Mammalian cells

bioreactor system

Introduction
In cell culture, the cell are selected and
maintained as independent unit.
The range of bioreactor available in the
market 1-10000 L.
- stirred tank (simplest & widely used)
- airlift bioreactors

Cont..,
Choice of bioreactor for particular cell
determined by:
- cell type (*animal cell more
fragile )
- the nature of the product
- the scale of the operation
- availability of space and services
- and the capital and operational
cost

Cont..,
A continuous mode is commonly applied for
mammalian cells (but, tends to application).

Batch
accumulation of inhibitor
depletion of an essential nutrient
Ideally nothing can be added or removed but
in practice addition of O2, acids bases are
made.

 In general, cell culture bioreactor can be

categorized into 2 types:
i- Cultivation of anchoragedependent
cells (primary culture from normal tissue)
ii- Cultivation of suspended mammalian
cells (cell line from cancerous cell,
hybridoma)
Ideally any cell culture, bioreactor able to
maintain in a sterile culture, which
maximum cell growth for production.

Limitation
Contamination in all stage of operation.
- early stage, inoculums preparation
Number of complex interaction (physical,
chemical and biochemical )
- Include mixing and aeration
- oxygen demand
-cell growth, waste and toxic produce
etc.
Animal cell more fragile and grow more
slowly than most bacteria.

Basic bioreactor configuration

An inoculums is seeded in specific volume
Equipments :
-pH probe
-Oxygen probe
-temperature probe
-hydrophobic air filter
-medium inlet
-air outlet
-sampling port
-..,

Principle features for hybridoma

cultivation
Mixing
- Small scale magnetic stirred commonly used
- For vessel up to 500L, low shear flat bladed
(marine propeller) used.
- For large vessels, high shear turbine type are used.
-typically, maximum stirring rate 100-150rpm for
cell in suspension.
Sterilization-1-10L vessels can be autoclave for 2030min at 121oc and 15psi.

 Temperature controlled by
thermocirculator or water
jacket (37OC)
pH optimal pH for animal cell
around 7.4, enriched CO2
decrease fluctuation.

 Oxygenation
- Supply O2 to satisfy cell
metabolism is one major problem
in scale-up.
- O2 consumption rate of mammalian
cells varies from 0.06-0.6mmol/L/h
for culture at 106cell/ml.
- OTR must above OUR or minimum
1:1

AERATION & AGITATION

IN ANIMAL CELL
CULTURES

SHEAR
Shear stress : force per unit area
acting on a body
Shear rate : a measure of how the
velocity change as we move away
from that body
The smaller the eddy and the greater
the velocity fluctuation then the
greater will be the level of shear

Are animal cells shear

sensitive?

YES & NO

NO
Unlike bacteria, animal cells do
not possess a cell wall which
should protect them from shear
forces.

YES
Their small size will protect
them from shear forces that
arise in the bioreactor liquid

Design considerations for

mammalian cell cultures
bioreactor :

The use of low shear axial flow

impellers
The use of shear protectorants
Ensuring that the medium
contains sufficient nutrients
Selecting cells that are not
shear sensitive
Minimize impeller speeds

Shear forces
Cells growing in stirrer tank
bioreactor subjected to
changing shear forces.
These changing have negative
effect on cell growth.
Cell produce higher biomass
yields in the more uniform shear
environment (airlift bioreactor)

How do bubbles damage

animal cells?

In two main ways

First:
When bubbles collapse at the
surface of a reactor, cells trapped in
the wake of the bubbles, relatively
high stress forces. The shear forces
originate from the velocity gradient.
Solution : add surface active agent
which stabilize the bubbles.

Second:
Cells can also be damaged when
trapped in foams. As the foams
move, they will tend to drag the
membranes of trapped cells with
them. When bubbles surrounding a
cell move in different directions, the
cell will be torn or sheared apart.
Solution : increasing the diameter of
the disengagement zone

Stirrers
Axial flow impellers, particular
spiral shaped ribbon turbines are
often used to agitate animal cell
cultures.
Produce less shear forces than
radial flow impellers and thus cells
are less susceptible to damage from
high or non-uniform shear forces.

Cont.
As animal cells grow slower
than bacterial cells, high
oxygen transfer rates facilitated
by direct sparging and high
shear radial flow impellers is
not necessary.

Protectorant
It protect cells from shear
damage and from bubble
damage.
It works by acting on the
surface properties of the cell
culture medium and possibly
the cell surface.

Cont.
It is believed to:
Make the bubbles slippery by providing a
highly mobile bubble boundary layer such
that the cells do not attach the bubbles.
Strengthen the cells membrane.
Stabilize foam. This allows the cells to
detach from the bubbles before they burst
and thus protecting them from the forces
released when the bubbles collapse.
Example: pluronic F-68 and serum.

Most important
bioreactor for animal
cell culture

Airlift Fermenter
Tall column with
an inner draught
tube (for fluid
circulation)
Long column for
minimize bubble
or foam damage

Ceramic Bioreactor
Consist of
multiple channels
which run through
the length of a
ceramic cylinder.
Channel is a
square and inner
surface area for
cell attachment.

A porous ceramic
for non-adherent
cell lines.
A non-porous for
anchorage cells.
The mode of
operation by
constant recirculation of
medium.

Hollow Fiber Bioreactor

Consist of
bundles of
synthetic,
semipermeable
hollow fibers
which allow
liquid to flow
through the
fibres.

Major limitation
is the pressure
difference along
the length of
fibre.
Suitable for both
anchoragedependent & in
dependent cells.

Hollow Fibre Bioreactor

Scaling-up microcarrier cultures of

mammalian cells for production
purposes
BY
D.BILIG
PHARMACIA AB,UPPSALA, SWEDEN

Introduction
Culturing of animal cells on
microcarriers provides
homogenous enviroment
This ensure an efficient
utilisation of costly culture
media component.

Cont.
Important to realise the full
potential this technology, especially
with respect to scaling-up culture
volumes
Some anchorage-dependant cells
are capable of bead to bead
migration' allowing culture volume
to be scale-up without requiring
enzymic harvesting of the cells

Cont.
This paper investigates
additional aspects involved in
optimising the subcultivation
steps for scaling-up
microcarrier cultures.

Materials and Methods

This work performed with Vero and
MRC-5 cells were cultured in DMEM
contained Cytodex 1 microcarriers
at a conc.2 g/l
Confluent microcarriers were
allowed to settle to the bottom of
the culture vessel, remove the
medium supernatant & wash the
microcarriers with PBS

Cont.
The confluent microcarriers were
maintained in gentle suspension
in the trypsin solution for approx
15min. The resultant mixture of
harvested cells and microcarriers
was transformed directly to the
subsequent, larger culture vessel
for scaling up.

Observations
100% of either Vero or MRC-5 cells
detached from Cytodex 1 within a
15min exposure to trypsin
The viability of the harvested cell
was greater than 95%
No loss of cells was incurred
during the transfer step to the
subsequent culture

Cont.
80-90% of the cell inocula
reattached to the fresh
microcarriers in the subsequent
culture.
Cultures could be successfully
initiated with as few as two
viable cells/bead

Cont.
Cultures of Vero cells could be
subcultivated with split ratios of
1:100; MRC-5 cells were split at
1:10
Residual trypsin and old
microcarriers did not effect the
initiation of the subsequent
culture

Conclusions
An optimum subcultivation
process requires maximised :
Cell recovery after trypsinisation
Retention of cell viability
Cell transfer of the inoculum
Plating efficiency of the
harvested cells

Cont.
Attachment and subsequent
growth of the inoculum.

Thank You!!!!!