- Email: [email protected]
Comparison of University of Wisconsin and ET-Kyoto Preservation Solutions for the Cryopreservation of Primary Human Hepatocytes
Comparison of University of Wisconsin and ET-Kyoto Preservation Solutions for the Cryopreservation of Primary Human Hepatocytes S. Illouz, T. Nakamura, M. Webb, B. Thava, J. Bikchandani, G. Robertson, D. Lloyd, D. Berry, H. Wada, and A. Dennison ABSTRACT Primary human hepatocytes are clinically used for transplantation or in bioartificial liver devices for the treatment of patients with liver failure. We aimed to assess whether an organ preservation solution containing trehalose, namely ET-Kyoto solution (ETK), could improve human liver cell viability when used for cryopreservation in comparison to the University of Wisconsin solution (UW). Our study showed beneficial effects of ETK when used in combination with other cryoprotectants on the viability of thawed hepatocytes. Indeed, no significant difference was seen between the viability of freshly isolated cells and cryopreserved cells when cryopreserved with ETK combined with other cryoprotectants. In contrast, a significant decrease of viability was observed in cells cryopreserved with UW or ETK combined with dimethysulfoxide (DMSO) only, or with UW combined with other cryoprotectants, compared to freshly isolated cells. No significant difference was observed between the four different groups of cryopreserved hepatocytes with regards to cell recovery rate or cell attachment after thawing. However, a significant decrease in cell metabolic activity was found in cells cryopreserved with UW 10% DMSO compared to the other groups. In conclusion, our study confirms the beneficial effect of ETK for the cryopreservation of human hepatocytes in combination with other cryoprotective agents.
O
PTIMIZING hepatocyte cryopreservation methods is still needed to increase the availability of primary isolated human hepatocytes, which are used for cell transplantation in the treatment of liver failure. It has been well demonstrated that cell death occurs during the cell isolation process and is further increased by the cryopreservation process.1 Freezing media that are generally used for the cryopreservation of human hepatocytes are composed of medium culture and University of Wisconsin (UW) preservation solution with a combination of different types of cryoprotectants such as dimethylsulfoxide (DMSO), bovine serum albumin (BSA), and polyvinylpyrrolidone (PVP).2,3 Particular interest has been shown in using disaccharides and especially trehalose for the cryopreservation of different cell types including hematopoietic cells,4 cord blood cells,5 pancreatic islets,6 and hepatocytes.7,8 The aim of the present study was to evaluate the effects of four different freezing solutions on cryopreserved primary human hepatocytes, including the use of an organ preservation solution containing trehalose. We tested whether the use of organ preservation solutions solely supplemented with 10% DMSO for the cryopreservation of
human hepatocytes should be more suitable for cell therapy applications. This study has direct clinical relevance for cell transplantation and the use of an optimal freezing solution. MATERIALS AND METHODS Chemicals Unless specified, all chemicals were purchased from Sigma-Aldrich (Gillingham, UK, or St. Quentin Fallavier, France). Culture medium, fetal bovine serum (FBS), and buffers were from Invitrogen (Paisley, UK, or Cergy-Pontoise, France). Plastic materials (BD Biocoat collagen I precoated culture plates) were purchased from VWR (Lutterworth, UK). Organ preservation University of Wisconsin solution (UW) was from Bristol-Myers Squibb (County Dublin, Ireland) and organ preservation ET-Kyoto solution (ETK), From the Department of Hepatobiliary and Pancreatic Surgery, Leicester General Hospital, Leicester, UK. This work was supported by research funds provided by the NHS Trust. Address reprint requests to Dr. Severine Illouz, UHL NHS Trust, Leicester General Hospital, Gwendolen Road, Leicester, Leicestershire LE5 4PW, United Kingdom. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.064
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1706
Transplantation Proceedings, 40, 1706 –1709 (2008)
COMPARISON OF PRESERVATION SOLUTIONS
1707
Cell Viability After Culture The mitochondrial dehydrogenase activity of viable cells—ie, the capacity of the cells to reduce tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a purple formazan product—was used to assess cell viability.10
Fig 1. Cell viability after thawing with BUW, BET, UW, or ET freezing solutions. Cell viability was evaluated with trypan blue staining. Each symbol and bar indicates the mean of six experiments (*P ⬍ .05; **P ⬍ .001). containing 0.12 mol/L trehalose, was provided by the Kyoto University Graduate School of Medicine (Kyoto, Japan). Human recombinant insulin at 3.5 mg/mL (Novonordisk) and dexamethasone at 3.8 mg/mL (Organon) injectable solutions were provided by the pharmacy of Leicester General Hospital.
Human Hepatocyte Isolations Adult normal liver samples were obtained from patients undergoing partial hepatectomy for primary or secondary tumors (Table 1). Ethical approval was granted by the Leicestershire Research Ethics Committee and by the French National Ethics Committee for the use of human tissue for research purposes in this study. Written informed consent was obtained from each patient prior to the surgery. Hepatocytes were isolated by a two-step collagenase perfusion as previously described.9 All experiments were carried out in duplicate unless otherwise stated. Cell viability and yield were evaluated with trypan blue staining on cells in suspension.
Hepatocyte Cryopreservation and Thawing Freshly isolated hepatocytes were cryopreserved at a final concentration of 5 ⫻ 106 viable cells/mL and thawed as previously described.3 Each batch of liver cells, from a single donor, was cryopreserved simultaneously with four different freezing solutions: (1) “BUW” consisted of DMEM high-glucose containing 2.5% BSA, 2% PVP, 20% UW solution, 20% FBS, and 10% DMSO; (2) “BET” was exactly the same formula as BUW except that UW was replaced by ETK; (3) “UW” consisted of UW with 10% DMSO; and (4) “ET” consisted of ETK with 10% DMSO.
Human Hepatocyte Culture Freshly isolated and cryopreserved cells were cultured in Dubelcco’s modified Eagle’s medium with 4.5 g/L glucose (DMEM) with 5% FBS, 0.1 mol/L human recombinant insulin, 1 mol/L dexamethasone, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 25 g/mL fungizone. Hepatocytes were seeded at 0.7 ⫻ 106 viable cells per well in a 12-well plate, or 3 ⫻ 104 viable cells per well in a 96-well plate. The protein content of both adherent and nonadherent cells was determined and used to calculate the plating efficiency as a percentage of initially plated cells.3
Fig 2. Recovery (A), cell attachment (B), and metabolic activity (C) of cryopreserved hepatocytes after thawing. Each symbol and bar indicates the mean of six experiments (*P ⬍ .05).
1708
ILLOUZ, NAKAMURA, WEBB ET AL
Statistical Analysis
DISCUSSION
All analyses were made using the GraphPad InStat software. Repeated-measures ANOVA were used to compare multiple groups, with P values of ⬍.05 considered significant. Results were expressed as mean ⫾ SEM.
Maximal viability was obtained when cells were cryopreserved in ETK combined with PVP, BSA, FBS, and culture medium, but not significantly different from the viability of freshly isolated cells. Furthermore, the use of ETK 10% DMSO with or without the combination of cryoprotectants gave comparable results on cultivated cell viability, demonstrating a beneficial effect of ETK for the cryopreservation of human hepatocytes. This is the first study showing that the trehalose-containing organ preservation solution ETK gave satisfactory results for the cryopreservation of primary human hepatocytes in terms of cell viability. This confirms the beneficial role of trehalose as an additional cryoprotective agent for cell cryopreservation,7 and it also guarantees for the first time the use of ETK as an appropriate solution for the cryopreservation of human hepatocytes. Conversely, we have compared the use of two different organ preservation solutions for the cryopreservation of human hepatocytes. The preservation solution UW is considered as the gold standard for organ preservation; however, its efficiency for the cryopreservation of human cells has been scarcely evaluated. Our study showed that the use of UW supplemented solely with 10% DMSO for the cryopreservation of human hepatocytes had a deleterious effect on cultivated cell viability. Our results are of clinical importance because UW is currently used for the cryopreservation of human hepatocytes either solely supplemented with 10% DMSO11 or supplemented with DMSO and human albumin12,13 for cell transplantation in the treatment of liver metabolic disease. We are currently using a complete freezing medium combining UW and DMEM containing 25 mmol/L glucose, FBS, and DMSO supplemented with other cryoprotectants such as albumin (BSA) and PVP, which gave optimal results, according to the regimens of human hepatocyte freezing media that have been reported to be most effective.3 The aim of this study was to test the efficiency of a trehalose-containing preservation solution for the cryopreservation of human
RESULTS
Beneficial effects of ETK were found on postthawed cell viability but only when used in combination with other cryoprotectants (‘BET’). Indeed, there was no significant difference on the viability of cells cryopreserved in ETK combined with other cryoprotectants (67.63 ⫾ 4.28%) compared to the viability of freshly isolated cells (78.33 ⫾ 2.08%) (Fig 1). In contrast, a significant decrease in postthawed viability was observed on cells cryopreserved with UW or ETK 10% DMSO (61.92 ⫾ 3.02, 63.17 ⫾ 5.40%), or with UW combined with other cryoprotectants (65.23 ⫾ 2.71%) compared to the viability of freshly isolated cells. However, no significant difference was observed on postthawed viability among the four different groups of cryopreserved cells. Also, no significant difference was observed among the four different groups of cryopreserved hepatocytes on cell recovery rate or on cell attachment after thawing (Fig 2A, B). In addition, no significant difference was found among the four groups concerning the percentage of apoptic cells, Annexin V-stained cells, evaluated by flow cytometer quantification (data not shown). Furthermore, a significant decrease in cell viability of cultivated cells was found in cells cryopreserved with UW 10% DMSO (0.231 ⫾ 0.047 unit of absorbance) compared to the one cryopreserved with UW combined with other cryoprotectants (0.416 ⫾ 0.065 unit of absorbance) (Fig 2C). Also, concerning cell viability of cultivated cells, the metabolic capacity of cells cryopreserved with ETK 10% DMSO (0.404 ⫾ 0.087 unit of absorbance) was not significantly different from those cryopreserved with ETK combined with other cryoprotectants (0.317 ⫾ 0.066 unit of absorbance).
Table 1. Patient Characteristics and Hepatocyte Isolation Results Liver Tissue Weight (g)
Isolation Duration (min)
Yield (106 viable cells/ g of tissue)
Donor
Disease
Gender
Age
BMI
WIT (min)
CIT (min)
D1
Colorectal metastases Hepatocellular carcinoma Colorectal metastases Colorectal metastases Colorectal metastases Colorectal metastases
F
66
22.5
25
60
25
145
9.30
M
64
26
18
35
45
180
15.00
M
50
24
48
60
94
200
8.01
F
25
18.5
71
45
115
210
16.1
M
70
21.5
27
85
22
140
12.16
F
68
20.5
25
60
49
170
1.8
57.2 ⫾ 7.0
22.2 ⫾ 1.1
35.7 ⫾ 8.2
57.5 ⫾ 6.92
58.3 ⫾ 15.5
174.2 ⫾ 11.6
10.4 ⫾ 2.1
D2 D3 D4 D5 D6 Mean ⫾ SEM
Abbreviations: BMI, body mass index; WIT, warm ischemia time; CIT, cold ischemia time.
COMPARISON OF PRESERVATION SOLUTIONS
hepatocytes. Trehalose solutions enhance cell viability after cryopreservation by stabilizing membranes.14 Indeed, trehalose stabilizes cell membrane by binding to the phospholipids.15 The combination of trehalose with PVP and human albumin has proven to improve the survival of cryopreserved red blood cells.16 Furthermore, a combination of trehalose with DMSO, a membrane-penetrating cytoprotective agent, has also proven to have a beneficial effect on cryopreserved embryos,17 spermatozoa,18 and trachea.19 Our study confirms the beneficial effect of trehalose for cryopreservation of human hepatocytes in combination with other cryoprotective agents. ACKNOWLEDGMENTS We would like to thank the Transplant Department, directed by Pr. Michael Nicholson, the theater staff of Leicester General Hospital and Leicester Royal Infirmary, and the Clinical Nurse specialists Sophie Noble, Cristina Pollard, and Jo Bishop for their help with patients’ recruitment.
REFERENCES 1. Fu T, Guo D, Huang X, et al: Apoptosis occurs in isolated and banked primary mouse hepatocytes. Cell Transplant 10:59, 2001 2. De Sousa G, Nicolas F, Placidi M, et al: A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology. Chem Biol Interact 121:77, 1999 3. Alexandre E, Viollon-Abadie C, David P, et al: Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiology 44:103, 2002 4. Limaye LS, Kale VP: Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium. J Hematother Stem Cell Res 10:709, 2001 5. Zhang XB, Li K, Yau KH, et al: Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells. Transfusion 43:265, 2003 6. Beattie GM, Crowe JH, Lopez AD, et al: Trehalose: a cryoprotectant that enhances recovery and preserves function of
1709 human pancreatic islets after long-term storage. Diabetes 16:519, 1997 7. Katenz E, Vondran FWR, Schwartlander R, et al: Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent. Liver Transpl 13:38, 2007 8. Miyamoto Y, Suzuki S, Nomura K, et al: Improvement of hepatocyte viability after cryoperservation by supplementation of long-chain oligosaccharide in the freezing medium in rats and humans. Cell Transplant 15:911, 2006 9. Richert L, Alexandre E, Lloyd T, et al: Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study. Liver Int 24:371, 2004 10. Finlay GJ, Baguley BC, Wilson WR: A semi-automated microculture method for investigating growth inhibitory effects of cytotoxic compounds on exponentially growing carcinoma cells. Anal Biochem 139:272, 1984 11. Dhawan A, Mitry RR, Hughes RD, et al: Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78:1812, 2004 12. Sokal EM, Smets F, Bourgois A, et al: Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up. Transplantation 76:735, 2003 13. Stephenne X, Najimi M, Smets F, et al: Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation. Am J Transplant 5:2058, 2005 14. Crowe JH: Trehalose as a “chemical chaperone”: fact and fantasy. Adv Exp Med Biol 594:143, 2007 15. Crowe JH, Crowe LM, Mouradian R: Stabilization of biological membranes at low water activities. Cryobiology 20:346, 1983 16. Quan G, Zhang L, Guo Y, et al: Intracellular sugars improve survival of human red blood cells cryopreserved at ⫺80 degrees C in the presence of polyvinyl pyrrolidone and human serum albumin. Cryoletters 28:95, 2007 17. Honadel TE, Killian GJ: Cryopreservation of murine embryos with trehalose and glycerol. Cryobiology 25:331, 1988 18. Terada T, Ashizawa K, Maeda T, et al: Efficacy of trehalose in cryopreservation of chicken spermatozoa. Jpn J Anim Reprod 35:20, 1989 19. Yokomise H, Inui K, Wada H, et al: Reliable cryopreservation of trachea for one month in a new trehalose solution. J Thorac Cardiovasc Surg 110:382, 1995
O
PTIMIZING hepatocyte cryopreservation methods is still needed to increase the availability of primary isolated human hepatocytes, which are used for cell transplantation in the treatment of liver failure. It has been well demonstrated that cell death occurs during the cell isolation process and is further increased by the cryopreservation process.1 Freezing media that are generally used for the cryopreservation of human hepatocytes are composed of medium culture and University of Wisconsin (UW) preservation solution with a combination of different types of cryoprotectants such as dimethylsulfoxide (DMSO), bovine serum albumin (BSA), and polyvinylpyrrolidone (PVP).2,3 Particular interest has been shown in using disaccharides and especially trehalose for the cryopreservation of different cell types including hematopoietic cells,4 cord blood cells,5 pancreatic islets,6 and hepatocytes.7,8 The aim of the present study was to evaluate the effects of four different freezing solutions on cryopreserved primary human hepatocytes, including the use of an organ preservation solution containing trehalose. We tested whether the use of organ preservation solutions solely supplemented with 10% DMSO for the cryopreservation of
human hepatocytes should be more suitable for cell therapy applications. This study has direct clinical relevance for cell transplantation and the use of an optimal freezing solution. MATERIALS AND METHODS Chemicals Unless specified, all chemicals were purchased from Sigma-Aldrich (Gillingham, UK, or St. Quentin Fallavier, France). Culture medium, fetal bovine serum (FBS), and buffers were from Invitrogen (Paisley, UK, or Cergy-Pontoise, France). Plastic materials (BD Biocoat collagen I precoated culture plates) were purchased from VWR (Lutterworth, UK). Organ preservation University of Wisconsin solution (UW) was from Bristol-Myers Squibb (County Dublin, Ireland) and organ preservation ET-Kyoto solution (ETK), From the Department of Hepatobiliary and Pancreatic Surgery, Leicester General Hospital, Leicester, UK. This work was supported by research funds provided by the NHS Trust. Address reprint requests to Dr. Severine Illouz, UHL NHS Trust, Leicester General Hospital, Gwendolen Road, Leicester, Leicestershire LE5 4PW, United Kingdom. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.064
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1706
Transplantation Proceedings, 40, 1706 –1709 (2008)
COMPARISON OF PRESERVATION SOLUTIONS
1707
Cell Viability After Culture The mitochondrial dehydrogenase activity of viable cells—ie, the capacity of the cells to reduce tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a purple formazan product—was used to assess cell viability.10
Fig 1. Cell viability after thawing with BUW, BET, UW, or ET freezing solutions. Cell viability was evaluated with trypan blue staining. Each symbol and bar indicates the mean of six experiments (*P ⬍ .05; **P ⬍ .001). containing 0.12 mol/L trehalose, was provided by the Kyoto University Graduate School of Medicine (Kyoto, Japan). Human recombinant insulin at 3.5 mg/mL (Novonordisk) and dexamethasone at 3.8 mg/mL (Organon) injectable solutions were provided by the pharmacy of Leicester General Hospital.
Human Hepatocyte Isolations Adult normal liver samples were obtained from patients undergoing partial hepatectomy for primary or secondary tumors (Table 1). Ethical approval was granted by the Leicestershire Research Ethics Committee and by the French National Ethics Committee for the use of human tissue for research purposes in this study. Written informed consent was obtained from each patient prior to the surgery. Hepatocytes were isolated by a two-step collagenase perfusion as previously described.9 All experiments were carried out in duplicate unless otherwise stated. Cell viability and yield were evaluated with trypan blue staining on cells in suspension.
Hepatocyte Cryopreservation and Thawing Freshly isolated hepatocytes were cryopreserved at a final concentration of 5 ⫻ 106 viable cells/mL and thawed as previously described.3 Each batch of liver cells, from a single donor, was cryopreserved simultaneously with four different freezing solutions: (1) “BUW” consisted of DMEM high-glucose containing 2.5% BSA, 2% PVP, 20% UW solution, 20% FBS, and 10% DMSO; (2) “BET” was exactly the same formula as BUW except that UW was replaced by ETK; (3) “UW” consisted of UW with 10% DMSO; and (4) “ET” consisted of ETK with 10% DMSO.
Human Hepatocyte Culture Freshly isolated and cryopreserved cells were cultured in Dubelcco’s modified Eagle’s medium with 4.5 g/L glucose (DMEM) with 5% FBS, 0.1 mol/L human recombinant insulin, 1 mol/L dexamethasone, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 25 g/mL fungizone. Hepatocytes were seeded at 0.7 ⫻ 106 viable cells per well in a 12-well plate, or 3 ⫻ 104 viable cells per well in a 96-well plate. The protein content of both adherent and nonadherent cells was determined and used to calculate the plating efficiency as a percentage of initially plated cells.3
Fig 2. Recovery (A), cell attachment (B), and metabolic activity (C) of cryopreserved hepatocytes after thawing. Each symbol and bar indicates the mean of six experiments (*P ⬍ .05).
1708
ILLOUZ, NAKAMURA, WEBB ET AL
Statistical Analysis
DISCUSSION
All analyses were made using the GraphPad InStat software. Repeated-measures ANOVA were used to compare multiple groups, with P values of ⬍.05 considered significant. Results were expressed as mean ⫾ SEM.
Maximal viability was obtained when cells were cryopreserved in ETK combined with PVP, BSA, FBS, and culture medium, but not significantly different from the viability of freshly isolated cells. Furthermore, the use of ETK 10% DMSO with or without the combination of cryoprotectants gave comparable results on cultivated cell viability, demonstrating a beneficial effect of ETK for the cryopreservation of human hepatocytes. This is the first study showing that the trehalose-containing organ preservation solution ETK gave satisfactory results for the cryopreservation of primary human hepatocytes in terms of cell viability. This confirms the beneficial role of trehalose as an additional cryoprotective agent for cell cryopreservation,7 and it also guarantees for the first time the use of ETK as an appropriate solution for the cryopreservation of human hepatocytes. Conversely, we have compared the use of two different organ preservation solutions for the cryopreservation of human hepatocytes. The preservation solution UW is considered as the gold standard for organ preservation; however, its efficiency for the cryopreservation of human cells has been scarcely evaluated. Our study showed that the use of UW supplemented solely with 10% DMSO for the cryopreservation of human hepatocytes had a deleterious effect on cultivated cell viability. Our results are of clinical importance because UW is currently used for the cryopreservation of human hepatocytes either solely supplemented with 10% DMSO11 or supplemented with DMSO and human albumin12,13 for cell transplantation in the treatment of liver metabolic disease. We are currently using a complete freezing medium combining UW and DMEM containing 25 mmol/L glucose, FBS, and DMSO supplemented with other cryoprotectants such as albumin (BSA) and PVP, which gave optimal results, according to the regimens of human hepatocyte freezing media that have been reported to be most effective.3 The aim of this study was to test the efficiency of a trehalose-containing preservation solution for the cryopreservation of human
RESULTS
Beneficial effects of ETK were found on postthawed cell viability but only when used in combination with other cryoprotectants (‘BET’). Indeed, there was no significant difference on the viability of cells cryopreserved in ETK combined with other cryoprotectants (67.63 ⫾ 4.28%) compared to the viability of freshly isolated cells (78.33 ⫾ 2.08%) (Fig 1). In contrast, a significant decrease in postthawed viability was observed on cells cryopreserved with UW or ETK 10% DMSO (61.92 ⫾ 3.02, 63.17 ⫾ 5.40%), or with UW combined with other cryoprotectants (65.23 ⫾ 2.71%) compared to the viability of freshly isolated cells. However, no significant difference was observed on postthawed viability among the four different groups of cryopreserved cells. Also, no significant difference was observed among the four different groups of cryopreserved hepatocytes on cell recovery rate or on cell attachment after thawing (Fig 2A, B). In addition, no significant difference was found among the four groups concerning the percentage of apoptic cells, Annexin V-stained cells, evaluated by flow cytometer quantification (data not shown). Furthermore, a significant decrease in cell viability of cultivated cells was found in cells cryopreserved with UW 10% DMSO (0.231 ⫾ 0.047 unit of absorbance) compared to the one cryopreserved with UW combined with other cryoprotectants (0.416 ⫾ 0.065 unit of absorbance) (Fig 2C). Also, concerning cell viability of cultivated cells, the metabolic capacity of cells cryopreserved with ETK 10% DMSO (0.404 ⫾ 0.087 unit of absorbance) was not significantly different from those cryopreserved with ETK combined with other cryoprotectants (0.317 ⫾ 0.066 unit of absorbance).
Table 1. Patient Characteristics and Hepatocyte Isolation Results Liver Tissue Weight (g)
Isolation Duration (min)
Yield (106 viable cells/ g of tissue)
Donor
Disease
Gender
Age
BMI
WIT (min)
CIT (min)
D1
Colorectal metastases Hepatocellular carcinoma Colorectal metastases Colorectal metastases Colorectal metastases Colorectal metastases
F
66
22.5
25
60
25
145
9.30
M
64
26
18
35
45
180
15.00
M
50
24
48
60
94
200
8.01
F
25
18.5
71
45
115
210
16.1
M
70
21.5
27
85
22
140
12.16
F
68
20.5
25
60
49
170
1.8
57.2 ⫾ 7.0
22.2 ⫾ 1.1
35.7 ⫾ 8.2
57.5 ⫾ 6.92
58.3 ⫾ 15.5
174.2 ⫾ 11.6
10.4 ⫾ 2.1
D2 D3 D4 D5 D6 Mean ⫾ SEM
Abbreviations: BMI, body mass index; WIT, warm ischemia time; CIT, cold ischemia time.
COMPARISON OF PRESERVATION SOLUTIONS
hepatocytes. Trehalose solutions enhance cell viability after cryopreservation by stabilizing membranes.14 Indeed, trehalose stabilizes cell membrane by binding to the phospholipids.15 The combination of trehalose with PVP and human albumin has proven to improve the survival of cryopreserved red blood cells.16 Furthermore, a combination of trehalose with DMSO, a membrane-penetrating cytoprotective agent, has also proven to have a beneficial effect on cryopreserved embryos,17 spermatozoa,18 and trachea.19 Our study confirms the beneficial effect of trehalose for cryopreservation of human hepatocytes in combination with other cryoprotective agents. ACKNOWLEDGMENTS We would like to thank the Transplant Department, directed by Pr. Michael Nicholson, the theater staff of Leicester General Hospital and Leicester Royal Infirmary, and the Clinical Nurse specialists Sophie Noble, Cristina Pollard, and Jo Bishop for their help with patients’ recruitment.
REFERENCES 1. Fu T, Guo D, Huang X, et al: Apoptosis occurs in isolated and banked primary mouse hepatocytes. Cell Transplant 10:59, 2001 2. De Sousa G, Nicolas F, Placidi M, et al: A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology. Chem Biol Interact 121:77, 1999 3. Alexandre E, Viollon-Abadie C, David P, et al: Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiology 44:103, 2002 4. Limaye LS, Kale VP: Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium. J Hematother Stem Cell Res 10:709, 2001 5. Zhang XB, Li K, Yau KH, et al: Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells. Transfusion 43:265, 2003 6. Beattie GM, Crowe JH, Lopez AD, et al: Trehalose: a cryoprotectant that enhances recovery and preserves function of
1709 human pancreatic islets after long-term storage. Diabetes 16:519, 1997 7. Katenz E, Vondran FWR, Schwartlander R, et al: Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent. Liver Transpl 13:38, 2007 8. Miyamoto Y, Suzuki S, Nomura K, et al: Improvement of hepatocyte viability after cryoperservation by supplementation of long-chain oligosaccharide in the freezing medium in rats and humans. Cell Transplant 15:911, 2006 9. Richert L, Alexandre E, Lloyd T, et al: Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study. Liver Int 24:371, 2004 10. Finlay GJ, Baguley BC, Wilson WR: A semi-automated microculture method for investigating growth inhibitory effects of cytotoxic compounds on exponentially growing carcinoma cells. Anal Biochem 139:272, 1984 11. Dhawan A, Mitry RR, Hughes RD, et al: Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78:1812, 2004 12. Sokal EM, Smets F, Bourgois A, et al: Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up. Transplantation 76:735, 2003 13. Stephenne X, Najimi M, Smets F, et al: Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation. Am J Transplant 5:2058, 2005 14. Crowe JH: Trehalose as a “chemical chaperone”: fact and fantasy. Adv Exp Med Biol 594:143, 2007 15. Crowe JH, Crowe LM, Mouradian R: Stabilization of biological membranes at low water activities. Cryobiology 20:346, 1983 16. Quan G, Zhang L, Guo Y, et al: Intracellular sugars improve survival of human red blood cells cryopreserved at ⫺80 degrees C in the presence of polyvinyl pyrrolidone and human serum albumin. Cryoletters 28:95, 2007 17. Honadel TE, Killian GJ: Cryopreservation of murine embryos with trehalose and glycerol. Cryobiology 25:331, 1988 18. Terada T, Ashizawa K, Maeda T, et al: Efficacy of trehalose in cryopreservation of chicken spermatozoa. Jpn J Anim Reprod 35:20, 1989 19. Yokomise H, Inui K, Wada H, et al: Reliable cryopreservation of trachea for one month in a new trehalose solution. J Thorac Cardiovasc Surg 110:382, 1995