Abstract
Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.
Highlights
Over the past three decades, the use of three dimensional cell culture has gained increased recognition as an important tool in biological research and in preclinical trials [1,2,3] over conventional organs or ex vivo cultures which are unsurprisingly in short supply
Despite the acknowledged reliability of this approach, Electron Paramagnetic Resonance (EPR) has not been applied to 3D culture systems until now to quantify oxygen gradients
One advantage which EPR based methodology has over normal micro-electrode measurements is that linewidth of spectra is based on pure physical interaction between paramagnetic probe molecules and oxygen within the biological system [27]
Summary
Over the past three decades, the use of three dimensional cell culture (e.g. spheroids) has gained increased recognition as an important tool in biological research and in preclinical trials [1,2,3] over conventional organs or ex vivo cultures which are unsurprisingly in short supply. There is an enormous body of literature on spheroid models and their use in cancer therapy orientated studies (3D tumour models) to bridge the gap between cell based assays and in vivo studies [6,7,8]. These systems can be used to model many characteristics of avascular tumours and micrometastases of large solid tumours, in addition to better replicating the barrier to drug penetration represented by native tumour tissue [9]. In order to use such a 3D system in non tumour models, we need to understand more about the mass transport limitations of the nontumour model, especially in respect of oxygen transport
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