Effect of one year of cryopreservation on the activity of lysosomal hydrolases from EBV-transformed lymphocytes.

BACKGROUND: The Epstein-Barr virus (EBV) was used as an agent of B lymphocyte proliferation for subsequent diagnosis of lysosomal storage disease. Due to the constant handling of long-preserved samples in our cell bank, we decided to observe the behavior and then compare cultured and frozen samples for at least one year's cryopreservation. METHODS: Twenty-five samples from healthy individuals were used to assess the possible changes in activity of enzymes ß-galactosidase, ß-glucosidase, α-iduronidase, α-galactosidase, and α-glucosidase. Transmission electron microscopy was used to confirm cell transformation of B lymphocytes into EBV-infected cells, generating lymphoblastoid cell lines. RESULTS: Transmission electron microscopy findings confirmed previous reports in the literature that is, significant and evident morphological changes in the nucleus occur after day 12 and the consequent cell transformation into EBV-infected cells. After thawing and subsequent treatment with the five enzymes utilized, we observed no significant changes in samples cryopreserved for more than one year, as compared to samples cultured for 12 days.

Feasibility of using cryopreserved lymphoblastoid cells to diagnose some lysosomal storage diseases.

OBJECTIVE: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. MATERIALS AND METHODS: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. RESULTS: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. CONCLUSIONS: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.