Liver Ultrasound Histotripsy: Novel Analysis of the Histotripsy Site Cell Constituents with Implications for Histotripsy Application in Cell Transplantation and Cancer Therapy.

Introduction: Allogenic hepatocyte transplantation is an attractive alternative to whole-organ transplantation, particularly for the treatment of metabolic disorders and acute liver failure. However, the shortage of human donor organs for cell isolation, the low cell yield from decellularisation regimes, and low engraftment rates from portal administration of donor cells have restricted its clinical application. Using ultrasound histotripsy to provide a nidus in the liver for direct cell transplantation offers a new approach to overcoming key limitations in current cell therapy. We have analysed the liver cavity constituents to assess their potential as a site for cell delivery and implantation. Methods: Using human organ retrieval techniques, pig livers were collected from the abattoir and transported in ice-cold storage to the laboratory. Following 2 h of cold storage, the livers were flushed with organ preservation solution and placed on an organ perfusion circuit to maintain viability. Organs were perfused with Soltran™ organ preservation solution via the portal vein at a temperature of 24-30 °C. The perfusion circuit was oxygenated through equilibration with room air. Perfused livers (n=5) were subjected to ultrasound histotripsy, producing a total of 130 lesions. Lesions were generated by applying 50 pulses at 1 Hz pulse repetition frequency and 1% duty cycle using a single element 2 MHz bowl-shaped transducer (Sonic Concepts, H-148). Following histotripsy, a focal liver lesion was produced, which had a liquid centre. The fluid from each lesion was aspirated and cultured in medium (RPMI) at 37 °C in an incubator. Cell cultures were analysed at 1 and 7 days for cell viability and a live-dead assay was performed. The histotripsy sites were excised following aspiration and H&E staining was used to characterise the liver lesions. Cell morphology was determined by histology. Results: Histotripsy created a subcapsular lesion (~5 mm below the liver capsule; size ranging from 3 to 5 mm), which contained a suspension of cells. On average, 61×104 cells per mL were isolated. Hepatocytes were present in the aspirate, were viable at 24 h post isolation and remained viable in culture for up to 1 week, as determined by phalloidin/DAPI cell viability stains. Cultures up to 21 days revealed metabolically active live hepatocyte. Live-dead assays confirmed hepatocyte viability at 1 week (Day 1: 12% to Day 7: 45% live cells; p < 0.0001), which retained metabolic activity and morphology, confirmed on assay and microscopy. Cell Titre-GloTM showed a peak metabolic activity at 1 week (average luminescence 24.6 RLU; p < 0.0001) post-culture compared with the control (culture medium alone), reduced to 1/3 of peak level (7.85 RLU) by day 21. Conclusions: Histotripsy of the liver allows isolation and culture of hepatocytes with a high rate of viability after 1 week in culture. Reproducing these findings using human livers may lead to wide clinical applications in cell therapy.

Synergy between Photodynamic Therapy and Dactinomycin Chemotherapy in 2D and 3D Ovarian Cancer Cell Cultures.

In this study we explored the efficacy of combining low dose photodynamic therapy using a porphyrin photosensitiser and dactinomycin, a commonly used chemotherapeutic agent. The studies were carried out on compressed collagen 3D constructs of two human ovarian cancer cell lines (SKOV3 and HEY) versus their monolayer counterparts. An amphiphilc photosensitiser was employed, disulfonated tetraphenylporphine, which is not a substrate for ABC efflux transporters that can mediate drug resistance. The combination treatment was shown to be effective in both monolayer and 3D constructs of both cell lines, causing a significant and synergistic reduction in cell viability. Compared to dactinomycin alone or PDT alone, higher cell kill was found using 2D monolayer culture vs. 3D culture for the same doses. In 3D culture, the combination therapy resulted in 10 and 22 times higher cell kill in SKOV3 and HEY cells at the highest light dose compared to dactinomycin monotherapy, and 2.2 and 5.5 times higher cell kill than PDT alone. The combination of low dose PDT and dactinomycin appears to be a promising way to repurpose dactinomycin and widen its therapeutic applications.

Therapeutic enhancement of a cytotoxic agent using photochemical internalisation in 3D compressed collagen constructs of ovarian cancer.

Photochemical internalisation (PCI) is a method for enhancing delivery of drugs to their intracellular target sites of action. In this study we investigated the efficacy of PCI using a porphyrin photosensitiser and a cytotoxic agent on spheroid and non-spheroid compressed collagen 3D constructs of ovarian cancer versus conventional 2D culture. The therapeutic responses of two human carcinoma cell lines (SKOV3 and HEY) were compared using a range of assays including optical imaging. The treatment was shown to be effective in non-spheroid constructs of both cell lines causing a significant and synergistic reduction in cell viability measured at 48 or 96 h post-illumination. In the larger spheroid constructs, PCI was still effective but required higher saporin and photosensitiser doses. Moreover, in contrast to the 2D and non-spheroid experiments, where comparable efficacy was found for the two cell lines, HEY spheroid constructs were found to be more susceptible to PCI and a lower dose of saporin could be used. PCI treatment was observed to induce death principally by apoptosis in the 3D constructs compared to the mostly necrotic cell death caused by PDT. At low oxygen levels (1%) both PDT and PCI were significantly less effective in the constructs. STATEMENT OF SIGNIFICANCE: Assessment of new drugs or delivery systems for cancer therapy prior to conducting in vivo studies often relies on the use of conventional 2D cell culture, however 3D cancer constructs can provide more physiologically relevant information owing to their 3D architecture and the presence of an extracellular matrix. This study investigates the efficacy of Photochemical Internalisation mediated drug delivery in 3D constructs. In 3D cultures, both oxygen and drug delivery to the cells are limited by diffusion through the extracellular matrix unlike 2D models, and in our model we have used compressed collagen constructs where the density of collagen mimics physiological values. These 3D constructs are therefore well suited to studying drug delivery using PCI. Our study highlights the potential of these constructs for identifying differences in therapeutic response to PCI of two ovarian carcinoma lines.