Method Of Media Exchange In A Sealed, Fixed Volume Bioreactor
Tech ID: 34884 / UC Case 2025-959-0
Background
Expansion Reservoirs in Cell Culture Systems
Most cell culture systems incorporate an expansion reservoir to allow media exchange - the removal and replacement of culture media—without drying out the cells.
This function is implemented in various ways, depending on the system design:
Conventional culture systems (e.g., wells, flasks, and basic bioreactors) use the culture chamber itself as an expansion reservoir. These chambers hold excess liquid media, which can be gradually removed and replaced. As liquid volume changes, it is balanced by air entering or exiting the chamber.
Circulating fluidic systems (e.g., perfusion bioreactors) typically include a dedicated inline expansion reservoir. In these systems, part of the media is actively circulated through the system (“circulation pathway”), while another part remains static in the reservoir. Media is exchanged from the non-circulating volume, with air compensating for the volume change.
Flexible bag systems and air-free flexible chamber systems (including recirculating setups) also support media exchange, but instead of displacing air, the flexible container itself changes shape to accommodate volume changes.
Despite their differences, all of these systems share key characteristics:
- They support variable liquid volumes during media exchange.
- They require separate actuators (e.g., pumps or valves) for draining and refilling media.
We define all these systems as cell culture systems that rely on an expansion reservoir.
Challenges of Expansion Reservoir-Based Systems
Mechanical and Control Complexity:
- Using separate mechanisms for draining and refilling media increases system complexity
- Multiple actuators must be precisely controlled and synchronized, which introduces more potential points of failure.
Media Dilution and Wastage:
- To avoid drying out the sample, the system must hold both:
- A minimum media volume to support the biological sample
- Additional dead volume (e.g., in pumps, sensors, and tubing) that must remain wetted
- In systems with large dead volumes, this can exceed the minimum required for biological homeostasis, leading to dilution of both:
- Endogenous substances (e.g., signaling molecules, hormones, biomarkers), which may affect cell behavior or impair data accuracy
- Exogenous additives (e.g., nutrients, growth factors), which are costly and may be wasted.
Reduced Sensitivity of Analytical Measurements:
- Diluted media lowers the concentration of analytes (e.g., metabolic byproducts, gene expression markers), reducing the sensitivity of diagnostic readouts. If some old media remains in the system during exchange, it may delay detection of biological changes.
Physical Bulk and System Complexity
- Expansion reservoirs add to both the physical size and operational complexity of the apparatus, which can reduce efficiency, increase cost, and complicate system management.
Air-Related Issues
- In systems where liquid volume is balanced by air volume, air introduces several risks:
- Air bubble formation (e.g., from surface exposure or in expansion tanks) can:
- Disturb gas concentrations
- Cause local precipitation or concentration of solutes
- Physically disrupt cells, sensors, or fluid flow (e.g., by turbulence, blockage, or altered mixing).
- Evaporation and osmotic imbalance occur when:
- Air space humidity is insufficient to prevent evaporation or airflow is great enough to cause loss through advection
- Temperature or pressure changes affect relative humidity or gas solubility.
- Gas concentration control is difficult:
- It either requires active feedback with gas lines and actuators to manage air composition in the dead space, or
- Continuous air replacement, which increases complexity and promotes further evaporation through water vapor loss due to advection.
Fluid Pressure:
- Having an expansion reservoir, which inherently involves equilibration of the internal pressure of a system with some surrounding environment makes it impossible to maintain the fluid pressure of the entire media compartment of a cell culture system at a pressure other than that of the expansion reservoir.
- Therefore, to achieve fluidic pressures other than this expansion reservoir pressure, more complicated methods of establishing fluid pressure must be used, such as ones which use active pumping and fluidic resistors or that require repletion of a gravity-pressurized reservoir.
- In fluid systems, achieving elevated or reduced pressures is crucial for controlling the flow and behavior of fluids within a model, especially when working with fluidic resistors and circulation pumps. Fluidic resistors are components that impede the flow of fluid, and by varying the resistance, they help regulate pressure within a system.
- When paired with circulation pumps, they provide a dynamic way to maintain or adjust pressure levels. However, using this setup introduces complexity in pressure control. To achieve and maintain the desired
- pressure, a pressure feedback control system becomes essential.
- This system monitors the pressure within the circuit and adjusts the pump's speed or resistance in real time to ensure the pressure stays within the desired range.
- Without this feedback loop, fluctuations in pressure could lead to inefficiencies or system malfunctions.
- Pressure feedback control itself adds complexity because it often requires sophisticated sensors, controllers, and algorithms to keep the pressure stable.
- These systems need to react quickly to changes in fluid dynamics, such as those caused by variations in fluid temperature, viscosity or flow rate.
- Moreover, the system must be calibrated to handle different conditions, such as varying load demands or system wear over time.
- Additionally, there is the risk of overpressure, which can occur if the pressure exceeds safe operating limits.
- In such cases, components like pressure relief valves or bypass lines become critical.
- Pressure relief valves open when pressure exceeds a certain threshold, allowing excess fluid to be diverted to prevent damage to the system.
- Bypass lines, on the other hand, allow fluid to flow around the system or around particular components to relieve pressure buildup.
- Both overpressure relief mechanisms add extra layers of complexity to the design and operation of the system. They must be carefully designed and sized to ensure proper operation under all possible conditions.
- Without them, there's a significant risk of damaging pumps, resistors, or other sensitive components due to excessive pressure, which could lead to system failure or safety hazards.
- Elevated or reduced pressures can be achieved in models containing fluidic resistors paired with circulation pumps
- however these require added complexity in pressure feedback control and may require additional complexity in overpressure relief and/or bypass line components to avoid damaging overpressure events.
- In a more complex design for expansion reservoirs, elevated or reduced pressures can be achieved in models containing an airliquid interface by simultaneous pressurization/depressurization of the air compartment as well as the liquid compartment, however this alters the relationship between gas partial pressure and gas molar concentration, requiring added complexity in gas provision feedback control
- The difficulty of these approaches is problematic because certain biological processes and tissues require specific pressure conditions that are not at ambient levels, and if these pressure conditions can't be modeled accurately, it is difficult to simulate real-life conditions of certain organ tissues.
- Some tissues experience pulsatile (regularly changing or "pumping") pressures, such as blood flow through arteries, or even pressure that changes dynamically over time due to body processes.
- These fluctuating pressures are important for mimicking real physiological conditions. Vascularized tissues (tissues with bloo vessels) rely on pressure gradients (differences in pressure) to function correctly. For example, blood pressure helps circulate nutrients and oxygen throughout tissues.
Technology Description
In its simplest form, the system comprises
- A cell culture fluid recirculation loop which has a fixed fluidic volume or at least an upper bound on said volume.
- A controllable recirculation pump.
- A controllable pump on an inflow line which may inject fresh media into the recirculation loop, combined with either a one-way valve, or a pump design which does not allow for backflow, to keep media contained at the injection port.
- A normally closed outflow valve on an outflow line which either is passively opened at a defined cracking pressure, or an actively controllable valve which can be selectively opened
- thereby acting as a one-way valve which is only opened during media change.
- This keeps media contained within the circulation loop as to not exit via the outflow line when media change is not being effected.
- The media change pump injects into the recirculation loop via a T junction
- The outflow line ejects from the recirculation loop via a T junction
- With respect to the culture well or the component of the circulation loop with the largest volume, the injection line should join the loop upstream and the ejection line should leave the loop downstream, relative to the normal direction of circulation, such that a media change effected by the media change pump will be capable of ejecting the largest possible volume of media contained between the two points.
- Media change is effected by injecting media via the controllable fresh media injection pump. This can be done with the recirculation pump either circulating or not circulating. Since the recirculation loop has a fixed or upper bounded volume, injection of fluid will cause an excess of volume to enter the loop
- requiring that, as a consequence of the outflow valve opening at a fixed or upper bounded fluidic pressure, liquid will be ejected from the loop to equalize pressure.
- This pressure regulation by fluid ejection can be effected either by a passive valve which opens at a certain pressure or by an actively controlled valve with feedback control on pressure
- Specifically, this media change occurs via continuous displacement and ejection of fluid from inside the loop by new fluid entering the loop. The fluid volume contained in the loop will remain the same during the media change.
- This is in contrast to traditional methods of media exchange where media is removed from and added to the loop sequentially, altering the fluid volume of the loop during the media change.
- Optionally, there may be an occlusive valve in the fluidic loop which redirects fluid out of the outflow port and prevents recirculation. This will allow the pump apparatus to replace fluid in the loop with a minimum of mixing.
- This valve might be a second role played by the recirculation pump itself, if the pump is of an occlusive design such as a peristaltic pump.
- Optionally, to perform gas exchange without needing a reservoir or open component containing an air volume, the system may contain a mechanism for gas exchange which may be permeable tubing (eg silicone, plasticised PVC) a hollow-fiber gas exchanger, a silicone or permeable plastic chamber or a membrane gas exchanger.
- This gas exchanger should have a fixed internal fluid volume
- Optionally, the system may contain a fluidic damper, a waveform generator, a pressure-generating accessory such as a syringe pump or other device to alter the pressure dynamically or statically,
- so long as these devices have a bounded internal fluid volume and/or can have their fluid volume restricted during a media change.
- Optionally, one or more of the inflow or outflow pumps may be used to dynamically or statically alter the pressure in the system separate from media change by addition or removal of fluid.
- Optionally, the system may contain multiple wells, in parallel or in series within the fluidic loop.
- Optionally, the system may consist of parallel replicates who share the same inflow or outflow pump actuators but which are fluidically isolated within their circuits.
Applications
Cell culture systems
Bioreactor systems
Advantages
1) In this fixed/bounded volume system, injection of a fluid bolus is all that is needed to effect a media change. A media change can occur in a single action, with simultaneous injection and ejection of media and unchanging fluid volume.
a. In other systems, such as those with fluid expansion tanks, those with open wells, those with robotized pipetting, separate media removal and media addition effectors (pumps, pipettes, etc) must operate sequentially to effect a media change.
b. In more complex systems, such as those with fluid expansion tanks or open wells, the approach is different. These systems often involve a separate sequential media removal and media addition process.
i. For example, in a system with a fluid expansion tank, you may need to first remove the old media (through a pump or pipette) before adding the new media.
ii. This ensures the system is emptied before the new media is introduced.
iii. In robotized pipetting systems, automated processes use pipettes or other robotic tools to carefully withdraw and dispense fluids. The addition and removal are performed one after the other, which can take longer but ensures precise control over the volumes involved.
2) The boundary layer of the fluid is not displaced during a media change because the volume of fluid does not change.
a. This avoids disturbing structures inside the bioreactor by changing the fluid boundary.
b. This avoids the possibility of drying out any sample within the device. This is a large problem in open welled microfluidic devices.c. This eliminates the possibility of underfilling or overfilling a well due to inaccuracy in pumps causing an imbalance between media addition and media extraction.
d. Any media change mechanism which involves sequentially removing and filling media from a well, or vice versa will alter the fluid boundary.
3) Since media change is performed by a single action and does not involve the coordination of multiple pumps, and because the fluid volume is fixed, it is possible to simultaneously perfuse and media change in the system without having to halt the normal operation of the bioreactor.
a. This is important for samples which depend on consistent physical cues.
b. Media change can be performed while maintaining a fixed perfusion rate equal to the rate when not media changing.
c. Media change can also be done in a continuous “trickle” fashion, eliminating the discrete media change intervals causing abrupt alterations in chemical environment seen in conventional cell culture.
4) Since the system is entirely closed and is completely filled, without any air liquid interface which might change based on the strength or direction of gravitational force, the apparatus can be used without restrictions or alterations to design in non-stationary formats.
a. The system to effect a complete media change without removal from a shaker, or from a rotating or rolling bioreactor apparatus.
b. This allows the system to be used in zero gravity.
5) Comparison to other closed perfusion bioreactors and closed bag culture systems:
a. Systems already exist which have the benefits of not having an air boundary, eliminating issues with air contact, but they still contain expansion reservoirs, adding complexity to media changes and making pressure control difficult.
b. The NASA Bioculture system also employs a closed perfusion bioreactor system with no air in the loop.
i. However, they use an inline reservoir with a deformable wall and variable volume, such that separate media injection and ejection steps are required to change media:
ii. there is an expansion reservoir, just sealed by a flexible skin.
iii. the deformable reservoir means that fluid pressure cannot remain fixed especially during the media change, because the deformable wall equalizes pressure with the environment.
iv. having a reservoir increases the fluid volume of the system, which causes dilution of added substances (wasting reagents) and dilution of circulating biological factors (altering biochemistry and diluting measurable biomarkers.)
c. Cell expansion systems such as the Xuri Cell Expansion System or Sartorius RM bioreactors use a sealed bioreactor which may be operated without an air-liquid interface and which may be run in perfusion or recirculatory perfusion,
i. but they employ fluid reservoirs which have deformable walls and do not possess minimum interior filled volumes.
ii. This has two consequences:
1. first, separate, sequential fluid addition and fluid removal mechanisms are required and second,
2. the internal pressure of the fluid may equalize with the surrounding ambient pressure.
Intellectual Property Information
Patent Pending
Related Materials
Contact
- University of California, Santa Cruz Industry Alliances & Technology Commercialization
- innovation@ucsc.edu
- tel: View Phone Number.
Other Information
Keywords
cell culture, sealed media system, bioreactor, tissue perfusion, organoids, fluid pressure, media exchange
