Three-dimensional paper-based model for cardiac ischemia

Abstract

In vitro models of ischemia have not historically recapitulated the cellular interactions and gradients of molecules that occur in a 3D tissue. This work demonstrates a paper-based 3D culture system that mimics some of the interactions that occur among populations of cells in the heart during ischemia. Multiple layers of paper containing cells, suspended in hydrogels, are stacked to form a layered 3D model of a tissue. Mass transport of oxygen and glucose into this 3D system can be modulated to induce an ischemic environment in the bottom layers of the stack. This ischemic stress induces cardiomyocytes at the bottom of the stack to secrete chemokines which subsequently trigger fibroblasts residing in adjacent layers to migrate toward the ischemic region. This work demonstrates the usefulness of patterned, stacked paper for performing in vitro mechanistic studies of cellular motility and viability within a model of the laminar ventricle tissue of the heart.

Keywords: 3D cell culture; cardiac ischemia; cardiomyocytes; co-culture; gradients.

Figure 1. Paper-based Model of Cardiac Ischemia

A) Image of a layer of wax-patterned paper and the top and bottom portions of a custom-made Delrin holder in which the cell containing papers are cultured. The Delrin material is non-toxic to cells and fully insulates the cell-containing stack from mass transport of outside nutrients. Scale bar: 8mm. B) Schematic of a single layer of paper containing 20 hydrophilic zones surrounded by a hydrophobic wax-printed border. Cells suspended in a hydrogel are pipetted into the hydrophilic zones. C) Schematic of a stack of paper in the Delrin holder comprising a top piece containing holes for access to culture media and a solid bottom piece. D) Heat map of the average fluorescence intensities of calcein-stained cells in a stack of six layers of paper. A darker color indicates higher levels of calcein, and is proportional to the number of viable cells. Each layer (clustered together as a stack of 20 slices) contains 20 replicate zones initially containing 100,000 neonatal rat cardiomyocytes suspended in matrigel. The stack was cultured for seven days prior to staining. E) Total number of viable cells in each zone of the stack for three different time points. Error bars are the S.D. of 20 zones. Means with different letters are significantly different based on Holm-Sidak method, (P = <0.001). Detailed statistics of each value available in the SI.

Figure 2. Effect of Ischemia on Morphology

A-B) Confocal, immunofluorescence images of the sarcomeric α-actinin in cardiomyocytes within a single zone of layer 1 (the top layer) and layer 6 (the bottom layer) of the stack after 7 days of culture. Scale bar: 50 μm. Inset Scale Bar: 10 μm. C) Graph of the average circularity of a set of 9-12 cells chosen at random from a single zone in each layer of the stack. Error bars are a S.D. of 9–12 cells in each layer, averaged between two different stacks. Means with different letters are significantly different based on Holm-Sidak method, (P = <0.001). Detailed statistics of each value available in the SI.

Figure 3. Migration of Cardiac Fibroblasts in Response to Varying Levels of Ischemia

A) Schematic of three stacks used to induce an environment of low ischemia (left), medium ischemia (middle), and high ischemia (right) for two layers containing cardiomyocytes (100,000 cells/zone) at the bottom of the stack. Each stack contains a different number of layers of immortalized 3T3 fibroblast (50,000 cells/zone) placed on top of the stack (the cells in these upper layers deplete nutrients diffusing toward the cardiomyocytes). Each stack contained a single layer of primary cardiac fibroblasts (10,000 cells/zone) between the 3T3 fibroblasts and cardiomyocytes. Cardiac fibroblasts were labeled with Cell Tracker Orange prior to seeding. Each stack was cultured for three days. B) Viability (determined with calcein AM) of the top layer of cardiomyocytes (e.g., cardiomyocytes in the second from bottom layer of the stack shown in A). C) Relative amount of migration of the cardiac fibroblasts into the top layer of cardiomyocytes, determined by the fluorescence intensity of Cell Tracker Orange in the second from bottom layer that contains either cardiomyocytes (in matrigel) or only matrigel. Error bars are the standard deviation of 10 replicates. Means with different letters are significantly different based on Holm-Sidak method, (P = <0.001). Detailed statistics of each value available in the SI.

Figure 4. Role of TGF-β Signaling in Invasion of Cardiac Fibroblasts

A) Schematic of a stack consisting of layers of cardiomyocytes and cardiac fibroblasts. B) Values of fluorescence intensity of fibroblasts, labeled with Cell Tracker Orange, that migrated into the adjacent layer of cardiomyocytes. Each condition is in the presence of a different concentration of TGF-β neutralizing antibody, for three days in a stacked culture. Error bars are the standard deviations for 20 replicates. Means with different letters are significantly different based on Holm-Sidak method, (P = <0.001). Detailed statistics of each value available in the SI.