Methods described in this protocol will provide a valuable platform to assess the functional maturation of ICMs for cardiac reprogramming studies. The calcium flux is exclusively observed in ICMs and provides a way to evaluate ICM functional maturation. This mouse strain simplifies the evaluation procedures and enhances the reproducibility of results.
This protocol is a new method to evaluate ICMs'functional maturation in cardiac reprogramming field. It can also be applied to evaluate cardiomyocyte differentiation and cardiac function. Begin by obtaining eight to 10 pups, P0 to P2 pups to isolate 10 million neonatal cardiac fibroblasts, or NCFs.
Deeply anesthetize the pups by hypothermia. Observe the hearts under the fluorescence microscope to ensure that the heart with the desired genotype shows calcium flux, indicated by GCaMP3 with the hearts beating. The other genotypes will not show fluorescence.
After isolation of the hearts, cut them into four pieces such that they're loosely connected. Wash them thoroughly several times in ice cold Dulbecco's phosphate-buffered saline, or DPBS, in a six centimeter plate to limit the blood cell pollution in the isolated cells. Digest the hearts with eight milliliters of warm 0.25%Trypsin-EDTA in 37 degree Celsius for 10 minutes.
Discard the Trypsin supernatant and add five milliliters of 0.5 milligrams per milliliter warm type two collagenase in Hank's balanced salt solution. Vortex the mixture thoroughly and incubated at 37 degrees Celsius for five minutes. After the incubation, again, vortex the mixture thoroughly and let the undigested tissue settle down by gravity.
Collect the supernatant in a 15 milliliter conical tube with five milliliters of cold fibroblast medium. Dilute the cells with fibroblast medium such that the seeding density is around at two to 2.5 times 10 to the fourth cells per cubic centimeter. Then seed the cells in dishes or plates.
The attached fibroblasts should have an oval to round shape on the second day after seeding. On day zero, seed the NCFs to the density of around one to two times 10 to the fourth cells per cubic centimeter in fibroblast medium. On day one one replace the culture medium with a virus containing medium for each well as desired.
On day 2, 24 hours after the virus infection, replace the virus containing medium with a regular ICM medium. Assess the calcium ion flux with an inverted fluorescence microscope at room temperature. Observe the GCaMP3 positive cells under 10X objective, ensuring that it shows spontaneous cells beating in the brightfield channel.
Hearts with the correct genotype showed calcium ion flux synchronized with beating, visualized as GCaMP3 flourescence. While no fluorescence was observed in control hearts. Isolated NCFs attached to the well within two hours and showed an oval to round shape one day after seeding.
The calcium ion oscillation showed GCaMP3 fluorescence change between the maximum and minimum and the calcium ion oscillation of such cells changed periodically. After introducing IMAP, the number of beating clusters was significantly higher than that in the controlled group as the number of GCaMP3 positive cells with calcium ion flux for a high power field was increased in the IMAP medium treated group. NCFs should be freshly prepared and healthy after isolation.
A rapid procedure of heart isolation, cutting, and proper digestion time can help to avoid reduction in cell viability and condition. This method provides a way to identify functional maturated ICMs for cardiac reprogramming researchers. With multiple further evaluation methods, one can get a more concise understanding about functional maturated ICMs.
