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Freezing induces artificial cleavage of apoptosis-related proteins in human bone marrow cells
J Schmidt-Mende1, E Hellström-Lindberg, B Joseph
1Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77, Stockholm, Sweden.
Abstract:
The aim of this study was to investigate whether freeze-thawing of freshly isolated human mononuclear bone marrow cells (MNC) influences the integrity of apoptosis-related proteins as determined by immunoblot analyses. Our results show that bone marrow is more sensitive to this process than either myelomonocytoid leukemic P39 or Jurkat T-lymphocyte cell lines. Specifically, bone marrow cells displayed a high level of intrinsic proteolytic activity in response to a single freeze-thaw cycle, which led to the cleavage of various proteins involved in apoptosis cell signaling. This effect was completely blocked by the inclusion of broad-spectrum protease inhibitors in the freezing medium and subsequently thawing the cells on ice. Since differences in the freezing conditions (-80 degrees C vs. liquid nitrogen) did not alter the proteins of interest, we suggest that the thawing process is the critical point when proteolytic enzyme activity is elevated.
Insights
Freeze-thawing human bone marrow cells (MNC) causes protein degradation due to proteolytic activity. This process, particularly during thawing, impacts apoptosis signaling proteins, but can be prevented with protease inhibitors.
Area of Science:
- Cell Biology
- Biochemistry
- Hematology
Background:
- Preserving cellular integrity during cryopreservation is crucial for research.
- Mononuclear bone marrow cells (MNC) are vital for various studies.
- Apoptosis-related proteins are key indicators of cellular health and function.
Purpose of the Study:
- To investigate the impact of freeze-thawing on apoptosis-related proteins in human bone marrow cells (MNC).
- To compare the sensitivity of bone marrow cells to freeze-thawing with leukemic and T-lymphocyte cell lines.
- To identify the critical stage and contributing factors to protein degradation during cryopreservation.
Main Methods:
- Immunoblot analyses were used to assess protein integrity.
- Freshly isolated human bone marrow cells (MNC), P39, and Jurkat cell lines were subjected to freeze-thawing.
- Protease inhibitors and varying freezing conditions (-80°C vs. liquid nitrogen) were tested.
Main Results:
- Human bone marrow cells (MNC) exhibited significant sensitivity to freeze-thawing, showing protein cleavage.
- This proteolytic activity led to the degradation of apoptosis signaling proteins.
- The observed effects were more pronounced in bone marrow cells compared to P39 and Jurkat cell lines.
- Protease inhibitors and thawing on ice completely blocked the protein degradation.
Conclusions:
- The thawing process, not the freezing temperature, is the critical step for elevated proteolytic activity in bone marrow cells (MNC).
- Cryopreservation protocols need optimization to preserve the integrity of apoptosis-related proteins in bone marrow samples.
- Understanding these effects is essential for reliable downstream analyses of bone marrow cells.