Alginate encapsulated liver cells (AELC) were incubated at different temperatures in increasing concentrations of dimethyl sulfoxide (Me2SO) to observe the effect of
temperature on Cryoprotectant agent (CPA) toxicity.
4.4.1.1 Me2SO toxicity at different temperatures and incubation times
AELC were incubated at 37°C in increasing concentrations of Me2SO (10-70% (v/v)).
A sudden viability drop was observed at 40% (v/v) for samples incubated at 37°C, but was delayed to 50% (v/v) Me2SO when the incubation temperature was set to 4°C. This
was independent of whether AELC were incubated for a period of 40 minutes or 6 hours. Recovery of AELC incubated for 6 hours was approximatly 20-30% lower than the recovery of AELC that were incubated for 40 minutes. When exposed to low concentrations of Me2SO (10-30% (v/v)) viabilities as high as 80% were maintained as
long as high incubation temperature (37°C) and long exposure time (6 hours) were not combined. In this case viability decreased to 60% before dropping to 0% at 40% (v/v) Me2SO. The viability of untreated samples was > 99% (Figure 4.10).
114 Figure 4.10 AELC viability at increasing Me2SO concentrations, incubation temperatures and time
Viabilities remained approximately constant until they suddenly dropped to nearly zero percent when reaching a concentration of either 40% or 50% (v/v) Me2SO at an incubation temperature of
37°C or 4°C, respectively. This was independent of whether the incubation time was set to 40 minutes or 6 hours. AELC were cultured for 24 hours in complete media at 37˚C before viability was assessed by FDA/PI staining.
4.4.1.2 Me2SO toxicity at different temperatures below zero
To verify the results of the previous experiment and to investigate the Me2SO toxicity at
subzero temperatures, AELC were incubated at temperatures between 37°C and -50°C. AELC 2-5 days after encapsulation were used as the purpose was to investigate Me2SO
toxicity. These beads contain less cells and no spheroids and therefore ice formation may have less impact on viability. Moreover lower cell density increases Me2SO
diffusion which is necessary to effectively prevent ice formation inside the cell. The viability of unfrozen samples measured each day was > 97%. Me2SO toxicity was
clearly decreased when exposure temperature was reduced. For example up to 50% (v/v) Me2SO was well tolerated when alginate encapsulated HepG2 cells were
incubated at -20°C but decreased to 0% for all temperatures between -10°C and +37°C. Temperatures lower than -20°C did not prevent further cell death. Some of the samples were frozen after the incubation process as the Me2SO concentration was too low to
0 20 40 60 80 100 0 10 20 30 40 50 60 70 80 V iabili ty (% ) Me₂SO concentration (%, v/v) 40 minutes at 4°C 40 minutes at 37°C 6 hours at 4°C 6 houts at 37°C
115 avoid ice formation. These samples showed lower viabilities than unfrozen samples of higher CPA concentration that were incubated at the same temperature (Figure 4.11).
Figure 4.11 Temperature impact on Me2SO toxicity
AELC were incubated at increasing Me2SO concentrations at decreasing temperatures for 40
minutes. From 37˚C to -20˚C viability increased, below -20˚C viability decreased. AELC were cultured for 24 hours in complete media at 37˚C before viability was assessed by FDA/PI staining.
* frozen samples
4.4.1.3 Dimethyl sulfoxide toxicity over time
To estimate the impact of exposure time on Me2SO toxicity, AELC were incubated for
1, 5, 15 and 40 minutes in 30% to 70% (v/v) Me2SO at 4°C. Solutions with 10-20%
(v/v) Me2SO were not further investigated as they had shown little effect on cell
viability for temperatures lower than 20°C (Figure 4.11). After 1 minute, AELC incubated in 30%, 40% and 50% (v/v) Me2SO still showed high viabilities of >90%.
Instead, recovery of cells incubated in 60% and 70% (v/v) Me2SO decreased to about 50% after 1 minute. After five minutes most of these cells incubated in 70% (v/v) Me2SO had died whereas those exposed to 60% (v/v) Me2SO still showed a recovery of
44%. Less toxic were solutions of 30-50% (v/v) Me2SO with AELC still showing
viabilities of approximately 83%. After 15 minutes the difference between the conditions became more evident showing a modest decline to 80% for 30% (v/v) Me2SO and to 70% for 40% (v/v) Me2SO. A stronger reduction was seen for 50% and
60% (v/v) Me2SO with viabilities being reduced to 50% and 14%, respectively. Finally
0 20 40 60 80 100 5 15 25 35 45 55 65 V iabili ty (% ) Me₂SO concentration (%, v/v) 37°C 20°C 4°C -10°C -20°C -30°C -40°C
116 after 40 minutes, viability of cells incubated in 60% (v/v) Me2SO was reduced to 0%.
AELC exposed to 50% and 40% (v/v) Me2SO still reached a viability of 12%, and 23%
respectively. The exposure to 30% (v/v) Me2SO showed little additional effect with
viabilities still reaching 77%. For 60% and 70% (v/v) Me2SO viability decreased
strongest during the first minute, for 30% and 40% (v/v) Me2SO during the first five
minutes and for 50% during the first 15 minutes (Figure 4.12).
Figure 4.12 Me2SO toxicity over time at 4˚C
The figure shows Me2SO toxicity at 4˚C over time. AELC were incubated in 1x PBS solutions
containing 10-70% (v/v) Me2SO. AELC viability slightly decreased over time when exposed to 30%
(v/v) Me2SO, but strongly decreased when 40% and 50% (v/v) Me2SO was used. Exposure to 60%
Me2SO (v/v) resulted in complete cell death after 40 minutes, whereas AELC incubated in 70%
Me2SO already showed very low recovery after five minutes. AELC were cultured for 24 hours in
complete media at 37˚C before viability was assessed by FDA/PI staining.
4.4.2 Determination of osmotic effects
AELC pre-incubated in 20% (v/v) Me2SO before being incubated in 40% (v/v) Me2SO
showed no significant difference in recovery to those directly exposed to 40% (v/v) Me2SO when the incubation temperature was set to 0.5°C. This changed when the
incubation temperature was set to 37°C. At this temperature the pre-incubation step 0 20 40 60 80 100 0 5 10 15 20 25 30 35 40 V iabili ty (% ) Time (minutes) 30% 40% 50% 60% 70%
117 significantly reduced cell death and improved recovery from 21% +/- 7% to 45% +/- 7% (n=5) for an incubation period of 15 minutes and from 3 +/- 2% to 36 +/- 2% (n=5) when AELC were incubated for 40 minutes (Figure 4.13).
Figure 4.13 Effect of temperature on osmotic injury
Columns in black: with pre-incubation - AELC first incubated for 2 minutes in 20% (v/v) Me2SO,
then in 40% (v/v) Me2SO for 15 or 40 minutes. Columns in white: without pre-incubation - AELC
directly incubated in 40% (v/v) Me2SO for 15 or 40 minutes. A: There was no significant difference
between the two conditions, (pre-incubation and without pre-incubation), when the incubation was carried out at 0.5°C. B: Viability was significantly higher for pre-incubated AELC when the incubation temperature was set to 37°C, with ** p<0.01, (n=5 +/-SD). AELC were cultured for 24 hours in complete media at 37˚C before viability was assessed by FDA/PI staining.
4.4.3 Thermocouple defined approximate freezing point (TdAFP) for dimethyl