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Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

Usta OB, Kim Y, Ozer S, Bruinsma BG, Lee J, Demir E, Berendsen TA, Puts CF, Izamis ML, Uygun K, Uygun BE, Yarmush ML - PLoS ONE (2013)

Bottom Line: Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification.Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation.We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture).

View Article: PubMed Central - PubMed

Affiliation: Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, Massachusetts, United States of America.

ABSTRACT
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

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Comparison of the supercooling preservation of rat hepatocytes with other preservation modalities in long term plate culture.Albumin secretion of cells preserved for A) 1 Days B) 3 Days and C) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. A) Cells preserved for one day in UW at +4oC show statistically higher (p<0.05) albumin secretion in culture beyond 4 days of culture compared to other preserved groups; this is similar to fresh cells. B) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of albumin compared to all other preserved groups yet significantly lower than fresh cells. C) Cells preserved for 5 days in HTS at -4oC produces statistically similar amount of albumin compared to cryopreserved groups which is significantly lower than fresh cells. Urea secretion of cells preserved for D) 1Days E) 3 Days and F) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. D) All nonfrozen groups show statistically similar urea production in culture for 5 days which is also similar to fresh cells yet significantly higher than cryopreserved cells. E) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of urea compared to all other preserved groups yet much lower than fresh cells. F) All preserved groups show significantly lower urea production than fresh cells; cryopreserved and HTS -4oC group show statistically similar production for all days in culture.
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pone-0069334-g004: Comparison of the supercooling preservation of rat hepatocytes with other preservation modalities in long term plate culture.Albumin secretion of cells preserved for A) 1 Days B) 3 Days and C) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. A) Cells preserved for one day in UW at +4oC show statistically higher (p<0.05) albumin secretion in culture beyond 4 days of culture compared to other preserved groups; this is similar to fresh cells. B) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of albumin compared to all other preserved groups yet significantly lower than fresh cells. C) Cells preserved for 5 days in HTS at -4oC produces statistically similar amount of albumin compared to cryopreserved groups which is significantly lower than fresh cells. Urea secretion of cells preserved for D) 1Days E) 3 Days and F) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. D) All nonfrozen groups show statistically similar urea production in culture for 5 days which is also similar to fresh cells yet significantly higher than cryopreserved cells. E) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of urea compared to all other preserved groups yet much lower than fresh cells. F) All preserved groups show significantly lower urea production than fresh cells; cryopreserved and HTS -4oC group show statistically similar production for all days in culture.

Mentions: In Figure 3 we present phase contrast microscopy images (48 hrs following plating and 24 hrs after top collagen gel application) of cells preserved for 1 day (first row), 3 Days (second row), and 5 Days (third row) along with fresh and cryopreserved cells (first column); this accompanies the analysis of the albumin and urea secretions (up to 7 days) for the same groups in Figure 4. The microscopic images show that 1 day preserved cells (Figure 4, first row) in either solution (HTS and UW) at either temperature (±4oC) show very similar morphology and attachment density comparable to fresh cells. This is confirmed by albumin secretion (Figure 4 A) and urea excretion (Figure 4 D) which are statistically same (p>>0.05) as the fresh culture for the first 3 days. The cryopreserved cells produce remarkably low secretions and excretions, and display low attachment and bad morphology. Although all of the non-frozen preserved cells show stable and high secretions, the group preserved at +4oC in UW shows statistically better secretions in culture beyond 5 days of culture.


Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

Usta OB, Kim Y, Ozer S, Bruinsma BG, Lee J, Demir E, Berendsen TA, Puts CF, Izamis ML, Uygun K, Uygun BE, Yarmush ML - PLoS ONE (2013)

Comparison of the supercooling preservation of rat hepatocytes with other preservation modalities in long term plate culture.Albumin secretion of cells preserved for A) 1 Days B) 3 Days and C) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. A) Cells preserved for one day in UW at +4oC show statistically higher (p<0.05) albumin secretion in culture beyond 4 days of culture compared to other preserved groups; this is similar to fresh cells. B) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of albumin compared to all other preserved groups yet significantly lower than fresh cells. C) Cells preserved for 5 days in HTS at -4oC produces statistically similar amount of albumin compared to cryopreserved groups which is significantly lower than fresh cells. Urea secretion of cells preserved for D) 1Days E) 3 Days and F) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. D) All nonfrozen groups show statistically similar urea production in culture for 5 days which is also similar to fresh cells yet significantly higher than cryopreserved cells. E) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of urea compared to all other preserved groups yet much lower than fresh cells. F) All preserved groups show significantly lower urea production than fresh cells; cryopreserved and HTS -4oC group show statistically similar production for all days in culture.
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Related In: Results  -  Collection

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pone-0069334-g004: Comparison of the supercooling preservation of rat hepatocytes with other preservation modalities in long term plate culture.Albumin secretion of cells preserved for A) 1 Days B) 3 Days and C) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. A) Cells preserved for one day in UW at +4oC show statistically higher (p<0.05) albumin secretion in culture beyond 4 days of culture compared to other preserved groups; this is similar to fresh cells. B) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of albumin compared to all other preserved groups yet significantly lower than fresh cells. C) Cells preserved for 5 days in HTS at -4oC produces statistically similar amount of albumin compared to cryopreserved groups which is significantly lower than fresh cells. Urea secretion of cells preserved for D) 1Days E) 3 Days and F) 5 Days at -4oC or +4oC compared to fresh and cryopreserved cells. D) All nonfrozen groups show statistically similar urea production in culture for 5 days which is also similar to fresh cells yet significantly higher than cryopreserved cells. E) Cells preserved for 3 days in HTS at -4oC produces statistically higher amount of urea compared to all other preserved groups yet much lower than fresh cells. F) All preserved groups show significantly lower urea production than fresh cells; cryopreserved and HTS -4oC group show statistically similar production for all days in culture.
Mentions: In Figure 3 we present phase contrast microscopy images (48 hrs following plating and 24 hrs after top collagen gel application) of cells preserved for 1 day (first row), 3 Days (second row), and 5 Days (third row) along with fresh and cryopreserved cells (first column); this accompanies the analysis of the albumin and urea secretions (up to 7 days) for the same groups in Figure 4. The microscopic images show that 1 day preserved cells (Figure 4, first row) in either solution (HTS and UW) at either temperature (±4oC) show very similar morphology and attachment density comparable to fresh cells. This is confirmed by albumin secretion (Figure 4 A) and urea excretion (Figure 4 D) which are statistically same (p>>0.05) as the fresh culture for the first 3 days. The cryopreserved cells produce remarkably low secretions and excretions, and display low attachment and bad morphology. Although all of the non-frozen preserved cells show stable and high secretions, the group preserved at +4oC in UW shows statistically better secretions in culture beyond 5 days of culture.

Bottom Line: Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification.Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation.We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture).

View Article: PubMed Central - PubMed

Affiliation: Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, Massachusetts, United States of America.

ABSTRACT
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Show MeSH