By Milica Radisic, Lauren D. Black III
Cardiac Tissue Engineering: tools and Protocols presents a suite of protocols on cardiac tissue engineering from pioneering and prime researchers all over the world. those comprise tools and protocols for cellphone practise, biomaterial instruction, telephone seeding, and cultivation in a variety of platforms. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key tips about troubleshooting and fending off identified pitfalls.
Authoritative and functional, Cardiac Tissue Engineering: equipment and Protocols highlights the most important options, either experimental and computational, for the research of cardiovascular tissue engineering.
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Extra info for Cardiac Tissue Engineering: Methods and Protocols
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. 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.
7. Centrifuge the tubes at 300 × g for 10 min (do not use centrifuge brakes). 8. Remove and discard the supernatant. Resuspend the cell pellets in a few ml of wash buffer and combine the cell pellet from each of the four tubes into a single tube. Bring the final volume up to 20 ml using wash buffer. 9. Centrifuge the tubes at 300 × g for 10 min (do not use centrifuge brakes). 10. Resuspend the pellet in 1 ml EBM culture medium. 2 Plating Fibronectin for Selection of CACs 1. Coat 4 × 10 cm plate with 3 ml of fibronectin solution (see Note 7).
This step is necessary to mix the blood with the EDTA and prevent clotting. If using the blood right away, mixing can be achieved by inverting the collection tubes multiple times, otherwise leave on the rocker. 4. Cannot have layers mix because it will affect the separation efficacy, and the buffy coat layer will not be as visible. 5. It is very important that braking is not applied during centrifugation, as this may disrupt proper separation of the layers. 36 Aleksandra Ostojic et al. 6. Make sure not to aspirate any cells that may be stuck to the side of falcon tube.