Ruiter, Floor A.A.
(2018)
Thermo-responsive electrospun scaffolds for enzymatic-free passage and mammalian cell phenotype support.
PhD thesis, University of Nottingham.
Abstract
Current conventional cell culture methods for the expansion of cells to large quantities for tissue engineering and regenerative medicine application relies on the use of enzymatic digestion passaging. However, this method significantly reduces the cell viability, due to the destruction of important cell-surface proteins and therefore considered undesirable for the subsequent cell therapies [1]. 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. Conventional cell culture is performed on a 2D platform, however this can result in undesired de-differentiation, due to lack of similarities to the natural extracellular matrix (ECM) environment. This thesis aims to develop a thermo-responsive 3D fibre-based scaffold, fabricated by electrospinning, to create an enzymatic-free 3D cell culture platform for the expansion of mammalian cells with the desired phenotype for clinical use. In particular the expansion of human corneal stromal cells (hCSCs), which have been observed to de-differentiate to their active myofibroblastic phenotype with conventional 2D cell culture methods were examined here.
Although experimentally the electrospinning method is relatively simple, at the theoretical level the interactions between process parameters and their influence on the fibre morphology is not yet fully understood. In this study a design of experiments (DoE) model was proposed to determine combinations of process parameters that result in significant effects on poly-D,L-lactic acid (PDLLA) fibre morphology. The process parameters used in this study were applied voltage, needle-to-collector distance, flow rate and polymer concentration. Data obtained for mean fibre diameter, standard deviation of the fibre diameter (stdev, measure of fibre morphology) and presence of ‘beading’ on the fibres (beads per µm2) were evaluated as a measure of PDLLA fibre morphology. Uniform fibres occurred at standard deviations of ± 500 nm, ‘beads-on-string’ morphologies were apparent between ± 500-1300 nm and large beads were observed at ± 1300-1800 nm respectively. Mean fibre diameter was significantly influenced by the applied voltage and interaction between flow rate and polymer concentration. Fibre morphology was mainly influenced by the polymer concentration, while bead distribution was significantly influenced by the polymer concentration as well as the flow rate. The resultant DoE model regression equations were tested and considered suitable for the prediction of parameter combinations needed for the desired PDLLA fibre diameter and additionally, provided information regarding the expected fibre morphology.
The thermo-responsive behaviour of the scaffolds was achieved by blend electrospinning of the thermo-responsive polymer poly(di(ethylene glycol) methyl ether methacrylate (PDEGMA) and the supportive polymer poly-D,L-Lactic Acid (PLA), which gives the support needed for cell culture. PDEGMA was synthesised by free radical polymerisation and has a lower critical solution temperature of 28°C. The optimised electrospinning process was taken forward for the fabrication of the blend electrospun PLA/PDEGMA scaffolds. Different weight (wt) % concentrations to PLA was incorporated and distribution of the PDEGMA was confirmed by fluorescent labeled PBIPDEGMA. Initial thermo-responsive passaging method was investigated with 3T3 fibroblasts (3T3s). No significant detachment of 3T3s was observed below 10 wt% of PDEGMA in the blend electrospun PLA/PDEGMA scaffolds, while a maximum of 25% of cells detached when cooled for 30 mins at 8°C as observed for 10 wt% PDEGMA. An increase in fibre diameter from 900 nm to 2-3 µm and different cooling methods (1 and 3 h at rt and controlled cooling of -1°C per minute) did not result in an increase of 3T3 detachment. These findings indicate possible insufficient presence of the PDEGMA thermo-responsive brushes at the surface of the fibres. However, when hCSCs were cultured on these scaffolds significantly higher cell detachment was observed (approx. 100% compared to 25% for 3T3s). These findings indicate the application of the blend electrospun PLA/PDEGMA scaffolds for the culture and passaging of hCSCs.
hCSCs were observed to not adhere and proliferate on the PLA/PDEGMA scaffolds. Therefore, a peptide thermo-responsive co-polymer conjugated by photo-initiated thiolene chemistry was proposed. Poly(di(ethylene glycol) methyl ether methacrylate-copoly(di(ethylene-glycol) vinyl ether (PDEGMA/ PDEGOH) was synthesised by free radical polymerisation and subsequently functionalised with a free thiol group by a condensation reaction. The obtained poly(diethylene glycol) methyl ether methacrylate co-poly(diethylene glycol) thiol (PDEGMA/PDEGSH) was blend electrospun and the presence of free thiols on the electrospun scaffolds were determined by ToF-SIMS and FITC labeling. These free thiol containing scaffolds were functionalised by a norbornene acid functionalised peptide sequence GGG-YIGSR by the photo-initiated thiolene reaction and its presence was confirmed by ToF-SIMS and ATTO labeling.
The biocompatibility of the peptide containing scaffolds was assessed by the adhesion, proliferation and immuno-staining of hCSCs. Significant increase in hCSCs 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 expressing a-SMA marker. This desired expression was observed to be maintained after thermo-responsive passaging and observed higher when cells were cultured on PLA scaffolds with 10 wt% PDEGMA/4 mole% PDEGS-Nor-GGG-YIGSR.
In this thesis, the fabrication and application of a first generation, biocompatible thermo-responsive peptide conjugate fibrous scaffolds is described. The ease of fabrication, successful adherence and expansion of a therapeutically relevant cell type make these scaffolds a promising new class of materials for the application of cell culture expansion platforms. The scaffolds developed and reported in this thesis are believed to represent a promising contribution to the fields of biomaterials and tissue engineering.
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