Cryoprotectant toxicity and hypothermic sensitivity among Anopheles larvae.

Laboratory rearing of mosquitoes is commonly practiced by researchers studying mosquito-borne infectious diseases and vector control methods. In the absence of cryopreservation methods to stabilize unique or genetically modified strains, mosquito lines must be continuously maintained, a laborious process that risks selection effects, contamination, and genetic drift. Towards the development of a cryopreservation protocol, several commonly used cryoprotectants were systematically characterized here both individually and as cocktails. Among first instar, feeding-stage An. gambiae and An. stephensi larvae, cryoprotectant toxicity followed the order of dimethyl sulfoxide > ethylene glycol > methanol. The resulting LD50 values were used as the basis for the development of cryoprotectant cocktail solutions, where formulation optimization was streamlined using Taguchi methods of experimental design. Sensitivity to hypothermia was further evaluated to determine the feasibility of cryoprotectant loading at reduced temperatures and slow cooling approaches to cryopreservation. The information described here contributes to the knowledge base necessary to inform the development of a cryopreservation protocol for Anopheles larvae.

Cryopreservation of infectious Cryptosporidium parvum oocysts achieved through vitrification using high aspect ratio specimen containers.

Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. While cryopreservation of Cryptosporidium oocysts by vitrification was recently achieved, the method is restricted to small sample volumes, thereby limiting widespread implementation of this procedure. Here, a second-generation method is described for cryopreservation of C. parvum oocysts by vitrification using custom high aspect ratio specimen containers, which enable a 100-fold increase in sample volume compared to previous methods. Oocysts cryopreserved using the described protocol exhibit high viability, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice. Importantly, the course of the infection is comparable to that observed in mice infected with unfrozen oocysts. Vitrification of C. parvum oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials.

Role of synthetic antifreeze agents in catalyzing ice nucleation.

Nature endows antifreeze (glyco)proteins (AF(G)Ps) with the excellent capability of inhibiting ice crystal growth. Recent years have also witnessed the emergence of many potent AF(G)P mimics such as poly (vinyl alcohol) (PVA). As researchers are revealing the molecular mechanisms of inhibiting ice crystal growth by AF(G)Ps and their synthetic substitutes, there remains no agreement about their effect on ice nucleation. In this study, we report the observation of ice nucleation catalyzed by PVA of different polymerization degrees using a freeze-on-a-chip platform which allows the monitoring of freezing and melting events over hundreds of monodisperse, picoliter-sized aqueous droplets. Aqueous droplets made of 1 mg/ml PVA solution exhibit a median freezing temperature of around -36 °C, two degrees higher than the observed homogeneous nucleation temperature of water. The findings in our study bring useful insights into the different roles of synthetic antifreeze agents in controlling ice formation.

Cryopreservation of infectious Cryptosporidium parvum oocysts.

Cryptosporidiosis in an enteric infection caused by Cryptosporidium parasites and is a major cause of acute infant diarrhea in the developing world. A major bottleneck to research progress is the lack of methods to cryopreserve Cryptosporidium oocysts, thus requiring routine propagation in laboratory animals. Here, we report a method to cryopreserve C. parvum oocysts by ultra-fast cooling. Cryopreserved oocysts exhibit high viability and robust in vitro excystation, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to what we observe with unfrozen oocysts. Oocyst viability and infectivity is not visibly changed after several weeks of cryogenic storage. Cryopreservation will facilitate the sharing of oocysts from well-characterized isolates and transgenic strains among different laboratories.

Ultra-fast vitrification of patient-derived circulating tumor cell lines.

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

Controlled ice nucleation using freeze-dried Pseudomonas syringae encapsulated in alginate beads.

The control of ice nucleation is of fundamental significance in many process technologies related to food and pharmaceutical science and cryobiology. Mechanical perturbation, electromagnetic fields and ice-nucleating agents (INAs) have been known to induce ice nucleation in a controlled manner. But these ice-nucleating methods may suffer from cumbersome manual operations, safety concerns of external fields, and biocompatibility and recovery issues of INA particles, especially when used in living systems. Given the automatic ice-seeding nature of INAs, a promising solution to overcome some of the above limitations is to engineer a biocomposite that accommodates the INA particles but minimizes their interactions with biologics, as well as enabling the recovery of used particles. In this study, freeze-dried Pseudomonas syringae, a model ice-nucleating agent, was encapsulated into microliter-sized alginate beads. We evaluated the performance of the bacterial hydrogel beads to initiate ice nucleation in water and aqueous glycerol solution by investigating factors including the size and number of the beads and the local concentration of INA particles. In the aqueous sample of a fixed volume, the total mass of the INA particles (m) was found to be the governing parameter that is solely responsible for determining the ice nucleation performance of the bacterial hydrogel beads. The freezing temperature has a strong positive linear correlation with log10m. The findings in this study provide an effective, predictable approach to control ice nucleation, which can improve the outcome and standardization of many ice-assisted process technologies.