Dataset Information

Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device.

ABSTRACT: Microtechnology offers great prospects for cellular research by enabling controlled experimental conditions that cannot be achieved by traditional methods. This study demonstrates the use of a microfluidic platform for long-term cultivation (3 weeks) of human mesenchymal stem-like cells (MSCs), a cell population of high interest for tissue engineering. The typical high motility of the MSCs required a strategy for preventing cells from inhabiting the feeding channels and thus interfere with a steady perfusion of medium to the cell cultivation chamber. Hence, a straightforward and long-term patterning method was developed and implemented for reliable cell positioning within the device. This method was based on the modification of a polystyrene substrate into cell supportive and non-supportive regions by the use of selective oxygen plasma treatment and the triblock copolymer Pluronic. Also, a novel and size-effective "flip-chip" set-up for operating the devices was invented. Successful and reproducible adipogenic and osteogenic differentiation of MSCs in the device was demonstrated, verifying that an adequate long-term microfluidic cultivation environment was obtained. Strengths of the experimental protocol include ease of fabrication and maintenance (gravity driven), good cell performance (viability/differentiation), as well as the possibility of exposing the culture to heterogeneous laminar flow for experimental purposes.

SUBMITTER: Tenstad E

PROVIDER: S-EPMC3877732 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device.

Tenstad Ellen E Tourovskaia Anna A Folch Albert A Myklebost Ola O Rian Edith E

Lab on a chip 20100309

Microtechnology offers great prospects for cellular research by enabling controlled experimental conditions that cannot be achieved by traditional methods. This study demonstrates the use of a microfluidic platform for long-term cultivation (3 weeks) of human mesenchymal stem-like cells (MSCs), a cell population of high interest for tissue engineering. The typical high motility of the MSCs required a strategy for preventing cells from inhabiting the feeding channels and thus interfere with a ste ...[more]

PMID: 20386793

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Project description:Human mesenchymal stem cells (hMSCs) have the potential to differentiate into different types of mesodermal tissues. In vitro proliferation and differentiation of hMSCs are necessary for bone regeneration in tissue engineering. The present study aimed to design and develop a fluid flow mechanically-assisted cartridge device to enhance the osteogenic differentiation of hMSCs. We used the fluorescence-activated cell-sorting method to analyze the multipotent properties of hMSCs and found that the cultured cells retained their stemness potential. We also evaluated the cell viabilities of the cultured cells via water-soluble tetrazolium salt 1 (WST-1) assay under different rates of flow (0.035, 0.21, and 0.35 mL/min) and static conditions and found that the cell growth rate was approximately 12% higher in the 0.035 mL/min flow condition than the other conditions. Moreover, the cultured cells were healthy and adhered properly to the culture substrate. Enhanced mineralization and alkaline phosphatase activity were also observed under different perfusion conditions compared to the static conditions, indicating that the applied conditions play important roles in the proliferation and differentiation of hMSCs. Furthermore, we determined the expression levels of osteogenesis-related genes, including the runt-related protein 2 (Runx2), collagen type I (Col1), osteopontin (OPN), and osteocalcin (OCN), under various perfusion vis-à-vis static conditions and found that they were significantly affected by the applied conditions. Furthermore, the fluorescence intensities of OCN and OPN osteogenic gene markers were found to be enhanced in the 0.035 mL/min flow condition compared to the control, indicating that it was a suitable condition for osteogenic differentiation. Taken together, the findings of this study reveal that the developed cartridge device promotes the proliferation and differentiation of hMSCs and can potentially be used in the field of tissue engineering.

Project description:Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs).Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation.RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.

Dataset Information

Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device.

Publications

Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device.

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