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 beta-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 beta-cells for clinical transplantation.

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture

“…Some authors have used specific reagents, such as iodoacetic acid (Rabinovitch et al 1980) or thimerosal (Bratten et al 1974), to eliminate fibroblasts; however, these reagents could have had a toxic effect on duct cells, as we observed in our cultures exposed to thimerosal (data not shown). A negative toxic effect of iodoacetic acid and thimerosal on fetal human pancreatic cells survival in vitro has also been reported by Simpson et al (1991).…”

“…More recently, several studies of fetal human or porcine pancreatic tissue in monolayer cultures have been published (Mandel et al 1982;Sandler et al 1985;Simpson et al 1990;Simpson et al 1991;Korsgren et al 1993;Kovarik et al 1995;Beattie et al 1996;Beattie et al 1997b;Hayek & Beattie 1997;Yoon et al 1999).…”

Growth-Promoting Effect of Rh(D) Antibody on Human Pancreatic Islet Cells

“…insulin and glucagon) was excluded by double staining the same sample of cells with different antibodies to distinguish the cell types (e.g. horseradish peroxidase for glucagon and alkaline phosphatase for insulin), as previously described (17). Dou-ble immunofluorescence staining for insulin (Linco Research, St. Charles, MO) and Ki67 (GenTex, Carbondale, PA), a cell proliferation marker, was carried out on unfixed cultures as described (18).…”

Expression of Glucokinase in Glucose-Unresponsive Human Fetal Pancreatic Islet-Like Cell Clusters1

“…Human fetal pancreas was obtained from the therapeutic termination of pregnancies between 16 -19 weeks gestation (40 -48% of term), and ICCs were isolated as described previously (11). Maternal consent for the use of fetal tissue was obtained, and ethical permission was given by the human ethics committees of both The University of New South Wales and the Eastern Sydney Area Health Service (Sydney, Australia).…”