With recent approaches to tissue engineering, cells face various stresses from physical, chemical, and environmental stimuli. For example, coating of cell membranes with nanofilms using layer-by-layer (LbL) assembly requires many cycles of centrifugation causing physical (gravity) stress. Damage to cell membranes can cause leakage of cytosol molecules or sometimes cell death. Accordingly, we evaluated the effectiveness of LbL films prepared on cell membranes in protecting cells from physical stresses. After 2 steps of LbL assembly using Tris-HCl buffer solution without polymers or proteins (4 centrifugation cycles including washing), hepatocyte carcinoma (HepG2) cells showed extremely high cell death and viability was ca. 15%. Their viability ultimately decreased to 6% after 9 step LbL assembly (18 cycles of centrifugation), which is the typical number of steps involved in preparing LbL nanofilms. On the other hand, significantly higher viability (>85%) of HepG2 cells was obtained after 9 step LbL assembly employing fibronectin (FN)-gelatin (G) or type IV collagen (Col IV)-laminin (LN) solution combinations, which are typical components of extracellular matrix (ECM), to fabricate 10 nm thick LbL films. When LbL films of synthetic polymers created via electrostatic interactions were employed instead of ECM films described above, the viability of the HepG2 cells after the same 9 steps slightly decreased to 61%. The protective effects of LbL films were strongly dependent on their thickness and the critical thickness was over 5 nm. Surprisingly, high viability of over 85% was achieved even under extreme physical stress conditions (10,000 rpm). We evaluated the leakage of lactate dehydrogenase (LDH) during the LbL assembly processes to clarify the protective effect, and a reduction in LDH leakage was clearly observed when using FN-G nanofilms. Moreover, the LbL films do not inhibit cell growth during cell culture, suggesting that these coated cells can be useful for other experiments. LbL nanofilm coatings, especially ECM nanofilm coatings, will be important techniques for protecting cell membranes from physical stress during tissue engineering.