DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

A microfluidic transistor for automatic control of liquids.

Microfluidics have enabled notable advances in molecular biology1,2, synthetic chemistry3,4, diagnostics5,6 and tissue engineering7. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity and scalability of electronic circuits8-10. Just as the electronic transistor enabled unprecedented advances in the automatic control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the automatic control of reagents, droplets and single cells on a microfluidic chip. Previous works on creating a microfluidic analogue to the electronic transistor11-13 did not replicate the transistor's saturation behaviour, and could not achieve proportional amplification14, which is fundamental to modern circuit design15. Here we exploit the fluidic phenomenon of flow limitation16 to develop a microfluidic element capable of proportional amplification with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. We then use this microfluidic transistor to directly translate fundamental electronic circuits into the fluidic domain, including the amplifier, regulator, level shifter, logic gate and latch. We also combine these building blocks to create more complex fluidic controllers, such as timers and clocks. Finally, we demonstrate a particle dispenser circuit that senses single suspended particles, performs signal processing and accordingly controls the movement of each particle in a deterministic fashion without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic-transistor-based circuits enable fluidic automatic controllers to manipulate liquids and single suspended particles for lab-on-a-chip platforms.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

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.

Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification.

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.

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Cryogenic enrichment of Plasmodium falciparum gametocytes from spiked whole blood.

Each individual cell type typically requires a unique set of conditions for optimal cryopreservation outcome, which relates to its specific response to cryoprotective agent (CPA) toxicity, osmotic behavior and sensitivity to ice crystallization. Cryopreservation of heterogenous cell populations is therefore exceedingly difficult as it requires separate and often conflicting conditions for each cell type. Conversely, these contrasting conditions could be utilized to favor cryogenic preference of a single cell population within a heterogenous sample, leading to its enrichment by elimination of remaining cells. To establish proof-of-concept for this overall approach, a protocol was developed for the cryogenic enrichment of Plasmodium falciparum gametocytes from whole blood. To accomplish this goal, we evaluated the effects of CPAs and cooling conditions during cryopreservation of whole blood samples spiked with P. falciparum gametocytes. We identified that cooling to -80 °C at a rate of -1 °C/min in the presence of 11 % glycerol selectively favors recovery of gametocytes. This protocol eliminates 95.3 ± 1.7 % of total blood cells and recovers 43.2 ± 6.5 % of parasites, leading to a 19-fold enrichment as assessed by microscopic examination of blood smears. This protocol is tunable, where gametocyte enrichment 900-fold may be feasible, however there is an apparent tradeoff in overall parasite recovery. Although translation of this protocol for point-of-care testing for malaria presents many challenges, the overall approach of cryogenic purification may prove useful for alternative diagnostic applications.

Polyethylene glycol and caspase inhibitor emricasan alleviate cold injury in primary rat hepatocytes.

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

Modeling the novel SERD elacestrant in cultured fulvestrant-refractory HR-positive breast circulating tumor cells.

PURPOSE: Metastatic hormone receptor-positive (HR+) breast cancer initially responds to serial courses of endocrine therapy, but ultimately becomes refractory. Elacestrant, a new generation FDA-approved oral selective estrogen receptor degrader (SERD) and antagonist, has demonstrated efficacy in a subset of women with advanced HR+breast cancer, but there are few patient-derived models to characterize its effect in advanced cancers with diverse treatment histories and acquired mutations. METHODS: We analyzed clinical outcomes with elacestrant, compared with endocrine therapy, among women who had previously been treated with a fulvestrant-containing regimen from the recent phase 3 EMERALD Study. We further modeled sensitivity to elacestrant, compared with the currently approved SERD, fulvestrant in patient-derived xenograft (PDX) models and cultured circulating tumor cells (CTCs). RESULTS: Analysis of the subset of breast cancer patients enrolled in the EMERALD study who had previously received a fulvestrant-containing regimen indicates that they had better progression-free survival with elacestrant than with standard-of-care endocrine therapy, a finding that was independent estrogen receptor (ESR1) gene mutations. We modeled elacestrant responsiveness using patient-derived xenograft (PDX) models and in ex vivo cultured CTCs derived from patients with HR+breast cancer extensively treated with multiple endocrine therapies, including fulvestrant. Both CTCs and PDX models are refractory to fulvestrant but sensitive to elacestrant, independent of mutations in ESR1 and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) genes. CONCLUSION: Elacestrant retains efficacy in breast cancer cells that have acquired resistance to currently available ER targeting therapies. Elacestrant may be an option for patients with HR+/HER2- breast cancer whose disease progressed on fulvestrant in the metastatic setting. TRANSLATIONAL RELEVANCE: Serial endocrine therapy is the mainstay of management for metastatic HR+breast cancer, but acquisition of drug resistance highlights the need for better therapies. Elacestrant is a recently FDA-approved novel oral selective estrogen receptor degrader (SERD), with demonstrated efficacy in the EMERALD phase 3 clinical trial of refractory HR+breast cancer. Subgroup analysis of the EMERALD clinical trial identifies clinical benefit with elacestrant in patients who had received prior fulvestrant independent of the mutational status of the ESR1 gene, supporting its potential utility in treating refractory HR+breast cancer. Here, we use pre-clinical models, including ex vivo cultures of circulating tumor cells and patient-derived xenografts, to demonstrate the efficacy of elacestrant in breast cancer cells with acquired resistance to fulvestrant.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

Point-of-care semi-quantitative test for adherence to tenofovir alafenamide or tenofovir disoproxil fumarate.

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.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

COX-2 mediates tumor-stromal prolactin signaling to initiate tumorigenesis.

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Evaluation of endocrine resistance using ESR1 genotyping of circulating tumor cells and plasma DNA.

PURPOSE: Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance. PATIENTS AND METHODS: In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness. RESULTS: High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies. CONCLUSION: Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.

Slow-delivery and distributed exchange of cryoprotective agents with hydrogel beads.

Intracellular loading of cryoprotective agents (CPAs) into target cells is a critical step for cryopreservation. However, biological membranes are usually much less permeable to CPAs than to water, resulting in high osmotic pressures and osmotic damage during the CPA loading and unloading phases of cryopreservation. Here, we show that calcium alginate hydrogel beads several millimeters in diamater containing CPAs can be admixed with a cell suspension to spontaneously release CPAs in a gradual and distributed manner. We demonstrate that beads containing cell media enable the gradual removal of CPA from Jurkat cells equilibrated in a typical cryopreservation solution of 15% glycerol, protecting the cells from hypotonic damage. We show that the dynamics of CPA exchange are accurately described by a numerical model of free diffusion within the gel. This approach may enable semiautomated and closed methods of gradual CPA exchange from large volume cell suspensions.

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.

Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos.

Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.