This protocol is designed to rapidly isolate cells from screening interfaces for genomic characterization and this capability is significant because it can combine macroscopically observable cell features with genomic information. The targeted bacterial cells from the screening interfaces can be isolated with high spatial position in an operationally simple manner using this technique. The hydrogels can be implemented in other screening interfaces.
The hydrogel material can be readily incorporated into microfluidic channels for on-demand retrieval of cells from microfluidic devices. Begin by inoculating the cross-linking buffer with the desired cell density. Prepare the hydrogel precursor solution in a 0.5 milliliter microcentrifuge tube by adding 12.5 microliters of the cross-linking buffer and 5.6 microliters of PEG ONB diacrylate solution.
And lastly, add 6.9 microliters of forearm PEG thiol solution to the mixture. For cell encapsulation in the hydrogel precursor solution, place the thiolated base coverslips on a clean Petri dish and position two spacers on the two opposing sides of the coverslip. Fix the spacers on the base coverslip by taping the spacers to the Petri dish.
After adding the desired volume of the precursor solution on a non-reactive perfluoroalkylated glass slide, place the slide on the base coverslip and allow the hydrogel formation to complete at room temperature for 25 minutes. Once the gelation is complete, gently remove the perfluoroalkylated glass slide. Place the substrate in the 60 by 15 millimeter Petri dish containing ATGN media supplemented with antibiotic.
Seed 700 microliters of bacterial cell suspensions with OD 600 of 0.1 over the microwell array substrate and make the hydrogel precursor solution as demonstrated previously using 12.5 microliters of phosphate buffered saline ATGN of pH eight. Pipette 12.5 microliters of the precursor solution on a non-reactive perfluoroalkylated glass slide and place two 38 micrometer steel spacers on two opposing sides of the microwell array substrate innoculated with cells. Then invert the perfluoroalkylated glass slide with the precursor solution droplet and place a droplet in the middle of the microwell substrate.
When the hydrogel is formed after an incubation of 25 minutes at room temperature, gently remove the glass slide from the microwell substrate and place the substrate in a Petri dish containing ATGN media supplemented with antibiotic. Place the sample in a PDMS holder and add the defined media on top of the sample to prevent sample dehydration and provide a carrier solution for released cells. After bringing the calibration mirror in position, adjust the microscope focus to get a sharp image of the colonies within the hydrogel or microwell array and inspect to identify colonies or wells of interest.
Here, design the light patterns while the camera view shows the colonies inside the sample to test different patterns for cell extraction and save the defined pattern. Then select the session control section, add the saved sequence under the tab titled with the patterned illumination tool product name. Choose the option for stimulating the pattern to view and adjust the desired exposure location.
Next, adjust the light intensity to 60%and the exposure time to 40 seconds under the LED control tab and start the exposure process. Monitor the hydrogel degradation in real time and Brightfield mode to ensure the cell release. To collect the released cell, change the microscope filter from Brightfield to TRITC to visualize the exposed area of the sample by the naked eye.
Once the exposed area is located, place the end of the tubing upon the irradiated spot, then change the microscope filter back to Brightfield to monitor cell retrieval in real time. Use the syringe attached to the other end of the tubing to carefully withdraw 200 microliters of solution containing the released cells and transfer the solution into a 1.5 millimeter centrifuge tube for DNA analysis or plating. Different UV light micro patterns were used for cell extraction from bulk hydrogels, which influenced the morphology of the released cells.
The UV exposure in a ring pattern resulted in the release of the entire colony encapsulated in a protective PEG hydrogel. In contrast, by exposing part or all of the colony to UV light, cells could be extracted either as aggregated cell clusters or as free individual cells. The cell seeding density and thickness of the hydrogel are important for cell encapsulation.
The thinner hydrogels resulted in micro colonies with minimal colony overlap, while the hydrogels with increased thickness resulted in overlapping colonies, which might result in the extraction of multiple colonies. Overlapping colonies can cause cross-contamination during extraction due to the two-dimensional nature of the light pattern. When a top colony was targeted, the underlying colony was also extracted with it.
The effect of varied UV light micro patterns on cell viability was also assessed. When micro colonies were exposed to controlled doses of UV light in either a circular or cross-pattern, cell recovery and DNA purity were not affected. The cells were successfully retrieved from microwell arrays using this approach, which was evident from the representative confocal microscopy images, where upon irradiation in the circular and ring pattern, the cells were removed in the respective pattern.
After extraction from microwell arrays, the cell viability and DNA quantity were assessed. The exposure patterns affected neither the viable cell count nor the DNA quality, indicating that viable bacteria cells could be selectively retrieved from microwells with minimal damage, and their DNA could be isolated at high purity for downstream genomic analysis. Always check that the hydrogel has formed before removing the perfluoroalkylated coverslip.
Precision and care are required for placing the spacers for hydrogel deposition and using the tubings for extraction of cells.
