The overall goal of this procedure is to introduce a technique which ensures cell modification with microRNA and in parallel cell magnetization. This method can address key challenges in the regenerative medicine field. Such as genetic engineering of cells prior to transplantation.
In addition to efficient cell modification with microRNA, they allowed it with iron oxide. This can be used for enhancing cell retention in the side of interest by magnetic field application. Cell therapy is very promising for the treatment of heart diseases, hypocell retention, and to cell survival urgently need to be improved.
Begin this procedure with human umbilical vein endothelial cell culture in preparation of transfection complexes as described in the text protocol. 48 hours prior to transfection, seed the HUVECs on plastic cell culture well plates of suitable size. With a starting cell density of approximately 13, 000 cells per square centimeter of growth surface.
Next, prepare fresh miR/polyethyleneimene/super paramagnetic iron oxide nanoparticles. First dilute 2.5 picomoles of microRNA per square centimeter of cell growth surface and five percent glucose solution to obtain a final concentration of 0.125 picomoles of microRNA per microliter. Next, dilute the PEI in an equal volume of five percent glucose solution.
Add the diluted PEI to the microRNA solution and vortex the obtained mixture for 30 seconds. Then incubate the miR/PEI mixture for 30 minutes at room temperature. Sonicate the MNP's for at least 20 minutes in the sonicating water bath at room temperature.
Then add the appropriate amount of MNP solution to the ready miR/PEI mixture. After vortexing for 30 seconds, incubate for 30 minutes at room temperature. Next add the prepared miR/PEI/MNP mixture drop-wise directly to the culture medium on the cells.
Replace this mixture with fresh culture medium six hours post-transfection. After analysis of vector safety as described in the text protocol, confirm and characterize the intracellular localization of transfection complexes by confocal and super-resolution structured illumination microscopy. To do so, seed the HUVECs on gelatin-coated glass cover slips placed in the wells of 24-well culture plates as before.
Transfect the cells with 3-color labeled miR/PEI/MNP and incubate for 24 hours at 37 degrees Celsius in a humidified atmosphere containing five percent carbon dioxide. Wash the cover slips first with two percent bovine serum albumin and phosphate buffered saline, and then with just PBS. Fix the cells by incubating in four percent paraformaldehyde or PFA at 37 degrees Celsius for 15 minutes.
Wash with PBS and then stain the nuclei with DAPI following standard protocols. Then wash the cells with PBS three times 15 minutes on the shaker to remove excess dye. Place the prepared cover slips on microscope slides using suitable mounting medium and let the slides dry for at least one hour before using them for microscopy.
Acquire images using a 63 times oil immersion objective in the SIM settings found in the text protocol. For cell targeting and simulated dynamic conditions in vitro, first transfect the HUVECs into 24-well plate as before. After incubating them for 24 hours, wash the cells.
Then add one x trips in EDTA diluted in PBS and incubate the cells for four minutes at 37 degrees Celsius before collecting the cells. Centrifuge the collected cells at 300 times G for 10 minutes. Mix the cell pellet obtained from one well with one milliliter of fresh culture medium.
And transfer the cells to a single well of a 12-well plate. Fix a small magnet locally using tape. Place the culture plate on the shaker and incubate the cell suspension for 12 hours at 150 RPM to simulate dynamic conditions.
Then wash the cells and fix them using four percent PFA. Stain the nuclei with DAPI. Record the cell attachment using laser scanning confocal microscopy with 405 and 504 teen-nanometer excitation lasers in z-stack mode to obtain the raw data.
Use maximum intensity projection image processing to create images for analysis. For quantitative analysis of magnetically reponsive and non-responsive cells, first transfect the HUVECs in a 24-well plate. Incubate the cells for 24 hours before washing and collecting the cells as described before.
Re-suspend the cell pellet obtained from each well with 500 microliters of warmed sterile cell-sorting buffer. Apply the cell suspension to a magnetic sorting column fixed by the supplied magnet. Then wash the columns on the magnet three times with fresh cell-sorting buffer.
Collect the flow-through as the magnetically unresponsive fraction. Remove the columns from the magnet and immediately collect the magnetically responsive cell fraction by pushing fresh cell-sorting buffer thought the column using a plunger. Centrifuge both Mag-and Mag+fractions at 300 times G for 10 minutes.
After re-suspending the cell pellet in PBS, count the cells and evaluate cell viability with a trypan blue exclusion assay. Isolated from umbilical cords, HUVECs are characterized by the expression of the endothelial marker CD31 and by formation of tubes on a basement membrane matrix. HUVECs transfected with miR/PEI/MNP all take up tagged miR.
And their survival remains unaffected 24 hours post-transfection. In addition super-resolution microscopy shows paranuclear localization of 3-color labeled transfection complexes. PEI toxicity is confirmed by the diminished functionality of miR/PEI transfected cells.
Importantly, MNP restores cell properties. miR/PEI/MNP modified cells do not differ from untreated cells in terms of tube formation. Moreover, these cells maintain intercellular gap junctional communication as revealed through fluorescence recovery after photo-bleaching.
Once a suspension of miR/PEI/MNP modified cells is seeded in the wells of a culture plate placed on the rotating shaker, cells tend to migrate and attach in the area of local magnet application. Representative images depict cell growth in the area with magnet application. Without magnet application.
And in the center of the culture well where the flow of liquid was concentrating the cells. Once mastered, cell transfection can be performed in two hours. After watching this video you should have a very good impression on how to transiently but highly-efficiently modify endothelial cells including the magnetization.
And also how to terminally characterize the cell product. While attempting this procedure, it is important to follow given numbers for cell-seeding amounts of components for the preparation of transfection complexes, incubation time, and the washing step after the transfection. We are very optimistic that this approach will be well-transferrable to other cell types for the treatment of other affected organs.
Following this procedure in vivo studies with modified magnetized cells can be performed. They could answer additional questions whether iron-loading of cells is efficient to ensure their retention in the side of interest. And also to define the limits of detection with MRI in vivo.
