Abstract
Cell culture typically employs inexpensive, disposable plasticware, and standard humidified CO2/room air incubators (5% CO2, ∼20% oxygen). These methods have historically proven adequate for the maintenance of viability, function, and proliferation of many cell types, but with broad variation in culture practices. With technological advances it is becoming increasingly clear that cell culture is not a “one size fits all” procedure. Recently, there is a shift toward comprehension of the individual physiological niches of cultured cells. As scale-up production of single cell and 3D aggregates for therapeutic applications has expanded, researchers have focused on understanding the role of many environmental metabolites/forces on cell function and viability. Oxygen, due to its role in cell processes and the requirement for adequate supply to maintain critical energy generation, is one such metabolite gaining increased focus. With the advent of improved sensing technologies and computational predictive modeling, it is becoming evident that parameters such as cell seeding density, culture media height, cellular oxygen consumption rate, and aggregate dimensions should be considered for experimental reproducibility. In this review, we will examine the role of oxygen in 3D cell culture with particular emphasis on primary islets of Langerhans and stem cell-derived insulin-producing SC-β cells, both known for their high metabolic demands. We will implement finite element modeling (FEM) to simulate historical and current culture methods in referenced manuscripts and innovations focusing on oxygen distribution. Our group and others have shown that oxygen plays a key role in proliferation, differentiation, and function of these 3D aggregates. Their culture in plastic consistently results in core regions of hypoxia/anoxia exacerbated by increased media height, aggregate dimensions, and oxygen consumption rates. Static gas permeable systems ameliorate this problem. The use of rotational culture and other dynamic culture systems also have advantages in terms of oxygen supply but come with the caveat that these endocrine aggregates are also exquisitely sensitive to mechanical perturbation. As recent work demonstrates, there is a strong rationale for the use of alternate in vitro systems to maintain physio-normal environments for cell growth and function for better phenotypic approximation of in vivo counterparts.
Highlights
Biomedical research of 3D cell culture has been ongoing for decades, increasing dramatically with the recent boom in stem cell research and associated cellular therapies (Moscona A., 1961; Knisely et al, 1969; Bruland et al, 1985; Li et al, 1992; De Moor et al, 2018; Gargotti et al, 2018; Jokinen et al, 2020; Qadir et al, 2020)
As 3D culture increasingly becomes a preferred mode of physiomimetic culture, historical culture methods on plastics need to be re-evaluated
Related to tissue oxygenation, the steep gradients that develop from apical to basal surface, both in the culture milieu and in the 3D organoids, lead to hypoxic and anoxic regions, inefficient nutrient metabolism and potentially, shifts in viability, function and gene expression that may deviate in comparison to the same tissue in vivo
Summary
Biomedical research of 3D cell culture has been ongoing for decades, increasing dramatically with the recent boom in stem cell research and associated cellular therapies (Moscona A., 1961; Knisely et al, 1969; Bruland et al, 1985; Li et al, 1992; De Moor et al, 2018; Gargotti et al, 2018; Jokinen et al, 2020; Qadir et al, 2020). Work by the group of and Colton, Papas, Avgoustiniatos, and Dionne utilized computational modeling to investigate the oxygenation limitations in conventional culture methods for islets of Langerhans (Dionne et al, 1989, 1991, 1993, 1996; Papas et al, 2005) Through their detailed study of OCR in endocrine cells and spheroids, they identified the Km value associated with islet OCR (0.4 mmHg), and pO2 values associated with anoxia (∼0.1 mmHg and below) and impaired insulin secretion (∼2.5 mmHg and below). Recent work has focused on addressing these challenges for the scale-up and implementation of clinical cell-based therapies
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