There is a need for functional tissue units that exhibit layered cellular architectures that have similarities with in vivo tissues and organs. Herein, we describe a method to produce mechanically stable self-standing composite polyelectrolyte multilayers (SSC-PEMs), and using them to create a three dimensional (3-D) cellular scaffold. The layer-by-layer (LbL) self-assembly technique was used to build multilayers of poly(acrylic acid) (PAA) and poly(ethylene glycol) (PEG) followed by multilayers of poly(diallyldimethylammonium chloride) (PDAC) and sulfonated poly(styrene), sodium salt (SPS) on a highly hydrophobic poly(dimethylsiloxane) (PDMS) substrate at a pH of 2.0. PAA/PEG multilayers produce a discontinuous film, of sub-micron scale pillars on hydrophobic PDMS, to support the assembly of multilayer PDAC/SPS on top. Mechanically stable SSC-PEMs were obtained under dry conditions by peeling the films from the PDMS substrate. Critical thicknesses (threshold number of bi-layers) exist for both the PAA/PEG and PDAC/SPS films in order for the assembly to be removed easily from the PDMS substrate. When SSC-PEMs are immersed in a solution at physiological pH, the PAA/PEG films degrade into biocompatible PAA and PEG components, leaving behind the PDAC/SPS films. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) studies were performed to confirm the presence of polyelectrolytes (with distinguishable functional groups) confined within the SSC-PEM. The thickness of the multilayers, surface topology and roughness were determined using atomic force microscopy (AFM). Surface roughness and topology of the SSC-PEMs with 'PDAC/SPS as the scanning surface' were found to be comparable to PDAC/SPS films built over a glass or silicon substrate. The smooth surface morphology of the PDAC/SPS films on SSC-PEM was also confirmed with scanning electron microscopy (SEM). Cells were cultured onto SSC-PEMs and found to sustain cellular growth. Plasma-treated SSC-PEMs attached easily to a layer of fibroblasts, whereupon Hela cells were subsequently added as a second layer of cells over the fibroblasts-SSC-PEM structure. Immunostaining results demonstrate the PDAC/SPS containing SSC-PEMs were permeable for cell culture applications. Our approach has the advantage of not having to deprive the growing cells of culture medium during the building of the scaffold, thus improving the cells' viability over previous methods.