Optimization of CRISPR dCas9 Lentiviral Transduction in Nucleus Pulposus Cells for Use in Tissue Engineering
TL;DR
An optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.
Abstract
Efficient delivery of large gene-editing plasmids, such as the mCherry-CRISPR dCas9 system, into nucleus pulposus (NP) cells is a key step in generating sufficient cells for tissue-engineered intervertebral disc (IVD) constructs and other regenerative therapies for degenerative disc disease (DDD). However, the transfection and transduction of these environmentally sensitive cells remain challenging. This study aimed to identify the best protocol for delivery with minimal cytotoxicity and greatest efficiency. Transfection conditions in HEK293T cells were evaluated using Lipofectamine 3000, Lipofectamine Classic, and ViaFect at different reagent-to-DNA ratios and DNA amounts. Transfection efficiency was quantified by flow cytometry based on mCherry expression. Lentivirus was produced and concentrated by comparing PEG8000, a commercial Lenti-X concentrator, and ultracentrifugation. For NP cell transduction, polybrene and protamine sulfate were tested at multiple concentrations to maximize efficiency and viability. The optimized protocol was validated by delivering a CRISPR/dCas9 Synergistic Activation Mediator (SAM) system to activate endogenous FOXA2, and by seeding the resulting cells onto membranes to assess in vitro NP-like tissue formation. Lipofectamine 3000 at a 2:1 reagent-to-DNA ratio with 0.5 µg DNA per well yielded the highest transfection efficiency in HEK293T cells while minimizing cytotoxicity. Coprecipitation methods for lentiviral concentration, particularly the in-house PEG8000 concentrator, were better than ultracentrifugation. Protamine sulfate at 30 µg/mL yielded efficient NP cell transduction with higher viability than polybrene, as assessed by survival after antibiotic selection. Application of this protocol upregulated endogenous FOXA2 mRNA and protein expression, demonstrating functional efficacy. SAM-FOXA2 cells produced thicker tissue on membrane inserts than SAM controls, confirming compatibility of the protocol with downstream tissue engineering applications. Therefore, an optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD. Impact Statement Generating sufficient numbers of phenotype-stable nucleus pulposus (NP) cells is a bottleneck in engineering NP tissues. CRISPR/dCas9 activation of FOXA2 produces cells that form superior NP tissue in vitro, but delivering the required 13.5 kb plasmid into environmentally sensitive NP cells is technically challenging. We describe a validated, step-by-step protocol covering transfection, lentivirus concentration, and transduction that achieves high ficiency with minimal cytotoxicity, and show that the resulting FOXA2-expressing cells generate NP-like tissue.
