In Three Dimensions: A Cell Culture Revolution

By Brandoch Cook, PhD

In a significant way, the entirety of modern biomedical research has depended on the ability to culture cells in a dish. Beginning in the late 19th and early 20th centuries, first Roux, then Harrison extracted embryonic and neural tissues and maintained them on glass. Alexis Carrel, whose scientific brilliance initially outshone his sordid involvement in the French Vichy regime, developed the first sterile techniques, invented tissue culture flasks, and observed that cells could grow in the serum of other animals, opening access to an unlimited supply of reagents. His frequent collaborator, Charles Lindbergh (yes, that one), helped him invent the perfusion pump and introduced the use of Pyrex glass.

In the ensuing years, the hunt for a polio vaccine and the resulting renaissance in virology drove innovations in culture paradigms, including the creation of primary cell cultures derived from different tissues, then the establishment of permanent cell lines such as HeLa that could be freely distributed. For most of the next five decades, cell culture largely consisted of the maintenance of these and derivative cell lines on flat plastic surfaces using serum-containing rich media.

Beginning in the late 1970s, researchers found that tumor cells displayed novel growth properties when cultured in soft agar. In 1985, Thomas Doetschman and his colleagues propagated embryonic stem cells (ESCs) as spherical, floating embryoid bodies with the capacity to form derivatives of all three embryonic germ layers, including cardiac muscle-like cells that beat rhythmically. Only within the past decade or so, however, has 3-D cell culture burgeoned into a field and an industry unto itself. The rise of biotech and big pharma as both drivers and consumers of biomedical innovation has resulted in a flood of drug discovery and validation.

Two-dimensional cultures have often served as drug discovery platforms because of their intrinsic amenability to high-throughput procedures such as chemical screening. However, the highly artificial constraint of an adherent monolayer culture is a poor predictive system for whether potential blockbuster medications will work in living animals, let alone patients. This is especially true of chemotherapeutics and other cancer drugs, which often show artificially high efficacy in monolayers compared to 3-D systems designed to recapitulate the cellular architecture of tumors. Major developments in academic science have also helped spur the 3-D culture revolution. A rapid expansion of stem cell research followed the isolation of human ESCs and the discovery of induced pluripotency. Additionally, the mission of regenerative medicine programs to understand the cellular basis of organ development with a view toward generating pure populations of transplantable cells resulted in a broad desire to more faithfully replicate tissue-specific conditions in which cells differentiate and reside. This resulted in ongoing attempts to define growth conditions under which cells of interest can flourish. For instance, cells such as cardiomyocytes or pancreatic beta cells can be derived from pluripotent cultures by replacing serum with instructive growth factors and cytokines, using time courses designed to mimic the progression of germ layer deposition and organogenesis. A three-dimensional framework is a naturally complementary system with which to probe the possibilities of these models. Unlike 2-D, it can provide realistic cell-cell and cell-ECM contacts, accommodating instructive and interactive niches and microenvironments engineered to replicate the physiological properties of gas and nutrient exchange.

Two-dimensional cultures’ long history has wrought an extensive record of publication and communication, informing strong standards and best practices concerning reagents, conditions, and investigations. Moreover, the uniformity of 2-D cultures naturally fits analyses such as microscopy and immunocytochemistry, with less variability to be expected between independent researchers performing the same assays.

Conversely, in the fledgling field of 3-D culture, there is still an absence of consensus concerning protocols and materials ideal for each system under study. There is, therefore, a bewildering array of possibilities to choose from when modeling cellular dynamics in three dimensions. A partial list of 3-D substrates and devices available from large manufacturers such as Thermo Fisher, Sigma-Aldrich, and Corning, and from niche firms such as Mimetris, Microtissues, and Creative Biolabs, includes 1) scaffold-based substrates made of bioactive natural materials, including silk, collagen, gelatin, and alginate; 2) hydrogel-based scaffolds that approximate an extracellular matrix with synthetic peptide polymers; 3) inert, synthetic scaffolds composed of polyethylene glycol and other industrial polymers; 4) scaffold-free systems utilizing agarose or Matrigel molds in combination with ultralow attachment microplates; and 5) proprietary microfluidics and bioreactor systems that can allow for controlled flow, laminar juxtaposition of different cell types, and variable mechanical stress on semisolid substrates.

Consequently, there is an extensive and unique process of optimization to be expected for almost every project that employs 3-D culture. To render some clarity regarding which system may best suit differing needs, the table on the right provides a list of the key advantages and disadvantages to both. In the balance, the potential power of 3-D, especially in preclinical studies, probably outweighs the drawbacks, and will almost certainly continue to tilt in favor of 3-D as the industry matures and procedures and reagents become more standardized.

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Published: November 12, 2018

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