- Email: [email protected]
Isolation of Human Hepatocytes after Hepatic Warm and Cold Ischemia: A Practical Approach Using University of Wisconsin Solution
Cryobiology 38, 165–168 (1999) Article ID cryo.1999.2155, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Isolation of Human Hepatocytes after Hepatic Warm and Cold Ischemia: A Practical Approach Using University of Wisconsin Solution Mario Caruana,* Thierry Battle,† Barry Fuller,* ,1 and Brian Davidson* *University Department of Surgery and Liver Transplant Unit, Royal Free and University College Medical School, London NW3 2QG, United Kingdom; and †Centre for Applied Microbiology and Research, Porton Down, Wiltshire SP4 0JG, United Kingdom A method is described for isolating human hepatocytes from tissue fragments after warm and cold ischemia as experienced during hepatic resections. Cells with a high trypan blue dye exclusion and good culture characteristics were isolated by employing an initial tissue perfusion with UW solution. The method could facilitate transfer of liver tissues between distant centers for cell isolation studies. © 1999 Academic Press
surgical team and the cell biologists, which tends to limit the use of such techniques to major centers. It would increase the availability of human hepatocytes if resected tissues could be pooled from several surgical centers and transported to a central cell biology facility, but this would obviously introduce hypoxic damage which may negatively impact on isolation of viable hepatocytes. The surgical technique of resection itself necessitates restriction of blood supply and thus warm ischemia to the affected portion of the liver, which would be additive with hypoxia experienced after removal from the body. As part of an ongoing collaboration in liver cell biology, we have developed a technique for preservation of resected specimens which has allowed consistent and successful culture of human hepatocytes after transfer of tissues between distant institutions. In this study, nine pieces of normal liver tissue were obtained from a group of patients undergoing partial hepatectomy at the Royal Free Hospital with fully informed patient consent and approval. Patient characteristics are shown in Table 1. All the patients had normal liver function tests immediately prior to surgery. The liver tissues were processed immediately after removal from the abdominal cavity, and the margins of the tissues were selected to avoid tumor tissues. Histological examinations
Isolated hepatocytes are a valuable resource for studying liver cell metabolism, xenobiotic transformation, and the development of biological liver support systems (4, 6). There is a particular demand for human hepatocytes, since metabolic capabilities may differ from those measured in cells derived from animal livers. However, the availability of human livers for cell isolation is limited. In previous years, some human donor livers harvested for transplantation purposes were subsequently available for hepatocyte isolation (3, 5) as they were considered unsuitable for transplantation. More recently, the continued expansion of liver transplantation and use of marginal grafts due to organ shortage have meant that there are virtually no surplus organs. Human hepatocytes have also been isolated from end ’wedges’ of tissue taken during partial hepatectomy operations (1), most often for tumor resection. By careful separation of macroscopically normal tissue from tumor in the resected material and perfusion via the cut surfaces of major blood vessels, it has been possible to isolate and culture normal hepatocytes. In practical terms, this has necessitated a close working relationship between the
Received December 7, 1998; accepted January 13, 1999. 1 To whom correspondence should be addressed. Fax: 0171-431-4528. E-mail: [email protected]. 165
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
166
BRIEF COMMUNICATION TABLE 1 Sources of Tissues for Hepatocyte Isolation
Patient code
Age (years)
Sex
Diagnosis
01 02 03 04 05 06 07 08 09
44 60 69 72 51 62 77 66 67
F F F F F M F F M
C Ga Co Met H Ca Co Met Co Met Panc Met H Ca Co Met Co Met
Site taken Segment Segment Segment Segment Segment Segment Segment Segment Segment
1 VI VI V VI VI II 1 VI
Mass (g wt) 10 14 22 15 32 23 25 23 27
Note. Abbreviations: C Ga, carcinoma of gallbladder; Co Met, colorectal metastasis; H Ca, hepatocellular carcinoma; Panc Met, metastatic pancreatic cancer.
on small biopsies taken from the selected tissues demonstrated that they exhibited normal liver architecture and were without evidence of tumor cells. The surgical resection procedures (which necessitated clamping of hepatic blood vessels in order to achieve a bloodless field) resulted in a mean warm hypoxic interval of 102 min (range 60 –190 min, Table 2). The normal liver tissues were divided from the tumor tissue following as closely as possible the segmental anatomy of the liver originally described by Couinaud and more recently applied by other workers (7). This was done to identify the major branches of the portal venous system running through the specimens. Effort was made to ensure that as many surfaces of the wedges were covered by the external Glisson’s capsule as normally cover the liver (to aid later perfusion). The hepatic tissue was then placed in a kidney dish containing University of Wisconsin (UW) solution (2) at 4°C, and venous catheters (between 14 and 22 gauge in size) were used to cannulate exposed vessels. Each cannula was shortened to about 1 cm in length. Care was taken to perfuse the smaller subcapsular vessels in order to obtain reflux. UW solution was delivered slowly by syringe and continued until an even washout of blood was achieved (100 –200 ml total). The most prominent vein was cannulated with a size-matched cannula and then sutured in place. UW solution was again perfused
and vessels which leaked effluent along the cut surface were ligated. The tissue specimen was then immersed in fresh sterile UW solution in sterile bags and packed in ice for transport. The total cold hypoxic time, defined as time between first perfusion with UW solution and the subsequent tissue rewarming for cell isolation, was a mean of 220 min (range 175–360 min; Table 2). After transport, the liver tissue was placed in 15 ml of Hepes-buffered salts solution (in g/liter; Hepes, 2.6; NaCl, 8; KCl, 0.2; NaH 2PO 4, 0.12; EDTA, 1.0 mM) at room temperature and allowed to warm for approximately 15 min. The liver tissue was then perfused via the implanted catheter with Hepes-buffered salts warmed to 37°C at a flow rate of 30 ml/min for 15 min. This was followed by perfusion of the buffered salts solution plus added CaCl 2 (0.23 g/100 ml) and dissolved collagenase (0.06 g/100 ml;Gibco hepatocyte qualified collagenase). At the end of perfusion, the softened tissue was placed in sterile phosphate-buffered saline (PBS) and massaged with a sterile glass rod to achieve disaggregation. The cell suspension was then centrifuged at 50g for 2 min at 4°C, the supernatant was discarded, and the pellet was resuspended in fresh sterile PBS. Further purification was achieved by placing the resuspended cells on a gradient of 60% Percoll (Pharmacia) in PBS and centrifuging at 400g for 5 min. Cell numbers and initial viabilities were assessed by microscopy using trypan blue (0.1% in PBS) and are shown in Table 2. The results show that TABLE 2 Tissue Ischemia Times and Cell Isolation Data
Patient code
Warm ischemia (min)
Cold ischemia (min)
Cell yield per g liver (310 5)
Trypan blue exclusion (%)
01 02 03 04 05 06 07 08 09
149 75 60 160 90 85 120 70 80
195 175 360 175 220 240 220 200 210
3.6 2.8 1.7 2.6 1.2 1.7 1.6 4.0 3.5
95 94 94 93 95 94 93 98 98
BRIEF COMMUNICATION
167
FIG. 1. Phase contrast micrograph taken 12 h after plating Percoll-purified human hepatocytes. Cells exhibit a uniform typical hepatocyte morphology with well-defined polyhedral shape and high nuclear to cytoplasmic ratio. (Original magnification x200).
a consistent yield of viable cells (mean of 2.3 3 10 5 hepatocytes/g of tissue) was obtained using this method. Since between 10 and 20 g of tissue was harvested from the resected specimens, this provides an ample number of replicates for biochemical or metabolic studies, particularly if molecular techniques are employed. Sufficient cells were also obtained to establish hepatocyte cultures (if required) on collagencoated dishes (see Fig. 1). The use of UW solution to minimize cold hypoxic damage in whole livers stored at hypothermia for transplantation is well established (2) and isolated hepatocytes have been successfully produced from intact human livers which have been flush cooled with UW solution (3). Our current studies demonstrate that the UW solution is also efficient at preserving hepatocyte viability in resection specimens which have experienced a con-
siderable warm hypoxic insult before cooling. In our experience, the establishment of good UW perfusion to clear blood from the liver tissue before cold storage and the careful technique of cannulation and ligation of subsidiary ’leaking vessels’ were important to the success of the subsequent collagenase digestion. In fact, in preliminary studies, two tissue specimens were taken under the same conditions of hypoxia and were not cannulated and flushed with UW solution but were simply immersed in cold solution. In these cases, the collagenase perfusion failed to yield adequate digestion, and few hepatocytes were recovered. A major reason appeared to be blockage of the hepatic microcirculation by blood cells because of either coagulation or swelling of the hypoxic liver cells, trapping red blood cells in the sinusoids. Using the UW flush has allowed cell isolation to be performed after several hours,
168
BRIEF COMMUNICATION
which potentially could increase the availability of human hepatocytes from a centralized expert cell biology facility handling specimens from several outlying clinics. Further studies are currently underway to assess long-term culture of human hepatocytes isolated by this method. ACKNOWLEDGMENTS This work was submitted by M.C. as a dissertation for the degree of MSc in Surgical Sciences, Royal Free and U.C. Medical School, and was funded in part by grants to T.B. from the Department of Health and from the EC Biotech Program (BIOCT 972148). The authors thank Mrs. Hilary Moulsdale for technical assistance in the perfusion studies and Mr. Paul Fuller for preparing the manuscript. REFERENCES 1. Ballet, F., Bouma, M., Wang, S., Amit, N., Marais, J., and Infante, R. Isolation, culture and characterisation of adult human hepatocytes from surgical liver biopsies. Hepatology 4, 849 – 854 (1984). 2. Belzer, F. O., and Southard, J. Principles of solid organ preservation by cold storage. Transplantation 45, 673– 676 (1988).
3. Dou, M., De Sousa, G., Lacarelle, B., Placidi, M., Lechene de la Porte, P., Domingo, M., Lafont, H., and Rahmani, R. Thawed human hepatocytes in primary culture. Cryobiology 29, 454 – 469 (1992). 4. Gouillouzo, A. Use of isolated and cultured hepatocytes for xenobiotic metabolism and cytotoxicity studies. In “Isolated and Cultured Hepatocytes” (A. Gouillouzo and C. Guguen-Gouillouzo, Eds.), pp. 313–332. John Libby Eurotext, Paris, 1986. 5. Moshage, H., Rijntjes, P., Hafkenscheid, J., Roelofs, H., Williams, F., and Yap, S. Primary culture of cryopreserved adult human hepatocytes on homologous extracellular matrix and the influence of monocytic products on albumin synthesis. J. Hepatol. 7, 34 – 44 (1988). 6. Rozga, J., Williams, F., Ro, M., Neuzil, D., Giorgio, T., and Gouillouzo, A. Development of a bioartificial liver: Properties and functions of a hollow fibre module innoculated with liver cells. Hepatology 17, 258 –265 (1993). 7. Ryan, C., Carter, E., Jenkins, R., Sterling, L., Yarmush, M., Malt, R., and Tompkins, R. Isolation and longterm culture of human hepatocytes. Surgery 113, 48 –54 (1993).
BRIEF COMMUNICATION Isolation of Human Hepatocytes after Hepatic Warm and Cold Ischemia: A Practical Approach Using University of Wisconsin Solution Mario Caruana,* Thierry Battle,† Barry Fuller,* ,1 and Brian Davidson* *University Department of Surgery and Liver Transplant Unit, Royal Free and University College Medical School, London NW3 2QG, United Kingdom; and †Centre for Applied Microbiology and Research, Porton Down, Wiltshire SP4 0JG, United Kingdom A method is described for isolating human hepatocytes from tissue fragments after warm and cold ischemia as experienced during hepatic resections. Cells with a high trypan blue dye exclusion and good culture characteristics were isolated by employing an initial tissue perfusion with UW solution. The method could facilitate transfer of liver tissues between distant centers for cell isolation studies. © 1999 Academic Press
surgical team and the cell biologists, which tends to limit the use of such techniques to major centers. It would increase the availability of human hepatocytes if resected tissues could be pooled from several surgical centers and transported to a central cell biology facility, but this would obviously introduce hypoxic damage which may negatively impact on isolation of viable hepatocytes. The surgical technique of resection itself necessitates restriction of blood supply and thus warm ischemia to the affected portion of the liver, which would be additive with hypoxia experienced after removal from the body. As part of an ongoing collaboration in liver cell biology, we have developed a technique for preservation of resected specimens which has allowed consistent and successful culture of human hepatocytes after transfer of tissues between distant institutions. In this study, nine pieces of normal liver tissue were obtained from a group of patients undergoing partial hepatectomy at the Royal Free Hospital with fully informed patient consent and approval. Patient characteristics are shown in Table 1. All the patients had normal liver function tests immediately prior to surgery. The liver tissues were processed immediately after removal from the abdominal cavity, and the margins of the tissues were selected to avoid tumor tissues. Histological examinations
Isolated hepatocytes are a valuable resource for studying liver cell metabolism, xenobiotic transformation, and the development of biological liver support systems (4, 6). There is a particular demand for human hepatocytes, since metabolic capabilities may differ from those measured in cells derived from animal livers. However, the availability of human livers for cell isolation is limited. In previous years, some human donor livers harvested for transplantation purposes were subsequently available for hepatocyte isolation (3, 5) as they were considered unsuitable for transplantation. More recently, the continued expansion of liver transplantation and use of marginal grafts due to organ shortage have meant that there are virtually no surplus organs. Human hepatocytes have also been isolated from end ’wedges’ of tissue taken during partial hepatectomy operations (1), most often for tumor resection. By careful separation of macroscopically normal tissue from tumor in the resected material and perfusion via the cut surfaces of major blood vessels, it has been possible to isolate and culture normal hepatocytes. In practical terms, this has necessitated a close working relationship between the
Received December 7, 1998; accepted January 13, 1999. 1 To whom correspondence should be addressed. Fax: 0171-431-4528. E-mail: [email protected]. 165
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
166
BRIEF COMMUNICATION TABLE 1 Sources of Tissues for Hepatocyte Isolation
Patient code
Age (years)
Sex
Diagnosis
01 02 03 04 05 06 07 08 09
44 60 69 72 51 62 77 66 67
F F F F F M F F M
C Ga Co Met H Ca Co Met Co Met Panc Met H Ca Co Met Co Met
Site taken Segment Segment Segment Segment Segment Segment Segment Segment Segment
1 VI VI V VI VI II 1 VI
Mass (g wt) 10 14 22 15 32 23 25 23 27
Note. Abbreviations: C Ga, carcinoma of gallbladder; Co Met, colorectal metastasis; H Ca, hepatocellular carcinoma; Panc Met, metastatic pancreatic cancer.
on small biopsies taken from the selected tissues demonstrated that they exhibited normal liver architecture and were without evidence of tumor cells. The surgical resection procedures (which necessitated clamping of hepatic blood vessels in order to achieve a bloodless field) resulted in a mean warm hypoxic interval of 102 min (range 60 –190 min, Table 2). The normal liver tissues were divided from the tumor tissue following as closely as possible the segmental anatomy of the liver originally described by Couinaud and more recently applied by other workers (7). This was done to identify the major branches of the portal venous system running through the specimens. Effort was made to ensure that as many surfaces of the wedges were covered by the external Glisson’s capsule as normally cover the liver (to aid later perfusion). The hepatic tissue was then placed in a kidney dish containing University of Wisconsin (UW) solution (2) at 4°C, and venous catheters (between 14 and 22 gauge in size) were used to cannulate exposed vessels. Each cannula was shortened to about 1 cm in length. Care was taken to perfuse the smaller subcapsular vessels in order to obtain reflux. UW solution was delivered slowly by syringe and continued until an even washout of blood was achieved (100 –200 ml total). The most prominent vein was cannulated with a size-matched cannula and then sutured in place. UW solution was again perfused
and vessels which leaked effluent along the cut surface were ligated. The tissue specimen was then immersed in fresh sterile UW solution in sterile bags and packed in ice for transport. The total cold hypoxic time, defined as time between first perfusion with UW solution and the subsequent tissue rewarming for cell isolation, was a mean of 220 min (range 175–360 min; Table 2). After transport, the liver tissue was placed in 15 ml of Hepes-buffered salts solution (in g/liter; Hepes, 2.6; NaCl, 8; KCl, 0.2; NaH 2PO 4, 0.12; EDTA, 1.0 mM) at room temperature and allowed to warm for approximately 15 min. The liver tissue was then perfused via the implanted catheter with Hepes-buffered salts warmed to 37°C at a flow rate of 30 ml/min for 15 min. This was followed by perfusion of the buffered salts solution plus added CaCl 2 (0.23 g/100 ml) and dissolved collagenase (0.06 g/100 ml;Gibco hepatocyte qualified collagenase). At the end of perfusion, the softened tissue was placed in sterile phosphate-buffered saline (PBS) and massaged with a sterile glass rod to achieve disaggregation. The cell suspension was then centrifuged at 50g for 2 min at 4°C, the supernatant was discarded, and the pellet was resuspended in fresh sterile PBS. Further purification was achieved by placing the resuspended cells on a gradient of 60% Percoll (Pharmacia) in PBS and centrifuging at 400g for 5 min. Cell numbers and initial viabilities were assessed by microscopy using trypan blue (0.1% in PBS) and are shown in Table 2. The results show that TABLE 2 Tissue Ischemia Times and Cell Isolation Data
Patient code
Warm ischemia (min)
Cold ischemia (min)
Cell yield per g liver (310 5)
Trypan blue exclusion (%)
01 02 03 04 05 06 07 08 09
149 75 60 160 90 85 120 70 80
195 175 360 175 220 240 220 200 210
3.6 2.8 1.7 2.6 1.2 1.7 1.6 4.0 3.5
95 94 94 93 95 94 93 98 98
BRIEF COMMUNICATION
167
FIG. 1. Phase contrast micrograph taken 12 h after plating Percoll-purified human hepatocytes. Cells exhibit a uniform typical hepatocyte morphology with well-defined polyhedral shape and high nuclear to cytoplasmic ratio. (Original magnification x200).
a consistent yield of viable cells (mean of 2.3 3 10 5 hepatocytes/g of tissue) was obtained using this method. Since between 10 and 20 g of tissue was harvested from the resected specimens, this provides an ample number of replicates for biochemical or metabolic studies, particularly if molecular techniques are employed. Sufficient cells were also obtained to establish hepatocyte cultures (if required) on collagencoated dishes (see Fig. 1). The use of UW solution to minimize cold hypoxic damage in whole livers stored at hypothermia for transplantation is well established (2) and isolated hepatocytes have been successfully produced from intact human livers which have been flush cooled with UW solution (3). Our current studies demonstrate that the UW solution is also efficient at preserving hepatocyte viability in resection specimens which have experienced a con-
siderable warm hypoxic insult before cooling. In our experience, the establishment of good UW perfusion to clear blood from the liver tissue before cold storage and the careful technique of cannulation and ligation of subsidiary ’leaking vessels’ were important to the success of the subsequent collagenase digestion. In fact, in preliminary studies, two tissue specimens were taken under the same conditions of hypoxia and were not cannulated and flushed with UW solution but were simply immersed in cold solution. In these cases, the collagenase perfusion failed to yield adequate digestion, and few hepatocytes were recovered. A major reason appeared to be blockage of the hepatic microcirculation by blood cells because of either coagulation or swelling of the hypoxic liver cells, trapping red blood cells in the sinusoids. Using the UW flush has allowed cell isolation to be performed after several hours,
168
BRIEF COMMUNICATION
which potentially could increase the availability of human hepatocytes from a centralized expert cell biology facility handling specimens from several outlying clinics. Further studies are currently underway to assess long-term culture of human hepatocytes isolated by this method. ACKNOWLEDGMENTS This work was submitted by M.C. as a dissertation for the degree of MSc in Surgical Sciences, Royal Free and U.C. Medical School, and was funded in part by grants to T.B. from the Department of Health and from the EC Biotech Program (BIOCT 972148). The authors thank Mrs. Hilary Moulsdale for technical assistance in the perfusion studies and Mr. Paul Fuller for preparing the manuscript. REFERENCES 1. Ballet, F., Bouma, M., Wang, S., Amit, N., Marais, J., and Infante, R. Isolation, culture and characterisation of adult human hepatocytes from surgical liver biopsies. Hepatology 4, 849 – 854 (1984). 2. Belzer, F. O., and Southard, J. Principles of solid organ preservation by cold storage. Transplantation 45, 673– 676 (1988).
3. Dou, M., De Sousa, G., Lacarelle, B., Placidi, M., Lechene de la Porte, P., Domingo, M., Lafont, H., and Rahmani, R. Thawed human hepatocytes in primary culture. Cryobiology 29, 454 – 469 (1992). 4. Gouillouzo, A. Use of isolated and cultured hepatocytes for xenobiotic metabolism and cytotoxicity studies. In “Isolated and Cultured Hepatocytes” (A. Gouillouzo and C. Guguen-Gouillouzo, Eds.), pp. 313–332. John Libby Eurotext, Paris, 1986. 5. Moshage, H., Rijntjes, P., Hafkenscheid, J., Roelofs, H., Williams, F., and Yap, S. Primary culture of cryopreserved adult human hepatocytes on homologous extracellular matrix and the influence of monocytic products on albumin synthesis. J. Hepatol. 7, 34 – 44 (1988). 6. Rozga, J., Williams, F., Ro, M., Neuzil, D., Giorgio, T., and Gouillouzo, A. Development of a bioartificial liver: Properties and functions of a hollow fibre module innoculated with liver cells. Hepatology 17, 258 –265 (1993). 7. Ryan, C., Carter, E., Jenkins, R., Sterling, L., Yarmush, M., Malt, R., and Tompkins, R. Isolation and longterm culture of human hepatocytes. Surgery 113, 48 –54 (1993).