Perfusion for live cell imaging: methods and systems

This short review presents one critical aspect of a perfusion cell culture system that is the choice of the fluid delivery method. Several types of pumping mechanisms are available.

The principle of this technique is to apply a controlled flow of air inside a reservoir containing the perfusion medium in order to create a pressure-driven flow.

The main advantage of this method is that it results in a very stable, pulseless flow rate, even at very low flow rates. Flow controller are well suited for dynamic cell culture.

In a peristaltic pump, the fluid is contained inside a flexible tube fitted inside a circular pump casing. A rotor, equipped with several rollers, turns and the roller compress the tube, forcing the fluid to move though the tube. One advantage of this technique is that peristaltic pumps can be miniaturized and integrated inside microfluidic chips to obtain autonomous perfusion cell culture systems. However, this type of pump is not able to generate a very stable flow rate, especially at low flow rates.

In a syringe pump, the fluid in held inside a syringe and a motor applies a pressure to a syringe plunger. This method is very effective to deliver small volume of fluid but, as peristaltic pump, is not suited for stable low flow rates.

The first live-cell imaging chambers were designed in the early twentieth century, shortly after mammalian cell culture techniques were developed. A wide range of perfusion chambers is now available, from simple sealed coverslip on a microscope glass to sophisticated perfusion chamber allowing a full control of the cells environment. Recently, the advance  of microfluidics has led to the development of integrated cell culture system.

Perfusions chambers are a critical aspect of live-cell imaging, and they must fulfilled two equally important requirements: maintain cells in a healthy state and allow the living cells to be observed with the highest possible resolution.

Two main categories of culture chambers are available. The first category is open chambers. They are more basic and very similar to Petri dishes. With open chambers, the medium surrounding the cells slowly approaches the equilibrium with the surrounding atmosphere during the experiment. They allow to easily access the cells, but exhibit a low level of control on the cells environment.

Closed chambers are sealed to avoid evaporation of the culture medium and ensure a large control over the environmental variables, such as temperature, pH or carbon dioxide concentration. Most closed chambers are designed with ports allowing the addition of fresh medium or drug during the experiment. Closed perfusion chambers are thus more suited to long term experiments.

For short term experiments, a coverslip can be attached onto a microscope slide using spacers. For longer experiments, more sophisticated microscope slides can be found. They include inlet and outlet allowing to refresh the culture medium or inject drugs during the experiment. These cell culture slides often include multiple chambers in order to multiplex the experiments.

Microscope slides can also be used with warmers, such as Peltier modules or air blowers, for experiments on longer periods of time. However, these simple culture chambers are restricted to short term experiments. For more complex experiments, closed perfusion chambers are usually preferable.

Dimensions of the chamber.  The depth of the perfusion chamber should be minimized to obtain the highest possible optical quality for transmitted light. Concerning the coverslip surface area, large viewing areas are more sensitive to leaks and physical damage, but allow the use of high numerical aperture objectives, that often have large diameter barrels.

Flow characteristics. For experiments involving the addition of other agents to the culture medium, such as drugs or growth factors, it is very important to obtain laminar flow, ensuring controlled media exchange. In order to maintain laminar flow inside the culture chamber, the chamber cross section should be very close to that of the inlet tube. Several fluids delivery techniques are available, but the uniformity of the flow should as be taken into account, since hydrodynamic pulses can harm the cells or produce coverslip flex. For more informations, see our review about perfusion systems.

Temperature control. For long term experiments, temperature control of the medium surrounding the cell should be ensured. Many commercial systems include heating elements coupled to the chamber or the mounting stage. However, some parts of the microscope, such as frame and objectives can act as heating sink and disrupt heat stability of the perfusion chamber. Moreover, small changes in ambient temperature can lead to thermal extension or contraction in some part of the microscope, modifying the plane of focus. Systems such as objective heaters or incubator box are available to tightly control the microscope temperature.

Microfluidics is currently a growing and promising field in biology. Microfluidics allows a high degree of control on cells environment, on flow dynamics and is compatible with existing high resolution microscopes. A lot of research is currently undertaken in this field, and simple microfluidics chips are already commercially available. Due to the versatility of microfabrication processes, numerous microfluidics lab-on-chips have been developed by researchers for a wide range of applications, from simple shear stress exposure experiments to complex organs on chip. For more information, consult our review about microfluidics and cell biology.