Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

Real-Time Temperature Mapping in Ultra-Low Freezers as a Standard Quality Assessment.

Adequate preservation of biospecimens has been proven to be critical to obtain reliable and reproducible results in genomics, transcriptomics, proteomics, and many other assays. Most biological assays can be performed on specimens preserved in -80°C ultra-low freezers, but their quality can be influenced by temperature variability within storage units. Thus, regulatory standards such as those from the College of American Pathologists (CAP), the federal Clinical Laboratory Improvement Amendments, and standards from the Food and Drug Administration require temperature mapping, a standard quality assessment for accreditation when using ultra-low freezers for long-term biospecimen storage. The current mapping methods, providing annual/periodic data, may not be adequate indicators of temperature stability within the different zones of the freezers. In addition, they frequently require manual handling of biospecimens periodically, as they require freezers to be emptied or rearranged temporarily for the installation of temperature probes, risking the integrity of biospecimen quality. In this article, we describe a novel monitoring methodology based on real-time temperature reading of multiple zones by permanently installing thermocouples. An online cloud-based application records temperature variations within 1 minute intervals, and its 24/7 alert system triggers text alarm messages to a predefined set of users when temperature values increase above preset defaults. This provides an opportunity to take remedial action and to obtain a better-quality assessment. Our results indicate that real-time temperature monitoring at multiple zones of a freezer with a 1 minute resolution is a stable and sustainable methodology and, most importantly, lowers the risk of compromising the quality of the biospecimen. The design and use of the real-time monitoring system for ultra-low freezers is one of the acceptable methods by CAP to ensure the stability of biospecimen quality during long-term storage.