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Human TNF-alpha Stability Testing

If you use cytokines in your research, you have probably seen the usual recommendations for storage and use:  Aliquot them out, avoid repeated freeze-thaw cycles, and replenish the culture medium frequently.  But what is this based on?  Here at Gene And Cell Technologies, we are committed to showing you the most detailed characteristics of our products you will find anywhere.  In this post, we share detailed test results regarding the stability of our human TNF-alpha preparation.

In all our TNF-alpha stability tests, the activity of the cytokine is measured by its effects to induce NF-Kb-dependent luciferase expression in our engineered HEK cell line.  Developing this reporter cell was a considerable upfront investment.  Now that we have it, we can easily test TNF-alpha activity, under a broad variety of conditions in high throughput format, and with very low costs.  Due to policies like these, we can deliver better characterized cytokines at lower prices than would be available elsewhere. 

There are three parts to the stability experiments reported here:  (1) Stability of the TNF-alpha lyophilizate.  (2) Stability of TNF-alpha against freeze-thaw cycles after dissolution.  (3) Stability of TNF-alpha in cell culture supernatant in the CO2 incubator.

 

(1) Stability of the TNF-alpha Lyophilizate

Our human TNF-alpha product ships as pure, lyophilized powder.  This makes it possible to ship and handle the cytokine at ambient temperature.  We had to invest upfront in developing a lyophilization process that preserves active and stable cytokine.  Now that we have that, we can realize considerable cost-savings against our competitors who need to ship their poorly characterized cytokines frozen on dry ice.  As you can see from our store prices, we pass those savings on to you.  

We tested the stability of the TNF-alpha lyophilizate by simply placing 8 fresh product vials at 37C in the incubator.  Every few days, we removed a vial and stored it at -80C freezer until the end of the experiment.  Then, we dissolved each vial's contents in sterile 10 mM Tris buffer pH 8.0 and diluted it as shown. 

We had our robot prepare a concentration gradient over our NF-Kb-luciferase reporter cell in 384-well format.  The next day, we lysed the cells and measured luminescence in each well.  Of course we took great care to keep the waiting time post luciferin addition short and consistent, to avoid biasing our results due to time decay of the short-lived light-producing reaction. 

As expected, increasing the TNF-alpha concentration resulted in increasing luciferase signal.  The half-maximally effective dose (ED50) for this effect was in the expected range of 0.5 - 1.0 ng/ml TNF-alpha in the culture medium.

There was no consistent effect of lyophilized storage time (shade of the curve, lighter = longer time) at 37C.  The very last data-point may show a small decline of both the maximal signal strength and the ED50 at 4 weeks of storage.  For all the other data-points we appear to be looking at random variance of a magnitude typical of cell based assays (+/- ~20%).  There is no detectable effect of storage time in as much as three weeks. 

Thus, our lyophilized TNF-alpha preparation is extraordinarily sturdy.  It can easily ship anywhere in the world, at ambient temperature, in hot climates, and can even survive customs delays if necessary.  In the continental USA, ambient shipping via our free 1-3 day priority service is certainly very safe, according to these results.

 

(2) Stability of TNF-alpha against freeze-thaw cycles after dissolution

Once you receive your human TNF-alpha product, you're going to want to dissolve it in sterile buffer, so that it can easily be dosed into cell cultures.  Our recommended buffer for dissolution of TNF-alpha is 10 mM Tris pH 8.0.  This liquid then must be frozen at -20C or -80C for longer-term storage. 

But to what degree does freezing and thawing damage TNF-alpha in solution?  To find out, we dissolved 100 ug TNF-alpha in 1 ml of 10 mM Tris pH 8.0.  Then, we subjected it to as many as 35 freeze-thaw cycles.  For each cycle, we placed the vial in a Styrofoam box filled with dry ice.  We waited at least 30 minutes for it to fully freeze, and go thorough all the different ice phase transitions all the way down to -80C.  Then, we thawed the vial in a water bath at 37C.  Every 5 cycles, we took a sample for analysis as above. 

There is no effect of freeze-thaw cycle count on activity at all.  We appear to be looking at random variance typical of cell-based assays.  The most frequently frozen and thawed sample (35 cycles!) happens to lie right in the middle (the lightest curve, not the easiest to see). 

We must conclude that TNF-alpha dissolved in 10 mM Tris pH 8.0 at 100 ug/ml is extremely resistant against freeze-thaw cycles.  We still recommend to prepare a few aliquots, to guard against issues like dropping a vial, or random acts of higher powers.  However, freeze-thaw cycling per se does not appear to cause any problem for this sturdy cytokine. 

 

(3) Stability of TNF-alpha in cell culture supernatant in the CO2 incubator.

Finally we asked:  How stable is human TNF-alpha in cell culture supernatant?  In other words, how frequently do you need to replenish the cytokine over your cells, in order to maintain constant exposure? 

To do so, we used a slow-growing primary human fibroblast line in DMEM with 10% FBS.  We seeded the cells to about 25% confluence.  Under these conditions, it takes them about 6 days to reach 100% confluence.  After that, the culture would overgrow with extreme media acidification.  So this experiment cannot run for longer than 6 days. 

Immediately upon seeding the culture, we added TNF-alpha to 20 ng/ml in the cell culture supernatant.  We took samples every day for an entire week.  Then, we did the same dilution experiment on our reporter cell line as above.  In this experiment, TNF-alpha supernatant is mixed into reporter cell suspension at a ratio of 1:1.  Therefore, the highest tested TNF-alpha concentration over the reporter cells is 10 ng/ml. 

In this experiment, we can clearly see a time-dependent decline of both the ED50 and the maximal signal intensity.  After the first 24-hours, TNF-alpha still seems to be as active as at the start of the experiment (two darkest shades).  After that, a noticeable decline sets in. 

There is also a control with no TNF-alpha added (lightest shade at the bottom).  This shows that the test culture does not produce its own TNF-alpha, but rather all the signal is attributable to our exogenously added TNF-alpha. 

TNF-alpha maintains some activity for the entire experiment.  The ED50 appears to shift ~5-fold during the 6-day incubation period.  Thus, if you can add 5x saturating concentrations, you should be able to maintain maximal TNF-alpha exposure for as long as a single cell culture passage can reasonably go. 

However, if you want to maintain a constant or submaximal exposure over time, these results suggests that daily media changes will be necessary to achieve that.

It is important to keep in mind that the present data show TNF-alpha stability only in one standard model system, a fibroblast culture growing slowly in DMEM 10% FBS.  If your culture conditions are different, your stability outcome might be different.  For example, the more the culture medium acidifies, the less stable TNF-alpha is going to be.  Some cell lines (e.g. HEK293) cause much more rapid acidification than these fibroblasts.  Also, if your cell has an unusually large amount of TNF-alpha receptors, or secretes soluble TNF-alpha receptors, your stability can be affected.  It is always safest to do your own studies. 

 

Conclusions

The results presented here suggest that our human TNF-alpha preparation is a sturdy cytokine that can take considerable abuse during shipping and storage, with no loss of activity.  However, once TNF-alpha is used in cell culture, it is advisable to either budget for a ~2x decline of TNF-alpha activity per day, or better perform daily media changes. 

 

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