Abstract
To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 μL of medium at 0–2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 μL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 103 cells/cm2 NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 103 cells/cm2 NWF-fiber and fold increase in cell density by 16–48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.
Similar content being viewed by others
References
Chua S, Yuen CM, Leu S, Lin YC, Sun CK, Yip HK (2010) Adipose-derived mesenchymal stem cells markedly attenuate brain infarct size and improve neurological function in rats. Eur Heart J 31:80
Nienow AW (2006) Reactor engineering in large scale animal cell culture. Cytotechnology 50:9–33
Sanford KK, Earle WR, Evans VJ, Waltz HK, Shannon JE (1950) The measurement of proliferation in tissue cultures by enumeration of cell nuclei. Anat Rec 106:243
Sato Y, Wakitani S, Takagi M (2013) Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells. J Biosci Bioeng 116:734–739
Schop D, Janssen FW, de Bruijn JD, Van Dijkhuizen-Radersma R (2008) Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism. J Tissue Eng and Regen Med 2:126–135
Takagi M, Okumura H, Okada T, Kobayashi N, Kiyota T, Ueda K (1994) An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture. J Ferment Bioeng 77:301–306
Takagi M, Sasaki T, Yoshida Y (1999) Spatial development of the cultivateon of a bone marrow stromal cell line carriers. Cytotechnology 31:225–231
Tomimori Y, Takagi M, Yoshida T (2000) The construction of an in vitro three-dimensional hematopoietic microenvironment for mouse bone marrow cells employing porous carriers. Cytotechnology 34(1–2):121–130
Yamahara K, Taguchi A, Soma T, Ogawa H, Ikeda T, Yoshimatsu J (2014) Clinical application of human amnion-derived mesenchymal stem cells for the treatment of acute GVHD. Placenta 35:A6
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Fu, B., Yamada, K., Fujiwara, M. et al. Protocol of mesenchymal stem cell inoculation to nonwoven fabric scaffold. Cytotechnology 71, 743–750 (2019). https://doi.org/10.1007/s10616-019-00320-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-019-00320-7