Incubators, Freezers & Cooling Equipment
Maintaining sample integrity during repeated freeze/thaw cycles Alexis MacLeod, Global Product Manager, Consumables and Instruments, Azenta Life Sciences.
Storage of biological materials or newly synthesised compounds in -80°C freezers is now commonplace. The need to retrieve aliquots from these compound libraries or bio stores means that individual tubes must fi rst be thawed and, following the aliquoting procedure, re-frozen. For popular, interesting, or useful samples this can result in multiple freeze/thaw cycles during the storage life of the sample. This paper sets out to discover if samples stored in quality screw-cap sample tubes rated for -80°C storage suffer any degradation from either very long-term storage or repeated freeze/thaw cycling.
To ascertain the facts about extended storage, representative sample storage tubes of four types were used and, in all cases, stored in -80°C freezer conditions. The testing had three main objectives:
• To confi rm any changes to the sample storage volume that may occur throughout its lifecycle in a freezer
• To confi rm no damage is likely to occur that could compromise the integrity of the tube during regular freeze/thaw cycles
• To re-affi rm that the dual-threaded cap design successfully prevents leakage and evaporation over long periods of time
The tests were conducted on externally threaded tubes designed and manufactured for Azenta of 1.0mL and 1.9mL working volume, and 1.0mL & 3.8mL working volume treated with electron-beam (e-Beam) radiation.
The experimental design was as follows: Apparatus and materials
Tubes selected for tests were Azenta Tri-Coded Tubes, External Thread, Pre-racked, Uncapped, or Capped (e-Beam treated only):
48 x 1.9mL Tri-coded Tube, 48-format, External Thread, e-Beam treated 48 x 3.8ml Tri-coded Tube, 48-format, External Thread 96 x 1.0mL Tri-coded Tube, 96-format, External Thread 96 x 1.0mL Tri-coded Tube, 96-format, External Thread, e-Beam treated Either 1 x 48- or 1 x 96-format Azenta External Cap Carrier
1 x -80°C Freezer 1 x Torque-controlled capper 1 x 12-channel pipettor 1 x 500ml bottle of Ringer’s Solution 1 x Precision laboratory balance
Method
Using the 12-channel pipettor, the maximum working volume (1,085µl) of Ringer’s solution was aspirated into each of 96 tubes in the SBS rack. The capper was adjusted to apply 0.08 Nm of torque to each cap as it was transferred from the cap carrier to its tube, capping it accordingly.
Using the precision balance, each tube was weighed and the weight and rack position from A1 to H12 was recorded. A visual check of each tube was made for any possible damage, such as grazing or cracking of the polymer surface and the condition of the was noted in a log. The completed rack of 96 tubes was then transferred to the -80°C freezer.
The tubes were then withdrawn from storage at -80°C, at defi ned predetermined intervals as follows:
• 2 Weeks • 1 Month • 3 Months • 6 Months • 1 Year • 2 Years • 3 Years
At each sampling interval designated above, the SBS rack of tubes was removed from the freezer. The rack was then left out on the bench until the tubes had returned to room temperature. After stability at room temperature was achieved, each tube was re-weighed, and data was recorded.
Figure 2: Variation between columns in the same 1.0mL tubes in a 96 rack over time.
There was no observed cracking, grazing, or other physical damage caused throughout the frozen storage period, nor by repeated freeze/thaw cycles over a three-year period.
This demonstrates the seal integrity of the 1.0mL Tri-Coded Tubes when correctly capped is adequate for long-term storage of aqueous-based substances, if the specifi ed working volume is adhered to.
Figure 1: Test samples stored in -80°C freezer.
At the same time, each tube was checked visually for any possible damage, grazing, or cracking, and anything noteworthy was recorded. After completion of all weighing and checks, the fi lled SBS rack of tubes was returned to the -80°C freezer.
The process was then repeated for each of the tube types at each incremental time point over three years, except the e-Beam sterile versions, which were only sampled after one year.
Results and observations
For the 1.0mL Azenta Tri-Coded Tubes, the total average weight loss of all 96 tubes was 0.010% (equal to 0.018mg) over the fi rst two-week sampling period and only increased to a loss of 0.045% (equal to 0.088mg) over the full three-year test period.
Table 1: Results for 1.0mL in 96-position rack. TIME OF WEIGHING
Before Storage 2 Weeks 1 Month 3 Months 6 Months 1 Year
2 Years 3 Years
-0.01% -0.01% -0.03% -0.03% -0.03% -0.04% -0.05%
AVERAGE % CHANGE 100%
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48
