Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane

Abstract

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, and disease development and progression. In this study we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 µm in diameter formed in hydrophobic polymer polydimethylsiloxane (PDMS) with ~60–100 µm circular openings and aspect ratio ~3.5. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells / MB) and cultured under standard conditions, adopt a compact 3-D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96 well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca2+ concentration and changes in Ecadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.

Gather Round: In vitro tumor spheroids as improved models of in vivo tumors

“…We showed that arrays of spherical cavities formed on PDMS [7] using this technique can be used for culturing cancer cells as spheroids [8]. The unique geometry of microbubbles allows the cells to rapidly condition their microenvironment by secreting soluble factors to influence their function [9]. Also, perfusion culture system using microbubble arrays has shown that Colo205 cells cultured as spheroids under flow are more resistant to doxorubicin treatment resembling the actual disease condition [10].…”

Characterization of cell seeding and specific capture of B cells in microbubble well arrays

“…Recently, we introduced microbubble (MB) well array technology and demonstrated its use to sustain single and small cell cultures for extended periods of time (>10 days) (Giang et al 2008; Chandrasekaran et al 2011). Microbubbles are spherical cavities formed in polydimethylsiloxane (PDMS) using the gas expansion molding (GEM) process (Giang et al 2007, 2012).…”

In vitro skin three-dimensional models and their applications

“…The critical role of enrichment of TGF-beta for sheeting behaviour was shown in a study comparing standard spheroid versus microbubble 3D cultures of keratinocytes [16]. While in the first, secreted molecules such as TGF-beta could diffuse freely, the compartmentation induced by the microbubble cultivation technique arguably led to a local increase in TGF-beta which induced a sheeting cell growth [16]. Using a similar chip-based technology featuring micro-fenestrated 300-m wide compartments (KIT Chip), we could recently confirm these findings and show a regular layered expression of keratinocyte differentiation markers, KRT14 and KRT10 (Fig.…”

“…Close to definitive wound healing, fibroblasts release TGF-beta, which shifts keratinocytes to the basal phenotype, i.e. suppressing hyperproliferation, activating the standard keratinocyte differentiation program, stimulating the production of ECM components, and inducing normal keratinocyte layering [6,16]. The critical role of enrichment of TGF-beta for sheeting behaviour was shown in a study comparing standard spheroid versus microbubble 3D cultures of keratinocytes [16].…”

“…suppressing hyperproliferation, activating the standard keratinocyte differentiation program, stimulating the production of ECM components, and inducing normal keratinocyte layering [6,16]. The critical role of enrichment of TGF-beta for sheeting behaviour was shown in a study comparing standard spheroid versus microbubble 3D cultures of keratinocytes [16]. While in the first, secreted molecules such as TGF-beta could diffuse freely, the compartmentation induced by the microbubble cultivation technique arguably led to a local increase in TGF-beta which induced a sheeting cell growth [16].…”