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.

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.

Ratiometric Nanoviscometers: Applications for Measuring Cellular Physical Properties in 3D Cultures.

New insights into the biomechanical properties of cells are revealing the importance of these properties and how they relate to underlying molecular, architectural, and behavioral changes associated with cell state and disease processes. However, the current understanding of how these in vitro biomechanical properties are associated with in vivo processes has been developed based on the traditional monolayer (two-dimensional [2D]) cell culture, which traditionally has not translated well to the three-dimensional (3D) cell culture and in vivo function. Many gold standard methods and tools used to observe the biomechanical properties of 2D cell cultures cannot be used with 3D cell cultures. Fluorescent molecules can respond to external factors almost instantaneously and require relatively low-cost instrumentation. In this review, we provide the background on fluorescent molecular rotors, which are attractive tools due to the relationship of their emission quantum yield with environmental microviscosity. We make the case for their use in both 2D and 3D cell cultures and speculate on their fundamental and practical applications in cell biology.

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.

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 µm; B: 175 ± 6 µm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018.

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.

Molecular basis for cytokine biomarkers of complex 3D microtissue physiology in vitro.

'Physiologically more-relevant' claims are readily made for cells cultured on any surface or in a scaffold that provides loosely defined 3D geometry. A set of tools to measure culture '3D-ness' more accurately are needed. Such tools should find applications in fields ranging from high-throughput identification of substrates for tissue engineering and regenerative medicine to cell-based screening of drug candidates. Until now, these fields have not provided a consensus for the most promising place to initiate the search. Here, we review recent advances in transcriptomic, proteomic, inflammation and oncology-related pathways, as well as functional studies that strongly point to cytokines as the most likely compounds to form the missing consensus.

Anthropometric characteristics of female smallholder farmers of Uganda--Toward design of labor-saving tools.

Sub-Saharan African women on small-acreage farms carry a disproportionately higher labor burden, which is one of the main reasons they are unable to produce for both home and the market and realize higher incomes. Labor-saving interventions such as hand-tools are needed to save time and/or increase productivity in, for example, land preparation for crop and animal agriculture, post-harvest processing, and meeting daily energy and water needs. Development of such tools requires comprehensive and content-specific anthropometric data or body dimensions and existing databases based on Western women may be less relevant. We conducted measurements on 89 women to provide preliminary results toward answering two questions. First, how well existing databases are applicable in the design of hand-tools for sub-Saharan African women. Second, how universal body dimension predictive models are among ethnic groups. Our results show that, body dimensions between Bantu and Nilotic ethnolinguistic groups are different and both are different from American women. These results strongly support the need for establishing anthropometric databases for sub-Saharan African women, toward hand-tool design.

Cultural Influences in Women-Friendly Labor-Saving Hand Tool Designs: The Milk Churner Case.

OBJECTIVE: The aim of this study was to highlight the importance of culture in sustainable, labor-saving solutions design for women in low-resource settings. BACKGROUND: One of the reasons behind the gender asset gap among Sub-Saharan African women is the higher labor burden these women face, making it difficult for them to produce for the home and markets. Hand tools are the simplest form and therefore the best first step to address this problem. But designing women-friendly (sustainable) hand tools calls for better understanding of the low-resource settings where these women reside. METHOD: A milk churner was redesigned using a human-centered (participatory) approach with groups of women from two dominant ethnolinguistic groups of Bantu and Nilotic of Uganda, and its usability was tested. RESULTS: The churner reduced labor up to eightfold and has potential to expand the range of uses to include children and husbands due to its simplicity. Also, the churner significantly reduced undesirable health effects, like pain in knee joints. Based on the experience with the churner, a six-item "survival guide" is proposed to complement human-centered design guiding principles for facilitating the generation of solutions in low-resource settings. CONCLUSION: By paying great attention to culture in relation to human factors, a labor-reducing churner has been successfully introduced among Ugandan women. The ultimate goal is to make the churner available to female smallholder dairy-farming households throughout Sub-Saharan Africa. APPLICATIONS: This study provides a survival guide for generating solutions to problems from low-resource settings.

Is time an extra dimension in 3D cell culture?

Time or the temporal microenvironment is a parameter that is often overlooked in 3D cell culture. However, given that the 3D system is a dynamic entity, there exists bidirectional signaling between the cells and their microenvironment and, in time, cells can develop the capacity to modulate their environment. We make this case here by illustrating the relation between the temporal dimension and other microenvironmental parameters and demonstrate how the exogenously incorporated microenvironmental factors (MEFs) can be rendered less significant with time. Such knowledge can guide construct design to make 3D platforms architecturally simpler by eliminating redundancy. We further show that there is a need to establish the point at which the construct is complex enough such that its use yields responses that more closely emulate in vivo outcomes.

A microwell pattern for C17.2 cell aggregate formation with concave cylindrical surface induced cell peeling.

We have developed a polydimethylsiloxane (PDMS) pattern with arrays of microwells for the formation of multicellular aggregates by C17.2 neural stem cells. Upon interfacing with the patterns, the neural stem cells would firstly attach to the microwell sidewalls, forming cellular strips on day 1 after plating. For channel connected microwells, cellular strips on the concave semi-cylindrical sidewall surfaces continued among wells and through channels, followed by strip peeling due to prestress arising from actin filaments and assembly of suspending cellular aggregates within the microwells in the following 1-2 days. Our results also suggested that a small microwell diameter of 80 and 100 µm and a narrow channel width of 20 µm would facilitate the aggregate formation among the structural dimensions tested. Finite element method (FEM) simulation revealed that cellular strips on the semi-cylindrical sidewall surfaces peeled under significantly smaller prestresses (critical peeling prestress, CPP), than cells on flat substrates. However, the CPP by itself failed to fully account for the difference in aggregate inducing capability among the patterns addressed, suggesting cell growth behaviors might play a role. This study thus justified the current patterning method as a unique and practical approach for establishing 3D neural stem cell-based assay platforms.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid).

INTRODUCTION: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). METHODS AND MATERIALS: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. RESULTS: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. CONCLUSION: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

Biophysical microenvironment and 3D culture physiological relevance.

Force and substrate physical property (pliability) is one of three well established microenvironmental factors (MEFs) that may contribute to the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in preclinical drug discovery. In 3D cultures, studies of the physiological relevance dependence on material pliability are inconclusive, raising questions regarding the need to design platforms with materials whose pliability lies within the physiological range. To provide more insight into this question, we examine the factors that may underlie the studies inconclusiveness and suggest the elimination of redundant physical cues, where applicable, to better control other MEFs, make it easier to incorporate 3D cultures into state of the art HTS instrumentation, and reduce screening costs per compound.

Performance evaluation of 3D polystyrene 96-well plates with human neural stem cells in a calcium assay.

In this study, we have generated a high-throughput screening (HTS)-compatible 3D cell culture platform by chemically "welding" polystyrene scaffolds into standard 2D polystyrene 96-well plates. The variability of scaffolds was minimized by introducing automation into the fabrication process. The fabricated 3D cell culture plates were compared with several commercially available 3D cell culture platforms with light and scanning electron microscopy. Voltage-gated calcium channel functionality was used to access the Z' factors of all plates, including a 2D standard plate control. It was found that with the No-Wash Fluo-4 calcium assay and neural progenitor cells, all plates display acceptable Z' factors for use in HTS. The plates with "welded" polystyrene scaffolds have several advantages, such as being versatile and economical, and are ready to use off the shelf. These characteristics are especially desired in HTS preclinical drug discovery applications.

Microtissue size and hypoxia in HTS with 3D cultures.

The three microenvironmental factors that characterize 3D cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. Although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. We make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in drug discovery. We further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. We offer guidance, depending on HTS objectives, for rational 3D culture platform choice for better emulation of in vivo conditions.

Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate). Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

Iron specificity of a biosensor based on fluorescent pyoverdin immobilized in sol-gel glass.

Two current technologies used in biosensor development are very promising: 1. The sol-gel process of making microporous glass at room temperature, and 2. Using a fluorescent compound that undergoes fluorescence quenching in response to a specific analyte. These technologies have been combined to produce an iron biosensor. To optimize the iron (II or III) specificity of an iron biosensor, pyoverdin (a fluorescent siderophore produced by Pseudomonas spp.) was immobilized in 3 formulations of porous sol-gel glass. The formulations, A, B, and C, varied in the amount of water added, resulting in respective R values (molar ratio of water:silicon) of 5.6, 8.2, and 10.8. Pyoverdin-doped sol-gel pellets were placed in a flow cell in a fluorometer and the fluorescence quenching was measured as pellets were exposed to 0.28 - 0.56 mM iron (II or III). After 10 minutes of exposure to iron, ferrous ion caused a small fluorescence quenching (89 - 97% of the initial fluorescence, over the range of iron tested) while ferric ion caused much greater quenching (65 - 88%). The most specific and linear response was observed for pyoverdin immobilized in sol-gel C. In contrast, a solution of pyoverdin (3.0 µM) exposed to iron (II or III) for 10 minutes showed an increase in fluorescence (101 - 114%) at low ferrous concentrations (0.45 - 2.18 µM) while exposure to all ferric ion concentrations (0.45 - 3.03 µM) caused quenching. In summary, the iron specificity of pyoverdin was improved by immobilizing it in sol-gel glass C.

Biomarkers for simplifying HTS 3D cell culture platforms for drug discovery: the case for cytokines.

In this review, we discuss the microenvironmental cues that modulate the status of cells to yield physiologically more relevant three-dimensional (3D) cell-based high throughput drug screening (HTS) platforms for drug discovery. Evidence is provided to support the view that simplifying 3D cell culture platforms for HTS applications calls for identifying and validating ubiquitous three-dimensionality biomarkers. Published results from avascular tumorigenesis and early stages of inflammatory wound healing, where cells transition from a two-dimensional (2D) to 3D microenvironment, conclusively report regulation by cytokines, providing the physiological basis for focusing on cytokines as potential three-dimensionality biomarkers. We discuss additional support for cytokines that comes from numerous 2D and 3D comparative transcriptomic and proteomic studies, which generally report upregulation of cytokines in 3D compared with 2D culture counterparts.