(579b) Cell Tracking and Cell Ratio Modulation for Cardiac Tissue Engineering

Introduction and Background

Recent work in the field of cardiac tissue engineering suggests that myocyte-nonmyocyte interactions may be crucial to engineering viable and functional heart tissues [1].  We previously showed that cultivation of cardiomyocytes (CM) on pre-formed networks (Pre-culture) of fibroblasts/endothelial cells (FB/EC), enhanced the structural and functional properties of cardiac ?organoids? engineered at the microscale compared to simultaneously seeding the three cell types (Simultaneous Tri-culture), which resulted in non-beating organoids with a clustered morphology and lacking connexin-43 expression[2]. We hypothesized that these observations suggested a potential soluble factor-mediated mechanism involving vascular endothelial growth factor (VEGF) signaling.  The goals of this study were to better visualize how these clusters formed with time and to further modulate the cell ratios used previously to select for better tri-culture conditions. To this end, we conducted cell tracking studies to get a visual picture of how cells organized both spatially and temporally into organoids when tri-cultured versus pre-cultured. We also assessed the effect of systematically varying the CM fraction (to improve the functional properties of the organoids) as well as the EC fraction (in order to improve capillary cord formation). 

Materials and Methods

Microchannels were patterned by photopolymerization of liquid poly(ethylene glycol) diacrylate pre-polymer (PEG, 700 Da) around a polypropylene master, resulting in discs with microchannel features 100-200µm diameter, 4mm in length, as previously described [2]. The channels were coated with 5µL of Matrigel® and allowed to gel at 37^oC prior to the addition of cells.  Primary cardiac FB, an EC cell line (D4T), and primary neonatal rat CMs were used for tri-culture and pre-culture. For cell tracking studies, CellTracker? Red, CellTracker? Green and 4'-6-diamidino-2-phenylindole (DAPI) were used to label EC, FB, and CM, respectively. FB/EC were incubated in the dyes for 30 minutes at a concentration of 5µM while CM were incubated in DAPI at a concentration of 20µg/mL for 1 hour. For Pre-cultures, the FB/EC alone were mixed and seeded followed by CM two days later, while for Simultaneous Tri-cultures, CM/FB/EC were simultaneously mixed and seeded. The discs were flipped over and imaged under fluorescence microscopy at days 1 and 4 after seeding the CM. CM ratios were also varied as 40%, 60%, and 80% of the total cell number, which was fixed at 2x10⁵ cells/disc.  In the case of the CM 100% group, all 2x10⁵ cells seeded were cardiomyocytes enriched by pre-plating the native heart isolate twice in T75 flasks to remove the more adherent non-myocytes, as previously described [2]. The percentages of each cell type seeded are given in Table 1 below.

Table 1: Percentages of cells seeded in Cell Tracking/CM ratio experiments

In the second part of the study, the fraction of EC was varied to promote capillary cord formation.  From our cell tracking studies, we observed that the 60% myocyte fraction had the best structural and functional properties (elongated morphology and largest contraction amplitude). Thus, the myocyte fraction was fixed at 60% and the EC fraction was varied to span a range of densities. Two densities at the lower end of the spectrum (16×10⁴ EC/disc and 31×10⁴ EC/disc) were chosen because they were in agreement with seeding densities previously reported to support capillary cord formation. The latter value of 62×10⁴ ECs/disc corresponded to the EC seeding density used in the Pre-culture 60% group (Table 1), and therefore served as a control for comparison to cell tracking studies. As the fraction of ECs increased, the fraction of fibroblasts seeded 24 hours later was correspondingly decreased to ensure the total cell density was maintained at 2x10⁵ cells/disc.  Finally, two days after EC seeding, CM were seeded at a fraction of 60%.  The final percentages of cells seeded in each group are given in Table 2 below.

Table 2 : Percentages of cells seeded in EC ratio / cord experiments

Results and Discussion

Figure 1 shows the results of cell tracking studies performed on Pre-cultures grown at three different myocyte fractions (40%, 60%, and 80% of the total cell number). Cardiac FB are labelled green, D4T EC are labeled red, and neonatal CM appear as blue nuclei. In general cells appear more elongated in both the 60% and 80% groups than in the 40% group.  As well, the cells appear more elongated on day 1 than on day 4.  As expected, a higher proportion of FB (green) is seen in the 40% group compared to more EC (red) and CM (blue nuclei) in both the 60% and 80% groups. In the 60% cells appear very elongated and display evidence of cords (white arrows) consisting of both EC and FB, suggesting that this condition might be more conducive to formation of vascular precursors.  Spontaneous contractions in the 60% and 80% were of a larger amplitude than in the 40% group.

Figure 2 shows the same data obtained from cell tracking studies on Simultaneous Tri-cultures. A comparison with Pre-cultures revealed marked differences in morphology. Cells appeared rounded and organized into spherical clusters (white arrows). Clusters were not apparent on day 1 but became more apparent on day 4, suggesting the cells organized into clusters over time.  The clusters also appeared to increase in size with an increase in myocyte fraction. For the 60% CM fraction, fibroblasts (green) organized on the periphery of clusters while for the 80% CM fraction, both CMs (blue) and also fibroblasts (green) organized on periphery of clusters.  In all three groups, EC (red) were primarily seen to organize within the centers of clusters, and this was more evident in the 60% and 80% groups. This localization of EC into clusters was also consistent with immunofluorescence data shown in previous studies[2]. We noted no spontaneous contractions in the Simultaneous Tri-culture 40% condition, consistent with our previous work [2], but were able to see sparse areas spontaneously contracting in the higher myocyte fractions (60%, 80%), suggesting that even in the case of Simultaneous Tri-culture it may be possible to restore contractile function by increasing the myocyte fraction.

To engineer vascularized organoids, we seeded D4T endothelial cells on Matrigel coated PEG microchannels to allow for capillary sprouts to form, a phenomenon reported to occur within 24 hours under these conditions.  The ratio of CM was fixed at 60%, while the ECs were seeded at varying densities from 16×10⁴to 62×10⁴ cells/disc. As shown in Figure 3 (top panel) 24 hours after seeding the endothelial cells, thin EC cords had formed in the 16k group (white arrows), but in the other groups, most of the cells had already proliferated into EC monolayers with only some evidence of cord formation at edges (single asterisks).

We then seeded cardiac FB 24 hours after cords had formed in order to stabilize them and allowed them to adhere for additional 24 hours.  Finally, we seeded Enriched cardiomyocytes at the 60% fraction (total cell number = 2×10⁵ cells/disc) and cultivated the cells for 7 days until organoids had formed. Figure 3 (bottom panel) shows phase contrast images of the resulting engineered organoids on Day 7 as a function of EC density.  The 16k group formed three-dimensional organoids (white arrows) characterized by elongated cells.  However in the higher EC fraction groups, we noted organoids that were extremely flat in morphology.  As the EC density increased from 31k to 62k per disc, we also noted fewer spontaneous contractions and more rounded cells. We observed that the 16k group had a low excitation threshold (comparable to enriched CM alone) and higher overall viability as determined by live/dead staining.

References

1. Naito H, Melnychenko I, Didie M, et al. Optimizing engineered heart tissue for therapeutic applications as surrogate heart muscle. Circulation. 2006 Jul 4;114(1 Suppl):I72-8.

2. Iyer RK, Chiu, L. L. Y., Radisic, M. Microfabricated poly(ethylene glycol) templates enable rapid screening of tri-culture conditions for cardiac tissue engineering. Journal of Biomaterials Research A. (2008, in press.)

Acknowledgements

The author wishes to thank Dr. P. W. Zandstra, Dr. M. V. Sefton,, and Dr. W. L. Stanford for cell lines, laboratory space, and use of their equipment.  Funding was provided by NSERC (DG), CFI (LOF), ORDCF (ARTEC), OGSST, OGS, NSERC USRA, and NIH grant R01HL076485.

Figure 1: Cell Tracking on Pre-cultured organoids at three myocyte fractions (40%, 60%, 80%). EC appear red, FB appear green, and CM nuclei appear blue.

Figure 2: Cell Tracking on Simultaneously Tri-cultured organoids at three myocyte fractions (40%, 60%, 80%). EC appear red, FB appear green, and CM nuclei appear blue.