Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.
The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.
Human peripheral blood mononuclear cells (PBMCs) are used for various applications in many scientific fields, including the study of immunological and bioenergetic issues, such as those related to aging processes or degenerative diseases1,2. PBMCs are heterogeneous in composition and consist of lymphocytes (B cells, T cells, and NK cells), monocytes, and dendritic cells. The cells sometimes show great individual differences and variations within a subject, so standardized procedures for handling these cells are required. Important parameters such as viability and purity of the isolation are the basic requirements for its handling and are additionally influenced by environmental factors such as the time of collection, the melatonin level, whether the subject is fasting, and others3,4.
Based on studies on bioenergetics of PBMCs, we describe here a method for the isolation, cryopreservation, and cultivation of PBMCs that is suitable for other methods as well. While muscle biopsy is considered the gold standard for mitochondrial energy metabolism5, the examination of blood cells is a rapid, minimally invasive procedure. In addition to this, more and more studies suggest that the changes in mitochondrial function in aging and Alzheimer's disease (AD) occur not only in the brain but also in the periphery6,7,8,9,10. The method also allows investigations of other conditions and diseases, including diabetes mellitus and obesity11,12,13. Gene expression patterns in multiple sclerosis patients can be analyzed, or immune function and influences on it in general14,15,16.
PBMCs generally rely on oxidative phosphorylation (OXPHOS) to generate adenosine triphosphate (ATP)17,18. Therefore, PBMCs cover a wide range of applications as surrogates. In previous reports, the energy metabolism of PBMCs has been used to address organ dysfunctions, such as in early heart failure19, septic shock20 or sex-associated differences4 in mitochondrial function. A generalized method for cryopreservation isolation and cultivation of PBMCs would have advantages in the comparability of results obtained at different institutes. There is a great deal of variation in the protocols for each step21,22, the goal of this method is to provide a guideline for bioenergetic measurements in PBMCs.
In this article we describe a method for measuring bioenergetic parameters in PBMCs. We explain the methods for isolating, cryopreserving and measuring bioenergetics of PBMCs from human blood. This method can be used to determine bioenergetic parameters in patients and evaluate them in a clinical context. To apply these measurements, researchers need access to a patient population from which fresh blood samples can be obtained.
All protocols described in this manuscript for blood collection, isolation and analysis have been reviewed and approved by the Institutional Review Board at the University of Giessen, Germany. The consent of the patients to include their samples in the study was obtained. All steps for isolation and cell culture are carried out under a biological safety cabinet.
1. Venipuncture
2. PBMC isolation
Figure 1: Schematic representation of a density gradient centrifugation to illustrate the different layers. Please click here to view a larger version of this figure.
3. Cryopreservation
4. Thawing
5. Cell culture
6. ATP assay
7. High-resolution respirometry
Figure 2: Schematic course of the O2 flux. The schematic course of the oxygen flux is shown. The curve is divided into the different phases after the addition of inhibitors and substrates from a-k. a: endogenous respiration; b: permeabilized cells; c: uncoupled complex I respiration; d: coupled complex I respiration; e: OXPHOS ; f: maximal uncoupled activity of CI and CII ; g: uncoupled respiration of complex II; h: leak respiration; i: residual respiration; j: CIV(U) uncoupled respiration and autooxidation of TMPD; k: autooxidation of TMPD. Please click here to view a larger version of this figure.
8. Citrate synthase activity
Cell viability and number
To achieve successful isolation and cryopreservation, cell count and viability should be as high as possible. Before and after cryopreservation, the cells are counted, and their viability is determined to ensure the health and quality of the cells. Figure 3 is a representative illustration of PBMCs before and after cryopreservation, cell count and viability hardly differ. This indicates successful isolation and preservation of PBMCs.
Figure 3: Effect of cryopreservation on cell number and viability. The test groups are divided into the control group with freshly isolated PBMCs and PBMCs after 1 month cryopreservation. The counting of the cells and the determination of their viability was carried out with an automated cell counter. Trypan blue was used to determine the viability. Each measurement was carried out as a triplicate of the mean value was calculated from the results of the individual measurements. (A) Determination of cell viability of PBMCs after fresh isolation and after 1 month cryopreservation. Values are given as mean ± SEM. Significances were determined with a paired t-test. (B) Determination of cell number of PBMCs after fresh isolation and after 1 month cryopreservation. Values are given as means ± SEM. Significances were determined with a paired t-test. The same shape and color show the same sample before and after one month of cryopreservation. Please click here to view a larger version of this figure.
ATP represents the major source of energy for eukaryotic cells. ATP is determined via a luminescence assay system. If cryopreservation is successful ATP between freshly isolated cells and cryopreserved cells should not differ. Figure 4 is a representative illustration of PBMCs before and after cryopreservation; ATP-levels are similar. This indicates successful isolation and preservation of PBMCs.
Figure 4: Comparison of ATP concentration of different cryopreservation periods. ATP concentration (µM / 100,000 cells) in PBMCs. The test groups are divided into the control group with freshly isolated PBMCs and PBMCs after 1 month cryopreservation. Samples were collected at the same time and then either cryo-stored or measured after isolation. A paired t-test was performed to test significant differences; the result showed that the differences were not significant. The values are given as mean values ± SEM (N = 13). The same shape and color show the same sample before and after one month of cryopreservation. Please click here to view a larger version of this figure.
Citrate synthase (CS) is a key enzyme of the citrate-cycle, located in mitochondria. Therefore, CS activity represents a robust marker for mitochondrial mass. CS activity is determined on the basis of conversion from DTNB into TNB. Figure 5 shows that before and after cryopreservation, CS values do not differ in PBMCs. Again, this indicates a successful isolation and preservation of PBMCs.
Figure 5: Effect of cryopreservation on citrate synthase activity. Citrate synthase activity in PBMCs after 1 month cryopreservation compared to the freshly measured control. Samples were collected at the same time and then either cryo-stored or measured after isolation. Values are expressed as mean ± SEM (N = 8). Significances were determined with a paired t-test. The same shape and color show the same sample before and after one month of cryopreservation. Please click here to view a larger version of this figure.
The bioenergetic profiles of PBMCs can be determined using polarographic oxygen sensors, e.g., in high-resolution oxygraph. Cellular oxygen consumption is measured in a closed-chamber system with very high resolution and sensitivity in biological samples, e.g., intact and permeabilized cells, tissues, or isolated mitochondria. The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per s per number of cells24. Within each oxygraph chamber polarographic oxygen electrodes measure the oxygen concentration and calculate oxygen consumption (flux) within each chamber. Oxygen consumption and concentration in the chamber is displayed in real time (Figure 6). With the addition of specific inhibitors and substrates, the individual complexes of the respiratory chain are targeted in order to measure their activity. After the assay, the data is analyzed with the software. The flux values were normalized to citrate synthase activity. The oxygraphy provided lower values for complex IV due to partially oxidized TMPD. In a series of tests, we found that the TMPD used deviated by a factor of 1.8. This factor was used to normalize the values in Figure 6.
Figure 6: Mitochondrial respiration in PBMCS after cryopreservation compared to freshly measured control. A solution containing 1.6 x 107 cells/mL was used to measure the oxygen consumption of cells in an oxygraph. Respiration was measured 1 month after cryopreservation. To investigate the activity of the complexes in the respiratory chain several inhibitors, substrates and uncouplers were added. The addition of a substance was done as follows: CI(L) = leak respiration of complex I; CI(P) = coupled respiration of complex I; CI&CII(P) = physiological respiration; CI&CII(U) = uncoupled respiration displaying maximum activity of complexes I and II; CII(U) = uncoupled respiration of complex II; CII(L) = leak respiration of complex II; CIV(U) = uncoupled respiration. A factor of 1.8 was used to normalize the values. This factor was determined experimentally for the present setup. Data are displayed as the means ± SEM (N = 10). Statistical significance was tested via Student's t-test (*p < 0.05). Please click here to view a larger version of this figure.
Endogenous respiration is determined by adding 2 mL of cell suspension into the chambers. The flux with endogenous substrates is measured. To further distinguish between complexes digitonin is added. Digitonin permeabilizes the plasma membrane while the mitochondrial membranes remain intact. To compensate for proton leak through the membrane, substrates glutamate and malate are added. The respiration rates at this point illustrate complex I-driven respiration in the absence of coupled respiration. To detect the oxidative phosphorylation (OXPHOS) capacity of complex I ADP is added, the respiration is now in a coupled state. The coupled respiration of CI and CII is achieved by addition of succinate. Now the respiratory chain works at maximal capacity. With the titration of carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) the electron transport chain (ETC) is uncoupled. With this uncoupling the maximal uncoupled activity of CI and CII is determined. To differentiate between CI and CII the complex I specific inhibitor rotenone is added. By the addition of oligomycin, the leak respiration of CII is determined. To exclude oxygen consumption by sources not involved in oxidative phosphorylation, the antibiotic antimycin A is added and this residual oxygen consumption is subtracted from all readings obtained in the experiment. The electron donor N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD; 0.5 mM) is an artificial substrate for CIV. To keep TMPD in a reduced state, ascorbate is used. Ascorbate and TMPD are used to measure the maximum uncoupled respiration of CIV. Since TMPD is subject to auto-oxidation, NaN3 is added after stabilization of the flux to inhibit CIV. The remaining oxygen consumption is subtracted from the raw CIV values. Figure 2 shows a typical measurement curve.
The parameters shown show the success of the technique. The technique was developed to perform bioenergetic measurements after cryopreservation. The results shown compare the cells before and after the measurement, as no statistically significant differences can be seen, it can be assumed that this method of preservation is suitable for storage over 1 month.
Table 1: Solutions, buffers, and consumables. Please click here to download this Table.
This protocol provides a means of isolating and cryopreserving peripheral blood mononuclear cells (PBMCs) from human blood in a manner suitable for bioenergetic analyses. The described method offers the possibility to isolate PBMCs gently and in large quantities, with high viability and sufficient cells for bioenergetic measurements. It has the disadvantage that even with minimal interruptions, long isolations occur, but subsequent cryopreservation allows a time-independent measurement of bioenergetics. With this method, samples can be collected and measured at later time points. It is also suitable to collect samples in multicenter trials and measure parameters at the same time in a central location.
Cryopreservation offers the possibility to store cells for a long period of time and to measure them afterwards. The isolation of PBMCs is a time-consuming process of about 2-3 h and only allows a small number of samples to be taken at once. It is not possible for a single experimenter to isolate fresh PBMCs from more than 2 individuals simultaneously without loss of quality. The need to perform follow-up experiments with fresh cells complicates the procedure and is thus associated with additional stress and time trouble. Separating isolation from the performance of the experiments can simplify studies and make tests more standardized. Specifically, blood collection, isolation, cryopreservation of a sample collective, followed by the performance of specific experiments. Samples can be collected from different locations and at different times and then measured at the same time in the same location.
Bioenergetics play an essential role in cell metabolism, disturbed bioenergetics leading to mitochondrial dysfunction are likely to play an important role in aging and subsequently in neurodegeneration and other diseases25,26. The methods described above can be used in freshly isolated and cryopreserved PBMCs to monitor changes. These bioenergetic measurements can be used as a basis for clinical studies and research.
There are several limiting factors to this technique and the advantages and disadvantages of fresh measurement versus measurement after cryopreservation must be weighed. Cryopreservation is generally very demanding for PBMCs; therefore, it is important to keep the number of stressors as low as possible. Time is of the essence as the cells should spend as little time as possible in DMSO which is toxic to PBMCs, each step should be prepared before thawing or freezing cells with DMSO. Also, for bioenergetic measurements, storage in a -80 °C freezer proved to be ineffective – the samples must be transferred to liquid nitrogen after 24 h. A controlled freezing rate is mandatory if cooling is too fast, the water remaining in the cells freezes and forms crystals damaging cell membranes and compartments. Too slow a cooling rate leads to prolonged contact with the toxic DMSO. Cell freezers using isopropanol can achieve a freezing rate of 1 °C/min in a -80 °C freezer. Thawing and refreezing of PBMCs should be avoided.
This protocol describes the isolation and cryopreservation of PBMCs. In order to confirm the functionality of the PBMCs after preservation for bioenergetic measurements, exemplary measurements of bioenergetics were carried out. This protocol has been optimized for the measurement of bioenergetic factors. Bioenergetic factors can be determined after cryopreservation, but it can also be determined for other protocols whether measurements are possible after cryopreservation.
The authors have nothing to disclose.
We would like to thank the clinical team of the University Hospital Giessen-Marburg for the blood collection. This work was funded by the Justus Liebig university.
0.1 M Triethanolamine-HCl-Buffer (pH = 8,0) | Self-prepared | – | |
0.5 M Triethanolamine-HCl-Buffer | Self-prepared | – | |
1.0 M Tris-HCl-Buffer (pH = 8,1) | Self-prepared | – | |
1.01 mM DTBB | Self-prepared | – | |
10 % Triton X-100 | Self-prepared | – | |
10 mM Oxalacetat | Self-prepared | – | |
14–20 G sterile blood draw needles Multi Adapter Sarstedt Safety-Multifly | Sarstedt | 156353_v | |
37% HCl | Carl Roth GmbH & Co. KG | – | |
70% Ethanol (EtOH) | Self-prepared | – | |
Acetyl-CoA | Pancreac Applichem | A3753 | |
ADP | Sigma-Aldrich | A5285 | |
Alcohol wipes | (70% isopropyl alcohol) | ||
Antimycin A | Sigma-Aldrich | A8674 | |
Aqua (bidest.) | With MilliQ Academic (self-made) | – | |
Ascorbate | Sigma-Aldrich | A4034 | |
ATP-Standard | Sigma-Aldrich | 6016949 | |
Biocoll Seperating Solution | Biochrom | 6115 | |
Biological safty cabinet MSC Advantage | Thermo Fisher Scientific Inc. | ||
Carbonylcyanid-p-trifluoromethoxy-phenylhydrazon (FCCP) | Sigma-Aldrich | C2920 | |
Cell counter TC20 Automated Cell Counter | Bio-Rad | ||
Centrifuge Heraeus Megafuge 16 R | Thermo Fisher Scientific Inc. | ||
Counting slides, dual chamber for cell counter | Bio-Rad | 1450016 | |
Cryotube Cryo.S | Grainer Bio-One | 126263-2DG | |
Digitonin | Sigma-Aldrich | 37008 | |
Dimethylsulfoxid (DMSO) | Merck | 102952 | |
Disinfection spray | |||
Disposable gloves latex, rubber, or vinyl. | |||
Distrips (12.5 ml) DistriTips | Gilson | F164150 | |
Dulbecco’s Phosphate Buffered Saline (DPBS; 10x) | Gibco (Thermo Scientific) | 15217168 | |
Ethanol (EtOH 100%) | Carl ROTH GmbH & Co. KG | 9065.3 | |
Fetal bovine serum (FBS) | Sigma-Aldrich | F9665 | |
Frezer (-80°C) | Thermo Fisher Scientific Inc. | ||
Glutamate | Sigma-Aldrich | G1626 | |
Holder/adapter | |||
Incubator Midi 40 CO2 | Thermo Fisher Scientific Inc. | ||
Injection syringe | Hamilton | ||
Malate | Sigma-Aldrich | M-1000 | |
MIR05 | Self-prepared | – | |
Mr. Frosty Freezing Container | Thermo Fisher Scientific Inc. | 10110051 | |
Multireader CLARIOstar | BMG Labtech | ||
Nitrogen tank Locator 6 plus | Thermo Fisher Scientific Inc. | ||
Oligomycin | Sigma-Aldrich | O4876 | |
Oxalacetate | Sigma-Aldrich | – | |
Oxygraph-2k | Orobororus Instruments | ||
Penicillin-Streptomycin | PAA | 15140122 | |
Pipettes Performance Pipettor 10 μL, 100 μL, 1000 μL | VWR | ||
Roswell-Park. Memorial-Institute-Medium (RPMI-1640) | Gibco (Thermo Scientific) | 11530586 | |
Rotenone | Sigma-Aldrich | R8875 | |
Saccharose | Carl ROTH GmbH & Co. KG | 9286.2 | |
Sodium azide | Sigma-Aldrich | S2002 | |
Succinate | Sigma-Aldrich | S2378 | |
Tetramethylphenylendiamin (TMPD) | Sigma-Aldrich | T3134 | |
Tourniquet/ Blood pressure cuff | |||
Tris(hydroxymethyl)amino-methane | Sigma-Aldrich | 108382 | |
Triton X-100 | Sigma-Aldrich | 108643 | |
Trypanblau | Biochrom | T6146 | |
Vacuum pump | Vaccubrand GmbH & Co. | ||
ViewPlate-96 | Perkin Elmer | 6005181 | |
Water bath WNB22 | Memmert GmbH & Co. KG |