[Isolation and purification of human islet cells with semi-automated digestion].
OBJECTIVE
To establish an semi-automated effective method for large-scale purification of islet cells from human pancreas.


METHODS
Human pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro.


RESULTS
The number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001).


CONCLUSIONS
The established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.
Zhang
Hua
H
Department of Endocrinology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. jimzhua@163.com
Cai
De-hong
DH
Han
Jun-ling
JL
Wei
Guo-hong
GH
Sun
Jia
J
Sun
Wei-ping
WP
Zhang
Jian
J
Chen
Hong
H
Zeng
Long-yi
LY
Weng
Jian-ping
JP
D002452:Cell Count; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002470:Cell Survival; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015203:Reproducibility of Results
D007328:Insulin; D005947:Glucose