Parameter optimisation for expansion of pluripotent stem cells
thesisposted on 23.02.2017, 04:00 by Gupta, Priyanka
Pluripotent stem cells have the unique properties of extensive replication and ability to differentiate into derivatives of all three germ layers. By virtue of this, they have been deemed to be of immense importance in the fields of tissue engineering, regenerative medicine, drug discovery, disease modeling, etc. The field of stem cell culture has undergone huge advancements over the past few years, with extensive studies being carried out for the various intrinsic and extrinsic parameters involved in pluripotent stem cell culture. This thesis looks at the effect of some of the extrinsic parameters involved in large scale expansion of pluripotent stem cells in vitro. The first work looks at the ability of two new materials for viability and pluripotency maintenance of mouse embryonic stem cells. Traditionally, the pluripotent stem cells are grown as co-culture on a feeder layer of inactivated mouse embryonic fibroblasts. Efforts are still being made to replace the feeder cells with appropriate biomaterials to avoid cross contamination of the pluripotent cells. In this work, tropoelastin was tested as a 2D coating material and compared with gelatin and matrigel coating. Cell count, pluripotency analysis by flow cytometry and visual inspection of colony morphology were carried out for mouse ES cells grown on all three materials to test the suitability of tropoelastin in comparison to the other two well known materials. It was observed that although tropoelastin was able to maintain cell growth and pluripotency, the number of viable cells obtained was lower in comparison to gelatin and Matrigel. PMVE- MA + gelatin composite was used for 3D scaffold preparation and compared with pure gelatin scaffold. Cell attachment, viability, and pluripotency maintenance measurement were undertaken for mouse ES cells on both the scaffolds; the novel scaffold on PMVE- MA+ gelatin was found to be suitable for the expansion of mouse ES cells. Preliminary comparison between commercially available Cytodex 3 and Hillex II microcarriers' ability to maintain mouse ES cells was also carried out. Cell attachment and expansion were found to be better on Cytodex 3 in comparison to Hillex II in static culture. Encouraged by the findings in the previous work, Cytodex 3 microcarrier were used for long term expansion of mouse induced pluripotent stem cells in a spinner flask. Optimization of the spin rate was first carried out for maximum carrier suspension without breaking them. The optimized range was then tested for attachment of iPS cells on the carriers. Long term culture of mouse iPS cells was then carried out followed by characterization of the cells growth, viability, pluripotency and multilineage differentiation capability. The results suggested that long term culture of mouse iPS cells in a spinner flask was possible by using Cytodex 3 without any prior coating of the carriers or prior adaptation of the cells to a feeder free culture at an optimized spin rate of 25 RPM. The third work looks at the effect of change in pH and increase in lactate concentration on mouse pluripotent stem cells. Cell viability, proliferation rate and pluripotency were investigated to see the effect of pH and externally added lactate on pluripotent stem cells, both feeder dependent and feeder independent. A pH of 7.5 was found to be optimum for feeder independent embryonic stem cells while the optimum range for feeder dependent pluripotent stem cells (both embryonic and induced) was between 7.0 and 7.5. Any change in medium pH from the optimum value had detrimental effects on cell proliferation, metabolic activity and pluripotency. The effect of increased lactate concentration in the medium was also studied. It was seen that cell proliferation decreased with an increase in lactate concentration in the medium while there was no significant change in cell pluripotency. Interestingly, cellular metabolic activity assay and analysis of spent medium suggested that the mouse pluripotent stem cells were able to metabolise the externally added lactate, a feature so far known to exist only in a few cell types eg. CHO cell line. Conditioned media from MEFs have been used for culturing of pluripotent stem cells for a long time. However, being a spent media, metabolite concentration and pH changes are expected. Hence, an experimental and theoretical analysis of MEF conditioned media was taken up to estimate changes in key metabolites like glucose and lactate. The study utilised the High-Performance Liquid Chromatography (HPLC) method for experimental estimation of glucose uptake and lactate production in the conditioned media at specific time intervals. At the end of 72 hours, glucose concentration decreased from 3.05 mg/ml to 2.03mg/ml. Accumulated lactate concentration reached a final value of 2.23mg/ml in the same time. Theoretical modeling of the same was performed to extend the study beyond the experimentally studied time points. All these studies highlight the importance of individual optimization of the various parameters involved in the large scale expansion of pluripotent stem cells. It needs to be kept in mind that it is possible that the effect of some of these parameters may be interlinked and calls for further studies in this field. Thesis submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy of the Indian Institute of Technology Bombay, India and Monash University, Australia.