Purification of cells Preparation of gradient solutions from general purpose media

Diatrizoate Nycodenz® Iodixanol Axis-Shield Iodinated Density Gradient Media (General Purpose)

I I I CH 3 COHN NHCOCH 3 COOH Diatrizoic acid (MWt 614) Hypaque  Axis-Shield Density Gradient Media I

Nycodenz (MWt 821) CONHCH 2 CHCH 2 OH OH NCH 2 CHCH 2 OH OHCO CH 3 CHCH 2 NHCO CH 2 OH OH II I Axis-Shield Density Gradient Media II

CONHCH 2 CHCH 2 OH OH CONHCH 2 CHCH 2 OH OH NCH 2 CHCH 2 N OHCO CH 3 CHCH 2 NHCO CONHCH 2 CH CH 2 OH OH I I II I I Iodixanol (MWt 1550) Axis-Shield Density Gradient Media III

Polysucrose MWt approx 450,000 Axis-Shield Density Gradient Media (General Purpose)

Concentration (% w/v) Density (g/ml) Sucrose Polysucrose Nycodenz® Iodixanol Density versus concentration

Use of density barriers I 1.06 g/ml 1.05 g/ml1.07 g/ml1.10 g/ml

1.06 g/ml 1.05 g/ml1.07 g/ml1.10 g/ml Use of density barriers II (mixer)

1.05 g/ml1.07 g/ml1.10 g/ml 1.07 g/ml 1.06 g/ml Barrier flotation strategy III

Axis-Shield Density Gradient Media for Cells Diatrizoic acid powder Nycodenz® powder OptiPrep™: a sterile 60% (w/v) solution of iodixanol in water, density = 1.32 g/ml Principal competition: Percoll®

Concentration (% w/v) Density (g/ml) Sucrose Polysucrose Nycodenz® Iodixanol Density versus concentration

Sucrose Polysucrose Percoll® Nycodenz® Iodixanol Concentration (% w/v) Osmolality (mOsm) ,000 Osmolality versus concentration

Isoosmotic solutions: cells (C01) Nycodenz®: Dissolve 27.6 g powder in 3 mM KCl 5mM Tricine/HEPES buffer (  = 1.15 g/ml); make further dilutions with buffered saline, culture medium etc. Iodixanol: Dilute OptiPrep™ with buffered saline, culture medium etc. Percoll®: first mix 9 vol. with 1 vol. of 10X saline

Synthesised as X-ray imaging agents Diatrizoate, Nycodenz® and iodixanol have been clinically tested as X-ray imaging agents Produced under pharmacological conditions: FDA and EU cGMP compliance Endotoxin: <1 EU/ml (European standard) Endotoxin: measured levels <0.13 EU/ml Axis-Shield Density Gradient Media

What is endotoxin? Endotoxin is a heat-resistant pyrogen (sometimes called lipopolysaccharide), which is released by Gram-negative bacteria.

What are the effects of endotoxin in vivo? A variety of inflammatory responses Hypotension Changes to leukocyte populations Intravascular coagulation Shock Death

What are the effects of endotoxin in vitro? Interacts with CD14 and other receptors Causes cytokine production in monocytes and macrophages Activates complement and coagulation cascades Acts as a B cell mitogen

Percoll® problems I High levels of endotoxin (6-60 EU/ml) Some free polyvinylpyrollidone (PVP) PVP toxic to cells More expensive versions with reduced endotoxin levels or PVP-free

Is Percoll® innocuous to cells? Wakefield, J.S. et al (1982) Biochem. J. 202, Peritoneal macrophages, rat liver Kupffer cells, rat testis Leydig cells ingest large numbers of colloidal silica particles at room temperature Colloidal silica particles adhere to cell surface at all temperatures Affects adherence of macrophages

Percoll® problems: II Toxicity to Type II lung pneumocytes: Viscardi RM et al (1992) Exp. Lung Res., 18, Poor recoveries of gastric parietal cells; stimul- ation of acid and cAMP secretion: Benn, SE et al (1987) J. Physiol. 391, 9P Toxicity to progenitor cells: Mayanagi, T et al (2003) Reproduction, 125, Adherence of silica to erythrocytes causes progressive haemolysis: Kamiyama M. (2002) Personal communication

Percoll® problems: III Poor recovery and function of soil bacteria: Robe, P et al (2003) Eur. J. Soil Biol., 39, Poor viability of plasmodium: Carter, EH et al (1987) Parasitology, 95, Poor yield and purity of intracellular bacteria: Henriquez, V. et al (2003) Appl. Environ. Microbiol., 69, Phagocytosed by sea urchin cells: Gross, PS et al (2000) Immunogenetics, 51,