Our finding suggests that optimizing the endothelial cell cardiomyocyte co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection against simulated ischemia reprofusion injury. Cell co-culture models have been used extensively to investigate the role of cell-cell interactions on cell function and differentiation. However, separate treatments between cell types and downstream analysis of a single cell type are not readily feasible in the mixed co-culture.
The current study aimed to create two separate cell layers with a meaningful distance between them where one can induce hypoxia reprofusion injury and study the result and effects on a single cell type. Begin the preparation by thawing both cell lines. Plate the cells in T25 flasks after washing them with fresh media.
On the next day, refresh the cells with media and use when confluent. Maintain the cell culture incubator at 37 degrees Celsius with 21%oxygen, 5%carbon dioxide, and 74%nitrogen. Keep it humidified.
Estimate the cardiomyocytes cell line confluency under a light microscope. Remove the media from the flasks containing confluent cell cultures and trypsinize with three to five milliliters of trypsin-EDTA per flask. Agitate the flask gently and incubate at 37 degrees Celsius for two to five minutes.
After that, assess the enzymatic progress under a light microscope. After detachment, inactivate the trypsin solution by adding the trysin or cell solution to a 50 milliliter tube containing 10 milliliters of media per T25 flask. Centrifuge the cell suspension at 120 x g for two minutes to obtain a soft cell pellet.
After centrifugation, remove the supernatant and resuspend the pellet in five milliliters of a media. Next, perform cell counting and confirm cell viability. Mix and aliquot of 10 microliters of resuspended cells and 10 microliters of trypan blue dye, then count the living cells using a cell counter.
Dilute the cells with fresh regular media to achieve the desired seeding densities. Take a 24-well plate pre-coat it with extracellular matrix and plate cardiomyocytes at seeding densities of 300, 000 per well onto the bottom of the plate. Maintain the cells at 37 degrees Celsius with 5%carbon dioxide overnight.
After 24 hours, plate endothelial cells into inserts at an optimal plating density of 100, 000 per insert. After 24 hours of endothelial cell plating, place endothelial cell inserts inside the cardiomyocyte wells and initiate co-culture. Allow cells to co-culture for 12 to 24 hours before performing the experiments.
Prepare the hypoxic media by pouring approximately 25 milliliters of media in a 50 milliliter conical tube. Air seal the top with a silicone membrane and use a sterilized pipette to punch a hole. After that, create another hole and leave approximately 2/3 of the pipettes submerged in the media.
Flush the media with hypoxic gas for five minutes at a flow rate of 30 liters per minute, then discard the media of the 24-well plates or culture inserts containing attached cells. Wash them very gently with 100 microliters of 10%PBS per well add 500 microliters of the freshly prepared hypoxic media to each well of the plates or inserts. Replace the original media with fresh normal media containing glucose and serum in the normoxic control group.
Place a Petri dish filled with sterile water into the hypoxia chamber to humidify the chamber. Then place the plates containing the hypoxic groups into the chamber. Flush the hypoxia chamber with hypoxic gas for five minutes at a flow of 30 liters per minute.
After that, place the chamber inside an incubator at 37 degrees Celsius for 24 hours. After hypoxia, discard the old media of the plates or inserts and add 500 microliters of normal media containing glucose and serum to each wall of the plates, then store the plates inside an incubator under normal culture conditions for two hours to mimic reperfusion. Transfer the cell culture media from the 24-well plate into a 96-well plate, then take 200 microliters of media from each well of the 24-well plate and equally distributed into four wells of the 96-well plate.
Measure the absorbance for LDH using a cytotoxicity assay kit in a 96-well plate and follow the manufacturer's instructions to determine the degree of cellular injury. In this study, three types of inserts were evaluated. All three inserts had the same pore size of 0.4 micrometers.
The only difference among them was the insert to base height, allowing the distances between the two co-cultured cell layers to be 0.5, 1.0, and 2.0 millimeters. Using this protocol, cells were cultured under normal oxygen conditions and then split into two groups a normoxic control group and the hypoxia group. After 24 hours, both groups were refreshed with media and cultured under normal oxygen conditions for another two hours before conducting the endpoint assays.
A comparison of the three types of culture inserts in assessing lactate dehydrogenase release for cardiomyocyte alone, endothelial cells alone, and co-culture of cardiomyocyte with endothelial cells is shown here under normoxic, hypoxia only, and hypoxia reoxygenation conditions. With a 0.5 millimeter distance between the cell layers, hypoxia led to a significantly increased LDH release compared to normoxia when cardiomyocytes were cultured alone. However, LDH was only increased slightly in the hypoxia reoxygenation group compared to the hypoxia only group.
When endothelial cells and cardiomyocytes were co-cultured together, the increase of LDH release under the hypoxia only conditions was not attenuated. This indicates that the endothelial cells did not have any protective effect compared to the cardiomyocytes alone group under the hypoxia only conditions. However, endothelial cells exerted mild but significant protection on cardiomyocytes during HR.When the distance between the cell layers was one millimeter, the LDH release was also significantly increased in cardiomyocytes only under the hypoxia only condition.
However, unlike in the 0.5 millimeter insert, the LDH increase in cardiomyocytes only was potentiated by HR over hypoxia only conditions. This increase was even more pronounced with the two millimeters insert. The concentration of endothelial cells plated in two millimeter inserts was titrated to 25, 000, 50, 000 and 100, 000 cells per insert, respectively.
it was found that increasing endothelial cell intensity in the co-culture led to a dose-dependent attenuation of LDH release caused by HR.However, no such effect was seen under normoxic conditions. This co-culture methodology is not limited to endothelial cells and cardiomyocytes. These methods should aid an investigation of specific cell-cell interactions amongst varied cell lines across multiple organs of interest.
