Optiprep

Intracellular RNA from transfected cells was extracted using the QIAGEN RNeasy kit as described by the manufacturer. For extracellular RNA extraction, 2 mL of purified vector supernatant was centrifuged at 22,000 × g for 3 h at 4°C in 8.4% Opti-Prep (STEMCELL Technologies). Pellets were resuspended in phosphate-buffered saline (PBS) (Thermo Fisher Scientific) and incubated in 200 μL of proteinase K extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM EDTA, 1% SDS, 100 μg/mL proteinase K [Ambion], and 100 μg/mL yeast tRNA [Sigma-Aldrich]) at 37°C for 30 min. vRNA was extracted with phenol-chloroform and chloroform:isoamyl alcohol, precipitated with 1/10^th volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, washed with ice-cold 70% ethanol, and air-dried.

The densities of glutaraldehyde treated cells were determined by adjusting the density of solutions they were suspended in. In an Eppendorf tube with treated cells, the percentage of OptiPrep (Stemcell Technologies, Canada) was adjusted slowly to match the density of treated cells. To determine the cell density values, tubes with known concentration of OptiPrep were centrifuged at 500 × g. In the case where the density of the solution matched the density of the cells, no sedimentation was observed after centrifugation.

Bone marrow (BM), spleen, and liver were harvested on days 9, 15, and 42. BM cells were isolated by flushing the femurs and tibias of tumor-bearing mice. The spleen was ground, and single-cell suspensions were prepared by filtering the cells through 200 um cell strainers. Livers were excised, minced, and passed through a cell strainer. Suspensions were centrifuged at 30g for 4 min at 4 °C to pellet nonhematopoietic cells. The supernatant was transferred to new tubes and spun at 460 g for 10 min. The cell pellet was resuspended in RPMI-1640, and lymphocytes were separated on a 25% Optiprep gradient (StemCell).

For gradient construction, we formulated solutions of 50%, 40%, and 10% (w/v) iodixanol by diluting OptiPrep (60% (w/v) aqueous iodixanol, Stemcell) with a mixture of 0.85% NaCl and 10 mM Tris-HCl (pH 7.4). The exosome pellet was resuspended in 50% iodixanol (3.8 mL) and layered into an 8 × 13.5 mL centrifuge tube (Beckman). Subsequently, 3 mL of 40% and 2.5 mL of 10% iodixanol solutions were carefully layered atop using a precision needle. This gradient underwent ultracentrifugation at 200,000 g for 2 h at 4 °C. Ten individual iodixanol fractions, subsequently diluted with PBS, were harvested top-down, centrifuged at 110,000 g for 70 min at 4 °C, and then resuspended in PBS. The density of each fraction was quantified gravimetrically. Exosomes from cells underwent morphological characterization by TEM, particularly those isolated from fraction no. 3, as previously cited [27 (link)]. Exosome size distribution and concentration were ascertained using the DKSH nanoparticle tracking analyzer (Zetaview, PMX). Expression levels of LAP-TGF-β1 in the exosomes were quantified via the TGF-β1 Quantikine ELISA kit (DB100B, R&D Systems) per manufacturer’s guidelines.

Generation of nanovesicles (NVs) was performed as described [140 (link)] with modifications. Briefly, adherent iPSCs (CL2 and CERA) were individually harvested (following PBS wash) using a solution of EDTA (10 mM) (3 × 3 min round incubation) and cell suspension spun at 500× g for 5 min. The pellet was resuspended in PBS and the cell suspension sequentially extruded through 10, 5, and 1 µm polycarbonate membranes (19 mm; Advanti Polar lipids, 610010) (13 times across each filter, Whatman). The extruded NVs were subsequently purified using 10% OptiPrep™ (Stemcell Technologies) density cushion (step gradient formed by overlaying extruded sample on 10% and 50% iodixanol) and centrifuged at 100,000× g for 2 h at 4 °C. Seven equal fractions were obtained, diluted in PBS (to 1.5 mL), centrifuged at 100,000× g for 2 h at 4 °C (TLA-55 rotor; Optima MAX-MP ultracentrifuge) and resuspended in PBS and stored at −80 °C until further use. The yield (protein) and density of each fraction was determined as described [56 (link)].
For comparison, natural extracellular vesicles (EVs) from each iPSC line were obtained as described previously [56 (link)], based on isopycnic (iodixanol density-based) ultracentrifugation [108 (link)].

HEK 293 T cells seeded in eighteen 10-cm dishes at 3 × 10⁶ cells in 9 mL of DMEM + 10% HI-FBS media were transfected by linear PEI. After 72 h, cells were harvested, pelleted at 1000 × g for 5 min at 4 °C, then resuspended in 14.4 mL of 50 mM Tris + 150 mM NaCl (pH 8.2). The cells were lysed by undergoing three freeze/thaw cycles, then incubated at 37 °C for 1 h with benzonase (10 U/mL; EMD Millipore). The lysate was then centrifuged at 13,200 × g for 10 min at room temperature. Supernatant was collected and stored at 4 °C until next step. The lysate supernatant was ultracentrifuged with iodixanol (OptiPrep; StemCell Technologies) density-gradient solutions (54%, 40%, 25%, and 15% w/v) at 76,900 × g for 18 h at 4 °C. Then, 4/5 of the 40% layer and 1/5 of the 54% layer were extracted from the polyallomer Quick-seal ultracentrifuge tube (Fisher) with an 18-gauge needle (Fisher) attached to a 10-mL syringe (VWR). The collected virus fraction was diluted in an equal volume of PBS + 0.001% Tween-20, applied to an Amicon Ultra-15 (EMD Millipore, 10 kDa NMWL) column, and centrifuged at 4000 × g for 20 min at 4 °C. The resulting virus fraction was diluted with PBS + 0.001% Tween-20 and centrifuged until 500 μL of the virus fraction remained in the column. Concentrated virus was stored at 4 °C for subsequent titer and use.