Staff member

JTD Keywords: performance, polarization, Magnetic-resonance

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

JTD Keywords: 3d cell culture, Biofabrication, Biomaterials, Carboxymethyl cellulose, Cryopreservation, Hepatocytes, Prevention, Scaffolds, Spheroids

Cryopreservation methods for cell and tissue storage have beenaround since 1954, where thawed sperm samples were used for aninsemination. Since then, the technology has evolved for cliniciansand researchers to cryopreserve tissue, cell lines and primary cells.While cryopreserving tissues helps maintain their physiological in-tegrity for study it does not assure the viability of the cells afterthawing. Furthermore, the cells cryopreserved in suspension losetheir dimensional anchors, forcing them to change their morphology.To solve this issue, tissue engineering allows researchers to create 3Dculture models, such as organoids and bioprinted cell clusters, thatmimic the physiological characteristics of the cells in tissue anddisease. Although this culture methods present promising results,there is a lack of methodology to cryopreserve 3D cell models andpatients’ samples for storage and transport in a way where they re-main viable after thawing. We propose a protocol that uses a car-boxymethyl cellulose scaffold and precise freezing and thawingconditions for spheroid survival. The scaffold provides structure forthe hepatocytes to create spheroids on their own as well as supportthroughout the freezing and thawing processes for optimal cell via-bility post-thawing. Furthermore, this method will achieve highercell viability than transporting the cells as a cryopreserved pellet formodel assembling after thawing, allowing the cells to settle and forma tissue beforehand to improve viability after cryopreservation. Thistechnique constitutes a step forward for it will facilitate the transportof already assembled 3D models from cell lines or primary cells frompatients.

JTD Keywords: Cryopreservation

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO2 and H2, and iii) H2/CO2 reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H2 showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from 13C-labelled DCM was marked at the methyl ([2–13C]acetate), carboxyl ([1–13C]acetate), and both ([1,2–13C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H2/CO2 acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 μM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater. © 2019 Elsevier Ltd

JTD Keywords: Bioremediation, Co-contaminants, Dehalobacterium, Dichloromethane, Inhibition