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 β-galactosidase, β-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.


Introduction
Inborn errors of metabolism (IEM) is the term used to refer to a set of genetically determined autosomal recessive diseases that include around 500 different dysfunctions and that account for 10% of genetic diseases. IEM have been divided into groups, according to the repercussions to cells and to the associated metabolic defect [1]. One of these groups of diseases includes lysosomal storage diseases (LSD), in which abnormal amounts of built-up substrates are stored in cells, usually leading to changes in cell architecture and function. These dysfunctions are generally caused by a deficiency in specific enzymes, among which β-galactosidase, β-glucosidase, α-iduronidase, α-galactosidase, and α-glucosidase, whose deficit may cause GM1-gangliosidosis, Gaucher, muccopolysaccharidosis type 1, and Fabry and Pompe diseases, respectively.
It is believed that early treatment may lessen the chances of irreversible damage like hepatomegaly and/or splenomegaly, skeletal changes, and significant alterations in the central nervous system [2]. Physicians establish a preliminary diagnosis based on clinical features. This initial diagnosis is then confirmed by laboratory analysis, which is usually performed using leukocytes, though fibroblasts and whole blood (blood spots) may also be used, and, in some conditions, plasma or urine. However, these biological specimens degenerate with time, compromising the enzyme test or its repetition [3].
When it is necessary to store cells for longer periods for enzyme activity analysis, cultivated fibroblasts are used, though these have limited lifespan, and suffer the effects of aging and modifications of enzyme activities during senescence [3,4]. Moreover, the discomfort as the collection, the lack of specific knowledge as to the correct collection procedure observed in some laboratories, and the even greater trouble in recollecting material are negative aspects of the utilization of fibroblasts obtained from skin fragments. In this scenario, the technique is used by the few professionals who are virtually acquainted with the standard procedures of this important diagnosis tool [5].
Alternatively, lymphoblastoid cell lines (LCLs) obtained from lymphocytes B in the peripheral blood flow incubated with the Epstein-Barr virus (EBV) may be used. In comparison with the skin fibroblast lines, this technique offers a few advantages: (a) faster collection of material, (b) lower cytogenetic variation, (c) faster cell proliferation with no senescence, (d) growth in suspension, (e) massive cell material proliferation, (f) freezing and subsequent reestablishment of cultures with higher success rates as compared to fibroblasts, (g) low volumes (2 to 3 mL, or less) of peripheral blood required, and (h) stability of the karyotype and of phenotypical expression [3,[6][7][8].
The EBV belongs to the Herpesviridae family and is the etiological agent of infectious mononucleosis [9], among other diseases such as Burkitt lymphoma, Hodgin's disease, and T-cell lymphoma [10]. Similarly to some LSD cells, EBVtransformed lymphocytes likewise form significant cytoplasm vacuoles caused by immune response [6].
Our research group has shown that no significant changes in enzyme activities are observed, before and after LSD freezing, within a 180-day cryopreservation period [11]. In addition to the shorter culture time (12 days), the maintenance of activities along time would guarantee the subsequent repetition of the procedure, if necessary.
The present study evaluates the activity of the five lysosomal hydrolases mentioned above after a 1-year cryopreservation period and tested the efficiency of the LCL transformation method [12] using transmission electron microscopy (TEM).

Blood Collection.
Twenty milliliter peripheral blood samples were collected from 25 healthy individuals using heparinized tubes for subsequent culture of peripheral blood mononuclear cells (PBMCs). These cells were obtained by Fycoll-Histopaque (Sigma, St. Louis, Mo, USA) gradient centrifugation. The protocol was approved by both scientific and ethics committees (Clinical Hospital of Porto Alegre, HCPA, Porto Alegre, Brazil), and written informed consent was obtained from all subjects.

Preparation and Conservation of EBV B95'8 Cells.
EBV B-95 8 cells were acquired from the Cell Bank of the State of Rio de Janeiro, from the Federal University of the State of Rio de Janeiro, and frozen in liquid nitrogen upon use. These cells were then cultured to the required volume in RPMI-1640 medium supplemented with 20% fetal calf serum and exposed to a CO 2 -free atmosphere by closing the culture flask for 5 to 7 days. Next, adherent cells were detached using Cell Scraper (Corning, New York, NY, USA) and then filtered in a 0.22-μm filter (Millipore, Bedford, Mass, USA).

Mononuclear Cell Culture.
After separation of PBMCs, cells were counted in a Neubauer chamber and adjusted to 9 × 10 6 cells per culture flask. Next, 2 μg/mL cyclosporine A (CyA) (Sandimmum, Sandoz, East Hannover, NJ, USA) and 1 mL of the viral particle were added. The viral particle was obtained using the EBV B-95'8 strain. The RPMI-1640 medium was replaced after 4 and after 8 days, with CyA 1 μg/mL supplementation. On day 12, cultures were centrifuged (400×g, 10 min) in PBS medium, after which cells were collected. Then, half of the collected cells were submitted to measurement of enzyme activity and half was cryopreserved for one year.

Freezing and
Thawing of Samples. Cells were gradually frozen in fetal bovine serum supplemented with 10% DMSO in a freezing container immersed in isopropanol (Nalgene Nunc Int., Rochester, NY, USA). Samples were kept in the freezer at −80 • C for 24 h and then conserved in liquid nitrogen upon enzyme activity evaluation.

Electron Microscopy.
After the separation of the mononuclear fraction by Fycoll-Histopaque (Sigma, St. Louis, Mo, USA) gradient centrifugation, cells were grouped as lymphocytes and EBV-infected lymphocytes. Pellet cells of the peripheral blood were fixed in a mixture formed by paraformaldehyde 4% and glutaraldehyde 2.5% buffered with 0.1 M phosphate (pH 7.3) at room temperature and postfixed in phosphate-buffered OsO 4 , dehydrated in ascending alcohol and acetone, and embedded in araldite (Durcupan ACM, Fluka). Ultrathin sections (70 nm) were stained with 2% uranyl acetate followed by lead citrate and examined under JEM 1200 EX II (CME -UFRGS).

Statistical
Analysis. Data were reported as mean ± SD. The activities of the five enzymes evaluated on day 12 in culture and immediately on day 325 in freezing were compared using the Student's t-test (P < .05) in the software GraphPad Prism 5.

Evaluation of Cell Transformation.
Cell transformation was successful in 100% of cases, since the material collected from all 25 subjects was successfully transformed. Figures 1(a), 1(b), and 1(e) show that lymphocytes presented a morphologically well-defined nucleus, with preserved mitochondria. Also, endoplasm reticuli, ribosomes, and Golgi apparatus were well organized in the cytoplasm. Figures 1(c), 1(d), and 1(f) from lymphocytes cultured with EBV show a higher number of cell organelles, apart from significant morphological changes such as larger mitochondrial having disorganized cristae, electrolucid structures (filled with material), numerous lysosomes, binucleated cells, and myelin bodies. These features confirm the transformation of lymphocytes by the EBV.

Discussion
Two previous studies conducted by our research group have demonstrated the effectiveness of this protocol in culturing lymphocytes transformed with EBV in a short period of time (12 days) [11,18]. Since the culturing of transformed lymphocytes is less labor intensive and the material (blood) is more easily obtained-as compared to fibroblasts [3], we decided to further investigate the influence of lymphocyte cryopreservation times on the activity of lysosomal hydrolases and to evaluate the efficiency of the transformation of these cells using the EBV.
In the present study, we evaluated five enzymes largely employed in the diagnosis of LSDs of common occurrence. According to Staretz-Chacham et al. [19], the methods used in our study are sensitive and selective for the measurement of these enzymes activities in the diagnosis of LSDs. The activity of all enzymes remained essentially constant after the 1-year cryopreservation period. This indicates that these samples may be safely handled after this period of time. Since blood collection is a less invasive approach as compared to skin biopsy, such result shows great promise. The consistency in enzyme activities has been demonstrated in 30-and 6month cryopreservation periods [11,18].
Cell culture was followed up throughout the cell culture period by observing the formation of clusters, which are, according to the literature, clear indicators of cell transformation [20]. These clusters increased with time, upon becoming visible to the naked eye.
Transmission electron microscopy afforded to detect myelinoid bodies, binucleated cells, nuclear invagination, and cytoplasm vacuoles in EBV-infected cell cultures (Figure 1). These findings confirm the successful transformation of lymphocytes by the EBV. This transformation, side by side with the fact that enzyme activities remained stable after the 1-year cryopreservation period cells were submitted to, affords an excellent approach to diagnose LSDs.
The presence of myelinoid bodies after the transformation of cells by the EBV has been observed in some LSDs, as reported in the literature [21][22][23]. This was also seen in normal cells in the present study. Disorders like Niemann-Pick type b and c [7,21] were also characterized by the formation of these myelin bodies in lymphocytes.
Lymphocyte binucleation (Figure 1(d)) has been reported in studies using lymphocytes of myeloma patients [24]. In our study, the cell cultures transformed by EBV B-95 8 strain originated from EBV-infected lymphocytes also exhibited the phenomenon.
The results obtained in the present study are in agreement with the findings obtained by our research group in previous studies [11,18]. This means that the freezing of samples according to a less invasive method which affords the easy obtainment of material allows to conduct a reliable enzyme analysis even when the sample is frozen for a long period of time.