The Effects of Solutes in the Cryopreservation of Adherent Neuroblastoma (Neuro-2a) Cells

A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to cell culture plates were incubated for 24 h at varying concentrations of trehalose, proline, sucrose and combinations of these compounds. Cells were cryopreserved for 24 h and cell viability post-freezing and thawing was quantified by trypan blue exclusion assay. On average, only 13.5% of adherent cells survived freezing in the presence of 10% Me2SO alone (control). Pre-incubation of cells with medium containing both trehalose and proline severely decreased cell proliferation, but increased cell recovery by 288% (overall recovery of 52.5%). Our results suggest that pre-incubation of Neuro-2a cells with trehalose and proline in combination provides cell protection resulting in increased cell survival post-freezing

Towards next generation cryopreservation utilising macromolecules and osmolytes

Complex cell preservation methods, such as attached monolayers, have failed to achieve a level of success that would provide insights and pathways for potential whole organ preservation. Ice crystal growth during freezing can cause both mechanical and osmotic damage to cells, and the ability to control this process by using ice recrystallisation inhibitors has been shown to result in enhanced cryopreservation outcomes. A variety of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) have been identified in organisms, of which all are icebinding proteins that are crucial for the species survival. Three different macromolecular cryoprotectants are evaluated: poly(vinyl alcohol) (PVA) due to its high ice recrystallisation inhibition activity, polyproline as a possible AF(G)P mimic, and a polyampholyte due to its scalable synthesis and precise 1:1 ratio of cationic/anionic groups. We also evaluated three potential osmoprotectants: alanine due to the heavy alanine rich regions of AF(G)Ps, betaine for its osmoprotecting properties, and proline due to its previous use as a cryoprotectant and its implications as an osmoprotectant. The macromolecular cryoprotectants and small molecule osmolytes were examined for their physical interactions with ice (Chapter 2), toxicity and proliferation impacts (Chapter 3), the ability to successfully cryopreserve mammalian cells along with post-freeze viability (Chapter 4), and finally, the membrane permeability at various steps throughout the freezing processes was evaluated (Chapter 5). Only PVA was found to have strong ice activity, minimal toxicity was found and proline was shown to down-regulate growth, osmolytes plus PVA or polyproline, and polyampholyte alone, were found to cryopreserve cell monolayers, and polyampholyte showed improved membrane permeability post-freeze. The application of these approaches could provide next generation cryopreservation strategies for many different cell types

Polyampholytes as emerging macromolecular cryoprotectants

Cellular cryopreservation is a platform technology which underpins cell biology, biochemistry, biomaterials, diagnostics, and the cold chain for emerging cell-based therapies. This technique relies on effective methods for banking and shipping to avoid the need for continuous cell culture. The most common method to achieve cryopreservation is to use large volumes of organic solvent cryoprotective agents which can promote either a vitreous (ice free) phase or dehydrate and protect the cells. These methods are very successful but are not perfect: not all cell types can be cryopreserved and recovered, and the cells do not always retain their phenotype and function post-thaw. This Perspective will introduce polyampholytes as emerging macromolecular cryoprotective agents and demonstrate they have the potential to impact a range of fields from cell-based therapies to basic cell biology and may be able to improve, or replace, current solvent-based cryoprotective agents. Polyampholytes have been shown to be remarkable (mammalian cell) cryopreservation enhancers, but their mechanism of action is unclear, which may include membrane protection, solvent replacement, or a yet unknown protective mechanism, but it seems the modulation of ice growth (recrystallization) may only play a minor role in their function, unlike other macromolecular cryoprotectants. This Perspective will discuss their synthesis and summarize the state-of-the-art, including hypotheses of how they function, to introduce this exciting area of biomacromolecular science

Opportunities for Self-Evaluation Increase Student Calibration in an Introductory Biology Course

Accurate self-evaluation is critical for learning. Calibration describes the relationship between learners’ perception of their performance and their actual performance on a task. Here, we describe two studies aimed at assessing and improving student calibration in a first-semester introductory biology course at a 4-year public institution. Study 1 investigated students’ (n = 310) calibration (the difference between estimated and actual exam performance) across one semester. Students were significantly miscalibrated for the first exam: their predicted scores were, on average, significantly higher than their actual scores. The lowest-performing students had the most inaccurate estimates. Calibration improved with each exam. By the final exam, students underestimated their scores. We initiated a second study in the following semester to examine whether explicitly teaching students about self-evaluation strategies would improve their calibration and performance. Instruction in the experimental section (n = 290) focused on students’ tendency to overestimate their abilities and provided retrieval-practice opportunities. Students in the experimental section showed better calibration and performance on the first exam compared with students in a control section taught by a different instructor during the same semester (n = 251). These findings suggest that simple instructional strategies can increase students’ metacognitive awareness and improve their performance

Extracellular antifreeze protein significantly enhances the cryopreservation of cell monolayers

The cryopreservation of cells underpins many areas of biotechnology, healthcare, and fundamental science by enabling the banking and distribution of cells. Cryoprotectants are essential to prevent cold-induced damage. Here, we demonstrate that extracellular localization of antifreeze proteins can significantly enhance post-thaw recovery of mammalian cell monolayers cryopreserved using dimethyl sulfoxide, whereas they show less benefit in suspension cryopreservation. A type III antifreeze protein (AFPIII) was used as the macromolecular ice recrystallization inhibitor and its intra/extracellular locations were controlled by using Pep-1, a cell-penetrating peptide. Flow cytometry and confocal microscopy confirmed successful delivery of AFPIII. The presence of extracellular AFPIII dramatically increased post-thaw recovery in a challenging 2-D cell monolayer system using just 0.8 mg·mL–1, from 25% to over 60%, whereas intracellularly delivered AFPIII showed less benefit. Interestingly, the antifreeze protein was less effective when used in suspension cryopreservation of the same cells, suggesting that the cryopreservation format is also crucial. These observations show that, in the discovery of macromolecular cryoprotectants, intracellular delivery of ice recrystallization inhibitors may not be a significant requirement under “slow freezing” conditions, which will help guide the design of new biomaterials, in particular, for cell storage

Engineering cell surfaces by covalent grafting of synthetic polymers to metabolically-labeled glycans

Re-engineering mammalian cell surfaces enables modulation of their phenotype, function, and interactions with external markers and may find application in cell-based therapies. Here we use metabolic glycan labeling to install azido groups onto the cell surface, which can act as anchor points to enable rapid, simple, and robust “click” functionalization by the addition of a polymer bearing orthogonally reactive functionality. Using this strategy, new cell surface functionality was introduced by using telechelic polymers with fluorescence or biotin termini, demonstrating that recruitment of biomacromolecules is possible. This approach may enable the attachment of payloads and modulation of cell function and fate, as well as providing a tool to interface synthetic polymers with biological systems

Polyproline is a minimal antifreeze protein mimetic and enhances the cryopreservation of cell monolayers

Tissue engineering, gene therapy, drug screening and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and requires large volumes of solvent 'antifreezes'. Herein we report polyproline is a minimum (bio)synthetic mimic of antifreeze proteins, which is accessible by solution, solid phase and recombinant methods. We demonstrate that polyproline has ice recrystallization inhibition activity linked to its amphipathic helix and that it enhances the DMSO- cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants

Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes

Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte L-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical (i.e. not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants

Synthetically scalable poly(ampholyte) which dramatically enhances cellular cryopreservation

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies

Completed Genome Sequence of the Anaerobic Iron-Oxidizing Bacterium \u3ci\u3eAcidovorax ebreus\u3c/i\u3e Strain TPSY

Acidovorax ebreus strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY