In autologous valved conduits (biovalves) using in-body tissue architecture, the limited area available for leaflet formation is a concern. In this study, we designed a novel biovalve mold with slits to enhance in vivo cell migration, regardless of size. As a control, the original mold without slits was used. When both types of molds were embedded into subcutaneous pouches in beagle dogs for 8weeks, the outer surfaces of all molds were completely covered with connective tissue to form conduit tissue. In the molds without slits, the leaflet size was limited to half of the design. In contrast, in the mold with slits, the complete leaflet area was formed. Upon trimming excess peripheral tissues, removing the mold, and cutting the connective tissue formed at the slits, completely autologous connective tissue biovalves with the designed leaflet area were obtained as type S (diameter, 6-28mm) biovalves. The slit structure customized to the mold was effective for allowing cells to enter, thereby facilitating cell migration and contributing to the successful preparation of reliable biovalves of various physiological sizes suitable for all clinical uses.