Two cell types were used for the experiments: canine epithelial kidney cells (MDCKII, a gift from F. Luton, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne) and human TIFs (a gift from E. Van Obberghen-Schilling, Institut de Biologie de Valrose). MDCKII cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (50 U ml−1), and streptomycin (50 μg ml−1). Fibroblasts were cultured in high-glucose DMEM and supplemented as previously described without glutamine. Once cells were detached from the tissue culture flask using a solution of 0.25% trypsin, cell suspension was centrifuged and supernatant was replaced by fresh medium. The fresh cell suspension (100 μl) was mixed with 100 μl of 0.4% trypan blue. Cells were counted using a glass hemocytometer and resuspended to prepare the desired cell concentration. Before cell seeding, scaffolds were kept submerged in cell medium for 2 hours at 37°C, allowing protein adhesion.

For the free-standing scaffold experiment, the medium was completely removed from the scaffold by placing it onto an absorber for 2 min. Cell seeding was done right after by dipping the dried scaffold into a cell suspension (MDCKII or TIF; 5 × 106 cells/ml), allowing cell penetration by capillarity forces. Then, the scaffold was kept at 37°C for 1 hour, allowing cell attachment and spread before changing the medium to remove nonattached cells. Cell culture maintenance was done by placing the scaffold into an Eppendorf tube filled with the medium for up to 3 days.

For in situ experiments, the medium was not removed to prevent any bubble formation inside the device, so cells were directly injected into the scaffold using the fluidic tubing at a velocity of 1.5 μl/min. Cell culture maintenance was done using cell medium supplemented with 5 mM Hepes and a continuous flow rate of 0.5 μl/min for 2 days.

For the cell adhesion experiments, two different cell suspensions were prepared: 5 × 106 and 5 × 104 cells/ml. A volume of 80 μl was injected inside the scaffold device, resulting in 4 × 105 and 4 × 103 cells, respectively. The electrical measurements were carried out before cell seeding and after incubating the devices for 1 hour at 37°C.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.