In vitro 3D culture platform for large-scale imaging by hybrid gel cube

In vitro three dimensional (3D) culture is essential to analyze morphogenesis, molecular interactions, and drug evaluations, as well as regenerate 3D tissue. However, the observation of developed 3D structure is often difficult, especially if the size of the sample is relatively large. The z-resolution of fluorescent imaging was low when low magnification lens (4× and 10×) was used due to large focal depth. We have developed a 3D culture device enabling the sample to be handled, rotated, and transferred during culturing, composed of two different hydrogels in a cubic frame. The target sample can be observed from six directions by rotating the device. By using the developed platform, the sample 3D structure was recognized without fluorescence by synthesizing multiple views. Here, we have achieved to recognize the 3D shapes of reconstructed small airway branching structure without fluorescence. In addition, the resolution of z-direction in laser fluorescent image was improved considerably by synthesizing multiple views. Millimeter depth imaging of developed bronchial tree was achieved with high z-resolution, which cannot be achieved by regular confocal microscopy.