Lentivirus-mediated reprogramming remains a workhorse for iPSC generation—particularly where efficiency, scalability, and broad donor compatibility matter. With deliberate MOI planning, defined feeder-free systems, and a rigorous QC gateway, you can achieve high-quality, low-VCN iPSC lines ready for differentiation, disease modeling, screening, or engineering.
Creative Biolabs is here to co-design your strategy and to carry you from donor cells to clean, banked iPSC clones with confidence.
Lentiviral reprogramming uses self-inactivating LV vectors to deliver canonical pluripotency factors, typically OCT4, SOX2, KLF4, c-MYC (OSKM), into somatic cells to induce an iPSC state.
Fig.1 Human iPSC technology.1,2
If genomic integration is a hard no, consider our Sendai virus, episomal plasmid, or mRNA protocols.
| Category | Reagents |
|---|---|
| Cells | Human dermal fibroblasts, blood-derived CD34+ cells, or other somatic cells (mycoplasma-free, low passage) |
| Vectors | Polycistronic OSKM (e.g., STEMCCA-like) or individual LV constructs for OCT4, SOX2, KLF4, c-MYC |
| Virus | Ready-to-use, titrated lentivirus |
| Media & supplements |
Somatic cell growth medium Reprogramming medium Extracellular matrix ROCK inhibitor Polybrene or protamine sulfate to enhance transduction |
Thaw and expand somatic cells to ~70–80% confluence; ensure mycoplasma-free status. Coat plates. Optional: pre-seed MEFs if using feeders.
Plate cells to ensure they are 50–70% confluent at transduction. Replace medium with fresh growth medium containing polybrene. Add lentivirus to achieve target MOI. Mix gently. Optional spinoculation and incubate 12–16 h at 37°C.
Replace with fresh growth medium (without polybrene). For difficult cell types, repeat a lower-MOI transduction to increase cassette integration while avoiding toxicity. Switch to iPSC medium. Add small-molecule enhancers if using for the first 7–10 days. If feeder-dependent, replating onto MEFs can occur here; otherwise continue feeder-free.
Daily medium changes. Watch for epithelial-like patches, compact cell borders, high nucleus: cytoplasm ratio, and nucleoli prominence. Mark colonies with classic iPSC morphology. Treat with ROCK inhibitor to pick. Manually pick colonies (clump passaging) to new plates. Expand each clone in separate wells; record clone IDs, passage numbers, and any phenotypic notes.
| Problem | Possible Cause | Solution |
|---|---|---|
| Low transduction efficiency |
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| High cell death post-transduction |
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| Few/no iPSC colonies |
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| Differentiated edges in colonies |
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| Persistent exogenous factor expression |
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| Abnormal karyotype |
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Because no two reprogramming projects are alike, we've built an integrated menu of add-on and alternative services that plug neatly into your lentiviral workflow—or replace it when an integration-free route is required.
Tell us your starting material, constraints, and downstream assays, and we'll assemble a fit-for-purpose plan and data package.
A: Human dermal fibroblasts, PBMC-derived erythroblasts, dental pulp cells, and urine-derived epithelial cells are reliable. Key is health: ≥90% viability, mycoplasma-free, and consistent growth. If your input is rare or fragile, we'll adjust seeding density, matrix, and enhancer windows.
A: P2–P6 is a sweet spot. Earlier passages reduce senescence-related variability and improve clonogenicity. Very late passages can reprogram, but expect lower efficiency and more QC attrition.
A: Check cell health, confirm titer/MOI, and verify enhancer concentrations. Try spinoculation if tolerated, switch to laminin-521, and ensure daily full medium changes. A short VPA/ascorbic window often rescues early epithelialization.
A: ≥2 × 10⁶ viable cells (or agreed equivalent), ≥90% viability, mycoplasma-negative, and accompanied by non-identifying donor/source metadata and culture history. Tell us about any prior edits or antibiotics.
A: We do. Neural progenitors, dopaminergic neurons, cardiomyocytes, hepatocyte-like cells, endothelial cells, and more.
A: Yes. RNP or mRNA CRISPR with clonal isolation, on-target confirmation, prioritized off-target shortlist, and post-edit karyotype. We strongly recommend producing isogenic control lines in parallel.
References
Created September 2025
For Research Use Only. Not For Clinical Use.
