Abstract
Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2’-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.
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Acknowledgments
The authors thank Dr. Wei Xu for the isolation of rabbit dermal fibroblasts. TEM samples were processed and imaged in the Electron Microscopy Core Facility, Department of Pathology, The University of Texas Health Science Center, San Antonio, Tex., USA. The authors are also grateful to Dr. Tao You for assistance with generating the SEM images.
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This work was presented, in part, at the Symposium on Advanced Wound Care (SAWC) and Wound Healing Society (WHS) Annual Meeting, May 1–5, 2013, Colorado Convention Center, Denver, Colo., USA.
This work was supported by the United States Army Medical Research and Material Command (W81XWH-10-2-0054). The authors are employees of the U.S. Government, and this work was prepared as part of their official duties.
The opinions or assertions contained herein are the private views of the authors and are not to be construed as being official or reflecting the views of the Department of Defense or U.S. Government.
No competing financial interests exist.
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Supplemental Figure 1
Confocal microscopic images of Live/Dead stain on ASC aggregates of three representative cell densities that have been maintained for 7 days in culture: (a) 250 cells/aggregate, (b) 1000 cells/aggregate, (c) 16000 cells/aggregate. Live or viable cells were stained green (calcein AM), whereas dead or compromised cells were stained red (ethidium homodimer-1). Non-viable cells were evident in ASC aggregates that were larger in size (greater than 4000 cells/aggregate). Magnification of images is 200×. (GIF 49 kb)
Supplemental Figure 2
Fluorescent images of ASC aggregates (at 500 cells/aggregate) labeled with EdU and counterstained with Hoechst after 7 days in culture (bottom panel, d-f). Images of ASC monolayers labeled with EdU and counterstained with Hoechst (top panel, a-c). Limited cell proliferation (low abundance of EdU-positive cells) was observed in ASC aggregates throughout the experiment, regardless of the size of the aggregates. Magnification of images is 100× (GIF 92 kb)
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Rettinger, C.L., Fourcaudot, A.B., Hong, S.J. et al. In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates. Cell Tissue Res 358, 395–405 (2014). https://doi.org/10.1007/s00441-014-1939-0
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DOI: https://doi.org/10.1007/s00441-014-1939-0