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.

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.

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.

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.

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.

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.

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.

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 [Ca] oscillation frequency as a CPR outcome that can demonstrate the in vivo-like behavior of 3D cultures and differentiate them from 2D monolayers.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Administration of BDNF/ginsenosides combination enhanced synaptic development in human neural stem cells.

Ginsenosides Rg1 and Rb1, major pharmacologically active ingredients from Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), were applied in the differentiation media for human neural stem cells (hNSCs), together with brain-derived neurotrophic factor (BDNF), a commonly used compound for neural stem cell (NSC) differentiation.

Characterization of micropatterned nanofibrous scaffolds for neural network activity readout for high-throughput screening.

Micropatterns were fabricated in nanofibrous poly-L-lactic acid (PLLA) films by laser micromachining and the resulting scaffolds were characterized with respect to architecture, thermal, mechanical, and mass transport properties. Also, human neural stem cells were successfully cultured in these micropatterned nanofibrous scaffolds (MNFSs). The scaffolds were incorporated in high-density well plates (e.

Effects of topography on the functional development of human neural progenitor cells.

We have fabricated a topographical substrate with a packed polystyrene bead array for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). Human neural progenitor cells (H945RB.3) cultured on both flat and topographical substrates were analyzed in terms of morphological spreading, neuronal commitment, resting membrane potential (V(m)) establishment and VGCC function development.

Exploring cellular adhesion and differentiation in a micro-/nano-hybrid polymer scaffold.

Polymer scaffolds play an important role in three dimensional (3-D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano-fibrous and micro-porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro-scale pores and nano-scale fibers.

Three-dimensional polymer scaffolds for high throughput cell-based assay systems.

Many whole cell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. In this study, a three-dimensional (3D) cell-based assay platform was established by integrating 3D synthetic polymer scaffolds with standard cell culture dishes and multi-well plates. This technology can be used to feasibly modify any traditional 2D cell-based assay vessels for 3D cell-based assay with currently used high throughput screening (HTS) systems.