Traditional cell cultures grown in two dimensions (2D cultures), on plastic plates or plates coated with extracellular matrix proteins, have long been the primary arena for studies in cell and developmental biology, genetics, and drug screening, among others. Over time researchers have honed their protocols and passed down culture lore, perfecting techniques for cell-based assays that generated data efficiently and reliably.
Despite the advantages of traditional 2D cultures—including familiarity with protocols, and a body of published results for reference and comparison—they are now rivaled by 3D cell cultures. In 3D cultures, cells are grown on structural scaffolds, hydrogel-based scaffolds, or in scaffold-free aggregates of cells such as spheroids and organoids. Thought to more closely approximate the in vivo cellular physiological environment, 3D cultures may give more relevant results in drug screening, safety, and toxicology studies.
However, 3D cultures pose new challenges. Their denser nature can make it harder to image cells deep within clusters, or to verify the concentration of dissolved compounds or even oxygen that might be reaching those cells. Assays using 3D cultures tend to be lower throughput, while 2D cultures are optimized for high-throughput assays (making them less expensive in drug screening, for example). This article focuses on 3D culture tools, and adapting cell-based assays from 2D to 3D.
Culturing in 2D versus 3D
Some researchers may elect to stick with 2D cultures despite the benefits of 3D cultures, whether for the sake of convenience, economy, throughput, or inability to perform a particular assay in 3D. “It is widely accepted that 3D cell cultures are better indicators of in vivo results,” says Mahesh Dodla, global product manager for cellular assays at MilliporeSigma. But the challenge now is to adapt important cell-based assays so that they can be conducted using 3D cultures. “The assays needed for 3D cell cultures will be like some of the 2D assays, but they need thorough validation and optimization because of the challenges with 3D culture systems,” says Dodla.
MilliporeSigma offers a range of technologies supporting 3D cell cultures, including hydrogels and scaffolds, organoid and spheroid culture systems. Their new 3D Live-Dead Cell viability assay kit uses three colors to help visualize and quantify the number of live, dead, and total cells. “This kit has been validated for analysis of cells in 3D matrices such as collagen and matrigel, as well as in 3D structures such as spheroids and organoids,” says Dodla. “We also offer other assays such as cytotoxicity, proliferation, and viability assays to help researchers understand how cell behave in these 3D environments.”
To make 3D cell culture easier, faster, and more user-friendly, CN Bio offers the PhysioMimix™ OOC, a benchtop microphysiological system (MPS) for organ-on-chip (OOC) studies. The system controls cell perfusion with microfluidics for long-term cell health. “3D perfused models of liver and other tissues can be easily generated on the platform and exhibit prolonged cell viability and function under serum-free conditions,” says Ovidiu Novac, senior scientist at CN Bio. “The platform allows culture of primary cells, stem cells, or even preformed tissues to create organ mimics.” One challenge when moving from 2D to 3D is figuring out the best culture protocol for the cell type, including optimization of seeding, cell density, and flow rate. PhysioMimix™ facilitates this, notes Novac, because “it enables continuous oxygenation and automatically controls microfluidics, providing round-the-clock cell culture.” Improving 3D using 2D cultures
Because 3D cultures are usually created from cells harvested from 2D cultures, quality control is key—cells from healthy 2D cultures will ultimately result in better 3D cultures. Anvajo’s fluidlab R-300, a handheld automatic cell counter with incorporated photospectrometer, is designed to make quality control faster and easier. It can measure cell viability without prior cell staining because living and dead cells refract light differently. “It uses a holographic microscopy signal, which is directly proportional to the refractive index of the cell,” says Felix Lambrecht, head of product at anvajo. Tools supporting better quality control of 2D cultures are especially important when using precious cell samples, such as from patient biopsies. “By increasing the quality of rare samples, [failed attempts at growing spheroids] are reduced and research is accelerated,” says Lambrecht.
Bovine collagen I, an extracellular matrix protein, is essential for growing cells in multiple formats, whether coating plates for 2D cultures or creating a collagen hydrogel for 3D scaffolding. Viscofan BioEngineering offers collagen I products for research and clinical applications. “Viscofan BioEngineering flexible extrusion technology allows manufacture of collagen membranes in different thicknesses, customizing parameters like permeability, tensile strength, stiffness, and resorption kinetics in vivo,” says Lluis Quintana Frigola from Viscofan. “The collagen membranes from Viscofan Bioengineering are characterized by the length and nativity of the collagen fibers, which resemble the natural extracellular collagen matrix in the body and confer high strength and biocompatibility to the scaffolds.”
Viscofan’s Collagen Cell Carrier® is a membrane that can be used to physically transfer an intact layer of cultured cells in sterile conditions, such as for transplantation studies. “Applications in the area of cardiac patches for cardiomyopathy patients, grafts for urethra stricture surgery and coating of blood vessel prostheses are already being developed,” explains Quintana Frigola.
Janny Marie Peterslund, scientific affairs manager at ChemoMetec, believes that 3D cell culture should be complementary to 2D culture, rather than a substitute. “Many 2D assays are being ‘converted’ into 3D assays where relevant in organoid research, but depending on the question asked, a combination of 2D and 3D assays will provide the best answers in any given field,” she says. ChemoMetec’s NucleoCounter® NC-200™ is used by organoid researchers for cell count and viability measurements. “With a slight modification to our protocol, 3D cultures are turned 2D prior to analysis, as we find this gives us the best accuracy and reliability of data,” she says, noting that making accurate and reliable measurements directly from 3D cultures can be challenging. ChemoMetec offers a range of cell-based assays, such as cell cycle analysis, caspase 3-7, Annexin-V, mitochondrial potential, redox potential, and cell counts and viability.
On the horizon for 3D cell culture
While 2D cultures aren’t going anywhere, technologies for 3D cultures will steadily evolve. Dodla believes that 3D culture advances will include greater “use of spheroids, organoids, 3D printing, organ-on-a-chip and multi-physiological systems, as well as 3D assays for cytotoxicity, proliferation, viability, apoptosis, oxidative stress, and metabolic assays.” Novac predicts we’ll soon see complex 3D cultures incorporating all cell types from a specific organ. “A promising area [is using] induced pluripotent stem cells to generate functional human cells from various patient populations on 3D platforms to create multi-organ models,” he says.
Quintana Frigola thinks that complex 3D organ models, constructed from multiple cell types and extracellular matrix, can be more effective at predicting the effects of drugs in human organs. “In our opinion, the predictability of drug effects in humans, based on animal studies alone, is an obsolete model,” he says. “The increased demand for human 3D lung models caused by the coronavirus epidemic is a first indication of this change.” One day, human 3D organ models may be used for testing pharmaceuticals and medical products in lieu of animal models.