I recently started working with fibrin hydrogels. I'm using bovine aortic endothelial cells and embedding them in a fibrinogen (10mg/mL) / thrombin (1.25U/mL) mixture. The hydrogels seem solid and stable when they first polymerize, but after 16 hours or so they rapidly degrade. I find cells and pieces of fibrin clot floating in the media.
I'm not sure what I'm doing wrong, because I'm following well-established protocols from a few different papers that have demonstrated stable long term fibrin hydrogel formation with endothelial cells. Has anyone encountered this problem? Any feedback about how I could troubleshoot would be very much appreciated. Thank you for your time!
Hi Jess,
so there are different factors that can contribute to instability of your scaffold.
1: Are you adding aprotinin to your medium? This is important to prevent cells from degrading your fibrin scaffold.
2: Which CaCl2 concentration do you use? A too high concentration results in a less stable fibrin scaffold. A final CaCl2 concentration of 40 mM, for instance, resulted in my experiments in a higher instability. 12 mM worked for me.
3: Cell concentration: test different cell concentrations to find a balance between scaffold stability and desired cell behavior.
4. Fibrinogen concentration. Have you tested higher concentrations or do you need the low Young´s modulus of the 10 mg/mL concentration?
Hi Amin and Ruben,
Thank you very much for your responses! I have some info for your questions:
I'm using EGM-2 media from Lonza and BAE cell density of 1x106 cells/mL. The protocol I'm using is derived from http://pubs.rsc.org/en/content/articlelanding/2017/bm/c7bm00223h#!divAbstract and is as follows: "The desired hiPS-EC (5×106 cells/mL) and hMSC (0, 1×105, or 1×106 cells/mL) cell densities were re-suspended in 37 °C, pre-warmed 10 mg/mL of fibrinogen (Sigma-Aldrich, St. Louis, MO), 1.25 U/mL of thrombin (Sigma-Aldrich, St. Louis, MO), and 1× phosphate buffered saline (PBS). Once the fibrinogen and thrombin came into contact, enzymatic crosslinking began, forming the fibrin gel within a few minutes. A 10 µL volume of the suspension was mixed and placed into individual wells of an Ibidi µ-slide (Martinsried, Germany) with delayed pipetting to ensure homogeneous distribution of encapsulated gels in the crosslinked fibrin gel."
The media for the hydrogel culture is as follows: "The iPS-ECs and hMSCs used in experiments were at passages 3- 6. When encapsulated in hydrogels, both the monoculture and co-culture conditions were cultured in VascuLife Medium. All cells were incubated at 37 °C and 5% CO2."
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Regarding your questions, Ruben:
1) I haven't added aprotinin to my medium. The protocols I'm following haven't specified it's use, do you have a suggestion for a protocol or publication using aprotinin?
2) I'm not sure the CaCl2 concentration for my media, I'm using Lonza's EGM-2 endothelial cell media. I've seen EGM-2 used with fibrin hydrogel experiments in literature, however.
3) I'm using similar cell densities as what I've found in literature, but I haven't optimized for my cell type
4) I'm also using a similar fibrinogen concentration to what I've found in literature (10mg/mL)
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It sounds like I need to optimize my thrombin, fibrinogen, and cell density ratios for my experiments. I will also look into adding aprotinin to my hydrogels and fabricate some acellular fibrin gels to make sure it's not my cells tearing up the gels! Please let me know if you have any other ideas, and thank you so much for your time!
Best,
Jess
I normally dilute the thrombin stock solution with CaCl2 to e.g. 1.25 U/mL, resulting in a 40 mM CaCl2 concentration. Then I mix my cells in 1:1 or 1:2 ratio with the thrombin solution and finally then 1:1 with fibrinogen.
Aprotinin:
Aprotinin should be also in your fibrinogen stock solution (normally done by the company). You can just google "fibrin gel medium aprotinin" and you will find papers about this.
In this paper, for instance, they observed that the fibrin gels dissolved within 2 days when cultured with medium w/o aprotinin.
https://www.ncbi.nlm.nih.gov/pubmed/10814924
Regarding your cell number, I would say it is ok and not too high. Your fibrinogen concentration is quite low but as long as you don´t want to apply mechanical strain (e.g., with a bioreactor) it should be fine.
I am pretty sure that the missing aprotinin in your medium is the reason. I used in my medium a concentration of 100 U/mL and had no problems.
Ruben Gerrit Scheuring Hi, have you found a problem for fibrinogen at lower concentration (10mg/ml) under the mechanical stimulation, will it collapse?
I never tested 10 mg/mL for this, but the scaffolds are definitely more fragile. I used 20 mg/mL with 3% baseline strain and 10% strain for 6h as training session without any issues. It always depends on what you are aiming for.
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