Maintenance of viability and proliferation of 3T3 cell aggregates incorporating fibroin microspheres into cultures
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ORIGINAL ARTICLE
Maintenance of viability and proliferation of 3T3 cell aggregates incorporating fibroin microspheres into cultures Da Yeong Ryu . Se Chang Kwon . Ji Young Kim . Won Hur
Received: 1 July 2019 / Accepted: 26 June 2020 / Published online: 14 August 2020 Ó Springer Nature B.V. 2020
Abstract This study investigated whether micronsized microspheres can be used as dispersed scaffolds where anchorage-dependent cells can proliferate and survive in suspension culture. Aggregates of murine 3T3 cells in a non-adherent plate cultured remained viable for more than 2 weeks by the presence of 0.5 mg/ml fibroin microspheres. A nucleoside incorporation assay confirmed the proliferation of 3T3 cells in the aggregates only when cultured with microspheres. Under these conditions, the glucose consumption rate of 3T3 cells increased to 66.5 nmol day-1 cell-1. Histological analysis demonstrated that the intercellular space of cell aggregates was larger in cultures supplemented with 0.5 mg/ml microspheres than in non-supplemented cultures. The cell aggregates with microspheres also exhibited a reduced arrest in G1 phase. Transmission electron microscopy verified the presence of microspheres in the space between cells in aggregates. Fibroin microspheres maintained the viability and proliferability of 3T3 cells cultured under non-adherent conditions and thus can be used to develop viable suspensions of anchorage-dependent cells.
D. Y. Ryu S. C. Kwon J. Y. Kim W. Hur (&) Department of Biotechnology and Bioengineering, Kangwon National University, Chuncheon 200-701, South Korea e-mail: [email protected]
Keywords 3T3 cells Anchorage dependence Fibroin microspheres Suspension culture
Introduction Anchorage dependence is a typical property of nontumorigenic cells and refers to the requirement of these cells for a solid surface on which to grow. Fibronectin and vitronectin usually mediate cell attachment to solid surfaces in serum-containing culture medium because cells prefer adhesion proteins on solid surfaces rather than directly interacting with other cells in vitro (Wilson et al. 2005). Loss of adhesion leads to a specific apoptotic response called anoikis (Gilmore 2005). For example, 3T3 cells, which originate from murine embryos, continue to proliferate until cell-to-cell contact inhibition occurs when cultured as a monolayer on a polystyrene surface, but form aggregates and eventually undergo apoptotic death when cultured under non-adherent conditions (Goundiam et al. 2010). Thus, suspensions of viable non-tumorigenic cells are mostly obtained using microcarriers (Kong et al. 1999) or scaffold particles (Bible et al. 2009), which provide a near-flat surface for cell adhesion due to their large size. Nanospheres, which are much smaller than cells, have been used to prepare a suspension of viable HEK293 cells (Ryu et al. 2003) and a viable
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suspension in serum-free media (Ryu et al. 2004). Similar results were achieved using nanofibers (Shin et al.
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