Development of matrices for 3D spheroid culture and assessment of their metabolic activity

Abstract

Major advancements being made for in vitro cellular studies on cancer are currently employing 3D spheroid models as these are capable of mimicking the behaviour of in vivo tumours and allow a better understanding of the complex mechanisms behind cancer cell progression. The matrix is a useful component as it can also promote biophysical and chemical cues which would resemble the behaviour of the natural extracellular matrix (ECM). The aim of this study was to compare the development of 3D spheroid cultures using different polymers as matrix supports and follow their progress by monitoring their metabolic activity. Specific objectives centred on the development of appropriate encapsulation techniques for 3D spheroid culture. Additionally, the extraction of collagen hydrogel from easily available raw materials was also attempted to reduce the cost. 3D spheroids were generated using the hanging drop technique and they were encapsulated in three different matrices, these being, calcium alginate, collagen and guar gum. The effect of these matrices on 3D spheroid growth and development was then investigated by measuring the diameter over a 7 day period, using 20 spheroids per sample in 2 independent trials. Additionally, the change in cell viability of the 3D spheroids was assessed using the Resazurin reduction assay, using 12 spheroids per sample in 2 independent trials. From the results obtained, it was shown that the calcium alginate promoted the largest increase in spheroid diameter, which on average resulted in a percentage increase of 40 % from days 1-7. This was followed by collagen with a percentage increase of 30 %. The values for the Resazurin reduction assay correspond with that of the growth curves as an increase in diameter was accompanied by a larger number of viable cells. Although collagen was extracted successfully, it was expected to promote a larger increase in size due to its protein content resembling that of the natural ECM. Additionally, the guar gum was not a successful matrix as it did not promote increase in spheroid size. Thus, for the latter two matrices further work is needed to make them optimal for 3D spheroid culture. However, this is still a step forward in bridging the gap for the development of spheroid encapsulation protocols.