engineered cardiac tissues  (10X Genomics)

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: Bioreactor design (A) Representation of the modular bioreactor incorporated with 24-well culture plates. The 3D printed bioreactor frame (c.) is placed between the 24-well plate (d.) and the lid (a.). It is possible to combine 6 independent mechanical stimulation modules (b.), allowing independent mechanical stimulation of 4 separate culture well rows (B and C). (D) Representation of ECTs cast in between 2 metal wires of the mechanical stimulation module. (E) Images of the cell-based fibrin gel generation in custom-made silicon mold. (F) Bioreactor’s modules functioning scheme: flexible wires provide resistance during auxotonic contraction and serve as sensors for measuring the contractile force F (a.); rigid wires are fixed in a moving shaft, connected to a driving stepper motor for delivery of active cyclical stretching (b.); the wire couple in between which the ECT is generated are used as conductive electrodes and connected to an electrical stimulator for application of electrical stimuli (c.).

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Generated

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: ECTs cardiac maturation (A) Representative immunofluorescence images of sarcomere organization in control ECTs and stimulated ECTs, stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan), F-actin (F-actin, green). Nuclei were stained in blue (DAPI, blue). Area of control was identified for the sarcomere length quantification analysis. Scale bar: 50 μm. (B) Image-analysis-based quantification of sarcomeric α-actinin positive area, cardiomyocytes (CMs) length and elongation (n = 2, from 2 independent experiments), and sarcomere length quantification (n = 2, from 3 independent experiments) ∗p ≤ 0.05; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (C) Schematic representation of the two identified zones within the stimulated ECTs: high resistance (HR) and low resistance (LR). (D) Representative immunofluorescence images of statically cultured constructs and stimulated constructs, with a detailed view of low-resistance (LR) and high-resistance (HR) zones, stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan). Nuclei were stained in blue (DAPI, blue). Scale bar: 100 μm. (E) Representative images of cardiomyocyte structures in control and stimulated tissues (HR and LR portion) acquired using transmission electron microscopy (TEM) technique. Mitochondria were indicated with M letter, nuclei with N, and the surrounding construct with C. White arrow heads pointed the sarcomeric myofibrils and yellow ones pointed Z-line, whereas read arrow heads indicated primitive desmosome structures. Scale bar: 1 μm. (F) Image-analysis-based quantification of sarcomeric α-actinin positive area, cardiomyocytes (CMs) length and elongation for the two zones within the stimulated ECTs; n = 2, from 1 independent experiment. (G) Graphical representation of the cardiomyocyte orientation within the tissue for statically cultured ECTs and stimulated ECTs (n = 2, from 2 independent experiments, ∗p ≤ 0.05) and schematic representation of the CMs alignment to the imposed passive force (H). Data were represented as mean ± SD.

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Immunofluorescence, Control, Staining, Marker, Cell Culture, Construct, Transmission Assay, Electron Microscopy

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: Gene and protein expression analysis (A) mRNA expression ratio of stimulated ECTs versus statically cultured ECTs. n = 10, from 3 independent experiments. (B) mRNA expression ratio of stimulated ECTs and statically cultured ECTs versus cardiac initial population. n = 4, from 1 independent experiment. Data were presented as mean ± SEM.

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Expressing, Cell Culture

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: ECTs remodeling (A) Images of ECTs at day 0 after cell-based fibrin gel generation (a.), and images of ECTs after 8 days of auxotonic stimulation (b.). (B) Representative brightfield images of different portions of ECTs at day 8: fixed wire portion (a.), middle portion (b.), and flexible wire side (c.). Scale bar: 500 μm. (C) Representative H&E staining showing cell distribution and orientation within control ECT and stimulated ECTs, distinguishing between low- and high-resistance zones. Scale bar: 200 μm. (D) Fibrosis markers of mRNA expression represented with stimulated-to-control ratio. (E) Representative immunofluorescence images of statically cultured ECT and stimulated ECTs stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan) and α-smooth muscle actin (α-SMA, red). Nuclei were stained in blue (DAPI, blue). Scale bar: 50 μm. (F) Representative immunofluorescence images of statically cultured and stimulated ECTs, low-resistance (LR) and high-resistance (HR) zones, stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan) and Ki67 (red). Nuclei were stained in blue (DAPI, blue). Scale bar: 50 μm. Data were represented as mean ± SEM.

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Staining, Control, Expressing, Immunofluorescence, Cell Culture, Marker

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: ECTs electrical functionality and evaluation of force of contraction Graphical representation showing the normalized excitation threshold (ET, A) and the maximum capture rate (MCR, B) of control and stimulated ECTs upon electrical stimulation (n = 18, from 4 independent experiments; ∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001). Measured stimulated ECT force upon electrical stimulation (C). Graphical representation of beating ECT strain upon electrical stimulation (F). (Response of a statically cultured ECT to an external electrical pacing) and (Response of a ECT culture under passive stimulation to an external electrical pacing) show examples of the beating constructs analysed. Strain peak values for control (n = 4) and stimulated (n = 4) ECTs (∗p ≤ 0.05, G). (Cantilever deflection upon electrical pacing of a ECT under passive) show the cantilever deflection analyzed to evaluate the construct strain. Representative contour plot from digital mage correlation (DIC) analysis of control (D) and stimulated (E) ECTs, during electrical stimulation. Electrical stimulation set-up of the bioreactor under live-imaging microscope (H). Detailed picture (I) and schematic representation (J) of the electrode set-up for one ECT replicate under live-imaging microscope. The electrical material parameters used to characterize the electrical stimulation module are summarized in Table 1 . Characterization of the electrical module. Data were represented as mean ± SEM.

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Control, Cell Culture, Construct, Imaging, Microscopy

Journal: iScience

Article Title: Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

doi: 10.1016/j.isci.2022.104297

Figure Lengend Snippet: hiPSC-based ECTs experiment (A) Representative immunofluorescence images of stimulated ECTs, high-resistance (HR) and low-resistance (LR) zones, stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan). Nuclei were stained in blue (DAPI, blue). Scale bar: 100 μm. (B) Representative immunofluorescence images of stimulated ECT, high-resistance (HR) and low-resistance (LR) zones, stained for sarcomeric α-actinin cardiac marker (α-actinin, cyan), homeobox protein Nkx2-5 (Nkx2-5, red), and octamer-binding transcription factor 4 (Oct4, green). Nuclei were stained in blue (DAPI, blue). Scale bar: 50 μm. (C) Graphical representation showing the normalized excitation threshold (ET) and the maximum capture rate (MCR) of stimulated (n = 2) ECTs upon electrical stimulation. (D) Measured stimulated ECT force upon electrical stimulation (n = 1). (E) Schematic representation of the two identified zones within the stimulated ECTs. All data were represented as mean ± SEM.

Article Snippet: Biomimetic approaches known to promote maturation ( ) of 3D engineered cardiac tissues (ECTs) typically provide during in vitro culture some of the key aspects of the multicellular and multibiophysical microenvironment typical of native heart tissue ( ; ; ).

Techniques: Immunofluorescence, Staining, Marker, Binding Assay