Hi everyone,

I am working with 3T3 fibroblasts currently and mixing them together into a hydrogel composed of fibrin, gelatin, and glycerol. I performed a live/dead cytotoxicity assay on them, and they appear to have undergone some trauma during the whole procedure. I have included an image at 10X to illustrate what I am specifically referring to.

In particular, I'm referring to the cells whose cell membrane appear tattered and ragged, like the one This is in additional to seeing many dead cells (red signal).

Below is my entire procedure for producing the hydrogel components leading up to culture of the cells:

1. Produce a 15% (w/v) porcine gelatin solution in 1X DPBS w/o Ca2+ or Mg2+ at 37 degrees C with stirring. The solution was pH adjusted to 7.4 with a 1M NaOH since the addition of the gelatin makes the solution acid (pH 5).

2. Produce a 50 mg/mL solution of bovine fibrinogen in 1X DPBS w/o Ca2+ or Mg2+. Fibrinogen solution is gently agitated in the saline solution at 37 degrees C for 4-6 hours. Afterwards, pass the fibrinogen solution through a 0.2-um syringe filter.

2. Add glycerol to the gelatin solution while continuing to stir for 1 hour.

3. Glycerol-gelatin mixture was filtered through 0.2-micron filter as well.

3. Right before cell were added, the fibrinogen solution was added to the glycerol-gelatin mixture.

4. Cells that were passaged were resuspended in a 1X DPBS solution w/o Ca2+ or Mg2+ with 1% (v/v) bovine calf serum at a concentration of about 5 million cells per mL. The cells were then added to the hydrogel mixture as 20% of the final hydrogel volume.

5. After all components were mixed together, the final concentrations for each component were: 10% (v/v) glycerol, 7.5% (w/v) gelatin, 10 mg/mL fibrinogen, and 1 million cells per mL.

6. The cells were extruded out of a syringe gently using a 24G tapered tip.

7. The whole gel was clotted using a 20 U/mL solution of bovine thrombin in 1X DPBS w/o Ca2+ or Mg2+ for 10 minutes.

8. The thrombin solution was removed and replaced with the complete medium for culture.