Immobilization of cells-For JNTUA B Pharmacy students

1. Introduction to
Immobilization of cells
Dr SV Suresh Kumar
Professor of Pharmacognosy
CES College of Pharmacy, Kurnool
Andhra Pradesh

2. Immobilization of cells
 Immobilization of cells means the encapsulation of cells in
culture by polymers like, sodium alginate, calcium
alginate, collagen, poly styrene, agar or cellulose
derivatives, so that cells are not able to divide but remain
viable for so many weeks.
 One of the major problems in cell culture based process
for secondary metabolite production is high production
cost due to slow growth of cells, low product yield, genetic
instability of the selected cells and intra cellular
accumulation of the product. Some of these problems can
be reduced by immobilized cell culture.
 In this technique cells are confined within a reactor
system, preventing their entry into the mobile phase
which carries the substrate and products/nutrients.

3.  Immobilization is only relevant where the production
involves two stages; in the first stage conditions are
optimized for biomass production by suspension culture
and in the second stage conditions are optimized for
product formation by immobilized cells.

4. The advantages of Immobilized cell culture are-
 It may enable prolonged use of biomass;
 By immobilization of cells the cell density in a bioreactor
can be increased 2-4 times that in suspension cultures (10-
30 g/l) and this enables the use of small reactors, reducing
the cost of medium, equipment installation and
downstream processing;
 The entrapped cells are protected against shear forces
and, consequently, a simple bioreactor design may be
used;
 It separates the cells from the medium and, therefore, if
the product is extracellular it can simplify downstream
processing;
 It uncouples growth and product formation which allows
product optimization without affecting growth;

5.  The non-dividing immobilized cells are less prone to
genetic changes and, therefore, provide a stable
production rate;
 It minimizes fluid viscosity, which in cell suspensions cause
mixing and aeration problems; and
 It promotes secondary metabolite secretion in some
cases.
 An immobilized system which could maintain viable cells
over an extended.
 period of time and release the bulk of the product into the
extracellular medium in a stable form could dramatically
reduce the cost of phytochemical production.

6. A wide range of bioreactors have been designed to culture
immobilized cells.The best design to use depends on the
method of immobilization.
 Entrapment of cells in gel or behind semi-permeable
membranes is the most popular method for
immobilization of plant cells.
 Some polymers used to entrap plant cells are alginate,
agar, agarose and carrageenan. Of these, alginate has
been most widely used because it can be polymerized at
room temperature using Ca 2+.
 Polyurethane foam has also been used to immobilize a
range of plant cells. Alternatively, plant cells can be
entrapped by inclusion within membrane reactors.

7.  A semi-permeable membrane is introduced between the
cells and the recirculating medium so that the cells can be
packed at a very high density under very mild conditions.
Some designs of membrane reactors are shown in Figure.
Fig: Flat plate membrane reactor with one side flow of nutrients

8. Fig B: Flat plate membrane reactor with two
side flow of nutrients

9. Fig C:Multi membrane reactor system.Fig C:Multi membrane reactor system.
Fig C:Multi membrane reactor system.

10. Immobilization of cells on the surface of an inert
support, such as fibreglass mats and unwoven short
fibre polyester, has also been examined for in vitro
production of secondary metabolites.
 For surface immobilization of cells, a bioreactor (air
lift or mechanically agitated design), provided with
the support matrix, is inoculated with a plant cell
suspension of suitable density and operated for an
initial period as a suspension bioreactor. During this
period virtually all cells spontaneously adhere to the
surface of the support.

12. Stirred tank rector and air lift reactor

13.  Binding of the cells to the immobilizing support is
regarded as a two-step process. In the first stage, cells are
spontaneously attracted to the support surface due to
vanderWaal's force aided by entrapment of cells in pores
or other irregularities on the surface of the support.
Subsequently, the cells appear to secrete a mucilaginous
substance which firmly cements them to the support
surface.
 The immobilized cells grow as a more or less continuous
layer or tissue-like structure on the surface of the support
matrix.
 Surface immobilization promotes the natural tendency of
plant cells to aggregate which may improve the synthesis
and accumulation of secondary metabolites.

14.  special advantage of this method over the other methods
of immobilization of cells is the absence of any physical
restriction to mass transfer between the culture medium
and the biomass surface.
 Since the surface immobilized cells grow on the surface of
the support matrix, it should facilitate visual monitoring of
the conditions, distribution and extent of the biomass and
to routinely sample the biomass, if desired.

15. Fig: Hollow fibre reactor for immobilized cell culture

16.  Surface immobilization promotes the natural tendency of
plant cells to aggregate which may improve the synthesis
and accumulation of secondary metabolites.
 A special advantage of this method over the other
methods of immobilization of cells is the absence of any
physical restriction to mass transfer between the culture
medium and the biomass surface.
 Since the surface immobilized cells grow on the surface of
the support matrix, it should facilitate visual monitoring of
the conditions, distribution and extent of the biomass and
to routinely sample the biomass, if desired.

17. Applications
 The accumulation of serpentine by C. Roseus and
anthraquinones by Morinda citrifolia were enhanced in the
immobilized state when compared with cell suspensions.
 Capsicum frutescens cells immobilized on polyurethane foam
produced 50 times more capsaicin than suspension cultures.
 Immobilization of the cells of Dioscorea deltoidea by passively
entrapping them into polyurethane foam cubes and growing
them in a medium containing 3% sucrose.This increased
diosgenin production by 40% over the suspension cultures.
 Immobilized cells can also serve as biocatalysts for
biotransformation.
 The immobilized cells of Digitalis lanata maintained their
capability for enzymatic conversion of β-methyldigitoxin to β -
methyldigoxin for a period of 61 days.

18. Some of the limitations of an immobilized cell system
are:
 Immobilization is normally limited to systems where
production is decoupled from cell growth;
 The initial biomass must be produced in suspension
cultures;
 Secretion of products into the external medium is
imperative;
 When secretion occurs there may be a problem of
extracellular degradation of product; and
 When gel entrapment is used, the gel matrix introduces an
additional diffusion barrier.