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Is Normothermic Perfusion a Feasible Option for Intestinal Preservation? Experience with a Porcine Model
TRANSPLANTATION AND TISSUE ENGINEERING
seeded with human aortic smooth muscle cells, and human umbilical vein endothelial cells. Constructs were imaged using multiphoton microscopy. Using Imaris software, the endothelial cell and pericyte fluorescence was visualized as a 3D volume, and each image channel was individually thresholded to generate 3D surfaces. Distance of individual pericytes from their nearest endothelial cell neighbor was then manually measured.
Bio-Artificial Liver Generation using Multilineage Xenogenic and Heterogenic Cells Wessam A Hassnein, MD, Mehmet C Uluer, MD, Jhade D Woodall, MD, Urmil Dhru, Arielle Cimeno, MD, Dawn M Parsell, Carlos Rivera-Pratts, Cinthia B Drachenberg, MD, Rolf N Barth, MD, FACS, John C LaMattina, MD, FACS University of Maryland, Baltimore, MD
RESULTS: MPM revealed an interconnected network of endothelial lined vessels with supporting perivascular pericytes and smooth muscle cells in anatomically appropriate orientation. At day 0 of culture, pericytes were randomly dispersed throughout the bulk. Following 14 days, pericyte coverage surrounding endothelial lined vessels dramatically increased with the highest density of pericytes localized within the 150-200 mm diffusion distance.
INTRODUCTION: Recent years have seen a proliferation of methods leading to successful organ decellularization. Nonetheless, the ability to reproducibly generate a robust recellularized construct remains challenging. Here we demonstrate the ability of rat liver scaffolds to support the growth of a variety of human and rat cells. METHODS: Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds, preserving the extracellular matrix. Initial studies were performed using a human tumor cell line (hepG2 through the bile duct). A second series utilized human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein). In the third series of experiments, a rat neonatal cell slurry isolated from 3-day old neonatal rats was seeded on the scaffold via the bile duct providing all cell types found in the liver.
CONCLUSIONS: We have fabricated hydrogel constructs with a complex hierarchical vasculature by combining our prior sacrificial technique with facilitated cellular self-assembly. A tissue engineered construct which contains the necessary environment to stimulate spontaneous vascular self-assembly of cells is a major step forward in the development of tissue engineering. Is Normothermic Perfusion a Feasible Option for Intestinal Preservation? Experience with a Porcine Model Alessandra Bertacco, Abedalrazaq O AlKukhun, MBBS, Alice Kitay, Moha Aldajani, MD, Francesco D’Amico, MD, PhD, Manuel I Rodriguez-Davalos, MD, FACS, David C Mulligan, MD, FACS, John P Geibel Yale University, Yale School of Medicine, New Haven, CT
RESULTS: Human HepG2 cells grew readily on the scaffold (n¼20). HepG2 cells co-cultured with HUVECs demonstrated viable endothelial lining with concurrent hepatocyte growth (n¼10). After neonatal cell slurry infusion (n¼9), obvious foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. CONCLUSIONS: A decellularized rat liver scaffold supports human HepG2 and HUVEC engraftment and survival of neonatal rat hepatocytes and endothelial cells isolated as a cell slurry in appropriately selected media.
INTRODUCTION: Normothermic preservation can potentially reduce ischemic injury caused by prolonged cold preservation, and maintain aspects of graft function throughout the preservation process. The aim of this study was to evaluate the effect of normothermic perfusion using normothermic machine perfusion in a porcine intestinal model.
Facilitated Self-Assembly of Pre-Vascularized Tissue Engineered Constructs Ross H Weinreb, Kerry A Morrison, Julia Jin, Xue Dong, Omer Kaymakcalan, MD, Rachel M Akintayo, MD, Sushmita Mukherjee, PhD, Jason A Spector, MD, FACS Weill Cornell Medical Center, New York, NY
METHODS: Yorkshire pigs were used to procure small intestines. Heparinization was achieved by intravenous injection before the sacrifice of the pig. Systemic perfusion using UW Solution was performed by cannulating the aorta 30-minutes after animal death. The small intestine was harvested, and flushed with isotonic saline. The intestinal lumen and the superior mesenteric artery were connected to cannulas and then to the Intestinal Perfusion Unit allowing continuous perfusion on the vascular and luminal side. Vascular side perfusion was performed with UW, while the lumen was perfused with 0.9% saline at 37 C for 6-8 hours. Control segments were collected at harvest and stored in saline and UW solutions at 4 C and 37 C. A pathologist, using the Park/Chiu-Scoring-System for grading intestinal ischemia, evaluated tissue samples.
INTRODUCTION: Fabrication of thicker tissue engineered skin substitutes with an intricate vascular bed of branching vessels recapitulating the complex hierarchical organization of human tissue continues to pose a major obstacle. In order to fabricate these higher order structures, we attempt to facilitate cellular self-assembly around our neovessels created using a sacrificial technique. METHODS: Pluronic F127 macrofibers 1.5 mm in diameter and 100-500 mm-interwoven microfibers were sacrificed within type I collagen with encapsulated human placental pericytes and human foreskin fibroblasts in bulk collagen. Channels were intraluminally
RESULTS: Intestine preserved in normothermic conditions showed a high degree of damage (G4-G5) without any significant
e50
http://dx.doi.org/10.1016/j.jamcollsurg.2016.08.145 ISSN 1072-7515/16
Vol. 223, No. 4S2, October 2016
difference between static or continuous preservation and time, in comparison to the static cold counterparts. CONCLUSIONS: There are potential advantages for preserving tissues under normothermic conditions such as restoring the tissue ATP content. This study shows that normothermic perfusion of the small intestine does not demonstrate superiority over static normothermic or hypothermic preservation. Furthermore, warm perfusion does not benefit the overall outcome as the intestine vulnerability to injury from ischemia increases.
Optimizing Recellularization of Decellularized Whole-Liver Graft: From Which Route and with Which Cell? Satoshi Ogiso, MD, Kentaro Yasuchika, MD, PhD, Ken Fukumitsu, Takamichi Ishii, MD, PhD, Hidenobu Kojima, MD, Ryoya Yamaoka, MD, Hokahiro Katayama, Sadahiko Kita, MD, Takayuki Kawai, MD, Shinji Uemoto, MD, PhD Kyoto University, Kyoto, Japan INTRODUCTION: Recently, decellularization technique has been successfully adapted to generate three-dimensional scaffolds for whole-liver engineering. However, there remains task of effective recellularization. We evaluated a new strategy for recellularization of the parenchyma using bile duct (BD) as a seeding route and fetal hepatocyte as a cell source. METHODS: Rat decellularized liver were repopulated with mice adult hepatocytes through two different routes, portal vein (PV) and BD. Distribution of the engrafted cells were analyzed by H&E staining of liver sections. Decellularized liver scaffolds were repopulated with two different cell sources, mice adult hepatocytes and E14.5 fetal hepatocytes, and cultivated for up to 1 week. Immunohistochemistry of liver sections, biochemical analyses of metabolic activity, quantitative RT-PCR, and quantification of DNA contents were undertaken. RESULTS: The majority of cells seeded via PV remained within portal branches but cells seeded via BD were deposited surrounding central veins in hepatic lobule. A higher proportion of cells were distributed in the parenchyma when seeded via BD (87.3 % versus 7.5 %). Gene expression levels of albumin, glucose-6phosphatase, transferrin, and ganmma-glutamyl-transpeptidase were higher for fetal hepatocytes cultivated in liver scaffolds compared to those cultivated in collagen-coated dishes. Immunohistochemistry revealed that fetal hepatocytes differentiated into hepatocytic and cholangiocytic lineages. Albumin production was greater for fetal hepatocytes, whilst urea synthesis was similar. The DNA content, a surrogate of cell viability, was higher in fetal hepatocytes-repopulating livers compared to adult hepatocytesrepopulating livers. CONCLUSIONS: The use of BD for recellularization is promising and fetal hepatocyte is a good candidate for a cell source of wholeliver engineering.
Scientific Forum: 2016 Clinical Congress
e51
Predicting Success in Septuagenarians Undergoing Liver Transplantation: A Single Center Analysis of 140 Recipients Keri E Lunsford, MD, PhD, Vatche G Agopian, MD, FACS, Ali Zarrinpar, MD, PhD, Fady M Kaldas, MD, FACS, Hasan Yersiz, MD, Douglas G Farmer, MD, FACS, Ronald W Busuttil, MD, PhD, FACS University of California, Los Angeles, CA INTRODUCTION: Recipient age is a significant predictor of mortality after liver transplantation (OLT). Despite an aging recipient population with increasing incidence of liver failure, data regarding predictors of successful OLT in septuagenarians are scarce. METHODS: Pretransplant comorbidities and factors affecting survival were retrospectively analyzed for 140 septuagenarian primary OLT recipients between 1984-2015. RESULTS: Overall 1-, 3-, and 5-year survival for septuagenarians were 76%, 66%, and 56%, significantly inferior to 82%, 73%, and 68% for adults age < 70 (n¼3600, p¼0.004). Septuagenarians suffering 1-year mortality were significantly more likely to have renal dysfunction, encephalopathy, pulmonary disease, diabetes with end-organ damage, poor functional status, malnutrition, and sepsis (Table). Multivariate predictors of mortality in septuagenarians included pretransplant creatinine (HR 1.5, p¼0.007), FEV1 (HR 0.98, p¼0.02), and sepsis within 30 days (HR 3.3, p¼0.04). Compared to younger recipients, septuagenarians with MELD>20 (p¼0.02) or pretransplant hospitalization (p¼0.001) demonstrated significantly inferior survival. Table. Univariate analysis of factors associated with 1 year mortality in OLT recipients with Age >70 years
Laboratory MELD at Transplant Renal dysfunction Serum creatinine (corrected for HD) Pre-transplant dialysis Pulmonary dysfunction FEV-1 (percent predicted) Pulmonary disease Functional status (Karnofsky score) Moderate e severe encephalopathy Diabetes with end-organ damage Malnutrition (poor oral intake) Sepsis within 30 d of OLT
Age >70 y Age >70 y with Death with Death >1 y (N¼100)* <1 y (N¼35)* p Value 15 36 0.07 1.2
1.9
0.04
13.3% (14)
28.6% (10)
0.04
89% 8.6% 70 18.1% 9.5% 31.4% 9.5%
74% 22.9% 50 34.3% 22.9% 54.3% 22.9%
0.001 0.02 0.01 0.004 0.03 0.02 0.04
(9) (19) (10) (33) (10)
(8) (12) (8) (19) (8)
*Median presented for continuous data, %(N) presented for categorical data.
CONCLUSIONS: With the largest single-center study evaluating OLT in septuagenarians, we demonstrate good pretransplant functional status results in survival equivalent to younger recipients after OLT; however, multiorgan comorbidities portend poor outcomes. Careful candidate selection is necessary to optimize resource utilization in older patients; however, excellent results may be achieved in appropriate recipients.
seeded with human aortic smooth muscle cells, and human umbilical vein endothelial cells. Constructs were imaged using multiphoton microscopy. Using Imaris software, the endothelial cell and pericyte fluorescence was visualized as a 3D volume, and each image channel was individually thresholded to generate 3D surfaces. Distance of individual pericytes from their nearest endothelial cell neighbor was then manually measured.
Bio-Artificial Liver Generation using Multilineage Xenogenic and Heterogenic Cells Wessam A Hassnein, MD, Mehmet C Uluer, MD, Jhade D Woodall, MD, Urmil Dhru, Arielle Cimeno, MD, Dawn M Parsell, Carlos Rivera-Pratts, Cinthia B Drachenberg, MD, Rolf N Barth, MD, FACS, John C LaMattina, MD, FACS University of Maryland, Baltimore, MD
RESULTS: MPM revealed an interconnected network of endothelial lined vessels with supporting perivascular pericytes and smooth muscle cells in anatomically appropriate orientation. At day 0 of culture, pericytes were randomly dispersed throughout the bulk. Following 14 days, pericyte coverage surrounding endothelial lined vessels dramatically increased with the highest density of pericytes localized within the 150-200 mm diffusion distance.
INTRODUCTION: Recent years have seen a proliferation of methods leading to successful organ decellularization. Nonetheless, the ability to reproducibly generate a robust recellularized construct remains challenging. Here we demonstrate the ability of rat liver scaffolds to support the growth of a variety of human and rat cells. METHODS: Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds, preserving the extracellular matrix. Initial studies were performed using a human tumor cell line (hepG2 through the bile duct). A second series utilized human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein). In the third series of experiments, a rat neonatal cell slurry isolated from 3-day old neonatal rats was seeded on the scaffold via the bile duct providing all cell types found in the liver.
CONCLUSIONS: We have fabricated hydrogel constructs with a complex hierarchical vasculature by combining our prior sacrificial technique with facilitated cellular self-assembly. A tissue engineered construct which contains the necessary environment to stimulate spontaneous vascular self-assembly of cells is a major step forward in the development of tissue engineering. Is Normothermic Perfusion a Feasible Option for Intestinal Preservation? Experience with a Porcine Model Alessandra Bertacco, Abedalrazaq O AlKukhun, MBBS, Alice Kitay, Moha Aldajani, MD, Francesco D’Amico, MD, PhD, Manuel I Rodriguez-Davalos, MD, FACS, David C Mulligan, MD, FACS, John P Geibel Yale University, Yale School of Medicine, New Haven, CT
RESULTS: Human HepG2 cells grew readily on the scaffold (n¼20). HepG2 cells co-cultured with HUVECs demonstrated viable endothelial lining with concurrent hepatocyte growth (n¼10). After neonatal cell slurry infusion (n¼9), obvious foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. CONCLUSIONS: A decellularized rat liver scaffold supports human HepG2 and HUVEC engraftment and survival of neonatal rat hepatocytes and endothelial cells isolated as a cell slurry in appropriately selected media.
INTRODUCTION: Normothermic preservation can potentially reduce ischemic injury caused by prolonged cold preservation, and maintain aspects of graft function throughout the preservation process. The aim of this study was to evaluate the effect of normothermic perfusion using normothermic machine perfusion in a porcine intestinal model.
Facilitated Self-Assembly of Pre-Vascularized Tissue Engineered Constructs Ross H Weinreb, Kerry A Morrison, Julia Jin, Xue Dong, Omer Kaymakcalan, MD, Rachel M Akintayo, MD, Sushmita Mukherjee, PhD, Jason A Spector, MD, FACS Weill Cornell Medical Center, New York, NY
METHODS: Yorkshire pigs were used to procure small intestines. Heparinization was achieved by intravenous injection before the sacrifice of the pig. Systemic perfusion using UW Solution was performed by cannulating the aorta 30-minutes after animal death. The small intestine was harvested, and flushed with isotonic saline. The intestinal lumen and the superior mesenteric artery were connected to cannulas and then to the Intestinal Perfusion Unit allowing continuous perfusion on the vascular and luminal side. Vascular side perfusion was performed with UW, while the lumen was perfused with 0.9% saline at 37 C for 6-8 hours. Control segments were collected at harvest and stored in saline and UW solutions at 4 C and 37 C. A pathologist, using the Park/Chiu-Scoring-System for grading intestinal ischemia, evaluated tissue samples.
INTRODUCTION: Fabrication of thicker tissue engineered skin substitutes with an intricate vascular bed of branching vessels recapitulating the complex hierarchical organization of human tissue continues to pose a major obstacle. In order to fabricate these higher order structures, we attempt to facilitate cellular self-assembly around our neovessels created using a sacrificial technique. METHODS: Pluronic F127 macrofibers 1.5 mm in diameter and 100-500 mm-interwoven microfibers were sacrificed within type I collagen with encapsulated human placental pericytes and human foreskin fibroblasts in bulk collagen. Channels were intraluminally
RESULTS: Intestine preserved in normothermic conditions showed a high degree of damage (G4-G5) without any significant
e50
http://dx.doi.org/10.1016/j.jamcollsurg.2016.08.145 ISSN 1072-7515/16
Vol. 223, No. 4S2, October 2016
difference between static or continuous preservation and time, in comparison to the static cold counterparts. CONCLUSIONS: There are potential advantages for preserving tissues under normothermic conditions such as restoring the tissue ATP content. This study shows that normothermic perfusion of the small intestine does not demonstrate superiority over static normothermic or hypothermic preservation. Furthermore, warm perfusion does not benefit the overall outcome as the intestine vulnerability to injury from ischemia increases.
Optimizing Recellularization of Decellularized Whole-Liver Graft: From Which Route and with Which Cell? Satoshi Ogiso, MD, Kentaro Yasuchika, MD, PhD, Ken Fukumitsu, Takamichi Ishii, MD, PhD, Hidenobu Kojima, MD, Ryoya Yamaoka, MD, Hokahiro Katayama, Sadahiko Kita, MD, Takayuki Kawai, MD, Shinji Uemoto, MD, PhD Kyoto University, Kyoto, Japan INTRODUCTION: Recently, decellularization technique has been successfully adapted to generate three-dimensional scaffolds for whole-liver engineering. However, there remains task of effective recellularization. We evaluated a new strategy for recellularization of the parenchyma using bile duct (BD) as a seeding route and fetal hepatocyte as a cell source. METHODS: Rat decellularized liver were repopulated with mice adult hepatocytes through two different routes, portal vein (PV) and BD. Distribution of the engrafted cells were analyzed by H&E staining of liver sections. Decellularized liver scaffolds were repopulated with two different cell sources, mice adult hepatocytes and E14.5 fetal hepatocytes, and cultivated for up to 1 week. Immunohistochemistry of liver sections, biochemical analyses of metabolic activity, quantitative RT-PCR, and quantification of DNA contents were undertaken. RESULTS: The majority of cells seeded via PV remained within portal branches but cells seeded via BD were deposited surrounding central veins in hepatic lobule. A higher proportion of cells were distributed in the parenchyma when seeded via BD (87.3 % versus 7.5 %). Gene expression levels of albumin, glucose-6phosphatase, transferrin, and ganmma-glutamyl-transpeptidase were higher for fetal hepatocytes cultivated in liver scaffolds compared to those cultivated in collagen-coated dishes. Immunohistochemistry revealed that fetal hepatocytes differentiated into hepatocytic and cholangiocytic lineages. Albumin production was greater for fetal hepatocytes, whilst urea synthesis was similar. The DNA content, a surrogate of cell viability, was higher in fetal hepatocytes-repopulating livers compared to adult hepatocytesrepopulating livers. CONCLUSIONS: The use of BD for recellularization is promising and fetal hepatocyte is a good candidate for a cell source of wholeliver engineering.
Scientific Forum: 2016 Clinical Congress
e51
Predicting Success in Septuagenarians Undergoing Liver Transplantation: A Single Center Analysis of 140 Recipients Keri E Lunsford, MD, PhD, Vatche G Agopian, MD, FACS, Ali Zarrinpar, MD, PhD, Fady M Kaldas, MD, FACS, Hasan Yersiz, MD, Douglas G Farmer, MD, FACS, Ronald W Busuttil, MD, PhD, FACS University of California, Los Angeles, CA INTRODUCTION: Recipient age is a significant predictor of mortality after liver transplantation (OLT). Despite an aging recipient population with increasing incidence of liver failure, data regarding predictors of successful OLT in septuagenarians are scarce. METHODS: Pretransplant comorbidities and factors affecting survival were retrospectively analyzed for 140 septuagenarian primary OLT recipients between 1984-2015. RESULTS: Overall 1-, 3-, and 5-year survival for septuagenarians were 76%, 66%, and 56%, significantly inferior to 82%, 73%, and 68% for adults age < 70 (n¼3600, p¼0.004). Septuagenarians suffering 1-year mortality were significantly more likely to have renal dysfunction, encephalopathy, pulmonary disease, diabetes with end-organ damage, poor functional status, malnutrition, and sepsis (Table). Multivariate predictors of mortality in septuagenarians included pretransplant creatinine (HR 1.5, p¼0.007), FEV1 (HR 0.98, p¼0.02), and sepsis within 30 days (HR 3.3, p¼0.04). Compared to younger recipients, septuagenarians with MELD>20 (p¼0.02) or pretransplant hospitalization (p¼0.001) demonstrated significantly inferior survival. Table. Univariate analysis of factors associated with 1 year mortality in OLT recipients with Age >70 years
Laboratory MELD at Transplant Renal dysfunction Serum creatinine (corrected for HD) Pre-transplant dialysis Pulmonary dysfunction FEV-1 (percent predicted) Pulmonary disease Functional status (Karnofsky score) Moderate e severe encephalopathy Diabetes with end-organ damage Malnutrition (poor oral intake) Sepsis within 30 d of OLT
Age >70 y Age >70 y with Death with Death >1 y (N¼100)* <1 y (N¼35)* p Value 15 36 0.07 1.2
1.9
0.04
13.3% (14)
28.6% (10)
0.04
89% 8.6% 70 18.1% 9.5% 31.4% 9.5%
74% 22.9% 50 34.3% 22.9% 54.3% 22.9%
0.001 0.02 0.01 0.004 0.03 0.02 0.04
(9) (19) (10) (33) (10)
(8) (12) (8) (19) (8)
*Median presented for continuous data, %(N) presented for categorical data.
CONCLUSIONS: With the largest single-center study evaluating OLT in septuagenarians, we demonstrate good pretransplant functional status results in survival equivalent to younger recipients after OLT; however, multiorgan comorbidities portend poor outcomes. Careful candidate selection is necessary to optimize resource utilization in older patients; however, excellent results may be achieved in appropriate recipients.