This video shows how to prepare two different types of cell culture substrates using type I collagen and two-dimensional and three-dimensional culture method. The three-dimensional culture method is considered to the biological environment, demonstrating how to easily prepare a three-dimensional culture substrate is useful for studies. In numerous cell types, despite using the same collagen, cell behavior considerably varies between two-dimensional and three-dimensional substrates.

Using three-dimensional culture substrate, cell behavior that is more similar to those of living organisms could be easily examined. This method is very simple and requires minimal special reagent. Visual demonstration is critical, because while we want to demonstrate that this method is easily attempted, the three-dimensional gel is fragile, and handling it properly is very important.

To begin, prepare the appropriate culture medium. To begin preparing the collagen, place 10x PBS, deionized water, collagen, a 96-well culture plate, and an empty two-milliliter tube on ice. Using a pipette, add 1.12 milliliters of deionized water to the empty two-milliliter tube.

Add 200 microliters of 10x PBS to the deionized water, and gently shake the tube several times. Immediately prior to use, add 0.66 milliliters of collagen to the two-milliliter tube. Gently and quickly shake the tube several times.

Pour 100 microliters of this collagen solution into each well of the 96-well culture plate, making sure to cover the entire surface of the wells. Gently shake the plate using a left-to-right motion. Incubate the culture plate in a carbon dioxide incubator at 37 degrees Celsius for one hour.

Then tilt the plate to confirm the gelation of the collagen, and move the culture plate to a clean bench. Gently pour 150 microliters of K110 onto the gels by pipetting along the wall of the well. Incubate in a carbon dioxide incubator at 37 degrees Celsius for one hour.

After this, move the culture plate to a clean bench. Immediately prior to cell culture, use a pipette to gently discard the K110. First use a pipette to add four microliters of collagen to 1.2 milliliters of one millimolar hydrochloric acid in a 1.5-milliliter tube, and gently mix.

Pour 100 microliters of this collagen mixture into each well of a new 96-well culture plate. Gently shake the plate in a left-to-right motion and incubate at room temperature for one hour. After this, discard the collagen solution.

Using a pipette, wash each well with PBS. Add 150 microliters of 1%BSA and PBS to each well of the culture plate, and incubate at room temperature for one hour. Prior to cell culture, discard the 1%BSA solution.

To begin, prepare the FEPE1L-8 cells and the K110, trypsin, and trypsin inhibitor as outlined in the text protocol. Carefully remove the medium from the culture dish, and use a pipette to add three milliliters of 0.05%trypsin. Place the dish back into the carbon dioxide incubator, and incubate at 37 degrees Celsius for five minutes.

Then use phase contrast microscopy at 10x magnification to check for cell detachment from the surface of the culture dish. Add three milliliters of trypsin inhibitor, and use a pipette to collect the detached cells in a 15-milliliter centrifuge tube. Centrifuge at 200 times g for five minutes.

After this, discard the supernatant, and resuspend the pellet in 10 milliliters of K110. Use phase contrast microscopy at 10x magnification to count the cells, and dilute the cells with K110 to a cell density of 50, 000 cells per milliliter. Gently seed 0.1 milliliter of the cell suspension into each well of the culture plate by pipetting along the wall of the well.

Incubate in a carbon dioxide incubator at 37 degrees Celsius for the appropriate length of time. First mix 130 microliters of tetrazolium salt and WST-8 with 1.3 milliliters of preheated K110 in a two-milliliter tube. Move the culture plate to a clean bench, and use a pipette to gently discard the K110 from the wells.

Gently wash away and nonadherent cells with K110. Then add 110 microliters of K110 mixed with WST-8 to each well. Incubate in a carbon dioxide incubator at 37 degrees Celsius for two hours.

After this, move the culture plate to a clean bench. Collect 100 microliters of the conditioned medium from each well, and move it into the wells of a new 96-well culture plate. Using a microplate reader, measure the absorbance at 450 nanometers, and estimate the number of viable cells.

The cell morphology is observed on the non-fibrous and fibril forms are shown here. In the initial two hours of culturing, the cells adhere and spread on both forms of collagen. Three days after seeding, cells on the non-fibrous form continue to spread, and cell numbers increase, while the cells on the fibril form show limited spreading.

FEPE1L-8 cells continue to proliferate on the non-fibrous form of type I collagen and on the untreated dish surfaces. In contrast, the cells do not proliferate on the fibril form. When attempting this procedure, it is important to remember that the three-dimensional gel is very fragile.

One must be careful to ensure that the solution or tips don't come into direct contact with the gels. This method can be modified in numerous ways. For example, to mix specimen's membrane components, one can make a cell culture substrate that mimic a basement membrane.

In numerous cases, the functions of the extracellular matrix in living bodies are unknown. To culture cells on the three-dimensional culture, one can examine the function of extracellular matrix component that is more similar to the living body. By applying three-dimensional gel culture techniques, one can effectively test the concentration of drugs in cells.

This protocol, based on one's purpose, could be used to develop complex three-dimensional culture substrates by mixing other EGM components or growth factors during gelations.

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