1. EVALUATION OF FINE SAND FILTRATION FOR
LARGE SCALE ALGINATE PURIFICATION FOR CLINICAL USE
T. GREGO,1 A. GANDER,2 DR C. SELDEN,2 PROF H. J. F. HODGSON,2 DR L. C. CAMPOS1
1 Civil, Environmental & Geomatic Engineering Department, University College London
2 Centre for Hepatology, Royal Free Campus, Royal Free & University College Medical School
INTRODUCTION
Alginate is a natural biopolymer extracted from brown seaweed, in this project, alginate was
used to microencapsulate HepG2 cells (a human hepatocyte immortal cell line) for use in a
Bioartificial Liver device (Figure 1.).
Alginate may contain “unknown” (e.g. micron particulates) and “known” (e.g. heavy metals,
endotoxins, proteins, pyrogens, and polyphones') contaminants [1-2]. Therefore,
biocompatibility of alginates for immobilising hepatocytes is crucial. Other purification methods
achieved a suitable biocompatibility of the polymer by removing the known impurities, but were
not appropriate for our application.
Figure 1. The three stages of the BAL system [3]
Stage 1 HepG2 cells are encapsulated in alginate beads achieving a 3D state, which provides better cell growth and function [4].
Stage 2 Encapsulated cells are proliferated in a Fluidised Bed Bioreactor (FBB) over 8-10 days;
Stage 3 patient plasma is passed over beads by fluidisation.
“(i.) FDA stained HepG2 cells after encapsulation. (ii.) FDA stained HepG2 cells after 8 days of proliferation. (iii.) A phase contrast alginate beads 8 days after initial
immobilisation. (iv.) Fluidised Bed Bioreactor (FBB) competence culture setup. (v.) FBB containing alginate” [3].
HYPOTHESIS AND AIMS
Hypothesis: For clinical application and regulatory authority approval, particulates
must be removed to prevent transit to the patient. Therefore, we seek to purify raw
sodium alginate solution removing micron particulate impurities by fine sand filtration
without altering the viscosity of the resulting solution.
The specific aims of the research project were:
•To remove micro particulates between 1μm and 10μm in size.
•To evaluate the effect of fine sand filtration on the sodium alginate properties.
•To determine and measure any changes in the physical properties of the non-Newtonian fluid
by rheology.
•Encapsulate empty alginate beads to assess morphology of beads after filtration.
•To immobilise encapsulated HepG2 cells in filtered alginate solution comparing cell growth,
viability and function.
METHODOLOGY
RESULTS
Figure 6. Proliferation of encapsulated HepG2
cells in 1.875% alginate beads over 14 days
20
15
10
5
CONCLUSION
(B)
(C)
10
8
6
4
2
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.35
0.30
0.25
0.20
0.15
0.10
0.05
• Filtering sodium alginate solution through a dual media filter could eliminate micron
particulates between 10 to 20μm, and reduced by 94% 2μm particulates compared to non
filtered Na-alginate solution.
• The filtration process had a major impact on the dynamic viscosity of the solution, even
though the micron particulates were removed to some extent. As a consequence of this, the
stability and the morphology of the beads were adversely affected.
• Immobilisation of HepG2 cells in fine sand filtered, aqueous sodium alginate did not provide
a coherent gel matrix to support cell growth.
RECOMMENDATIONS
The experimental works were evaluated not only by engineering and medical science aspects
but also the efficiency of the currently publish purification processes were taken into
consideration [2]. Therefore, a key question was addressed:
“Is the liquid-solid based purification method the most appropriate for the purification of “micron
particulates free” alginate biopolymer suitable for cell encapsulation?”
The research outcomes highlighted the need for an alternative approach for alginate
purification. One possibility would be utilising dry alginate powder in a cyclonic separation from
gas stream and possible production of “micron particulate free” alginate for cell encapsulation
[5].
REFERENCES
Figure 2. Life cycle of the experimental work
Phase I. Fine sand filtration (single and dual media filter): to remove macro particulates
from raw Na-alginate.
Phase II. Lyophilisation 0.2% filtered alginate solution was shell frozen in a dry ice
acetone bath, to be lyophilised to sublimate water, leaving dry alginic acid salt.
Phase III. Reconstitution of alginate to final concentration of 1.0%, 1.25%, 1.5%, 1.75%,
1.875% and 2%, and autoclaving for 10 mins at 121°C.
Phase III-B. Physical analysis: to determine the particle size distribution and to measure
the viscosity of the filtered 2% NaAlg solutions.
Phase IV. Biological analysis: HepG2 cell encapsulation in 1.875% alginate beads using
Inotech® encapsulator.
Figure 3. Particle Size and Number distributions of
alginate powder with Different Filtration Methods
Figure 4. Effects of fine sand filtration on the viscosity of
0.2% Na-alginate solutions
Figure 5. Effects of fine sand and dual (fine and coarse sands)
media filtration on the viscosity of 0.2% Na-alginate solutions
(A)
(A) Encapsulated HepG2 cells in fine sand (RH110) filtered
1.875% sodium alginate solution, phase contrast on day 9;
(B) Viability of cells on day 9, estimated by vital dye staining
(C) Dead cells as estimated by propidium iodide
(cell permeability).
Green shows live cells, and red represent dead cells.
0.000
0.00 0.09 0.17 0.25 0.34 0.44 0.51 0.70 0.68
Shear stress (Pa)
Dinamic viscosity (Pa s)
0.2% Control NaAlg solution (non filtered,
freeze-dried, and autoclaved)
0.2% NaAlg filtered (RH-70) solution,
freeze-dried and autoclaved
0.2% NaAlg filtered (RH-110) solution,
freeze-dried and autoclaved
0.00
1 29 59 89 118 148 177 207 237
Shear rate (1/s)
Dinamic viscosity (Pa s)
0.2% Unfiltered NaAlg (control) solution
0.2% Filtered (fine sand RH-110) NaAlg
solution
0.2% Filtered (fine and coarse sands)
NaAlg solution
1. Dusseault, J., et. al. ‘Evaluation of alginate purification methods: Effect on polyphenols, endotoxin, and protein contamination’ (2005) DOI: 10.1002/jbm.a.30541
2. van de Ven, W. J. C., et. al. ‘Hollow fibre dead-end ultrafiltration: Influence of ionic environment on filtration of alginates. (2008)
Journal of Membrane Science pp 218-229
3. Jones, J. The removal of toxic lactate levels using Immobilised lactate oxidase within a Bioartificial Liver. Research Department of Structural and Molecular Biology,
Division of Bioscience (unpublished work)
0
2 5 10 15
Particle size (μm)
Particle Number in 1g Dry Alginate (x 10 6)
non filtered non autoclaved
non filtered autoclaved
Dual Media Filter-I.
Mono Media Filter-III
RH110/VI Sand
RH70/VII Sand
0
0 2 4 6 8 10 12 14
Days
Cell number (x 10 6)
Dual Sand Media
Filter
Mono Layer Media
Non-Filtered Alginate
4. Damelin, L. H., et. al. (2007) ‘Fat-Loaded HepG2 Spheroids Exhibit Enhanced Protection From Pro-Oxidant and Cytokine Induced Damage’. Journal of Cellular
Biochemistry. 101:723-734.
5. Hoffmann, A. C. and Stein, L. E. (2007) ‘Gas Cyclones and Swirl Tubes: Principles, Design, and Operation’. Springer.