3D cell culture

3D cell cultures have been used in research for several decades.[2] One of the first recorded approaches for their development was at the beginning of the 20th century, with the efforts of Alexis Carrel to develop methods for prolonged in vitro tissue cultures.[3] Early studies in the 80's, led by Mina Bissell from the Lawrence Berkeley National Laboratory, highlighted the importance of 3D techniques for creating accurate in vitro culturing models. This work focused on the importance of the extracellular matrix and the ability of cultures in artificial 3D matrices to produce physiologically relevant multicellular structures, such as acinar structures in healthy and cancerous breast tissue models. These techniques have been applied to for in vitro disease models used to evaluate cellular responses to pharmaceutical compounds.[4]

Eric Simon, in a 1988 NIH SBIR grant report, showed that Electrospinning could be used to produced nano- and submicron-scale polystyrene and polycarbonate fibrous mats (now known as scaffolds) specifically intended for use as in vitro cell substrates. This early use of electrospun fibrous lattices for cell culture and tissue engineering showed that various cell types including Human Foreskin Fibroblasts (HFF), transformed Human Carcinoma (HEp-2), and Mink Lung Epithelium (MLE) would adhere to and proliferate upon the fibers. It was noted that as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more histotypic rounded 3-dimensional morphology generally observed in vivo.[5]

In living tissue, cells exist in 3D microenvironments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells.[6][7][8][9][10][11][12][13][14] Standard 2D, or monolayer, cell cultures are inadequate representations of this environment, which often makes them unreliable predictors of in vivo drug efficacy and toxicity.[15][12] 3D spheroids more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices.[1] These matrices help the cells to be able to move within their spheroid similar to the way cells would move in living tissue.[8] The spheroids are thus improved models for cell migration, differentiation, survival, and growth.[13] Furthermore, 3D cell cultures provide more accurate depiction of cell polarization, since in 2D, the cells can only be partially polarized.[8] Moreover, cells grown in 3D exhibit different gene expression than those grown in 2D.[8]

The third dimension of cell growth provides more contact space for mechanical inputs and for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signalling.[16][17] Normal solute diffusion and binding to effector proteins (like growth factors and enzymes) is also reliant on the 3D cellular matrix, so it is critical for the establishment of tissue scale solute concentration gradients [18][19]

For the purposes of drug toxicology screening, it is much more useful to test gene expression of in vitro cells grown in 3D than 2D, since the gene expression of the 3D spheroids will more closely resemble gene expression in vivo. Lastly, 3D cell cultures have greater stability and longer lifespans than cell cultures in 2D.[20] This means that they are more suitable for long-term studies and for demonstrating long-term effects of the drug. 3D environments also allow the cells to grow undisturbed. In 2D, the cells must undergo regular trypsinization in order to provide them with sufficient nutrients for normal cell growth.[21] 3D spheroids have been cultured in a lab setting for up to 302 days while still maintaining healthy, non-cancerous growth.[20]

There are a large number of commercially available culturing tools that claim to provide the advantages of 3D cell culture. In general, the platforms can be classified in two types of 3D culturing methods: scaffold techniques and scaffold-free techniques.

A model showing three examples of techniques used for culturing cells in a 3D environment.

Spheroids

Electron microscopy of a mesothelioma spheroid (NCI-H226).[26] Scale bars, 200 μm.

Spheroids are a type of three-dimensional cell modeling that better simulate a live cell's environmental conditions compared to a two-dimensional cell model, specifically with the reactions between cells and the reactions between cells and the matrix.[27] Spheroids are useful in the study of changing physiological characteristics of cells,[28] the difference in the structure of healthy cells and tumor cells, and the changes cells undergo when forming a tumor.[29] Spheroids co-cultured with tumor and healthy cells were used to simulate how cancerous cells interact with normal cells.[30] Spheroids can be grown with a few different methods. One common method is to use low cell adhesion plates, typically a 96 well plate, to mass-produce spheroid cultures, where the aggregates form in the rounded bottom of the cell plates.[26][31] Spheroids can also be cultured using the hanging drop method[32] involving forming cell aggregates in drops that hang from the surface of a cell plate.[27] Other methods under investigation include the use of rotating wall vessel bioreactors, which spins and cultures the cells when they are constantly in free fall and forms aggregates in layers [33] Recently, some protocols have been standardized in order to produce uniform and reliable spheroids.[34] Researchers had also explored standardized, economical and reproducible methods for 3D cell culture. [35]

The various cell structures in the human body must be vascularized to receive the nutrients and gas exchange help that they need to survive. Similarly, 3D cell cultures in vitro require certain levels of fluid circulation, which can be problematic for dense, 3D cultures where cells may not all have adequate exposure to nutrients. This is particularly important in hepatocyte cultures because the liver is a highly vascularized organ. One study cultured hepatocytes and vascular cells together on a collagen gel scaffold between microfluidic channels, and compared growth of cells in static and flowing environments, and showed the need for models with tissues and a microvascular network.[39]

Advanced development of 3D models for high-throughput screening in high density formats has recently been achievable due to technological achievements related to increased microplate density. These can be found in 384 and 1536-well formats that are cell repellent, cost effective, and amenable to fully automated screening platforms.[40] Two options that afford 1536-well formats are available from either Greiner Bio-One utilizing the m3D Magnetic 3D bioprinting[41] and Corning Life Sciences which incorporates a ultra-low attachment surface coating, along with a microcavity geometry and gravity to create 3D models.[42][43] Due to the rapid and affordable methods and technologies that have been developed for 3D screening, parallel high-throughput screening approaches to test isogenic pairs of oncogene related mutants versus wildtype have been enabled.[44]

A primary purpose of growing cells in 3D scaffolds and as 3D cell spheroids in vitro is to test pharmacokinetic and pharmacodynamic effects of drugs and nanomaterials in preclinical trials.[13][45][46][47][48] Toxicology studies have shown 3D cell cultures to be nearly on par with in vivo studies for the purposes of testing toxicity of drug compounds. When comparing LD50 values for 6 common drugs: acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid, the 3D spheroid values correlated directly with those from in vivo studies.[49] Although 2D cell cultures have previously been used to test for toxicity along with in vivo studies, the 3D spheroids are better at testing chronic exposure toxicity because of their longer life spans.[50] The matrix in 3D Spheroids causes cells to maintain actin filaments and is more relevant physiologically in cytoskeletal organization and cell polarity and shape of human cells.[51] The three-dimensional arrangement allows the cultures to provide a model that more accurately resembles human tissue in vivo without utilizing animal test subjects.[52]

Existing 3D methods are not without limitations, including scalability, reproducibility, sensitivity, and compatibility with high-throughput screening (HTS) instruments. Cell-based HTS relies on rapid determination of cellular response to drug interaction, such as dose dependent cell viability, cell-cell/cell-matrix interaction, and/or cell migration, but the available assays are not optimized for 3D cell culturing. Another challenge faced by 3D cell culturing is the limited amount of data and publications that address mechanisms and correlations of drug interaction, cell differentiation, and cell-signalling in these 3D environments. None of the 3D methods have yet replaced 2D culturing on a large scale, including in the drug development process; although the number of 3D cell culturing publications is increasing rapidly, the current limited biochemical characterization of 3D tissue diminishes the adoption of new methods.

There are also problems using spheroids as a model for cancerous tissue. Although beneficial for 3D tissue culture, tumor spheroids have been criticized for being challenging or impossible to “manipulate gradients of soluble molecules in [3D spheroid] constructs, and to characterize cells in these complex gradients”, unlike the paper-supported 3D cell culture for tissue-based bioassays explored by Ratmir et al.[37] Further challenges associated with complex 3D cell culture techniques include: imaging due to large scaffold sizes and incompatibility with many fluorescence microscopes, flow cytometry because it requires the dissociation of spheroids into a single-cell suspension, and the automation of liquid handling.[53]

• Cell culture
• Cell lines
• Cell culture assay
• Hydrogel
• Madin-Darby Canine Kidney cell line
• Microphysiometry