A standard procedure for lentiviral‐mediated labeling of murine mesenchymal stromal cells in vitro

Abstract

Tracking of stem cells after transplantation is effectively performed in vivo with imaging systems, assuming the cells are adequately labeled to facilitate their recognition. This study aimed to optimize a protocol for fluorescent labeling of mesenchymal stromal cells (MSCs) in vitro, by using a third-generation lentiviral system. Basically, 293T cells are seeded in high-glucose Dulbecco's modified Eagle medium with 10% FBS one day before transfection. Transfection is done for 24 h using a mix of transfer, packaging, regulatory, and envelope plasmids, in molar ratio of 4:2:1:1, respectively. After transfection, the cells are further cultured for two days. During this period, the viral medium is harvested two times, at 24-h intervals, with the first round being stored at 4 • C until the second round is completed. The pooled viral medium is frozen in single-use aliquots. MSCs are transduced with 25 multiplicity of infection (MOI) and one day later the cells are passaged at standard seeding density and further grown for three days, when the fluorescence reach the maximum level. Our protocol provides particular experimental details for permanent MSC labeling that makes the procedure highly effective for therapeutic purposes, without affecting the functional properties of stem cells.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation

“…The presence of MSC characteristic markers (Sca‐1, CD105, CD44), the absence of haematopoietic markers CD45 and CD11b, and the in vitro differentiation potential of cells into osteogenic, adipogenic and chondrogenic lineages were evaluated to confirm the MSC attributes 4 . These attributes were retained for at least 10 passages after completing the selection process 21 . Cells were used between the 8th and 13th passages.…”

“…4 These attributes were retained for at least 10 passages after completing the selection process. 21 Cells were used between the 8th and 13th passages. The 3D aggregates were obtained by assembling various number of cells (from 10 4 to 3 × 10 5 ) for 3 days using the hanging-drop method as previously described.…”