TY - JOUR

T1 - Video analysis of osmotic cell response during cryopreservation

AU - Spindler, Ralf

AU - Rosenhahn, Bodo

AU - Hofmann, Nicola

AU - Glasmacher, Birgit

N1 - Funding information: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy), [EXC 62/1].

PY - 2012/2/16

Y1 - 2012/2/16

N2 - Cellular response during the freeze-thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to -60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (T n=-8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R 2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

AB - Cellular response during the freeze-thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to -60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (T n=-8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R 2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

KW - Computer vision

KW - Cryomicroscopy

KW - Cryopreservation

KW - Dimethyl sulfoxide

KW - Ethylene glycol

KW - Image processing

KW - Osmotic response

UR - http://www.scopus.com/inward/record.url?scp=84861234321&partnerID=8YFLogxK

U2 - 10.1016/j.cryobiol.2012.02.008

DO - 10.1016/j.cryobiol.2012.02.008

M3 - Article

C2 - 22342926

AN - SCOPUS:84861234321

VL - 64

SP - 250

EP - 260

JO - Cryobiology

JF - Cryobiology

SN - 0011-2240

IS - 3

ER -