[Comparison of different isolation methods on adult Sertoli cells co-transplanted with islets].
OBJECTIVE
To establish a more convenient and effective method for isolating adult Sertoli cells and apply the method to islet transplantation.


METHODS
Trypsin, DNase, collagenase and hyaluronidase were used for a one-step digestion in group A. A two-step digestion was used in group B, in which testis tissues were first digested by trypsin and DNase and then were digested by collagenase and hyaluronidase. In group C, trypsin and DNase were used in the first step of digestion, hyaluronidase was used in the second step and collagenase was used in the third step. Sertoli cells were identified by morphology and immunohistochemistry and the viability and purity of Sertoli cells were detected by MTT and flow cytometry (FCM). Expression of Fas-L was detected by Western blot and the effects of the co-transplantation of islets and Sertoli cells were compared between the three groups.


RESULTS
Typical Sertoli cells were seen after isolation using the three different methods. Sertoli cells isolated by method A and method B were large in number while those isolated by method B and method C were pure. The results of MTT showed that the viability of Sertoli cells in group B was significantly higher than that in group A and group C (P<0.05) and Western blot results showed that expression of Fas-L on Sertoli cells in group B was significantly stronger than that in group A and group C (P<0.05). The purity of Sertoli cells detected by FCM in group B and group C were significantly higher than that in group A (P<0.05). The survival of islets co-transplanted with Sertoli cells to renal capsule of diabetic mice was significantly longer in group B compared with that in control group as well as in group A and group C (P<0.05).


CONCLUSIONS
Sertoli cells obtained by the two-step digestion method are of higher purity and viability, which significantly prolong the survival of islet graft by co-transplantation.
Li
Yang
Y
Department of Renal Transplantation, Center of Nephropathy, First Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710061, China.
Xue
Wu-jun
WJ
Song
Huan-jin
HJ
Feng
Xin-shun
XS
Tian
Xiao-hui
XH
Ding
Xiao-ming
XM
Song
Yong
Y
Ding
Chen-guang
CG
D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental; D053222:Fas Ligand Protein / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012708:Sertoli Cells / Q000378:metabolism / Q000637:transplantation*
D053222:Fas Ligand Protein