- Email: [email protected]
Cryopreserved human hepatocytes from cell bank: In vitro function and clinical application
Hepatocytes Cryopreserved Human Hepatocytes From Cell Bank: In Vitro Function and Clinical Application U. Baccarani, A. Sanna, A. Cariani, M. Sainz, G.L. Adani, D. Lorenzin, D. Montanaro, M. Scalamogna, G. Piccolo, A. Risaliti, F. Bresadola, and A. Donini ABSTRACT We Aimed to analyze the in vitro function of isolated and cryopreserved human hepatocytes (CHH) from a cell bank and to define their potential clinical application in a bioartificial liver (BAL) device. Methods. Over 24 months, 103 not transplantable livers were utilized for human hepatocytes isolation and cryopreservation. Hepatocytes isolated by collagenase were analyzed for yield, viability, diazepam metabolism, and production of human albumin after isolation and cryopreservation in LN2. Results. The causes for refusal for transplantation were macrosteatosis ⬎60%, ischemic damage due to donor hypotension, and nonviral cirrhosis in 60%, 11%, and 8%, respectively. Cell yields averaged 7 million hepatocytes per gram of liver of mean viability of 80% ⫾ 13%. The viability of CHH after thawing averaged 50%. Thawed hepatocytes showed diazepam metabolism, and human albumin synthesis comparable to fresh cells. CHH were utilized as the biological component of a BAL for temporary support as three applications of two patients affected by fulminant hepatic failure awaiting urgent transplant. Ten to 13 billion viable CHH were loaded into each BAL. Liver function showed bilirubin and ammonia reduction at the end of each treatment. One patient was successfully bridged to emergency OLTx after one BAL; in the second case there was spontaneous recovery of liver function after two BAL. Conclusions. Recovery of donor human livers unwanted for transplantation allowed isolation and cryopreservation of viable and functionally active human hepatocytes, which have been banked and successfully used for clinical applications of a BAL device.
L
IVER TRANSPLANTATION is currently limited by the shortage of organs. Many patients who could benefit from the transplant die because they do not get a chance to be included in a liver transplant waiting list.1 Parallel to the development of new surgical strategies, such as partial liver transplantation from both living and cadaveric donors aimed at increasing the number of transplantable organs, techniques of cell transplantation and cellbased therapy are potential tools for the treatment of terminal liver disease.2 However, large scale clinical application of cell-based liver therapy, such as hepatocyte transplantation and bioartificial liver use, which have shown interesting results in animal models, is limited by the lack of 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.12.230 256
a reliable source of liver cells.3 The development of facilities dedicated to the isolation, preparation, and banking of human-hepatocytes is considered a possible solution to this From the Department of Surgery & Transplantation, University of Udine (U.B., M.S., G.L.A., D.L., D.M., A.R., F.B.); Bank of Human Hepatocytes (A.S., A.C.), Udine; NITp, Ospedale Maggiore Milano (M.S., G.P.); Department of Emergency Surgery, University of Perugia (A.D.), Ferrara, Italy. Address reprints requests to Umberto Baccarani, MD, PhD, Department of Surgery and Transplantation Unit, University Hospital, Pad. Petracco, P.le S.M. della Misericordia, 33100, Udine Italy. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 256 –259 (2005)
CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK
Fig 1.
257
Diazepam metabolism of cryopreserved human hepatocytes.
problem. The aims of this paper were to (1) analyze the in vitro function of isolated, cryopreserved human hepatocytes (CHH) from a cell bank obtained on a national basis from livers deemed unusable for transplantation at harvesting; and (2) define their potential clinical application in a bioartificial liver (BAL) device as a bridge to liver transplantation. MATERIALS AND METHODS Aim 1 One-hundred three not transplantable livers in the Nord Italia Transplant area (NITp and AIRT) were utilized for human hepatocyte isolation and cryopreservation from July 2001 to December 2003. Organs were judged not usable for transplantation during harvesting based on gross appearance and hepatic biopsy. The main causes for refusal were macrosteatosis ⬎60%, ischemic damage due to cardiac arrest or prolonged donor hypotension, and nonviral cirrhosis in 60%, 11%, and 8%, respectively. Liver retrieval was performed according to the usual surgical technique. The organs were always flushed and cooled with preservation solutions and placed on ice for transportation to the laboratory for hepatocyte isolation. The method of liver digestion and hepatocyte isolation has already been described.4 Cell yield and viability was measured by a trypan blue dye exclusion test using a Neubauer chamber. Functional integrity and activity of the isolated hepatocytes, before and after cryopreservation, was measured by a diazepam metabolism assay (HPLC) and albumin production by immunonephelometry. Hepatocytes were progressively cryopreserved using DMSO 10% to 20% as cryoprotectant at ⫺80°C and finally placed in LN2. For thawing, the hepatocytes were removed from LN2 and placed in a 37°C water bath. DMSO was gradually removed by centrifugation at 40g for 3 minutes.
Aim 2 Thawed hepatocytes were loaded into a BAL device used as a bridge to emergency liver transplantation in two cases of fulminant hepatic failure. Thirteen billion cells with a 70% postthawing viability as determined by a trypan blue dye test that were ABO compatible and functional based on cytochrome P450 metabolic
reactions5 were loaded into two hollow-fiber bioreactors (Dideco, Mirandola, Italy) with a molecular weight cut-off of 0.6 m. Bioartificial liver treatment was performed using the Performer BAL (Rand, Medolla, Italy) device, which integrates an external BAL circuit, complete with bioreactors loaded with the hepatocytes, a Plasorba cartridge (Dideco, Braun-Carex), the heater, and an oxygenator (Lilliput Dideco, Mirandola, Italy) with continuous plasmapheresis over a plasma filter. Plasma obtained from patients at a flow rate of 50 mL/min was recirculated through the BAL circuit at a flow rate of 200 to 250 mL/min with the interposition of a plasma reservoir. Heparin was used as the anticoagulant (2000 IU/h). Every treatment lasted 4 hours until a liver became available for transplantation or there was clinical improvement.
RESULTS
Overall hepatocyte yield averaged 9 ⫾ 3 ⫻ 109 with mean viability of 80% ⫾ 13%. Viable hepatocyte yield, per gram of liver tissue digested averaged 7 million cells. The best results in terms of cell yield and viability were obtained from steatotic organs, while cirrhotic livers yielded fewer cells/g liver (3 ⫻ 106 vs 7 ⫻ 105, P ⬍ .043 and 81% vs 66%, P ⫽ .27, respectively). The viability of cryopreserved hepatocytes after thawing averaged 50%. Cryopreserved hepatocytes maintained normal diazepam clearance and metabolic production on days 0 and ⫹1 after thawing (Fig 1). Moreover, fresh and thawed hepatocytes produced a comparable amount of human albumin when maintained in culture conditions on collagen coated dishes (Fig 2). The morphology of cultured human hepatocytes showed typical features of confluent colonies of normally appearing viable liver cells. Two cases of fulminant hepatic failure were treated with a BAL device loaded with cryopreserved/ thawed human hepatocytes as a bridge to emergency liver transplantation. Both patients had encephalopathy grade IV and were intubated with mechanically assisted ventilation. The etiology of fulminant hepatic failure were acute HBV infection and cryptogenic. The first patient, affected by acute HBV infection, underwent a single BAL treatment
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Fig 2. Albumin synthesis fresh vs cryopreserved human hepatocytes.
resulting in reduction of the bilirubin (from 11.9 mg/dL pretreatment to 6.9 mg/dL at the end of BAL) and ammonia level (from 216 mcg/dL pretreatment to 193 mcg/dL at the end of BAL). At the end of this BAL procedure, a liver became available for transplantation that was performed successfully. The second patient had two BAL procedures over a 2-day course. The bilirubin level decreased (from 8.7 mg/dL to 5.4 mg/dL) during the first treatment, while increasing during the delay between the first and the second application to 12.3 mg/dL. At the end of the second BAL procedure, the bilirubin dropped to 8.9 mg/dL. Also the ammonia level dropped at the end of the second BAL (130 mcg/mL from 177 mcg/mL). Clinical conditions improved after the second BAL treatment; the patient finally recovered without the need for liver transplantation. Renal function remained stable in both patients with normal urine output. Hemodynamic and respiratory stability was maintained throughout the treatment period. Both patients did not receive mannitol or hyperventilation therapy during the treatment. Neurological conditions remained stable with no clinical evidence of developing brain edema. Intracranial pressure was not measured due to coagulopathy and the risk of bleeding. No adverse events were recorded. DISCUSSION
Great controversy still exists about what is the best source of hepatocytes to be loaded into bioartificial liver devices.3 The ideal hepatocyte should have a detoxification function. Moreover, biotransforming properties of hepatocytes, such as phase I and II reactions, should be assured by the hepatocytes loaded into the bioartificial liver. Both xenogenic (porcine) and human hepatocytes derived from tumor cell lines or immortalized have been shown to be able to catalyze the requested functions, although at different levels. Moreover tumor-derived or immortalized human hepatocytes can potentially show a risk of neoplastic transmission to patients, requiring selective filters that hamper passage of molecules from the patients’ circulation to the hepatocytes. Porcine xenogenic hepatocytes have been considered a possible solution to the problem of the biological component of BAL devices because they are easily available in large quantities,6 but they have known disadvantages.7–9
Isolated primary human hepatocytes would probably be the best source of cells for BAL devices because no concerns exist regarding immunogenicity, zoonosis, and biocompatibility. However, the major drawbacks to the widespread use of primary isolated human hepatocytes are their limited availability, difficult isolation, maintenance, and expansion in culture as well as cryopreservation. Actually the available source of live human liver cells is represented by organs that are discarded from transplantation during harvesting. In the Nord Italia Transplant area of 18 million inhabitants, the number of unused livers is approximately 100 per year.10 The rescue of those discarded livers for cell isolation on a national basis would probably offer to the scientific community a definite amount of hepatic tissue available for cell transplantation. Moreover, rescue of those organs, otherwise lost, might be viewed as an improvement in the utilization of the scarce donor organ resources. Therefore, we have developed a national Bank of Human Hepatocytes that would isolate and cryopreserve human hepatocytes from all marginal livers unwanted for transplantation at harvesting. We have been able to develop standardized methods of isolation, cryopreservation, and thawing of primary human hepatocytes,6 obtaining a sufficient amount of live and functioning cryopreserved human hepatocytes for use in bioartificial liver devices to treat patients affected by FHF. More data are needed to confirm this preliminary report, but we suggest that donor livers deemed unusable for transplantation should be rescued for isolation, cryopreserved, and banked as a source of hepatic tissue for research and clinical purposes. REFERENCES 1. Adam R, McMaster P, O’Grady JG, et al: European Liver Transplant Association. Evolution of liver transplantation in Europe: report of the European liver Transplant Registry. Liver Transpl 9:1231, 2003 2. Horslen SP, Fox IJ: Hepatocyte transplantation. Transplantation 77:1481, 2004 3. Tsiaoussis J, Newsome PN, Nelson LJ, et al: Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems. Liver Transpl 7:2, 2001 4. Donini A, Baccarani U, Piccolo G, et al: Hepatocyte isolation using human livers discarded from transplantation: analysis of cell yield and function. Transplant Proc 33:654, 2001
CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK 5. Baccarani U, Sanna A, Cariani A, et al: Isolation of human hepatocytes from livers rejected for liver transplantation on a national basis: results of a 2-year experience. Liver Transpl 9:506, 2003 6. Demetriou AA, Brown RS Jr, Busuttil RW, et al: Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg 239:660, 2004 7. Baquerizo A, Mhoyan A, Kearns-Jonker M, et al: Characterization of human xenoreactive antibodies in liver failure patients exposed to pig hepatocytes after bioartificial liver treatment: an ex
259 vivo model of pig to human xenotransplantation. Transplantation 67:5, 1999 8. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3:282, 1997 9. van der Laan LJ, Lockey C, Griffeth BC, et al: Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 407:90, 2000 10. Ministero della salute, Centro Nazionale Trapianti: Available at www@ministerosalute. it/trapianti/trapianti.jsp. Accessed 2004
L
IVER TRANSPLANTATION is currently limited by the shortage of organs. Many patients who could benefit from the transplant die because they do not get a chance to be included in a liver transplant waiting list.1 Parallel to the development of new surgical strategies, such as partial liver transplantation from both living and cadaveric donors aimed at increasing the number of transplantable organs, techniques of cell transplantation and cellbased therapy are potential tools for the treatment of terminal liver disease.2 However, large scale clinical application of cell-based liver therapy, such as hepatocyte transplantation and bioartificial liver use, which have shown interesting results in animal models, is limited by the lack of 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.12.230 256
a reliable source of liver cells.3 The development of facilities dedicated to the isolation, preparation, and banking of human-hepatocytes is considered a possible solution to this From the Department of Surgery & Transplantation, University of Udine (U.B., M.S., G.L.A., D.L., D.M., A.R., F.B.); Bank of Human Hepatocytes (A.S., A.C.), Udine; NITp, Ospedale Maggiore Milano (M.S., G.P.); Department of Emergency Surgery, University of Perugia (A.D.), Ferrara, Italy. Address reprints requests to Umberto Baccarani, MD, PhD, Department of Surgery and Transplantation Unit, University Hospital, Pad. Petracco, P.le S.M. della Misericordia, 33100, Udine Italy. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 256 –259 (2005)
CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK
Fig 1.
257
Diazepam metabolism of cryopreserved human hepatocytes.
problem. The aims of this paper were to (1) analyze the in vitro function of isolated, cryopreserved human hepatocytes (CHH) from a cell bank obtained on a national basis from livers deemed unusable for transplantation at harvesting; and (2) define their potential clinical application in a bioartificial liver (BAL) device as a bridge to liver transplantation. MATERIALS AND METHODS Aim 1 One-hundred three not transplantable livers in the Nord Italia Transplant area (NITp and AIRT) were utilized for human hepatocyte isolation and cryopreservation from July 2001 to December 2003. Organs were judged not usable for transplantation during harvesting based on gross appearance and hepatic biopsy. The main causes for refusal were macrosteatosis ⬎60%, ischemic damage due to cardiac arrest or prolonged donor hypotension, and nonviral cirrhosis in 60%, 11%, and 8%, respectively. Liver retrieval was performed according to the usual surgical technique. The organs were always flushed and cooled with preservation solutions and placed on ice for transportation to the laboratory for hepatocyte isolation. The method of liver digestion and hepatocyte isolation has already been described.4 Cell yield and viability was measured by a trypan blue dye exclusion test using a Neubauer chamber. Functional integrity and activity of the isolated hepatocytes, before and after cryopreservation, was measured by a diazepam metabolism assay (HPLC) and albumin production by immunonephelometry. Hepatocytes were progressively cryopreserved using DMSO 10% to 20% as cryoprotectant at ⫺80°C and finally placed in LN2. For thawing, the hepatocytes were removed from LN2 and placed in a 37°C water bath. DMSO was gradually removed by centrifugation at 40g for 3 minutes.
Aim 2 Thawed hepatocytes were loaded into a BAL device used as a bridge to emergency liver transplantation in two cases of fulminant hepatic failure. Thirteen billion cells with a 70% postthawing viability as determined by a trypan blue dye test that were ABO compatible and functional based on cytochrome P450 metabolic
reactions5 were loaded into two hollow-fiber bioreactors (Dideco, Mirandola, Italy) with a molecular weight cut-off of 0.6 m. Bioartificial liver treatment was performed using the Performer BAL (Rand, Medolla, Italy) device, which integrates an external BAL circuit, complete with bioreactors loaded with the hepatocytes, a Plasorba cartridge (Dideco, Braun-Carex), the heater, and an oxygenator (Lilliput Dideco, Mirandola, Italy) with continuous plasmapheresis over a plasma filter. Plasma obtained from patients at a flow rate of 50 mL/min was recirculated through the BAL circuit at a flow rate of 200 to 250 mL/min with the interposition of a plasma reservoir. Heparin was used as the anticoagulant (2000 IU/h). Every treatment lasted 4 hours until a liver became available for transplantation or there was clinical improvement.
RESULTS
Overall hepatocyte yield averaged 9 ⫾ 3 ⫻ 109 with mean viability of 80% ⫾ 13%. Viable hepatocyte yield, per gram of liver tissue digested averaged 7 million cells. The best results in terms of cell yield and viability were obtained from steatotic organs, while cirrhotic livers yielded fewer cells/g liver (3 ⫻ 106 vs 7 ⫻ 105, P ⬍ .043 and 81% vs 66%, P ⫽ .27, respectively). The viability of cryopreserved hepatocytes after thawing averaged 50%. Cryopreserved hepatocytes maintained normal diazepam clearance and metabolic production on days 0 and ⫹1 after thawing (Fig 1). Moreover, fresh and thawed hepatocytes produced a comparable amount of human albumin when maintained in culture conditions on collagen coated dishes (Fig 2). The morphology of cultured human hepatocytes showed typical features of confluent colonies of normally appearing viable liver cells. Two cases of fulminant hepatic failure were treated with a BAL device loaded with cryopreserved/ thawed human hepatocytes as a bridge to emergency liver transplantation. Both patients had encephalopathy grade IV and were intubated with mechanically assisted ventilation. The etiology of fulminant hepatic failure were acute HBV infection and cryptogenic. The first patient, affected by acute HBV infection, underwent a single BAL treatment
258
BACCARANI, SANNA, CARIANI ET AL
Fig 2. Albumin synthesis fresh vs cryopreserved human hepatocytes.
resulting in reduction of the bilirubin (from 11.9 mg/dL pretreatment to 6.9 mg/dL at the end of BAL) and ammonia level (from 216 mcg/dL pretreatment to 193 mcg/dL at the end of BAL). At the end of this BAL procedure, a liver became available for transplantation that was performed successfully. The second patient had two BAL procedures over a 2-day course. The bilirubin level decreased (from 8.7 mg/dL to 5.4 mg/dL) during the first treatment, while increasing during the delay between the first and the second application to 12.3 mg/dL. At the end of the second BAL procedure, the bilirubin dropped to 8.9 mg/dL. Also the ammonia level dropped at the end of the second BAL (130 mcg/mL from 177 mcg/mL). Clinical conditions improved after the second BAL treatment; the patient finally recovered without the need for liver transplantation. Renal function remained stable in both patients with normal urine output. Hemodynamic and respiratory stability was maintained throughout the treatment period. Both patients did not receive mannitol or hyperventilation therapy during the treatment. Neurological conditions remained stable with no clinical evidence of developing brain edema. Intracranial pressure was not measured due to coagulopathy and the risk of bleeding. No adverse events were recorded. DISCUSSION
Great controversy still exists about what is the best source of hepatocytes to be loaded into bioartificial liver devices.3 The ideal hepatocyte should have a detoxification function. Moreover, biotransforming properties of hepatocytes, such as phase I and II reactions, should be assured by the hepatocytes loaded into the bioartificial liver. Both xenogenic (porcine) and human hepatocytes derived from tumor cell lines or immortalized have been shown to be able to catalyze the requested functions, although at different levels. Moreover tumor-derived or immortalized human hepatocytes can potentially show a risk of neoplastic transmission to patients, requiring selective filters that hamper passage of molecules from the patients’ circulation to the hepatocytes. Porcine xenogenic hepatocytes have been considered a possible solution to the problem of the biological component of BAL devices because they are easily available in large quantities,6 but they have known disadvantages.7–9
Isolated primary human hepatocytes would probably be the best source of cells for BAL devices because no concerns exist regarding immunogenicity, zoonosis, and biocompatibility. However, the major drawbacks to the widespread use of primary isolated human hepatocytes are their limited availability, difficult isolation, maintenance, and expansion in culture as well as cryopreservation. Actually the available source of live human liver cells is represented by organs that are discarded from transplantation during harvesting. In the Nord Italia Transplant area of 18 million inhabitants, the number of unused livers is approximately 100 per year.10 The rescue of those discarded livers for cell isolation on a national basis would probably offer to the scientific community a definite amount of hepatic tissue available for cell transplantation. Moreover, rescue of those organs, otherwise lost, might be viewed as an improvement in the utilization of the scarce donor organ resources. Therefore, we have developed a national Bank of Human Hepatocytes that would isolate and cryopreserve human hepatocytes from all marginal livers unwanted for transplantation at harvesting. We have been able to develop standardized methods of isolation, cryopreservation, and thawing of primary human hepatocytes,6 obtaining a sufficient amount of live and functioning cryopreserved human hepatocytes for use in bioartificial liver devices to treat patients affected by FHF. More data are needed to confirm this preliminary report, but we suggest that donor livers deemed unusable for transplantation should be rescued for isolation, cryopreserved, and banked as a source of hepatic tissue for research and clinical purposes. REFERENCES 1. Adam R, McMaster P, O’Grady JG, et al: European Liver Transplant Association. Evolution of liver transplantation in Europe: report of the European liver Transplant Registry. Liver Transpl 9:1231, 2003 2. Horslen SP, Fox IJ: Hepatocyte transplantation. Transplantation 77:1481, 2004 3. Tsiaoussis J, Newsome PN, Nelson LJ, et al: Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems. Liver Transpl 7:2, 2001 4. Donini A, Baccarani U, Piccolo G, et al: Hepatocyte isolation using human livers discarded from transplantation: analysis of cell yield and function. Transplant Proc 33:654, 2001
CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK 5. Baccarani U, Sanna A, Cariani A, et al: Isolation of human hepatocytes from livers rejected for liver transplantation on a national basis: results of a 2-year experience. Liver Transpl 9:506, 2003 6. Demetriou AA, Brown RS Jr, Busuttil RW, et al: Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg 239:660, 2004 7. Baquerizo A, Mhoyan A, Kearns-Jonker M, et al: Characterization of human xenoreactive antibodies in liver failure patients exposed to pig hepatocytes after bioartificial liver treatment: an ex
259 vivo model of pig to human xenotransplantation. Transplantation 67:5, 1999 8. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3:282, 1997 9. van der Laan LJ, Lockey C, Griffeth BC, et al: Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 407:90, 2000 10. Ministero della salute, Centro Nazionale Trapianti: Available at www@ministerosalute. it/trapianti/trapianti.jsp. Accessed 2004