Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca²⁺ ions at a concentration of 5–10 mM. The present study aimed to determine the Ca²⁺ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca²⁺ is required to maintain an optimal Ca²⁺ concentration during the various phases of the islet isolation process.

Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca²⁺ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca²⁺ to reach a Ca²⁺ of 5 mM.

Ca²⁺ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca²⁺ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca²⁺ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas.

Ca²⁺ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca²⁺ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca²⁺ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.