Purification of cells Preparation of gradient solutions from general purpose media.

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