An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

Abstract

SUMMARYIschemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs.

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

“…This was most likely attributed to the accumulation of cytotoxic byproduct, namely gluconic acid and H 2 O 2 . This is not a unique problem to our system as other GOX/CAT-induced hypoxia culture systems (e.g., soluble or membrane-bound enzyme) also exhibited decreased cytocompatibility after 24 hours of culture (Askoxylakis et al, 2011;Huang et al, 2013;Zitta et al, 2012). One potential solution for this drawback is to replace the GOX-immobilized hydrogel and the CAT supplemented media frequently.…”

“…This system has been used to induce hypoxia in solutions and in microfluidic devices (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). The use of GOX/CAT is beneficial in that the system provides a rapid onset of hypoxia (usually within a few minutes) (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). One drawback to any GOX system, however, is the production of hydrogen peroxide, a reactive oxygen species (ROS) (Fruehauf and Meyskens, 2007) whose accumulation would not only cause undesired cellular response but also inactivate both GOX and CAT (Hielscher and Gerecht, 2015;Pal et al, 2000;Trachootham et al, 2009;Tse and Gough, 1987).…”

“…Some recent work has started to explore the ability of GOX/CAT reactions to induce hypoxia for in vitro cell culture (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013). The GOX/CAT system has also been adapted to 3D printed inserts (Li et al, 2016) where GOX and CAT were coated on printed disks and the degrees of solution hypoxia were controlled by the distance between the enzyme-immobilized disks and the solution in the culture plate.…”

Human monocytes subjected to ischaemia/reperfusion inhibit angiogenesis and wound healing in vitro

“…Moreover, using this system pH and glucose concentration in the culture medium decrease, resembling well the in vivo situation of I/R. 18,19 Hypoxic conditions were verified by using a tissue oxygen pressure monitor (LICOX CMP Oxygen Catheter; Integra). Pretests considering the increase in ischaemia-inducible factors in monocytes and progression of cell damage during I/R were performed to evaluate the optimal duration of ischaemia (3 hours) and reperfusion (24 hours beginning of ischaemia, T(0) directly after ischaemia and T(24) the end of reperfusion ( Figure 1).…”

Portable, low‐cost hypoxia chamber for simulating hypoxic environments: Development, characterization and applications

“…Apart from these, there are many type of commercial hypoxia simulating devices that are available in the market (Catalog #27310; Z736848; X3CYTOCENTRIC; C21HYPOXIA). The available commercial hypoxia chambers are designed as per specific application and requirements (Hsu & Chen, 2013; Huang, Zitta, Bein, Steinfath, & Albrecht, 2013; Wang, Jin, & Zhong, 2014). Hsu and Chen developed a hypoxia chamber with a microprobe that continuously monitors the electrical and optical properties of neuronal cells for drug screening and/or fundamental research related to ischaemic condition (Hsu & Chen, 2013).…”