Industrial Biotechnology Conservation Processes: Similarities with Natural Long-Term Preservation of Biological Organisms

Abstract

Cryopreservation and lyophilization processes are widely used for conservation purposes in the pharmaceutical, biotechnological, and food industries or in medical transplantation. Such processes deal with extremely low temperatures (e.g., –196 °C) and multiple physical states of water, a universal and essential molecule for many biological lifeforms. This study firstly considers the controlled laboratory/industrial artificial conditions used to favor specific water phase transitions during cellular material cryopreservation and lyophilization under the Swiss progenitor cell transplantation program. Both biotechnological tools are successfully used for the long-term storage of biological samples and products, with reversible quasi-arrest of metabolic activities (e.g., cryogenic storage in liquid nitrogen). Secondly, similarities are outlined between such artificial localized environment modifications and some natural ecological niches known to favor metabolic rate modifications (e.g., cryptobiosis) in biological organisms. Specifically, examples of survival to extreme physical parameters by small multi-cellular animals (e.g., tardigrades) are discussed, opening further considerations about the possibility to reversibly slow or temporarily arrest the metabolic activity rates of defined complex organisms in controlled conditions. Key examples of biological organism adaptation capabilities to extreme environmental parameters finally enabled a discussion about the emergence of early primordial biological lifeforms, from natural biotechnology and evolutionary points of view. Overall, the provided examples/similarities confirm the interest in further transposing natural processes and phenomena to controlled laboratory settings with the ultimate goal of gaining better control and modulation capacities over the metabolic activities of complex biological organisms.

Development of Lyophilized Eukaryotic Cell-Free Protein Expression System Based on Leishmania tarentolae

“…The superiority of trehalose as a solo lyoprotectant is similar to that observed in lyophilization of E. coli CFPS and is perhaps unsurprising since multicellular organisms such as tardigrades rely on trehalose to survive freeze thawing …”

“…13 Antioxidants (N-acetyl-Lcysteine (NALC) and β-mercaptoethanol (βME)) were also tested due to their established roles in preserving biological tissues, cell lysates, and pharmaceuticals. 19 Lyoprotectants added to nonlyophilized LTE did not affect eGFP expression significantly except for 20 mM NALC, which decreased expression by 39% relative to the positive control (Figure 3A). Trehalose (100 mM) and the polymers PEG (1% w/v) and PVP (1% w/v) proved to be the best solo lyoprotectants, with protein expression efficiencies of >70% relative to positive controls (Figure 3A).…”

“…20 The superiority of trehalose as a solo lyoprotectant is similar to that observed in lyophilization of E. coli CFPS 13 and is perhaps unsurprising since multicellular organisms such as tardigrades rely on trehalose to survive freeze thawing. 19 Polymers such as PEG and PVP inhibit the unfolding of proteins in the frozen state during lyophilization due to the high binding affinity for water. Although sometimes considered to be cryoprotectants only, they have been associated with stabilization for enzymes such as lactate dehydrogenase during lyophilization.…”

“…coli CFPS that included sugars (maltose, trehalose, and sucrose), organic osmolytes (betaine), and polymers (PEG, molecular weight 3350 g·mol –1 , and poly­(vinylpyrrolidone) (PVP)) . Antioxidants ( N -acetyl- l -cysteine (NALC) and β-mercaptoethanol (βME)) were also tested due to their established roles in preserving biological tissues, cell lysates, and pharmaceuticals …”