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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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Related in: MedlinePlus

Representative SEM images of fresh and preserved cells.A) Wide angle view of freshly isolated cells B) High magnification view of a freshly isolated cell. C) Intact preserved Cell D) Intact preserved cells with very minor blebbing E) Mild and F) severe blebbing damage on preserved cell membranes G) and H) Complete loss of ultrastructure on cell membranes which occur only on cold stored (+ 4oC) cells beyond 3 days.
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pone-0069334-g007: Representative SEM images of fresh and preserved cells.A) Wide angle view of freshly isolated cells B) High magnification view of a freshly isolated cell. C) Intact preserved Cell D) Intact preserved cells with very minor blebbing E) Mild and F) severe blebbing damage on preserved cell membranes G) and H) Complete loss of ultrastructure on cell membranes which occur only on cold stored (+ 4oC) cells beyond 3 days.

Mentions: In an effort to understand cell membrane injury to the hepatocytes during the different preservation processes we have conducted scanning electron microscopic imaging of all groups for up to 5 days of preservation. In Figure 7 we present representative images of this study corresponding to the different modes and extent of damages. Figure 7 A shows a wide angle view (500 X) of freshly isolated hepatocytes; in Figure 7 B we present two levels (1000 X (left) and 5000 X (right)) of magnification on these fresh cells to show the overall intact membrane structure and the microvilli as a reference for all preserved groups. In Figure 7 C–F) we present images of cells preserved under different conditions with different damage patterns. In C) we show an intact cell with no visible membrane damage whereas in D) we present a cell with very minor blebbing. In E and F we show increasing levels of blebbing, which are also visible even at very low magnifications. In the last row (G-H) we depict a different mode of damage where the ultrastructure (microvilli) on the cell membrane are completely lost; we have observed this mode of damage only in the cold stored (+4oC) cells beyond 3 days of storage and this constitutes the major mode of damage for those groups.


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)

Representative SEM images of fresh and preserved cells.A) Wide angle view of freshly isolated cells B) High magnification view of a freshly isolated cell. C) Intact preserved Cell D) Intact preserved cells with very minor blebbing E) Mild and F) severe blebbing damage on preserved cell membranes G) and H) Complete loss of ultrastructure on cell membranes which occur only on cold stored (+ 4oC) cells beyond 3 days.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0069334-g007: Representative SEM images of fresh and preserved cells.A) Wide angle view of freshly isolated cells B) High magnification view of a freshly isolated cell. C) Intact preserved Cell D) Intact preserved cells with very minor blebbing E) Mild and F) severe blebbing damage on preserved cell membranes G) and H) Complete loss of ultrastructure on cell membranes which occur only on cold stored (+ 4oC) cells beyond 3 days.
Mentions: In an effort to understand cell membrane injury to the hepatocytes during the different preservation processes we have conducted scanning electron microscopic imaging of all groups for up to 5 days of preservation. In Figure 7 we present representative images of this study corresponding to the different modes and extent of damages. Figure 7 A shows a wide angle view (500 X) of freshly isolated hepatocytes; in Figure 7 B we present two levels (1000 X (left) and 5000 X (right)) of magnification on these fresh cells to show the overall intact membrane structure and the microvilli as a reference for all preserved groups. In Figure 7 C–F) we present images of cells preserved under different conditions with different damage patterns. In C) we show an intact cell with no visible membrane damage whereas in D) we present a cell with very minor blebbing. In E and F we show increasing levels of blebbing, which are also visible even at very low magnifications. In the last row (G-H) we depict a different mode of damage where the ultrastructure (microvilli) on the cell membrane are completely lost; we have observed this mode of damage only in the cold stored (+4oC) cells beyond 3 days of storage and this constitutes the major mode of damage for those groups.

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
Related in: MedlinePlus