RESOURCES

Abstract
Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.

Abstract
The demand for high-throughput and scalable cell expansion platforms that can accommodate diverse cell types remains a critical requirement across various biomedical fields. Fibronectin (Fn), an essential component of the extracellular matrix (ECM), has been used as a conformal surface coating for two-dimensional (2D) cell culture systems. However, the soluble, globular Fn used for 2D coatings differs structurally from the native Fn, which possesses a three-dimensional (3D) fibrillar structure. Herein, a large-scale engineered ECM (EECM) cell expansion platform based on a 3D fibrillar Fn network spanning over centimeters is presented. Extended fibrillar networks are formed by shearing dilute Fn solutions over tessellated polymeric scaffolds, which are conveniently prepared by 3D printing. The structure and size of the Fn-based 3D EECM scaffold are optimized by evaluating the proliferation of a colorectal tumor cell line, CT26, commonly used in the in vivo tumor immunotherapy models. The 3D EECM scaffolds support a fourfold more efficient tumor cell expansion than a conventional 2D culture system, demonstrating the potential efficacy in supporting the robust expansion of cancer cells ex vivo with an eye on cancer immunotherapy.