Microfluidic devices are well-suited for the study of metabolism and paracrine and autocrine signaling because they allow steady or intermittent perfusion of biological cells at cell densities that approach those in living tissue. They also enable the study of small populations of rare cells. However, it can be difficult to introduce the cells into a microfluidic device to achieve and control such densities without damaging or clumping the cells. We describe simple procedures that address the problem of efficient introduction of cells and cell culture media into microfluidic devices using small bore polyetheretherketone (PEEK) tubing and Hamilton gastight syringes. Suspension or adherent cells grown in cell culture flasks are centrifuged and extracted directly from the centrifuge pellet into the end of the PEEK tubing by aspiration. The tube end is then coupled to prepunched channels in the polydimethylsiloxane microfluidic device by friction fitting. Controlled depression of the syringe plunger expels the cells into the microfluidic device only seconds following aspiration. The gastight syringes and PEEK tubing with PEEK fittings provide a non-compliant source of pressure and suction with a rapid response time that is well suited for short-term intramicrofluidic cellular studies. The benefits of this method are its simplicity, modest expense, the short preparation time required for loading appropriate numbers of cells and the applicability of the technique to small quantities of rare or expensive cells. This should in turn lead to new applications of microfluidic devices to biology and medicine.