[Influence of different thawing temperature on the morphology and type I collagen metabolism of the human fibroblasts processed at - 10 degrees C in vitro].
OBJECTIVE
To explore the influence of different thawing temperatures on the morphology and type I collagen metabolism of the human fibroblasts processed at - 10 degrees C in vitro.


METHODS
In vitro cultured human fibroblasts were randomly divided into control, 20 degrees C thawing, and 37 degrees C thawing groups. After being frozen at -10 degrees C, the cells in the latter two groups were thawed at 20 degrees C and 37 degrees C, respectively. The cell proliferation was assessed with MTT method and was expressed by absorption under 570nm (A ), The morphological change of the cells was observed with inverted phase contrast microscope, the change in the intracellular content of collagen was determined with immunohistochemistry, and the extracellular content of collagen was assayed with ELISA.


RESULTS
In 20 degrees C thawing group, the absorbance decreased at first and increased thereafter, and they were obviously lower than that before freezing (0.95 +/- 0.16, P < 0.05 or 0.01). Cell dehydration and shrinking, cytoplasm loss and increased ratio of cytoplasm to nucleus were found in the survived fibroblasts. The cells proliferated actively at 72 and 96 hours after injury, with increased mitotic index and disordered arrangement. Compared with that before freezing (96.4 +/- 2.9) , the extracellular collagen content increased at first, decreased thereafter, and increased again slowly later (P < 0.05), while the intracellular collagen content decreased at first and increased thereafter (P < 0.05). The collagen metabolism in 37 degrees C thawing group was no difference compared with that in control group. Some cells undergone a floating period before adhering to the culture dish walls.


CONCLUSIONS
Cell dehydration after low temperature treatment could protect the cells from damage. Proper thawing temperature could be beneficial to the cell resuscitation. Comparing with slow thawing, rapid thawing could minimize the cell damage.
Guan
Hao
H
Center of Burns and Plastic Surgery of PLA, Xijing Hospital, The Fourth Military Medical University 710032, P, R. China.
Jia
Chi-yu
CY
Chen
Bi
B
Meng
Qing-yan
QY
He
Fei
F
Luo
Ning
N
D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D015925:Cryopreservation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005615:Freezing; D006801:Humans
D024042:Collagen Type I