Generation of Oxygenating Fluorinated Methacrylamide Chitosan Microparticles to Increase Cell Survival and Function in Large Liver Spheroids

Abstract

Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 μm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

Modulatory Contribution of Oxygenating Hydrogels and Polyhexamethylene Biguanide on the Antimicrobial Potency of Neutrophil-like Cells

“…Additionally, due to the presence of oxygen-rich domains within MACF hydrogels, they can be seen as oxygen transport enhancers that are constantly reabsorbing and redissolving oxygen in the culture to benefit metabolically active cells, rather than simply serving as a limited reservoir for oxygen. 12,14 After the confirmation of successful oxygenation from MACF treatment, the study of the release profile of PHMB from MACF substrates was vital to understand the amount of antibiotic in contact with cells over time. Accordingly, the release kinetics of PHMB from the MACF substrates showed a burst phase profile during the first 30 h followed by a slowrelease phase (up to 51 h in Figure 1C).…”

“…These data demonstrated that our hydrogels are able to attract and dissolve molecular oxygen to enhance the local oxygen levels in aqueous buffered cell cultures, , which we again confirmed with our newest data (Figure, E). Additionally, due to the presence of oxygen-rich domains within MACF hydrogels, they can be seen as oxygen transport enhancers that are constantly reabsorbing and redissolving oxygen in the culture to benefit metabolically active cells, rather than simply serving as a limited reservoir for oxygen. , …”

“…7−12 We have previously shown the application of MACF hydrogels as potent oxygenating platforms to improve wound healing responses in rodent and porcine models 7,8,11,13 as well as to overcome oxygen transport limitations to enhance cell survival in vitro. 9,12,14 Clinically speaking, the treatment of an infection at a wound site is usually achieved using a multimethod approach that most often includes using chemical antibacterial agents. Antibiotics are known to interact with leukocytes 15 to stimulate their intracellular production of ROS, 16 while altering the overall oxidative mechanisms.…”

“…Hypoxia in the wound environment is known to promote the persistence of neutrophils at the site and act as a secondary inflammatory signal to attract more neutrophils while upregulating pro-inflammatory cytokine levels at the site of injury. , To counteract the negative effects of hypoxia, the delivery of oxygen to injuries can alleviate inflammatory conditions by regulating the antibacterial functions of immune cells . Accordingly, we have focused on utilizing the excellent oxygen delivering properties of perfluorocarbon-based chitosan hydrogels in the form of our previously developed fluorinated methacrylamide chitosan (MACF) polymers to alleviate local hypoxic conditions. − We have previously shown the application of MACF hydrogels as potent oxygenating platforms to improve wound healing responses in rodent and porcine models ,,, as well as to overcome oxygen transport limitations to enhance cell survival in vitro . ,, …”

Microwell-Based Flow Culture Increases Viability and Restores Drug Response in Prostate Cancer Spheroids

“…To demonstrate the utility of the MFD culture platform, we generated large (4k cells/well) spheroid models from the Lymph Node Carcinoma of the Prostate (LNCaP) cell line. Large spheroids achieve better physiological relevance in terms of growth, cell function, and drug responses, but have been underutilized in in vitro studies due to challenges in viability and long-term growth [38, 39]. We investigate that LNCaP spheroids in the MFD exhibit reduced necrosis and hypoxic stress and maintain cellular structural integrity throughout the spheroid.…”