Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

Summary

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.

Abstract

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.

Introduction

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 diseases^1,2. 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 requireme....

Protocol

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

1. Prepare all equipment needed for blood collection including disinfection spray, sterile .......

Discussion

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,.......

Materials

Name	Company	Catalog Number	Comments
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