Hello everyone.
I am trying to achieve 3D culture of primary human dermal fibroblasts in Matrigel. I tried working on different concentration variants of Matrigel- Normal (Basement membrane matrix) and High concentration (HC). Normal one doesn't seem to suit my application most of the times as it does not end up getting a rigid gel. I tried diluting the HC variant (approx 21-25 mg/ml concentration) to 1:2 or 1:3 with media.
But I am getting differently sized cells of primary human dermal fibroblasts in the matrix. Also I can observe spheroid like clumps in certain areas. I am not sure whether the large sized cells (which may seem to be individual) are actually clumps. On the other hand, keratinocyte cell line forms uniform distrubution throughout the matrix and cell size increases with increase in matrix concentration.
I intend to achieve uniform distribution of cells throughout the matrix with proper networks to form the dermis. Please help me troubleshoot this. Thanks in advance.
Give some time to gel first add gel with pbs not media as solutioN. Give 24 hour prior settin time. It will be done. Then go for 3D culturE. I am sure it will work. Thanks
Mohammad Tariq Ali Khan : Many thanks four your reply. However, I am entrapping my cells in the gel while seeding itself. Then how should I let my blank Matrigel form a gel first and then add the cells? Please let me know.
We use hanging drop method for 3D cell culture it takes 48 to 72 hr to form spheroiD. 3 D cell can live up to 5 days no problEm. Firstly make 3D then transfer in matrigel. After 2 or 3rd day. Good luck.
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