Fig 1.
Formation of cardiac spheroids, spheroid pairs, and elongated microtissues.
(A) 3D spheroids were generated using non-adhesive agarose gels with cylindrical recesses with hemispherical bottoms to guide self-assembly. CFs and CMs in suspension were added separately or co-seeded together to the center of the hydrogel seeding chamber to generate monoculture CM or CF spheroids or coculture CM:CF spheroids, respectively. The cells were allowed to settle into the recesses and self-assemble. (B/C) After 3 days, individual spheroids were transferred with a capillary tube attached to a microdispenser and micromanipulator to (B) cylindrical microwells containing a preformed spheroid to form homotypic (X-X where X = CM, CF, or CM:CF) or heterotypic (X-Y where X = CM and Y = CF) spheroid pairs or (C) to non-adhesive troughs, in which they were positioned with spheroids of variable composition in the center (CM-CM-Z-CM-CM where Z = CM or CF). Over time the spheroid building blocks fused together into longer microtissues. The phase contrast images on the right were acquired 3 days after cell seeding (A) and 19–20 hrs after spheroid transfer (B/C). Scale bars: 800 μm.
Fig 2.
Schematics of 3D computer modeling.
(A/B) Schematics of 3D box modeling. One spheroid was modeled with a cube composed of 20×20×20 cells. Five spheroids were connected in series to have 20×20×100 cells. The middle spheroid was composed of CFs only, the boundary was composed of mixed CM and CF cells, and the remainder of the cells were CMs. The density profile is shown in panel B. The CF distribution is followed with Gaussian distribution in both border regions. (C) Example of an AP propagation map in the 3D box model. The simulated tissue was stimulated from the bottom layer as indicated. (D) Representative Vm traces from a CM at cell location 1 (CM @1), a CF at location 50 (CF @50), and a CM at location 100 (CM @100) along the propagation line.
Fig 3.
CM and CF cell size changes with passage and 3D culture.
(A) Cell diameter of CMs and CFs that were freshly isolated (P0) or precultured for 3 days (P1), recorded with a Countess Automated Cell Counter (n = 7–9 isolations). Mean ± SD. *,# p<0.05 (ANOVA). (B) P1 CFs or CMs were plated in 24-well hydromolds with 35 recesses at 0.6x106 cells/mold. After 3 days in 3D culture, spheroids were imaged and analyzed (N = 178–209 spheroids) for circular cross-sectional area. (C) Assuming spherical cell shape, spheroid volume was calculated from the cell diameters in A (solid bars) or spheroid areas in B (hatched bars). (D) Internuclear distance in DAPI-stained cryosections of spheroids of indicated composition (generated as in B), expressed as chord length across the spheroid/number of intersected nuclei (N = 28–30 spheroids). (E) H&E stained cryosections of monoculture and coculture spheroids of indicated compositions. Scale bar: 100 μm. (F) Representative Western blots of spheroid lysates (10 μg protein/lane) obtained after 2 days in 3D culture and probed with indicated antibodies. α-SA– α sarcomeric actinin; Vim–vimentin; N-Cad–N-cadherin; Pan-Cad–pan-cadherin; GAPDH–glyceraldehyde 3-phosphate dehydrogenase. Total protein visualized by Stain Free Technology (Bio-Rad) was used as loading control.
Fig 4.
Time course of self-directed assembly of homo- and heterotypic pairs of cardiac spheroids.
After 3 days in 3D culture as individual spheroids, CM, CM:CF (at 9:1 and 1:1 ratios), and CF microtissues were replated in pairs and allowed to fuse. Representative phase contrast images acquired over 19 hrs post assembly of homotypic (X-X) pairs, in which X are either CM, CM:CF(9:1), CM:CF(1:1), or CF spheroids, and heterotypic (X-Y) pairs comprised of a CM and a CF spheroid. Scale bar: 200 μm.
Fig 5.
Time course of changes in intersphere angle and length of homotypic and heterotypic spheroid pairs.
Intersphere angle (A/B) and long axis length (D/E) of indicated spheroid pairs that were generated as for Fig 4. Length is also presented normalized to pair length at 1 hr after replating (G/H). Homotypic (X-X) pairs: CM pairs (black, n = 51–52), CM:CF(9:1) pairs (dark grey, n = 21), CM:CF(1:1) pairs (light grey, n = 26), CF pairs (red, n = 31); heterotypic (X-Y) pairs: CM-CF pairs (blue, n = 37). Mean (solid line) ± SD (dashed lines). The original values behind the means and SD are provided in S2 Appendix. Linear regression analysis was carried out on each data set, and the regression coefficients are presented in bar graphs (C, F). * p<0.05 vs. ALL, % p<0.05 vs. CM:CF(1:1) pairs, # p<0.05 vs. CM-CF pairs.
Fig 6.
CM and CF distribution in homotypic and heterotypic spheroid pairs.
Cryosections of homotypic (X-X) and heterotypic (X-Y) pairs of spheroids of indicated cellular compositions were fluorescently double-stained with antibodies recognizing α-sarcomeric actinin (α-SA) and vimentin (Vim) to visualize the CMs (green) and CFs (red), respectively. Merged max projections of confocal image z-stacks are shown from representative spheroid pairs 7 hrs and 15 hrs after they were assembled. Scale bar: 50 μm. See S1 Fig for individual max projections plus DAPI staining.
Fig 7.
Electrotonic coupling and action potential propagation in elongated CM-CM-Z-CM-CM microtissues with a CM or a CF spheroid in the center.
CM and CF spheroid building blocks were assembled in hydrogel troughs to generate elongated microtissues comprised of 2 CM spheroids on each end (labeled 1, 2, 4, 5) separated by either another CM spheroid (panels A, C, and E) or by a CF spheroid (panels B, D, and F) in the center (labeled as Z). CM denotes the inclusion of 5% CFs in the CM spheroids. (A/B) Representative merged phase contrast and immunofluorescent images that were acquired 1–20 hrs after assembly of the indicated spheroids (shown in a schematic to the left). Scale bars: 200 μm. The corresponding movie for the fusing microtissue shown in panel B is provided as S3 Movie. To better visualize the two configurations and spheroid fusion over time, some CMs were infected with Ad-GFP (green) and the CFs with Ad-RFP (red) prior to generation of the individual spheroids. For all subsequent optical mapping experiments, non-infected CMs and CFs were used. (C/D) Space-time plots of AP propagation acquired from indicated locations of elongated microtissues 7 hrs after assembly of indicated spheroids. The Y axis indicates the pixel locations and the X axis indicates time. The color bar represents depolarization of membrane potential (ΔF/F). The elongated tissue was stimulated from the bottom with a microelectrode. (E/F) The corresponding AP traces from panels C and D. Note that the activation pattern across the center CF spheroid (red trace) shows discontinuous conduction with two consecutive depolarizations in the AP trace: the first one is in synchrony with the AP recorded from the bottom CM (#2), while the second one is in synchrony with the AP from the top CM (#4).
Fig 8.
Action potential propagation maps and conduction delay in elongated CM-CM-Z-CM-CM microtissues with a CF or a CM spheroid in the center.
CM and CF spheroid building blocks were assembled in hydrogel troughs as described for Fig 7. (A/B) Fluorescent images (left) and AP propagation maps (right) from elongated microtissues assembled from indicated spheroids. The corresponding movies are provided in S4 Movie. The color bar shown to the right of panel B represents time (in ms) for the AP propagation maps in both panels. (C) Time delay across a CM center spheroid (n = 7) and a CF center spheroid (n = 24). Mean±SD and individual data points. * P<0.05.
Fig 9.
Computer modeling of action potential propagation across CF spheroids.
(A) Sodium channel conductance (gNa) in CFs facilitates AP conduction: Conduction failure at the CF cube when CF gNa = 0 but conduction across CF cubes when CF gNa = 0.6 nS (see also S5 Movie). (B) Conduction time delay shortens with increasing gNa in CFs. (C) Relative mRNA expression of TTX-sensitive (SCN2A) and TTX-resistant (SCN5A) Na+ channel α subunits assessed by qPCR in CF spheroids and expressed relative to CM spheroids. Mean±SD plus individual data points (n = 7 samples each). (D/E) Role of the spatial pattern of CM-CF boundary in AP conduction. CF portion profiles were generated using Gaussian function. (D). The CF density profiles of non-conduction cases are drawn with dotted lines and conduction cases are drawn with red lines. Note that the spread of the CM-CF boundary caused by infiltration of CFs paradoxically promotes conduction across CFs. The first conduction across CFs occur when σ = 6.0 (red star in E).