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Lai Y.,Cellular Bioengineering | Asthana A.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Drug Discovery Today | Year: 2011

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. © 2011 Elsevier Ltd.


Asthana A.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Drug Discovery Today | Year: 2016

'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. © 2016 Elsevier Ltd. All rights reserved.


Asthana A.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Drug Discovery Today | Year: 2016

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. © 2015 Elsevier Ltd. All rights reserved.


Asthana A.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Drug Discovery Today | Year: 2013

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. © 2012 Elsevier Ltd. All rights reserved.


Asthana A.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Drug Discovery Today | Year: 2012

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. © 2012 Elsevier Ltd.


Wang L.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Journal of Neuroscience Methods | Year: 2011

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. Cell locomotion and neurite extension were observed by time-lapse microscopy and analyzed by ImageJ software. The expression of synaptic formation was confirmed by immunostaining of synaptophysin (SYN) or/and the co-localization of synapsin I and microtubule associated protein-2 (MAP-2). Effects of cell density on neural differentiation were also examined. Results have shown that administration of BDNF/ginsenosides (Rg1 and Rb1) combination in differentiation medium promoted cell survival, enhanced neurite outgrowth and synaptic marker expression during differentiation. High cell density enhanced synaptic marker expression in BDNF/ginsenosides combination medium. In all, this study established a condition for hNSCs synaptic development in early differentiation, which is a crucial step in applying this cell line in neural network-based assay. © 2010 Elsevier B.V.


Gil V.,Cellular Bioengineering | Gil V.,University of Barcelona | Gil V.,CIBER ISCIII | Del Rio J.A.,Cellular Bioengineering | And 2 more authors.
Nature Protocols | Year: 2012

This protocol uses rat tail-derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail-derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks. © 2012 Nature America, Inc. All rights reserved.


Wang L.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Journal of Biomedical Materials Research - Part B Applied Biomaterials | Year: 2010

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.g., 96-well plates), creating a platform for high-throughput screening of drugs with physiologically more relevant networked neural cultures. Through mathematical modeling of the transport of model stimulants, the feasibility of stimulating neural networks cultured in MNFSs was demonstrated. More work is needed to establish biological network activity-MNFS architecture relationships. © 2010 Wiley Periodicals, Inc.


Zhao H.,Cellular Bioengineering | Schuck P.,Cellular Bioengineering
Acta Crystallographica Section D: Biological Crystallography | Year: 2015

Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design. © 2015.


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