Current cell expansion methods for tissue engineering and regenerative medicine applications rely on the use of enzymatic digestion passaging and 2D platforms. However, this enzymatic treatment significantly reduces cell quality, due to the destruction of important cell-surface proteins. In addition, culture in 2D results in undesired de-differentiation of the cells caused by the lack of 3D similarity to the natural extracellular matrix (ECM) environment. Research has led to the development of thermo-responsive surfaces for the continuous culture of cells. These thermo-responsive materials properties can be used to passage cells from the surface when the cell culture temperature is reduced. Here we report the development of a PLA/thermo-responsive (PDEGMA) blend 3D electrospun fibre-based scaffold to create an enzymatic-free 3D cell culture platform for the expansion of mammalian cells with the desired phenotype for clinical use. Human corneal stromal cells (hCSCs) were used as an exemplar as they have been observed to de-differentiate to an undesirable myo-fibroblastic phenotype when cultured by conventional 2D cell culture methods. Scaffolds were functionalised with a cell adherence peptide sequence GGG-YIGSR by thiol-ene chemistry to improve cell adherence and phenotype support. This was obtained by functionalising the thermo-responsive polymer with a thiol (PDEGMA/PDEGSH) by co-polymerisation. These incorporated thiols react with the norbornene acid functionalised peptide (Nor-GGG-YIGSR) under UV exposure. Presence of the thiol in the scaffold and subsequent peptide attachment on the scaffolds were confirmed by fluorescence labelling, ToF-SIMS and XPS analysis. The biocompatibility of the peptide containing scaffolds was assessed by the adhesion, proliferation and immuno-staining of hCSCs. Significant increase in hCSC adherence and proliferation was observed on the peptide containing scaffolds. Immuno-staining showed maintained expression of the desired phenotypic markers ALDH, CD34 and CD105, while showing no or low expression of the undesired phenotype marker α-SMA. This desired expression was observed to be maintained after thermo-responsive passaging and higher when cells were cultured on PLA scaffolds with 10 wt% PDEGMA/4 mol% PDEGS-Nor-GGG-YIGSR. This paper describes the fabrication and application of a first generation, biocompatible peptide conjugated thermo-responsive fibrous scaffold. The ease of fabrication, successful adherence and expansion of a therapeutically relevant cell type makes these scaffolds a promising new class of materials for the application of cell culture expansion platforms in the biomaterials and tissue engineering field.