(This article is the basis of an article published at BioProcess International)
|To develop a system that automates a continuous culture process for anchorage dependent cells, avoiding cellular stress cycles such as discontinuous medium replacement and intermittent cell detachment. The system should provide homeostatic culture conditions throughout the culture process and programming and manual control options to modify culture conditions. Relevant parameters should be recordable.|
To address Challenge #2 we first need to look into the process we want to automate.
Initially a reduced amount of cells suspended in culture medium is added on top of the
attachment surface (whether a continuous flat surface, a roller bottle or particulated
supports). Gentle agitation is used to achieve an even distribution of cells. Normally
attachment surface has been previously equilibrated with culture medium and culture
conditions have been reached before cells are seeded. Then some time is allowed for cells
to settle and attach to the surface. When cells are properly attached, cell growth and
expansion begins. In agitated systems, such as roller bottles or stirred tanks, process
speed is reached at this point. From this point on, process parameters are controlled and
actions, such as medium replacement, increasing gas inflow, pH regulation or thermostating
are triggered as a response to deviation of the process parameters from defined setpoints.
Continuous and automated systems differ from discontinuous and manual systems mainly in
the metabolic stress suffered by cells in discontinuous processes and the contamination
risk introduced by manual operations. All this factors affecting the quality of the
adherent cell culture.
Although much of the process retains similarities with the culture of suspension cells,
adherent cells culture has some particularities arising from the cells depending on
available attachment surface to grow in nice monolayers (a warning note on this: although
some systems provide means to grow 3D cultures from adherent cells, at this moment we are
focusing on 2D cultures) but overall, we can summarize process requirements as providing a
gentle and homeostatic system for the optimum growth of cells attached to the culture
This concise sentence implies that a number of parameters must remain constant around the
cells: temperature, pH, medium composition (including oxygen and other gases and excreted
metabolites), surface geometry and composition, agitation rate and others that might have
an effect on the culture conditions.
Let us now summarize the parameters that require control to achieve a gentle and
homeostatic system in adherent cell culture:
|Culture medium||Gaseous phase||Attachment surface||Other parameters|
From existing culture systems we can learn methods to achieve the stability of these
parameters. For example, we know that providing a constant concentration of CO2 in the
gaseous phase works well to stabilize pH to a certain extent when using CO2-bicarbonate
based buffers. We also know that the change in pH derives from the metabolic activity of
growing cells, i.e., the more active the cell population, the faster the changes in medium
pH. Therefore, controlling the composition of the gaseous phase and/or continuously
replacing the culture medium could be a good strategy for pH control. We could also choose
to control pH through the controlled addition of acid or base. To make things more
interesting, we should bear in mind how some parameters affect others. For example,
temperature has an effect on the solubility of gases, and therefore in oxygen
Having an ample choice of strategies, we want to be sure that the chosen ones are in best
agreement to the solution to the other three challenges faced by The Bolt-on Bioreactor
Culure medium composition
Cells need nutrients to maintain their metabolic activity. Nutrients are provided by the
culture medium which will become depleted of nutrients as cells use them up. As it happens
the consumption rate of each nutrient is different, and these rates change at different
stages in the culture process. Glucose is a major example. On the other hand, cells excrete metabolites that have an effect on the culture. The effect can be negative (for example, changes in pH) or positive, as is the case of growth factors. Dissolved oxygen is a component of major concern since efficient gas transfer
mechanisms have to be ensured to achieve oxygen availability to cells.
A common strategy to deal with culture medium composition in sophisticated bioreactors is
to monitor the concentration of each component considered relevant (glucose, lactate,
ammonia, dissolved oxygen, pH, ...) and then control its concentration to reach desired
levels through the addition of different components. Some systems go as far as to
eliminating deleterious metabolites in a sort of dialysis that results in enormous
More tempting to us is the strategy followed on traditional cell culture on plates, t-
flasks and roller bottles: replacing used medium with fresh medium. In these systems, a
certain amount of medium is added to the device. The volume of medium added depends on the
amount of culture surface since gas transfer and therefore oxygen uptake, is affected by
the depth of the liquid layer atop the cell layer (this is not the case in roller bottles,
where gas transfer occurs when the cell layer is exposed to the gaseous phase). The volume
of medium added is about 0.5 ml per square centimeter and it is replaced every 24-48
hours, replacement rate depending on pH change which in turn depends on the size of cell
population. But... we do not want cells to suffer stress cycles and hence we do not want
to wait until pH has dropped to unbearable levels before replacing culture medium.
Considering a 10,000 cm2 (1 m2) culture device, maximum culture medium replacement rate
when confluence is reached, will round 3.5 ml per minute, easily provided even by very
small peristaltic pumps. Therefore, a control system that regulates culture medium
replacement rate based on the level of a chosen indicator, such as pH, will result in a
robust alternative to control culture medium composition. Additionally, some buffering
system, such as a reservoir of medium within the culture device will help reduce the
sudden composition change as fresh medium is added and used medium withdrawn. From the
solution to Challenge #1, we have chosen to use a rolled membrane as cell attachment
surface. In this geometry, oxygen uptake will occur when cells are exposed to the gaseous
phase, and therefore measuring dissolved oxygen in the culture medium would be a
misleading indicator for oxygen availability (this is not the case when using particulated
supports as the attachment surface, where dissolved oxygen is a most important parameter).
Gaseous phase composition is a more robust indicator for oxygen availability in our
As cell population increases, culture medium replacement rate increases to maintain a constant pH.
Temperature is most relevant in adherent cell culture. In contrast to suspension cells,
where culture medium temperature determines the temperature at which cells are cultured,
in adherent cell culture, cells are exposed to three distinct environments: the solid cell
attachment surface, the liquid culture medium and the gaseous phase. These three
environments, each with its particular heat transmitting properties, must be kept at the
same temperature for optimum culture conditions. The temperature of the cell growing
environment will be mainly affected by the temperature of the newly introduced culture
medium, the temperature of the gaseous phase and the heat transfer to the environment
surrounding the culture chamber.
In existing culture systems such as t-flasks, following the stressing medium replacement
step, culture takes place in a thermostated environment, the incubator, where cell
attachment surface, culture medium and gaseous phase get the heat from the surrounding
environment within the incubator. The same system is used with rollers. As long as the
door of the incubator (or the thermostated room) is closed, this thermostating system
works well. In automated bioreactors for suspension cells it is more common to find
heating jackets surrounding the culture chamber. This is an advantage when you want a
temperature within the chamber different from that of the surrounding environment. Using
heating sources within the culture chamber is rare due to the negative effect of the
extreme temperature gradient as cells get closer to the heat source. Conditioning the
medium and the gas before they enter the culture chamber is also used in existing systems
to reduce sudden temperature changes.
Having analysed the different options available, we favor the combination of three
elements to thermostatize the system: i) a thermostating detachable jacket surrounding the
culture chamber to heat the culture medium and prevent heat loses; ii) a culture medium
reservoir within the chamber to act as a heat accumulator and; iii) a heating system for
A thermostated jacket will maintain a constant temperature within the culture chamber while the culture medium reservoir absorbs temperature difference on incoming fresh medium. Inflowing gas will be conditioned before entering the culture chamber.
Metabolic activity of growing cells modifies the acidity of the culture medium. Minute
deviations of pH from optimum can be deleterious to growing cells and therefore a control
system for pH must be in place. Some media for cell culture contain buffering systems that
successfully regulate pH to a certain extent. CO2 helps maintain the buffering capacity
for many of these media. Others require acid/base adition. We have seen earlier how
replacing culture medium as pH changes is a tempting strategy to control culture medium
composition. This medium replacement will serve as well as a pH control system. Therefore,
we propose to control pH through the combination of CO2-containing gas circulation and
culture medium replacement.
Gaseous phase composition
Traditionally, air or CO2 enriched air are used in adherent cell culture. Oxygen addition
is normally not necessary for adherent cell culture since metabolic requirements for
oxygen are usually satisfied with the oxygen contained in air. However, both CO2 and
oxygen are subject to close control, especially in suspension cell culture, due to the
high impact that both of them have on the culture. When necessary, for example when using
stirred tanks, oxygen is also introduced in the culture chamber, normally through
spargers. When using laboratory devices such as t-flasks, culture plates, roller bottles
or plate-stacks, gas difussion between the inside of the device and the incubator through
filter membranes is enough. However, we are aiming at a high cell density compared to
traditional laboratory scale devices, and therefore metabolic demand of the culture will
demand, in most cases, forced gas circulation.
When considering temperature control, we have considered conditioning incoming gas before
it enters the culture chamber. When more than one gas is to be part of the gaseous phase,
an option is to have each of them enter the chamber separatedly. However, since a
temperature conditioning step is to be made on the gas phase, this option would force
condioning each of the gases separatedly. Therefore, mixing the gases before conditioning
makes the alternative of choice. The gas mixture will be provided directly from an
external source while gas temperature, flow and composition will be controlled by the
system. Gas flow will be kept to the minimum necessary to satisfy metabolic needs and,
given the large contact area with the culture medium soaked membrane, the gaseous phase
will be sufficiently humid.
The deviation of CO2 concentration within the culture chamber from a defined set-point will trigger forced circulation of the gas mixture from an external source.
Attachment surface availability
When culturing adherent cells in plates or roller bottles, culture starts with a
relatively low cell population that propagates and colonizes all available attachment
surface. Once confluence has been reached, cells are harvested and divided into two or
more devices to start a new culture cycle, the so-called culture pass, passage or sub-
culture. The process is repeated until the desired amount of cells has been obtained.
In the Bolt-on Bioreactor, however, the amount of available culture area is enough to
provide the desired amount of cells, and therefore, once the culture starts, and given the
continuity in the attachment surface, cells propagate and colonize the whole surface. This
effect will not take place in systems where culture surface is not continuous, as is the
case of particulated supports. In those systems, cells will have a much harder time
colonizing particles other than the one they are growing in.
A major concern when culturing cells in stirred tanks is shear stress. Shear stress is
mainly induced by mechanical agitation of the liquid (mainly due to rotatory impellers)
although air bubbling and foaming are also major sources of shear forces. Impeller design
and antifoaming agents are common strategies to reduce shear stress.
When using small laboratory scale culture devices, shear forces come mainly from regular
medium replacement and sub-culture cycles, since pipetting and cell detachment and shaking
are very agressive to cells. In the case of roller bottles, rotatory speed is also to be
considered, since cells require some time to establish a strong interaction with the
attachement surface. Therefore control of rotatory speed is important because a balance
has to be found to ensure a frequent exposure of the cells to both culture medium and
gaseous phase, and to provide the cells with conditions gentle enough for attachment,
especially during the initial seeding step. Given a sufficiently slow rotation, foaming is
not a consideration in roller bottles.
The previous factors considered, we find that rotatory speed is an important parameter to
be controlled in order to provide optimum culture conditions and, since cell detachment
requires more vigorous agitation, inversion of rotatory direction and fast speed should
also be a option at desired points in the process. Rotatory movement of the rolled membrane has also an important effect as medium distribution and mixing system.
During the initial seeding stage, rotatory speed is kept to a minumum to facilitate cell attachment to the surface. Once cells have attached to the surface, rotation speed is set to process speed.
As for the effect of light in the culture, deleterious effects have been reported, whether due to the direct effect of certaing wavelengths on cells or due to the light-mediated decomposition of culture media components. In anycase, nothing seems to be gained from exposing the cells to light, and therefore, the thermostating jacket will protect the culture from light while allowing enough room for visual inspection of the process.
In adherent cell culture, it is not rare the need for the control of particular parameters
for special strains, culture media or culture conditions, neither is it rare the need for
additional components to be added to the culture at particular stages in the process, or
the need for pH control through acid / base addition. Therefore, the system must include
the option for the user to expand the number of controlled parameters.
We conclude that control of:
rotation speed and direction,
medium replacement rate,
thermostating jacket temperature,
incoming gas temperature and flow,
CO2 concentration in gaseous phase,
liquid phase temperature,
liquid phase pH,
an additional, user defined, parameter in liquid / gaseous phase and
a culture medium reservoir within the culture chamber
will result in an automated and continuous culture process for adherent cells that meet Challenge #2
FOLLOWING A MARKET SURVEY CONDUCTED TO FIND OUT THE OPINION OF FINAL USERS ON THE PARAMETERS TO BE CONTROLLED, WE HAVE INCLUDED DISSOLVED OXYGEN AS ONE OF THE STANDARD PARAMETERS TO BE CONTROLLED IN THE BOLT-ON BIOREACTOR.
Below we summarize how the different issues related to Challenge #2 are addressed by the proposed design solution of The Bolt-on Bioreactor.
Homogeneous cell and medium distribution
Controlled rotation speed helps distribute cells and culture medium homogeously throughout the attachment surface. Reversing direction combined with faster rotation speed will be used in cell detachment and cell washing steps.
Culture medium composition
Culture medium replacement rate will be regulated based on variations on pH. Culture medium replacement is also used to harvest extracellular products. Culture medium reservoir helps absorb sudden composition changes.
Process temperature control
The combined effect of the thermostating jacket, the culture medium reservoir within the culture chamber, and the heated gas inflow will maintain a constant temperature within the chamber. Temperature of both liquid and gaseous phases will be continuously measured. The thermostating jacket shields growing cells and culture medium from the damaging effects of light.
A sustained gas inflow guarantees that the oxygen contained in the external gas source is available to the cells.
For CO2 / bicarbonate buffered media, CO2 contained in the incoming gas flow will help regulate pH. Gas flow will be influenced by CO2 concentration in the gaseous phase. Medium replacement will be the major mechanism for pH control.
User defined control
Base addition, aditional medium components, specialty gases or other comoponents will be added based on the reading from user defined parameters.
The following video summarizes the findings expalined above.
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