Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Formation of mouse primary cardiomyocyte clusters in agarose-coated wells. ( a ) Schematic drawing of the conventional dish cultivation of cardiomyocytes. The dispersed cells were cultured on the bottom of a 35-mm non-agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells attached on the bottom of the 35-mm cultivation dish dispersedly. The cells started to beat 2–3 days after cultivation started. ( b ) A micrograph of dispersed cardiomyocytes in a 35-mm non-agarose-coated dish. ( c ) Schematic drawing of the cultivation of dispersed cells in a 35-mm agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells dispersed on the bottom of the agarose layer in the agarose-coated 35-mm cultivation dish. Even after 2–3 days of cultivation, the cells remained isolated with a round shape, and no clusters formed on the bottom. ( d ) A micrograph of cardiomyocytes in an agarose-coated 35-mm cultivation dish. ( e ) Schematic drawing of the cultivation of dispersed cells in a 15.5-mm agarose-coated cultivation well (in a 24-well cultivation plate). After spread of the 1.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5\times 10^{4}\hbox { cells/mL}$$\end{document} 5 × 10 4 cells/mL isolated single cardiomyocytes, dispersed cells gathered and formed small clusters; finally, they gathered into a single large cluster in the 15.5-mm agarose-coated cultivation well. ( f ) A micrograph of a cardiomyocyte cluster in a 15.5-mm agarose-coated cultivation well.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Cell Culture, Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Micrographs of single cells and clusters of mouse primary and hES-derived cardiomyocytes. ( a ) Mouse primary cardiomyocytes (primary) in a 35-mm non-agarose-coated dish (single cell), ( b ) primary cells in a 24-well agarose-coated plate (cluster), ( c ) hES cardiomyocytes in a 35-mm non-agarose-coated dish (single cell), and ( d ) hES in a 24-well agarose-coated plate (cluster).

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Analysis of interbeat interval (IBI) distribution of single and clustered mouse primary and hES cardiomyocytes. ( a )–( d ): Method of measuring interbeat interval (IBI) of single cardiomyocytes and clusters. Temporal change of luminance in the red square area for single cell ( a ) and cluster ( c ) caused by their beating was recorded, as shown in the time-course intensity profiles ( b ) and ( d ), respectively. IBIs of their beating were acquired from the time intervals between two neighboring peaks in the time-course intensity profiles. ( e ), ( f ): Distribution of IBIs of mouse primary cardiomyocytes. ( e ) The relationship between mean IBIs and fluctuations of beating [coefficient of variability (CV) of IBIs] of single isolated primary cardiomyocytes (blue open circles, n = 73) and primary clusters (red filled triangles, n = 6). ( f ) A histogram of all plots in ( e ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean value and standard deviation (SD) of single-cardiomyocyte IBIs, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters. ( g ), ( h ): Distribution of IBIs in hES cardiomyocytes. ( g ) The relationship between mean IBIs and CV of IBIs in single isolated hES cardiomyocytes (blue open circles, n = 125) and hES clusters (red filled triangles, n = 27). ( h ) A histogram of all plots in ( g ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean values and SD of single cardiomyocytes, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distribution of IBIs and fluctuation of IBI distribution of the hES cardiomyocyte clusters and their constituent cells. ( a )–( c ): Micrographs of hES cardiomyocyte clusters. ( d )–( f ): Distribution of IBIs and the CV of IBIs in the clusters ( a )–( c ) and isolated constituent cells from each cluster (n=50 from among re-cultivated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.0\times 10^{3}\hbox { cells}$$\end{document} 1.0 × 10 3 cells ). These plots ( d )–( f ) correspond to each cluster ( a )–( c ). The red filled triangles indicate the cardiomyocyte clusters, and the blue open circles indicate constituent cardiomyocytes of each cluster. Each cluster was measured 2 days after the beating started. Single cardiomyocytes were isolated from each cluster by trypsinization. IBIs of single constituent cardiomyocytes were measured 3 days after their isolation. Median and 95% confidence interval of single cardiomyocytes were 0.971 s and 0.825–1.25 s ( d ), 1.19 s and 1.00–1.28 s ( e ), and 1.12 s and 0.844–1.22 s ( f ), respectively. ( g )–( i ): Histograms of IBIs of each cluster and its isolated constituent cells. The blue filled bars indicate the ratio of frequency for single constituent cardiomyocytes; the blue arrows and error bars indicate the mean IBIs and SDs, and the red arrows also indicate the mean IBIs of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Influence of trypsinization on interbeat intervals in dispersed individual hES cardiomyocytes. ( a ) Distribution of the IBIs and the CV of IBIs in single hES cardiomyocytes before and after trypsinization. The blue open circles indicate the single hES cardiomyocytes (n = 50) before trypsinization. The orange open circles indicate the single cardiomyocytes (n = 50) after trypsinization. ( b ) Histograms of IBIs of hES single cardiomyocytes before and after trypsinization. The blue filled bars indicate the mean IBIs of single cardiomyocytes before trypsinization; the blue arrow and error bar indicate their mean value and SD. The orange filled bars indicate the frequency of mean IBIs of trypsinized single cardiomyocytes; the orange arrow and error bar indicate their mean value and SD.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distributions of interbeat intervals (IBIs) and fluctuations of the two hES cardiomyocyte clusters before and after their connection and after re-separation. ( a )–( e ): Micrographs of cardiomyocyte clusters. Micrographs of the large cluster ( a ) and small cluster ( b ) before contact. These clusters were measured when they had been cultivated for 7 days. The two hES cardiomyocyte clusters were connected ( c ). The measurement was performed 3 days after the two clusters contacted each other. Micrographs of the large cluster ( d ) and small cluster ( e ) after separation. The measurements were taken within 5 min of separation. ( f ): Distribution of IBIs and fluctuations of two clusters before contact, during contact, and after separation. Blue filled bar and error bar indicate the mean IBIs and SD of the large cluster. Green filled bar and error bar indicate the mean IBIs and SD of the small cluster.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Nature Communications

Article Title: 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels

doi: 10.1038/s41467-021-21029-2

Figure Lengend Snippet: a (i) Development of healthy and scarred spheroids through mixing iPSC-derived cardiomyocytes (iPSC-CMs) with primary adult cardiac fibroblasts (CFs) at defined ratios of cell numbers (4:1 for healthy; 1:4 for scarred). Top: Images (cardiac troponin-T (cTnT) (red; iPSC-CMs); vimentin (green; CFs)) taken 3 days after cell seeding. Scalebar 50 µm. Bottom: Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) in healthy and scarred spheroids at 3 days. Scalebar 10 µm. (ii) Quantification of cellular composition through staining for cTnT (iPSC-CMs) and vimentin (CFs) ( n = 3 biologically independent samples, mean ± s.d, two-sided student t test, healthy - cTnT + vs. Vimentin + p = 5.6 × 10 −5 , scarred - cTnT + vs. Vimentin + p = 6.7 × 10 −4 ). b (i) Contraction profiles, (ii) contraction amplitude (a.u. absolute units), and (iii) peak-to-peak time (ms) of healthy and scarred cardiac spheroids at 3 days ( n = 3 biologically independent samples, mean ± s.d, two-sided student t test, (ii) p = 5.0 × 10 −4 ). c Quantification of calcium activation parameters from calcium mapping experiments in healthy and scarred spheroids at 3 days, including (i) calcium transient duration (ms), (ii) time-to-peak (ms), and (iii) calcium flux amplitude (F/Fo) ( n = 5 biologically independent samples, mean ± s.d, two-sided student t test, (i) p = 0.0014, (ii) p = 5.0 × 10 −6 , (iii) p = 2.0 × 10 −4 ). Note for n = 3 scarred spheroids a significant calcium activation peak was not observed, and these samples were not included in the statistical analysis and are denoted by x mark on the graph. All experiments are from a single iPSC-CM donor (donor A). (n.s. not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Human iPSC-CMs were purchased from Fujifilm Cellular Dynamics (iCell 01434 (Donor A) and 11713 (Donor B), purity >97%).

Techniques: Derivative Assay, Immunofluorescence, Staining, Activation Assay

Journal: Nature Communications

Article Title: 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels

doi: 10.1038/s41467-021-21029-2

Figure Lengend Snippet: a (i) Schematic of 3D bioprinting of healthy and scarred cardiac microtissue rings and (ii) immunofluorescence staining for cTnT and vimentin in healthy and scarred cardiac microtissues after 5 days of fusion within the support hydrogel. Scalebar 100 µm (insets 50 µm). (iii) Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) and (iv) connexin-43 (green; gap junctions) and cTnT (red; iPSC-CMs), in healthy and scarred regions of microtissues after 5 days of fusion within the support hydrogel. Scalebar 10 µm. b (i) Contraction profiles of healthy and scarred cardiac microtissues following removal from the support hydrogel after 5 days of culture. (ii) Contraction amplitude (a.u. absolute units) and (iii) Peak-to-peak time (ms) at 5 days ( n = 3 biologically independent samples, mean ± s.d, two-sided student t test, (ii) p = 0.061). c (i) Calcium mapping in healthy and scarred cardiac microtissues after 5 days culture, each image represents a 20 ms frame. Scalebar 100 µm. (ii) Representative calcium traces from regions 1 and 2 (see methods) in healthy and scarred cardiac microtissues. Scalebars 0.5 ΔF/F o (y), 500 ms (x). (iii) Activation maps of healthy and scarred cardiac microtissues, and activation delay (ms) (difference in activation time (ms) between regions 1 and 2) in healthy and scarred cardiac microtissues ( n = 3 biologically independent samples, mean ± s.d, two-sided student t test, p = 0.0035). d (i) Activation maps of scarred cardiac microtissues with 1 or 2 scars after 5 days of culture and (ii) quantification of activation delay (ms) ( n = 3-4 biologically independent samples, mean ± s.d, two-sided student t test, p = 0.0066). e Regional quantification of (i) calcium transient duration (ms), (ii) time-to-peak (ms), and (iii) calcium flux amplitude (F/Fo), in healthy and scarred regions of microtissues (scarred 1x) after 5 days of culture ( n = 4 biologically independent samples, mean ± s.d, two-sided student t test, (i) p = 0.010, (ii) p = 0.0098, (iii) p = 0.0029). Note full calcium transient duration, time-to-peak, and activation maps can be found in Supplementary Fig. . All experiments from a single iPSC-CM donor (donor A). (n.s. not significant, * p < 0.05, ** p < 0.01).

Article Snippet: Human iPSC-CMs were purchased from Fujifilm Cellular Dynamics (iCell 01434 (Donor A) and 11713 (Donor B), purity >97%).

Techniques: Immunofluorescence, Staining, Activation Assay

Journal: Nature Communications

Article Title: 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels

doi: 10.1038/s41467-021-21029-2

Figure Lengend Snippet: a (i) Schematic of cholesterol modified miR302 (chol-miRNA 302 b/c) delivery to scarred cardiac spheroids for 0, 0–2, 0–4, and 0–7 days. (ii) Contraction amplitude (a.u) and (iii) peak-to-peak time (ms) within scarred spheroids measured after 2, 4, and 7 days for each treatment period ( n = 4, 6, 5, 5, 7, 5, 5, 5, 5 biologically independent samples (from left to right), mean ± s.d, one-way ANOVA, (ii) day 4: 0 vs. 0–4 days treatment p = 0.014, (ii) day 7: 0 vs. 0–7 days treatment p = 0.0045, (iii) day 4: 0 vs. 0–4 days treatment p = 1.6 × 10 −7 , (iii) day 7: 0 vs. 0–7 days treatment p = 4.1 × 10 −9 ). b (i) Immunofluorescence staining for cTnT (red; iPSC-CMs), vimentin (green; cardiac fibroblasts), and EdU (proliferation marker) in scarred spheroids at day 7 for each treatment condition. Top panel scalebar 50 µm, and bottom panel scalebar 40 µm. (ii) Quantification of cardiomyocyte proliferation (EdU + and cTnT + ) and (iii) fibroblast proliferation (EdU + and Vimentin + ) at day 7 for each treatment condition. ( n = 3, 4, 4, 4 biologically independent samples (from left to right), mean ± s.d, one-way ANOVA, 0 vs. 0–4 days treatment p = 0.044, 0 vs. 0–7 days treatment p = 0.026). Scalebar 50 µm. c (i) Experimental outline where scarred microtissues are bioprinted in the support hydrogel as previously described, followed by 4 days treatment with miR302, and analysis compared to non-treated controls. (ii) Calcium mapping in scarred cardiac microtissues at 9 days (5 days culture in support hydrogel; (+) and (−) 4 days miRNA treatment) each frame 40 ms. Scalebar 100 µm. (iii) Representative calcium traces from regions 1 and 2 in treated and non-treated scarred cardiac microtissues. Scalebars 0.5 ΔF/F o (y), 500 ms (x). (iv) Activation time maps across scarred regions for treated and non-treated scarred cardiac microtissues, and quantification of activation delay (ms) ( n = 5 biologically independent samples, mean ± s.d, two-sided student t test, p = 0.019). d Immunofluorescence staining for cTnT (iPSC-CMs), vimentin (CFs), and EdU (proliferation) in scarred cardiac microtissues at day 9 for treated and non-treated scarred cardiac microtissues. Scalebar 50 µm. All experiments from a single iPSC-CM donor (donor B). (n.s. not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Human iPSC-CMs were purchased from Fujifilm Cellular Dynamics (iCell 01434 (Donor A) and 11713 (Donor B), purity >97%).

Techniques: Modification, Immunofluorescence, Staining, Marker, Activation Assay