Retroviral replicating vectors (RRVs) have shown efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the previous study, we developed the spRRV system (sRRVgp-yCD & spRRVe-TK) encoding HSV1-TK and human codon-optimized yeast CD genes as for cancer therapeutic agents, respectively. In the present study, to achieve a successful clinical trial using RRVs, we were willing to establish the virus producer cells (VPCs) that produce high-titer spRRVs. First, each of the spRRVs (sRRVgp-yCD8 & spRRVe-TK) transduced separately into retrovirus packaging cell line PG13. And then, PG13/env-HSV1-TK and PG13/gagpol-yCD8 VPCs were selected clonally and amplified for the screening of VPCs producing high-titer retroviral vector particles. The characteristics of established VPCs were accomplished by validation of genome stability, infectivity, cytotoxicity, productivity, and mycoplasma test. Finally, the validated PG13/env-HSV1-TK and PG13/gagpol-yCD8 VPCs were manufactured in the GMP facility. Also, the MCBs can be used for mass production and purification of retroviral vectors in GLP or GMP facilities for nonclinical and clinical trials.