Biophysical investigation of novel cryoprotectants
Creators: name: Raju, Rekha
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 510405
subject: Soft condensed matter

description: This thesis aims to examine the critical molecular properties that lead to good cryoprotective performance and use this knowledge to test novel non-toxic compounds which can be optimized to use as cryoprotectants (CPAs). The key properties needed to make a good CPA are: low toxicity; the ability to pass through membranes to get inside cells; the ability to form glasses at relatively high sub-zero temperatures; and the ability to inhibit ice recrystallisation during thawing (the growth of larger ice crystals from smaller ones). This thesis presents a systematic investigation of these important properties, for both traditional CPAs and novel compounds with cryoprotective potential. Cryopreservation trials with some of these compounds are carried out to assess their cryoprotective potential.<br><br>First, this thesis presents a Langmuir monolayer study of the effects of four common cryoprotective agents (dimethyl sulfoxide, ethylene glycol, glycerol and dimethyl formamide) on phospholipid monolayers. Four different phospholipids (DOPC, DPPC, POPC, POPE) are studied to determine if the head group and level of chain saturation influence the interactions. It is shown that the phospholipid species can have a significant effect. DMSO showed interesting lipid specific effects - causing expansion of DPPC monolayers but compression for POPC monolayers, while having little effect for DOPC and POPE. The results highlight the importance of studying more than one model lipid system as well as the need to study concentrations relevant to cryopreservation.<br><br>Second, this thesis investigates the potential of carbohydrate-based surfactants such as n- octyl(thio)glycosides to be a novel and accessible class of penetrating CPAs. A series of eight n-octyl (thio)glycosides (1alpha/beta - 4alpha/beta) with D-glucose or D-galactose-configured head groups and varying anomeric configurations were evaluated for glass transition behavior and membrane permeability. Of these, n-octyl beta-D-glucopyranoside (2beta) exhibited high glass transition temperature (Tg), both as a neat sample and 20wt-% aqueous solution. Membrane permeability studies of this compound revealed cellular uptake at concentrations relevant to the inhibition of intracellular ice formation, thus presenting a promising lead candidate for further biophysical and cryopreservation studies.<br><br>Third, this thesis presents similar studies on four trehalose derivatives in order to understand their cryoprotective potential. Permeability trials on these molecules showed that trehalose-di acetate has no cell permeability, whereas both trehalose di pivotate and trehalose tetra acetate have moderate permeability. The most promising candidate with high glass transition temperature and relatively good permeability was trehalose di pivotate, so this compound was used for further cryopreservation studies on THP-1 cells.<br><br>Finally, this thesis presents the development of a novel microfluidic cell trapping device for cell permeability studies, which has considerable advantages over traditional techniques. Two microfluidic cell trapping structures were produced, combining direct laser writing (DLW) and soft lithography techniques. These structures were used for the hydrodynamic capturing of single human monocyte (THP-1) cells and tested by studying the cell volume kinetics upon the addition of DMSO. Compared to existing technologies, the method allowed: (i) rapid capture of single cells without surface functionalization; (ii) the rapid exchange of solvent to generate osmotic gradients across the cell membrane; and (iii) real-time imaging of cells during the shrinkage and swelling phases due to the imaging stability provided by the traps.<br><br>To date, the search for new CPAs has been reliant on educated guesswork and trial and error. This thesis systematically investigates the critical molecular properties relevant for cryopreservation and provides a systematic method for assessing novel molecules as alternative cryoprotectants.