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The development of technologies for the biopreservation of infectious organisms requires careful analysis of benefits and risks. This article reviews the regulatory landscape and oversight responsibilities in the United States in respect to pathogen biopreservation. Focused on two globally significant pathogens, Cryptosporidium and Plasmodium , the article explores advantages and potential risks of biopreservation concerning biosafety, biosecurity and biocontainment. [ABSTRACT FROM AUTHOR]

Earth's biodiversity is increasingly threatened and at risk. We propose a passive lunar biorepository for long-term storage of prioritized taxa of live cryopreserved samples to safeguard Earth's biodiversity and to support future space exploration and planet terraforming. Our initial focus will be on cryopreserving animal skin samples with fibroblast cells. An exemplar system has been developed using cryopreserved fish fins from the Starry Goby, Asterropteryx semipunctata. Samples will be expanded into fibroblast cells, recryopreserved, and then tested in an Earth-based laboratory for robust packaging and sensitivity to radiation. Two key factors for this biorepository are the needs to reduce damage from radiation and to maintain the samples near –196° Celsius. Certain lunar sites near the poles may meet these criteria. If possible, further testing would occur on the International Space Station prior to storage on the Moon. To secure a positive shared future, this is an open call to participate in this decades-long program. [ABSTRACT FROM AUTHOR]

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine. [ABSTRACT FROM AUTHOR]

Cryptosporidium hominis is a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The human C. hominis TU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiring C. hominis. Here, we report cryopreservation of C. hominis TU502 oocysts by vitrification using specially designed specimen containers scaled to 100 μL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens. Author summary: We report here on the first successful method of cryopreservation of Cryptosporidium hominis parasites, one of the most common infectious causes of pediatric diarrhea in the developing world. This is significant as lack of cryopreservation methods for this species of parasite hinders progress of scientific and clinical research. In the absence of these methods, a reference isolate of C. hominis must be maintained periodically by passage in gnotobiotic piglets. Cryopreservation of this unique isolate is therefore a priority to increase sharing and availability for research, and to prevent catastrophic loss. Cryopreservation is also necessary to facilitate human challenge studies and clinical trials for the evaluation of therapeutics. Here, we successfully cryopreserved the parasite by vitrification using rapid cooling rates achieved in specially designed specimen containers. We showed that the thawed parasite retained viability and infectivity in gnotobiotic piglets. Notably these methods allowed us to establish a biobank of C. hominis oocysts of reference isolate TU502. The availability of biobanked optimized/standardized oocyst preparations may facilitate drug and vaccines evaluation. [ABSTRACT FROM AUTHOR]

Objectives: Objective measurement of antiretrovirals may aid clinical interventions for improving adherence to HIV prevention or treatment regimens. A point-of-care urine test could provide real-time information about recent adherence to regimens containing tenofovir disoproxil fumarate or tenofovir alafenamide. We developed a lateral flow immunoassay (LFA) and ELISA for urinary tenofovir.Methods: The intensity of the LFA test line was quantified using an optical reader and visually scored 0-5 by two independent people, using a reference card. The sensitivity and specificity of both the ELISA and LFA were determined for two different tenofovir concentration cut-offs for tenofovir disoproxil fumarate and tenofovir alafenamide adherence-1500 and 150 ng/mL, respectively. To validate the assays, we measured 586 urine samples from 28 individuals collected as part of a study of tenofovir pharmacokinetics in adults, which were also measured by MS for reference.Results: Both the LFA signal and ELISA signal were each strongly correlated with drug concentrations (0.91 and 0.92, respectively). The LFA signal and ELISA were highly sensitive and specific at both thresholds (LFA sensitivity/specificity: tenofovir disoproxil fumarate, 89%/96%; and tenofovir alafenamide, 90%/96%) (ELISA sensitivity/specificity: tenofovir disoproxil fumarate, 94%/94%; and tenofovir alafenamide, 92%/84%). Visual scoring of the LFA was also highly sensitive and specific at both the tenofovir disoproxil fumarate threshold and the tenofovir alafenamide threshold (sensitivity/specificity: tenofovir disoproxil fumarate, 91%/94%; and tenofovir alafenamide, 87%/90%).Conclusions: Our rapid semi-quantitative test can measure tenofovir concentrations relevant to both tenofovir alafenamide and tenofovir disoproxil fumarate adherence, which may support adherence-promoting interventions across a range of HIV care settings. [ABSTRACT FROM AUTHOR]

The article discusses the need for objective tools to assess the quality of stored red blood cells (RBCs) for transfusion medicine. The authors highlight the potential of lab-on-a-chip (LOC) technologies to quantitatively evaluate RBC quality using multidimensional metrics. They acknowledge the development of a point-of-care device called Erysense, which assesses the flow properties and morphological abnormalities of stored RBCs. However, they also mention limitations and challenges that need to be addressed, such as the need for extensive validation, automation of the workflow, and addressing storage lesions. The authors suggest that while scalable strategies for in vitro erythropoiesis are promising, other steps can be taken to address blood shortages and improve transfusion practices. [Extracted from the article]

A lipid blend solution comprised of mono- and diglycerides is deposited onto an aqueous surface to generate synthetic neutral lipid droplets (SNLDs), which might have function other than energy storage molecules in Plasmodium falciparum during the intraerythrocytic stage of the parasite life cycle. The results indicated that neutral lipid structures within the digestive vacuole are not only the location of in vivo hemozoin formation but is also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.

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. [ABSTRACT FROM AUTHOR]

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics. [ABSTRACT FROM AUTHOR]

Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of β-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate β-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent's molecular structure and the corresponding kinetics of β-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent's ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote β-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation, Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemoeoin formation but are also essential for the survival of the parasite by functioitieg as a kinetically competent and site specific mediator for heme detoxification. [ABSTRACT FROM AUTHOR]

The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.

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. Research on Cryptosporidium parasites, a major cause of acute infant diarrhea, is hampered by the short shelf life of oocysts, thus requiring routine propagation in laboratory animals. Here, the authors report a method to cryopreserve viable and infectious Cryptosporidium oocysts by ultra-fast cooling. [ABSTRACT FROM AUTHOR]

Cryopreservation is of significance in areas including tissue engineering, regenerative medicine, and organ transplantation. We investigated endothelial cell attachment and membrane integrity in a microvasculature model at high subzero temperatures in the presence of extracellular ice. The results show that in the presence of heterogeneous extracellular ice formation induced by ice nucleating bacteria, endothelial cells showed improved attachment at temperature minimums of -6 °C. However, as temperatures decreased below -6 °C, endothelial cells required additional cryoprotectants. The glucose analog, 3- O -methyl-D-glucose (3-OMG), rescued cell attachment optimally at 100 mM (cells/lane was 34, as compared to 36 for controls), while 2% and 5% polyethylene glycol (PEG) were equally effective at -10 °C (88% and 86.4% intact membranes). Finally, endothelialized microchannels were stored for 72 h at -10 °C in a preservation solution consisting of the University of Wisconsin (UW) solution, Snomax, 3-OMG, PEG, glycerol, and trehalose, whereby cell attachment was not significantly different from unfrozen controls, although membrane integrity was compromised. These findings enrich our knowledge about the direct impact of extracellular ice on endothelial cells. Specifically, we show that, by controlling the ice nucleation temperature and uniformity, we can preserve cell attachment and membrane integrity. Further, we demonstrate the strength of leveraging endothelialized microchannels to fuel discoveries in cryopreservation of thick tissues and solid organs., Competing Interests: Notes The authors declare the following competing financial interest(s): S.N.T., M.T., S.L.S., K.U., and L.W. are inventors on several provisional patents on the topic of cryopreservation of cells, tissues, and organs, including high subzero preservation. Further, K.U. has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. The interests of all researchers are managed by the MGH and Partners HealthCare in accordance with their conflict of interest policies.

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.

The deterioration of whole blood ex vivo represents a logistical hurdle in clinical and research settings. Here, a cocktail preservative is described that stabilizes leukocyte viability and erythrocyte morphology in whole blood under ambient storage. Neutrophil biostabilization was explored using a sophisticated microfluidic assay to examine the effectiveness of caspase inhibition to stabilize purified neutrophils. Following 72 h ambient storage, neutrophils remained fully functional to migrate towards chemical cues and maintained their ability to undergo NETosis after stimulation. Furthermore, stored neutrophils exhibited improved CD45 biomarker retention and reduced apoptosis and mortality compared to untreated controls. To stabilize erythrocyte morphology, a preservative solution was formulated using Taguchi methods of experimental design, and combined with the caspase inhibitor to form a whole blood cocktail solution, CS WB . CS WB was evaluated in blood from healthy donors and from women with metastatic breast cancer stored under ambient conditions for 72 h. CS WB -treated samples showed a significant improvement in erythrocyte morphology compared to untreated controls. Leukocytes in CS WB -treated blood exhibited significantly higher viability and CD45 biomarker retention compared to untreated controls. This 72 h shelf life under ambient conditions represents an opportunity to transport isolates or simply ease experimental timelines where blood degradation is problematic.

The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of β-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of β-hematin formation. The resulting assay exhibited a favorable Z' of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 μM, resulting in the identification of 161 previously unreported β-hematin inhibitors. Of these, 48 also exhibited ≥ 90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 μM. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

The growing drug resistance of Plasmodia spp. to current antimalarial agents in the quinine and artemisinin families further asserts the need for novel drug classes to combat malaria infection. One approach to the discovery of new antimalarials is the screening of natural product extracts for activity against the formation of hemozoin, a biomineral essential to parasite survival. By mimicking the in vivo lipid-water interface at which native hemozoin is found, hemozoin can be synthesized outside the parasite. In this work, a variety of lipophilic mediators was used to determine the optimal platform for in vitro hemozoin formation and then tested for efficacy in preliminary screens containing crude natural product extracts. The complete optimization and validation of a NP-40 detergent-mediated assay provide a screening template with an expedited 4-hour incubation time and identical IC50 values to those measured from the parasite's native lipid component.