The overall goal of this procedure is to encapsulate neonatal rat cardiomyocytes in a FI hydrogel for cardiac tissue engineering. This is accomplished by first isolating the hearts of two to three day old neonatal rat pups. Next, the hearts are digested in collagenase.
To separate the cardiac cells from the extracellular matrix, the cardiac cells are then encapsulated in a fibrin hydrogel. Finally, the constructs are cultured for two weeks. Ultimately, the results show that the constructs have the ability to generate a twitch force of approximately 1.3 milli Newtons in response to electrical stimulation.
Furthermore, the aligned structure of the construct can be observed via histological staining from myocyte proteins. Engineered myocardial constructs can serve as a potential therapy for myocardial infarction by allowing for the repair or replacement of scarred tissue with engineered tissue, thereby improving cardiac function, following injury or disease. These constructs can serve as a valuable in vitro model system for studying the effects of various stimuli on contractile forces and normal and pathophysiological cardiac development.
After preparing medium and sterilizing surgical instruments for each person, place an absorbent bench underpad on the hood, work surface and cover it with a sterile drape. Place the sterile surgical instruments and a four by four gauze pad onto the sterile drape. Without touching the instruments, open a sterile number 20 scalpel blade and place it onto the drape.
After decapitating a pup, swab the chest with Betadine soaked gauze. Secure the pup by pinching the shoulder blades together. Perform a partial thoracotomy to expose the heart, increase the pressure applied to the shoulder blades, which will force the heart past the ribs.
For scapular dissection, run the scalpel blade behind the heart to sever the great vessels and remove the organ. Place the heart on ice in a Petri dish containing PBS glucose. Once the hearts have been isolated, remove any residual blood and connective tissue by rinsing them in ice cold PBS glucose.
Remove the top one third of each heart, so the bottom two thirds remain. To isolate the majority of the ventricular tissue only, and place the tissue into a fresh Petri dish with ice cold PBS glucose. Using sterile micro scissors and forceps, carefully mince the hearts into roughly one cubic millimeter pieces.
Transfer the tissue to a 50 milliliter conical tube and remove all but 10 milliliters of the buffer. Add seven milliliters of collagenase solution. Shake the collagenase tissue mixture at 37 degrees Celsius for seven minutes.
Gently pipette 10 times to break up the tissue pieces. Allow the pieces to settle and then aspirate the liquid and discard it. Add seven milliliters of collagenase to the tissue pieces and digest again for seven minutes.
For each remaining step, gently pipette 10 times to break up the tissue pieces. After they settle, draw off the supernatant and collect it In a separate 50 milliliter conical tube, add seven milliliters of collagenase to the tissue pieces and digest again for seven minutes. To each supernatant tube, add 10 milliliters of stop solution with a different serological pipette after each addition of supernatant from the digestion.
To prepare for casting fibrin gel constructs, please refer to the written portion of the protocol. Place the previously constructed mandrels into the six cc syringe casings Using the three cc syringe as a plunger to obtain a tight seal between the O-rings and Teflon washers. To make one milliliter of fibrin gel, create F solution in a conical tube by adding 112 microliters of the fibrinogen stock to 558 microliters of 20 millimolar heaps.
Buffer in 0.9%saline solution in a separate conical tube. Create a T solution by adding 17 microliters of the thrombin stock and 1.3 microliters of two normal calcium solution to 135 microliters of DMEM. In a third conical tube, spin down the cells and resuspend them to a concentration of 29.4 million cells per milliliter, or six times the concentration of the desired final concentration of cells in the construct.
Generally, individuals new to this method will struggle with the construct fabrication step. After mixing the fibrinogen and thrombin solutions together, the polymerization of fibrin occurs rapidly. Thus, individuals should work quickly to ensure a homogeneous solution while preventing the incorporation of air bubbles into the hydrogel upon injection into the mold casting.
When ready to cast the fibrin gel construct, prep a one milliliter syringe with an 18 gauge one and a half inch needle. Have a 21 gauge one inch needle ready as well. To make one milliliter of fibrin gel solution add 667 microliters of F solution into a 50 milliliter centrifuge tube, followed by 167 microliters of cell solution.
And lastly, 167 microliters of T solution. Carefully pipette the mixture so as not to introduce air bubbles. At this point, the reaction has started and the injection of the construct should be done immediately.
Use the syringe and 18 gauge needle and draw up the fibrin solution. Take care not to invert the syringe to prevent bubbles from getting into the needle. Replace the 18 gauge needle with a 21 gauge needle.
Tap the syringe gently to force out any air bubbles. Next, insert syringe into the mold between the stopper encasing following the groove in the Teflon O-ring and inject the solution into the mold. Tilt the mold to ensure complete filling.
Then fill the remaining molds. Wrap the molds in para film in groups of three, and place them in the incubator or oven at 37 degrees Celsius. Incubate the gels for 20 minutes to allow the fibrin enough time to fully polymerize.
Fill each culture jar with 21 milliliters of myocardial construct medium per construct. Use the sterile three cc syringe casing as a plunger to force the mandrel with the construct into a large Petri dish with DMEM. Then place the construct into the sample jar.
Incubate the jars at 37 degrees Celsius for two weeks. To begin analysis of a fibrin construct clamp, the alligator clips on the lead coming from the stimulator to the wires on the electrodes in the bath. Power up the data acquisition board, pulse generator and force transducer.
Set the force transducer to the 5G setting and zero it. Open a custom lab view program that displays and saves the data from the force transducer. Create a new empty text file in the data folder for each sample using tweezers, remove the construct off the mandl support by gently sliding the construct ring from the Teflon support and placing it over the fixed metal post in the force measurement system.
Medium bath. Use the tweezers to push and lift the construct around the force transducer post. Place the other end of the construct over the transducer arm and tighten until the transducer reads.
1.0 G force on the cardiac stimulator. Set the pulse voltage to 20 volts, duration to six milliseconds and rate to one hertz. Start the electrical pacing by pressing the output on off button.
Start recording until the waveform becomes regular. Analyze the waveform in MATLAB to determine the force rate of contraction and rate of relaxation. The cardiomyocyte fibrin construct initially covers the entire width of the mold.
After two weeks of culture, it contracts to approximately one fifth of the initial width. Electrical pacing of the construct generates twitch force data as shown here. Twitch forces of roughly 1.3 millinewtons are expected.
Cell viability of the construct is dependent on how far the cells are from the surface of the construct that is in contact with the culture medium due to the diffusion limitations of oxygen into the construct. On the surface of the construct, high cell viability observed using confocal microscopy. The aligned structure of the construct is observed by staining for myosin heavy chain in red Connexion 43, which is necessary for cell coupling between myocytes is in green.
Once mastered, the entire protocol should take approximately five hours. The I isolation portion will take approximately three hours. While the construct fabrication portion will take approximately two hours While attempting this procedure, it is important to always keep the needle pointing downwards to prevent the incorporation of air bubbles into the fibrin network.
Furthermore, tilting the mold will always keep the needle hole at the highest point during injection, which will ensure that the mold casting is fully filled with the fibrin solution. After watching this video, you should have a good understanding of how to encapsulate cardiomyocytes into ring shaped fibrin hydrogels by first isolating the cardiomyocytes from neonatal rats, and then injection molding the fibrin solution into the easily assembled cylindrical molds.
