Characterization of Endocrine-Rich Monolayers of Human Fetal Pancreas That Display Reduced Immunogenicity

Abstract

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14–20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 ± 13 and 43 ± 8%, respectively, with the number of β-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6–1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of β-cells for clinical transplantation.