Optimization of neutral protease to collagenase activity ratio for islet of Langerhans isolation.
OBJECTIVE
The optimal neutral protease to collagenase activity ratio has not been determined for islet isolation. We evaluated a new highly purified collagenase that can be blended with predetermined amounts of neutral protease (NP).


METHODS
Islets were isolated from 7 groups of Sprague-Dawley rats. In group I, collagenase type XI (Sigma) at 2 mg/mL, and, in group II, Liberase at 0.6 mg/mL (2.4 PZ- U/mL; Roche) were used as controls. In groups III to VII, collagenase NB1 0.6 mg/mL (2.4 PZ-U/mL; Serva Electrophoresis) was used with increasing amounts of added NP. The NP to collagenase activity ratio (DMC-U/PZ-U) increased from 0.5% in group III to 2.0% in group VII.


RESULTS
Mean islet equivalent (IE) yields per rat were 1367, 1755, 597, 895, 1712, 1043, and 905 in groups I to VII. IE yields were maximal at DMC-U/PZ-U = 1.2%. Islet morphology was influenced by NP concentration with decreasing numbers of trapped islets and increasing numbers of fragmented islets as NP contents increased. Cytokine release, islet cell apoptosis, and in vitro function were significantly better in groups III to VII as compared with groups I and II.


CONCLUSIONS
NP is a crucial additive to collagenase for islet isolation. Optimization of the NP to collagenase activity ratio (1.2% in this model) improves yields and morphology after islet isolation.
Bucher
P
P
Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals, Geneva, Switzerland.
Bosco
D
D
Mathe
Z
Z
Matthey-Doret
D
D
Andres
A
A
Kurfuerst
M
M
Rämsch-Günther
N
N
Bühler
L
L
Morel
P
P
Berney
T
T
D000818:Animals; D002176:Candida albicans / Q000302:isolation & purification; D002469:Cell Separation / Q000379:methods*; D005658:Fungi / Q000302:isolation & purification*; D006090:Gram-Negative Bacteria / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000302:isolation & purification*; D007515:Islets of Langerhans / Q000166:cytology* / Q000382:microbiology; D010179:Pancreas; D051381:Rats; D017207:Rats, Sprague-Dawley; D020858:Tissue and Organ Harvesting / Q000379:methods