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

Paper ID Volume ID Publish Year Pages File Format Full-Text
7667 559 2011 10 PDF Available
Title
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, 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 polydimethylsiloxane (PDMS) with ∼60–100 μm circular openings and aspect ratio ∼3.0. 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 3D 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 undergo a 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 E-cadherin 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.

Keywords
Cell spreading; Polydimethylsiloxane; Cell culture; Transforming growth factor (TGF)
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Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 32, Issue 29, October 2011, Pages 7159–7168
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us