Isolation of human peripheral blood mononuclear cells John Graham

Introduction

Using a density barrier

The density of most human peripheral blood mononuclear cells (PBMCs) is <1.078 g/ml. In order to separate these cells from the denser erythro-cytes and polymorphonuclear leukocytes ch3

Polysucrose
Figure 3.2 Molecular structure of iodinates density gradient media: (a) metrizoate, (b) diatrizoate, (c) Nycodenz®, (d) iodixanol

(1.077 g/ml) and contain 9.6% (w/v) dia-trizoate and 5.6% polysucrose (or Ficoll®). The only difference between the media is the endotoxin level - this is lower (<1 EU/ml) in Lymphoprep™ than in other comparable commercial media.

There is, however, evidence that the polysaccharide may interact with the surface of lymphocytes [42]. Moreover, the presence of an impermeant ion (diatri-zoate) in the medium may also affect the Gibbs-Donnan equilibrium of ions across the membrane. An alternative medium was therefore developed which was non-ionic and contained no polysaccharide. This isoosmotic medium comprises 14.1% (w/v) Nycodenz®, 0.44% NaCl and 5 mM Tricine-NaOH, pH 7.0 [43,44]. It is available commercially as NycoPrep™ 1.077; it has the same density as Lymphoprep™ and separation of the PBMCs is achieved in exactly the same manner, although in the absence of a polysaccharide a slightly longer centrifugation time is required to achieve satisfactory pelleting of the ery-throcytes.

The density barrier can alternatively be produced by dilution of one of the commercial general-purpose media, NycoPrep™ Universal (60%, w/v Nycodenz®) or OptiPrep™ (60% w/v iodixanol). See Figure 3.2 for the molecular structure of Nycodenz® and iodixanol.

Using a mixer technique

An alternative strategy, devised by Ford and Rickwood [45], considerably simplifies the procedure. A 19% (w/v) Nycodenz® solution (p = 1.100 g/ml) was added to an equal volume of whole blood in order to raise the density of the plasma in whole blood to 1.077 g/ml. During cen-trifugation at 1500g for 30 min at 20 °C the erythrocytes and polymorphonuclear leukocytes (PMNs) will sediment while the PBMCs float to the top and they are recovered from the meniscus and the medium below it. The ease of operation makes the mixer-flotation technique particularly attractive when handling large numbers of potentially pathogenic samples.

Recoveries and purity of PBMCs isolated by flotation are almost identical to those obtained with Lymphoprep™. Kaden et al. [46] compared Lymphoprep™ with a mixer based on Nycodenz®; these workers found that the PBMC harvests were essentially identical by both techniques.

A commercial solution (NycoPrep™ Mixer) is no longer produced and the high-density solution for mixing with the blood must be produced by dilution of either NycoPrep™ Universal or OptiPrep™. A convenient alternative is simply to add a smaller volume of OptiPrep™ (1.32 g/ml) itself - the lower osmolality of OptiPrep™ compared to NycoPrep™ Universal allows the use of this strategy and it is the one that is described below.

An interesting observation is that if the blood has to be stored before fractionation, then, as long as the density of the blood is raised by addition of the dense medium immediately after drawing, the loss of recovery and purity of the PBMCs that is observed with density barrier techniques is much less marked. This is probably related to the fact that once the density of the plasma has been raised, the PBMCs do not settle out upon standing [47].

Using a barrier flotation technique

In a third alternative (a variation of the mixer technique), the blood is adjusted to a density considerably higher than that of the PBMCs (p = 1.090 g/ml) and layered beneath a p = 1.078 g/ml density barrier. As with the mixer, the PBMCs float to the surface, but this is the only system in which the cells do not band adjacent to the plasma-containing sample layer. In both of the other techniques the PBMCs are heavily contaminated by platelets (thrombocytes). In this method the great majority of platelets remain in the plasma layer [48]. The low-density barrier thus acts as a 'buffer-zone' between the PBMCs and all the other blood components - erythrocytes, PMNs, platelets and plasma proteins.

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