Proliferation of pancreatic endocrine cells using disaggregation-expansion-reaggregation technology in isolated rat islets.
Donor scarcity is a major obstacle for clinical islet transplantation. Hence, the effective use of the limited number of available islets is necessary for successful islet transplantation. We have developed a new technology that could produce pseudo-islets. Morphologic and functional evaluation was performed to test the feasibility of using these cells for transplantation. A 3-step procedure known as disaggregation-expansion-reaggregation (DER) was employed for pseudo-islet preparation. Islets isolated from 200 to 250-g male Lewis rats by collagenase digestion were separated into single cells by trypsinization. These pancreatic endocrine cells (PECs) were expanded by serial passages in culture before being aggregated at a high cell-density in a suspended state. After DER, cells were morphologically analyzed over time, and gene expression evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Through expansion by passage for 2 weeks in continuous cultures, approximately 1 million PECs were recovered after aggregation. By phase-contrast microscopy, they presented with spherical shapes and similar sizes compared with naïve islets (50-800 microm). RT-PCR results indicated expression of insulin, glucagon, and pancreatic and duodenal homeobox gene 1, which were observed in primary isolated islets as well. The insulin secretion capacity of pseudo-islets was confirmed by enzyme-linked immunosorbent assay. In conclusion, PECs treated with DER showed potential to serve as a cell source for pseudo-islet generation after in vitro cellular expansion. These cells were both morphologically and genetically similar to naïve islets. Our new technique could be a potential method to overcome the scarcity of donor islets in the near future.
Jeong
J H
JH
Research Institute for Transplantation, Yonsei University, Graduate Program of Nanoscience and Technology, Department of Surgery, Yonsei University College of Medicine, Yonsei University, Seoul, South Korea.
Lee
J-I
JI
Ju
M K
MK
Joo
D J
DJ
Huh
K H
KH
Kim
M S
MS
Kim
J Y
JY
Cho
Y
Y
Kim
Y S
YS
D000818:Animals; D002449:Cell Aggregation / Q000502:physiology*; D002499:Centrifugation, Density Gradient / Q000379:methods; D017931:DNA Primers; D005934:Glucagon / Q000235:genetics; D005947:Glucose / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008858:Microscopy, Phase-Contrast / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics
D017931:DNA Primers; D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D012313:RNA; D005934:Glucagon; D005947:Glucose