Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation

Abstract

During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

Fig 1. Diagram of the experimental procedure used and the analysis performed on L. bulgaricus CFL1 samples.

DSC, FITR, viability and acidification activity measurements were performed on at least three independent biological replicates. Steps involving different conditions are in blue. Similar experimental procedure was used for S. cervisiae 338.

Fig 2. DSC traces of S. cerevisiae 338 cells during cooling.

(a) DSC heat flow traces at 50°C.min^-1 (red curves), 10°C.min^-1 (green curves) and 2°C.min^-1 (blue curves) and (b) DSC first derivative of the heat flow during warming at 10°C.min^-1. In all figures the effects of a repeat freeze thaw were also determined. Capital letters A, B and C in Fig 2A correspond to exothermic events indicated in Table 1.

Fig 3. CryoSEM of S. cerevisiae 338 cells following cooling.

(a) At 50°C.min^-1 and (b) at 2°C.min^-1. Note the large voids in cells cooled at 50°C.min¹, caused by the formation of intracellular ice.

Fig 4. DSC first derivative of heat flow of L. bulgaricus CFL1 with cryoprotective additives and aqueous solutions of cryoprotectants.

(a) sucrose, (b) glycerol and (c) DMSO are all additives at 0.58 M. The vitrification temperatures of incubated (light blue curve) and subsequently washed cells (grey curve) are indicated. Samples were cooled below −90°C and then warmed at 10°C.min^-1. Control samples (washed with peptone water) (black curves) and aqueous solutions of cryoprotectants (dark blue curves) are also presented for comparison.

Fig 5. The effects of cryoprotective agents on the loss of specific acidification activity and viability upon freeze-thawing of L. bulgaricus CFL1.

Cells were grown in either whey or MRS medium, harvested in stationary culture phase and frozen to -80°C at 2°C min^-1 with sucrose, glycerol or DMSO (all additives at 0.58 M) and no additive (control: 1 g.L^-1 peptone water). Superscripts letters represent statistical contrasts (significant differences) between samples at the 95% confidence level. Control whey grown cells displayed an extreme loss in cell function (64.8 ± 3.5 (min.log(CFU.mL^-1)^-1)) and viability upon thawing (3.3 ± 0.3 log(CFU.mL^-1)), and for clarity are not included in this figure.

Fig 6. The loss of specific acidification activity on thawing of L. bulgaricus plotted against intracellular vitrification temperature (Tg’).

Cells were grown in either whey (dark grey) or MRS medium (light grey), harvested in stationary culture phase and frozen to -80°C at 2°C min^-1 with sucrose (triangles), glycerol (diamonds) or DMSO (squares), (all additives at 0.58 M) and MRS control (circle). Dotted arrow indicates the direction of control whey grown cells loss in cell function (64.8 ± 3.5 (min.log(CFU mL^-1)^-1)).

Fig 7. Schematic of the transitions which occur during cryopreservation of L. bulgaricus CFL1.

Temperature transitions correspond to cells grown in whey medium and protected with glycerol (0.58 M).