细胞分化和牵引力的相关分析高通量细胞芯片平台

The overall goal of this cell microarray platform is to correlate measurements of both cell differentiation and traction forces as a function of microenvironmental context. This method can help answer key questions in the field of tissue engineering enabling both fundamental investigations of stem cell biology and optimization of differentiation protocols. The main advantages of this technique include its throughput, ability to vary biochemical and biophysical cues, and endpoint readouts by both immunoflourescents and traction force microscopy, or TFM.

Though have used these method, those understanding liver progenitor differentiation it can be readily applied to other arterial cell types and tissue contexts. Visual demonstration of this method is critical, as experimental success depends on integration of the arraying protocol with both high-quality hydrogel substrates and traction force microscopy. To fabricate fluorescent bead containing polyacrylamide hydrogels on a silanized 35 mm glass-bottom petri dish for live evaluation of cell substrate interactions using TFM, first, prepare glass substrates in solutions as described in the text protocol.

Place silanized 35 mm glass bottom petri dishes into a glass drying tray and pipette 20 microliters of 9-to-1 pre-polymer bead to photo initiator solution onto the center of each dish. Gently cover each dish with a 12 mm circular cover slip while avoiding the creation of bubbles. In order to distribute the fluorescent beads to the surface of the hydrogel, invert the dishes and leave them at room temperature for 20 minutes.

While still inverted, expose the dish to 365 nanometer UVA for 10 minutes. Optimize the polymerization time as needed. Next, immerse the hydrogels in 1 molar HEPES buffer and leave them at room temperature in the dark overnight.

Remove the cover slips carefully with a razor, taking care not to damage the polymerized hydrogels. Dehydrate the hydrogels at 50 degrees Celsius on a hot plate until dry. Hydrogels can be stored at room temperature in the dark for three months.

Prepare buffers to print the bio-molecules and for the source plate as described in the text protocol. After loading the clean pins as described in the text protocol, prepare the microarrayer and program it using the manufacturer software. Next, turn on the humidifier unit.

Adjust the set point to 65%relative humidity and wait until the rheometer matches the set point. Place the source plate in the appropriate adapter. Then place the dehydrated hydrogel substrates into the appropriate adapter.

Adjust the parameters of the program to accurately reflect the layout of the source plate, array design and desired format. Start the array fabrication. Check no less frequently than once per hour that the humidity has not dropped below 65%relative humidity and that the pins are not clogged.

If the humidity has dropped unexpectedly, pause arraying to fill the humidifier and clear associated tubes of condensation. If the pins are clogged, pause arraying to clean the pins or otherwise replace with pre-cleaned pins. Once the program is complete, place fabricated arrays in a slide box or micro plate covered with aluminum foil.

Leave the arrays at room temperature at 65%relative humidity overnight. In our experience, the most common difficulties are related to array fabrication. We recommend confirming the technical quality and robustness of fabricated arrays using flourescently labeled molecules, general protein stains, and immunoflourescents.

The day after fabrication, immerse the array 35 mm petri dishes in 3 mL of 1%volume per volume penicillin-streptomycin in PVS. Expose the arrayed substrates in UVC for 30 minutes. Then exchange the penicillin-streptomycin solution for cell culture media.

After collecting and counting the cells, seed the cells onto arrays at 3 mL per 35 mm petri dish. Incubate the array cultures at 37 degrees Celsius and 5%CO2 for two to 24 hours, or until formation of well-populated cell islands. The seeding density and time can be adjusted depending on cells and particular application.

After allowing for the formation of cell islands, wash the array cultures twice with 3 mL of pre-warmed cell culture media. At this point, the appropriate controls and treatments of interest can be added to the biological system. Change the media of the arrays every one to two days to maintain the concentration of any treatments.

Within one to five days of initiating array cultures, perform live evaluation of cell substrate interactions using TFM. Move the 35 mm petri dishes containing array cultures to an incubated inverted fluorescent microscope with a robotic stage for TFM measurements. In one dish, mark the positions and focus planes of individual cell islands using phase contrast microscopy.

Switch to far red fluorescent microscopy to visualize the beads. Then return to each of the positions saved in the previous step and correct the z-coordinate of the focus plane, so that only the first layer of beads below the cell island is in focus. Save the new coordinates and proceed to automated imaging of all cell islands to capture the pre-dissociation phase contrast and far red fluorescent images.

Next, carefully add 150 microliters of BSA/SDS solution to the dish and wait five minutes to allow for complete cell dissociation from the substrate. Monitor cell dissociation using phase contrast microscopy. After the cell islands have been dissociated from the substrate, return to the marked positions and check that the first layer of beads are still in focus.

If these beads are out of plane due to deformation induced by cell-generated traction, correct the z-coordinate of the focused plane so that they are again in focus. Save the corrected z-coordinates and repeat automated imaging of all islands to capture the post-dissociation far red fluorescent images. Repeat these steps for the remaining dishes.

Protein A/G conjugated notch ligands jagged one and delta like one showed improved retention in hydrogels. The notch ligand presentation also drove differentiation of liver progenitors towards a bile duct cell fate as indicated by the presence of the green bile duct cell marker. The response to notch ligands was quantified for five extra-cellular matrix, or ECM, proteins and showed that the response of liver progenitors to ligands depends on the ECM context.

Small hairpin RNA knockdown was utilized to generate progenitors without the ligands delta like one and jagged one. Cells were then presented with the notch ligands jagged one and delta like one and delta like four. Response to the arrayed notch ligand varied depending on the cell-intrinsic expression of either ligand.

These images show the differentiation of liver progenitors as dependent on both substrate stiffness and ECM composition. Quantitative analysis revealed that collagen four is supportive of differentiation on both soft and stiff substrates, while fibronectin only supports differentiation on stiff substrates. Representative heat maps suggest that sustained traction stress at low substrate stiffness on collagen four promotes differentiation into bile duct cells.

This finding was confirmed by quantification of average root mean square values of traction stress. This video and the accompanying protocol have provided the main steps for fabricating hydrogels and arrays for performing cell culture on the arrayed substrates and for measuring cell substrate interactions using traction force microscopy. After becoming familiar with the techniques, each experiment can be completed in as little as one to two weeks if performed properly.

In contraction with this method, cell cultures should be used to validate high-scoring array conditions using qualitative PCR, immuno blotting, standard scale mechanobiology axes or other complementary molecular biology techniques. This versatile platform can be applied towards the high throughput investigation of cell functions in a broad number of cell and tissue contexts, including stem cell differentiation and cancer cell biology.