In traditional two-dimensional (2D) monolayer culture, cells adhere and stretch across a rigid, flat substrate adopting an abnormal morphology. Cellular processes including proliferation, differentiation, mechanosensing, and protein expression are impacted by this divergence, and cells excised from their native tissues and grown in monolayer often fail to recapitulate in vivo behavior. Further, these cells are exposed to homogeneous concentrations of nutrients, growth factors, and oxygen, resulting in cellular heterogeneity below what is observed in physiological three-dimensional (3D) cellular structures. Consequently, the usage of 2D monolayer culture is a significant contributor to instances of false discovery during drug development and repositioning.
The introduction of 3D culture mediums and scaffolds, including Matrigel, resolved many of these issues by promoting the growth of cellular structures which better resemble their in vivo counterparts. However, the introduction of these scaffolding mediums also presented a new problem: diffusion-limited transport.
In conventional, liquid-cover culture, media freely convects and mixes which prevents the accumulation of cellular waste near metabolically active samples. Scaffolding mediums significantly inhibit this mechanism, resulting in stagnant microenvironments and a steady buildup of toxicity. Samples must be positioned near the liquid-solid interface of the scaffolding and culture medias to be within the diffusion limit.
Darcy™ perfusion devices reintroduce nutrient and metabolite transport in Liquid-Like Solids (LLS™) using perfusion, a form of convection first characterized by the French engineer Henry Darcy, which describes fluid flow through a porous medium. Darcy’s law governs the interstitial flow of liquid culture media through LLS™ and is practically a function of the induced pressure gradient and both the LLS™ permeability and system geometry. Because the effective flow rate is heavily dependent on the permeability of the system, including both the LLS™ and the supporting membrane, culture media transport through the system can be optimized for any desired culture. Flow is also proportional to pressure, and can be passively maintained using constant-pressure devices such as Jackson-Pratt drains, or actively controlled using low-vacuum pumps. Tissues often include stromal cells which secrete proteins to remodel the microenvironment, and pressure regulation provides a means to compensate for the potential decline in LLS™ permeability.
