Why 3D cell culture?

In 1907, Dr. Ross Harrison, an Anatomy Professor at Yale, developed a technique for growing three-dimensional frog embryonic nerve fragments in a small droplet suspended from a coverslip. This “hanging-drop” technique allowed researchers, for the first time, to maintain and study living tissues outside of the body, marking the birth of the field of tissue culture. Eighteen years later, while researching new tissue culture techniques, the French surgeon Alexis Carrel noticed that when tissue explants were grown in a glass flask, the cells spread out into thin, transparent layers, making imaging the cells much simpler than in the three-dimensional tissues in the hanging drop. Additionally, the flask could be designed to greatly reduce rates of culture contamination. Since then, life science researchers have relied primarily upon adherent, two-dimensional (2D) cell culture techniques to model human cells, tissues, and diseases.While traditional 2D tissue culture techniques have been thoroughly studied, standardized, and have contributed to the development of life-saving therapies, 2D tissue models have some fundamental flaws. First, cells grown in 2D monolayers interact primarily with a plastic or glass substrate and do not form intercellular interactions like cells within tissues in the body. Additionally, cells grown in a monolayer are exposed to uniform culture conditions, failing to account for the complex nutrient and metabolic transport processes and cellular heterogeneity found within three-dimensional (3D) tissues. These conditions lead to an unnatural mechanical and biochemical state that causes cells in monolayers to express genes and proteins differently than they would in the body. In contrast to 2D cell culture, cells grown in three-dimensions exhibit a significantly higher degree of intercellular interactions, assume more physiologically relevant morphologies, and can better recapitulate higher-order tissue processes such as the epithelial-to-mesenchymal transition or hypoxia-induced apoptosis. Even at the micro-scale, multicellular aggregates comprised of just 10s-100s of cells can replicate higher-order tissue properties. A great resource outlining some of the physiological differences between cells grown in two and three dimensions can be found here.Despite the advantages of 3D cell culture, 2D cell culture has traditionally been simpler, cheaper, more reproducible, and more scalable, making it more suitable for high volume drug discovery and toxicity assays. Many believe, however, that the limited physiological relevance of 2D cell-culture models are partially to blame for the high failure rates (~90%) associated with drug development, and a growing number of researchers hypothesize that utilizing more relevant, 3D disease models can yield more accurate drug toxicity and efficacy data in-vitro (see Figure 1). The value proposition is clear: with drug development timelines lasting up to 10 years and development costs north of $2.5B ( Tufts study), even just small improvements in drug development efficiency could translate into savings of hundreds of millions of dollars and years of time.

Recently, tools, technologies, and protocols have been developed that make in-vitro 3D cell culture and analysis simple-, cheap-, reproducible-, and scalable-enough for practical use in drug development. As these tools have become more accessible and validated, researchers in both academia and biopharma are starting to shift to their attention from 2D to 3D cell culture. While 2D cell culture still dominates the cell-based assay landscape, early adopters in the biopharma industry ( Merck , Bayer, Novartis , Genentech , GSK , Roche, and L’Oreal, just to name a few) have begun to integrate 3D tissue models into their R&D. With increasing interest in phenotypic screening and promising data showing a high correlation between the drug response of in-vitro 3D tissue cultures and clinical drug response, we expect more companies to follow suit.

Key Technologies and Players in the 3D Cell Culture Industry

As awareness and acceptance of the benefits and potential of 3D cell culture grows, many research tools companies are competing to capture share of the ~$6B cell-based assay market in anticipation of a shift in research spend from 2D to 3D cell-based assays. While it’s clear that 3D cell culture will stake a claim in the preclinical research market, no single technology or company has yet to emerge as the clear leader in this space. Below I have highlighted some of the key technologies and companies to keep an eye on as this space evolves.

Scaffold/Hydrogel Techniques

Cells within tissues grow within a network of proteins, glycoproteins, proteoglycans, and polysaccharides called the extracellular matrix (ECM) which provides mechanical and biochemical signaling support to the surrounding cells. In order to mimic the structural and biochemical properties of the ECM as well as provide a framework for supporting cell growth in three-dimensions, multiple companies have developed a variety of synthetic and naturally-derived matrices and scaffold materials for 3D cell culture. Products range from purely biomolecular hydrogel systems (e.g., Corning Matrigel ®, Trevigen Cultrex® gels, QGel , Lonza/TAP Biosystems RAFT , Celenys Biomimesys®) to fully synthetic scaffold inserts for multiwell plates (e.g., Reinnervate Alvetex Scaffold, 3D Biotek 3D Insert, Synthecon NanobioMatrix Scaffolds).Advantages of scaffold/gel-based techniques include the ability to integrate the scaffold/gel into an automated 96-well or 384-well assay workflow, relatively simple assay protocols, ease of imaging, and the ability to tune the mechanical and biochemical properties of the gel/scaffold. Disadvantages of these techniques can include additional prep-time and lot-to-lot variability associated with purely bimolecular hydrogel matrices, the use of unnatural scaffold materials (e.g., polystyrene, PCL, PLGA) for synthetic inserts, a relatively high-degree of cell-matrix vs. cell-cell interactions, and limited ability to control cell growth or architecture within the scaffold.

Spheroid-Based Techniques

Cell spheroids are compact, multicellular aggregates with sizes of up to ~1000 µm in diameter. While cell spheroids have been used in basic research for decades (traditional spheroid culture techniques utilize Harrison’s hanging-drop technique), recent advancements in spheroid generation and assaying have led to a resurgence in interest in these simple yet powerful tissue models. Spheroids are formed in-vitro by creating conditions that minimize cell-substrate interactions and favor cell-cell interactions. Spheroids can be generated using a variety of techniques including hanging-drop plates (InSphero, 3D Biomatrix), low-attachment plates ( Scivax, Corning, Thermo Scientific, NOF America), rotary vessels ( Synthecon), and magnetic levitation ( n3D biosciences). Some of the previously mentioned scaffold/gel materials can also be used to induce spheroid formation.The advantages of spheroid-based assays can include a high-degree of intercellular interactions within the spheroid, compatibility with 96- and 384-well workflows (for plate-based techniques), precise control over spheroid size, simple assay protocols, the ability to provide some degree of control over co-culture morphology and architecture, and validation by decades of characterization in literature. However, spheroids present unique imaging challenges, require precise culture conditions, are susceptible to mass transport limitations (which can be favorable for some tissue models but unfavorable for others), and, for non-automated culture techniques, can require labor intensive culture and assay protocols.Because their resemblance to small, avascular tumors and the simplicity of the culture workflow, spheroids are currently considered the most favored in-vitro 3D tissue model. One of the leaders in the 3D cell-based assay space is InSphero, a private Swiss startup that has developed 96- and 384-well plates that support automated hanging-drop spheroid culture and analysis. InSphero offers a broad portfolio of spheroid culture reagents and consumables, ready-to-use pre-grown spheroids, and contract toxicology and DMPK testing services. According to its website, InSphero claims the title of the leading provider of assay-ready 3D microtissues and 3D-focused contract testing services and counts the world’s top 15 pharmaceutical companies, the number one cosmetics company, and 3 of the top 10 chemical companies amongst its customers. As the #1 Swiss Startup of the Year in 2014 and the winner of the 2014 Academic Enterprise Awards (ACES) for life sciences, InSphero is one of the key players to watch as the 3D cell culture market evolves.

Bioprinting Techniques

Three-dimensional bioprinting is an emerging trend in tissue engineering, and while much of the fanfare is focused on the clinical applications of organ/tissue printing, the technology has applications in the research space as well. To oversimplify greatly, bioprinting involves using an automated instrument, similar to an inkjet printer, to pattern cells and/or extracellular matrix materials in precise spatial arrangements, layer-by-layer, to construct a three-dimensional tissue in-vitro. These tissues can be comprised of multiple cell-types and possess functional, organ-specific architectures to provide an advanced in-vitro model of virtually any tissue type.The main advantages of bioprinting are the abilities to control the structure and composition of the tissue, to pattern almost any cell type, and to produce tissues across a relatively large range of length scales, all with a high-degree of reproducibility. Some potential drawbacks of bioprinting include the need for specialized equipment and expertise (if performing assays in-house) or else reliance on contract service providers, relatively long experiment set-up times, limited validation or standardization of tissue designs/models, and comparatively lower throughput/scalability compared to other 3D culture techniques.Organovo is undoubtedly the biggest name in bioprinting today. As the only public bioprinting company, and having been named one of The Scientist Magazine’s Top 10 Innovations of 2014 and Fast Company’s Top 10 Most Innovative Healthcare Companies in the World for 2015, Organovo has garnered a lot of media and investor attention over the past year. Organovo currently offers bioprinted liver tissues and a toxicology testing service and has plans to launch bioprinted kidney tissue products/services in 2016. Additionally, Organovo is also pursuing opportunities in skin and oncology tissues. While Organovo has received most of the attention and accolades, multiple other bioprinting companies, including Aspect Biosystems, 3Dynamic Systems, regenHu, n3D Biosciences, OxSyBio , and Cyfuse Biomedical, offer different bioprinting technologies but a similar value proposition in the R&D space.

Microfluidics / Organs-on-Chips Techniques

Organ-on-chip systems are now emerging from the lab into the marketplace. Organs-on-chips technologies combine microfabrication with tissue engineering to create devices that can precisely manipulate cells, tissues, and the extracellular microenvironment in a way that mimics physiological conditions. These microfluidic techniques enable functionalities and capabilities that are difficult or impossible to achieve using traditional screening techniques, such as electrical or mechanical stimulation, continuous perfusion, localized and programmable temperature control, particle/bead sorting and manipulation, multi-tissue fluidic interconnection, and minimal reagent consumption. However, as early-stage and highly-specialized techniques, organs-on-chips technologies can also present a number of drawbacks such as limited throughput compared to other 3D assay techniques, complex chip/device fabrication protocols, specialized equipment requirements, costly consumables, and lack of standardization/validation.Thus far, only a few companies have commercialized, or are in the process of commercializing, organ-on-chip technologies. Both EMD Millipore’s CellAsic and Mimetas’ Organoplate platforms incorporate microfluidic elements into a multiwell-plate architecture to allow for the continuous perfusion of cells encapsualted in a gel or scaffold material. Mimetas is also currently developing a kidney-on-a-chip in collaboration with pharmaceutical partners. Solidus Biosciences has developed a 3D-cellular microarray chip, which allows for the encapsulation of cells within up to 532 hydrogel pillars on a single chip. Emulate Bio, TissUse GmbH , and InSphero utilize proprietary microfluidic chips to model different tissue systems. For example, Emulate Bio, a spin-out from the Harvard Wyss Institute, has developed a flexible microfluidic device that uses air pressure to periodically expand and contract a membrane seeded with lung cells to mimic the mechanical and biochemical conditions of a breathing human lung. Emulate has also partnered with Johnson & Johnson to develop a thrombosis-on-chip device that will be used in early drug testing to identify which drug candidates might pose a thrombosis risk. German company TissUse has launched a microfluidic chip that enables the culture and fluidic connection of two different 3D tissue types on a single chip, and has plans to launch a 10-tissue chip by 2017, potentially allowing for a complete body-on-chip platform. Similarly, InSphero is leading a collaborative effort alongside AstraZeneca and German research institutions to develop a body-on-chip platform that would allow the testing of a drug across multiple different 3D microtissues on a single chip.

Going Forward

Today, over 100 years after Harrison’s initial hanging-drop experiments, cell-based assays appear to be returning to their three-dimensional origins. With numerous different technologies and companies competing for adoption in an industry that favors standardization, the development of the 3D cell culture market is bound to be interesting. Fortunately, regardless of who wins or loses, if competition breeds innovation, we all stand to benefit.Stay tuned for future posts as I track the progress and key developments of the 3D cell culture market.Disclaimer: Some of the companies listed above may be DeciBio clients or customers.Author: Andrew Aijian, Sr. Associate at DeciBio Consulting, LLCaijian@decibio.comConnect with Andrew on LinkedInhttps://www.linkedin.com/pub/andrew-aijian