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Affinity Bio separation Column. Wuraola Akande. School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK. Cryogel preparation. Cryogel was produced by free radical polymerisation at sub zero temperature from HEMA (hydroxyethyl methacrylate) and

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affinity bio separation column

Affinity Bio separation Column

Wuraola Akande

School of Pharmacy and Biomolecular Sciences,

University of Brighton, Brighton, UK

cryogel preparation
Cryogel preparation

Cryogel was produced by free radical polymerisation at sub zero temperature from

HEMA (hydroxyethyl methacrylate) and

AGE (allyl glycidyl ether) monomers

MBAA as a cross linker

At the presence of APS/TEMED

as initiator

Cryogel in 5cm x 1cm glass tube

Ice crystals & initial forming polymers

Monomers & initators

Macroporous gel

characteristics of cryogel
Characteristics of cryogel

Cryogel , possess the following characteristics.

  • High mechanical and chemical stability
  • Low flow resistance
  • Good pore interconnection
  • Ease of functionalisation and attachment of biological ligands
  • Large surface area
objectives were achieved by
Objectives were achieved by
  • Producing Macroporous Monolithic polymer cryogels with a range of interconnected pore sizes
  • Investigating the mechanical stability
  • Investigating haemocompatibility
  • Functionalising the surface of the matrix
slide6

Creep test of 8% PHEMA cryogel, at approximately 8 minutes the cryogel had 100% strain recovery , while with 10% and 16% PHEMA cryogel there was 98% and 96% strain recovery

slide7

8% PHEMA had approximately 97% recovery after applying a constant load of 0.2N for one minute, cryogel was compressed to approximately 60% and left to recovery for two minutes in a repeated cycle five times . The difference in recovery in the first cycle compared to the fifth cycle is less than 1%.

slide8

After approximately 6ml of elution from the column 100% of the red blood cells were eluted .

Heamolysis was calculated according to Blankey and Dinewoodie and no haemolysis was observed

slide9

There is no significant difference between the platelet loss in tube with adsorbent and without adsorbent, but there is a significant platelet protection in EDTA to control.( n= 3) *P> 0.05 as analysed by one way ANOVA

slide10

There is no significant difference between the platelet loss in tube with adsorbent and without adsorbent, but there is a significant platelet protection with EDTA compared to control.( n= 3) *P> 0.05 as analysed by one way ANOVA

slide11

There is no significant difference between the platelet activation in tube with adsorbent and without adsorbent in sigmacote tube, but there is a difference in platelet activation in plain tube with and without adsorbent. EDTA protected activation of PAC-1. ( n= 3) *P> 0.05 as analysed by one way ANOVA

slide12

There is no significant difference between the platelet activation in tube with adsorbent and without adsorbent in both plain tube and sigmacote tube, But there is a significant difference compared to control

( n= 3) *P> 0.05 as analysed by one way ANOVA

summary of results
Summary of results
  • The PHEMA monolithic cryogel column consists from ~13% of polymer phase and ~87% of the interconnected pores within the range of 10-120 µm
  • Column can withstand a maximum pressure of 180mmHg without deviation in linearity
  • Compression release cyclic test showed approximately 97% strain recovery after been compressed to about 60% and thus cryogel mechanically stable
  • ~100% of Red Blood Cells passed through the column
  • No evidence of haemolysis was found in blood eluted
slide14

6. Results from Sysmex cell counter correlates with data obtained from the FACS analysis of free platelets

7. EDTA protect the loss of platelet in all surface

8. PHEMA cryogel is a material of low thrombogenicity.

PHEMA cryogel can be an appropriate matrix for further functionalisation and use as affinity bio separation column.

future work
Future work

1.Functionalisation of cryogel matrix surface

2. In vitro biocompatibility of cryogel matrix in an extracorporeal circulation

slide16

Acknowledgements

The team at Krems Donau University

  • Prof .Dr. Dieter Falkenhagen
  • Dr Viktoria Weber
  • Ute Fichtinger
  • Ingrid Linsberger
  • Anita Schildberger
  • Stephan Harm

The team at Polymerics

  • Dr Aneila Leistner
  • Andre Leistner
  • Anke Lehmann
  • Marina Stier
acknowledgements

This work was funded by the FP7 MONACO EXTRA project and University of Brighton

Acknowledgements
  • Dr Lyuba Mikhalovska
  • Dr Irina Savina
  • Dr Stuart James
  • Prof Sergey Mikhalovsky
  • Dr Lyuba Mikhalovska
  • Dr Irina Savina
  • Dr Stuart James
  • Prof Sergey Mikhalovsky
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