Compared with two-dimensional (2D) cell cultures, 3D cultures provide varying degrees of physiologic relevance. In 3D, cells interact with their environment and with each other or, through co-cultures, with secondary cells, in ways that are impossible for “flat” plated or suspension cultures.

3D cell culture has given rise to several useful terms: organoids and spheroids, scaffolded and un-scaffolded, microfluidics-enabled or not, organ-like and tissue-like cultures—all with special relevance to the main act, namely the assay that follows successful culturing.

Basic tools

As with conventional cell culture, the success of 3D culture relies on basic reagents and culture tools.

Corning® Matrigel® matrix, which its eponymous creator describes as “the most widely used natural bioactive ECM for 3D cultures,” behaves in culture like living extracellular matrix. Rich in laminin, collagen IV, enactin, heparin sulfate proteoglycans, and growth factors, Matrigel has found applications in 3D stem cell culture as well as for organoids and spheroids.

“To create better models for testing drugs and understanding complex cell biology, we need to reproduce the in vivo environment as much as possible,” says Hilary Sherman, senior applications scientist. “Since Matrigel is a biologically derived ECM it better reflects the in vivo environment compared with synthetic hydrogels.”

Any other ECM preparation could, of course, be supplemented with the main ingredients in Matrigel, in any desired quantities, which would approximate the Matrigel formula. The benefit from using the natural starting materials is found in nature’s “secret ingredients,” Sherman adds. “Since Matrigel is naturally derived it contains other undefined components that may be necessary for proper cell function.”

Collagen Type I, another material that supports 3D cell culture, supports life-like 3D growth and differentiation by providing physiological interactions with receptors that modulate gene expression related to cancer cell invasion, susceptibility to anticancer drugs, cell proliferation, and cell migration. Corning Collagen Type 1 has been used to culture intestinal, primary mammary, and salivary organoids.

Sherman explains the significance of collagen type 1. “Collagen is the most abundant protein in the body and is responsible for the architecture and biomechanical properties of tissues and organs. Additionally, collagen serves as a regulatory molecule in which cells interact with binding sites on the protein, which can affect cell behavior.”

Corning also supplies cultureware, a critical component of any 3D cell culture workflow. Its Spheroid microplates combine the Corning Ultra-Low Attachment surface with an innovative U-shaped well geometry, providing an accessible, highly reproducible platform for culturing and assaying spheroids. These microplates, which also support matrix-free 3D cultures, are available in 96-, 384-, and 1536-well automation-friendly formats.

Generating 3D cultures

Magnetic levitation, a specialty of Greiner Bio-One, is an emerging platform for generating native 3D culture tissue environments and moving organoids or spheroids around at will. Using a magnet positioned above for levitating or below for bioprinting cells within the culture well, levitation creates an “invisible scaffold” that gently and rapidly brings cells together, while inducing cell-cell interactions and synthesis of an extracellular matrix. Long-surviving 3D cultures thus form without any artificial substrate, specialized media, or extraneous equipment. The gentleness of magnetic levitation allows cultures to acquire microscale morphology that closely resembles its tissue of origin. Studying the resulting 3D structures, for example through immunohistochemistry or western blotting, occurs through the bottom of the plate. According to Greiner this technique has been applied to a wide variety of 3D culture types, for example from stem cells, primary cells, and co-cultures.

For example, a study appearing in Nature described a toxicity assay using magnetic 3D bioprinting, while a group at Rice University has described robust methods for 3D culturing using magnetic levitation.

Four Key Takeaways

  • 3D cell culture provides greater depth and breadth for nearly any cell-based assay
  • Obtaining optimal results from 3D cultures requires using the right tools
  • Consult with suppliers beforehand, to understand better the capabilities of their culture systems and consumables, and their suitability toward the problems you're trying to solve
  • The more complex the assay or system used to generate it, the more critical it will be to consider automation tools for reducing variability and inconsistencies, and to improve throughput

Magnetization occurs through another Greiner product, NanoShuttle™-PL, a reagent kit consisting of gold and iron oxide nanoparticles cross-linked withpoly-L-lysine. NanoShuttle magnetizes cells through electrostatic attachment to cell membranes during an overnight incubation. Poly-lysine attaches non-covalently to the cell membrane and attracts the magnetically susceptible iron oxide, which serves as the hook to push and pull cells in any desired direction. The gold particles are a component of the labeling kit that carried over from previous iterations to enable fluorescence, surface-enhanced Raman scattering (SERS), or contrast studies. The magnetized complex, which appears under a microscope as peppered with dark nanoparticles, remains attached to cells for up to eight days, which is sufficient for most 3D experiments.

“Key is the ability to use very small forces to move cells within a magnetic field,” says Glauco Souza, Ph.D., director of global business development at Greiner. “When cells interact they produce extracellular matrix, which forms an in vivo-like environment. The idea is not to keep cells separate, but to bring them together in controlled fashion. The unique culture environment fosters 3D model formation within hours with no specialized media, and is applicable to co-cultures.”

With all the buzz surrounding 3D cell culture it’s easy to forget that forming spheroids or organoids is only the first step in the experiment. “If you want to do immunofluorescence, or test a drug, and you need to manipulate cells in predictable, controllable ways,” Souza says. “With monolayer cultures cells stick to a surface, so they’re easy to manipulate. All rules are off in 3D, where even media exchange is difficult because the spheroids are just floating. By applying magnetic field you can hold the spheroid, perform media exchange, and do immunofluorescence experiments similarly to how you’d carry them out in 2D. That’s why we refer to our products as operating in a more traditional 2D workflow.”

Magnetic manipulation also permits easy scalability: The workflow is identical whether users are working with 24 wells or 1536-well plates. It also allows utilization of flat-bottomed culture vessels. Round-bottom wells, which are more common, introduce optical distortions when 3D cultures are imaged from below.

Automation: Key to consistency

BioTek Instruments, which specializes in automated liquid handling, has kept up with the times by adapting its line of culture and liquid-dispensing tools, where applicable, to 3D cell culture.

As the spheroids grow, they consume increasingly more nutrients from the media, which must be exchanged over time, and at more frequent intervals. BioTek’s newest product, MultiFlo™ FX, provides repeated, automated media exchanges for spheroid cultures in such applications as spheroid proliferation, where kinetic studies can go on for weeks. MultiFlo FX performs extremely gentle aspirate and dispense steps such that the non-adhered spheroids are not disturbed during the exchange process.

MultiFlo FX is an automated multi-mode reagent dispenser for 6- to 1536-well microplates. The product incorporates several interesting technologies, such as Parallel Dispense, RAD™ Random Access Dispense, and the most recent addition, the patent-pending AMX™ Automated Media Exchange modules. AMX facilitates liquid-handling applications from 2D and 3D cell culture for concentration normalization assays, ELISA, bead-based assays, and others. When fully configured, MultiFlo FX replaces up to five liquid handlers, saving space, time, and instrumentation budgets, according to Peter Banks, scientific director at BioTek.

MultiFlo FX integrates with BioTek’s BioSpa™ 8 Automated Incubator and an imager or multi-mode reader, to provide complete workflow automation for cell imaging and biochemical studies.

BioTek’s AMX module for the MultiFlo FX enables gentle automated media exchange to protect and encourage proliferation of spheroids, tumoroids, and other 3D cell structures, 2D and suspension cells in microplate-based assays The AMX module uses both peristaltic pumps, one to dispense and one to aspirate media from the plate wells.

The software operates the pumps slowly and gently so cells and cell structures aren’t disturbed during the media exchange, and the x-y-z positions for the aspirate and dispense tubes are adjustable to avoid contact with cell structures in the wells.

“Automation is critical for robust 3D applications, particular for those involving spheroid proliferation,” Banks says. “Typical assay design calls for long-term kinetic monitoring of spheroid growth over a period of days to weeks. When screening several plates of different compounds for effects on spheroid growth, manual operation is tedious and prone to both systematic and random error, particularly during the repetitive need for media exchange as the spheroid consumes its nutrients. Automation provides true walk-away automation of the 3D spheroid workflow.”

Safe spaces for cells

In discussing 3D cell culture, the term environment or “microenvironment” comes up all the time. The physical, chemical, and physiologic surroundings of organoids and spheroids, also known as the culture’s niche, determines how closely cells and cell assemblies cultured in vitro resemble, and behave like, the cell’s normal in vivo environment. Cellular or organoid confinement—the physical restrictions imposed by the culture method—plays a critical role in this context.

Confinement provides an environment where the thickness of plated cultures are more easily controlled and defined, and where imaging-based assays are facilitated. Confinement also improves prospects for imposing geometric features on the cells, controlling cellular adhesion, environmental elasticity, and surface chemistry.

“For example, it’s difficult to study cell migration of immune cells because they are usually non-adherent,” notes Lamia Moubakir, research engineer at 4D Cell. “Confinement allows you to visualize cells in a 3D environment by giving them walls to catch, so they can move freely between the two surfaces and interact with each other.”

4D Cell offers the one-well dynamic confiner (featuring control of confinement speed and reversible confinement), one-well static confiner (confines cells within two surfaces with micrometer precision), six-wells static confiner (higher-throughput version of the one-well model), and six-wells static confiner with the CO2 plug (allowing gas exchange), all of which are compatible with high-resolution imaging systems.

Compression or contractility studies, based on how 3D cultures respond to controlled squeezing between two surfaces (for example hydrogels), are an attractive way to compare two cultures, for example how normal and cancer cells respond to drug treatment. Confinement allows such studies, as well as adds a new twist to the characterization of 3D co-cultures. “Instead of using two types of cells on the same substrate, you could put one cell on one surface, another on an opposing surface, and control spacing between them to study intercellular chemical interactions,” Moubakir says.

More complex embodiments

Mimetas, which operates under the tagline, “the organ-on-a-chip company,” has enjoyed widespread adoption of its 3D cell culture products. In 2018 alone, the company or its customers published ten peer-reviewed articles on 3D cultures utilizing one or more Mimetas cultureware products. Among these are a Nature paper on 3D brain cell cultures for organophosphate toxicity screening, a study on nephrotoxicity and kidney transport assessment in 3D perfused proximal tubules, and a report on 3D cultures of Parkinsons disease-specific dopaminergic neurons.

For example, Mimetas’ OrganoPlate 2-lane 400 features 96 independent culture compartments, each supporting one in-gel culture and a perfusion channel, for studying perfused tubular tissue. Four wells of the 384-well-plate connect to each microfluidic network. The OrganoPlate 3-lane plate features 40 independent culture cells, each supporting adjacent in-gel culture and two perfusion channels for perfused tubular tissues. This model supports apical and basal access to epithelial and endothelial tubules, allowing barrier integrity and transport assays.