心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

The overall goal of this procedure is to create human engineered cardiac tissues of a defined cellular composition. This method can address key challenges in the cardiac tissue engineering field, including controlling for variability and cardiac differentiation efficiencies and understanding how specific cell types impact cardiac contractile function. The main advantage of this technique is that the end result is human engineered cardiac tissue of a controlled and defined composition.

Replications of this technique extend toward therapy for heart disease because defined tissues can provide a novel, biologically relevant and biomedic screening platform capable of evaluating therapeutic interventions. Though this method can provide insight into novel cardiac therapeutics, the human engineered cardiac tissue system can also be used to understand mechanisms of cardiac cell therapy. Beginning with human embryonic stem cell-derived cardiomyocyte cultures, wash the cells one time with PBS and add one milliliter of 0.04%trypsin-EDTA.

Incubate the cells for 10 minutes at 37 degrees Celsius and 5%CO2. When the cells are incubating, add 12 microliters of ROCK inhibitor to six milliliters of trypsin neutralization solution. Remove the plates from the incubator and add one milliliter of the neutralization solution to each well of the six-well plate.

Transfer all of the cells from each well into a single 15-milliliter centrifuge tube. Wash all six wells with one three-milliliter volume of PBS and combine this wash with the cells in the tube. Centrifuge the tube at 300 times G for five minutes at four degrees Celsius to pellet the cells.

To prepare the cells for live cell sorting by FACS, prepare the staining buffer by adding five milliliters of FBS and 50 microliters of ROCK inhibitor to 45 milliliters of PBS on ice. Remove the supernatant from the pelleted cells and resuspend them in 1.2 milliliters of staining buffer. Transfer 200 microliters of the suspended cells to a new prechilled 50-milliliter centrifuge tube on ice.

Next, transfer the remaining one milliliter of the cell suspension to a new prechilled 50-milliliter centrifuge tube on ice and add two microliters of SIRP alpha-PE/Cy7 and four microliters of CD90-FITC antibodies. Gently mix the cell suspension with a transfer pipette and place the tube on ice. Incubate the two 50-milliliter tubes containing the negative control and the sample on a rocker shaker on ice at four degrees Celsius for one hour.

While the cells are incubating, transfer three milliliters of differentiation medium two to each of two 15-milliliter centrifuge tubes. Then add three microliters of ROCK inhibitor, and store these FACS collection tubes on ice prior to use. Next, pellet the stained cells at 300 times G for five minutes at four degrees Celsius.

And wash them twice with 10 milliliters of ice-cold PBS. Gently resuspend the sample pellet with one to three milliliters of staining buffer containing DAPI. Add 500 microliters of staining buffer without DAPI to the negative control pellet.

Filter both of the cell suspensions through a 40-micron cell strainer to remove cell clumps, and then transfer them to polystyrene FACS tubes on ice. Immediately bring the samples to the cell sorter. Beginning with the negative staining control sample, establish the gating parameters.

Then, while running the sample cells, select for the DAPI negative live cell population. Collect both the FITC positive and PE/Cy7 positive cell populations independently at 20 PSI. After culturing and reaggregating cells, as indicated in the text protocol, remove the cell culture plates from the incubator and transfer the medium to a 50-milliliter centrifuge tube.

Wash the plates with three milliliters of PBS and transfer the wash to the same 50-milliliter centrifuge tube. Then add three milliliters of 0.04%trypsin-EDTA to the plates and incubate at 37 degrees Celsius and 5%CO2. After five minutes, examine the plates for cell detachment using a microscope.

Once all of the cells have detached, add three milliliters of trypsin neutralization solution to the plates. Gently mix and transfer the cells to the original 50-milliliter centrifuge tube. Wash the plates with five milliliters of PBS and add this wash to the tube as well.

Pellet the cells by centrifugation at 300 times G for five minutes at room temperature. Remove the supernatant and resuspend the pellet in one milliliter of neonatal bovine serum, or NBS medium. Next, transfer the cells to a 1.5-milliliter microcentrifuge tube.

Pellet the cells at 300 times G for five minutes at room temperature and remove the supernatant. To begin generating the six engineered cardiac tissues, dilute 60.0 microliters of the five milligrams per milliliter collagen stock to 3.125 milligrams per milliliter with 1.5 microliters of one molar sodium hydroxide, 9.6 microliters of 10X PBS, and 24.9 microliters of sterile ultra pure water. Here, it is critical to avoid the introduction of air bubbles into the collagen mix as the air bubbles are slow to release from the viscous solution and can interfere with tissue formation during tissue compaction.

Pipette down the side of the 15-milliliter tube to add 12.0 microliters each of 10X MEM and 0.2 normal heaps at pH 9 to dilute the collagen mixture. Then add the basement membrane matrix to the collagen mixture to a final concentration of 0.9 milligrams per milliliter and mix gently. Store this final tissue mix on ice.

Next, transfer the entire tissue mix to the cell pellet and add supplemental cells as indicated in the text protocol. Fill the tube to a final volume of 150 microliters with cell-free NBS media and mix gently. Carefully pipette 25 microliters of the cell suspension into each one of the six wells in the bioreactor base plate.

Repeat the mixing procedure between each well to resuspend any cells that may have settled. Pipette only one tissue per well at a time to ensure a consistent volume and cell number per tissue. Separately, push two rows of polydimethylsiloxane, or PDMS, force sensors onto either side of the polysulfone frame to form six pairs of opposing posts.

Invert the frame on top of the base plate so that one pair of posts enters each well containing the cell suspension. Then place the bioreactor into a 60-millimeter dish and place that dish without its cover inside of a 10-centimeter dish. Place the lid on the 10-centimeter dish and move the entire bioreactor assembly into the tissue culture incubator.

After two hours of incubation, remove the bioreactor assembly and add 14 milliliters of NBS medium to cover the base plate. Return the bioreactor to the cell culture incubator and change half of the medium every day. After 48 hours, remove the base plate gently a few millimeters at a time.

Then return the bioreactor with the tissue facing down to the medium. If a tissue falls off of a post when removing the base plate, gently reattach the tissue onto the post. Differentiation of human embryonic stem cells results in a mixed culture of primarily cardiomyocytes and fibroblasts that self-organize into clusters and form robustly beating webs throughout the dish.

FACS sorting of these cultures revealed that this differentiation method results in a population that contains at least 65%SIRP alpha positive cells and 10%CD90 positive cells. After removing the base plate, the human engineered cardiac tissues, or HECTs, are allowed to self-assemble within the bioreactor. Mature and compacted HECTs visibly deflect the integrated force sensing posts with an average of five to 15 micronewtons of force.

Twitch force measurements reveal how isolated variables alter the contractile force in these defined engineered tissues. Here, when the tissues are supplemented with 10%human mesenchymal stem cells, a substantial enhancement of the contractile force is observed at both the one-hertz and two-hertz pacing frequencies. Once mastered, the differentiation will take approximately 20 days, the cell sorting about five hours, and the tissue construction about three hours.

Another seven days will be required for proper tissue formation after construction. Following this procedure, other methods, including cell labeling and supplementation of the tissue mix, can be performed in order to answer additional questions regarding the effects of specific cell-cell interactions or to determine the mechanisms of cell therapy. After watching this video, you should have a good understanding of how to create human engineered cardiac tissues with a known and controlled cellular composition.