Pranav Joshi

High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology.

Biocompatible Hydrogels for Microarray Cell Printing and Encapsulation

Conventional drug screening processes are a time-consuming and expensive endeavor, but highly rewarding when they are successful. To identify promising lead compounds, millions of compounds are traditionally screened against therapeutic targets on human cells grown on the surface of 96-wells. These two-dimensional (2D) cell monolayers are physiologically irrelevant, thus, often providing false-positive or false-negative results, when compared to cells grown in three-dimensional (3D) structures such as hydrogel droplets. However, 3D cell culture systems are not easily amenable to high-throughput screening (HTS), thus inherently low throughput, and requiring relatively large volume for cell-based assays. In addition, it is difficult to control cellular microenvironments and hard to obtain reliable cell images due to focus position and transparency issues. To overcome these problems, miniaturized 3D cell cultures in hydrogels were developed via cell printing techniques where cell spots in hydrogels can be arrayed on the surface of glass slides or plastic chips by microarray spotters and cultured in growth media to form cells encapsulated 3D droplets for various cell-based assays. These approaches can dramatically reduce assay volume, provide accurate control over cellular microenvironments, and allow us to obtain clear 3D cell images for high-content imaging (HCI). In this review, several hydrogels that are compatible to microarray printing robots are discussed for miniaturized 3D cell cultures.

Chapter 3:High-throughput Screening of Toxic Chemicals on Neural Stem Cells

Exposure to environmental toxicants such as heavy metals, pesticides, and nanoparticles poses a severe threat to both the developing and the adult human brain, causing various neurodegenerative disorders. Detection and quantification of neurotoxicity induced by such toxicants represent a major challenge due to the complexity of neuronal pathways involved and a lack of understanding of mechanistic actions of toxicants in vivo. While the role of neural stem cells (NSCs) in this process is becoming increasingly evident, outcomes from conventional in vitro assays explored thus far are curtailed by the relative high-cost and low throughput (number of bioassays per time), physiologically-irrelevant 2D cell cultures, and unavailability of the requisite cell populations. This chapter aims to highlight the various mechanisms involved in neurotoxicity and neuronal differentiation of NSCs, and summarizes various in vitro assays currently being used for the detection of neurotoxicity along with their limitations.

Deconvolution of images from 3D printed cells in layers on a chip

Layer‐by‐layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D‐printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries. With epifluorescence microscopy, the throughput can be maintained at a cost of blurred cell images from printed tissue constructs. To rapidly acquire in‐focus cell images from bioprinted tissues using an epifluorescence microscope, we created two layers of Hep3B human hepatoma cells by printing green and red fluorescently labeled Hep3B cells encapsulated in two alginate layers in a microwell chip. In‐focus fluorescent cell images were obtained in high throughput using an automated epifluorescence microscopy coupled with image analysis algorithms, including three deconvolution methods in combination with three kernel estimation methods, generating a total of nine deconvolution paths. As a result, a combination of Inter‐Level Intra‐Level Deconvolution (ILILD) algorithm and Richardson‐Lucy (RL) kernel estimation proved to be highly useful in bringing out‐of‐focus cell images into focus, thus rapidly yielding more sensitive and accurate fluorescence reading from the cells in different layers.

High-Content Image Analysis

High-content imaging (HCI) and image processing of cells grown in 3D pose a significant challenge because 3D cells are not grown in a single focal plane, and the cell culture systems are often incompatible with traditional microscopes. Confocal microscopy is an important tool for imaging 3D cells due to its ability to acquire high definition images at various optical sections. However, the low scanning rate and depth induce low throughput in image acquisition and also incur additional problems such as photobleaching or phototoxicity [1–3]. To alleviate these issues, miniaturized 3D cell cultures on a micropillar/microwell chip platform have been demonstrated, which facilitate high-throughput spheroid cultures in hydrogels while offering better imaging capabilities. The whole sample depth can fit within the focus depth of a normal objective lens due to the small dimensions.

3D-cultured neural stem cell microarrays on a micropillar chip for high-throughput developmental neurotoxicology

&NA; Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high‐throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell‐specific biomarkers, which is often time‐consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three‐dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high‐throughput promoter‐reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self‐renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self‐renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC‐specific biomarkers including sex determining region Y‐box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter‐reporter assay system. HighlightsOptimum cell viability and spheroid formation observed in 3D cultured NSC microarrays.Lentiviruses constructed for measuring the expression levels of NSC biomarkers.Differentiation of ReNcell VM assessed after growth factor removal and with additives.High‐throughput assessment of self‐renewal and differentiation demonstrated in 3D cell culture.

High-Content Cell Staining

Fluorescence-based cell imaging is an important technology for analyzing various biological processes at cellular and molecular levels [1]. Morphological and functional features in a cell can be labeled with multiple fluorescent probes/reagents, imaged with automated fluorescence microscopes, and quantified with image analysis algorithms [2]. In particular, high-content imaging (HCI) has gained popularity for systematic and accurate evaluation of drug candidates [3, 4] because of its capability to assess specific signals, including changes in the nucleus, organelle structure, protein translocation, oxidative stress, apoptosis/necrosis, mitochondrial impairment, calcium homeostasis, morphology, and phenotype profiling as readouts [2, 4]. Three different types of cell labeling are typically used for HCI assays, which include fluorescent dyes for direct cell staining, antibodies for immunofluorescent labeling, and genetically expressed fluorescent proteins such as green fluorescent protein (GFP) [1, 5]. This chapter summarizes basic cell staining protocols with various fluorescent dyes and antibodies for HCI assays on the micropillar/microwell chip platform.

3D-Cultured Cell Image Acquisition

Acquiring high-content images of 3D-cultured cells for analyzing multiple cellular events is a daunting task, requiring an automated fluorescent microscope and high-throughput image analysis software. High throughput is an important feature for high-content imaging (HCI) devices to enable rapid image acquisition. Various factors come into play when dealing with the speed of image acquisition. For example, capturing large number of cells with lower magnification or reducing sample volume/size can increase data acquisition speed. In addition, reducing exposure time with the use of high intensity light sources, optimizing fluorescence staining protocols for brighter colors, and using an objective lens with relatively high numerical aperture also significantly increases the image acquisition speed [1].