Further Applications of Ficoll-Paque PLUS and Ficoll-Paque PREMIUM

Extracted from Isolation of Mononuclear Cells, Methodology and applications (PDF)
GE Healthcare, 2010

A great many modifications and extensions of the method have come into use following the introduction of the technique described by Bøyum in 1968 and its subsequent widespread adoption. For example, monocytes (which are recovered in the mononuclear cell fraction, using the standard procedure described in this booklet) can be removed, if desired, by incubating the blood sample with iron (or iron carbonyl) before separation on Ficoll-Paque PLUS. The monocytes phagocytose the iron particles and become denser, with the result that they sediment through the Ficoll-Paque PLUS layer on centrifugation and collect in the red blood cell pellet at the bottom of the tube3.

An important and widely used extension of the original technique is its application, in combination with selective rosetting (clustering), to the isolation of lymphocyte subclasses. Selective rosetting is presented in RosetteSep™ products marketed by StemCell Technologies Inc 19, 20, 21, 59. These products contain tetrameric antibody complexes (TAC) that crosslink unwanted cells to multiple red blood cells present in the sample, forming immunorosettes. Upon centrifugation over Ficoll-Paque product, the immunorosettes sediment to the bottom of the tube together with the red blood cells. Alternatively, the purified lymphocytes obtained by the standard procedure (with or without monocyte removal) can be incubated with an excess of sheep red blood cells (ratio of red blood cells to lymphocytes at least 50:1), whereupon the T lymphocytes spontaneously form rosettes with the sheep red blood cells. On centrifugation for a second time over Ficoll-Paque PLUS, the T lymphocyte rosettes sediment to the bottom of the tube together with the excess red blood cells, leaving the other (non-rosetting) lymphocytes at the interface3.

Such techniques for the separation of lymphocyte subclasses, as well as the standard method for isolating the entire lymphocyte population, have been widely applied to studies of lymphocyte functions and surface markers in disease states as compared to normal controls. Caution is, however, necessary in applying the Ficoll-Paque PLUS technique to pathological blood specimens, since it has been found that the resulting mononuclear cell layer may be contaminated with immature granulocytes in patients with certain infections 22, and particularly cancer 9, 23. In the latter case, elevated numbers of monocytes may also be present 24.

Ficoll-Paque PLUS has been used with success to separate cells from a variety of sources other than peripheral blood, even though its properties have been optimized specifically for blood mononuclear cell isolation. Thus, separation over Ficoll-Paque PLUS facilitated detection and identification of malignant cells in abdominal and pleural fluids 23,25. Separation on Ficoll-Paque PLUS has also been reported to assist in establishing cultures of amniotic fluid cells and to facilitate their subsequent cytogenic analysis 26. Ficoll-Paque PLUS and Ficoll-Paque PREMIUM have also been used for the isolation of cord blood derived hematopoietic stem cells, cord blood derived pluripotent stem cells for regenerative medicine clinical applications, and bone marrow mesenchymal stem cells 27, 28, 60, 61. Ficoll-Paque products are also suitable for granulocyte isolation 29, 30, 31.

Ficoll-Paque PLUS can also be used to isolate mononuclear cells from species other than man. In some cases (e.g. cow, goat, and rabbit) it may be necessary to alter the standard procedure to achieve good results 3 and it should be remembered that the density of Ficoll-Paque PLUS (1.077 g/mL), although optimized for the isolation of human mononuclear cells, may not give optimal yield and purity of mononuclear cells from other species. However, isolation methods using Ficoll-Paque PLUS for preparation of mononuclear cells from peripheral blood (PB) and bone marrow (BM) have been described for mouse ((32(PB), 33 (BM)), dog (34 (PB), 35(BM)), monkey (36 [PB], 37 [BM]), cow (38 [PB], 39 [PB]), rabbit (36 [BM], 40 [PB]), horse (41 [PB]), pig (42 [PB]), and even fish (43). Where it is desired to work with solutions of densities other than 1.077 g/mL it may be convenient to use Ficoll-Paque PREMIUM 1.084, Ficoll-Paque PREMIUM 1.073, or the alternative centrifugation media Percoll/Percoll PLUS since iso-osmotic solutions of different densities are very easily prepared with these media, facilitating the optimization of a particular separation. In research studies, separation with Percoll has also been reported to give improved lymphocyte yields and purities in some cases 44, 45, 46, 47.


Applications using Ficoll-Paque PREMIUM 1.084

Ficoll-Paque PREMIUM 1.084 can be used for isolating higher density cells. It has for instance been shown that the lymphocytes in rodents have a slightly higher average density than lymphocytes in humans 50, 51 and a fraction of these will therefore be lost into the pellet after a standard 1.077 g/mL density gradient centrifugation, contaminating the granulocyte layer and decreasing the mononuclear cell recovery. Dog, bovine, rabbit, mouse, and rat blood cells have successfully been separated using a 1.084 g/mL density gradient media such as Percoll or Ficoll-diatrizoate solutions 52, 53.

It has been reported that selective loss of lymphocytes that form rosettes with autologous blood cells (as described above) may occur by the standard procedure using a 1.077 g/mL Ficoll-Paque products 14, 15 and these lymphocytes have been shown to be recovered almost quantitatively by resuspending the red cell pellet in medium and recentrifuging over a gradient of slightly higher density than normal (i.e., 1.083 g/mL) 15. Thus, Ficoll-Paque PREMIUM 1.084 could potentially be used for this application, although this remains to be shown.


Applications using Ficoll-Paque PREMIUM 1.073

Ficoll-Paque PREMIUM 1.073 can be used for enriching lower density cells such as monocytes from peripheral blood, or mesenchymal stem cells from bone marrow or placenta 54, 55, 56, 57.

Grisendi et al. directly compared Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073 for isolating and expanding mesenchymal stem cells from human bone marrow aspirate and convincingly demonstrated that density gradient separation using the lower density Ficoll-Paque PREMIUM 1.073 was associated with an enrichment of mesenchymal stem cell (MSC) subtypes characterized by a higher proliferation potential. Thus, Ficoll-Paque PREMIUM 1.073 can ultimately benefit clinical applications based on these cells. Brooke et al. recently described a successful manufacturing process for isolating and expanding placenta-derived human MSC using Ficoll-Paque PREMIUM 1.073 for a clinical trial 57.

 Materials

     

 References

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