Browsing by Author "Wu, Ze-Zhi"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
Item A microwell pattern for C17.2 cell aggregate formation with concave cylindrical surface induced cell peeling(Biomaterials, 2014) Zhang, Li-Guang; Zhong, Dong-Huo; Zhang, Yiguo; Li, Chen-Zhong; Kisaalita, William S.; Wu, Ze-ZhiWe 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 platformItem Effects of Topography on the Functional Development of Human Neural Progenitor Cells(Biotechnology and bioengineering, 2010) Wu, Ze-Zhi; Kisaalita, William S.; Wang, Lina; Zachman, Angela L.; Zhao, Yiping; Hasneen, Kowser; Machacek, Dave; Stice, Steven L.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 (Vm) establishment and VGCC function development. We found, by SEM imaging, that arrayed substrates, formed with both sub‐micrometer (of 0.51 µm in mean diameter) and micrometer (of 1.98 µm in mean diameter) beads, were capable of promoting the spreading of the progenitor cells as compared with the flat polystyrene surfaces. With the micrometer beads, it was found that arrayed substrates facilitated the neural progenitor cells' maintenance of less negative Vm values upon differentiation with bFGF starvation, which favored predominant neuronal commitment. Almost all the progenitor cells were responsive to 50 mM K+ depolarization with an increase in [Ca2+]i either before or upon differentiation, suggesting the expression of functional VGCCs. Compared to the flat polystyrene surfaces, microbead arrayed substrates facilitated the development of higher VGCC responsiveness by the progenitor cells upon differentiation. The enhancement of both VGCC responsiveness and cell spreading by arrays of micrometer beads was most significant on day 14 into differentiation, which was the latest time point of measurement in this study. This study thus rationalized the possibility for future substrate topography engineering to manipulate ion channel function and to meet the challenge of low VGCC responsiveness found in early drug discovery. Biotechnol. Bioeng. 2010;106: 649–659. © 2010 Wiley Periodicals, Inc.Item Interfacing SH-SY5Y human neuroblastoma cells with SU-8 microstructures(Colloids and Surfaces B: Biointerfaces, 2006) Wu, Ze-Zhi; Zhao, Yiping; Kisaalita, William S.Microwell structures were fabricated using SU-8 photoresist for engineering a quasi-three-dimensional (quasi-3D) microenvironment for cultured neuronal cells. SH-SY5Y human neuroblastoma cells were successfully integrated into microwells of a nominal diameter of 100 μm, with or without 10-μm wide microchannels connecting neighboring microwells, in an aspect ratio (ratio of structure depth over width) of approximately 1. With the help of polyethylene glycol stamping and laminin coating, a neuronal-like network was achieved by integrating populations of SH-SY5Y cells with a microwell network pattern. Resting membrane potential establishment was evaluated with confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester. It was found that the intra/extracellular fluorescent intensity ratio (R) was 2.4 ± 1.4 [n (number of cells measured) = 112] for SH-SY5Y cells on flat SU-8 substrates on day 5 into differentiation, which was not significantly different from the ratio on day 13 into differentiation, 2.0 ± 1.8 (n = 104) (P > 0.05). For cells in the microwell network structures, R was 4.8 ± 4.7 (n = 51) and 3.9 ± 3.2 (n = 62) on days 5 and 13 into differentiation, respectively (P > 0.5). Cells within the network structures had higher R ratios than on flat substrates, for either day 5 or 13 into differentiation (P < 0.01). These results demonstrated that the well network structures, or topographically patterned substrates, were more suitable formats for promoting SH-SY5Y cell resting membrane potential establishment than flat substrates, suggesting the potential to control cellular function through substrate topography engineering.Item Micro and Nanofabrication : Is a Practical Three-Dimensional Cell Culture Platform for Drug Discovery Achievable?(International Journal of Medical Nano Research, 2015) Wu, Ze-Zhi; Kisaalita, William S.Direct and indirect evidence, in support of the notion that biological activity of three-dimensional (3-D) cultures may more closely mirror what happens in vivo, has appeared in the literature for the past three decades. This is probably best exemplified in the field of experimental oncology that adopts 3-D multicellular tumor spheroids (MCTS) to mimic the in vivo situation. Early seminal work in this section was represented by Sutherland and colleagues who used MCTS as an in vitro model for systematic studies on tumor cell responses to therapeutic treatments [1]. Multicellular spheroids have been able to fully recapitulate the multicellular mediated drug resistance of EMT6 tumors, which was inherently induced in vivo but completely lost when cancer cells were dissociated and cultured as monolayers. It has been shown that the phenotypic transformation of malignant cells in a 3-D collagen gel configuration is achievable upon treatment with integrin antibodies, while this has never been possible in monolayer cultures. Researchers have also noted that HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells showed protease independent amoeboid movement within 3-D collagen matrix while, in 2-D cultures, this movement is totally dependent on proteases like matrix metalloproteinases. This challenges the traditional screening for anti-metastatic agents against proteolytic activity with 2-D monolayer cultures. In fact, Mueller-Klieser [2] has actually proposed that 3-D spheroids should become mandatory test systems in cancer therapeutic screening programs.Item Microstructured Topography Enhanced the Responsiveness of Voltage-gated Calcium Channels in H945RB.3 Human Neural Progenitor Cells(International Conference on Bioinformatics and Biomedical Engineering, 2009) Wu, Ze-Zhi; Kisaalita, William S.; Wang, Lina; Zhao, YipingA topographical substrate with a packed polystyrene microbead (1.98 plusmn 0.20 mum in diameter) array was fabricated for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). We found that the microbead arrayed substrates enhanced the attachment and spreading of the cultured human neural progenitor cells (H945RB.3) as compared to the flat polystyrene surfaces. Microbead arrayed substrates also facilitated the development of higher VGCC responsiveness upon neuronal differentiation than flat substrates. The enhancement of both VGCC responsiveness and cell spreading were most significant until day 14 into differentiation.Item Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)(International Journal of Nanomedicine,, 2013) Wu, Ze-Zhi; Wang, Zheng-Wei; Zhang, Li-Guang; An, Zhi-Xing; Zhong, Dong-Huo; Huang, Qi-Ping; Luo, Mei-Rong; Liao, Yan-Jian; Jin, Liang; Li, Chen-Zhong; Kisaalita, William S.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).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.Item SU-8 microstructure for quasi-three-dimensional cell-based biosensing(Sensors and Actuators B: Chemical, 2009) Wang, Lina; Wu, Ze-Zhi; Xub, Bingqian; Zhaoc, Yiping; Kisaalita, William S.A quasi-three-dimensional (quasi-3-D) cell-based biosensor platform microfabricated from SU-8 has been developed and characterized. In this work, SH-SY5Y human neuroblastoma cells were integrated with SU-8 microfabricated microwells with diameters of 100μm. SH-SY5Y cells were differentiated with 1mM dibutyryl cAMP and 2.5μM 5-bromodeoxyuridine. Voltage-gated calcium channel (VGCC) function of SH-SY5Y cells cultured within the microwells (quasi-3-D) versus those cultured on the SU-8 planar substrates (2-D) was evaluated by confocal microscopy with a calcium fluorescent indicator, Calcium Green-1. In response to 50mM high K+ depolarization, cells in microwells were less responsive in terms of increase in intracellular Ca2+ in comparison to cells on 2-D substrates. This study shows that VGCC function of cells within SU-8 microwells was indeed different from that of cells on planar SU-8 surfaces, suggesting that SU-8 microstructure did affect SH-SY5Y cell differentiation with respect to VGCC function and that high-aspect-ratio microstructures are not merely “folded” 2-D structures. Furthermore, these results are consistent with previous 2-D/3-D comparative studies carried out in polymer scaffolds and support the hypothesis that cell calcium dynamics on 2-D substrates may be exaggerated. Overall, this work is supportive of SU-8 micropattern as a viable platform for engineering a quasi-3-D cell culture system for cell-based biosensing against drugs for VGCCs.