The use of engineered keratinocytes for cell therapy has long been an attractive option to treat various dermatological, oral, and aural disorders. Keratinocytes are easily adapted to in vitro culture and expanded from even small tissue specimens, making them an ideal cell type for a range of autologous and allogeneic cell therapy products. However, engineered keratinocyte-based cell therapies have been limited by the lack of efficient transfection methods for adult keratinocytes. Here we aimed to develop a GMP-compliant, scalable cell engineering process using the MaxCyte® cell electroporation platform to transfect neonatal and adult primary keratinocytes from four distinct anatomical locations.
Using MaxCyte electroporation, we efficiently delivered mRNA or CRISPR-Cas9 ribonucleoproteins (RNPs) to primary keratinocytes from the foreskin, arm, tonsils, and tympanic membrane at two different scales. This was accomplished without compromising cell viability, morphology, or growth capability. Furthermore, delivery of multiple CRISPR RNPs in a single electroporation achieved highly efficient, multiplexed gene editing in a simple, adaptable process. These improvements in transfection efficiency and cell viability reduced keratinocyte engineering times by up to 4 weeks, with a significantly higher success rate than a standard chemical transfection method. Finally, we demonstrate the scalability of the MaxCyte electroporation process, enabling the engineering of millions of primary keratinocytes without any loss of efficacy. In summary, the MaxCyte ExPERTTM platform provides efficient, GMP-compliant, multiplexed transfection for the development and scalable production of engineered keratinocyte cell therapy products.