Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study

Cryopreservation, shipment, and storage of lymphocytes.

Ninety-milliliter blood samples were collected at 6-month intervals in heparinized tubes (Becton Dickinson Inc., Rutherford, N.J.). The method used was essentially that described previously in detail (8). Whole blood was centrifuged for 15 min at 300 × g. Next, the buffy coats for each sample were placed in a 50-ml polypropylene tube, and an equal volume of isotonic saline was added. This was overlaid onto Ficoll-Hypaque density gradients. Mononuclear cell suspensions were isolated by centrifugation at 700 × g for 10 min. The cell pellets were washed twice at room temperature with Hanks buffered salt solution by centrifugation at 300 × g for 10 min. The final pellets were resuspended in 2 ml of RPMI 1640 containing 20% human AB serum or fetal calf serum, and the suspensions were gently mixed and chilled on ice. Cells were counted on a Coulter Counter in the centers in Baltimore and Los Angeles by using 20 μl of cell suspension in 10 ml of isotonic buffered saline plus 3 drops of Zap-oglobin2 lysing solution or on a hemocytometer in the centers in Chicago and Pittsburgh. Cells were diluted to 2 × 10⁷ cells per ml in cold RPMI 1640 plus 20% serum. Then, an equal volume of RPMI 1640 plus 20% dimethyl sulfoxide (DMSO) as a cryoprotectant was added. Next, the cells were quickly aliquoted into freezing vials (1 ml per vial). Usually six vials were saved for each blood specimen, and each vial contained 10⁷ PBMC. The final freezing solution consisted of RPMI 1640 containing 10% serum and 10% DMSO. All blood samples were processed and frozen by sterile techniques, within 24 h (range, 5 to 24 h) of collection, at the center where they were obtained. For the vast majority of samples, freezing occurred in less than 10 h after collection.

For the initial part of the cryopreservation procedure, cells were placed overnight either in controlled-rate freezers (Gordinier Cryogenics Electronics, Roseville, Mich.), used in the center in Los Angeles from the initiation of the study and in the center in Pittsburgh after August 1988, or in insulated containers (Sigma, St. Louis, Mo.) filled with isopropanol, used in the centers in Baltimore, Chicago, and Pittsburgh before August 1988. Cryovials from the controlled-rate freezers were transferred directly to −135°C freezers (Queue Systems Pacific Sciences, Inc., El Segundo, Calif.) or into the vapor phase of liquid nitrogen for long-term storage. Cryovials within the insulated containers were placed in a −70°C freezer overnight and transferred to the long-term, colder storage the next morning. Frozen tubes were shipped in a customized TA-60 dry-nitrogen shipper (MVE, New Prague, Minn.) to Biomedical Research Incorporated (BRI) (Rockville, Md.), which, under contract to the National Institutes of Health Division of AIDS, received, catalogued, stored, and dispersed clinical specimens from MACS participants in Division of AIDS studies. MACS samples have now been stored at BRI in vapor-phase freezers for up to 12 years. Data in this report include those for samples collected through 1995.

Thawing cryopreserved cells and determining viability.

Samples for this study were shipped from BRI in dry-nitrogen shippers (see above) and were then stored continuously in liquid nitrogen or at −135°C until immediately prior to their use. The method used was essentially that described previously (8). Cells were removed from storage and rapidly thawed by immersion in a 37°C water bath with gentle agitation, by trained technical staff in two MACS pathogenesis laboratories. Each tube was thawed individually until only a small amount of ice crystals remained, a procedure that took about 40 s. Samples were immediately transferred to round-bottom tissue culture tubes. Then, 10 to 14 ml of RPMI 1640 containing 10% human AB serum or fetal calf serum was added over a period of 4 min with agitation, and the tubes were centrifuged immediately at 100 to 400 × g for 10 min. After the DMSO-containing liquid had been removed by aspiration, the cells were washed once in medium containing at least 10% serum or an alternate protein source. Next, the cells were resuspended in about 1 ml of buffer appropriate to the assay to be done, and counts and viability were determined with a hemocytometer and the trypan blue dye exclusion technique. With this method, dead cells appear blue and are therefore distinguishable from viable cells. Viable cells for this project were defined as PBMC that had survived the freeze-thaw process. They were used (i) to evaluate cell function, (ii) for immunophenotype analysis by flow cytometry, (iii) to isolate HIV for subsequent analysis, or (iv) for immortalization by Epstein-Barr virus transformation.

Statistical analysis.

Data reported to the Center for the Analysis and Management of the MACS included (i) the date the sample was originally collected, processed, and frozen (freeze date); (ii) the date the sample was thawed for laboratory testing (thaw date); (iii) the total number of cells recovered regardless of viability; and (iv) the percent cell viability. The number of viable cells recovered was calculated by multiplying items iii and iv. We addressed two main questions: first, whether cell recovery and viability differed over time according to the duration of freezing, and second, whether cell viability differed by center, the original processing or freeze date, the HIV serostatus of the donor, and the CD4⁺-lymphocyte count of the donor.

The number of viable cells and cell viability were summarized via percentiles, overall and across centers. Simple linear regression models were used to evaluate trends in viability over time with regard to the length of time frozen (thaw date − freeze date) and the initial date of sample collection and processing. Nonparametric Kruskal-Wallis tests (4) were applied to assess the significance of observed differences in viability by center, serostatus, and CD4⁺-cell count.

Viability testing.

A total of 596 PBMC samples from 147 individuals were tested for cell viability. They had initially been stored at one of the four MACS centers (179 samples in Baltimore, 170 in Chicago, 88 in Pittsburgh, and 159 in Los Angeles). The center where the sample was originally collected and stored may or may not coincide with the city or laboratory where the sample was thawed, evaluated for cell viability, and used for a pathogenesis study. A total of 197 specimens were from HIV-seronegative donors, and 399 specimens were from HIV-seropositive donors.

Of the 596 PBMC samples evaluated, the median number of total cells recovered per vial across all centers was 7.75 × 10⁶. The 10th and 90th percentiles were 3.9 × 10⁶ and 14.0 × 10⁶, respectively, and the range was 1 × 10⁶ to 25 × 10⁶ total cells recovered per vial. The viability assessment of these cells by trypan blue dye exclusion resulted in a median of 92% of the cells recovered remaining viable (range, 24 to 100%). The median number of viable cells recovered per vial across all centers was 6.9 × 10⁶ (Table 1). The 10th and 90th percentiles were 3.5 × 10⁶ and 12.9 × 10⁶, respectively, and the range was 0.4 × 10⁶ to 23 × 10⁶ viable cells recovered per vial. The distributions of numbers of total and viable cells recovered and of percent cell viability were significantly different across centers (P < 0.05 [Kruskal-Wallis test]). However, the median number of viable cells was at least 5 × 10⁶ for each center, and the median viability was at least 90%. Recovery was often considerably lower than the expected 6 × 10⁶ cells. At the same time, recovery from some vials, especially those stored in the center in Chicago early in the study, was higher than 10⁷ cells, indicating that more cells than prescribed by the protocol must have been stored.

In order to address the center differences and analyze the effect of time on the viability and number of viable cells recovered, the data were evaluated separately by center. Four specimens from the center in Pittsburgh which yielded viabilities of less than 35% occurred early in the study and were excluded from the analysis. Scatter plots by center of the 596 individual cell viabilities (Fig. 1) and counts of viable cells (Fig. 2) over the length of time frozen (from 0 to 12 years), together with a corresponding regression line, were used to illustrate changes over time. There were statistically insignificant decreases in viability in all centers, with estimated losses per year of 0.10% in Baltimore, 0.47% in Chicago, 0.73% in Pittsburgh, and 0.09% in Los Angeles (Fig. 1). Overall, there was an estimated decrease (average slope) in viability of 2.9% over the entire 12 years of the study (0.24% per year), which was of borderline statistical significance (P = 0.06). Viabilities did not vary according to the year the sample was collected at the clinic center (P = 0.36 [data not shown]). Additionally, the median viabilities were 93% for seronegatives and 92% for seropositives, while the median numbers of viable cells were 6.4 × 10⁶ and 7.2 × 10⁶ for seronegatives and seropositives, respectively. These differences were not statistically significant.

The rate of change for the number of viable cells recovered with respect to the number of years frozen differed by center. However, the directionality of the trends was not consistent for all centers (Fig. 2). Data from the centers in Baltimore and Pittsburgh revealed estimated losses of 5.1 × 10⁵ and 1.5 × 10⁵ cells per year, respectively. In contrast, the numbers of cells at the centers in Chicago and Los Angeles increased by an estimated 5.4 × 10⁵ and 2.3 × 10⁵ cells per year, respectively. The rates of change observed in the specimens from the Baltimore and Chicago centers were significantly different from 0 (P < 0.01).

The median number of CD4⁺ lymphocytes from the 399 samples from HIV-seropositive donors was 607/μl (range, 8 to 1,784/μl). After adjusting for the number of years frozen, there was a relatively strong positive association between the number of viable cells recovered and the CD4⁺-cell counts in the Chicago and Los Angeles samples (P ≤ 0.001). There was no apparent relationship between CD4⁺-cell count and total cell viability in the other two centers or between CD4⁺-cell count and percent viability in any center. Therefore, we conclude that our data do not indicate decreased viability in samples due to patients being immunosuppressed.

Data from the Pittsburgh center were used in order to assess the effect of using a controlled-rate freezer on viability by analyzing the results for two groups selected according to the method of cryopreservation within the same laboratory: (i) an isopropanol-filled insulated container used prior to August 1988 and (ii) a controlled-rate freezer used after August 1988. By using one laboratory for this assessment, we were able to control for differences by center that may be unrelated to the type of freezer container. There was a significant improvement in percent viability over time, with an estimated loss of 2.23% per year when alcohol-filled containers were used for freezing, compared to only a 0.01% loss per year with the controlled-rate freezer (P = 0.04). In order to assess whether the differences that we observed in the Pittsburgh center may be due to a learning curve, we repeated the analysis in the Los Angeles center, because a controlled-rate freezer had been used there consistently since the beginning of the study. We found no suggestion of improvement over time and therefore conclude that the trends seen in the center in Pittsburgh were not entirely due to a technique improved over time.

We also classified all 596 samples into two groups depending on the freezing process, that using a controlled-rate freezer (n = 198) and that using an alcohol-filled container (n = 398). Overall, there were no significant differences in the median viability (93 versus 92%) or the number of cells recovered (6.6 × 10⁶ versus 7.0 × 10⁶) between the two freezing methods. However, using cutoffs of 85, 80, 75, and 50%, there were consistently more samples below the cutoffs when the alcohol-filled container was used. For example, when we applied a cutoff of 80%, there were more samples with less than 80% viability when the alcohol-filled container was used (9.5%) than when the controlled-rate freezer was used (2.5%). Although consistent with the literature, these data alone are not sufficient to conclude that a controlled-rate freezer is better than an alcohol-filled container for the cryopreservation of cells.

In addition to the issues related to cell freezing, it is worth noting that after controlling for the center that cryopreserved the cells, significant differences in the median viable-cell recovery persisted between the two pathogenesis laboratories that thawed the samples (8.4 × 10⁶ [n = 167] versus 6.2 × 10⁶ [n = 429]).

Use of cryopreserved PBMC for pathogenesis studies.

One example of a typical use of the samples in this repository for pathogenesis studies is a study that included a subset of the 596 samples, a total of 112 samples from all time points between seroconversion and T-cell inflection (12), a span of up to 10 years. In all cases, one vial per person visit yielded sufficient cells to complete flow cytometry, analysis of cytotoxic-T-lymphocyte precursors, proviral HIV load measurements, and HIV sequencing (16). In another study, 37 of the 596 samples were from donors who were severely immunosuppressed (CD4⁺-cell counts, <50 per mm³). Every sample provided enough cells to obtain immunophenotyping results, and culturable virus was recovered from all except one of the samples (6). In another series of studies (1, 14), 118 samples from HIV-seropositive donors were tested for cell surface markers and immune function.

In other studies requiring viable cells where similar protocols for freezing and thawing were used but viability and recovery were not measured, comparably good results were obtained. For example, in a MACS virology laboratory, virus has been isolated from frozen viable cells in more than 90% of attempted isolations (13). In addition, 1,185 of 1,312 (90%) PBMC specimens used were transformed with Epstein-Barr virus for development of B lymphoblastoid cell lines used to extract DNA for genetic typing. Despite being cultured for up to several weeks these samples showed little or no bacterial contamination.