The overall goal of the following experiment is to prepare micro fabricated channels to study spontaneous confined cell migration. This is achieved by first preparing the poly polymethyl SUBOXANE or PDMS chip containing the channels. As a second step, the channels are coated with fibronectin or another coating substrate, which allows the functionalization of the device.
After several washes, dendritic cells are loaded into the microsystem to study cell migration. Results are obtained that show differences in velocity for different cell types or treatments based on microscopy and image analysis. The main advantage of this technique over existing methods like classic two migration, is that we confront the geometry and confinement of the cells.
Generally, individuals new to this method will struggle because of the precision needed to achieve every step. To begin mixed PDMS oil and curing agent at a weight ratio of 10 to one in a plastic cup mix, both compounds thoroughly cast the mix over the mold bearing micro channels. The total height must be between 0.5 and one centimeter.
Remove any air bubbles in a vacuum bell jar for one hour, then harden the PDMS in the mold by placing it in an oven for two hours at 65 degrees Celsius. After the PDMS has cooled to room temperature, cut a large piece around the structure with a surgical blade and peel it off from the mold. Next drill holes where cells will be injected, then resize the PDMS with a surgical blade to fit in glass bottom dishes in which migration will be assessed Clean.
The resized PDMS chip containing the channels by sticking and peeling adhesive tape on the structure sides sonicate the PDMS pieces. 30 seconds in 70%ethanol to remove dust and small PDMS particles before proceeding. Remove residual dust from the dish using lens cleaning paper.
Activate the PDMS and culture dishes by air plasma treatment for 30 seconds at 300 Milit, place both activated surfaces in contact to permanently stick the PDMS to the substrate if needed. Use metallic forceps to press lightly on top of the PDMS to force the contact between the polymer and the glass of the dish. Incubate the chip in the oven at 65 degrees Celsius for one hour.
To strengthen the binding, activate the whole structure by air plasma at 300 millitorr for one minute. This will promote the entry of liquid into the channels at the next step. Next, fill the entry holes of the chip rapidly with fibronectin at 10 micrograms per milliliter in water.
Other substrates, such as polyethylene glycol may be used to modify cell adherence to the channel walls. Pay attention that the liquid spreads throughout the entire structure. This can be easily checked by eye under regular light, or using a regular brightfield microscope.
Then incubate the structure for one hour at room temperature to allow absorption of the fibronectin or any other coating substrate to the walls of the channels. Finally, wash the structures to remove the non bound substrate by removing all the liquid from the channels and dish before soaking in fresh phosphate buffered saline or PBS for five minutes. Remove the PBS from the plate and fill the microsystem with cell medium.
Let the plate incubate for one hour to saturate the channels with the medium to collect cells for imaging. Remove floating dendritic cells and recover semi adherence cells by flushing the plate with culture medium. Before counting the cells.
Using a hemo cytometer centrifuge the cells at 300 times G for five minutes and discard the medium. Dilute the pellet to reach a concentration of 20 million cells per milliliter. Remove the excess of medium from the plate and empty the entry holes in the PDMS structure.
Using a micro pipette. Then fill the entries with five microliters of cell solution to reach an amount of one times 10 to the fifth cells in each entry hole. After incubating the microchip for 30 minutes at 37 degrees Celsius in the incubator, add two milliliters of complete medium to the experiment dish for imaging.
First, clean the external bottom surface of the dishes with lens cleaning tissue before placing the plates under the microscope. To analyze migration in a large number of cells, use 10 x magnification and wide field illumination in a CO2 and temperature equipped video microscope to facilitate cell tracking. Hirsch staining and UV light can be used as discussed in the text protocol for time lapse microscopy at 10 x magnification.
Choose a time frequency according to the expected cell speed. This is typically two minutes for dendritic cells migrating with the speed of five microns per minute. In each experiment, the surface of the PDMS is coated with a molecule adapted to the interest of the study.
In this figure, channels are coated with a fluorescent molecule. Polylysine grafted polyethylene glycol before and after washing. Such an experiment allows the control of the homogeneity of the coating in the channels after cell loading video microscopy can be performed to follow cell migration.
Here is an example of dendritic cells migrating in micro channels at a density appropriate to track the cells. Both phase contrast and hersch staining can be used for this purpose. The technique is also compatible with fluorescent confocal microscopy at higher resolution and can be used to track organelles or cellular structures as shown in the dynamics of polymerized actin in migrating dendritic cells expressing life act GFP.
An example of quantification of cell motility analyzed in migrating dendritic cells is shown here. Kraft generated from wild type or Y 27 632 dihydrochloride treated dendritic cells show that the drug is a well-characterized inhibitor of cell contractility and migration, and was used to verify the ability of the system to detect changes in speed. The kog can be further analyzed to extract position and size of cells as a function of time, allowing the study of different parameters to describe cell migration.
The speed of migration was tracked by imaging the nucleus position using homemade software. Dendritic cells have a mean velocity close to six microns per minute. Treatment with the drug significantly decreases their velocity.
This example shows the power of the technique, which allows the quantification of large numbers of cells in single experiments and the utility of the system to detect differences induced by drug treatments Once mastered, the technique demonstrated here can be done in three hours if it is performed properly After its development. This technique paved the way for researchers in the field of migration to study cell locomotion in controlled confined environments.
