Bio-acoustic levitational assembly of heterocellular 3D constructs: 3D model establishment for cells radiation effect studies in 3D microenvironment

Abstract

Introduction Many tissues are comprised of multilayered structures for which spatial organization and inter-layers cellular communications are essential to sustain their biological functions. Generation of multilayered constructs is motivated by the need to obtain tissue-like structures for many applications including radiobiology studies, where cells microenvironment is playing an important role. Purpose While current studies are using 2D monolayer cells culture and recent progress in genomic to study cells molecular response to RX, these 2D structures fail to represent 3D complex cells behavior depending on their surrounding environment, and could also impact radiation effects. We aimed at developing different 3D constructs such as neuronal or epithelial constructs that could be useful for radiobiology studies. Materials and methods To generate 3D constructs with native-like spatial organization, a novel strategy was developed using bulk acoustic standing waves, enabling easy, rapid and biocompatible 3D multilayers tissue constructs formation with tunable interlayer spacing and layers thickness. To show the device tunability, different cells types were used. Results First, we demonstrate the ability to generate multilayers 3D neural constructs assembling human ESC in fibrin hydrogels and differentiating them in neural cells. Then, to demonstrate the ability to bioengineer heterogeneous multilayers of cells in a single construct, three cell types (HeLa cells with different fluorochromes) were assembled subsequently via layer-by-layer acoustic levitation. Conclusion We have developed an acoustic technique to bio-compatibly assemble, within minutes, multilayered constructs with multiple cell types in fibrin hydrogels. This acoustical method is potentially useful for broad fields including radiobiology, where 3D constructs could be used to further explore effects of RX in native-like tissue. Many tissues are comprised of multilayered structures for which spatial organization and inter-layers cellular communications are essential to sustain their biological functions. Generation of multilayered constructs is motivated by the need to obtain tissue-like structures for many applications including radiobiology studies, where cells microenvironment is playing an important role. While current studies are using 2D monolayer cells culture and recent progress in genomic to study cells molecular response to RX, these 2D structures fail to represent 3D complex cells behavior depending on their surrounding environment, and could also impact radiation effects. We aimed at developing different 3D constructs such as neuronal or epithelial constructs that could be useful for radiobiology studies. To generate 3D constructs with native-like spatial organization, a novel strategy was developed using bulk acoustic standing waves, enabling easy, rapid and biocompatible 3D multilayers tissue constructs formation with tunable interlayer spacing and layers thickness. To show the device tunability, different cells types were used. First, we demonstrate the ability to generate multilayers 3D neural constructs assembling human ESC in fibrin hydrogels and differentiating them in neural cells. Then, to demonstrate the ability to bioengineer heterogeneous multilayers of cells in a single construct, three cell types (HeLa cells with different fluorochromes) were assembled subsequently via layer-by-layer acoustic levitation. We have developed an acoustic technique to bio-compatibly assemble, within minutes, multilayered constructs with multiple cell types in fibrin hydrogels. This acoustical method is potentially useful for broad fields including radiobiology, where 3D constructs could be used to further explore effects of RX in native-like tissue.