William S. Kisaalita

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.

Glutamate‐induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium‐dependent pathways and the microtubule cytoskeleton

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium-dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate-induced changes in the pattern of dendrite outgrowth. We used Fura-2 AM and inhibitors of the calcium-dependent proteins, calmodulin and calpain, to identify the role of specific calcium-dependent pathways in glutamate-regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate-induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca(2+)](i)) changes in a biphasic manner over a 12-h period in the presence of glutamate. A transient increase in [Ca(2+)](i) over the first hour of glutamate exposure correlated with a calmodulin-associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca(2+)](i) was correlated with calpain-associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin-associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain-associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity-dependent regulation of dendrite morphology during development and after pathology.

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.

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. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later.

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.

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. 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.

Three Dimensional Neuronal Cell Cultures More Accurately Model Voltage Gated Calcium Channel Functionality in Freshly Dissected Nerve Tissue

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells’ functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.

Determination of Resting Membrane Potential of Individual Neuroblastoma Cells (IMR-32) Using a Potentiometric Dye (TMRM) and Confocal Microscopy

The potentiometric dye, Tetramethylrhodamine methyl ester (TMRM) has been extensively used with fluorometry or optical microscopy to evaluate the electric potential across plasma or mitochondrial membranes. We present here a TMRM confocal microscopy-based potential measurement technique. Corrections are introduced to minimize nonspecific dye binding and insensitivity to low background levels. We have used this technique to compare the resting membrane potential of proliferating and differentiated human neuroblastoma cells (IMR-32).

GABAA receptor currents recorded from Müller glial cells of the baboon (Papio cynocephalus) retina

The effect of gamma-aminobutyric acid (GABA) application on acutely isolated, non-cultivated Muller glial cells from the baboon retina was studied using the whole-cell voltage-clamp technique. Application of GABA (0.1 mM) generated inward currents at a holding potential of -80 mV as well as an increase in current noise. The GABA-activated current had a reversal potential of 18.6 mV and was therefore supposed to be a Cl- current (ECl = 5 mV). The GABAA receptor agonist muscimol (0.1 mM) elicited an inward current and bicucullin (0.5 mM), a blocker of the GABAA receptor, diminished the GABA responses in our experiments completely. Baclofen (0.1 mM), a GABAB agonist, neither had an effect when applied under conditions where the dominant Muller cell K+ currents were unblocked, nor when the K+ currents were blocked by application of Ba2+ (1 mM). Glycine (0.1 mM) was ineffective as well. From these results we conclude that the baboon retinal Muller cells possess GABAA receptors. However, these have recently been discovered on skate Muller cells whereas GABAA receptors could not be found on Muller cells of guinea pig, pig, mouse, rat and rabbit.

Effects of 60 Hz electromagnetic field exposure on APP695 transcription levels in differentiating human neuroblastoma cells

Epidemiological studies have suggested that workers with primary occupation that are likely to have resulted in the medium-to-high extremely low frequency (ELF) electromagnetic field (EMF) exposure are at increased risk of Alzheimers disease (AD) pathogenesis. As a first step in investigating the possibility of an association between the ELF-EMF exposure and AD at the cellular level, we have used the differentiating IMR-32 neuroblastoma cells. In double-blind experiments, IMR-32 cells were exposed to the magnetic field intensities of 50, 100, and 200 microT at a frequency of 60 Hz for a period of 4 h at the three ages of differentiation (2, 10, and 16 days after incubation in differentiation medium). We used a custom-made Helmholtz coil setup driven by a 60-Hz sinusoidal signal from a function generator and an in-house built power amplifier. Total RNA extracted from the exposed cells was separated by the agarose gel electrophoresis and transferred to a nylon membrane for the northern hybridization. Digoxygenin-labeled APP695 RNA probes were used to detect changes in the APP695 mRNA levels in response to the ELF-EMF exposure. The results reported herein provided no support for any relationship between the APP695 gene transcription and IMR-32 differentiation age, as well as the magnetic field exposure. This study constitutes the first step towards investigating the possibility of an association between the ELF-EMF exposure and AD manifestations at the cellular level.