This protocol describes a noble and easy method for creating mesh-shaped engineered cardiac tissues derived from human induced body parts and STEM cell derived cardiac cells. The flexibility of the tissue geometry and the scalability of the preserved tissue function are the main advantages of this technique. The implanted mesh shape ECTs can restore a cardiac structure and function in a rat myocardial infarction model.
Confirming its feasibility for use in cardiac regenerative therapy. These techniques require reproducible human STEM cell processing and the tissue culture skills for consistent results. Begin by cutting a cured PDMS sheet to the appropriate size to allow the sheet to be bonded with silicone adhesive to fabricate a 21 by 20.5 millimeter rectangular tray with seven millimeter long 0.5 millimeter wide and 2.5 millimeter high rectangular posts at staggered positions.
The horizontal spacing between two lines of posts should be 2.5 millimeters. Autoclave the tray at 120 degrees Celsius for 20 minutes before coating the mold with 1%Poloxamer 407 in PBS for one hour. Then thoroughly rinse the mold with PBS and place the mold into one well of a six-well plate.
For lineage analysis, combine the cells from the cardiomyocyte plus endothelial and vascular mural cell protocols. So that the final concentration of mural cells is 10 to 20%in a total cell number of six times 10 to the six cells per construct. Mix 133 microliters of Acid Soluble Rat Tail collagen type one solution and 17 microliters of Alkali Buffer to 17 microliters of 10X Minimum Essential Medium on ice.
Then add 67 microliters of Basement Membrane Matrix to the collagen solution. Next, centrifuge the cells and resuspend the pellet in 167 microliters of High Glucose Dulbecco's Minimal Essential Medium supplemented with fetal bovine serum and antibiotics. Mix the cells on the matrix solution on ice.
Then carefully pour 400 microliters of the cell matrix suspension onto the poloxamer 407 coated PDMS tissue mold. Incubate the cell matrix mixture in a standard cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for 60 minutes. When the tissue has formed, carefully soak the mold with four milliliters of ECT culture medium and return the plate to the cell culture incubator for 14 days.
Before ECT implantation, you sterilized fine forceps to carefully remove the ECT from the loading posts. Tissue molds can be fabricated with various patterns, post lengths and post spacing to generate different final ECT geometries. For this analysis, a mold with seven millimeter long posts with 2.5 millimeter wide intervals was used.
Poloxamer 407 prevents adhesion of the cells to the mold and enables the formation of a characteristic mesh structure, following rapid gel compaction in 14 days. The structure is maintained even after the ECT is released from the mold. The fabricated tissue construct is approximately 1.5 centimeters wide and 0.5 millimeters thick.
And the width of each bundle in the mesh is approximately 0.5 millimeters in average. It is possible to generate a 3 centimeter final width mesh ECT containing 24 million cells from a four times larger mold with the same staggered post design. This larger mesh ECT can also be easily removed from the mold and generates a local act of force equivalent to the smaller mesh ECT.
It is important to pour the cell matrix mixture evenly into the tissue mold without generating bubbles to prevent feeling defects in the final gel. Functional characterization of the contractor. and fourth frequency relations can be performed using in-built systems.
Our ultimate goal is the translation of ECT paradigms into clinical therapies that utilize large animal preclinical studies.
