Co-culture Model Using Two Types of Adherent Cell Lines

As the name of our institution, Maternal-Fetal Medicine Institute suggests, our research mainly focus on female reproductive biology. We aim to understand the mechanism of reproductive disorders and identify potential targets for treating them. One key advantage of this approach is the substantial reduction in time and expenditure compared to commercially available co-culture systems.

The homemade scaffold is constructed from readily available materials, significantly lowering the cost of the setup. This multi-cell in vitro models better represent a complex in vivo anatomy of the implantation region. In the future, our lab will introduce other cell types to this mixed seeding multi-cell in vitro model, and investigate more both implantation studies and uterine function in the pathogenesis of diseases such as endometrial injury and intrauterine adhering.

To begin, obtain the 3D-printed scaffold for model construction. For cell culture, place a clean and sterile 18-millimeter square coverslip at the bottom of a 12-well plate. Coat the coverslip with 200 microliters of extracellular matrix at a concentration of 200 to 300 micrograms per milliliter.

Keep the plate at 37 degrees Celsius for one hour. Then remove the extracellular matrix from the plate. Next, inspect the previously cultured human embryonic stem cells and Ishikawa cells under a microscope to confirm appropriate confluency.

Then remove the spent medium from the wells, and wash the cells twice with three milliliters of PBS. Now incubate the cells with two milliliters of dissociation enzyme at 37 degrees Celsius for two minutes to detach them. Neutralize the reaction using a complete culture medium.

Then create a single-cell suspension by pipetting up and down. Now mix 100 microliters of the cell suspension with trypan blue, maintaining a dilution factor of two. Then slide the coverslip over the hemocytometer chamber.

Fill both the side chambers with the cell suspension mixed with trypan blue. Count the number of cells in squares on both sides under a 20X microscope, and calculate the cell density based on the counts. Now seed the human embryonic stem cells at a concentration of 10 to the power of five cells per milliliter in the prepared plate containing fresh medium, and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.

For the Ishikawa cell line, seed the cells into the well without a coverslip the following day. Thoroughly soak the scaffolds with ethyl alcohol and double distilled water for two days. After soaking, remove the double distilled water and seal the metal can.

Then sterilize the scaffolds using an autoclave at 121 degrees Celsius. After that, allow them to dry. Add approximately two milliliters of complementary medium to the well with the Ishikawa cells.

Transfer the coverslip with attached human embryonic stem cells onto the scaffold, positioning the cell side down. Plug the scaffold into the well containing Ishikawa cells. For the second setup, place the coverslip with Ishikawa cells onto the scaffold and plug it into the well containing embryonic stem cells.

Incubate the co-culture systems at 37 degrees Celsius with 5%carbon dioxide. Carefully remove the culture medium from the wells, and gently rinse the cells with sterile PBS. Using fine forceps or tweezers, remove the coverslips with attached cells from the culture dishes.

Optionally, place the coverslips in a new dish with PBS to keep them hydrated during the transfer process. Add a drop of PBS on a clean microscope slide, and gently transfer the coverslip containing the cells onto the drop, ensuring the cells are face down onto the slide. Position the prepared slide on the stage of an inverted microscope.

Select the appropriate objective lens, and use the coarse and fine focus knobs to adjust the focus. Set microscope parameters, including illumination intensity and camera settings. Use the microscope software to capture images at desired magnifications and fields of view.

The density of both cell types remained low in the co-culture condition after 72 hours, while independently cultured cell densities increased significantly. The length of co-cultured human embryonic stem cells was shorter than those cultured independently.