By Milica Radisic, Lauren D. Black III
Cardiac Tissue Engineering: equipment and Protocols presents a suite of protocols on cardiac tissue engineering from pioneering and best researchers worldwide. those contain equipment and protocols for telephone coaching, biomaterial coaching, telephone seeding, and cultivation in a variety of platforms. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and key tips about troubleshooting and warding off identified pitfalls.
Authoritative and sensible, Cardiac Tissue Engineering: equipment and Protocols highlights the key recommendations, either experimental and computational, for the learn of cardiovascular tissue engineering.
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Extra info for Cardiac Tissue Engineering: Methods and Protocols
Allow 10 min for the aggregates to settle. Aspirate the supernatant and resuspend the aggregates in Stage 3 Induction Medium. Using a 5 mL serological pipette, redistribute the aggregates into a 24 well ULA plate at 1 mL per well. 3. Incubate under hypoxic conditions until day 12. 5 Cardiac Induction Stage 3 (Day 12 to Harvest) 1. Prepare the necessary volume of Stage 3 Induction Medium (volume = number of wells × 1 mL) and place in a 37 ºC water bath for 10–15 min. 2. Use a 5 mL serological pipette to transfer the aggregates to 15 mL conical tubes, pooling up to 10 mL of aggregates per tube.
Also reexamine wells which have theoretically <1 cell for any growing colonies. 11. One 96-well plate typically yields 5–10 clonal candidates. 12. Continue to maintain the clonal candidate numbering for future identification. 13. Expansion is not critical until after characterization has been performed, but starting to expand at this point maintains the cells at a lower passage. 14. Expand as much as possible at this point. 15. Clones typically maintain c-kit for >15 passages; characterization can be repeated at later passages to confirm that cells are >90 % c-kit positive.
Aspirate the excess liquid and air dry for 15 min. 4. Rinse the plates 2× with PBS: leave PBS on for 5 min, aspirate, and repeat. 5. Add 10 ml EBM medium with supplements, warmed to room temperature. 6. Add ¼ of the cells to each of the four plates and culture at 5 % CO2 at 37 °C for 4 days without changing the medium. 7. After 4 days, the adherent population is considered to be CACs (Fig. 3). Fig. 3 Adherent population of CACs generated from the culture of PBMCs on fibronectin-coated plates for 4 days 32 Aleksandra Ostojic et al.