Micropost arrays for measuring stem cell-derived cardiomyocyte contractility

Abstract

Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes.

Keywords: Cardiomyocytes; Cell mechanics; Induced pluripotent stem cells; Microposts; Soft lithography.

Figure 1

Structural and force differences in (A) immature SC-CMs and (B) adult cardiomyocytes. In the top schematics, the cytoplasm is colored dark red, the sarcomeric Z-discs are white, and the nucleus is gray. The arrows in the bottom schematics indicate the primary directions of contractile forces. Stem cell-derived cardiomyocytes have a smaller, more round morphology, with unaligned myofibrils, resulting in less force generation than their adult counterparts.

Figure 2

(A) SU-8 lithography is used to create an SU-8 master of the microposts. (B) Manufacturing of the PDMS microposts uses a double-casting process with a PDMS negative mold. (C) Microcontact printing is used to coat the tips of microposts with extracellular matrix (ECM) proteins before seeding cells.

Figure 3

SEM image of PDMS microposts used in estimating the spring constant. Length and diameter are measured from the image, accounting for the rounded edges, and used in Eq. 1 to determine the stiffness of the microposts.

Figure 4

A phase contrast microscopy video is taken at an image plane that contains the tips of the microposts. A reference image is taken at an image plane near the base of the microposts. The video frames and reference image are analyzed with a series of image analysis steps using custom GUI. Adapted from [39].

Figure 5

(A) Phase contrast image of a cardiomyocyte on microposts. The image is segmented into a grid such that each grid-box contains a single micropost. Adapted from [39]. (B) The centroid of the micropost is measured for each video frame (red ●), and the distance from the reference position (blue ×) is used to measure its displacement. (C) For each micropost, the deflection is calculated at each frame to produce a waveform. Posts near the edge of the cell (blue) typically deform much more than posts near the middle (red). (D) The calculated force waveform from a single contraction from the cardiomyocyte and the characteristic times.

Figure 6

(A) Composite immunofluorescent image showing microposts (red), sarcomeres (green), and nucleus (blue). Adapted from [40]. (B) A linescan (shown in yellow) is drawn across at least four sarcomeres, and the distance between the peaks is averaged to determine sarcomere length.