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

Biopreservation and biobanking (2020-04-18)
Andy He, Samantha Powell, Mason Kyle, Michael Rose, Edgar Masmila, Valeria Estrada, Jason K Sicklick, Alfredo Molinolo, Sharmeela Kaushal
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
CryoStor® 细胞冻存培养基, CS10
Sigma-Aldrich
MEM 非必需氨基酸溶液 (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
重组人表皮细胞生长因子, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Sigma-Aldrich
氢化可的松, BioReagent, suitable for cell culture
Sigma-Aldrich
胰岛素 人, recombinant, expressed in yeast (proprietary host)