Takara RetroNectin Pro (Recombinant Human Fibronectin Fragment). 2.5 mg

RetroNectin Pro is a 63-kD fragment of recombinant human fibronectin (also referred to as rFN-CH-296) that enhances the efficiency of lentiviral- and retroviral-mediated gene transduction. This is particularly important for hematopoietic cells and other hard-to-transfect cell types.

RetroNectin Pro uses the same protocol and performs the same as RetroNectin reagent (Cat. #T100A & T100B) but is produced with more stringent manufacturing practices. Additionally, it can be used with gas-permeable containers such as g-Rex to perform transduction and cell expansion in the same container/vessel.

Treatment with RetroNectin Pro reagent increases transduction efficiency.Transduction efficiency was quantified as the percentage of T cells (both CD8+and CD4+) that expressed the modified chimeric antigen receptor (CAR). Methods A and B were conducted at MOIs of 0, 5, and 15.Panel A.Transduction efficiency on Day 4.Panel B.Transduction efficiency on Day 10. At both Days 4 and 10, Method A shows higher transduction efficiency than Method B, indicating that the addition of RetroNectin Pro reagent effectively increases the percentage of modified T cells.

Diagram of interactions that colocalize target cells and viral particles using RetroNectin reagent. RetroNectin reagents contain three functional domains: the cell-binding domain (green), the heparin-binding domain (purple), and CS-1 sequence (orange). Viral particles (purple) bind via interaction with the H-domain; target cells mainly bind through the interaction of cell surface integrin receptor VLA-5 and VLA-4 with the fibronectin C-domain and CS-1 site, respectively.

Gene transfer and expansion workflow using a g-Rex 24-well plate.Panel A.Standard g-Rex workflow. Two days before gene transfer, T cells are activated with CD3/CD28 for ~48 hours. Following T-cell activation, cells are transduced using a promotor and spinculation. Two days after transduction, the T cells can be transferred to the g-Rex system, expanded, and collected after 10 days. This whole workflow requires four hands-on steps (activation, transduction, transfer, and collection).Panel B.Addition of RetroNection Pro reagent to the standard g-Rex workflow reduces hands-on steps. One day before gene transfer, T cells are activated with anti-CD3 antibodies for ~24 hours. Following activation, T cells and virus are added to the g-Rex system, expanded, and collected after 10 days. The addition of the RetroNectin Pro reagent requires one less hands-on step and removes one day from the workflow.

Number of cells per well following 10 day culture with Methods A and B.Total cells per well (2 cm2) were measured for Method A (orange) and Method B (blue). These data indicate that the addition of RetroNectin Pro reagent does not significantly impact the cell number.

Ratio of naive T cells is higher under the addition of RetroNectin Pro reagent.The naive T-cell ratio was analyzed for cells collected on Day 10. Naive cells were defined as cells expressing CD62L and CD45RA. Naive T cell ratio among CD8+(Panel A) and CD4+(Panel B) cells was higher with Method A (with RetroNectin Pro treatment) condition regardless of MOI value (5 and 15). This indicates that the addition of RetroNectin Pro reagent to a g-Rex system can produce high numbers of edited naive T cells.