Fast Post-thaw Recovery of Cryopreserved Cells for Use in Bioassays

Introduction

Large-scale bioassays are frequently used in the biotechnology and pharmaceutical industries for a wide variety of testing, including drug screening, vaccine development, and quality control. There are several requirements for these systems that ensure reliability and effectiveness. Standardization, reproducibility, and automation compatibility are just a few system attributes that facilitate development of a successful bioassay. Any inconsistencies in the biological material input poses the greatest challenge to creating a robust bioassay system featuring these attributes.

Typically, the generation of sufficient biomaterial (e.g., cell lines) requires large-scale cell culture and demands extensive time and resource commitments. Additionally, long-term cell culture faces several added risks such as genetic drift, phenotypic changes, and chance of contamination. A high-content, highly standardized cell material that can be stored for optimal durations could replace the requirement for large-scale cell culture and quickly plug into a large-scale bioassay systems.

Here, we employ a fetal bovine serum (FBS)-free cryoformulation and strict process parameters to cryopreserve ATCC cells for use in assays after minimal post-thaw culture time. The thawed cells have high post-thaw viability and display similar bioassay sensitivity to cultured cells. 

Methodology

Due to their ability to mimic human monocyte and macrophage activity, THP-1 cells are used extensively in biopharmaceuticals to assess anti-inflammatory and immunomodulatory compounds. THP-1 (parent) and THP-1-NFkB-Luc2 (reporter) cells were cultured using ATCC standard protocols and then cryopreserved in a proprietary cryoformulation. The reporter cell line is a clone of THP-1 and expresses bioluminescent proteins when NFkB pathways are activated in the cells. These cells were used to quickly and sensitively assess the functionality of THP-1 cells post-thaw.

Post-thaw characterization experiments included evaluating viability and growth using an automated counting instrument. For the reporter, NFkB activation via stimulation with LPS was evaluated using a commercially available bioluminescent detection system. All experiments were performed within two-hours post-thaw.

Data

A post-thaw viability of >80% and a viable cell number of >9 million was achieved for both cell lines. There was minimal lag in post-thaw growth lag, and normal growth resumed within 48 hours. The post-thaw luciferase response of the reporter cells was >100-fold higher than the negative control, and the cells demonstrated less variability than the culture controls.

Conclusion

The cryopreserved cells demonstrated very high post-thaw viability, quick growth recovery, and similar functionality as compared to culture control cells. Further, the FBS-free cryoformulation reduced the batch-to-batch variability commonly exacerbated by the freeze concentration of constituents. These improvements address some of the long-standing pain-points around cellular work in industry. Therefore, it is feasible that cells in this thaw-and-go format can replace the need for long-term culture to save time and valuable resources.