Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression.

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.

Fluorescence In Situ Hybridization: Trisomy 12 Follow-up In Hairy Cell Leukemia.

Peripheral white blood cells from five patients with hairy cell leukemia (HCL) were studied applying fluorescence in situ hybridization (FISH) using heparinized peripheral blood, biotin-labeled chromosome 12-specific α satellite DNA probe and biotin fluorescein-labeled avidin to detect hybridization. This methodology is ideal to investigate the incidence of numerical chromosomal abnormalities of both interphase and metaphase cells. Blood samples from 28 normal subjects were included as control samples. Trisomy 12 was considered to be present when ≥4% of cells had three hybridization spots. Trisomy 12 was detected in three of the five patients at different stages of their evolution. Trisomy 12 was also evident in two patients upon relapse of the HCL. When treatment with interferon alfa (IFN) and steroids was started, clinical and analytical remission were achieved and trisomy 12 disappeared. In one patient trisomy 12 persisted regardless of treatment with IFN and a good clinical evolution. Our results show that trisomy 12 was detected in HCL with FISH at a higher frequency than that previously reported by cytogenetic analysis in either peripheral blood or in cultivated cell lines. These results indicate that the presence of trisomy 12 may be a useful marker for the presence of HCL cells, to check the percentage of trisomic cells and that trisomy 12 is also a useful marker to indicate the activity of the disease.