Our protocol can answer key questions in the field of regenerative medicine such as whether cells can be improved for the beneficial properties as well as whether they can be targeted to the site of interest. A main advantage of our protocol is the fact that the cells can be transiently, genetically engineered using our non viral transfection method, thereby retaining the cells'properties. Sauter's method can provide insight into the modification of hematopoietic stem cells that can be applied to other cell types used for transplantation such as mesenchymal stem cells.
To begin the protocol, prewarm human lymphocyte medium to room temperature and thaw Collagenase B and DNase. Collect the bone marrow, or BM, into a 50 milliliter conical tube. Discard any existing clots.
Take a BM sample of 200 microliters for subsequent flow cytometric analysis. Store the sample at four degrees Celsius until needed. Next, transfer ten milliliters of BM into a new 50 milliliter conical tube and add six milliliters of PBS EDTA, 20 milliliters of human lymphocyte medium, 175 microliters of Collagenase B, and 175 microliters of DNase.
Gently mix the solution and incubate it for 30 minutes at room temperature on a shaker. Prime a 50 milliliter density gradient centrifugation tube by applying 15 milliliters of human lymphocyte separating medium into the 50 milliliter tube and centrifuge it at 1, 000 times G for 30 seconds. Carefully layer 35 milliliters of diluted BM on top of the density gradient centrifugation tube filter and centrifuge the tube at 445 times G for 35 minutes.
Gently remove the tube from the centrifuge without shaking. Carefully discard a maximum of 20 milliliters from the upper-clear solution, without touching the cloudy layer directly on top of the filter. Carefully transfer the cloudy layer containing the mononuclear cells, or MNCs, into a new 50 milliliter conical tube and fill it up with PBS EDTA to a final volume of 50 milliliters.
Count the MNCs by transferring 10 microliters of the 50 milliliter cell suspension into a 1.5 milliliter tube. Add 10 microliters of 3%acetic acid with methylene blue. Gently mix the contents, and apply 10 microliters into a counting chamber.
Calculate the number of MNCs. Centrifuge the MNC suspension at 300 times G for 10 minutes. Discard the supernatant.
To prepare a magnetic selection for one times 10 to the eighth total cell number, carefully re-suspend the MNCs in 300 microliters of MACS buffer. Add 100 microliters of FCR blocking reagent and 100 microliters of CD133 antibody linked superparamagnetic iron dextran particles. Then, gently mix the cell suspension and incubate it for 30 minutes at four degrees Celsius.
Gently shake the cell suspension during incubation two to three times. After incubation, add two milliliters of MACS buffer per one times 10 cells. Gently mix the cell suspension and centrifuge the mix at 300 times G for 10 minutes at four degrees Celsius.
Set up the MACS magnet holder and attach the MACS permanent magnet. Install the MACS column and apply the pre-separation filter on top. Equilibrate the first MACS column and pre-separation filter with MACS buffer.
Discard the supernatant and re-suspend the pellet in 500 microliters of MACS buffer. Apply the cell suspension to the pre-separation filter. Wash the MACS column and pre-separation filter three times and discard the pre-separation filter.
Elute the cell fraction directly onto the second MACS column. Immediately transfer the first MACS column above the second MACS column and push the cell suspension through the MACS column using the supplied plunger. Wash the MACS column 3 times with MACS buffer.
Elute the cell fraction from the column by adding MACS buffer onto the second MACS column and removing the MACS column from the MACS permanent magnet. Immediately transfer the second MACS column above a tube and push the cell solution suspension through the MACS column. Centrifuge the tube.
Carefully discard the supernatant and re-suspend the pellet in 100 microliters of MACS buffer. Take a sample of one times 10 to the fourth cells for subsequent flow cytometric analysis. Re-suspend remaining cells in culture medium and seed five times 10 to the fourth cells in a 24 well plate for transfection.
Incubate the cells at 37 degrees Celsius, 5%CO2, and 20%O2. Use two 10 microliter samples BM aliquots and one sample of freshly isolated CD133 positive SCs for analysis. Transfer the cells into a 1.5 milliliter tube.
Add 10 microliters of FcR blocking reagent and fill up the tube with MACS buffer to a volume of 33 microliters. Add the antibodies onto the inner side of the tube. After all antibodies have been added, shake down the antibodies side of the tube.
Gently mix the solution, which has a final volume of 50 microliters, and incubate it for 10 minutes at four degrees Celsius. Add one milliliter of one X red blood cell, or RBC lysis buffer. Gently mix the suspension and incubate the solution on ice for 10 minutes.
Then centrifuge the suspension. Discard the supernatant and re-suspend the obtained pellet in 100 microliters of PBS. Evaluate purity and viability of cells by flow cytometric measurements.
Use 20 picomoles of MIR per well of a 24 well plate. Dilute MIR in the 5%glucose solution to a final concentration of 0.25%picomoles MIR per microliter. Dilute the PEI in an equal amount of 5%glucose solution.
Next, add the prediluted PEI into the prediluted MIR and vortex for 30 seconds. While incubating the MIR PEI complexes for 30 minutes at room temperature, sonicate the magnetic nanoparticles, or MNPs for 20 minutes at 35 kilohertz in a room temperature sonicating water bath. Ensure that the particles are in suspension and not aggregating.
Add the sonicated MNPs into the MIR PEI complexes and vortex the solution for 30 seconds. Incubate the MIR PEI MNP complexes for 30 minutes at room temperature. Add the MIRNA PEI MNP complex drop-wise directly into the appropriate well containing the freshly isolated CD133 positive SCs in culture medium.
Mix gently by rocking the plate back and forth. Incubate the cells. 18 hours after transfection, collect the contents of each well in a separate 1.5 milliliter tube.
Wash each well once with 500 microliters of PBS and transfer this cell suspension to the same tube. Then, centrifuge the cells at 300 times G for 10 minutes at four degrees Celsius. After the spin, discard the supernatant and re-suspend the pellet in 100 microliters of MACS buffer.
Add 0.5 microliters of an amine reactive dye to distinguish between live and dead cells. Gently mix the suspension and incubate the cells for 10 minutes at four degrees Celsius. Add one milliliter of PBS and centrifuge the cells again at 300 times G for 10 minutes at four degrees Celsius.
Discard the supernatant, re-suspend the obtained pellet in 100 microliters of PBS, add 33 microliters of 4%PFA, and mix vigorously. Evaluate the MIR uptake efficiency and transfection mediated cytotoxicity by flow cytometric measurements. Following the method outlined in the protocol, flow cytometry determined that the magnetically enriched CD133 positive cell fraction yielded a viability and purity higher than 80%The transfection method described here allows a highly effective introduction of MIR in these freshly isolated human CD133 positive SCs.
With an uptake efficiency of approximately 80%of viable cells. In addition, there are no significant cytotoxic effects evident 18 hours after transfection compared to control cells. Moreover, a sufficient delivery and usual distribution of the transfection complex compounds can be observed within the cytoplasm of cells using structured illumination microscopy, or SIM.
Following this procedure, other methods like in vivo application of modified stem cells can be performed. This will allow to investigate the actual survival and effects under dynamic conditions. After watching this video, you should have a good understanding of how to apply polymer and magnetic nanoparticle based transfection to efficiently introduce microRNA into freshly hematopoietic stem cells.
