Diffusion dependent cell behavior in microenvironments

Abstract

Understanding the interaction between soluble factors and cells in the cellular microenvironment is critical to understanding a wide range of diseases. Microchannel culture systems provide a tool for separating diffusion and convection based transport making possible controlled studies of the effects of soluble factors in the cellular microenvironment. In this paper we compare the proliferation kinetics of cells in traditional culture flasks to those in microfluidic channels, and explore the relationship between microchannel geometry and cell proliferation. PDMS (polydimethylsiloxane) microfluidic channels were fabricated using micromolding methods. Fall armyworm ovarian cells (Sf9) were homogeneously seeded in a series of different sized microchannels and cultured under a no flow condition. The proliferation rates of Sf9 cells in all of the microchannels were slower than in the flask culture over the first 24 h of culture. The proliferation rates in the microchannels then continuously decreased reaching 5% of that in the flasks over the next 48 h and maintained this level for 5 days. This growth inhibition was reversible and influenced only by the cell seeding density and the channel height but not the channel length or width. One possible explanation for the observed dimension-dependent cell proliferation is the accumulation of different functional molecules in the diffusion dominant microchannel environment. This study provides insights into the potential effects of the diffusion of soluble factors and related effects on cell behavior in microenvironments relevant to the emerging use of microchannel culture systems.

Induction of alternative fate other than default neuronal fate of embryonic stem cells in a membrane-based two-chambered microbioreactor by cell-secreted BMP4

“…Moreover, inherent Marangoni convection (caused by surface tension differences in the free liquid-air interface) in these culture systems rapidly sweeps the soluble factors away from the cell neighborhood. 6 However, in enclosed micro-scale static culture systems, cellular microenvironment is more diffusion dominant than the macro-scale owing to the small dimension as well as the absence of free liquid-air interface. 6 Therefore, in these culture systems, cellsecreted soluble factors can accumulate in a small volume as well as remain in the cell neighborhood for long-time because of the diffusion dominant nature of the microenvironment.…”

“…4 Therefore, for establishing an in vitro differentiation system, it is essential to consider influence of exogenously added soluble factors as well as cell-secreted ones (i.e., endogenous soluble factors) on cell behavior. 5 A cell-secreted soluble factor in a conventional open macro-scale static culture system, e.g., 6-well plate (6WP), becomes diluted in a large volume compared to cellular volume. Moreover, inherent Marangoni convection (caused by surface tension differences in the free liquid-air interface) in these culture systems rapidly sweeps the soluble factors away from the cell neighborhood.…”

Generating steep, shear-free gradients of small molecules for cell culture

“…Attempts at eliciting sharp graded cell responses to diffusible cues have been limited by the fact that many signals (including signals secreted by the cells themselves, such as Lewis acids, bases and small peptides) diffuse rapidly in cell culture (Gurdon and Bourillot 2001;Lander et al 2002;Yu et al 2005). One of the most extreme cases, the hydroxyl ion, has a diffusion constant greater than: ∼5.28×10 −9 m 2 /s in water (Cussler 1997).…”

“…Since controlling and sensing the gradients of signaling molecules provides a basis for understanding many patterning/developmental processes, there has been much interest in microfluidic devices for generating gradients of such molecules (Campbell and Groisman 2007;Dertinger et al 2001;Irimia et al 2006;Jeon et al 2002;Keenan and Folch 2008;Takayama et al 2001). These devices have attempted to recreate in vivo cellular chemical environments in vitro by exploiting microscale mass transfer phenomena, including manipulation of laminar flows Irimia et al 2006;Jeon et al 2000;Saadi et al 2006;, generation of mass-transfer limitations in microchannels (Abhyankar et al 2006;Keenan et al 2006;Yu et al 2005), and microscale dosing (Bansal et al 2007;Chung et al 2006). These devices have the potential to generate microenvironments that more closely approximate the in vivo milieu (El-Ali et al 2006;Khademhosseini et al 2006) than traditional, two chamber techniques such as Boyden (Boyden 1962), Zigmond (Zigmond 1977), and Dunn (Zicha et al 1991) chambers.…”

“…However, existing microfluidic gradient generation devices have known drawbacks; those that depend on convective flow and diffusive mixing within channels populated by cells tend to remove secreted chemicals even as they generate gradients and expose the cells to shear forces Irimia et al 2006;Jeon et al 2000;Saadi et al 2006;. Microfluidic methods that do not employ flow eliminate or reduce shear, but tend to generate shallow gradients and lack the ability to produce complex gradient profiles (Abhyankar et al 2006;Keenan et al 2006;Yu et al 2005). Recently, a class of devices has emerged which cultures cells across a diffusible membrane so that the interference with hydrodynamic flow is minimized (Abhyankar et al 2006;Chueh et al 2007).…”