0963-6897/04 $20.00 + .00 Copyright 2004 Cognizant Comm. Corp. www.cognizantcommunication.com
Cell Transplantation, Vol. 13, pp. 497–502, 2004 Printed in the USA. All rights reserved.
The Use of Multiparametric Monitoring During Islet Cell Isolation and Culture: A Potential Tool for In-Process Corrections of Critical Physiological Factors Chris Fraker, Jorge Montelongo, Joel Szust, Aisha Khan, and Camillo Ricordi Diabetes Research Institute, Cell Transplant Center, University of Miami School of Medicine, Miami, FL
Variables such as pH, pCO2, and pO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted. This report describes a highly accurate continuous multiparametric monitoring system and its evaluation for continuous monitoring of physiological variables during critical steps of the islet isolation procedure as well as during in vitro culture of the insulin-producing cells. Close monitoring of these variables could be of assistance to improve the outcome of islet cell processing, allowing to identify as soon as possible problems that could be corrected during the procedure, as well as during in vitro preservation, or shipment to remote sites. Key words: Assessment; Monitoring; Islet; Isolation; Cell culture; Optical probes
INTRODUCTION Islet cell transplantation represents a potentially valuable therapeutic approach for the treatment of type I diabetes mellitus. Recent clinical trials carried out at several institutions, including our own, have clearly indicated that islet transplantation can result in a high success rate in patients with type I diabetes (19,20). With the recent clinical advances in islet cell transplantation, there has been an increased need for standardization and further improvement of islet cell processing technologies. In this direction, close monitoring of critical physiological variables during the isolation procedure would be highly desirable. Given the elevated sensitivity of islet cells to variations in oxygen partial pressures and pH, it would be important to assess deviations from the physiological range in pH, pO2, pCO2, and temperature, as they could affect the outcome of islet isolations and purification procedures. Besides islet yield, viability and function of the islet cells after isolation procedures could also be affected by nonphysiological conditions in the solutions used throughout processing and preservation. Careful monitoring of these variables during the isolation procedure could be of assistance not only to document potential deviations, but also to correct
them in a timely fashion, towards further improvement of islet isolation outcomes. MATERIALS AND METHODS Multiparametric Monitor: Setup and Calibration The TrendCare/Paratrend 7+ (Diametrics Medical Inc., Minneapolis, MN) is an automated fiber optic probe system that monitors in real time pH, pCO2, pO2, and temperature, as well as calculating bicarbonate concentration and base excess values. The probes are FDA approved for intra-arterial (femoral, inguinal, cranial) and brain tissue monitoring. The Paratrend probes are currently used extensively in continuous patient multiparametric monitoring during surgical procedures and in the intensive care setting (9,16,28,29). The Paratrend 7+ probes are prepackaged, sterile units that are attached by means of an in-line connector to the fiber optic conduction lines of the monitor. The probe packaging allows for the maintenance of sterility during a three-point automated 30-min gas calibration with varying gas mixtures, delivered through calibration tanks connected in the base of the unit. At the end of calibration, the flexible sensor is introduced into the environment to be monitored by a control dial that extends and retracts the probe. Immedi-
Accepted February 10, 2004. Address correspondence to Camillo Ricordi, M.D., Stacy Joy Goodman Professor of Surgery and Medicine, Chief, Division of Cellular Transplantation, Scientific Director and Chief Academic Officer, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136. Tel: (305) 243-6913; Fax: (305) 243-4404; E-mail:
[email protected]
497
498
ately following the calibration, the sensor must be hydrated and remain hydrated at all times, either in the monitored environment or in its sterile luer-locked storage tubing. When the sensors were used during the islet isolation procedure, they were disposed of immediately after, unless monitoring was continued on the same isolated cells during in vitro culture. In addition, for continuous monitoring of selected culture media conditions, the probes were utilized for up to 3 days. Published data regarding long-term monitoring using the Paratrend sensors indicate that there is some negligible drift over extended monitoring time, but substantially less than would be observed in conventional fluorescence and electrochemical electrodes. As well, the system can be manually adjusted to compensate for deviations from blood gas values measured on a laboratory gas analyzer. The sensors have a recommended use period of 30 days or less in patients and have been safely used and have demonstrated comparable accuracy to blood gas analysis for periods of time much greater than the 3-day monitoring periods of our study (28). Data are collected in the unit for 24 h and can be displayed on the monitor screen and printed onto a chart strip. The data can be shuttled via a built-in communications port to a secondary collection computer at predetermined rates of collection, varying from every second to every 30 min, therefore bypassing the 24-h limitation of the built-in data module. At all times, the algorithms of the unit make corrections for vapor pressure and deviations of temperature, related to gas solubility. The user has the ability to view values of monitored parameters adjusted to body temperature (37°C) or measured environmental values. Multiparametric Monitoring of Islet Isolation Ten consecutive human pancreata were obtained from OPOs to be utilized for the islet cell isolation procedure using a modification of the automated method (17). Multiparametric fiber optic probes (Diametrics Inc.) were inserted, after sterile calibration, by means of a Y-connector into the tubing approximately 10 cm downstream from the Ricordi chamber. The probe was extended approximately 2.5–3 inches into the tubing system to insure that all four parametric monitors were exposed to the circulating cells and media for the duration of the procedure. The monitor was connected via a communications port to a laptop computer utilizing a HyperTerminal protocol to collect real-time and 37°C adjusted data every second. The monitoring was initiated at the start of the isolation procedure and carried through the end of the dilution phase, when the cells were purified on Ficoll density gradients. Single point monitoring was also performed during and after Ficoll purification on several pancreata. At the conclusion of
FRAKER ET AL.
each procedure, the real-time data were plotted to observe the changes of all four parameters during the procedure. Multiparametric Monitoring of Islet Cells in Culture Following two islet isolation procedures, the Paratrend 7+ probe was introduced into a flask with the cap removed containing isolated islets in different culture conditions. In particular, variations in culture temperature and carbon dioxide concentration and their effects on culture conditions were tested. The probe was left to monitor the cells overnight or for longer periods. At the end of the culture period, the data were collected, as above, and graphed to monitor the variation of the culture parameters over time. Multiparametric Monitoring of Culture Media To assess physiological parameters in selected media formulations, the Paratrend 7+ probe was introduced into flasks (at the bottom of the flask) containing medium alone. Studies were performed with different media recipes and with different culture conditions, related to gas concentrations, temperature, and culture vessel. The probe was utilized to simulate the shipment of islets in order to observe the variations in physiological parameters over the course of an overnight shipment period to other centers. At the conclusion of each study the data were graphed to assess fluctuations in the selected physiological variables over time. RESULTS Multiparametric Monitoring of Islet Isolation Temperature was the most consistent variable, rising to near 37°C during digestion and then dropping to levels below 10°C during the dilution phase, as per protocol. In all isolations, despite variations in the length of digestion time, this temperature shift was comparable (Fig. 1). As for the other three measured parameters, variations were significant and followed no clearly predictable pattern. A common trend was a drop in pH during the phase 1 digestion of the islet isolation procedure, which was paralleled by an expected increase in pCO2 most likely due to the activity of cells during digestion. Oxygen levels varied significantly as well. Some of the variations could be attributed to changes in temperature that affect oxygen solubility, although changes in oxygen solubility were minimal across the range of temperatures during islet cell processing. It is conceivable that cell respiratory and metabolic activity could affect the observed fluctuations in pO2 and pCO2. In Figure 1A, pH remained relatively stable during the digestion phase (14) at 37°C. This was made possible by interchamber correction of the pH by addition of small amounts of NaOH to the chamber. In-process correction of the ob-
MONITORING OF ISLET CELL ISOLATION & CULTURE
Figure 1. Monitored physiological parameters from a representative “good” (A) and “poor” (B) islet isolation.
499
500
FRAKER ET AL.
Figure 2. Multiparametic monitoring of physiological parameters in cultured cells. (A) Islets were cultured in conventional culture medium at 22°C in room air to simulate islet shipment to a remote center. (B) Islets were cultured at 37°C in the presence of room air with 5% carbon dioxide.
served drop in pH (from 7 to less than 6.8) most likely prevented a further drop in pH levels that could have been associated with a negative islet isolation outcome, as observed in the processing monitored in Figure 1B. In this case, a poor quality pancreas with prolonged is-
chemia time required an extended digestion time at 37°C. This was associated with a significant drop in pH from approximately 7 to less than 6.3. In this isolation, there was no attempt to perform in-process corrections of the pH level during the procedure and the pH dropped
MONITORING OF ISLET CELL ISOLATION & CULTURE
to levels inferior to 6.5 for approximately 15 min. This isolation was completely unsuccessful, with less than 7000 IEQ obtained, even prepurification. Multiparametric Monitoring of Culture Conditions In culture, the multiparametric monitor proved a particularly useful tool. Given the sensitivity of the islet cells to slight variations in physiological variables, we were able to utilize this monitoring system to assess over time selected culture conditions, simulations of islet shipment conditions, and to determine the effect of different cellular compositions on selected variables monitored during culture for prolonged periods (Fig. 2). The utilization of this multiparametric monitoring system for the assessment of our media formulations was very effective, allowing us to measure the stability of physiological parameters in selected culture media and in different culture conditions. In one media formulation we observed a progressive, significant drop in the pH levels. In other formulations, shifts in pH were observed following the addition of select additives to the base medium. In the experiments on the simulation of islet shipment, we observed that physiological parameters, most notably the pH, were stable for long durations of time, validating the stability of culture conditions during islet shipment for transplant applications (11). DISCUSSION Close monitoring of variables that could affect the outcome of islet cell processing could become highly desirable to identify as soon as possible problems that could be corrected during islet processing, in vitro preservation, or shipment to remote sites. Variables such as pH, pCO2, and pO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted (2–8,12–15,18,21,22). Given the recent and ongoing successes of islet cell transplant programs, it would seem likely that the multiparameteric monitoring of such variables could become an important tool in the continued improvement of the field of islet cell transplantation (1,10,23–26).
501
6. 7. 8. 9.
10.
11.
12.
13.
14. 15.
16.
17. 18.
REFERENCES 1. Baidal, D. A.; Ferreira, J. V.; Khan, A.; Alejandro, R.; Ricordi, C. The bag method for islet cell infusion. Cell Transplant. 12:809–813; 2003. 2. Biancone, L.; Ricordi, C. Pancreatic islet transplantation: An update. Cell Transplant. 11(4):309–311; 2002. 3. Burghen, G. A.; Murrell, L. R. Factors influencing isolation of islets of Langerhans. Diabetes 38(Suppl. 1):129– 132; 1989. 4. Carlsson, P. O.; Palm, F. Markedly decreased oxygen tension in transplanted rat pancreatic islets irrespective of the implantation site. Diabetes 50(3):489–495; 2001. 5. Carlsson, P. O.; Mattsson, G. Oxygen tension and blood
19. 20. 21. 22. 23.
flow in relation to revascularization in transplanted adult and fetal rat pancreatic islets. Cell Transplant. 11(8):813– 820; 2002. Carlsson, P. O.; Palm, F.; Mattsson, G. Low revascularization of experimentally transplanted human pancreatic islets. J. Clin. Endocrinol. Metab. 87(12):5418–5423; 2002. Carlsson, P. O.; Palm, F. Oxygen tension in isolated transplanted rat islets and in islets of rat whole-pancreas transplants. Transpl. Int. 15(11):581–585; 2002. Carlsson, P. O.; Nordin, A.; Palm, F. pH is decreased in transplanted rat pancreatic islets. Am. J. Physiol. Endocrinol. Metab. 284(3):E499–504; 2003. Coule, L. W.; Truemper, E. J.; Steinhart, C. M.; Lutin, W. A. Accuracy and utility of a continuous intra-arterial blood gas monitoring system in pediatric patients. Crit. Care Med. 29(2):420–426; 2001. Froud, T.; Yrizarry, J. M.; Alejandro, R.; Ricordi, C. Use of D-StatTM to prevent bleeding following percutaneous transhepatic intraportal islet transplantation. Cell Transplant. 13:55–59; 2004. Goss, J. A.; Schock, A. P.; Brunicardi, F. C.; Goodpastor, S. E.; Garber, A. J.; Soltes, G.; Barth, M.; Froud, T.; Alejandro, R.; Ricordi, C. Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center. Transplantation 74(12):1761– 1766; 2002. Hering, B. J.; Matsumoto, I.; Sawada, T.; Nakano, M.; Sakai, T.; Kandaswamy, R.; Sutherland, D. E. Impact of two-layer pancreas preservation on islet isolation and transplantation. Transplantation 74(12):1813–1816; 2002. Lu, S.; Jaeger, C.; Wolff-Vorbeck, G.; Khalaf, A. N.; Kirste, G. Increase of islet yields obtained from human pancreas donor following change of pH value in collagenase digestion medium. Transplant. Proc. 27(6):3202–3204; 1995. Lynch, A.; Best, L. Cytosolic pH and pancreatic beta-cell function. Biochem. Pharmacol. 40(3):411–416; 1990. Matsumoto, S.; Rigley, T. H.; Qualley, S. A.; Kuroda, Y.; Reems, J. A.; Stevens, R. B. Efficacy of the oxygen-charged static two-layer method for short-term pancreas preservation and islet isolation from nonhuman primate and human pancreata. Cell Transplant. 11(8):769–777; 2002. Palmer, T. E.; Cumpston, P. H.; Foster, W. J.; Jones, R. D. Continuous intravascular blood gas analysis during aortic aneurysm repair: The Paratrend 7. Anaesth. Intensive Care 23(2):200–202; 1995. Ricordi, C.; Lacy, P. E.; Scharp, D. W. Automated method for isolation of human pancreatic islets. Diabetes 37(4): 413–420; 1988. Ricordi, C.; Fraker, C.; Szust, J.; Al-Abdullah, I.; Poggioli, R.; Kirlew, T.; Khan, A.; Alejandro, R. Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the coldstorage solution. Transplantation 75(9):1524–1527; 2003. Ricordi, C. Islet transplantation: A brave new world. Diabetes 52(7):1595–1603; 2003. Ricordi, C.; Strom, T. Clinical islet transplantation: Advances and immunological challenges. Nat. Rev. Immunol. 4:259–268; 2004. Ronner, P. Transmembrane ion distribution and insulin secretion. Ann. NY Acad. Sci. 488:341–355; 1986. Rose, N. L.; Palcic, M. M.; Lakey, J. R. Evaluating the effect of serine proteases on collagenase activity during human islet isolation. Cell Transplant. 11:821–826; 2002. Ryan, E. A.; Lakey, J. R.; Paty, B. W.; Imes, S.; Korbutt,
502
G. S.; Kneteman, N. M.; Bigam, D.; Rajotte, R. V.; Shapiro, A. M. Successful islet transplantation: Continued insulin reserve provides long-term glycemic control. Diabetes 51(7):2148–2157; 2002. 24. Shapiro, A. M.; Lakey, J. R.; Ryan, E. A.; Korbutt, G. S.; Toth, E.; Warnock, G. L.; Kneteman, N. M.; Rajotte, R. V. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343(4):230–238; 2000. 25. Shapiro, A. M.; Ryan, E. A.; Lakey, J. R. Diabetes. Islet cell transplantation. Lancet 358(Suppl.):S21; 2001. 26. Shapiro, A. M.; Ricordi, C.; Hering, B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 362(9391):1242; 2003.
FRAKER ET AL.
27. Sweet, I. R.; Khalil, G.; Wallen, A. R.; Steedman, M.; Schenkman, K. A.; Reems, J. A.; Kahn, S. E.; Callis, J. B. Continuous measurement of oxygen consumption by pancreatic islets. Diabetes Technol. Ther. 4(5):661–672; 2002. 28. Venkatesh, B.; Clutton-Brock, T. H.; Hendry, S. P. Evaluation of the Paratrend 7 intravascular blood gas monitor during cardiac surgery: Comparison with the C4000 inline blood gas monitor during cardiopulmonary bypass. J. Cardiothorac. Vasc. Anesth. 9(4):412–419; 1995. 29. Zollinger, A.; Spahn, D. R.; Singer, T.; Zalunardo, M. P.; Stoehr, S.; Weder, W.; Pasch, T. Accuracy and clinical performance of a continuous intra-arterial blood-gas monitoring system during thoracoscopic surgery. Br. J. Anaesth. 79(1):47–52; 1997.
