Extreme Anoxia Tolerance in a Cell Line Derived from Embryos of the Annual
Anoxia tolerance is the exception rather than the rule among vertebrates (Podrabsky et al., 2007). On the low end of the spectrum, the naked mole rat (Heterocephalus glaber) is the most anoxia tolerant mammal; it can survive without oxygen for 18 min (Storz & McClelland, 2017). This tolerance is owed to the naked mole rat’s subterranean burrow lifestyle (Larson & Park, 2009). On the other end of the spectrum are two of the most well studied anoxia tolerant vertebrates: the crucian carp (Carassius carassius) and the freshwater turtle (Chrysemys picta bellii), each of which can routinely survive several months of anoxia at 0-4?°C. Such conditions would be lethal to the vast majority of vertebrates (Boutilier, 2001).
The anoxia tolerance of the crucian carp and freshwater turtle likely evolved in response to the winters in their natural habitats, where they are submerged in waters that become ice-locked at the surface, limiting oxygen availability to the water underneath (Krivoruchko & Storey, 2015; Sollid et al., 2003). However, the champion of anoxia tolerance during development is A. limnaeus, whose anoxia tolerance is 2 orders of magnitude greater than that of any other vertebrate during 7development (Figure 1; Podrabsky et al., 2012).
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This observation runs counter to the long- appreciated observation that animals in development have a higher tolerance for low O2 conditions compared to their adult counterparts (Podrabsky et al., 2012). Because of the exceptional anoxia tolerance and unique life history of the annual killifishes, it is highly possible that there are undiscovered mechanisms of anoxia tolerance in the species. Establishing a novel cell line for the study of anoxia tolerance Here we report for the first time the establishment of an extremely anoxia tolerant continuous cell line named AL4 derived from the head region of a 12 dpd A. limnaeus embryo, which survives anoxia for approximately 16 days (Podrabsky et al., 2012).
Through proteomic analysis, we conjecture that AL4 consists of undifferentiated neuroepithelial cells. AL4 shows an unprecedented ability to survive in anoxia for 49 days, longer than any other vertebrate cell line in the literature. Additionally, AL4 is able to undergo mitosis and proliferate for one week under anoxic conditions. The establishment of AL4 and other cell lines from A. limnaeus expands the tools—e.g., localization of RNAs (in situ hybridization) and proteins (immunohistochemistry), and proteomics—for studying the molecular mechanisms of anoxia tolerance in A. limnaeus and further develops the species as a model for extreme anoxia tolerance.
The new research opportunities yielded by the development of an anoxia tolerant cell line may allow researchers to uncover entirely novel cellular mechanisms not yet discovered in other organisms. Ultimately, it is our hope that such studies will aid in developing therapies for anoxia-sensitive tissues such as in the heart and brain, especially in patients susceptible to heart attacks or strokes.
The cell line AL4—named for being our fourth attempted cell line of the species Austrofundulus limnaeus—was established from the head region of a 12 dpd embryo (data not shown). AL4 was maintained in complete medium containing Leibovitz L-15 cell medium (Gibco, Life Technologies, Burlington, ON, Canada) supplemented with 100 U/mL penicillin- streptomycin (pen-strep) (Gibco), 5 mM glucose (Corning, Corning, NY), and 8.5% fetal bovine serum (FBS; Gibco). 8The subculture protocol for the cells is as follows: spent media is removed and the cells are rinsed three times with sterile PBS to remove cell debris. Enough TrypLE Express?„? (Gibco) is added to cover the surface of the plate.
The plate is incubated at 30?°C for 10 min to dissociate the cells. The same amount of complete media, as TrypLE, is added to the cells to dilute and thereby deactivate the TrypLE. The cell suspension is transferred to a 15 mL conical tube then centrifuged at 100 RCF for 7 min at 25?°C to pellet the cells. The supernatant is removed from the pellet and the cells are re-suspended in complete media and added to a sterile tissue culture (TC) plate (CytoOne, USA Scientific, Ocala, FL) with enough complete media to ensure the working volume is met. Cells are then returned to a 30?°C incubator. Cells adhere to the plate surface within 3-4 h of being plated. The cells were subcultured according to this protocol at least twice a week, typically at a split ratio of 1:3-8.
The Vero cells were obtained from the Laboratory of Professor Kenneth Stedman at Portland State University. They were maintained in growth medium containing DMEM (Corning, catalog number 45000-312) with 10% FBS in an incubator at 37?°C with 5% CO2. Vero cells were subcultured similarly to AL4 cells, except Vero cells were kept in 25 cm2 TC flasks with 0.2 ?µm vent caps. The N2A cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, catalog number CCL-131) and required growth medium containing EMEM with 10% FBS. N2A cells were thawed and subcultured according to ATCC’s recommended protocols.
Anoxic L-15 was prepared under sterile conditions. Pen-strep and glucose were added to L-15 to achieve final concentrations of 100 U/mL and 5 mM, respectively. FBS was injected into a hydrated Slide-A-Lyzer?„? dialysis cassette (ThermoFisher Scientific, Waltham, MA) using a sterile syringe and 22G needle to achieve a final concentration of 8.5%. The cassette was added to the prepared L-15 solution in a graduated cylinder.
The L-15 on either side of the cassette was bubbled with high purity N2 gas (Matheson, Basking Ridge, NJ) through a sterile scintered glass diffuser for 60 min then equilibrated with the anaerobic chamber environment (Bactron III, Sheldon Laboratories) by bubbling chamber atmosphere (95% N2, 5% H2) into the media overnight with an aquarium pump. Afterwards, FBS was withdrawn from the cassette and added to the L-15 containing pen-strep and glucose, within which it was bubbled. The media type and concentrations were adjusted for Vero and N2A, but the same procedure was followed.
Cells were seeded at 47,430 cells per well were seeded in triplicate in 24-well TC plates and grown for two days to reach nearly 100% confluence before being introduced to the anaerobic chamber at 30?°C. Upon introduction, oxygenated medium was discarded and fresh anoxic medium (see above) was added. Spent anoxic medium was replaced with fresh anoxic medium at least once a week to avoid cytotoxic acidosis from the buildup of lactic acid and increased osmolarity from evaporation. Three wells of cells were sampled at 7, 14, 21, 35, and 49 d of anoxia.
At each sampling point, cells were stained with a LIVE/DEAD?„? 10viability/cytotoxicity kit (catalog number L3224, ThermoFisher Scientific) according to manufacturer instructions. For AL4, the optimal concentration of calcein AM (stains live cells) and ethidium homodimer-1 (stains nonliving cells) was found to be 1.0 ?µM and 2.5 ?µM, respectively. Stained cells were photographed with a Leica DM IRB inverted microscope and Leica DFC450C camera.
Ice-cold PBS containing 2.7 mM EDTA (1700 ?µL) was added to confluent cells in 100 mm TC plates (CytoOne) and incubated at room temperature for 5 min before the cells were collected by scraping using a plastic cell lifter. The cells were kept on ice when not being centrifuged. The cell suspensions were centrifuged at 200 RCF for 7 min to pellet the cells. The supernatant was removed and the cells were subsequently rinsed and pelleted at 200 RCF for 7 min twice with ice cold PBS. After the third and final rinse, cells were pelleted at 600 RCF for 5 min to form a tighter cell pellet. Final pellets were collected in low-retention microcentrifuge tubes. All remaining liquid was removed before flash freezing the pellets in liquid nitrogen and storing at -80?°C prior to processing for proteomics analysis.
AL4 grow to the greatest numbers when grown in media supplemented with 20% FBS, but cells with 8.5% grew at a similar rate until day 6, after which the cells somewhat plateaued at a lower number than the cells with 20% FBS (Figure 2). When FBS was excluded from the media, the cells rarely reached numbers that were greater than the initial value on day 1. This serum dependence carries over to anoxia as well; cells that are supplied with only pure L-15 die and detach within 3-4 d (Claire Riggs, personal communication).
Long term anoxia tolerance of AL4 AL4 cells maintain confluence and can even proliferate at confluence for the first 7 d of anoxia (Figure 3). A substantial number of cells still adhered to the plate up to 21 d of anoxia (Figure 3). After 21 d, there was a noticeable decrease in the number of adherent and live cells. At 49 d, few cells remained attached to the plate, but those that did stained positively for calcein AM, indicating they were alive. The normoxic cells overgrew and stacked upon each other over the 49 d (Figure 3, bottom row).
AL4 cells have the ability to undergo mitosis in anoxia for up to 7 d (Figure 4). After this time, the cells are unable to continue growing and many detached from the plate, as evidenced by 12the reduced number of cells by 14 d. By 21 d, even fewer cells remained. The ability of AL4 to grow in anoxia seems to be dependent upon the density of seeded cells (Rosey Le, personal observation); cells are less successful in proliferating when cell density is low upon exposure to anoxia.