Hello everyone,
I was hoping someone might have some insight into a problem I have with filtering fibrinogen solutions. I use bovine fibrinogen from Sigma Aldrich (F8630), which is Type I-S, 65-85% protein (≥75% of protein is clottable). I want to sterilize it using a 0.2 micron filter but I find that it is very difficult to pass through using a syringe.
I am using it to make fibrin hydrogels. Many researchers use a 10 mg/mL fibrin gel for cell culture, and we have designed our protocols to include other components, meaning that the concentration of the sterile fibrinogen solution we want to make is at least 25 mg/mL or above. Most frequently, I am making a 35 mg/mL stock solution.
I dissolve the fibrinogen currently in 1X DPBS, pre-warmed to 37 degrees Celsius before adding the fibrinogen. I then place the solution in a 37 degree incubator for 45 minutes to 3 hours until I get a hazy, opaque solution before trying to pass through a 0.2 micron regenerated cellulose filter. I use the ones from Corning. I have tried different diameters of filter (4 mm & 25 mm). I typically make 1 mL of stock solution and if I use the 0.2-micron 4-mm diameter filter, I am only able to pass only a few microliter before it seemingly clogs and the fibrinogen solution stops passing through. When I use the 25-mm diameter one, it passes much faster and more easily, but I have to push the syringe plunger all the way down before I start to see fibrinogen solution coming out the exit side of the filter (I use a 1 mL syringe for all sizes of filter). The majority of it stays trapped in the syringe filter. What I have to do to get more of it out is disconnect the filter from the syringe, pull the syringe plunger up, reconnect the filter and syringe back together, and push down again. I am at most able to get out 500-600 microliters, which means I am losing about 40%-50% of my solution.
Does anyone know of a way to consistently sterile filter concentrated fibrinogen solutions? Sigma Aldrich suggests to dissolve the fibrinogen in a warmed 0.85% - 0.9% saline solution. Is this equivalent or similar to a 1X DPBS solution? The techs at Corning suspected that if the syringe filter was "clogging", then the fibrinogen was not all going into solution. One tech I spoke with suggested to allow 6 hours for dissolution, and I have not tried that yet.
Also, does anyone know how to improve stability of fibrinogen solution long term. I notice I start to get precipitate in my solution just after a couple days. After I make it, I keep it in the fridge.
I would greatly appreciate any protocols people could share. Thank you.
Hello Sebastian,
I would first suggest that you use a highly purified fibrinogen. Any trace of protease would favor aggregation and polymerization. Use any buffer without calcium (to limit polymerization and the formation of agregates.
For your routine experiments I woul rather store frozen aliquotes and when needed quickly unfreeze at 37 °C (oherwise you will favour precipitation since fibrinogen is a cryoglobulin).
You could also buy some highly and purified firinogen from your local blood transfusion Center.
Thank you, Florence. I had thought about freezing it, but I worried whether the freezing would negatively affect the fibrinogen. How long can you keep the fibrinogen in the freezer and what concentrations do you make?
Well I am out of Lab so, you will have to wait for precise storage concentration value and buffer untill nex year!
Yet i had a glance at the data sheet from sigma: The powder is in ~10% sodium citrate (calcium chelator) and ~15% sodium chloride. Purity is for sure below what you would expect from a blood transfusion product. 25 % of unclottable fibrinigen at best means 25 % of altered fincnogen and contaminating proteins (fibronectin, for instance).
1- The reconstituted fibrinogrn stock solution should contain about 150 mM of all salts (f.c.), meaning that you have to take int consideration the amount of salts brought by the added DPBS volume to the powder (or by any other reconstituting buffer or solution).
1-bis Also warm the powder in a 37°C waterbath, and gently roll the flask between your palms to avoid foam when you add the fluid.
2-. Additionally, PBS is really not recommended : it prompts hydoxyapatite cristals in the presence of calcium.( check if calcium is present in your cell cultutre medium). Replace by non phosphate buffer or HBSS (although in the latter case pH will not be controlled in your stock solution, but can be after addition of a small volume of the fibrinogen stock solution to the final medium).
3- When using calcium (in the polymerozation tepof fibrinigen into fibrin, or for other purpose) you will have to take ito account the citrate concentration of the stock solution. 1 mole of citrate binds 1.5 mole of calcium that will not be free and will remain ineffective. Therefore you will have to add the amount of calcium known to be bound to citrate to the calculated amount of calcium needed to reach the desired final calcium concentration,
Seasons greetings!
Hi Florence,
The DPBS that I resuspend the fibrinogen in contains 2.67 mM of potassium chloride and 137.93 mM of sodium chloride, along with 1.47 mM of potassium phosphate monobasic and 8.06 mM sodium phosphate dibasic.
What is the desired calcium concentration for fibrin polymerization? Is this mechanism different than when fibrin is polymerized by thrombin? Just curious, although I don't seem to have any problems with polymerization for fibrin gels.
Hi Florence Toti Sebastian Freeman !
I notice precipitates forming when I freeze my fibrinogen stock solutions. However, they seem to eventually dissolve away when I thaw them at RT (I'll switch to thawing at 37C in the future!). Have y'all observed this phenomenon? I'm wondering if the solution is still good for use if the precipitates eventually dissolve away.
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