O2-controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo

Abstract

Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro. The hydrogel polymeric network is formed via a laccase-mediated cross-linking reaction. In this reaction, laccase catalyzes diferulic acid (diFA) formation to form hydrogels with an O2-consuming reaction. Cells, such as cancer or endothelial cells, as well as tumor/tissue grafts, can be encapsulated in the hydrogels during hydrogel formation and then analyzed for cellular responses to 3D hypoxic gradients and to elucidate the underlying mechanisms governing these responses. Importantly, oxygen gradients can be precisely controlled in standard cell/tissue culture conditions and in vivo. This platform has been applied to study vascular morphogenesis in response to hypoxia and to understand how oxygen gradients mediate cancer cell behavior. Herein, we describe the means to validate the assay from polymer synthesis and characterization—which take 1–2 weeks and include verification of ferulic acid (FA) conjugation, rheological measurements, and O2 monitoring—to the study of cellular responses and use in rodent models. Time courses for biological experiments using this hydrogel are variable, and thus they may range from hours to weeks, depending on the application and user end goal.

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…The oxygen consumption is a result of the cross-linking reaction and hydrogel formation, resulting in an oxygen gradient from the top layer of the culture to deeper, more oxygen-deprived regions. Adapted with permission according to (165). (D) Preformed oxygen-consuming hydrogel immersed in a 3D culture creates a hypoxic environment with an oxygen gradient toward the hydrogel.…”

“…Several novel approaches and techniques have emerged tackling the challenges of pO 2 in 3D tissue structures. Analogically to adherent cell cultures, oxygen-sensing microelectrodes have been employed to measure pericellular oxygen gradients in thicker hydrogel-based tissues (164,165). However, the disadvantages of this approach, such as its invasive nature, time demands and technical challenges requiring repetitive calibrations and measurements in different spots inside the tissue construct, motivated the search for alternative approaches.…”

“…A unique feature of 3D cell culture systems is represented by the possibility of actively inducing a controlled oxygen gradient across the model, based on the experimental needs. Such gradients can be induced by perfusion with an oxygen scavenger in the medium (159); by positioning the culture between two micro-channel circuits perfused with gas, each with a different oxygen level (Figure 4B) (169,172); or by incorporation of an oxygen-consuming reaction of specific hydrogel materials, either encapsulating ( Figure 4C) (164,165) or being in close vicinity of the cells (Figure 4D) (170), and thus regulating the pericellular oxygen levels.…”

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“…1a) [16][17][18] . Such a steep O 2 gradient in living organisms has been challenging to mimic with a man-made device 19 . Designs of hydrogel and microfluidic systems have been employed to create steady-state concentration profiles, yet these delicate setups require a continuous gas purging or supply of O 2 scavengers, which are not readily tunable or attainable with long-term stability [19][20][21] .…”

“…Such a steep O 2 gradient in living organisms has been challenging to mimic with a man-made device 19 . Designs of hydrogel and microfluidic systems have been employed to create steady-state concentration profiles, yet these delicate setups require a continuous gas purging or supply of O 2 scavengers, which are not readily tunable or attainable with long-term stability [19][20][21] . While biofilms hosting diazotrophs have been recently reported to exhibit N 2 fixation in air 22 , their complex and less-well-defined nature renders certain challenges (Supplementary Note 1).…”