Figure 3.

Generalized protocol flowchart. The steps in this protocol can generally be divided into (i) preparation (Steps 1–58), (ii) scaffold seeding (Step 59), (iii) perfusion cultivation and (iv) construct assessment. During preparation steps, we would prepare, sterilize and assemble the perfusion loop (Steps 1–27), which can take up to 4 d. We would also prepare the appropriate scaffold (Step 28), which, depending on the scaffold type, can take up to 7 d. (Note that generalized loop preparation (Steps 1–27) and scaffold preparation (Step 28) can be performed in parallel, if desired). The cell isolation (Steps 29–58) should start ~12–18 h before scaffold seeding. It is required that Steps 1–28 be completed before cell isolation. Subsequently, the scaffolds are seeded with cells (Step 59), placed into the perfusion chamber of the generalized perfusion loop (Steps 60 and 61), and cultivated in perfusion for a desired time period (Steps 62 and 63) followed by assessment of the construct’s structural and functional properties (Step 64).