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Assessment of some porcine strains as donors of islets of langerhans
Assessment of Some Porcine Strains as Donors of Islets of Langerhans M. Sabat, E. Godlewska, J. Kinasiewicz, A. Urbanowicz, and T. Orłowski ABSTRACT Mass isolation of viable porcine islets is a difficult task because of their fragility, and because of donor variability with respect to strain, age, sex, feeding, and methods of slaughtering. Not all strains are equally suitable for islet separation. The aim of this study was to evaluate porcine pancreata as an alternative source of islets for clinical transplantation. Pancreata were digested from pig strains available in Poland: 248 market weight slaughterhouse pigs and 42 pigs, belonging to the Polish Large White (WBP, 14 sows and 3 males), Polish White Pendant-Ears (PBZ; 16 sows), Pietrain (8 sows), and Yorkshire (1 sow) races. Prepurification data of recoverable islets/g and islet equivalents/g were considered as representative for the number of recoverable islets. Acceptable results namely, islet and/or islet-equivalent (IE) number of at least 1000/g, were obtained from only 56 of 248 slaughterhouse pigs, namely 2073 ⫾ 137.4 SE (median 1767/g) islets with values of IE of 2994 ⫾ 303 SE (median 1874/g). Our data support Krickhahn et al suggesting that only pancreata with an average islet size exceeding 199 m should be digested and that only from 1 of 3 to 5 porcine pancreata is an adequate amount of islets generated.
X
ENOTRANSPLANTATION may be the solution to the shortage of organ donors. Due to close similarities between human and porcine insulins and blood glucose levels, porcine islets are considered the best alternative for treatment of type 1 diabetes in humans. The availability of potential porcine donors is unlimited. Pigs have large litters and can be genetically modified using transgenetic technology. Their breeding and maintainance costs are relatively low. However, mass isolation of viable porcine islets is difficult because of their fragility, and because of donor variability with respect to strain, age, sex, type of feeding, and method of slaughtering. Not all strains are equally suitable for islet separation. The present study investigated pigs from strains available in Poland. MATERIALS AND METHODS
and infused with digestion medium (1–3 mL/g; 4°C) containing collagenase with the addition of 10% porcine serum. Immediately after distension, the organ was transported (4°C, cold ischemia time ⬍190 minutes) to the laboratory. Only the tail of the pancreas was processed using a standard method (73 cases) or with minimal shaking (217 cases). The digestion was ceased when free islets were detected under the microscope. After filtration through 300-m mesh, the islets were washed, suspended in Hanks’s solution, and quantitated for yield. Their number was converted1 into 150-m diameter islet equivalents (IE). Islets ⬍50 m in diameter were not counted. The viability of islets was confirmed by dithizone staining. To eliminate the influence of variations in gradient purification, only the prepurification data on recoverable islets/g and IE/g were considered representative of the number of recoverable islets. The value of a particular strain was assessed according to the frequency of acceptable donors, namely those whose yield of islets and/or IE were ⬎1000/g.
The pancreata were harvested from 248 market weight slaughterhouse pigs (156 males, 92 sows) and from 42 pigs weighing 100 kg, acquired from the Institute of Zootechnics breeding farm and belonging to the following strains: Polish Large White (WBP, 14 sows, 3 males), Polish White Pendant-Ears (PBZ, 16 sows), Pietrain (8 sows), and Yorkshire (1 sow). In the slaughterhouses, the pancreata were dissected ex situ immediately after killing, exsanguination, water [40°C] bathing, and skin shaving (warm ischemia time, 15–20 minutes). The main pancreatic duct was cannulated
From the Transplantation Institute, and Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland. Address reprint requests to M. Sabat, Polish Academy of Sciences, Pawinski Street 5, Warsaw 02-106 Poland.
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00793-0
Transplantation Proceedings, 35, 2343–2344 (2003)
2343
2344
RESULTS
In all specimens, cell groups with diameters ⬎100 m contained the smallest number of islets. The acceptable results (islet and/or IE number ⱖ 1000/g) were obtained from only 56 of 248 slaughterhouse pigs, namely, 2073 ⫾ 137.4SE (median, 1767/g) islets. Most of them were big (IE 2994 ⫾ 303 SE; median, 1874/g). Only 3 of 17 WBP and 2 of 16 PBZ pancreata yielded ⬎ 1000 islets/g. Pancreata of Pietrain and Yorkshire strains yielded ⬍1000 islet/g and clusters of 100 m in diameter contained the smallest number of islets. Pancreata of WBP, PBZ, and Pietrain donors tended to have islets of ⬍100-m diameter (⬎82%). DISCUSSION
Xenotransplantation of porcine islets may be the solution for the shortage of human organ donors. We have confirmed the great variability of islet yield, not only between
SABAT, GODLEWSKA, KINASIEWICZ ET AL
particular strains, but also between animals belonging to the same race. We identified slaughterhouse sources as the best substrain of pigs available in Poland. The rather poor yields obtained in our studies probably result from the elaborate method of slaughtering, namely a hot water bath. The main reason was the prevalence of little islets (80%) in digested organs, a finding in pig pancreata that was already noticed by others.2 For this reason, Krickhahn et al recommended histological screening of each organ after harvesting such that only pancreata with an average islet size ⬎199 m should be digested.
REFERENCES 1. Ricordi C (ed): Pancreatic Islet Cell Transplantation. Austin: R.G. Landes Company; 1992, p 132 2. Krickhahn M, Meyer T, Buehler C, et al: Cell Transplant 11:827, 2002
X
ENOTRANSPLANTATION may be the solution to the shortage of organ donors. Due to close similarities between human and porcine insulins and blood glucose levels, porcine islets are considered the best alternative for treatment of type 1 diabetes in humans. The availability of potential porcine donors is unlimited. Pigs have large litters and can be genetically modified using transgenetic technology. Their breeding and maintainance costs are relatively low. However, mass isolation of viable porcine islets is difficult because of their fragility, and because of donor variability with respect to strain, age, sex, type of feeding, and method of slaughtering. Not all strains are equally suitable for islet separation. The present study investigated pigs from strains available in Poland. MATERIALS AND METHODS
and infused with digestion medium (1–3 mL/g; 4°C) containing collagenase with the addition of 10% porcine serum. Immediately after distension, the organ was transported (4°C, cold ischemia time ⬍190 minutes) to the laboratory. Only the tail of the pancreas was processed using a standard method (73 cases) or with minimal shaking (217 cases). The digestion was ceased when free islets were detected under the microscope. After filtration through 300-m mesh, the islets were washed, suspended in Hanks’s solution, and quantitated for yield. Their number was converted1 into 150-m diameter islet equivalents (IE). Islets ⬍50 m in diameter were not counted. The viability of islets was confirmed by dithizone staining. To eliminate the influence of variations in gradient purification, only the prepurification data on recoverable islets/g and IE/g were considered representative of the number of recoverable islets. The value of a particular strain was assessed according to the frequency of acceptable donors, namely those whose yield of islets and/or IE were ⬎1000/g.
The pancreata were harvested from 248 market weight slaughterhouse pigs (156 males, 92 sows) and from 42 pigs weighing 100 kg, acquired from the Institute of Zootechnics breeding farm and belonging to the following strains: Polish Large White (WBP, 14 sows, 3 males), Polish White Pendant-Ears (PBZ, 16 sows), Pietrain (8 sows), and Yorkshire (1 sow). In the slaughterhouses, the pancreata were dissected ex situ immediately after killing, exsanguination, water [40°C] bathing, and skin shaving (warm ischemia time, 15–20 minutes). The main pancreatic duct was cannulated
From the Transplantation Institute, and Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland. Address reprint requests to M. Sabat, Polish Academy of Sciences, Pawinski Street 5, Warsaw 02-106 Poland.
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00793-0
Transplantation Proceedings, 35, 2343–2344 (2003)
2343
2344
RESULTS
In all specimens, cell groups with diameters ⬎100 m contained the smallest number of islets. The acceptable results (islet and/or IE number ⱖ 1000/g) were obtained from only 56 of 248 slaughterhouse pigs, namely, 2073 ⫾ 137.4SE (median, 1767/g) islets. Most of them were big (IE 2994 ⫾ 303 SE; median, 1874/g). Only 3 of 17 WBP and 2 of 16 PBZ pancreata yielded ⬎ 1000 islets/g. Pancreata of Pietrain and Yorkshire strains yielded ⬍1000 islet/g and clusters of 100 m in diameter contained the smallest number of islets. Pancreata of WBP, PBZ, and Pietrain donors tended to have islets of ⬍100-m diameter (⬎82%). DISCUSSION
Xenotransplantation of porcine islets may be the solution for the shortage of human organ donors. We have confirmed the great variability of islet yield, not only between
SABAT, GODLEWSKA, KINASIEWICZ ET AL
particular strains, but also between animals belonging to the same race. We identified slaughterhouse sources as the best substrain of pigs available in Poland. The rather poor yields obtained in our studies probably result from the elaborate method of slaughtering, namely a hot water bath. The main reason was the prevalence of little islets (80%) in digested organs, a finding in pig pancreata that was already noticed by others.2 For this reason, Krickhahn et al recommended histological screening of each organ after harvesting such that only pancreata with an average islet size ⬎199 m should be digested.
REFERENCES 1. Ricordi C (ed): Pancreatic Islet Cell Transplantation. Austin: R.G. Landes Company; 1992, p 132 2. Krickhahn M, Meyer T, Buehler C, et al: Cell Transplant 11:827, 2002