This protocol describes a novel and easy mold based method for three dimensional cardiac tissue creation, which uses spheroids as a tool for scaffold free cardiac tissue creation. In our experiments three types of cells, human induced pluripotent stem cell-derived cardiomyocytes, human cardiac fibroblasts, and human umbilical vein endothelial cells were isolated and used to generate a cell suspension. With 70%cardiomyocytes, 15%fibroblasts, and 15%endothelial cells, in a 50ML conical tube.
We then dispense the cell suspension into each well of an ultra low attachment hanging drop system, which will result in the spontaneous formation of hundreds of beating spheroids after three days. The spheroids are then harvested and seated into a novel mold cavity. The tissue is then maintained in a sterile container with medium, on a shaker in the incubator for several days.
The mold is subsequently removed, resulting in a scaffold free tissue patch with mechanical integrity. The hanging drop device is a reusable, ultra low attachment well with 850 micro pores, each with a diameter of 350 microns. The hanging drop is compatible with standard six well plates.
After making the cell suspension we dispense four milliliters of cell suspension containing 10 million cells, to each well of the hanging drop system. The devices are cultured at 37 degrees celsius, 5%carbon dioxide and 95%humidity. After 72 hours spontaneous formation of beating spheroids, with an average diameter of 350 microns is observed.
The spheroids are harvested by placing the hanging drop device into another dish with medium, and swirling gently to release the spheroids and allow them to drift through the pores at the bottom of the hanging drop system. This is a schematic of the novel mold for three dimensional, scaffold free, cardiac tissue creation. It includes the handling mat, filling base and novel mold.
The novel mold is comprised of a base, a bottom square plate, a bottom net, 10 layers of side nets made with fine stainless steel prongs. A top net, a top square plate, the holding tubes, and stopper pins. The Novel Mold system can create different sizes of tissues, including two by two millimeters, four by four millimeters, and six by six millimeters.
The base bottom square plate and bottom net are stacked and aligned using the corner posts. 10 layers of side nets are then stacked in alternating directions to create a net of fine stainless steel prongs, with the aid of a pair of sterile forceps. The filling base is then flushed with 1X phosphate buffer solution, or PBS, to reduce the surface tension prior to seating the spheroids into the novel mold.
After wetting the novel mold cavity in the same methane the spheroids are seated into the presupposed cavity of desired size. Upon seating the excess medium drains through the net mold, allowing the spheroids to fill and assume the shape of the predefined cavity. The top net and top square plate are then assembled onto the corner posts and the stopper pins and the holding tubes are fixed, to prevent washout of the spheroids.
The filled novel mold system is transferred to a six well plate, with six millimeters of B-27 supplemented medium, and then maintained in the incubator on a swinging shaker for seven to ten days, at 30 to 40 RPM. The duration of incubation can be altered, depending on the size of the cardiac patch. After incubation the system is taken out and the holding tube, stoppers, the top square plate and the top net are removed.
The layered side nets are then carefully slid out one by one, taking care not to disrupt the tissue. After removal of all side nets, an intact cardiac patch is obtained on top of the bottom net. Functional synchronous beating is observed within 24 hours.
This novel mold based method is an efficient and easy method of creating three dimensional scaffold free cardiac tissues, with minimal manual effort. The resulting multi-layered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This novel mold based method presents a cost effective and scalable alternative to current bio-fabrication methods of engineered tissues.
