The cell medium was added on the top of the cell-hydrogel. For bright-field OPT imaging, the cell culture medium was removed from the FEP tube, and cell-laden hydrogels were taken by pumping a portion of the sample into an FEP tube. research. In TE, there is an urgent need for methods to image actual three-dimensional (3D) cell cultures and access the living cells. This is hard using established optical microscopy techniques such as wide-field or confocal microscopy. To address the problem, we have developed a new protocol using Optical Projection Tomography (OPT) to extract quantitative and qualitative measurements from hydrogel cell cultures. Using our tools, we demonstrated the method by analyzing cell response in three different hydrogel formulations in 3D with 1.5?mm diameter samples of: gellan gum (GG), gelatin functionalized gellan Vc-seco-DUBA gum (gelatin-GG), and Geltrex. We investigated cell morphology, density, distribution, and viability in 3D living cells. Our results showed the usability of the method to quantify the cellular responses to biomaterial environment. We observed that an elongated morphology of cells, thus good material response, in gelatin-GG and Geltrex hydrogels compared with basic GG. Our results show that OPT has a sensitivity to assess in actual 3D cultures the differences of cellular responses to the properties of biomaterials supporting the cells. strong class=”kwd-title” Subject terms: Biophysics, Cell biology, Materials science, Optics and photonics Introduction Tissue engineering (TE) is usually a fast-growing field that is designed to restore the structure and function of diseased or damaged tissue through the use of cells, supportive biomaterials, and biologically Vc-seco-DUBA active molecules1. In TE, various Rabbit Polyclonal to Cytochrome P450 24A1 types of biomaterials are used as scaffolds. Among these, hydrogels are becoming progressively attractive due to their high quantity of water and biocompatibility, while their mechanical and structural properties mimic many soft tissues1. Extracellular matrix (ECM)-mimicking hydrogels are thus the key to the progression of cell culture models from smooth 2D surfaces to Vc-seco-DUBA 3D structures that are more representative of human tissues2. Hydrogels have recently received attention in drug testing and have been used as 3D culture microenvironments in vitro to predict drug response in vivo3. In this paper, we developed a 3D quantitative imaging process based on optical projection tomography (OPT) and demonstrate its applicability for the quick and effective screening of 3D hydrogel cell cultures utilized for TE applications. A variety of hydrogels can be produced from synthetic or natural biopolymers or their combinations and can be selectively applied for specific applications based on their physical and biological properties4. This creates a need to systematically study their overall performance as macroscopic scaffolds for cell culturing5. During culturing, cell properties can be Vc-seco-DUBA influenced by a variety of factors, such as interactions with scaffold biomaterials, cell culture times, the density of cells, and cell signaling processes6. The microenvironment of cells, such as the surrounding ECM and neighboring cells, define the cell morphology, i.e., size and shape, through adhesive causes and cell-to-cell interactions7. Most of our understanding of such biological processes, however, comes from cells cultured on a 2D substrate8. Yet, it is well known that there is a significant variance in cell behavior when cells are encapsulated in a 3D environment compared with 2D surface culturing. When cells do not have enough attachment sites, they remain round and inactive7. Changes in cell morphology from spherical to a spread or elongated shape are, therefore, a strong indication that this cells prefer their culturing environment7,9. Hence, methods to evaluate cell and scaffold properties in Vc-seco-DUBA 3D in the mesoscopic level are needed to facilitate the generation of functional tissue in vitro. To image cells in a 3D cell culture environment, most optical methods can only image samples to a limited depth, which is the major challenge in imaging TE scaffolds10. For example, confocal microscopy (CM) has been a useful tool for the high resolution functional imaging of cells11. However, CM can only image samples to a depth of up to 300?m12. Although two-photon fluorescence microscopy (TPFM) can provide high-resolution fluorescence images of cell samples at a higher penetration depth (~?500?m) and is less phototoxic to live samples when compared with CM, the technique is still limited by its velocity and depth of imaging13. The development of the selective plane illumination microscopy.