Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

It is critical to effectively combine capture efficiency with dynamic live cell image observation to understand the effect of cell-cell interaction on cell behaviors. This tube can highly efficiently capture multiple single cells in a larger chamber, and provide a sufficient cultures base, for dynamic tracking of multiple single-cell interaction behaviors. Start by preparing the PDMS device.

Place a wafer mold in the weighing dish with 100 microliters of trichlorosilane in a desiccator and apply a vacuum for 15 minutes. Stop the vacuum and salinize the wafer mold in the desiccator at 37 degrees Celsius for at least one hour. Mix the PDMS base and PDMS curing agent at a ratio of ten to one, then pour a total of 20 grams of the mixture onto the wafer mold in a 15-centimeter dish.

Place the 15-centimeter dish into a desiccator and apply a vacuum for one and a half minutes. Then remove the dish from the desiccator and eliminate residual air bubbles in the PDMS with nitrogen gas. Cure the PDMS by placing the dish in an oven at 65 degrees celsius for two to four hours.

When finished, remove the PDMS replica from the wafer mold, and punch a 1.5-millimeter inlet and 0.5-millimeter outlet on the PDMS. Clean the PDMS replica and the slide surface with removable tape. Treat the surface with oxygen plasma.

Then, manually align the PDMS replica with the slide and bring them into contact with each other. Leave the PDMS slide in the 65 degree Celsius oven for one day. The next day, immerse the PDMS device in a container filled with PBS.

Place it into the desiccator, and apply vacuum for 15 minutes. Move the device to a cell culture hood and sterilize it with UV light for 30 minutes. Replace the PDMS device buffer with medium and incubate it at four degrees Celsius for one day.

When the cells achieve 70 to 80%confluency, remove the culture medium and gently wash them three times with five milliliters of PBS. Add one milliliter of DMEM/F-12 medium containing one micromolar fluorescent dye to the WNT5B sh4 and pLKO-GFP cells, and incubate them for 30 minutes at room temperature. After the incubation, gently wash the cells three times with five milliliters of sterile PBS, remove the PBS, and add two milliliters of 0.25%Trypsin-EDTA.

Incubate the cells for four minutes at room temperature, then gently tap the culture dish to promote cell detachment. Add four milliliters of DMEM/F-12 medium to disperse WNT5B sh4 and pLKO-GFP cells, transfer the cells into a 15-milliliter tube and centrifuge them at 300 xg for three minutes. Remove the supernatant and gently re-suspend the cell pellet in one milliliter of DMEM/F-12 medium.

Prepare one milliliter of cell suspension at a concentration of three times ten to fifth cells per milliliter and keep it on ice to prevent cell aggregation. Connect the polytetrafluoroethylene tube between the outlet of the device and the syringe pump. Remove the medium, and add one microliter of the prepared cell suspension into the inlet of the PDMS device.

Next, load the cell suspension into the device using a syringe pump at a flow rate of 0.3 microliters per minute. Add one microliter of DMEM/F-12 medium into the inlet of the device, then load the medium into the device using a syringe pump. Load 0.3 microliters of DMEM/F12 medium into the device with the syringe pump at a flow rate of 10 microliters per minute.

After the cells are loaded, remove the tube, and seal the inlet and outlet with polyolefin tape to create a closed culture system. Move the PDMS device to a 10-centimeter culture dish and add 10 milliliters of sterile PBS around the device to avoid evaporation of medium. Transfer the culture dish to an incubator for triple single-cell culture.

The PDMS device has a three-layer structure with a capture site that connects to the culture chamber and the bypass channel. The difference in flow resistance between the culture chamber and the bypass channel allows the cell to flow into the capture position, which causes the following cell to go through the bypass channel to the next capture site. The reflux step is used to release the cell from the capture site and send it into the culture chamber.

Cell capture efficiency of this protocol was evaluated on three different cell types. Human lymphatic endothelial cells, or LECs, human oral squamous cell carcinoma T2.6 cells expressing WNT5B-specific short hairpin RNA, and pLKO-GFP vector control cells. The single-cell capture efficiencies of the three demonstrated cells were approximately 71%for the LECs, 74%for WNT5B sh4 cells, and 78%for the pLKO-GFP cells.

The triple single-cell capture efficiency was 48%This method can be used for multiple single-cell co-cultures of lymphatic endothelial cells and squamous cancer cells, and the cell's proliferation and morphology can be observed under a microscope. It is important to make sure the prepared cell suspensions have minimal cell aggregates, because cell aggregates not only reduce single pairing efficiency, but can also clog the micro channels. Cell-cell interaction between individual cells plays an important role in controlling cell behaviors.

Our method can provide an efficient tool to help researchers study cell-cell interaction at a single cell level, which may lead to the discovery of new mechanisms which cannot be easily obtained from population-based studies.