Cell Culture


Considerations for engineering composable, disease-tunable 3D human microtissues for drug discovery


Life operates in three spatial dimensions (3D), as do the patients for whom we are developing therapies and cures. Earlier limitations in cell culture and analysis methods constrained us to 2D cell culture substrates, but the amount of valuable information we can extract from such systems is quite limited, and largely exhausted. In this article, we will share some of what we have learned over the past 10 years as pioneers in the 3D cell culture space and provide guidance for those who are just getting started with 3D models and end-point assays.


T


he human organism is composed of cells, tissues and organs with defined functions and a variable capacity for regeneration.


Adult, mature cells perform the bulk of functions while regenerative capacity is mediated either by rare, specialised progenitor cells or adult, differen- tiated cells that can reactivate regenerative pro- grammes. Primary, differentiated adult cells, isolat- ed from donor material, are therefore the perfor- mance gold standard for building 3D models. Even though not an inexhaustible source, cell stocks lasting months to years can be built up from a sin- gle donor for some organ systems as, for example, the liver. An alternative to working directly with adult cells is starting with stem or progenitor cells, such as human pluripotent stem cells-derived pro- genitors or Lgr5+ cells isolated from adult tissue. This permits for an in vitro expansion, at the cost of often questionable maturity as the degree of het- erogeneity among microtissues increases as they grow. Tissues are heterotypic in nature and composed


of multiple different cell types arranged in 3D. While most cell types in solid organs are organ-spe-


Drug Discovery World Winter 2018/19


cific (eg, liver hepatocytes, cholangiocytes and hep- atic stellate cells), others are specialised versions of cell types also found in other organs, such as liver sinusoidal endothelial cells (LSECs) and Kupffer cells, the resident macrophages of the liver. Organs are also permeated by many other immune cell types that patrol and, importantly, modulate inflammatory activation as well as tolerance and quiescence within tissues. These immune cell pop- ulations play a critical part in the development of many diseases and must be considered when estab- lishing advanced disease models. The isolation process of cells from solid organs is


not without stress. In our experience, we have observed that tissues generated from adult cells require a few days of resting to downregulate stress as, for example, inflammation, extracellular matrix or adhesion markers. This brief resting period serves as a good indicator for the plasticity poten- tial, which is also a hallmark of complex, organ- otypic tissues. Picture cells being ripped out of their in vivo environment and forced to adapt for their new in vitro environment. While cells, in vivo, are embedded in complex, organ-specific extracellular


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By Dr Patrick Guye, Dr Eva Thoma and Dr Olivier Frey


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