Purification of cellsPurification of cells

Preparation of gradient Preparation of gradient solutions from general solutions from general

purpose mediapurpose media

• Diatrizoate

• Nycodenz®

• Iodixanol

Axis-Shield Iodinated Density Axis-Shield Iodinated Density Gradient Media (General Purpose) Gradient Media (General Purpose)

II

I

CH3COHN NHCOCH3

COOHDiatrizoic acid (MWt 614)Hypaque

Axis-Shield Density Gradient Media I

Nycodenz (MWt 821)CONHCH2 CHCH2 OH

OH

NCH2 CHCH2 OH

OHCO

CH3

CHCH2 NHCO

CH2 OH

OH

II

I

Axis-Shield Density Gradient Media II

CONHCH2 CHCH2 OH

OH

CONHCH2 CHCH2 OH

OH

NCH2 CHCH2 N

OHCO CO

CH3 CH3

CHCH2 NHCO CONHCH2 CH

CH2 OH CH2 OH

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)

0 10 20 30 40 50 601

1.1

1.2

1.3

SucrosePolysucrose

Nycodenz®Iodixanol

Density versus concentration

Use of density barriers I

1.06 g/ml

1.05 g/ml 1.07 g/ml 1.10 g/ml

1.06 g/ml

1.05 g/ml 1.07 g/ml 1.10 g/ml

Use of density barriers II (mixer)

1.05 g/ml 1.07 g/ml 1.10 g/ml

1.07 g/ml

1.06 g/ml

Barrier flotation strategy III

Axis-Shield Density Gradient Axis-Shield Density Gradient Media for CellsMedia 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)

0 10 20 30 40 50 601

1.1

1.2

1.3

SucrosePolysucrose

Nycodenz®Iodixanol

Density versus concentration

SucrosePolysucrosePercoll®

Nycodenz®

Iodixanol

Concentration (% w/v)

Osmolality (mOsm)

0 10 20 30 40 50 600

200

400

600

800

1,000

Osmolality versus concentration

Isoosmotic solutions: cells (C01)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, 795-797

• 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, 225-245

• 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, 667-675

• 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, 183-190

• Poor viability of plasmodium: Carter, EH et al (1987) Parasitology, 95, 25-30

• Poor yield and purity of intracellular bacteria: Henriquez, V. et al (2003) Appl. Environ. Microbiol., 69, 6268-6271

• Phagocytosed by sea urchin cells: Gross, PS et al (2000) Immunogenetics, 51, 1034-1044