Extracellular matrix (ECM) is related with various cellular signaling pathways and functions. We have proposed an alternative method for preparing ECM enriched human adipose derived stem cells (hADSCs) with pH-sensitive polymer layer to enhance the therapeutic efficacy. We synthesized enzyme free pH-sensitive polymer (EFP) and cultured hADSCs on EFP-coated tissue culture plates. The hADSCs were detached from EFP-coated tissue culture plate by simply adding low pH solution. The hADSCs detached from EFP and reattached to normal plates showed more ECM and angiogenesis factors secretion compared to hADSCs detached from and reattached on normal plates. Our novel EFP based cell culture system might suggest new platform for stem cell harvesting that can replace the conventional proteolytic enzyme treatment.