Centrifugal elutriation cells

Isolation of alveolar type II cells by centrifugal elutriation. In Vitro 15(9) 673-684... 

DNA flow cytometry is a very useful tool that permits rapid, objective assessment of a large number of cells but may not be readily available. Comet assay, when combined with centrifugal elutriation, can provide a useful in vitro model to study differences in metabolism and the susceptibility of different te,sticular cell types to DNA-damaging compounds. Thus, new findings using these systems should lead to greater knowledge about why a chemical or class of chemicals can cause testicular toxicity. 

Lutz, M.P. Geadick, G. Hartmann, W. Large-scale cell separation by centrifugal elutriation. Anal. Biochem. 1992, 200, 376. 

Chicken testis cells at different stages of spermatogenesis, separated by centrifugal elutriation as described in (7), and spermatozoa obtained from the vas deferens, were treated with bleomycin for 1 hr and the poly(ADP-ribose) polymerase activity was assayed in nuclei isolated from the different cell t)rpes. Incubation with bleomycin resulted in stimulation of the enzymatic activity in premeiotic and meiotic cells, round spermatids and in elongated spermatids and was ineffective in spermatozoa (Fig. 1). 

Fig. 1. (left) ADP-ribose incorporation by nuclei after treatment of chicken testis cells with bleomycin. Intact chicken testis cells, separated by centrifugal elutriation, were incubated for 1 hr in a medium with (hatched bars) or without bleomycin (dotted bars). Nuclei were isolated from treated and untreated cells and incubated at 37°C for 2 min in a medium containing [ -NAD. The incubation was terminated by adding 20% trichloroacetic acid and the precipitates were collected on Whatman GF/A filters. I n, premeiotic and meiotic cells HI, round spermatids IV, elongated spermatids V, spermatozoa from the vas deferens. 

Several methods have been employed to isolate and purify type II cells, including enzymatic digestion of pulmonary tissue to free lung cells from the pulmonary epithehum and purification of type II pneumocytes by discontinuous density gradients (Kikkawa and Yoneda 1974), primary culture (Dobbs and Mason 1979), centrifugal elutriation (Jones et al. 1982), or unit gravity sedimentation (Brown et al. 1984). 

Figure 6.3.1. Separation systems where the bulk flow is parallel to the direction offorce (a) elutriation (particle separation in liquids) (b) capillary electrophoresis (c) centrifugal elutriation (d) inertial impaction in particle filtration (e) distillation (flash/batch) (f) liquid extraction (separating funnel) (g) zone melting (h) normal freezing (i) cake filtration (j) ultri tration separation of proteins (dead-end) (k) batch cell reverse osmosis separation of brine (1) gas permeation.

F are 6.3.7. (a) Schematic of a truncated cone as an elutriation chamber, with bulk flow toward the axis of rotation of the centrifuge, (b) Mixture of cells separated according to their size in centrifugal elutriation. (After Figdor et aL (1998).)... 

Beckman Elutriation Method. The Beckman elutriation method uses a chamber designed so that the centrifugal effect of the radial inward fluid flow is constant (Fig. 3). The separation chambers are made of transparent epoxy resin which faciUtates observation of the movements of the cell boundary in strobe light illumination. This enables detection of the radius at which the cells are separating. When a mixture of cells, eg, mononuclear white cells, enters the chamber, separation can be achieved by fine tuning centrifuge speed and inward fluid flow to the specific cell group. This is a laboratory method suitable for relatively small numbers of cells. Chambers are available in sizes to handle 2-3 x 10 , 1 2 x 10 , and 1 x 10 ° cells. The Beckman chambers can be appHed to collect mononuclear cells from bone marrow aspirates. 

A continuous centrifugal bioreactor, in which cells are fluidized in balance with centrifugal forces, has been designed to allow high density cell cultivation and superior aeration without elutriation of the suspended cells (van Wie et al., 1991). Reactor performance was hampered by elutriation of biomass by evolved gas in an anaerobic fermentation, indicating that it may not be suitable in its present state for three-phase fermentations. Immobilization of the cells on denser particles may overcome this problem. 

Isolation of Cells for Transport Studies Use of nonequilibrium thermodynamics in the analysis of transport general flow-force relationships and the linear domain, 171, 397 cell isolation techniques use of enzymes and chelators, 171, 444 cell separation by gradient centrifugation methods, 171, 462 cell separation by elutriation major and minor cell types from complex tissues,... 

Several useful CCC systems have been developed from these centrifuge schemes. The nonplanetary scheme has been used for toroidal coil CCC [3,4], centrifugal precipitation chromatography [5], and on-line apheresis in the blood bank [6,7]. The nonsynchronous scheme has been applied to partition of cells with polymer phase systems and also to cell elutriation with physiological solutions [8,9]. The type J synchronous scheme is further developed into a highly efficient CCC system called high-speed CCC (HSCCC) [10]. 

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