Flow Cytometric Assessment of the Influence of Cryopreservation on Vitality, Recovery and Immunophenotype of Camel Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC), including cells of the innate and adaptive immune system, represent a valuable sample source for the evaluation of the immune system in health and disease (Pourahmad and Salimi 2015; Alexovic et al., 2024). The major cell subpopulations that fall within the PBMC population include blood monocytes in addition to the lymphoid cells alpha beta (αβ) T cells, gamma delta (γδ)T cells, B cells (Ambade et al., 2023; Shitikova et al., 2024). Immunophenotyping of PBMC using flow cytometry and fluorescent antibodies represents an essential procedure in diagnostic and research immunology laboratories (Heubeck et al., 2023; Preglej et al., 2023). The technique enables the simultaneous measurement of cell viability, cell count, shape change and marker antigen expression (Rawat et al., 2019; Huang et al., 2024). In addition, several innate and adaptive functions, such as phagocytosis, proliferation and cytokine production could be analyzed using this single-cell analytic methodology (Ammann et al., 2020). However, due to the limited availability of flow cytometry devices because of their expensive prices, samples cannot always be analyzed immediately after separation and need therefore to be stored for later analysis in core facility laboratories (Juhl et al., 2021). In addition, in long-time clinical trials collecting and storing samples to be analyzed under the same conditions is usually advantageous to minimize variation in test results.

Previous studies reported using several cell storage procedures for the preservation of PBMC for later analysis by flow cytometry (Hope et al., 2021; Browne et al., 2024). This includes storage of fixed unstained cells, fixed and stained cells, or unfixed and unstained cells (Pinto et al., 2005). For several species, cryopreservation of PBMC directly after their separation has been proven an effective procedure for storing cells and preserving their epitopes for later staining with mAbs and by flow cytometry (Li et al., 2021; Efthymiou et al., 2022). In a recent study, Hong et al., reported that storing human PBMC at -80°C in a DMSO-based cryoprotectant medium followed by thawing at 37°C is associated with high cell viability and recovery (Honge et al., 2017).

As no preservation methods have been investigated for storing camel PBMC for later analysis by flow cytometry, the present study was undertaken to comparatively analyze cell apoptosis, necrosis, shape change, staining patterns with monoclonal antibodies (mAbs) and cellular composition of freshly separated and cryopreserved camel PBMC.

Collection of blood samples from camels
 
Blood samples were collected from nine dromedary camels (Camelus dromedarius. The camels were healthy camels selected from the animals admitted during February 2024 for normal slaughtering at Al-Omran Slaughterhouse (Al-Ahsa, Eastern Province, Saudi Arabia). The animals included five males and four females with ages ranging from 4 to 8 years (mean age 6.1±0.5). Blood (30 mL per animal) was collected from the jugular vein in EDTA-containing tubes. Collected blood samples were kept refregrated and transported within one hour to the Immunology Laboratory at King Faisal University for further processing.
 
Separation and cryopreservation of blood mononuclear cells
 
Separation of peripheral blood mononuclear cells (PBMC) was performed within one hour after blood collection. For this, 15 mL camel blood was diluted in 15 mL cold PBS and the mixture was carefully overlaid on 15 mL Lymphoprep™ (STEMCELL Technologies Inc. Vancouver, BC, Canada) in a sterile 50 mL falcon tube without mixing the blood with the Lymphoprep™. The tubes were then centrifuged at 800 x g for 30 min at 10°C. After centrifugation, the PBMC-containing interphase between the Lymphoprep™ and the plasma layers was collected into new sterile 50 mL tubes. Separated PBMC were washed in three rounds with cold PBS at 400, 200 and 100 x g, each for 10 min to remove platelets. In the last centrifugation step, the cell suspension was divided into two parts in two 15 mL tubes. The washed cell pellet in one tube was suspended in cold RPMI 1640, adjusted to 2.1 x 10⁶ cells/mL and further processed for flow cytometry (Hussen et al., 2024).

The pellet in the other tube was suspended in (2°C to 8°C) cooled cryopreservation medium (Recovery™ Cell Culture Freezing Medium, gibco by Life Technologies), adjusted to 2.1 x10⁶ cell/mL, dispensed in 1.5 mL cryovials and kept at -80°C until analysis. For cell recovery, the tubes were thawed by incubation in a water bath (37°C) for 5 min. Thawed cells were washed two times (300xg for 10 min each) with pre-warmed RPMI 1640 and finally suspended in RPMI medium.
 
Analysis of cell vitality
 
The percentage of apoptotic, necrotic and viable cells within fresh and preserved camel PBMC were measured by flow cytometry (Hussen et al., 2023b) using the Annexin V-FITC Apoptosis Kit according to the kit protocol (Abcam; ab14085). The washed cell pellet was stained in cell culture plates (96-wells) with Annexin V-FITC and propidium iodide (PI) diluted 1:100 in Kit buffer (100 mL /well) for 5 min at RT in the dark. Cells were classified upon excitation at 488 nm into Annexin V+/PI- apoptotic cells, Annexin V+/ positive/PI+ necrotic cells and Annexin V-/ PI- viable cells.
 
Analysis of shape change
 
Shape change in camel PBMC was measured by calculating the change in side scatter (SSC) area, indicating cell granularity and forward scatter (FSC) area, indicating cell size by flow cytometry (Hussen et al., 2023a).
 
Membrane immunofluorescence
 
Fresh separated and cryopreserved camel PBMC were labeled with monoclonal antibodies (Table 1) to camel leukocyte antigens and analyzed by flow cytometry (Hussen et al., 2018). Cells (1x 10⁶ cell/well) were incubated in the wells of a 96-well plate with mAbs to camel antigens: a cluster of differentiation (CD)45, CD44, CD11a, major histocompatibility (MHC) class-I, MHC-II, BAQ44A, workshop cluster (WC)1, CD4, CD172a, CD14 and CD163 (Hussen 2021; Hussen et al., 2023b). After incubation (15 min; 4°C), cells were washed twice and were incubated with mouse secondary antibodies (IgM, IgG1, IgG2a; Invitrogen) labeled with different fluorochromes or with mouse isotype control antibodies (Becton Dickinson Biosciences). After washing, cells were analyzed on a Becton Dickinson Accuri C6 flow cytometer (Becton Dickinson Biosciences, San Jose, California, USA). Data from at least 100,000 cells were collected and analyzed with the flow cytometric software C-Flow (Becton Dickinson Biosciences, San Jose, California, USA).


 
Statistical analyses
 
The software program C-Flow (BD) was used for the analysis of flow cytometric data. Surface molecule abundance was calculated using the mean fluorescence intensity of the corresponding cell type and molecule. Means and standard error of the mean (SEM) were calculated using the column statistic function of the Prism software (GraphPad). Data normality was calculated using the Shapiro-Wilk test. For the comparison between fresh separated and cryopreserved cells, the Student’s t-test (paired t-test) was performed; and the differences were considered significant if the p-value was less than 0.05. The correlation between molecule abundance on fresh and preserved cells, the linear regression function of GraphPad Prism was performed and the R Square was calculated.

Impact of cryopreservation on cell vitality of camel mononuclear cells
 
Cell apoptosis and necrosis of camel PBMC were analyzed using flow cytometry (Fig 1A). The comparison between fresh separated and cryopreserved cells revealed comparable percentages of apoptotic, necrotic and viable cells within camel blood lymphocytes and monocytes (Fig 1B). The absolute numbers of viable lymphocytes (1.30 x10⁶ ± 0.11 cell/mL) and monocytes (0.79 x 10⁶±0.09 cell/mL) within fresh PBMC showed no significant differences (p> 0.05) to the numbers of viable lymphocytes (1.30 x 10⁶ ±0.15 cell/mL) and monocytes (0.76 x 10⁶ ± 0.09 cell/mL) within cryopreserved PBMC (Fig 1C).


 
Shape change of cryopreserved camel mononuclear cells
 
Cryopreserved lymphocytes showed significantly (p< 0.05) reduced side scatter (SSC; 8.92 x 10⁴ ± 0.08) values,  and increased (p<0.05) forward scatter (FSC; 21.39 x 10⁵ ± 0.10) values (Fig 2A) in comparison to the SSC (9.41 x 10⁴ ± 0.19) and FSC (20.80 x 10⁵ ± 0.22) values of freshly separated lymphocytes. For monocytes, however, both SSC and FSC parameters did not show significant (p>0.05) differences between fresh and cryopreserved cells (Fig  2B).

​
 
Impact of cryopreservation on the staining pattern of camel mononuclear cells with monoclonal antibodies (mAbs)
 
Monoclonal antibodies (mAbs) to the pan-leukocyte marker CD45, the cell adhesion molecules CD44 and CD11a as well as the major histocompatibility complex (MHC) class stained all camel PBMC positively with no significant differences in the percentage of stained cells between fresh and preserved PBMC (Fig 3 A, B). Similarly, the percentages of CD14-negative cells (lymphocytes), CD14-positive cells (monocytes), CD163-positive cells (monocyte subset) and CD172a-positive cells (myeloid cells) within PBMC did not differ significantly (p> 0.05) between fresh and preserved cells (Fig 3 A, B).


 
Impact of cryopreservation on camel lymphocyte composition
 
The analysis of lymphocyte composition (Fig 4A) revealed a higher (p<0.05) percentage of MHC-II-positive lymphocytes (B cells) within cryopreserved (26.9 %±2.4) compared to fresh separated PBMC (18.6 %±2.7) (Fig 4B). In contrast to this, the percentage of lymphocytes positively stained with BAQ44A mAb (B cell subset) was significantly lower (p<0.05) within preserved (13.3%±1.7) than fresh (17.4 %±2.3) PBMC (Fig 4B). On the other hand, the fractions of CD4-positive (T helper) cells, WC1-positive gd T cells and CD11ahigh lymphocytes (activated lymphocytes) were comparable (p>0.05) between fresh and cryopreserved PBMC (Fig 4B).



Impact of cryopreservation on the expression of activation markers on camel monocytes
 
In comparison to freshly separated monocytes, cryopreserved monocytes showed higher (p<0.05) mean fluorescence intensity (MFI) of the cell activation marker MHC-II molecules (7939±1532 versus 2757±205 on fresh monocytes), while the abundance of CD163 did not differ significantly between fresh and preserved monocytes (Fig 5A). For the two monocyte activation markers, however, there was a positive correlation between the MFI values of fresh cells and those of preserved cells (R square = 0.7 for CD163 and 0.6 for MHC-II) (Fig 5B).



Immunophenotyping of peripheral blood mononuclear cells (PBMC) has been proven a valuable tool to evaluate the immune status in health and disease (Maecker et al., 2012). Single-cell analysis by flow cytometry is the most commonly used technique for immunophenotyping of PBMC (Perfetto et al., 2004). Due to the limited availability of flow cytometry devices in veterinary laboratories, cells are usually preserved to be analyzed in central or core facility laboratories. The present study aimed to investigate the influence of cryopreservation of camel PBMC on their vitality and immunophenotype.

In the present study, the comparable percentages of dead, apoptotic and necrotic, cells within fresh and preserved camel lymphocytes and monocytes indicate that PBMC cryopreservation for 4 weeks did not impact their cell vitality. The similarity in absolute numbers of lymphocytes and monocytes between fresh and preserved PBMC also in support of this.

Cell light scatter parameters, including side scatter (SSC), which is indicative of cell granularity and forward scatter (FSC), which is indicative of cell size, are usually used for the identification of cell populations in flow cytometry (Stanciu et al., 2016). In the present study, although cryopreservation did not impact monocytes SSC or FSC values, the reduced SSC and increased FSC of preserved lymphocytes indicate a cryopreservation-induced shape change in this population. This change, however, did not interfere with the identification of cryopreserved lymphocytes based on their SSC and FSC characteristics.

The protein tyrosine phosphatase CD45 (Meza Guzman et al., 2024), the cell adhesion molecules CD44 (hyaluronic acid receptor) (Wu et al., 2024) and CD11a (Leukocyte function-associated antigen 1) (Lacouture et al., 2024) as well as the major histocompatibility complex (MHC) class I are common leukocyte markers expressed on all leukocyte populations (Kuzilkova et al., 2022), while the expression of the surface antigens CD14 (LPS receptor), CD163 (the receptor for hemoglobin-haptoglobin complexes) and CD172a (SIRPa) is limited to myeloid cells (mainly monocytes) within PBMC (Elnaggar et al., 2019). In the present study, the comparable percentages of marker-positive cells within fresh and preserved PBMC indicate no impact of cryopreservation on the staining pattern of camel PBMC with monoclonal antibodies to the indicated markers.

Blood lymphocytes population encompass several cell subsets, mainly including CD4+ alpha-beta (ab) helper T cells, CD8+ ab cytotoxic T cells, gamma delta gd T cells, B cells and natural killer cells (NK cells), (Heubeck et al., 2023). In the present study, the commercially available monoclonal antibodies to camel CD4, WC1 (gd T cell marker) (Gillespie et al., 2021), MHC-II and BAQ44A (B cell markers) (Stabel et al., 2022) were used to investigate the impact of cryopreservation on camel lymphocyte composition. The results indicate a significant increase in the proportion of B cells with a reduced BAQ44A+ B cell subset. This effect may lead to misinterpretation of results when B cells are comparatively analyzed in fresh and cryopreserved samples using the mentioned antibodies.

The expression pattern of the monocyte markers CD163 and MHC-II are commonly used for the characterization of monocyte subsets and the functional phenotypes of macrophages (Elnaggar et al., 2019). In the present study, the enhanced expression of MHC-II, which is a marker of inflammatory monocytes (Hussen et al., 2020), leads to the assumption that the cryopreservation or the thawing process either resulted in monocyte activation or higher accessibility of MHC-II epitopes on monocytes to the anti-MHC-II mAbs.

Comparative analysis of fresh-separated and cryopreserved camel PBMC by flow cytometry revealed no significant changes in cell viability four weeks after preservation. In addition, the reactivity of camel PBMC with mAbs to the cell markers antigens CD45, CD44, CD11a, MHC-I, CD14, CD163 and CD172a remained unchanged for cryopreserved cells. However, mAbs to MHC-II and BAQ44A displayed modified binding to cryopreserved PBMC leading to significant changes in the positively stained B cells and increased the abundance of MHC-II on cryopreserved monocytes. Collectively, the results of the current study show that storing camel PBMC in cryopreservation media at -80°C is an appropriate procedure for preserving the epitopes for most of the used antibodies.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.