Hello everyone, when exploring the in vitro cell culture system recently, I paid attention to collagen (bought from MCE, Cat.NO.: HY-NP010) as a natural extracellular matrix (ECM) component.
Its gelation application can effectively promote the maintenance of cell-specific morphology and functional expression (such as directional differentiation, metabolic activity or secretory function enhancement, etc.) by simulating the in vivo microenvironment.
When used as a gel, can collagen help in vitro culture and enhance the expression of cell-specific morphology and function?
What are the specific methods of use?
Thanks at first!
Collagen I can be made to gel when its pH reaches alkaline by following the steps below.
1) Prepare an ammonia vapor chamber by attaching a sterile 5 cm gauze sponge to the lid of a 150 mm culture dish. Saturate the gauze with ammonium hydroxide, place the lid on the 150 mm culture dish, and set aside.
2) Spread collagen I evenly on the surface of the object to be coated. The thickness can be varied as needed. Collagen I (50-100 μL) is sufficient to coat a 22 mm coverslip. For a 100 mm diameter culture dish, add about 6 mL to each; for a 60 mm culture dish, add about 2.3 mL, and for a 35 mm culture dish, add about 1 mL.
3) Transfer the collagen-coated coverslip or culture dish with lid to the ammonia vapor chamber and expose for three minutes.
4) Soak the coated coverslip or dish in sterile distilled water (5 mL for 35 mm dishes, 10 mL for 60 mm dishes, etc.) for 30 min. Aspirate the original distilled water and add 0.5-1.0 mL of sterile distilled water and place in a laminar flow hood overnight.
5) Aspirate the distilled water and replace with a balanced salt buffer solution containing serum and store at 2-8°C.
Alternate gelling procedure:
1) Place the following on ice: Collagen I, sterile 10× PBS, sterile distilled water, sterile 1M NaOH
2) Determine the volume of collagen I solution required for the final concentration to be used.
3) Place a sterile tube on ice to store the collagen I.
4) Perform the following steps under sterile conditions.
4.1 Add 10×PBS (final volume/10) mL
4.2 Calculate the volume of collagen I to be used (do not add to the tube until step 4.6). At this point, final volume x final collagen I concentration (mg/mL) = amount of collagen to be added.
4.3 Add (volume of collagen to be added x 0.023) mL of sterile ice-cold 1M NaOH to the 10×PBS solution.
Hi, you can use PureCol products according to manufacturer instruction.
Moreover, not sure what you mean "by simulating in vivo environment". In vivo collagen fibers have orientation (anisotropic) and is not disorganized. In vitro simple systems usually are disorganized (isotropic). If you want to make it anisotropic in vitro, you need to use sophisticated crosslinking systems.
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