Exosomal microRNAs from mesenchymal stem/stromal cells: Biology and applications in neuroprotection

Table 1 Summary of the techniques used to manipulate the content of mesenchymal stem/stromal cells and their exosomes

Technique	Description	Direct/indirect exosomal loading
Transfection	A lentiviral vector or a plasmid is encoded with the desired miRNA and introduced to the MSCs[57,85,100,102]	Indirect
Electroporation	Electrical pulses in microseconds to milliseconds durations are applied to cause a temporary loss of the stability of the membranes of both MSCs and exosomes, which allows cargo to pass into the cell or exosomes[45,47,81,97]	Direct and indirect
Sonication	Low-frequency ultrasound is applied to disrupt the membrane integrity of the exosomes and form small pores in their membrane to allow small RNAs into the exosomes[114,116]	Direct
Modified calcium chloride transfection	Phosphate-buffered saline is slowly mixed with a CaCl2 solution containing the desired small RNA which leads to formation of RNA-calcium phosphate precipitates on the cell/exosomes . A heat shock is added to the solution to change the fluidity of the exosomes' plasma membranes for introducing[118]	Direct
Co-incubation of exosome with hydrophobically modified RNA	Conjugating the small RNA with a cholesterol moiety enhances hydrophobicity of the RNA and allows for diffusing the exosomal membrane during simple incubation[119]	Direct

miRNA: MicroRNA; MSCs: Mesenchymal stem/stromal cells.