The influence of adherent cell morphology on hydrodynamic recruitment of leukocytes.

Innate immunity is characterized by the coordinated activity of multiple leukocytes mobilizing at or near the site of tissue injury. Slow rolling and/or adherent leukocytes have been shown to hydrodynamically recruit free-stream leukocytes to a model of inflamed tissue. In this paper, we numerically investigate the hydrodynamic recruitment of free-stream leukocytes due to the presence of a nearby adherent, deformed leukocyte by using a computational model developed from first principles to simulate these types of interactions. For free-stream cells at least one diameter above the surface and subsequently involved in a glancing (out-of-plane) collision with one or more adherent cell, the simulation indicated that the free-stream cell was driven closer to the surface as a function of increasing glancing distance. Further, with increasing deformation of the adherent cell a similar effect was observed beginning at smaller glancing offsets. The influence of binary interactions on the trajectories of free-stream cells that were less than one diameter above the surface was also examined. For fixed glancing distance, increased adherent cell deformation led to enhanced recruiting effectiveness which was quantified by determining the time needed for the free-stream cell to enter the reactive zone; that is, a membrane separation distance such that receptor-ligand binding was possible. This effectiveness was only moderately influenced by variations in shear rate and cell buoyancy. Finally, for large glancing offset the domain of influence of the adherent cell diminished and the trajectory of the free-stream cell was unaffected by the adherent cell, with regard to hydrodynamic recruitment.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

Cryopreservation of hepatocyte (HepG2) cell monolayers: impact of trehalose.

A simple method to cryogenically preserve hepatocyte monolayers is currently not available but such a technique would facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. We investigated the effect of trehalose and dimethyl sulfoxide (Me2SO) in cryopreservation of human hepatocellular carcinoma (HepG2) cells in suspension and monolayer formats. HepG2 cell monolayers were incubated for 24h at varying concentrations of trehalose (50-150 mM) prior to cryopreservation to identify the optimum concentration for such preincubation. When trehalose alone was used as the cryoprotective agent (CPA), cells in monolayer format did not survive freezing while cells in suspension demonstrated 14% viability 24h after thawing. Only 6-13% of cells in monolayers survived freezing in cell culture medium supplemented with 10% Me2SO, but 42% of cells were recovered successfully if monolayers were preincubated with 100 mM trehalose prior to freezing in the Me2SO supplemented medium. Interestingly, for cells frozen in suspension in presence of 10% Me2SO, metabolic activity immediately following thawing did not change appreciably compared to unfrozen control cells. Finally, Raman spectroscopy techniques were employed to evaluate ice crystallization in the presence and absence of trehalose in freezing solutions without cells because crystallization may alter the extent of injury observed in cell monolayers. We speculate that biomimetic approaches of using protective sugars to preserve cells in monolayer format will facilitate the development of techniques for long-term preservation of human tissues and organs in the future.

High-Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures.

3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid-like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light-sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high-throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min-1 . The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.

Culture of Adherent Cancer Cell Lines.

Adherent cell lines grow attached to the surface of a cell culture vessel. Due to the adherent nature of the cells, enzymes, such as trypsin, are required to lift the cells from the cell culture vessel for harvesting or subculturing. Many cancer cell lines are adherent, rendering adherent cell culture a critical experimental method in the fields of cell biology, biochemistry, and cancer research. In this chapter, we outline the protocols for culturing and maintaining adherent cells. We detail the procedures for preparing cell culture medium, thawing and reviving frozen adherent cells, subculturing adherent cells, freezing cells, and counting cells. Most notably, we outline the best techniques and practices for optimal growth of healthy adherent cells while diminishing the risk of contamination.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Development of non-adherent cell-enclosing domes with enzymatically cross-linked hydrogel shell.

Non-adherent cells, such as hematopoietic cells and lymphocytes, are important research subjects in medical and biological fields. Therefore, a system that enables the handling of non-adherent cells in solutions in the same manner as that of adhering cells during medium exchange, exposure to chemicals, washing, and staining in imaging applications would be useful. Here, we report a 'Cell Dome' platform in which non-adherent cells can be enclosed and grown in the cavities of about 1 mm diameter and 270µm height. The domes consist of an alginate-based hydrogel shell of 90µm thickness. Cell Domes were formed on glass plates by horseradish peroxidase-mediated cross-linking. Human leukaemia cell line K562 cells enclosed in Cell Domes were stable for 29 days with every 2-3 days of medium change. The enclosed cells grew in the cavities and were stained and differentiated with reagents supplied from the surrounding medium. Additionally, K562 cells that filled the cavities (a 3D microenvironment) were more hypoxic and highly resistant to mitomycin C than those cultured in 2D. These findings demonstrate that the 'Cell Dome' may be a promising tool for conveniently culturing and evaluating non-adherent cells.

Effective Capacitance from Equivalent Electrical Circuit as a Tool for Monitoring Non-Adherent Cell Suspensions at Low Frequencies.

Analyzing the electrical double layer (EDL) in electrical impedance spectroscopy (EIS) measurement at low frequencies remains a challenging task for sensing purposes. In this work, we propose two approaches to deal with the EDL in measuring impedance for particles and non-adherent cells in an electrolytic suspension. The first approach is a simple procedure to compute a normalized electrical impedance spectrum named dispersed medium index (DMi). The second is the EIS modeling through an equivalent electric circuit based on the so-called effective capacitance (Cef), which unifies the EDL phenomena. Firstly, as an experiment under controlled conditions, we examine polymer particles of 6, 15, and 48 µm in diameter suspended in a 0.9% sodium chloride solution. Subsequently, we used K-562 cells and leukocytes suspended in a culture medium (RPMI-1640 supplemented) for a biological assay. As the main result, the DMi is a function of the particle concentration. In addition, it shows a tendency with the particle size; regardless, it is limited to a volume fraction of 0.03 × 10-4 to 58 × 10-4. The DMi is not significantly different between K-562 cells and leukocytes for most concentrations. On the other hand, the Cef exhibits high applicability to retrieve a function that describes the concentration for each particle size, the K-562 cells, and leukocytes. The Cef also shows a tendency with the particle size without limitation within the range tested, and it allows distinction between the K-562 and leukocytes in the 25 cells/µL to 400 cells/µL range. We achieved a simple method for determining an Cef by unifying the parameters of an equivalent electrical circuit from data obtained with a conventional potentiostat. This simple approach is affordable for characterizing the population of non-adherent cells suspended in a cell culture medium.

A bioimaging system combining human cultured reporter cells and planar chromatography to identify novel bioactive molecules.

For the first time, a human cancer cell line was shown to grow and be functionally active on the particulate porous adsorbent surface of separated sample mixtures. This allowed the novel combination of chromatographic separations with human cells as biological detector. As exemplary screening for cancer treatment drugs, cytotoxic substances were directly discovered in Saussurea costus and ginseng samples using the Cytotox CALUX® osteosarcoma cells (with luciferase expressing reporter gene) as detector. In addition, rosiglitazone and pioglitazone were detected as luminescent zones upon binding to the PPARγ receptor expressed in the respective CALUX cell line that was grown on the surface of the adsorbent. This demonstrates the ability to address receptor-mediated signaling with this method, and opens the perspective to use our novel bioimaging method to identify bioactive molecules targeting a wide range of pathways with toxicological, pharmaceutical and nutraceutical relevance. The new bioimaging directly pointed to individual effective compounds in multi-component mixtures. Furthermore, discovered effective compounds were directly characterized by online elution to high-resolution mass spectrometry and fragmentation.

Functional Pathway Identification With CRISPR/Cas9 Genome-wide Gene Disruption in Human Dopaminergic Neuronal Cells Following Chronic Treatment With Dieldrin.

Organochlorine pesticides, once widely used, are extremely persistent and bio-accumulative in the environment. Epidemiological studies have implicated that environmental exposure to organochlorine pesticides including dieldrin is a risk factor for the development of Parkinson's disease. However, the pertinent mechanisms of action remain poorly understood. In this study, we carried out a genome-wide (Brunello library, 19 114 genes, 76 411 sgRNAs) CRISPR/Cas9 screen in human dopaminergic SH-SY5Y neuronal cells exposed to a chronic treatment (30 days) with dieldrin to identify cellular pathways that are functionally related to the chronic cellular toxicity. Our results indicate that dieldrin toxicity was enhanced by gene disruption of specific components of the ubiquitin proteasome system as well as, surprisingly, the protein degradation pathways previously implicated in inherited forms of Parkinson's disease, centered on Parkin. In addition, disruption of regulatory components of the mTOR pathway which integrates cellular responses to both intra- and extracellular signals and is a central regulator for cell metabolism, growth, proliferation, and survival, led to increased sensitivity to dieldrin-induced cellular toxicity. This study is one of the first to apply a genome-wide CRISPR/Cas9-based functional gene disruption screening approach in an adherent neuronal cell line to globally decipher cellular mechanisms that contribute to environmental toxicant-induced neurotoxicity and provides novel insight into the dopaminergic neurotoxicity associated with chronic exposure to dieldrin.

Scale-Up Technologies for the Manufacture of Adherent Cells.

Great importance is being given to the impact our food supply chain and consumers' food habits are having on the environment, human health, and animal welfare. One of the latest developments aiming at positively changing the food ecosystem is represented by cultured meat. This form of cellular agriculture has the objective to generate slaughter-free meat products starting from the cultivation of few cells harvested from the animal tissue of interest. As a consequence, a large number of cells has to be generated at a reasonable cost. Just to give an idea of the scale, there were billions of cells just in a bite of the first cultured-meat burger. Thus, one of the major challenges faced by the scientists involved in this new ambitious and fascinating field, is how to efficiently scale-up cell manufacture. Considering the great potential presented by cultured meat, audiences from different backgrounds are very interested in this topic and eager to be informed of the challenges and possible solutions in this area. In light of this, we will provide an overview of the main existing bioprocessing technologies used to scale-up adherent cells at a small and large scale. Thus, giving a brief technical description of these bioprocesses, with the main associated advantages and disadvantages. Moreover, we will introduce an alternative solution we believe has the potential to revolutionize the way adherent cells are grown, helping cultured meat become a reality.

Rapid quantification of cellular proliferation and migration using ImageJ.

Cellular proliferation and migration are crucial during development, regeneration and disease. Methods to quantify these processes are available; however, many are time consuming and require specialized equipment and costly reagents. Simple cell counts (proliferation analysis) and the scratch assay (migration analysis) are favorable methods due to their simplicity and cost-effectiveness; however, they rely on subjective and labor-intensive manual analysis, resulting in low throughput. We have developed optimized protocols to rapidly and accurately quantify adherent cell number and wound area using ImageJ, an open-source image processing program. Notably, these adaptable protocols facilitate quantification with significantly greater accuracy than manual identification.

Quantification of Carbon Nanotube Doses in Adherent Cell Culture Assays Using UV-VIS-NIR Spectroscopy.

The overt hazard of carbon nanotubes (CNTs) is often assessed using in vitro methods, but determining a dose-response relationship is still a challenge due to the analytical difficulty of quantifying the dose delivered to cells. An approach to accurately quantify CNT doses for submerged in vitro adherent cell culture systems using UV-VIS-near-infrared (NIR) spectroscopy is provided here. Two types of multi-walled CNTs (MWCNTs), Mitsui-7 and Nanocyl, which are dispersed in protein rich cell culture media, are studied as tested materials. Post 48 h of CNT incubation, the cellular fractions are subjected to microwave-assisted acid digestion/oxidation treatment, which eliminates biological matrix interference and improves CNT colloidal stability. The retrieved oxidized CNTs are analyzed and quantified using UV-VIS-NIR spectroscopy. In vitro imaging and quantification data in the presence of human lung epithelial cells (A549) confirm that up to 85% of Mitsui-7 and 48% for Nanocyl sediment interact (either through internalization or adherence) with cells during the 48 h of incubation. This finding is further confirmed using a sedimentation approach to estimate the delivered dose by measuring the depletion profile of the CNTs.

A solution to prevent secondary flow in adherent cell cultures.

High quality cell cultures require reliable laboratory practices. Today's small-scale in vitro cell culture format is dominated by circular topology vessels, with the inherent disadvantage of secondary flow induced each time the cell cultures are repositioned. The secondary flow generates uneven sedimentation and adherence that negatively impacts cell culture quality. Here we show a modification of the circular culture vessel that abrogates these disturbances. Cell culture wells were augmented with a central column to diminish secondary flow. Human carcinoma cell lines (BeWo, JEG-3), mesenchymal stem cells [human umbilical cord perivascular cells (HUCPVC)] and mouse embryonic fibroblasts (MEF) were cultured in both column-augmented and regular culture wells. Human carcinoma cell cultures showed even cell densities and significantly more viable cells in column-augmented vessels. In FTM HUCPVC cultures, cell surface MSC marker (CD90, CD105) expression and cell differentiation-related gene expression patterns were significantly more homogeneous in column-augmented vessels. MEF cells in column-augmented culture vessels showed a more consistent expression of IGF-1. Column-augmented cell culture vessels significantly improve the homogeneity of adherent cell cultures by mitigating the adverse effect of the secondary flow.This article has an associated First Person interview with the first author of the paper.

Measuring the Nutrient Metabolism of Adherent Cells in Culture.

Metabolite extraction from cells cultured in vitro enables the comprehensive measurement of intracellular metabolites. These extracts can be analyzed using techniques such as liquid chromatography-mass spectrometry (LC-MS). This chapter describes in detail a method for metabolite extraction from cultured adherent mammalian cells to collect both polar and nonpolar intracellular metabolites. This chapter also describes experimental design considerations for performing stable isotope labeling experiments, and the use of chemical derivatization to increase the number of compounds that can be detected using one chromatography method.

In Vitro Cytogenetic Assays: Chromosomal Aberrations and Micronucleus Tests.

Chromosome damage is a very important indicator of genetic damage relevant to environmental and clinical studies. Detailed descriptions of the protocols used for detection of chromosomal aberrations induced by genotoxic agents in vitro both in the presence or absence of rat liver-derived metabolizing systems are given in this chapter. Structural chromosomal aberrations that can be observed and quantified at metaphases are described here. For the detection of chromosomal damage (fragments or whole chromosome) in interphase, the micronucleus test can be used, and a description of this test is also presented. Criteria for determining a positive result using appropriate statistical methods are described.

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines.

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge-s-ARU-method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.

Single-Cell Ca2+ Imaging.

In rheumatological studies, visualization of Ca2+ dynamics in intact cells as direct experimental evidence of Ca2+-dependent signal pathways is generally used to monitor the function of immune cells at first glance. Ability to monitor Ca2+ signaling in living cells would greatly facilitate advances in the functional dissection of immune cells. In this chapter, we describe a basic technique and methods of data analysis for single-cell real-time Ca2+ monitoring using Fluo-4 labeling, which is a single-wavelength Ca2+ indicator.

CellNO trap: Novel device for quantitative, real-time, direct measurement of nitric oxide from cultured RAW 267.4 macrophages.

Nitric oxide (NO), is arguably one of the most important small signaling molecules in biological systems. It regulates various biological responses in both physiological and pathological conditions, often time producing seemingly contradictory results. The details of the effects of NO are highly dependent on the level of NO that cells experience and the temporal aspect of when and how long cells are exposed to NO. Herein, we present a novel measurement system (CellNO trap) that allows real-time NO measurement via chemiluminescence detection from general adhesive cultured cells using standard cell culture media and reagents that does not perturb the cells under investigation. Highly controlled light-initiated NO releasing polymer SNAP-PDMS was used to characterize and validate the quantitative data nature of the device. The NO generation profile from the macrophage cell-line RAW264.7 stimulated by 100ng/ml LPS and 10ng/ml IFN-γ was recorded. Measured maximum NO flux from RAW264.7 varied between around 2.5-9pmol/10(6)cell/s under 100ng/ml LPS and 10ng/ml IFN-γ stimulation, and 24h cumulative NO varied between 157 and 406 nmol/10(6)cell depending on different culture conditions, indicating the conventional report of an average flux or maximum flux is not sufficient to represent the dynamic characters of NO. LPS and IFN-γ's synergistic effect to RAW264.7 NO generation was also directly observed with the CellNO trap. The real-time effect on the NO generation from RAW264.7 following the addition of arginine, nor-NOHA and L-NAME to the cultured cells is presented. There is great potential to further our understanding of the role NO plays in normal and pathological conditions clearly understanding the dynamic production of NO in response to different stimuli and conditions; use of CellNO trap makes it possible to quantitatively determine the precise NO release profile generated from cells in a continuous and real-time manner with chemiluminescence detection.

Microchamber device for detection of transporter activity of adherent cells.

We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells over-expressing MDR1, an ATP-binding cassette transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level.