Tuesday, October 23, 2018

There’s No Place Like Home, Even (and Especially) for a Cell

Traditional monolayer cell culture in plastic dishes has been the standard for many assays and high throughput screens for decades, in large part due to its ease and low cost. Unfortunately, established cell lines grown on flat, plastic surfaces cannot truly recapitulate the phenotypes, signaling, and microenvironment of their native tissue, leading many to question the relevance of scientific conclusions drawn from monolayer assays. In response, cell culture methods are increasingly moving toward improving our recreation of a cell’s “home” environment [1].

Cells in their native tissue are surrounded by other cells and a complex web of proteins that provide a tissue-specific stiffness optimized by evolutionary pressure over millennia. Cells are bathed in a biochemical cocktail of carefully balanced signaling molecules and nutrients that dynamically changes in response to the status and needs of the overall tissue. This microenvironment drives the cell to adopt a specific morphology and perform a specific task in the context of the cells and signals around it, ultimately cooperatively regulating complex biological functions. This is the environment that carefully directs development, balances health and disease, and dictates response to drugs.

By contrast, cells grown on tissue culture plastic are forced to adapt to growth against a rigid structure on one side and bathed in liquid media on the other. In this setting, most cells adopt a flat, strained morphology against the stiffness of the plastic, are less able to form traditional cell-cell junctions, and, as all cells in the culture are equally exposed to the same media for days, there is no dynamic biochemical microenvironment to direct specificity in phenotype or function. Everything from gene expression to proliferation to metabolism and drug sensitivity have been shown to be altered by monolayer culture as compared with in vivo measurements.

So how do we encourage cultured cells to behave as they do in the body? Three dimensional (3D) cell culture, pioneered in the 1980s, has matured over the past 3 decades to provide both manual and automated tools for accurate artificial modeling of physiologically relevant structures that can be adapted for both highly intricate low throughput investigations and improved accuracy for high throughput drug screening. 3D methods, though not perfect, more accurately reproduce in vivo physiological conditions resulting in cells that respond to experimental stimuli with better correlation to clinical expectations. 3D culture methods can be grouped into 3 categories of differing complexity and physiological relevance:

SUSPENSION CULTURE: The simplest design for creating a 3D cell culture model is to plate the cells in a low-adherence environment or hanging-drop assay and allow them to self-assemble into aggregates. Cells cultured in this way will secrete their own extracellular matrix and form appropriate cell-cell junctions and interactions. Aggregates may be tight (to form spheroids) or loose (looking instead like clusters) depending upon the characteristics and needs of the cells themselves.
Benefits: Suspension culture can maintain the ease and low cost of traditional monolayer culture, making it suitable and scalable for high throughput applications.
Drawbacks: Cells in aggregates will not fully model the desired tissue, but can mimic some aspects of the cell’s home environment. Additionally, cell aggregates are floating and thus for imaging applications may pose some obstacles with excess movement and high variability of their focal plane.
When using a BioTek Lionheart or Cytation imager, increase the delay after stage movement to account for the floating aggregates and try acquiring a z-stack with a large step size in order to capture a broader depth of cells.
Lionheart FX
Lionheart FX Automated Microscope
  • SCAFFOLD CULTURE: Hydrogels, electrospun membranes, bioprinted scaffolds, or other matrices can be artificially engineered to provide additional structure and biochemical recapitulation of the natural tissue environment. Cells can be cultured on or embedded within the matrix, which can also be loaded with nutrients, growth factors, drugs, or other signaling compounds, depending upon the needs of the assay or the requirements of the cell type.
    Benefits: Physical characteristics of the scaffold can be optimized to more accurately model the cell’s native influences. By promoting appropriate stiffness, cell-cell interactions, and cell-matrix interactions, scaffold-based 3D cell culture can drive organotypic gene expression and cellular organization that more accurately mimics activity in the body.
    Drawbacks: Scaffold or matrix-based 3D culture can be costly in both time and money, making it less desirable for high throughput applications, though not impossible. Preparing the matrix/scaffold can be technically demanding (even some commercially available hydrogel-based matrices can be difficult to manipulate) which can result in poor structure formation and/or difficult imaging conditions.
    When working with hydrogel-based matrices for 3D culture, lay down 90% of the total volume and allow it to gel before adding the final 10%. This will result in a more consistent gel layer, which will result in improved imaging conditions and uniformity of 3D structures. In Gen5, by optimizing the autofocus parameters in Experiment Mode, you can easily maintain reliable focus during automated acquisition of scaffold-based 3D assays. 
  • ORGAN ON A CHIP: One of the newest and most intriguing applications of 3D cell culture is the development of Organ on a Chip, which takes 3D cell culture to the next level, utilizing co-culture and microfluidic chambers to fully mimic in vivo functionality as well as model the interfaces between tissues [2]. This approach has successfully simulated heart, lung, kidney, skin, and bone, and it is hoped that this technology may one day reduce or eliminate the need for animal studies.
    Benefits: Organ on a Chip is the in vitro method able to most accurately model multiple organ systems as well as the dynamic balance between health and disease.
    Drawbacks: Organ on a Chip is still in early stages of development for many organ systems and is not yet scalable to mid- or high throughput. Much work remains to be done on this technology.
    Custom labware can be created within Gen5 to create specific Plate Definitions for Organ on a Chip or any other unique cell culture vessel. Simply use a picture of the vessel in the appropriate stage adapter (such as the multi-vessel adapter for Organ on a Chip) to drag and drop imaging regions where needed.
  1. Ye Fang, Richard M. Eglan. Three Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discovery, 2017. DOI: 10.1177/1087057117696795 
  2. Dongeun Huh, Benjamin D. Matthews, Akiko Mammoto, Martin Montoya-Zavala, Hong Yuan Hsin, Donald E. Ingber. Reconstituting Organ-Level Lung Functions on a Chip. Science, 2010. DOI: 10.1126/science.1188302 

By: BioTek Instruments, Diane M. Kambach, FAS Team Leader

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