Tissue-engineered liver microreactor as an in vitro surrogate assay for gene delivery

Abstract

The lack of correlation between in vitro and in vivo gene delivery experiments presents a significant obstacle in the progress of gene therapy studies by preventing the extrapolation of successful cell culture results into animals. This phenomenon has also been documented in the specific case of liver where standard hepatocyte culture systems fail to reliably predict the in vivo performance of gene delivery vectors. This is possibly a consequence of the loss of differentiated phenotype that these cells undergo when they are dissociated from their in vivo environment and cultured in vitro. This problem underscores the necessity for better in vitro models that can mimic the physiological environment and responses of in vivo liver tissue. This thesis aimed at developing an alternative in vitro gene delivery assay based on the Tissue-Engineered Liver Microreactor, a culture system designed to facilitate the morphogenesis of three-dimensional tissue-like structures from isolated liver cells under continuous perfusion, maintain cell viability and hepatic functionality for long-term culture periods and enable repeated in situ observation with microscopy. We developed experimental assays to non-invasively detect and quantify gene delivery efficiency in the 3D environment of the microreactor culture based on the application of 2-photon microscopy and spectroscopy.

(cont.) These techniques provide a convenient platform for comparative analysis of different vectors. Our main objective was to compare the gene delivery efficiency of an adenoviral vector (Ad5-CMV-EGFP) in the microreactor system and 2D hepatocyte monolayer culture. Quantitative assays were developed based on Real-Time PCR and RT-PCR to measure the levels of Ad vector uptake and transgene expression. The Ad mass transport in both systems was mathematically modeled to estimate the Ad uptake constant as a basis for comparison of delivery efficiency. This parameter was found to be significantly higher in the microreactor system, suggesting a more efficient mechanism of Ad internalization. Moreover, gene expression was measured in terms of transgene mRNA levels; the ratio of gene expression relative to Ad uptake was estimated as the basis for comparison of vector transcription efficiency. No significant difference was found between the 2 systems. These results provide some evidence that a more physiological culture system can yield different information (potentially more relevant to the in vivo situation) compared to standard in vitro culture.