Inst of liver studies
kings' college hospital
Neonatal DCD livers with long warm ischemic times can produce good quality clinical grade hepatocytes.
Sharon C Lehec1, Ragai R Mitry1, Shirin-Elizabeth Khorsandi2, Charlotte Lee1, Celine Filippi1, Emer Fitzpatrick1, Simon Walker1, Raquel Fernandez-Dacosta1, James Hardie 3, Nigel Heaton2, Anil Dhawan1.
1Dhawan Lab, Institute of Liver Studies, Kings' College Hospital, London, United Kingdom; 2Liver Transplantation, Institute of Liver Studies, Kings' College Hospital, London, United Kingdom; 3NHS Blood and Transplant , London, United Kingdom
Introduction: Neonatal livers are being explored as a source of cells as these organs are not used in solid organ transplantation. In the context of donation after circulatory death (DCD), solid organ and cell transplantation warm ischemic time (WIT) is typically kept under 30 minutes for good clinical results. However, neonatal livers may be less sensitive to prolonged ischemia time.
Aim: To assess the feasibility of hepatocyte isolation from neonatal DCD livers with a WIT over 30 minutes.
Methods: DCD Donor Procedure: After withdrawal of life support, asystole and a 5min stand off period a rapid thoraco-abdominal incision is made, with venting into the chest. The aorta is cannulated, followed by cross clamp in the chest. The portal vein (PV) is cannulated either in the hilus or via the root of the small bowel mesentery. Ice is applied to cool the organ in situ. The gall bladder and bile duct are then flushed in situ with cold saline. Perfusion fluid used throughout is University of Wisconsin Solution (UW) with heparin 20,000 IU/L. In situ 500ml UW is perfused through the aorta and PV followed by rapid hepatectomy. A further 250 -500mls of UW is perfused through PV and hepatic artery until the canal effluent becomes clear. The biliary tree is flushed again. WIT is the time period when oxygen saturation reaches 70% or a systolic pressure of 50mmHg to aortic cannulation.
Donor liver 1: Female, blood group O+, weight 3.1kg, delivered by caesarean section at 39 weeks + 5 days. No maternal history of note and normal baby. Cause of death was hypoxic brain injury on day 2 after delivery from antenatal asphyxia of unknown aetiology. Serum total bilirubin 93 (3-20)IU/L, alkaline phosphatase (ALP) 119 (30-130)IU/L, aspartate transaminase (AST) 47 (10-50) IU/L, sodium 127 (135-145)mmol/L. WIT was 46 min and cold ischemia time was 7h.
Donor liver 2: Male, blood group O+, weight 4.2kg, one month old. Cause of death was intracranial haemorrhage after elective embolization vein of Galen malformation. Serum total bilirubin 127 (3-20)IU/L, ALP (30-130)IU/L 82, AST 37 (10-50) IU/L. WIT was 67min and cold ischemia time 4h.
Hepatocytes were isolated from the two neonatal livers under sterile conditions using a collagenase perfusion technique . Assessment of viability was made using trypan blue exclusion. The cells were cryopreserved in UW/10%DMSO/5% glucose, using a controlled rate freezer. After thawing, the cell viability was measured again, cell attachment and functions were analysed for donor liver 2.
Results: Donor liver 1: Weight=110g, and cell yield of 9.55 x 106 hepatocytes per gram (93% viability). On thawing the cell viability was 85% cells used successfully for clinical hepatocyte transplantation.
Donor liver 2: Weight =277g, and cell yield 5.42 x 106 hepatocytes per gram (85% viability). On thawing the cell viability was 66%, functional assays MTT (OD: mean ± SEM; 0.477±0.019) a 3 fold increase compared to DCD donors (>1 year old), urea 2.675µg/mg protein, gave an albumin concentration of 454.94µg/mg protein, SRB (OD: 2.76±0.04) equal to DCD donors (>1 year old) .These cells have been stored for clinical transplantation.
The average cell yield for DCD donors (>1 year old) at Kings’ is 2.2 x 106 cells per gram with 51% viability.
Conclusions: Neonatal DCD livers can experience long warm ischemic times and still produce good quality clinical grade hepatocytes. In the future, further analysis of cell functions would highlight potential differences between adult and neonatal donor hepatocytes.
 Mitry et al. (2003), Cell Transplantation 12(1), 69-74.
 Mitry et al. (2005), Transplantation Proceedings 37, 2391-2394.
 Hughes et al. (2006), Liver Transplantation 12(5), 713-7
11:00 - 12:30
|Hepatocyte Transplantation||Neonatal DCD livers with long warm ischemic times can produce good quality clinical grade hepatocytes.||Room 111-112|