J Korean Diabetes Assoc.  2002 Oct;26(5):383-395.

3-Dimensional Long Term Culture of Monolayer Cultured Dispersed Neonatal Porcine Pancreas Cells (NPCC)

Affiliations
  • 1Division of Endocrinology and Metabolism, Department of Internal Medicine, The Catholic University of Korea, Seoul, Korea.
  • 2Immunology and cell Biology core laboratory, The Catholic University of Korea, Seoul, Korea.

Abstract

BACKGROUND: We have reported porcine neonatal pancreas cell clusters (NPCCs) to be useful clinical alternative due to their growth potential and convenience. However, to apply the porcine NPCCs in human islet transplantation, there is a need to achieve in vitro maturation of porcine pancreas duct cells for the immediate cure of diabetes, and to escape hyperacute rejection. We have established a long-term 3D culture system of porcine pancreas duct cells for their in vitro induction in differentiated beta-cells. METHOD: For making NPCCs, pancreata from 1~3 days old pigs were minced, digested and cultured for 8 days. After 8 days, the cells were layered with Matrigel. After 50 days, the 3 dimensional cultures, the components of the reconstructed cell clusters were confirmed by three approaches: immunofluorescent staining, mea-surement of glucose stimulated insulin secretion and semiquantitative RT-PCR. RESULT: The monolayers of epithelial cells formed three-dimensional structures of cysts from which 50~200 micro meter diameter islet-like clusters of pancreas cells budded. The insulin and DNA contents, and the ratio of insulin/DNA, did not change significantly, even after 50 days of culturinge. The levels of insulin and galactosyl transferase mRNA showed a tendency to increase in the monolayer culture of the duct cells until day 8, after which the levels significantly decreased. However, the level of glucagon mRNA was maintained until day 50. Compared with their basal secretion at 5mM glucose, the cysts/cultivated porcine islet buds exposed to stimulatory 20mM glucose did not show difference in insulin secretion.
CONCLUSION
We have shown the expansion of dispersed porcine neonatal pancreas cells in vitro, and the reconstruction of a three-dimensional structure, following Matrigel overlaying, but were unable to observe the transition of duct cells to beta cells, as observed in human duct cells. Further studies will be required to elucidate this difference.

Keyword

Porcine; Beta-cell; Three-dimensional Culture; Duct cell

MeSH Terms

DNA
Epithelial Cells
Glucagon
Glucose
Humans
Insulin
Islets of Langerhans Transplantation
Pancreas*
RNA, Messenger
Swine
Transferases
United Nations
DNA
Glucagon
Glucose
Insulin
RNA, Messenger
Transferases
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