Hydrogel preparation
Léa Lagny
Hi everyone,
We are trying to set up the BMO generation and we do not have experience. We meet a problem during the embedding stage. We are not certain that our hydrogel is behaving as expected.
We would like to clarify that we are not using exactly the same protocol as the one described in the article previously shared in this group. In our case, we are using type I collagen (5 mg/mL) mixed with Matrigel.
To better understand the problem, we have prepared a cell-free hydrogel and would greatly appreciate your feedback on its appearance and consistency (I will attach the images)
Here is the protocol we used:
1. Preparation is done on ice using pre-cooled tips
2. In an Eppendorf tube, we mix:
1. 25 µL DMEM 1X
2. 68.75 µL ddH₂O
3. 6.25 µL sodium bicarbonate
4. 8 µL Hams F-12
5. 4.8 µL HEPES
6. 2.4 µL Glutamax
7. 150 µL Collagen type I (5 mg/mL)
3. Matrigel is added last
4. The mixture is gently homogenized
5. Place 1 mL of pbs 1X and remove it
6. 500 µL of the gel is plated per well (plate 12 wells)
7. Incubation for 30 minutes at 37°C
Do you think the appearance of this hydrogel meets our expectations, or does it suggest it could be improved? We're not entirely sure of the optimal structure. If the gel doesn't appear correct, do you think this could be due to the incubation time or rather to the preparation itself? Furthermore, could the absence of collagen IV mean that our organoid culture conditions are not optimal?
The difference between “Hydrogel 1 COL I Matrigel” and “Hydrogel 2 COL I Matrigel” are:
“Hydrogel 1 COL I Matrigel” we used directly a collagen type I (5 mg/ml) and matrigel without leaving it overnight at 4 degrees. And we tried to adjust a pH by adding 1 µL NaOH 1M after to add a collagen and matrigel.
“Hydrogel 2 COL I Matrigel” we leave collagen I (5 mg/mL) overnight at 4 degrees on rocker and a matrigel solution overnight at 4 degrees and we followed the steps above.
Thank you in advance for your help.
Best regards
Lea
