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 anchorage–dependent 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 20-

30min 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 re-circulation 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 anchorage-dependent & in dependent cells.

Hollow Fibre Bioreactor

BYD.BILIG

PHARMACIA AB,UPPSALA, SWEDEN

Scaling-up microcarrier cultures of mammalian cells for production

purposes

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 subcultivationprocess 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!!!!!