Chondrogenic differentiation of human adipose‑derived stem cells using microcarrier and bioreactor combination technique

Kang,Hongjun; Lu,Shibi; Peng,Jiang; Yang,Qiang; Liu,Shuyun; Zhang,Li; Huang,Jingxiang; Sui,Xiang; Zhao,Bin; Wang,Aiyuan; Xu,Wenjing; Guo,Quanyi; Song,Qing

doi:10.3892/mmr.2014.2820

Chondrogenic differentiation of human adipose‑derived stem cells using microcarrier and bioreactor combination technique

Authors:
- Hongjun Kang
- Shibi Lu
- Jiang Peng
- Qiang Yang
- Shuyun Liu
- Li Zhang
- Jingxiang Huang
- Xiang Sui
- Bin Zhao
- Aiyuan Wang
- Wenjing Xu
- Quanyi Guo
- Qing Song
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Affiliations: Key Laboratory of PLA, Department of Orthopaedics, Chinese PLA General Hospital, Beijing 100853, P.R. China, Department of Spine Surgery, Tianjin Hospital, Hexi, Tianjin 300211, P.R. China, Critical Care Medicine, Chinese PLA General Hospital, Beijing 100853, P.R. China
Published online on: October 30, 2014 https://doi.org/10.3892/mmr.2014.2820
Pages: 1195-1199

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Abstract

The aim of the current study was to explore the application of microcarrier technology in the rapid amplification and chondrogenic differentiation of human adipose‑derived stem cells (ADSCs) in a rotating bioreactor. Human ADSCs were cultivated with Cytodex 3 microcarriers in a rotary cell culture system (RCCS), and using inverted and scanning electron microscopes, the ADSCs were observed on the surface of the microcarriers. The harvested ADSCs were stained with safranin‑O or toluidine blue histochemical stains, and type II collagen immunohistochemical stain. ADSCs were adherent to the surface of Cytodex 3 microcarriers by 24 h. They became short and spindle‑shaped, and as time progressed, the adherence of the cells to the microcarriers gradually improved. By the end of the culture period, the cell densities were ~19 times that of the initial cell density. The harvested cells on microcarriers were safranin-O and toluidine blue staining and collagen Ⅱ‑positive staining, which were stronger than the control group. The application of microcarrier technology is able to rapidly amplify human ADSC proliferation and successfully implement chondrogenic differentiation in vitro.

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1	Zuk PA, Zhu M, Mizuno H, et al: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7:211–228. 2001. View Article : Google Scholar : PubMed/NCBI
2	Zuk PA, Zhu M, Ashjian P, et al: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 13:4279–4295. 2002. View Article : Google Scholar : PubMed/NCBI
3	Planat-Benard V, Silvestre JS, Cousin B, et al: Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 109:656–663. 2004. View Article : Google Scholar : PubMed/NCBI
4	Ogawa R, Mizuno H, Watanabe A, et al: Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. Biochem Biophys Res Commun. 313:871–877. 2004. View Article : Google Scholar : PubMed/NCBI
5	Ogawa R, Mizuno H, Watanabe A, et al: Adipogenic differentiation by adipose derived stem cells harvested from GFP transgenic mice including relationship of sex differences. Biochem Biophys Res Commun. 319:511–517. 2004. View Article : Google Scholar : PubMed/NCBI
6	Fujimura J, Ogawa R, Mizuno H, et al: Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice. Biochem Biophys Res Commun. 333:116–121. 2005. View Article : Google Scholar : PubMed/NCBI
7	Marlovits S, Tichy B, Truppe M, et al: Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor. Int J Artif Organs. 26:319–330. 2003.PubMed/NCBI
8	De Ugarte DA, Morizono K, Elbarbary A, et al: Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 174:101–109. 2003. View Article : Google Scholar : PubMed/NCBI
9	Koch RJ and Gorti GK: Tissue engineering with chondrocytes. Facial Plast Surg. 18:59–68. 2002. View Article : Google Scholar : PubMed/NCBI
10	Winter A, Breit S, Parsch D, et al: Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum. 48:418–429. 2003. View Article : Google Scholar : PubMed/NCBI
11	Ogawa R, Mizuno S, Murphy GF and Orgill DP: The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells. Tissue Eng Part A. 15:2937–2945. 2009. View Article : Google Scholar : PubMed/NCBI
12	Costa AR, Withers J, Rodrigues ME, et al: The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody. Springerplus. 2:252013. View Article : Google Scholar : PubMed/NCBI
13	Chu L and Robinson DK: Industrial choices for protein production by large scale cell culture. Cur Opin Biotechnol. 12:180–187. 2001. View Article : Google Scholar
14	Rudolph G, Lindner P, Gierse A, et al: Online monitoring of microcarrier based fibroblast cultivations with in situ microscopy. Biotechnol Bioeng. 99:136–145. 2008. View Article : Google Scholar
15	Saris DB, Sanyal A, An KN, et al: Periosteum reponds to dynamic fluid pressure by proliferating in vitro. J Orthop Res. 17:668–677. 1999. View Article : Google Scholar : PubMed/NCBI
16	Griffiths B: Scale-up of suspension and anchorage-dependent animal cells. Mol Biotechnol. 17:225–238. 2001. View Article : Google Scholar : PubMed/NCBI
17	Dan JL, Xu JZ, Zhou Q, et al: Culturing human mesenchymal stem cells in vitro in large scale on microcarriers. Orthop J Chin. 12:1158–1160. 2004.(In Chinese).
18	Parkkinen JJ, Ikonen J, Lammi MJ, et al: Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants. Arch Biochem Biophys. 300:458–465. 1993. View Article : Google Scholar : PubMed/NCBI
19	Lammi MJ, Inkinen R, Parkkinen JJ, et al: Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure. Biochem J. 304:723–730. 1994.PubMed/NCBI
20	Simon WH, Mak A and Spirt A: The effect of shear fatigue on bovine articular cartilage. J Orthop Res. 8:86–93. 1990. View Article : Google Scholar : PubMed/NCBI
21	Tomatsu T, Imai N, Takeuchi N, Takahashi K and Kimura N: Experimentally produced fractures of articular cartilage and bone. The effects of shear forces on the pig knee. J Bone Joint Surg Br. 74:457–462. 1992.PubMed/NCBI
22	van der Kraan PM, Buma P, van Kuppevelt T and van den Berg WB: Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering. Osteoarthritis Cartilage. 10:631–637. 2002. View Article : Google Scholar : PubMed/NCBI
23	Dickinson SC, Sims TJ, Pittarello L, et al: Quantitative outcome measures of cartilage repair in patients treated by tissue engineering. Tissue Eng. 11:277–287. 2005. View Article : Google Scholar : PubMed/NCBI
24	Li Z, Kupcsik L, Yao SJ, Alini M and Stoddart MJ: Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. J Cell Mol Med. 14:1338–1346. 2010. View Article : Google Scholar
25	Li Z, Yao SJ, Alini M and Stoddart MJ: Chondrogenesis of human bone marrow mesenchymal stem cells in fibrin-polyurethane composites is modulated by frequency and amplitude of dynamic compression and shear stress. Tissue Eng Part A. 16:575–584. 2010. View Article : Google Scholar