Development of polycaprolactone microcarrier with surface treatment using ultraviolet/ozone system

Abstract

Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the main challenge remains at ensuring the surface biocompatibility with cells. Polycaprolactone (PCL), a biodegradable polymer, has received considerable attention because of its good mechanical properties and degradation kinetics that suit various applications, but its non-polar hydrocarbon moiety renders it sub-optimal for cell attachment. In this present study, the effects of the preparation conditions on the size and morphology of the microspheres were investigated. The result showed that high stirring speed (300 rpm), the emulsion is easily dispersed therefore producing smaller droplets. The size of microspheres was independent of surfactant at low concentrations (0.05-0.01%) as the surfactant is not sufficient to stabilize the droplet formation. However, the size of microspheres decreased from 384.5 µm to 190.8 µm as the surfactant concentration is increased. The microspheres size was not only affected by those two parameters, but also by the amount of PCL in dichloromethane. At high ratio (1:10) of the polymer matrix to the organic solvent, small microspheres size (94.6 µm) was observed and as the ratio decrease (1:30), the microspheres size increase (293.3 µm). Convincingly, the size of microspheres can be controlled by manipulating the parameter’s variables by mean of solvent emulsion method. In order to promote cell attachment and proliferation on PCL microcarrier, the surface of microcarriers were modified using an ultraviolet/ozone (UV/O3) treatment system to introduce functional groups (COOH). Subsequently, the modified microcarriers surface were coated with gelatin using zero length cross-linker reagents. Two stages of optimisation were conducted in order to optimise COOH concentration and amount of gelatin immobilised on the microcarrier surface. One-factor-at-a-time (OFAT) method and face centred central composite design (FCCCD) were employed in the optimisation process. Optimum oxygen containing functional group (1495.9 nmol/g) was introduced via ultraviolet irradiation and ozone aeration (UV/O3) system to allow covalent immobilisation of gelatin on the PCL microcarrier surface using zero-length cross linker reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDAC/NHS). Meanwhile, the optimum amount of gelatin immobilised (1797.3 µg/g) on the microcarrier surface was obtained by statistically optimised variables in protein immobilisation namely the carboxyl group to EDAC molar ratio (COOH:EDAC), NHS concentration, and gelatin concentration. The immobilisation of gelatin was confirmed using the attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) where the presence of peak at wave number 1654 cm-1 and 1544 cm-1 are attributed to amide I and amide II, respectively ensure the successful immobilization of gelatin. Meanwhile, the decreased water contact angle (86.93o for untreated PCL to 49.34o for post-gelatin immobilisation) confirmed the enhancement of hydrophilicity of the surface. Cytocompatibility of the develop PCL microcarrier was tested with few cell lines namely human keratinocytes cells (HaCaT), rat amniotic fluid stem cell (AFSC) and human skin fibroblast cell (HSFC) that represent immortalised cells, stem cells and primary cells respectively. All types of cells were cultured on gelatin coated PCL microarrier, UV/O3 PCL microcarrier and untreated PCL microcarrier (as control). All cells show favourable biocompatibility towards gelatin coated microcarrier surface except for HaCaT cell that achieved high cell density towards both gelatin coated PCL and UV/O¬3 treated PCL. The pluripotency of AFSC after cultured on microcarrier were confirmed by spontaneous differentiation of AFSC to form embryiod bodies post-trypsinization from microcarrier culture. Meanwhile, the ability of HSFC to migrate from microcarrier to culture flask demonstrate the usability of the biodegradable microcarrier in tissue applications and may benefit skin wound healing. As a conclusion, this study has successfully develop two functional microcarriers which are i) UV/O3 microarrier (charge microarrier) that are meant for immortalized and continuous cell lines and ii) gelatin coated microcarrier for low plating efficiency cells that require supplementary growth factor to attach and proliferate in-vitro. To this end, this study provides a novel model system for the in vitro study of cell proliferation ability on microcarrier that may benefit skin wound healing and provide insights into the potential mechanisms of application of stem cell transplantation in therapeutic setting.