|CINCINNATI February 2000
Our collaborator Dr. Horacio Rilo decided to move to the University of Cincinnati in 1999. He invited us to come to his new laboratory as soon as it was outfitted and the experiments had been approved by the animal research authorities at UC. This turned out to be mid-February. His laboratory is in the biotechnology incubator center right on the medical center campus (left).
Our first collaboration with Dr. Rilo's group at the University of Cincinnati was productive and exhausting. We made and implanted fifteen test sheets, some with islets of Langerhans from three dogs. One diabetic dog received six Islet Sheets made from the dog's own islets. The other diabetic recipient received six Islet Sheets made from the islets of two donors. (The other sheets were various controls without purified islets.)
We arrived late on Tuesday, February 15. Arrangements had been handled by Georgeanna Adams (right), to whom we are grateful. We stayed in Covington, Kentucky, Cincinnati's twin city across the February-brown, debris-filled Ohio river.
We arrived Wednesday at Horacio's new laboratory. He had been up all night unpacking and preparing equipment! Our experiment the very first use of the new Cincinnati islet laboratory. We were honored and somewhat rattled.
In addition to Georgeanna, Horacio had hired one technician, Lisa, left. (His staff continues to expand.) It was her second day on the job and also her first islet isolation. Fortunately, another scientist experienced in islet isolations was visiting Horacio's laboratory and he was able to help out. Thus we had enough hands -- barely.
The plan was to do the allograft on Wednesday and the autograft on Thursday. So on Wednesday Horacio did two total canine pancreatectomies to isolate the pancreases. Pancreatectomy requires skill and experience because the vascular supply to the organ needs to be preserved so that the islets inside remain healthy.
The second step in islet isolation is the digestion of the pancreas. A special enzyme preparation (LiberaseTM collagenase) is used to make the ducts and exocrine cells fall apart, leaving only the islets as clumps of tissue. The fresh canine pancreas is inflated with a solution of collagenase (right).
The next step is physical disruption of the pancreas. At left is a Ricordi-style digester (named after Camillo Ricordi of the University of Miami DRI). The pancreas is digested for 10 to 20 minutes, and the result is a suspension of cells and cell clusters including islets of Langerhans. The undigested debris remains is the Ricordi chamber.
The suspension is purified using a Cobe cell purification machine. This device basically separates tissue by density. The density of islets of Langerhans is lower than the density of other cells in the pancreas.
All of these manipulations are performed under conditions designed to prevent contamination. The fluids are kept in closed systems or, if open, handled in biological safety hoods with curtains of filtered air.
After all that work, you end up with a small vial of pure islets of Langerhans.
Preparation of sheets from these islets went well. In fact, we have made so many test sheets over the past nine months that sheet fabrication was uneventful (in contrast to the near panic during our first experiment at the University of Chicago).
To the right Dr. Rilo (back to you) and UC staff are implanting the sheets into the diabetic dog's abdominal cavity. All the sheets were put on the omentum, a large membrane stretching from the stomach to the spleen. Each sheet was secured with four sutures in the corners.
All went according to plan. We completed the allograft on Wednesday and the autograft on Thursday, and had a planning meeting on Friday. The dogs recovered well from surgery.
Our main concern was that we had islets with some bacterial contamination. This was because, like any human activity, islet isolation is hard to do right the first time in a new setting. So we decided to treat the dogs with antibiotics (common enough post-surgically). We were also concerned that the donor dogs were young. The quality of islets isolated from young dogs is usually inferior.
The crew on Friday is below, looking tired because we had not slept much in three days.
Horacio Rilo MD, Rick Storrs, Randy Dorian, Scott King
(I am on the right. They let me operate a centrifuge to pellet the islets, proving we were shorthanded.)
These two experiments were Cincinnati dogs #1 and 2. Click here to see how the dogs are doing.