Sviluppo di nanovescicole mesenchimali avvolte nella membrana delle cellule staminali per un migliorato apporto genico

In this protocol, we introduce a method to prepare mesenchymal stem cell-derived nanovesicles, which resemble extracellular vesicles and encapsulate AAVs for improved gene delivery. We call this AAV-containing nanovesicles CME-AAVs. Compared with the emerging gene delivery vector EV-AAV, CME-AAVs offer greater flexibility in cell sources and higher yield.

To begin, remove the culture medium from a culture dish containing human mesenchymal stem cells at 70 to 90%confluency. Using a cell scraper, detach the cells from the culture dish and transfer them into a conical tube containing PBS. Centrifuge the collected cells at 300g for five minutes at four degrees Celsius to pellet the cells.

Then, wash the cell pellet twice with PBS and centrifuge again. Resuspend the final cell pellet in PBS supplemented with a protease inhibitor cocktail. Then, subject the cell suspension to three freeze-thaw cycles to disrupt the plasma membrane and generate a crude lysate.

Using a pipette, homogenize the lysate thoroughly. Then, centrifuge at 1, 000g for 20 minutes at four degrees Celsius to remove cellular debris. Transfer the supernatant to a new tube.

Centrifuge at 18, 000g for one hour at four degrees Celsius to pellet the membrane fraction. Discard the supernatant and resuspend the membrane pellet in PBS to obtain the crude mesenchymal stem cell membrane fraction. Now, homogenize the membrane suspension by sequentially passing it through syringes fitted with 20 to 27 gauge needles.

Determine the protein concentration of the homogenized membrane suspension using a bicinchoninic acid assay. Aliquot the suspension and store at minus 80 degrees Celsius until needed. Re-homogenize the suspension using a 27 gauge syringe after each thaw cycle.

Mix the prepared membrane suspension with adeno-associated virus, or AAVs, at a ratio of 150 micrograms protein per one times 10 to the power of 10 genome copies. Incubate the mixture at 37 degrees Celsius for 30 minutes with gentle shaking. Load the membrane and virus mixture into a mini extruder fitted with a 400 nanometer polycarbonate membrane.

Pass the mixture through the extruder 20 times to promote encapsulation. Replace the 400 nanometer membrane with a 200 nanometer membrane and repeat extrusion. Collect the process suspension containing encapsulated virus, along with unencapsulated virus and empty vesicles.

Pool multiple batches, if required, and proceed immediately to purification. Dilute the stock iodixanol solution with 10x PBS and sterile deionized water to prepare 40 and 25%solutions. Using a pipette, add one milliliter of 40%iodixanol to the bottom of a 13.2 milliliter open-top thin-wall ultra-clear tube.

Then, overlay three milliliters of 25%iodixanol above the 40%layer to form a discontinuous gradient. Gently overlay seven milliliters of the processed suspension onto the top of the gradient. Balance the tubes and load them into a swing bucket ultracentrifuge rotor.

Centrifuge at 150, 000g for three hours at four degrees Celsius, using fast acceleration and slow deceleration. Then, carefully remove the tubes without disturbing the gradient. Collect one milliliter fractions from top to bottom into labeled 1.5 milliliter microcentrifuge tubes.

Keep all fractions on ice. Now, identify and pool fractions seven and eight containing purified CME-AAVs into a new open-top ultracentrifuge tube. Make up the volume to 11 milliliters with PBS.

Then, gently mix with a one milliliter pipette. Using fast acceleration and deceleration, centrifuge the pooled fractions at 150, 000g for three hours at four degrees Celsius. Discard the supernatant carefully.

Resuspend the pellet in 50 to 200 microliters of sterile PBS, depending on the desired concentration. Aliquot the purified preparation. Then, store at 80 degrees Celsius until further use.

Prepare working solutions of encapsulated virus and control virus in sterile PBS. Add equal genome copies of encapsulated virus or control virus directly to the culture medium in each well. Include negative control wells with an equivalent volume of PBS or empty virus particles.

Gently swirl the plate to ensure uniform distribution and incubate for 24 to 72 hours, depending on experimental design. Next, gently aspirate the culture medium. Wash the cells twice with PBS without dislodging them.

Stain nuclei with Hoechst 33342 according to the manufacturer's instructions. Perform live-cell fluorescence imaging using an inverted fluorescence microscope with appropriate filter sets for enhanced green fluorescent protein and Hoechst. Select 10x or 20x objectives for quantification and 63x objective for high-resolution imaging, if needed.

Acquire at least five fields of view per well, using identical imaging parameters. Save all images in raw and grayscale formats for quantitative analysis. Transmission electron microscopy revealed that CME-AAVs exhibited size dimensions and membrane-bound structures similar to extracellular vesicles.

The mesenchymal stem cell membrane markers, CD73 and CD90, were detected in CME-AAV by Western blot analysis. The three major adeno-associated virus capsid proteins, VP1, VP2, and VP3, were identified in CME-AAV by SDS-PAGE. NanoSight analysis showed that CME-AAV9.

EGFP had a mean diameter of 128.6 nanometers and a total particle concentration of 1.32 times 10 to the power of 10 particles per milliliter. Fluorescence microscopy demonstrated that CME-AAV9. EGFP produced a markedly higher level of enhanced green fluorescent protein expression in HEK293T cells.

The membrane-enveloped virus achieved approximately 2.5-fold higher gene expression, suggesting enhanced cellular uptake and potentially improved intracellular trafficking. In vivo bioluminescence imaging showed that the liver exhibited the strongest luminescent signal following CME-AAV administration. Quantitative analysis of liver regions revealed that CME-AAV administration resulted in significantly higher gene expression levels.

These protocols describe steps to obtain purified cell membrane-enveloped AAVs, or CME-AAV, via extrusion and density gradient ultracentrifugation. Two key factors require careful consideration:Preprocessing cell fragments for extrusion and optimizing membrane fragment-to-AAV mixing ratio to control CME-AAV particle-to-genome ratio. Future studies can further explore cell membrane enclosure to enhance immune protection and functional surface properties, optimizing CME-AAV as a versatile platform for AAV delivery.