To maintain the cellular functions of a stem cells for therapeutic tissue engineering, an advanced cell culture method for safe cell dissociation is necessary. We developed a novel cell dissociation method by applying pH-responsive bioreducible polymer on the surface of tissue culture plates (TCPs). We applied acid-responsive bioreducible poly (amino ester) (PAE) as a new candidate for surface coating method to develop alternative cell dissociation method against conventional enzyme (trypsin-ethylene diamine tetra acetic acid (EDTA)) treatment. Human adipose derived stem cells (hADSCs) were cultured on and dissociated from PAE-coated TCPs to compare cell adhesion, cell proliferation, cell viability, and functionality to those of the cells cultured on and dissociated with trypsin-EDTA from normal TCPs without PAE coating. To confirm the in vivo therapeutic efficacy of the hADSCs retrieved from PAE-coated TCPs compared to that of the cells retrieved from normal TCPs with trypsin-EDTA, we induced skin defects at the dorsal area of mice and injected the cells collected from both conditions. With the PAE coating method, cell adhesion, cell proliferation, cell viability, and functionality, especially the angiogenic efficacy, were well preserved when compared to those of the cells treated with trypsin-EDTA. In addition to in vitro results, injecting hADSCs retrieved from PAE-coated TCPs showed similar in vivo angiogenesis and wound closing efficiency compared to those of injecting hADSCs retrieved from normal TCPs with trypsin-EDTA treatment at 2 weeks after the transplantation into mouse skin wound models. We proposed the alternative method for the cell dissociation with pH-responsive bioreducible polymer, PAE. This PAE coating method may lead to the development of alternative cell dissociation method without using enzyme for future regenerative medicine and stem cell therapy.