Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional collagen constructs.
Mechanical signal is important for regulating cellular activities, including proliferation, metabolism, matrix production, and orientation. Bioreactors with loading functions can be used to precondition cells in three-dimensional (3D) constructs so as to study the cellular responses to mechanical stimulation. However, full-scale bioreactor is not always an affordable option considering the high cost of equipment and the liter-sized medium with serum and growth factor supplements. In this study, a custom-built loading system was developed by coupling a conventional camera-equipped inverted research microscope with two micromanipulators. The system was programmed to deliver either cyclic compressive loading with different frequencies or static compressive loading for 1 week to investigate the cellular responses of human mesenchymal stem cells (hMSCs) entrapped in a 3D construct consists of reconstituted collagen fibers. Cellular properties, including their alignment, cytoskeleton, and cell metabolism, and properties of matrix molecules, such as collagen fiber alignment and glycosaminoglycan deposition, were evaluated. Using a MatLab-based image analysis program, reorientation of the entrapped cells from a random distribution to a preferred alignment along the loading direction in constructs with both static and cyclic compression has been demonstrated, but no such alignment was found in the free-floating controls. Fluorescent staining on filamentous actin cytoskeleton also confirmed the finding. Nevertheless, the collagen fiber meshwork entrapping the hMSCs remained randomly distributed, and no change in cellular metabolism and glycosaminoglycans production was noted. The current study provides a simple and affordable option toward setting up a mechanoregulation facility based on existing laboratory equipment and sheds new insights on the effect of mechanical loading on the alignment of hMSCs in 3D collagen constructs.
The University of Hong Kong , Hong Kong Special Administrative Region, China .
Kwan Lok
KL
Au-Yeung
Kam Yim
KY
Sze
Mai Har
MH
Sham
Barbara Pui
BP
Chan
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D003094:Collagen / Q000737:chemistry*; D019245:Compressive Strength; D003599:Cytoskeleton / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005583:Fourier Analysis; D006025:Glycosaminoglycans / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008846:Micromanipulation / Q000295:instrumentation* / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D013314:Stress, Mechanical
D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; D003094:Collagen