Engineering Decellularized Matrices to Support Adherent Cell Therapy

Whole-organ perfusion decellularization was performed with rat hearts on a modified chromatography apparatus. Analysis of the flow properties and effluent material over time provided insights into the decellularization process, and allowed non-destructive testing of perfused cardiac tissue. Decellularized matrices were stored for up to 1 year at -80°C and then conditioned to remove residual detergent and cryoprotectant. Tissue was reseeded with canine blood outgrowth endothelial cells (BOECs) and cultured in an autoclavable closed-circuit bubble-free reactor. The entire process was considered in the context of eventual scale-up in equipment design, the use of disposable components, and extracellular matrix (ECM) product storage. Tissue patch substrates for cell growth were studied for cytotoxic effects towards process development. Decellularization protocols were compared. Extracellular matrix derived coatings and gels were investigated as process assays and potential cell delivery vehicles. Peracetic acid and UV disinfection were tested. Micronized ECM carriers were developed for scalable culture, with considerations to carrier morphology, cell attachment, and egress. Micronized ECM carriers were tested with a novel in vitro assay to simulate the support of adherent cells for gene-modified cell therapy

Transgene-host Cell Interactions Mediate Significant Influences On The Production, Stability, And Function Of Recombinant Canine Fviii.

Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII