Imaging and microscopy: Perfusion chambers

Introduction about perfusion chambers for live-cell imaging and microscopy

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 chambersare 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 systems.

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.

This short review will describe different types of perfusion chambers and their requirements. For more general information about perfusion for live-cell imaging, you can check our review. If you are interested in a perfusion system for live-cell imaging, check our Perfusion Pack.

Closed perfusion chambers

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.

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.

Microscope slides for cell culture

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 oftenly 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.

Microfluidic chips

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 microfluidic 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.

Cultivating cells inside perfusion chambers

Maintaining cells in a healthy state on the microscope stage can be very challenging. Culture techniques depends on the cell line used during the experiment.

One very important requirement is the attachement of the cells to the coverslip, in order to obtain a stable focus plan. Adherent cell lines will be naturally immobilized on the glass covership, whereas non adherent cell lines will have to be stabilized. Several techniques have been used, such as adhesion promoting agents (poly-L-lysin or collagen), or thin agar pad.

Cell culture require a carbon dioxide and bicarbonate buffer system in order to regulate pH. In conventional cell culture, a controlled atmosphere is created in dedicated incubators. For live-cell imaging, some perfusion chambers are specifically designed to create a regulated atmosphere. However, it is possible to use synthetic biological buffers, such as TRIS or HEPES to control the pH in absence of a controlled atmosphere.

Discover how to do dynamic cell culture perfusion in a microfluidic chip.