• +1-513-348-4677
  • info@bluekick.com

Yearly Archive 2014

Hypoxia enhances tumor cell resistance to cell stress caused by starvation and hypothermia

by Emilio Barbera-Guillem, MD, Ph.D., Chief Scientist and Founder of Celartia in Hypoxia Cell Cultures

Hypoxia enhances tumor cell resistance to cell stress by starvation and hypothermia
Since many years ago it is well known that tumor cells cultured in hypoxia conditions show higher survival performances when exposed to radiation and diverse drugs (for example: I. TANNOCK and P. GUTTMAN, 1980, J.M. BROWN 1999). That was observed with certain perplexity because exposure to oxygen is essential for nearly all living vertebrates. However it is also true that some vertebrates can survive hours in absence of oxygen. Actually, even in close to absence of molecular oxygen (anoxia), some vertebrate enter a state of reversible suspended animation where all observable cell macroscopic and microscopic movement ceased, including cell division and cell motility. The cell-cycle looks arrested in S and G2 phases in this “dormant state” (P.A. Padilla and M.B. Roth, 2001). Moreover, some invertebrate cells have the ability to survive in anoxia for months (G.L. ANDERSON, 1978). Many research results support the idea that the survival in anoxia depends on the organism’s ability to restrain energy usage by shutting down nonessential cellular functions, maintaining stable/low permeability of membranes, and synthesizing ATP though glycolytic processes (P.W. HOCHACHKA, 1996) As a Decreasing the aerobic energy production, cells increase anaerobic energy production and decrease energy demand. In mild hypoxia, oxidative phosphorylation is low but active, therefore maintaining some aerobic energy production, and simultaneously up regulating the activity of hypoxia-inducible transcription factor 1 (HIF-1), together with other genes involved in anaerobic energy production, such as glycolytic enzymes, glucose transporters, and antioxidants (catalase, superoxide dismutase, etc) which protect free radical-induced damage.

Experiments of chemically induce expression of HIF with deferxamine or ClCo, mimicking hypoxia, show that this factor is involved in the induction of a chain of events that protect cells from apoptosis and death extending the life of cells implanted in the peritoneal cavity for example (H. SAUER, 2003; B.E. TUCH, 2010).When cultured adherent cells reach confluent monolayer may drop the media pH below 6.8 units in spite of the controlled 5% CO2, due to the nutrients deprivation (starvation) and excess of metabolically produced Lactic Acid and CO2. These conditions, when maintained for several hours at 37OC generate cell stress and induce cell death.

First here it is challenged the hypothesis that cells in culture in starvation may survive longer periods of time when exposed to deep hypoxia (DO= 1mg/L, equivalent to 2% O2 in the incubator), compared with regular open atmosphere cultures in plates and flaks.

Eighty T75 flasks with filtered cap at a density of 2×105 CHO Cells/cm2 (over 107 cells per flask) in high glucose DMEM, in open cultures at 37OC, exposed to 21% O2 atmosphere without media change, 90% of the cell become unviable (conventional culture death) when were confluent within 36 h. and 80 hours (Median survival 72 hours). However cultures in parallel in Petaka G3 LOT (80 unit total) with internal 17.2 mmHg Oxygen of partial pressure, equivalent to 2.3 % oxygen in hypoxia incubator cultures loss more than 90% cell viability in a period within 80 and 288 hours (Fig 1a) with a median survival of 252 hours. The comparative Mantel-Haenszel test showed a Chi square value =36.12, proving that survival curves were significant different for P<0.0001.
Second it is challenged the hypothesis that cells in culture in mild hypothermia (within 15 and 22OC) may survive longer periods of time when exposed to deep hypoxia as before (DO= 1mg/L, equivalent to 2% O2 in the incubator), compared with regular open atmosphere cultures in plates and flaks.

Eighty T75 flasks with filtered cap at a density of 2x105HeLa Cells/cm2 (over 107 cells per flask) in high glucose DMEM, in open confluent cultures, exposed to at 22OC and 21% O2 atmosphere without media change, 74% of the cultures become unviable in 5 days 90% were dead in 7 days (Median survival 7 days). However parallel cultures in Petaka G3 LOT (80 unit total) with internal 17.2 mmHg Oxygen of partial pressure, equivalent to 2.3 % oxygen in hypoxia incubator, 50% cultures die (cell viability <10%) in 17 days and 90% in 31 days (Fig 1b) with a median survival of 30 days. The comparative Mantel-Haenszel test showed a Chi square value =19.98, proving that survival curves were significant different for P<0.0001.

tumor resist  hypoxya p
Conclusion: Two models of tumor cell lines (both carcinomas) exposed to starvation and mild hypothermia show statistical significant increase of survival rate when the available oxygen in the media is within the range of the physiological tissue levels (currently called Hypoxia), and are less resistant when exposed to the not physiologic atmosphere standard 21% O2 level in open cultures (currently called Normoxia).
Cells are normally cryopreserved for shipping; however cryopreservation process is strenuous for cells. Cryopreservation media needs the addition of toxic chemicals, such as DMSO and glycerol, to avoid ice crystals formation that kills cells. Moreover, transportation of cryovials needs about 5 to 10 pounds of dry ice per day of transportation. Therefore, for a 3 days distance between sender and receiver the shipping package needs 15 to 30 pounds (7 to 15 Kg) of dry-ice. This is heavy, expensive and hazardous (http://ehs.missouri.edu/haz/pdf/dry-ice-training.pdf). Keeping the cells in hypoxia in specific vials (like Petaka) constitute a safe alternative for cell shipping without dry-ice.

Collaborative articles by Celartia staff writers.

A low cost version of GlycoStation Reader series

A new version of current GlycoStation Reader series will be released shortly with a moderate price.

Major differences between this one and GlycoStation Reader 1200 are:

(1) Resolution: 5um -> 15um

(2) Fluorescence color: Multi-colors -> Cy3 only

The user interface has been greatly improved, so that simple analysis can be done on the LCD panel.

http://www.glycotechnica.com/images/technology/NewGSR.jpg