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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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Scaling up, Comparison of cells preserved in small cryo vials (1.5M cells/vial) and big centrifuge tubes (50M cells/tube).A * and O means statistically different (p<0.05) than small vials (SV) and cryopreserved cells respectively. A) Viability of cells after 1 hour of suspension rewarming post-preservation. Cells preserved in big tubes show significantly (p<0.05) higher 1 hr viability compared to both small vials and cryopreserved cells B) Viability of cells after 6 hours of suspension culture. Cells preserved in small vials show statistically higher 6 hr viability after 3 days of preservation, cells preserved in small vials and big tubes show similar viabilities that is statistically better (p<0.05) compared to cryopreserved cells. C) Albumin production by cells during 6 hours of suspension culture D) Urea production by cells during 6 hours of suspension culture; cells preserved in big tubes show statistically higher urea production (p<0.05) on both days.
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pone-0069334-g005: Scaling up, Comparison of cells preserved in small cryo vials (1.5M cells/vial) and big centrifuge tubes (50M cells/tube).A * and O means statistically different (p<0.05) than small vials (SV) and cryopreserved cells respectively. A) Viability of cells after 1 hour of suspension rewarming post-preservation. Cells preserved in big tubes show significantly (p<0.05) higher 1 hr viability compared to both small vials and cryopreserved cells B) Viability of cells after 6 hours of suspension culture. Cells preserved in small vials show statistically higher 6 hr viability after 3 days of preservation, cells preserved in small vials and big tubes show similar viabilities that is statistically better (p<0.05) compared to cryopreserved cells. C) Albumin production by cells during 6 hours of suspension culture D) Urea production by cells during 6 hours of suspension culture; cells preserved in big tubes show statistically higher urea production (p<0.05) on both days.

Mentions: The cells in big tubes along with small vials were preserved for 3, 5, and 7 days at -4oC. In Figure 5 we present the viability and functionality comparison of these two groups after 1 hour and 6 hours of suspension rewarming. Cells preserved in big tubes consistently show a statistically higher 1 hr viability (Figure 5 A) and the decline in viability for this group is even slower compared to the small vials. In fact, the cells preserved in big tubes show a significantly higher viability (~75%) compared to the cryopreserved cells (~70%) (p<0.05) even at the end of a week. After 6 hours of rewarming both big tube and small vial preserved cells show almost identical viabilities (Figure 5 B) significantly higher (p<0.05 for both) than the cryopreserved group. Albumin production (Figure 5 C) via 6 hours suspension studies reveal similar functionality for both big tubes and small vials. Big tube preserved cells do show significantly better (p<0.05) urea genesis (Figure 5 D) after both 3 and 5 days preservation in the 6 hour suspensions 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)

Scaling up, Comparison of cells preserved in small cryo vials (1.5M cells/vial) and big centrifuge tubes (50M cells/tube).A * and O means statistically different (p<0.05) than small vials (SV) and cryopreserved cells respectively. A) Viability of cells after 1 hour of suspension rewarming post-preservation. Cells preserved in big tubes show significantly (p<0.05) higher 1 hr viability compared to both small vials and cryopreserved cells B) Viability of cells after 6 hours of suspension culture. Cells preserved in small vials show statistically higher 6 hr viability after 3 days of preservation, cells preserved in small vials and big tubes show similar viabilities that is statistically better (p<0.05) compared to cryopreserved cells. C) Albumin production by cells during 6 hours of suspension culture D) Urea production by cells during 6 hours of suspension culture; cells preserved in big tubes show statistically higher urea production (p<0.05) on both days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713052&req=5

pone-0069334-g005: Scaling up, Comparison of cells preserved in small cryo vials (1.5M cells/vial) and big centrifuge tubes (50M cells/tube).A * and O means statistically different (p<0.05) than small vials (SV) and cryopreserved cells respectively. A) Viability of cells after 1 hour of suspension rewarming post-preservation. Cells preserved in big tubes show significantly (p<0.05) higher 1 hr viability compared to both small vials and cryopreserved cells B) Viability of cells after 6 hours of suspension culture. Cells preserved in small vials show statistically higher 6 hr viability after 3 days of preservation, cells preserved in small vials and big tubes show similar viabilities that is statistically better (p<0.05) compared to cryopreserved cells. C) Albumin production by cells during 6 hours of suspension culture D) Urea production by cells during 6 hours of suspension culture; cells preserved in big tubes show statistically higher urea production (p<0.05) on both days.
Mentions: The cells in big tubes along with small vials were preserved for 3, 5, and 7 days at -4oC. In Figure 5 we present the viability and functionality comparison of these two groups after 1 hour and 6 hours of suspension rewarming. Cells preserved in big tubes consistently show a statistically higher 1 hr viability (Figure 5 A) and the decline in viability for this group is even slower compared to the small vials. In fact, the cells preserved in big tubes show a significantly higher viability (~75%) compared to the cryopreserved cells (~70%) (p<0.05) even at the end of a week. After 6 hours of rewarming both big tube and small vial preserved cells show almost identical viabilities (Figure 5 B) significantly higher (p<0.05 for both) than the cryopreserved group. Albumin production (Figure 5 C) via 6 hours suspension studies reveal similar functionality for both big tubes and small vials. Big tube preserved cells do show significantly better (p<0.05) urea genesis (Figure 5 D) after both 3 and 5 days preservation in the 6 hour suspensions 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.

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