High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

The overall goal of this procedure is to rapidly characterize genetically engineered stem cells using a high content screening or HCS system. This is accomplished by first designing and preparing experiments for a 96 well plate, and then plating the different populations of engineered stem cells. Next, the high content screening system is prepared for live cell imaging and then the time-lapse imaging is initiated for the desired time period.

Then after completing the time-lapse imaging, the culture plate is prepared for cell staining procedures such as propidium iodide, staining for cell death, or KI 67 immuno labeling to characterize cell proliferation. Finally, the 96 well plate is reloaded into the high content screening system for fluorescence imaging and image acquisition. Ultimately, the image analysis software is used to conduct data analysis for determination of different cell growth parameters.

This methodology can help address key issues in the field of stem cell biology, such as the identification and characterization of factors that stimulate stem cell differentiation. The implications of this technique extend towards cell-based therapeutic strategies because of the importance of characterizing the cell types before they are used in vivo. This method can provide insight into cell behaviors and can be applied to virtually any other cell type that grows well in culture.

Additionally, these approaches can potentially be used on whole organisms such as zebrafish larva. To begin create a map as shown here of the 96 well plate outlining the different substrates and cell types to be examined under a sterile culture hood. Prepare a workstation with different substrates and a 96 well plate.

Add 100 microliters of substrate solution to each, well according to the map. Then use a strip of perfil to seal the lid and store at four degrees Celsius overnight after isolating mouse mesenchymal stem cells and infecting them with lentiviral vectors according to the text protocol, remove the substrate solutions from the 96 well plate and use about 200 microliters of sterile PBS to wash the wells two times. Once the final rinse has been removed at 200 microliters of cell culture medium and equilibrate the plate in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.

In the meantime, under sterile conditions, harvest the MSCs by first collecting the growth medium, now referred to as the conditioned medium from the flask into a 15 milliliter conical tube. Then add eight milliliters of sterile PBS to the flask and gently swirl before aspirating the buffer To detach the cells from the flask, add one milliliter of 0.05%trypsin and 0.01%EDTA solution. When the cells have detached, add eight milliliters of the conditioned medium before collecting the cell suspension and replating the cells at about 300 cells per well.

According to the text protocol, incubate the plate for two hours to allow the MSCs to attach to the substrate while the cells are incubating. Start the HCS system and wait two hours for it to equilibrate. Set the environmental controller to 37 degrees Celsius and carefully turn on the mixed gas cylinder containing 5%carbon dioxide in air to the HCS system, environmental chamber that supplies a constant air source.

Next, after the two hour incubation period, remove the 96 well plate from the incubator and place it directly into the environmental chamber of the HCS system. After allowing the plate to equilibrate for 30 minutes, start the software to configure the plate settings. Once the imaging parameters have been set up, according to the text protocol, use laser autofocus to focus on the well bottom and take test images from multiple sites and multiple wells to find an optimized focal plane.

Once the focus has been established, begin capturing images every five minutes for 48 hours for all 60 wells. At the end of the experiment, remove the 96 well plate from the HCS system under sterile conditions, collect conditioned medium samples from each well and transfer them to a fresh 96 well plate. These samples can later be used for an ELIZA to carry out a KI 67 cell proliferation assay.

Use 0.1 molar phosphate buffer to rinse the cell cultures for one minute. After repeating the wash, use 4%para formaldehyde or PFA at room temperature for 20 minutes to fix the cultures following fixation. Remove the PFA and use PBS to rinse the wells three times for seven minutes each.

After blocking the cells according to the text protocol, apply 100 microliters of primary antibody solution to each. Well cover the plate and incubate at four degrees Celsius overnight. After washing the cells three times for seven minutes.

For each wash, apply secondary antibody. Place the cells in the dark and incubate at room temperature for 90 minutes following another three washes. Cover the plate and store it at four degrees Celsius until imaging.

To perform automated imaging, load the immuno labeled plate into the HCS system and allow the plate to equilibrate for 20 minutes. Open the HCS system image acquisition and analysis software. Choose the acquisition settings for the 10 x objective using camera bending at one and a gain setting of two.

Use the auto exposure function to find the Z plane in which the cells reside and calculate the offset for each wavelength of interest for the analysis demonstrated here. Capture images for DAP EGFP and S3.Choose the maximum intensity level at which the negative control wells show no signal for image acquisition. Use the same threshold settings for positive wells.

Finally, capture images and save them to a database before performing image analysis. According to the text protocol, as shown here, five different populations of MSC subtypes were plated into 96 Well tissue culture plates pre-coded with different substrates in this figure, anti KI 67, which identifies proliferating cells, and DAPI was used to evaluate whether the different substrates influenced proliferation of the different populations of engineered MSCs as illustrated Here, although there was variation in the percentages of proliferating MSCs, all substrates supported considerable cell proliferation for each MSC subtype. This plot shows that the percentage of cells with propidium iodide or PI staining, which identifies dead cells in a population is low on all substrates examined cells treated with 70%ethanol, which kills most cells, exhibited a high rate of PI labeling and serves as a positive control to investigate the behavior of MSCs on different substrates.

Cell migration over a 29 hour period was analyzed using time-lapse digital microscopy. The migration path for cell one and cell two are marked by the green and blue line respectively. As shown here, all subtypes of MSCs showed the fastest migration rate on the extracellular matrix, coated surfaces, and the slowest on non coated polystyrene surfaces.

While attempting this procedure, it's important to remember to carefully plan and design the experiment for a multi-well plate format, such as a 96 well plate Following this procedure. Other methods like Eliza S can be performed on condition media in order to answer additional questions concerning the production and secretion of therapeutic factors by the engineered stem cells After its development. High content screening procedures have paved the way for researchers in the fields of stem cell biology and drug discovery to explore complex biological systems in a high throughput manner.