Despite being a gold standard for the treatment of end-stage liver diseases, orthotopic liver transplant (OLT) is limited due to a shortage of high-quality donor organs, lifelong immunosuppression, and organ rejection. Hepatocyte transplantation is an alternative for the treatment of inherited metabolic liver diseases, which could potentially overcome the limitations of OLT. However, it is important to source high-quality donor cells that potentially engraft post-transplantation, allowing us to develop an off-the-shelf treatment regimen. Off-the-shelf therapeutics require the preservation of gene-edited primary hepatocytes that could readily be transplanted into the recipient, thus eliminating the need for instantaneous cell isolation and ex vivo gene therapy. To achieve this treatment regime, the Cottle lab is developing an optimized protocol for electroporation-assisted cryopreservation of gene-edited primary hepatocytes. CPAs such as DMSO and Glycerol have been shown to be remarkable for long-term preservation of primary cells. However, their inherent toxicity post-transplantation, beyond certain considerations, has limited their clinical application. The Cottle is investigating nontoxic CPAs for cryopreservation of hepatocytes. Despite being nontoxic, their non-penetrating nature has limited their application, necessitating delivery into cells for cryopreservation. Electroporation is a technique that uses short, high-voltage electrical pulses to temporarily create pores in cell membranes, enabling the entry of external substances such as DNA, RNA, proteins, or drugs. This method can co-deliver RNPs and non-toxic CPAs into cells, facilitating ex-vivo gene editing and the long-term preservation of primary cells. The Cottle group has demonstrated that systemic delivery of RNPs via electroporation is highly effective for ex-vivo gene therapy. By combining electroporation-based gene therapy with cryopreservation of primary hepatocytes, we are developing a protocol for electroporation-mediated cryopreservation of gene-edited primary hepatocytes. This aims to create off-the-shelf therapeutics for treating inherited metabolic liver diseases.