Fig 1.
Different devices for cryo-preservation by fast-freezing.
A: From top to bottom: SPRF-tube, open pulled straw (OPS), cryotop, and mini straw. B: The devices are shown with their casing for storage in liquid nitrogen, if available. C: Higher magnification of the sample storage area. The sample is pipetted on the black area of the cryotop. All other devices are tubes, in which the sample is sucked into. In OPS and mini straw, the sample is filled in the tip only, whereas the SPRF tube is completely filled. Scale Bars: A, B: 15 mm; C: 5 mm.
Fig 2.
Ultrastructure of different organisms and cultured cells after SPRF.
A-E: Electron micrographs after SPRF fixation without cryoprotectant, subsequent freeze substitution, and ultrathin sectioning of E. coli (A), C. elegans (B,C), S. cerevisiae (D), and Cos7 cells (E). F: Representative cryo-electron microscopy of vitrified section of a mammalian cell after SPRF in the presence of 30% dextran. Diffraction pattern of the sample is shown in the insert verifying that the sample is vitrified. Note acceptable ultrastructural preservation in A-D and E, with several recognizable cellular components: mitochondria (M), Golgi fields (G), vacuoles (V), nucleus (N), cell wall (CW), microtubules (MT). The mammalian cell frozen without cryoprotectant in E is severely damaged, its outer shape is not discernable and membranes are highly disordered (white arrows).
Fig 3.
SPRF tube during re-warming under a stereomicroscope.
After SPRF, an aluminum tube filled with EAFS medium was cut open under liquid nitrogen and observed under a stereomicroscope during re-warming. Shown are images before ice expansion (A), and 130 ms (B), 195 ms (C), 260 ms (D), 520 ms, and (E) 1495 ms after first visible expansion occurs (see also S1 Video). Ice expands out of the tube (white arrowheads in B and C) and melts subsequently (white arrowheads D-F). Afterwards liquid drops out of the tube (black arrowheads in E and F).
Fig 4.
Viability of mammalian cells in SPRF tubes.
A: Viability of MDCK (○), HeLa (■) and Cos7 (△) cells in PBS after indicated presence in copper SPRF tubes at room temperature, quantified by their ability to re-adhere (n = 5). B: Viability of HeLa cells suspended in cryoprotectants (EAFS and DES) after 60 sec in sliver or aluminum tubes at room temperature quantified by their ability to re-adhere. Cells suspended in the according media served as controls (n = 5). C: Membrane integrity of HeLa cells suspended in cryoprotectants (EAFS and DES) assessed by PI-staining after cryopreservation in sealed SPRF tubes (SPRF), in tubes that were sealed, plunge-frozen, opened under liquid nitrogen and then thawed (opened tubes) and tubes that were not sealed before plunge-freezing (open tubes). All tubes were thawed in air at room temperature instead of a water bath at 37°C (n = 10); D: HeLa cells suspended in indicated CPA mixtures were frozen in aluminum or silver tubes. Their viability was quantified by their ability to re-adhere after thawing. Additionally to the cryoprotectant mixtures DES and EAFS, which lead to high viability rates, a mixture of 27% dextran and 10% ethylene glycol (Dextran + EG) was used (n = 6). E: HeLa cells suspended in different dilutions of DES medium were cooled in aluminum tubes either by plunging into liquid ethane with the help of a plunge-freezer (grey bars) or by directly plunging into liquid nitrogen by hand (white bars). Their viability was quantified by their ability to re-adhere after thawing (n = 5). F: A suspension of HeLa cells in PBS was filled in SPRF-tubes. The tubes were immersed in a 70% ethanol bath for 30 s, and re-cultured afterwards for quantification of viability. Controls were treated the same, except for immersion into ethanol. All data are represented as mean ± s. d.; significance was tested using student’s t-test; **: p<0.01; ns: p>0.05.
Table 1.
Comparison of sample volumes vs. storage space of cryopreservation devices.
Shown is the typical sample size in different vitrification devices compared to the minimum storage space as needed in liquid nitrogen tanks. The storage space is calculated from the outer dimensions of the devices, for OPS and cryotop the casing is taken into account (compare Fig 1).
Fig 5.
Comparison of viability rates of HeLa cells in mini straws, OPS, cryotop and SPRF tubes.
Viability of HeLa cells determined by their ability to re-adhere. A HeLa cells suspended in DES (dark grey) or EAFS (light grey) have been cryopreserved using different vitrification devices: mini straw, OPS, cryotop, and SPRF. (n = 12); *** significantly different to SPRF in the same medium (p<0,001 using student’s t-test). B HeLa cells were suspended in different dilutions of DES, indicated on the x-axis. They were cryopreserved using SPRF or cryotop (n = 5). Data is represented as mean ± s. d.