Bioengineering
A subscription to JoVE is required to view this content.
You will only be able to see the first 2 minutes.
The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.
If that doesn't help, please let us know.
Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair
Summary June 9th, 2020
The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.
Transcript
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.
Related Videos
You might already have access to this content!
Please enter your Institution or Company email below to check.
has access to
Please create a free JoVE account to get access
Login to access JoVE
Please login to your JoVE account to get access
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Please enter your email address so we may send you a link to reset your password.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Your JoVE Unlimited Free Trial
Fill the form to request your free trial.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Thank You!
A JoVE representative will be in touch with you shortly.
Thank You!
You have already requested a trial and a JoVE representative will be in touch with you shortly. If you need immediate assistance, please email us at subscriptions@jove.com.
Thank You!
Please enjoy a free 2-hour trial. In order to begin, please login.
Thank You!
You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free.
To get started, a verification email has been sent to email@institution.com. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.
