BIOREACTOR-BASED STRATEGY FOR DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS TOWARDS NUCLEUS PULPOSUS CELLS

Abstract

Intervertebral disc degeneration (IVDD) experienced by ~80% of worldwide population initiates in the nucleus pulposus (NP). This research aimed to establish a validated method for obtaining NP-like cells from adipose-derived stem cells (ASCs) for NP regeneration, through optimizing culture conditions inside a bioreactor. Five conditions were proposed to be tested in a full 25-factorial design, based on their physiological values: loading amplitude (0.8 vs. 0.2 MPa), strain frequency (1 vs. 0.25 Hz), oxygen tension (20 vs. 2%), presence of NP-specific extracellular-matrix-particles (DNPT) and immortalized human chordoma U-CH1 cells. Human ASCs encapsulated within reversible Ca-alginate hydrogels were subjected to these factors within a commercial bioreactor obtained from CellScale Biomaterials Testing Inc. Firstly, the encapsulation and cell retrieval processes were optimized, by testing different protocols on crosslinking and decrosslinking of Ca-alginate gels, the centrifugation step in cell retrieval from the gels, and purification of the Na-alginate solution. The best cell recovery obtained following de-crosslinking the gels was 66.5 ± 20.8 % with a cell viability of 69.1 ± 15.0 % for human ASCs. In addition, a 2-time charcoal purification of Na-alginate, and a seeding density of 1.7 x 106 cells/mL with 3 gels to be harvested generated sufficient high quality RNA samples from the ASCs for qPCR analyses. Thirdly, the bioreactor design was optimized with respect to valves used, flow pattern within the wells, and flow conditions that allowed for medium perfusion between loading cycles for nutrient and oxygen exchange, and also for re-crosslinking of Ca-alginate gels to maintain their integrity, while withstanding up to 0.9 MPa with no medium leakage during loading. Lastly, the flow rate (FR) through the bioreactor was also optimized with respect to cell viability. The highest cell viability of 52.1 ± 6.1 % was obtained at no perfusion, and best cell viability of 16.1 ± 3.7 % under a FR of 1 mL/min. Overall, this research provides the first few steps in the design of a bioreactor-based strategy for ASC differentiation towards NP cells.