises from connectivity among neuroanatomical structures. Therefore, development of brain-on-a-chip microsystems must shift to multiple region neuron network designs to be relevant in brain functionality and deficit modeling. However, in vitro formation of multiregional networks on microdevices presents several challenges that we illustrate using a few neurological disorders; and we offer guidance, depending on objectives (HTS, disease modeling, etc.) for rational design of microfluidic systems and better emulation of in vivo conditions.

3D nerve cell cultures and complex physiological relevance.

The field of tissue engineering has not yet provided knowledge on which a consensus for the complex physiological relevance (CPR) of neuronal cultures could be established. The CPR of 3D neuronal cultures can have a profound impact on the drug discovery process through the validation of in vitro models for the study of neuropsychiatric and degenerative diseases, as well as screening for neurotoxicity during drug development. Herein, we assemble evidence in support of the potential of [Ca2+]i oscillation frequency as a CPR outcome that can demonstrate the in vivo-like behavior of 3D cultures and differentiate them from 2D monolayers. We demonstrate that [Ca2+]i oscillation frequencies in 2D cultures are significantly higher than those found in 3D cultures, and provide a possible molecular explanation.