A simple two-step protocol for the purification of human pancreatic beta cells.
OBJECTIVE
Isolated pure human beta cells would be helpful for a number of research purposes. However, lack of beta cell-specific surface antigens has been a major problem. We aimed to develop a simple method for human beta cell isolation based on the initial elimination of ductal cells by their expression of carbohydrate antigen 19-9 (CA19-9), followed by positive selection of beta cells by their expression of polysialic acid-neural cell adhesion molecule (PSA-NCAM).


METHODS
Cell type-specific expression of CA19-9, NCAM and PSA-NCAM was studied in sections of adult human pancreas and in cultured primary endocrine and exocrine cells. Dispersed human islet cells were purified in two steps, after 4 days of suspension culture, by binding to magnetic microbeads coupled to antibodies against CA19-9 and PSA-NCAM.


RESULTS
NCAM expression was detected in ducts and islets in the human pancreas. In contrast, PSA-NCAM immunoreactivity was detected only in islets. PSA-NCAM staining in dispersed cells revealed that the marker is expressed in all endocrine cell types, but not in duct cells. Purification of dispersed islet cells using PSA-NCAM microbeads alone did not completely eliminate contaminating duct cells. However, elimination of the duct cells by CA19-9 microbeads followed by positive sorting of the PSA-NCAM-positive cells in five consecutive islet preparations resulted in 90 to 98% pure endocrine cells, of which 89 to 97% were beta cells.


CONCLUSIONS
We describe a simple and reproducible method for purification of viable human pancreatic beta cells devoid of exocrine acini and ducts.
Banerjee
M
M
Biomedicum Stem Cell Center, University of Helsinki, Room C507b, PO Box 63, 00014, Helsinki, Finland. meenal2005@gmail.com
Otonkoski
T
T
D018395:CA-19-9 Antigen / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D004492:Edetic Acid; D006801:Humans; D007150:Immunohistochemistry; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000502:physiology; D039842:Neural Cell Adhesion Molecule L1 / Q000032:analysis; D019006:Neural Cell Adhesion Molecules / Q000032:analysis; D010183:Pancreatic Ducts / Q000166:cytology; D012794:Sialic Acids / Q000032:analysis; D014357:Trypsin
D018395:CA-19-9 Antigen; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules; D012794:Sialic Acids; C103469:polysialyl neural cell adhesion molecule; D004492:Edetic Acid; D014357:Trypsin