Dataset Information

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D.

ABSTRACT: OBJECTIVE:Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D. APPROACH:We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy. MAIN RESULTS:We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. SIGNIFICANCE:Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

SUBMITTER: Ren Y

PROVIDER: S-EPMC6040821 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D.

Ren Yuan Y Mlodzianoski Michael J MJ Lee Aih Cheun AC Huang Fang F Suter Daniel M DM

Journal of neural engineering 20180124 3

<h4>Objective</h4>Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devi ...[more]

PMID: 29363623

Similar Datasets

Project description:Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.

Project description:Background:Advances in 3D shape capture technology have made powerful shape analyses, such as geometric morphometrics, more feasible. While the highly accurate micro-computed tomography (µCT) scanners have been the "gold standard," recent improvements in 3D surface scanners may make this technology a faster, portable, and cost-effective alternative. Several studies have already compared the two devices but all use relatively large specimens such as human crania. Here we perform shape analyses on Australia's smallest rodent to test whether a 3D scanner produces similar results to a µCT scanner. Methods:We captured 19 delicate mouse (Pseudomys delicatulus) crania with a µCT scanner and a 3D scanner for geometric morphometrics. We ran multiple Procrustes ANOVAs to test how variation due to scan device compared to other sources such as biologically relevant variation and operator error. We quantified operator error as levels of variation and repeatability. Further, we tested if the two devices performed differently at classifying individuals based on sexual dimorphism. Finally, we inspected scatterplots of principal component analysis (PCA) scores for non-random patterns. Results:In all Procrustes ANOVAs, regardless of factors included, differences between individuals contributed the most to total variation. The PCA plots reflect this in how the individuals are dispersed. Including only the symmetric component of shape increased the biological signal relative to variation due to device and due to error. 3D scans showed a higher level of operator error as evidenced by a greater spread of their replicates on the PCA, a higher level of multivariate variation, and a lower repeatability score. However, the 3D scan and µCT scan datasets performed identically in classifying individuals based on intra-specific patterns of sexual dimorphism. Discussion:Compared to µCT scans, we find that even low resolution 3D scans of very small specimens are sufficiently accurate to classify intra-specific differences. We also make three recommendations for best use of low resolution data. First, we recommend that extreme caution should be taken when analyzing the asymmetric component of shape variation. Second, using 3D scans generates more random error due to increased landmarking difficulty, therefore users should be conservative in landmark choice and avoid multiple operators. Third, using 3D scans introduces a source of systematic error relative to µCT scans, therefore we recommend not combining them when possible, especially in studies expecting little biological variation. Our findings support increased use of low resolution 3D scans for most morphological studies; they are likely also applicable to low resolution scans of large specimens made in a medical CT scanner. As most vertebrates are relatively small, we anticipate our results will bolster more researchers in designing affordable large scale studies on small specimens with 3D surface scanners.

Dataset Information

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D.

Publications

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets