Functionalized Carbon Nanotubes Increase the Viability of Post-Thaw Cord Blood Cells and Enhance the Overall Hematopoietic Progenitor Cell Expansion in Ex Vivo Culture
Authors: Chu Pat PY , Bari Sudipto , Ling Leong U , Chen Yee W , Lim Andrea , Fan Xiubo , Gay Florence PH , Ang Justina ML , Chiu Gigi NC , Hwang William YK 
Published: October 2019

Abstract
Abstract 1327
The clinical application of cord blood (CB) cells for transplantation is often hampered by the low cell number and viability from frozen cell sources. This problem could be partly resolved by ex vivo expansion and the improvement of CB viability through the use of a mesenchymal feeder cell layer or an appropriately designed material scaffold. Carbon-based nanomaterials have gained immense attention in biomedical applications in recent years, owing to their unique physicochemical characteristics. These novel materials have been applied as scaffolds in tissue engineering to increase the proliferation and differentiation of a wide range of cell types that include neurons and osteoblasts. In this study, we carried out a hitherto unreported use of functionalized carbon nanotubes to support the viability and ex vivo expansion of umbilical cord blood – mononucleated cells (UCB-MNCs). Specifically, the use of single-walled carbon nanotubes functionalized with carboxylic acid groups (fSWCNT-COOH) was selected, based on its improved aqueous dispersibility which constitutes better biocompatibility.
UCB-MNCs cultured in a cocktail of human stem cell factor, Flt-3 ligand, thrombopoietin and insulin-like growth factor-binding protein 2 were exposed to fSWCNT-COOH at various concentrations (0 to 1 mg/mL). The effects of fSWCNT-COOH treatment were characterized based on UCB-MNC viability, CD45+CD34+CD38− phenotypic-based progenitor cell number, total nucleated cell count and functional progenitor cell number.
In a concentration-dependent manner, fSWCNT-COOH was found to support the viability of UCB-MNCs, an effect that is similar to those observed with mesenchymal feeder co-culturing. After 3 days in culture, 0.5 and 1.0 mg/mL fSWCNT-COOH significantly (p<0.01) increased the percentage of viable CD45+ UCB-MNCs from 23.1±0.6% to 58.4±0.9% and 72.7±1.5%, respectively, as defined by Annexin-V/7AAD double negativity. Significant fold increases (p<0.01) in the phenotypically-defined progenitor cell numbers were observed after UCB-MNC exposure to 1 mg/mL fSWCNT-COOH for 11 days, with increases of 7.3±0.5-fold, 92.1±3.5 fold and 183.7±6.1-fold obtained respectively for viable CD45+, CD45+CD34+ and CD45+CD34+CD38− progenitor cell numbers. This finding was further supported by a significantly lower (p<0.01) side scatter value of 346.9±13.3 in the fSWCNT-COOH treated UCB-MNC as compared to the control treated with the cytokine cocktail alone (side scatter value of 405.6±4.9). Furthermore, a significant increase (p<0.01) in the expansion fold of functional CFU from 35.2±3.7 to 120.9±7.6 was obtained when UCB-MNC were treated with 1 mg/mL fSWCNT-COOH. Our results demonstrated that concentrations of fSWCNT-COOH as high as 1 mg/mL were non-toxic to UCB-MNCs. Similar to the mesenchymal feeder co-culture system, carbon-based nanomaterials such as fSWCNT-COOH could support UCB-MNC viability and progenitor expansion.
In conclusion, we have demonstrated a novel method of ex vivo expansion of UCB-MNCs using carbon-based nanomaterials. Further investigation is required to elucidate the exact biological mechanism of the cytoprotective effect of carbon-based nanomaterials coupled with validation of the ex vivo expanded UCB-MNCs using in vivo immunodeficient mice models.

Disclosures:
No relevant conflicts of interest to declare.