for applications such as drug toxicology, with the reduction in

unstirred layers leading to an improved, more physiologically rele-

vant transfer of drugs to the cells [7].

There are many techniques which can be used to introduce

media perfusion into cell culture systems. These can range from

very simple systems such as placing standard culture ware onto an

electronic shaker or rocker, to complex systems consisting of mul-

tiple components such as pumps and medium reservoirs. The

higher complexity typically comes with a greater level of control

over the fluid flow properties such as shear stress and mass transfer,

however, can also lead to greater spatial requirements, increased

levels of user expertise required, and higher cost.

The system utilized for this protocol uses a magnetic stirrer to

induce perfusion in a 3D cell culture system based around porous

Alvetex^® membranes. This allows for a simplistic setup, which can

be used with minimal expertise required while still giving control

over properties of the fluid across the cells. A completely self-

contained vessel with a large volume of medium means that cells

and tissues can be cultured for prolonged periods without the need

for media changing, leading to more consistent culture conditions

over time. As shown by Fig. 2, this system consists of a glass beaker

to contain the culture, a polytetrafluoroethylene (PTFE)-coated

magnetic stir bar and PTFE stand which holds an Alvetex^® insert

as well as providing control over the fluid flow, and a PTFE lid.

Fig. 2 The stirred bioreactor system. This design incorporates includes the conical stand to hold a 6- or

12-well Alvetex insert. A stir bar placed at the bottom provides continuous fluid recirculation. (a) Exploded

diagram of the major components of the bioreactor system. (b) The system when fully assembled. The vessel

can hold up to 120 mL of cell culture medium

Applying Stirred Perfusion to 3D Tissue Equivalents to Mimic the Dynamic In. . .

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