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-Glo^TM 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.

Non-invasive therapeutic-focused ultrasound (US) can be used for the mechanical dissociation of tissue and is described as histotripsy. We have performed US histotripsy in viable perfused ex vivo porcine livers as a step in the development of a novel approach to hepatocyte cell transplantation. The histotripsy nidus was created with a 2 MHz single-element focused US transducer, producing 50 pulses of 10 ms duration, with peak positive and negative pressure values of P+ = 77.7 MPa and P− = –13.7 MPaat focus, respectively, and a duty cycle of 1%. Here, we present the histological analysis, including 3D reconstruction of histotripsy sites. Five whole porcine livers were retrieved fresh from the abattoir using human transplant retrieval and cold static preservation techniques and were then perfused using an organ preservation circuit. Whilst under perfusion, histotripsy was performed to randomly selected sites on the live. Fifteen lesional sites were formalin-fixed and paraffin-embedded. Sections were stained with Haematoxylin and Eosin and picro-Sirius red, and they were also stained for reticulin. Additionally, two lesion sites were used for 3D reconstruction. The core of the typical lesion consisted of eosinophilic material associated with reticulin loss, collagen damage including loss of birefringence to fibrous septa, and perilesional portal tracts, including large portal vein branches, but intact peri-lesional hepatic plates. The 3D reconstruction of two histotripsy sites was successful and confirmed the feasibility of this approach to investigate the effects of histotripsy on tissue in detail.