Cryopreservation of Freshly Isolated Porcine Islet Cells P. Stiegler, V. Stadlbauer, S. Schaffellner, H. Florian, C. Lackner, F. Iberer, and K. Tscheliessnigg ABSTRACT Introduction. The use of xenogenic islet cells may be a possibility to overcome the shortage of human donor organs to treat diabetes. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor intensive, cryopreservation has emerged as an attractive system of islet banking. The aim of this study was to determine the influence of three different freezing media (FM) on viability of freshly isolated porcine islet cells (FIPIC). Methods. FIPIC were isolated using a modified Ricordi method and purification performed using a Lymphoprep density gradient. Viability of FIPIC prior to freezing and after thawing was determined using the MTT-based Cell Growth Determination Kit. Insulin production was detected using enzyme-linked immunosorbent assay. Three different FM containing dimethylsulfoxide (DMSO) or glycerol and sucrose were used for cryoprotection of FIPIC. Results. Isolation and purification of FIPIC resulted in 95% ⫾ 1.3% viability and 97% ⫾ 1.4% purity. Cryopreservation with FM I (containing DMEM, FCS, DMSO) yielded 98.4% and FM III (containing DMEM, FCS, glycerol) 93.1% viability, whereas only 85.6% were alive when cryoprotection is performed with FM II (containing DMSO, BM). Glucose stimulation revealed a loss of 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P ⬍ .05) when FIPIC were stored in FM II. Discussion. Low concentrations of DMSO or the use of glycerol and sucrose seem to be equivalent to cryopreserve FIPIC.
HE USE OF XENOGENIC islet cells to physiologically replace the endocrine pancreas seems to be an alternative to alleviate the shortage of human donor organs.1,2 However, xenogenic cells have to be isolated from the host immune system to avoid hyperacute rejection. Microencapsulation is an effective method for immunoisolation.3,4 The process of isolating, purifying, microencapsulation, and transplanting islets in a single day is labor intensive and difficult for routine use. Therefore, cryopreservation has emerged as an attractive system of islet banking.3,5 As cryopreservation can facilitate clinical islet transplantation by providing a possibility to store supplemental islets to augment marginally adequate grafts, protocols are needed for minimal loss of viable ␤ cells.6 In order to survive freezing and thawing, cells must be treated with different cryoprotective agents. The aim of this study was to determine the influence of three freezing media (FM) on viability and functionality of freshly isolated porcine islet cells (FIPIC).
MATERIALS AND METHODS Isolation and Purification of FIPIC Porcine pancreata were harvested at the local slaughterhouse according to EU guidelines of the slaughtering process. Pancreas (4 U/g) of Collagenase NB1 (Serva, Heidelberg, Germany) and 80 U of Neutral Protease NB (Serva, Heidelberg, Germany) were used for enzymatic digestion of the pancreas. A novel method with a modified Ricordi chamber7 of stainless steel, an 800-m mesh, and four glass balls was developed in our laboratory for islet isolation. Purification was performed using Lymphoprep (AxisShield PoC AS, Oslo, Norway) density gradient. Isolation success was assessed using conventional dithizone staining for purity and From the Department for Transplantation Surgery, University Clinic for Surgery, Graz, Austria. Address reprint requests to Dr Philipp Stiegler, MD, Department for Transplantation Surgery, University Clinic for Surgery, Auenbruggerplatz 29, Graz, Austria 8036, Austria. E-mail: [email protected]
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0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.02.076
Transplantation Proceedings, 39, 1609 –1611 (2007)
STIEGLER, STADLBAUER, SCHAFFELLNER
Fig 1. (A) Decrease in viability after cryopreservation of FIPIC determined by MTT-based Cell Growth Determination Kit. Cells cryopreserved in FM II had significantly poorer viability after overnight culture. (B) Percentage decrease in insulin secretion per microgram DNA after cryopreservation. Cells stored in FM II showed significantly lower insulin secretion compared to FIPIC cryopreserved with FM I and FM III. FDA/PI staining for viability testing immediately after isolation and purification.
Freezing and Thawing of FIPIC FIPIC were rinsed with basal medium (BM) containing 2,5% fetal calf serum (FCS), 7% horse serum, 0.4% glutathione, and 89% HAM F12 supplemented with 1 mL on antibiotic/antimycotic supplement (10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate, and 25 g/mL amphotericin B). All reagents were obtained from Sigma Aldrich (Vienna, Austria). FM I consisted of 70% Dulbecco’s modified Eagle medium high glucose (DMEM, Gibco, Invitrogen Corporation, Paisley, UK) with 4.5 mg/L D-glucose, 20% FCS (Sigma-Aldrich, Vienna, Austria), 10% dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) and 0.4 mol/L sucrose (␣-D-glucopyranosyl ␤-D-fructo-furanoside) (Sigma-Aldrich, Vienna, Austria). FM II consisted of 40% DMSO and 60% basal medium. FM III consisted of 75% DMEM high glucose, 25% FCS, and 1 mol/L glycerol (Merck, Darmstadt, Germany) and 0.4 mol/L sucrose. For freezing, 10 mL of cold FM I or II was added to FIPIC and 1 mL of this suspension was transferred in 1.2-mL freezing ampules (Corning, SZABO-SCANDIC HandelsgmbH & Co KG, Vienna, Austria) and frozen slowly with a temperature decrease of 1°C per minute using a freezing container (5100 Cryo 1°C Freezing Container, “Mr. Frosty,” Nalge Nunc International, Rochester, NY, USA). Using FM III, FIPIC were taken in 4.5 mL cold FCS and put on ice for 10 minutes. FM III (1.5 mL) were added and the procedure continued as described above. For thawing, the cryovials were put in a water bath at 37°C until it was possible to drain the semifrozen liquid in 15 mL BM. FIPIC were sown in a culture flask after washing with BM several times. After overnight culture, cell viability and functionality was tested as described below.
Cell Viability and Functionality The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT)based Cell Growth Determination Kit (Sigma-Aldrich, Vienna, Austria) was used for viability assessment exactly as described by the manufacturer before freezing and after overnight culture following thawing. FIPIC were exposed to 200 mg/dL glucose for 60 minutes to assess islet functionality. Insulin concentration was measured using the Mercodia High Range Porcine Insulin ELISA (Mercodia AB, Uppsala, Sweden) as described by the manufacturer. DNA isolation was performed using the DNeasy-Kit (Quiagen, Vienna, Austria) as described by the manufacturer before freezing and after overnight culture following thawing.
Statistics All experiments were performed in quadruplicates. Descriptive statistics and one-way analysis of variance were used with P ⱕ .05 considered significant.
Isolation and purification of FIPIC resulted in 95% ⫾ 1.3% viability and 97% ⫾ 1.4% purity. Islet yields of isolations used for these experiments were 3479 ⫾ 2029 islet-like equivalents. After cryopreservation FM I yielded 98.4% and FM III 93.1% viability, whereas only 85.6% were alive when cryoprotection was performed with FM II (P ⬍ .05, Fig 1A). Stimulation with 200 mg/dL glucose for 60 minutes prior to freezing resulted in insulin secretion of FIPIC at a level of 15.1 ⫾ 0.3 ng insulin/g DNA. After cryopreservation and overnight culture, glucose stimulation revealed a loss of only 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P ⬍ .05) when FIPIC were stored in FM II (Fig 1B). DISCUSSION
Isolation, purification, microencapsulation, and transplantation of FIPIC is labor intensive and time-consuming. Therefore cryopreservation of FIPIC has emerged as an attractive system of islet banking. In this study two FM containing different amounts of DMSO and one medium containing glycerol and sucrose were tested to assess their cryoprotective ability when working with FIPIC. Best results in viability after cryopreservation were obtained when working with FM I, whereas glucose-dependent insulin secretion had a tendency to decrease less when using FM III (1.9%) compared to 2.8% applying FM I. Using higher concentrations of DMSO as a cryoprotective agent resulted in 14.4% cell loss and 7.8% decrease in insulin secretion. Low concentrations of DMSO or the use of glycerol and sucrose seemed to be equivalent in cryopreserving FIPIC. REFERENCES 1. Daar AS: Ethics of xenotransplantation: animal issues, consent, and likely transformation of transplant ethics. World J Surg 21:975; 1997
CRYOPRESERVATION OF PORCINE ISLET CELLS 2. Smith RM, Mandel TE: Pancreatic islet xenotransplantation: the potential for tolerance induction. Immunol Today 21:42; 2000 3. Charles K, Harland RC, Ching D, et al: Storage and microencapsulation of islets for transplantation. Cell Transplant 9:33; 2000 4. Stadlbauer V, Stiegler P, Schaffellner S, et al: Morphological and functional characterization of a pancreatic ␤-cell line microencapsulated in sodium cellulose sulphate/poly(diallyldimethylammoniumchloride). Xenotransplantation 13:337–344; 2006
1611 5. Stiegler P, Stadlbauer V, Schaffellner S, et al: Cryopreservation of insulin producing cells encapsulated in sodium cellulose sulfate (NaCS). Transplant Proc 38:3026, 2006 6. Korbutt GS, Rayat GR, Ezekowitz J, et al: Cryopreservation of rat pancreatic islets: effect of ethylene glycol on islet function and cellular composition. Transplantation 64:1065, 1997 7. Ricordi C, Finke EH, Lancy PE: A method for mass isolation of islets from the adult pig pancreas. Diabetes 35:649 – 652, 1986