112 VIABILITY AND APOPTOSIS OF BOVINE FIBROBLASTS FROZEN IN A CONVENTIONAL (−20°C) OR LOW-TEMPERATURE (−80°C) FREEZER
L. Chacón A B , M. C. Gómez C D , J. Jenkins E , S. P. Leibo B C , G. Wirtu C F , B. L. Dresser B C and C. E. Pope CA School of Veterinary Medicine, Colombian National University, Bogotá, Colombia
B Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA
C Audubon Center for Research of Endangered Species, New Orleans, LA 70131, USA
D Genetic Institute, Colombian National University, Bogotá, Colombia
E U.S. Geological Survey, National Wetlands Research Center, Lafayette, LA 70506, USA
F School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
Reproduction, Fertility and Development 19(1) 173-174 https://doi.org/10.1071/RDv19n1Ab112
Submitted: 12 October 2006 Accepted: 12 October 2006 Published: 12 December 2006
Abstract
Fibroblasts are commonly frozen in dimethyl sulfoxide (DMSO, 10% v/v) at a cooling rate of 1°C min−1 in cryo-containers placed in a low-temperature (−80°C) freezer (LTF) overnight before storage in liquid nitrogen (LN2). LTFs are not always available and cell viability may be altered by freezing at different cooling rates. Therefore, the purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen at different cooling rates in a −80°C freezer or conventional −20°C freezer. Fibroblasts were generated from skin tissue collected from an adult female cow. Tissue was cut and cultured in DMEM for 7 to 10 days and passaged 2 or 3 times before being cryopreserved. Cells were frozen by one of 6 procedures; cells that were not frozen served as a control. To be frozen, cells were resuspended in DMEM + 10% FBS and 10% DMSO in 2-mL cryo-vials before being placed into freezing containers (Mr. Frosty, Nalgene). For groups 1 and 2, the container was placed in a −20°C freezer and cooled at ≈0.6°C min−1 to −7°C; ice formation was induced by seeding, after which cooling was continued at ≈0.6°C min−1 to −20°C. Vials in groups 1 and 2 were placed in LN2 after 1 h and 16 to 18 h, respectively. Group 3 and 4 vials were also cooled to −20°C at 0.6°C min, except that these samples were not seeded. After being held at −20°C for 16 to 18 h, group 3 vials were thawed; group 4 vials were stored in LN2. Vials in groups 5 and 6 were placed into a Mr. Frosty container and cooled at ≈1°C min−1 in a −80°C freezer. After being held at −80°C for 16 to 18 h, group 5 vials were thawed, and group 6 vials were placed into LN2. Group 1, 2, 3, and 4 vials were thawed in a 37°C water bath; group 5 and 6 vials were thawed in a 40°C water bath. Cell viabilities were quantified by using TB exclusion (TB) with microscopy. At least 20 000 cells from each group were thawed, and caspase 3, a key enzyme involved in apoptotic processes, was detected using a Caspase 3-FLICA apoptosis detection kit; propidium iodide-stained nuclei were assessed with cytometry (FCM) to determine viability (Table 1). Data were analyzed by ANOVA (P < 0.05). Although there were viability differences among groups, freezer type, seeding, holding time, and LN2 had no significant impact, as assessed by TB or FCM; however, no LN2 significantly increased the percentage of viable cells displaying apoptosis. In summary, bovine fibroblasts were successfully frozen in a conventional (−20°C) freezer before storage in LN2.
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