Table 2 Methods for the depletion of EVs in serum additives for cell culture medium

Ultracentrifugation

120,000 g, 18 h, 4 °C, SW32 Ti rotor (Beckman Coulter, Brea, CA, USA)

[66]

Ultrafiltration

Amicon ultra-15 centrifugal filters (UFC910024, 100 K filters and benchtop Merk Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, Ireland), 3,000 g, 55 min, 4 °C

[62]

Tangential flow filtration (TFF)

hollow fiber-modified polyethersulfone (mPES) membrane filter column (area 1,600 cm², 500 kDa molecular weight cut off) operated on a KR2i TFF System (Repligen, USA)

[67]

Commercially available exosome-depleted serum or medium

MesenCult™-ACF Plus (STEMCELL Technologies, China);

[68]

Exo-FBS™ (System Biosciences, Mountain View, CA, USA);

[69]

Oxium^TMEXO (patent No. PCT/CL2019/100175);

[70]

RoosterCollect EV Pro™ (RoosterBio Inc., Frederick, MD, USA)

[71]

Fibrinogen and fibrin depletion

Hydrogel formation was facilitated for 4 h at room temperature (RT) followed by overnight incubation at 4 °C. The resulting coagulated medium was heated to 37 °C for 1 h to enable a complete fibrin clotting. Afterward, a collapse was induced by vigorous shaking followed by centrifugation at 3000 g for 10 min at RT. Finally, the clear medium supernatant was filtered through a 0.22 µm filter (Merck Millipore, Billerica, MA, USA)

[72]