Comparison of Different Media for Short-Term Preservation of Testicular Cells.

Abstract

The study and manipulation of testis cells require their preservation for various length of time. There are situations where it is necessary to maintain cells in suspension for up to several days (e.g., shipment of cells between collaborating labs). In such cases, culturing the cells at 37°C may not be suitable and resorting to cryopreservation will compromise the overall cell viability. This study was designed to develop strategies for maintaining the viability of isolated testis cells at room or refrigeration temperatures. Testes were collected by castration of one-week-old piglets and transferred to the lab within 1 hour in ice-cold PBS. After removal of the tunica albuginea and connective tissue, testis parenchyma was minced using fine scissors and cells were dissociated following a two-step enzymatic digestion procedure (with 0.2% collagenase-IV + 0.1% hyaluronidase + 0.01% DNase-I at 37°C for 15 min, followed by 0.25% trypsin with 2.21 mM EDTA for an additional 5 min). Cells were filtered (40μm pore size) and depleted of erythrocytes with lysis buffer (0.156 M NH4Cl, 0.01 M Na2EDTA, 0.1 M KHCO3, Ph=7.4). The resultant cells were then kept at either room or refrigeration temperatures in polypropylene tubes containing one of the following media: PBS, DMEM, DMEM with FBS (10%, 20%, or 50%), FBS, or Leibovitz (L-15) (n≥8 replicates each). Cell viability was assessed daily for 6 consecutive days (day of cell isolation as Day 0) using trypan blue exclusion (0.4% solution). By Day 3, storage at refrigeration temperature maintained significantly more live cells than at room temperature in all preservation media, except in PBS. At this time, while under room temperature conditions no significant differences in cell viability were observed among different media, cells stored at refrigeration temperature in DMEM (76.4±2.6% of live cell compared to Day 0, Mean±SEM), DMEM+50% FBS (76.7±2.7), FBS (77.6±3), or L-15 (81±5.7) had significantly better viability than those in PBS (60.3±1.9). Similarly on Day 6, all media preserved significantly more live cells at refrigeration temperature than those at room temperature, with the exception of L-15 in which cells at room temperature had maintained a surprisingly high viability similar to those at refrigeration temperature. On Day 6 under room temperature, cells maintained in DMEM+50% FBS (51.3±2.6), FBS (40.7±6.2), or L-15 (53.7±9.1) had maintained significantly more viable cells than those in PBS (0±0), or DMEM+10% FBS (14.6±3.1). At refrigeration temperature, cells kept in DMEM+50% FBS (64±4.5), FBS (74.4±3.8), or L-15 (72.2±5.6) also survived significantly better than those in PBS (42.9±3.5). Based on these results, for refrigeration of porcine testis cells, DMEM+50% FBS, FBS, or L-15 are equally suitable media. This research was supported by NSERC and SHRF grants (AH).