Porcine neonatal pancreatic cell clusters in tissue culture: benefits of serum and immobilization in alginate hydrogel.

Porcine neonatal pancreatic cell clusters (NPCCs) may be a suitable source of insulin producing tissue for transplantation in diabetic patients. The possible beneficial effect of serum on maturation of NPCCs in vitro is difficult to achieve because of cell clumping, which can be avoided by immobilization in alginate hydrogel matrix. Collagenase treated pancreata, cultured for 4 days, formed NPCCs that were embedded in alginate cross-linked with CaCl2 and cultured in modified Ham's F10 medium with 10% fetal calf serum (FCS) for 10 days. NPCCs cultured as suspension in F10+ with 0.5% bovine serum albumin or with 10% FCS were used as control. To prevent the aggregation when cultured with serum, NPCCs were kept as a very diluted suspension. At the beginning and end of the culture, samples were taken for insulin and DNA content and immunostained for beta and non-beta cells. The culture of NPCCs immobilized in alginate resulted with 3-fold increase in insulin content and 9-fold increase in insulin/DNA ratio. Histology revealed evident increase of number of insulin- and other hormone-positive cells compared with the control. Even though 2 weeks in culture resulted in impaired glucose-induced insulin release, the amount of insulin secreted by clusters cultured in the presence of serum was 4-fold higher than in serum-free conditions. After transplantation, NPCCs retrieved from alginate reversed hyperglycemia similarly to NPCCs cultured in standard conditions. In conclusion, this study shows the feasibility of in vitro immobilization of NPCCs in alginate three-dimensional matrix, allowing cell clusters to be cultured at least two times higher density compared with culture in suspension.