Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.
Total time
~3–4 days (includes collagen preparation, cell culture, and viability assays)
Model organism
FEPE1L-8 keratinocytes
Steps
1
Prepare keratinocyte culture medium and fibril collagen
Formulate keratinocyte-specific culture medium and prepare type I collagen in fibril form by controlling pH and temperature to promote reassembly into three-dimensional fibrils.
▶ 01:12
2
Prepare non-fibrous form of type I collagen
Treat type I collagen to maintain its two-dimensional, non-fibrous molecular form for use as an alternative culture substrate.
▶ 02:53
3
Culture FEPE1L-8 keratinocytes on collagen substrates
Seed keratinocytes onto both fibril and non-fibrous collagen-coated surfaces and maintain cultures under standard conditions.
▶ 03:50
4
Estimate viable cell number using viability assays
Quantify the number of live cells at specified timepoints using appropriate cell viability techniques.
▶ 05:20
5
Evaluate keratinocyte proliferation across collagen forms
Analyze and compare cell proliferation rates between keratinocytes cultured on fibril versus non-fibrous collagen substrates.
▶ 06:20