Current artificial tissue-substitutes have limited clinical applications due to unmatched complex combination of cells and extracellular matrix (ECM) as seen in native tissues. From a developmental perspective, the construction of effective biomimetic tissues is from the bottom (one-dimensional nanoparticles or two-dimensional membranes) up (three-dimensional scaffolds or more complex composite). In a hierarchical architecture, each sub-structure can be assembled in a flexible way with specific regulators and cells, which overcomes the deficiency of one-for-all scaffold. Here, a cell-compatible cell-lined layered nano-membrane is developed. Bioactive molecules are mounted on a nano-membrane and later released to its lined cell sheet. The cell-lined membrane is in a free-standing form to regulate cellular functions. The major advantage of this methodology is to provide a versatile approach to construct biomimetic tissues for clinical applications.