A comparison of media supplement methods for the extended culture of human islet tissue.
BACKGROUND
The preservation of sufficient quantities of islets for human transplantation has proven to be a tenacious problem for researchers and transplant programs. Beyond the variables associated with islet procurement, there is the problem of tissue storage before transplantation. Cryopreservation has been adopted as a method for long-term islet storage that allows for recovery of viable tissue. However, there is significant tissue loss during the process and the possibility that long-term viability may be compromised. An alternate method of prolonged culture at 24 degrees C was initially introduced as a means of reducing islet antigenicity. Although successful in the short term, prolonged culture with serum-based media has also resulted in a significant loss of tissue. In this study, we report the successful use of an ITS+ Premix-supplemented serum-free media for prolonged islet culture and its comparison to fetal bovine serum-supplemented media and to cryopreservation.


METHODS
Pancreata were procured from cadaveric organ donors, and islets were isolated using our own modification of the automated method of Ricordi. Aliquots from a series of human islet isolations were cultured in parallel in (A) CMRL + ITS (serum-free media; SFM) or (B) CMRL +10% fetal bovine serum (standard media) and compared with cryopreserved and thawed tissue.


RESULTS
Our results show that SFM allows for the long-term culture of islet tissue. For time points up to 2 months, islets cultured in SFM showed recovery ratios greater than those for standard serum-supple. mented media. At 1 week and 1 month, islet recovery ratios were greater for SFM-cultured islets than for cryopreserved tissue. Viability studies confirmed that the SFM-cultured islets were able to respond to glucose stimulation (stimulation index 0.8-21.2). Additionally, in vivo results using cultured islets in a patient demonstrated good islet function, with a 1-month stimulation index of 4.02 in response to an intravenous glucose tolerance test.


CONCLUSIONS
We conclude that this culture modification represents a method by which functional islet tissue can be maintained in long-term culture and successfully transplanted.
Fraga
D W
DW
Department of Surgery, University of Tennessee, Memphis 38163-2116, USA.
Sabek
O
O
Hathaway
D K
DK
Gaber
A O
AO
D000241:Adenosine; D000293:Adolescent; D000328:Adult; D000368:Aged; D000493:Allopurinol; D000818:Animals; D001769:Blood; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods; D003470:Culture Media; D005978:Glutathione; D006801:Humans; D007328:Insulin; D007515:Islets of Langerhans / Q000166:cytology*; D007552:Isotonic Solutions; D008875:Middle Aged; D019188:Organ Preservation Solutions; D011887:Raffinose; D013997:Time Factors; D014019:Tissue Donors; D014021:Tissue Preservation / Q000379:methods*
D003470:Culture Media; C034229:ITS calcifying solution; D007328:Insulin; D007552:Isotonic Solutions; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose