Scalable Culture Strategies for the Expansion of Patient-Derived Cancer Stem Cell Lines

Abstract

Cancer stem cells (CSCs) have recently raised great interest as a promising biological system for designing effective cancer therapies. The scarcity of CSCs in vivo and the consequent low numbers obtained from biopsies represent a major hurdle to the development of such strategies. It is therefore necessary to design robust scalable methods to enable efficient expansion of bona fide CSCs in vitro. Here, we evaluated the applicability of computer-controlled bioreactors combined with 3D aggregate culture and microcarrier technology, widely used in stem cell bioprocessing, for the expansion and enrichment of CSCs isolated from different types of solid tumors—colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC) from two patients. Results show that these culture strategies improved cell expansion and CSC enrichment. Both patient-derived CSC lines were able to grow on microcarriers, the best results being achieved for PPlus 102-L, Pro-F 102-L, Fact 102-L, and CGEN 102-L beads (5-fold and 40-fold increase in total cell concentration for CRC and NSCLC cells, respectively, in 6 days). As for 3D aggregate culture strategy, the cell proliferation profile was donor dependent. NSCLC cells were the only cells able to form aggregates and proliferate, and the flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller was the most efficient configuration for cell growth (21-fold increase in cell concentration achieved in 8 days). Serum-free medium promotes CSC enrichment in both 3D aggregate and microcarrier cultures. The protocols developed herein for CSC expansion have the potential to be transferred to clinical and industrial settings, providing key insights to guide bioprocess design towards the production of enriched CSC cultures in higher quantity and improved quality.

Inhibitory Effect and Mechanism of Ursolic Acid on Cisplatin‐Induced Resistance and Stemness in Human Lung Cancer A549 Cells

“…Studies have shown that CSCs can be identifed in tumors by their mammosphere formation capacity [47]. CSCs from epithelial organs can be expanded as sphere-like cellular aggregates in a serum-free medium containing epidermal growth factor and basic fbroblast growth factor [85][86][87]. In the present study, the spheres of A549-CisR cells were more numerous and larger than those of the parental cells after being cultured with the previous medium, meaning the ability to form spheres was increased after a low-dose cisplatin induction, which illustrates a more observable characteristic of CSCs.…”

Polymer Thin Film Promotes Tumor Spheroid Formation via JAK2-STAT3 Signaling Primed by Fibronectin-Integrin α5 and Sustained by LMO2-LDB1 Complex

“…For example, cellular surface markers such as CD24, CD44, CD133, epithelial cell adhesion molecule (EpCAM), leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5), and CD117 have been used to isolate CSCs from cancer cells using fluorescence-activated cell sorting (FACS) and magnetic cell sorting [ 10 , 11 , 12 , 13 ]. In addition, serum-free culture conditions supplemented with various combinations of growth factors have been applied to enrich or maintain isolated CSCs from patients diagnosed with cancer [ 14 , 15 , 16 ].…”

Antiproliferative Effect of Colonic Fermented Phenolic Compounds from Jaboticaba (Myrciaria trunciflora) Fruit Peel in a 3D Cell Model of Colorectal Cancer

“…In vitro 3D cell culture models using human and patient-derived CRC cell lines have been described as to be promising tools to evaluate the role of many antitumoral compounds, since these models provide cell–cell and cell–matrix interactions and, in this way, a cellular context similar to the cancer microenvironment. In particular, spheroid cultures of human CRC cells produced in stirred culture systems have been shown to be enriched in cancer stem cells and better recapitulate the in vivo tumor behavior [ 4 , 5 ]. Of note, these models have been used to ensure an accurate evaluation of the anticancer potential of phytochemicals derived from brassicas, nuts and citrus fruits [ 4 , 6 , 7 , 8 ].…”