The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes
| Autor: | William De Loecker, Jacques Gruwez, Barry Fuller, Robert De Loecker |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Male
Cell Survival ATPase Biological Transport Active Buffers In Vitro Techniques Biology General Biochemistry Genetics and Molecular Biology Cryopreservation medicine Animals Amino Acids Incubation chemistry.chemical_classification Cell Membrane Rats Inbred Strains General Medicine Metabolism Membrane transport Rats Amino acid medicine.anatomical_structure Liver Biochemistry chemistry Protein Biosynthesis Hepatocyte biology.protein Sodium-Potassium-Exchanging ATPase General Agricultural and Biological Sciences Intracellular |
| Zdroj: | Cryobiology. 27:143-152 |
| ISSN: | 0011-2240 |
| Popis: | The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me 2 SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me 2 SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na + , K + )ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of l -[U- 14 C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of α-amino[1- 14 C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled α-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined. |
| Databáze: | OpenAIRE |
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