Dextran Poster 3rd IPITA 1991 • Michel van der Burg • Miracles.Media • 20250104_1 • TakeNode 1c05b2a5-de41-4185-b304-fee2335100f3
Poster (remastered edition, 2025) presented at the 3rd IPITA Congress, June 1991, in Lyon, France (1). One of three posters presented at this congress on purification during the isolation of pancreatic islets (1,2,3,4).
Background
Purification during the isolation of pancreatic islets was greatly improved in 1989 by replacing the conventional islet isolation solutions with the UW organ preservation solution.
The UW-Solution was used as the isolation solution both before , and after the purification of islets — but not yet during the purification step, during centrifugation of the pancreatic tissue in a density gradient.
A conventional (Hanks’ balanced salts) solution was used for preparing density gradients with dextran, the Minneapolis method introduced in 1988 in our laboratory (5).
We had demonstrated previously, that the use of the UW organ preservation solution for isolation of canine pancreatic islets prior to dextran gradient purification, consistently results in >90% purified islets. Further 4 out of 5 islet transplants purified using this gradient had been successful.
Following our islet transplants series June-August 1989 , next a working visit in Minneapolis Sep-Oct ’89, and presentations at the Nov ’89 ESOT congress in Barcelona , we had started a new series of islet isolation experiments from December 1989.
During the first two isolations, Dec 1989, of this new series, I wondered whether the dextran gradient could be optimized , for example by layering the islet suspension in UW-Solution (the pancreatic digest suspension) on top of the dextran bottom layer, or perhaps layering the digest on top of a bottom layer of dextran dissolved in UW-Solution…
The new , much shorter, Labcraft density hydrometers, I had purchased during the Minneapolis visit Nov. 1989 , would greatly facilitate designing new density gradients.
The same lot of dextran (Sigma’s industrial-grade dextran) had been used for all these studies. That stock ran out, so a switch was made Jan 2, 1990 to using a new batch of that dextran powder, resulting however in a significantly impaired recovery of islets during purification , as shown in the poster shown here.
Thus, designing a new density gradient became urgent January 1990.
Dextran UW Gradient • First density gradient in UW-Solution
March 6, 1990 , new density gradient research was started during islet isolation , splitting the suspension of digested pancreatic tissue (‘digest’) for comparison of either dextran of Ficoll (a similar product) in Hanks’ solution , and also a dextran gradient in UW-Solution – the first ‘UW gradient’…
Later that month, purification research was started with a different, essentially pure, dextran product : ‘dextran T70′ from Pharmacia, in Hanks’ solution — with results reported in the poster here.
From April 1990, dextran T70 was used also in dextran-UW gradients, and islet purification research was further extended that month, by designing a density gradient of Percoll in UW Solution (3), and from May 1990, designing a Pentastarch-UW gradient (4).
Notes
1) Van der Burg MPM, Guicherit OR, Scherft JP, Frölich M, Gooszen HG. Sigma’s industrial grade dextran is not suitable for islet isolation. 3rd International Congress on Pancreatic and Islet Transplantation, Symposium on Artificial Insulin Delivery System, Lyon (France) June 6–8, 1991.
2) Dextran UWS Pancreatic Islet Purification • 20250104 • Michel van der Burg • michelvanderburg•com
3) Percoll UWS Pancreatic Islet Purification • 20240731 • Michel van der Burg • michelvanderburg•com
4) PentaStarch UWS Pancreatic Islet Purification • 20240802 • Michel van der Burg • michelvanderburg•com
5) Pancreas & Islet Transplantation Program • 20240912 • Michel van der Burg • michelvanderburg•com
Citation info : Dextran UWS Pancreatic Islet Purification • Michel van der Burg • Miracles.Media • @1MEMO 20250104 • URL michelvanderburg.com/2025/01/04/
Pancreatic Islet Transplantation • Isolation & Transplantation of Islets of Langerhans • 1988 to early 1990s • Islet Transplant Laboratory • Leiden University Hospital • 20240912_1 (Note 1)
Grants
In 1988, we started a project with unique integration of two important trends in research into the treatment of diabetes by transplantation at the Leiden University Hospital (AZL) in Leiden, with major grants from the dutch Ministry of Welfare, Health and Culture (WVC) and the dutch Diabetes Fund (Diabetes Fonds Nederland).
Grants diabetes research • Newspaper Leidsch Dagblad, June 24, 1988 (Note 2)
The generous grants were received — as reported June 24, 1988 in both the dutch newspaper Leidsch Dagblad (2) and in Cicero, the biweekly publication of the Leiden Medical Faculty and University Hospital (3), as a result of our preliminary research over the previous five years into improving the technique of pancreas transplantation, and more recently the development of a technique for isolating the Islets of Langerhans from the pancreas, which produce hormones such as insulin. The idea behind this is, that these isolated islets, after injection into the body, will restart and maintain insulin production.
Questions
What is the consequence of the loss of normal nerve connections to the islets during transplantation? What are the consequences of transplantation of only a segment of the pancreas, or islet transplantation, when fewer islets are available? What is the consequence after transplantation of the different insulin drainage route, where the insulin does not follow the normal path directly to the liver? To what extent do transplantation procedures disrupt the normal architecture and interactions of islets, the pancreas, and other parts of the gastro-intestinal system, and the blood sugar regulation in the body?
Project
With the hope of answering these questions, the grants supported an ambitious new project supervised by Hein Gooszen, integrating two concomitant PhD projects for detailed islet function studies in both experimental pancreas transplantation, by Onno Guicherit (4) , as well as islet isolation by Michel van der Burg (5) in the same model.
A special feature of the experimental design in this preclinical model is that the results of functional studies in the experimental animal can be compared with such functional studies with the isolated islets from the pancreas, where the direct effect on the islets is investigated during perifusion tests in our laboratory (6).
That same month, June 1988, we began research to improve the method of islet isolation in our laboratory, with the assistance of Jane Field, who had come over that month at our invitation from Minneapolis, where she was a key member of the Surgical Research lab, University of Minnesota (led by David Sutherland).
The introduction of this Minneapolis method for islet isolation significantly improved the yield of isolated islets in our Islet Transplant Lab.
New concept : University of Wisconsin solution (UWS) for islet isolation
Next , early 1989 , our Islet Transplant Lab took a major step forward in a new approach to isolation and purification of the islets by replacing the islet isolation solution (the general used basic physiological salt solution or tissue culture solution) with a novel organ preservation solution : the University of Wisconsin solution (UWS). The method of choice nowadays in clinical islet transplantation centers. More on this introduction of UWS will soon be posted here. A reprint of a first poster presented in Minneapolis, Sep. 1989, is now online here (7).
Program
June 1989 our Surgery Department (Leiden University) started an additional project, for ‘auto’ transplantation of isolated islets : Isolation of the islets from the canine pancreas and transplantation of the isolated islets ca 4 hours later in the same animal (8).
Thus, from 1989, we studied the insulin secretion of the isolated islets in perifusion experiments in the laboratory (‘in vitro’) in comparison with both the islet function ‘in vivo’ after transplantation of the isolated islets, and in the concomitant pancreas transplantation experiments — in support of our clinical pancreas transplantation program in Leiden.
Notes
1) Pancreatic Islet Transplantation • Isolation & Transplantation of Islets of Langerhans • 1988 to early 1990s • Islet Transplant Laboratory • Leiden University Hospital • 20240912_1 • michelvanderburg•com | TakeNode 5b5ca362-2adf-497a-b2be-a83e47617017 | Image edit based on : Pancreatic islet transplantation • Michel van der Burg • Miracles.Media • Thesis Repository Leiden University https://hdl.handle.net/1887/3486604
Captions in Note 9
2) Subsidie voor onderzoek suikerziekte. Leidsch Dagblad, June 24, 1988 (leiden.courant.nu) • 20240912_2 • michelvanderburg•com
3) Diabetesonderzoek | AZL krijgt grote subsidies voor diabetesonderzoek | by Sylvia van Leeuwen in Cicero, June 24th, 1988 (ISSN 0920-2900), the biweekly publication of the Academic Hospital and Leiden Medical Faculty | 20240912_3 • michelvanderburg•com | Download PDF article below : file 20240912_3_CICERO
4) Onno R. Guicherit. Long-term metabolic sequelae of beta cell mass reduction, systemic venous drainage and denervation of the canine pancreas : experimental studies in relation to clinical pancreas transplantation. (Doctoral Thesis, Faculty of Medicine, Leiden University Medical Center (LUMC) , Leiden University)(S.l.: s.n.), (ISBN print: 9789090072319, 9090072314), 1994: 149 p. URL WorldCat https://search.worldcat.org/en/title/69433794
5) Michael P.M. van der Burg. Pancreatic islet transplantation: studies on the technique and efficacy of islet isolation and transplantation. (Doctoral Thesis, Faculty of Medicine, Leiden University Medical Center (LUMC) , Leiden University), Boskoop: M.P.M. van der Burg | Miracles.Media (ISBN electronic, pdf, 9789080216402 ; ISBN print 9789080216419, 9080216410), 1994: 192 p. URL Thesis Repository Leiden University https://hdl.handle.net/1887/3486604
9) Pancreatic Islet Transplantation • Isolation & Transplantation of Islets of Langerhans 1988 to early 1990s • Islet Transplant Laboratory • Leiden University Hospital • 20240912_1 • michelvanderburg•com | TakeNode 5b5ca362-2adf-497a-b2be-a83e47617017 |
Captions Image 20240912_1 :
1. Paul Langerhans
2. Section of the normal dog pancreas showing islets stained reddish-brown (immunostained for insulin)
3. Michel van der Burg, watching the dog islet isolation procedure in the Minneapolis surgical research laboratory (University of Minnesota), September 1989, with Philippe Morel and Pericles Tzardis
4. Experimental Surgery Laboratory in Leiden, June 1988, with (left to right) Hein Gooszen, Jane Field (Minneapolis), and Onno Guicherit, starting the surgical procedure for canine islet isolation
5. The pancreas (segment) is removed
6. Islet Transplant Laboratory in Leiden, at the start of islet isolation, with infusion of the collagenase solution via the ducts in the dog pancreas (whole gland for islet transplantation) – collagenase leaking from the pancreas is recirculated using a roller pump
7. During collagenase digestion at 37-39°C the pancreas falls apart, shown here in a low magnification microscopy image of pancreatic exocrine tissue, and a free-ed small blood vessel
8. Dissociated pancreatic tissue, on ice
9. Tissue is further dispersed in the cold isolation solution (here RPMI tissue culture solution is used), by aspiration in a syringe, and sieved to remove undigested fragments, ducts and vessels ~ demonstrated here by Jane Field (Minneapolis), with the introduction of the Minneapolis isolation procedure, June 1988, in our Islet Transplant Laboratory in Leiden
10. Microscopy of tissue suspension, with a low purity of the islets (stained red by dithizone) obtained by density separation in Dextran gradients (in Hanks’ solution) after islet isolation in the RPMI tissue culture solution (dark-brown exocrine fragments remain unstained)
11. Pure islet suspension obtained by density separation in Percoll gradients (in University of Wisconsin solution) after islet isolation in the University of Wisconsin solution (islets are only slightly stained due to poor diffusion over the cell membrane in the preservation solution)
12. Section of highly purified islets obtained by Dextran density separation after isolation in the University of Wisconsin solution
13. Islets are autotransplanted by infusion in the spleen of the dog
14. Transplanted islet in section of the spleen of one of the dogs shortly after the onset of fasting hyperglycemia at three months posttransplant (immunostained for insulin), 1989
15. Highly purified human islets obtained by Percoll density separation after isolation in the University of Wisconsin solution (immunostained with gold for insulin; not counterstained), July 1990
Pancreatic islet transplantation • Michel van der Burg • Miracles.Media | QR code (link to Thesis Repository Leiden University) https://hdl.handle.net/1887/3486604
Citation info : Pancreas & Islet Transplantation Program • 20240912 | Michel van der Burg | michelvanderburg•com
Pancreatic islet transplantation Thesis Repository Leiden University | 20221125 | Michel van der Burg | Miracles•Media
Pancreatic islet transplantation , the doctoral thesis by Michel van der Burg – digital publication ISBN 9789080216402 – is freely available (Open Access) now in the Scholarly Publications repository of Leiden University. Persistent URL of this record https://hdl.handle.net/1887/3486604
The dissertation ‘Pancreatic islet transplantation’ was published first as a paperback in 1994 ( ISBN paperback 9789080216419 ) — and obviously that is considered virtually inaccessible in this digital age. Though it is still available ‘new’ — collectors item ;)
Last October the digital edition ISBN eBook (pdf) 9789080216402 has been produced and first released by the author / publisher Michel van der Burg at Miracles•Media as a free download , available from October 25th, 2022.
