Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells

Abstract

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

Inter-donor variability of extracellular matrix production in long-term cultures of human fibroblasts

“…The strength of the fresh CAM sheets was evaluated using a perforation assay in accordance with the ISO 7198:1998/2001 standard, as previously described. 29 The CAMs cultured within six-well plates were manually detached from the plastic and positioned on a custom-made clamping device machine. A nine-mm-diameter spherical Teflon® indenter was used to perform a perforation test.…”

Immunomodulation Strategies for the Successful Regeneration of a Tissue‐Engineered Vascular Graft

“…[ 62 ] The fibrous collagen served as the backbone of the sheets to provide the required mechanical performance. [ 63 ] The autologous CAM‐TEVGs were first evaluated in clinical trials as arteriovenous access devices, which functioned successfully for up to 20 months in high‐risk populations and maintained high patency rates with no access‐related infections. [ 64 ] However, the high cost of production (greater than $15,000) and the long waiting time (6–9 months) prevented its application in clinical settings.…”

“…And as a result, the optimized culturing conditions were studied and identified to support animal cell‐assembled ECM sheets. [ 63 ]…”

“…Torres et al also investigated the reproducibility of CAM cell sheets using animal cells. [63] Both human and ovine fibroblasts were able to generate reproducible robust cell sheets, with the human CAM being threefold stronger than the ovine CAM. On the other hand, canine and porcine fibroblasts had difficulty producing cohesive sheets.…”

“…And as a result, the optimized culturing conditions were studied and identified to support animal cell-assembled ECM sheets. [63] In comparison to Niklason and L'Heureux, who adopted the deposition of ECM protein as the primary graft material, Breuer and colleagues studied synthetic biodegradable vascular grafts seeded with autologous bone marrow-derived mononuclear cells (BM-MNCs) in small [68] and large animal models [69] as well as in a clinical trial. [17] The graft was composed of a PGA knitted mesh sealed by a 50:50 co-polymer of l-lactide and 𝜖-caprolactone, designed to degrade within 6 months.…”