Single Cell Transcriptome Conservation in Cryopreserved Cells and Tissues
Ontology highlight
ABSTRACT: Sample preservation method that maintains transcripts in viable single cells and so allows to disconnect time and place of sampling from subsequent processing steps.
Project description:The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.
Project description:Archaeological bone material is increasingly being sampled for various molecular analyses. Minimally invasive sampling protocols should therefore be utilised whenever possible. We compare the proteomic output of 10 Early Neolithic Bos sp. humeri, from two different preservation contexts, of seven sampling protocols, namely retrieving bone chips (with and without demineralisation), acid etching (FASP and chemical neutralisation), eraser, membrane box, and polishing film. We note that minimally invasive sampling methods can extract a substantial amount of protein groups and provide sufficient taxonomic identifications if preservation conditions are favorable. However, if preservation conditions are poorer more invasive sampling techniques should be considered.
Project description:Ex vivo studies with first trimester placental tissues are crucial for understanding human placental development, and culturing placental villi in floating conditions is vital to mimic the natural setting. Moreover, being able to cryopreserve these scarce materials for ex vivo studies would open unprecedented avenues, as they are very limited to research and the need to culture freshly adds further hurdles while limiting efficient use. Here we showed that hanging drop method is a simple yet effective approach to culture placental floating villi. We also revealed that these functional units of the early-stage human placenta can be cryopreserved for culturing, and that frozen-thawed villi can regenerate the syncytial layer following denudation. We further illustrated the utility of frozen-thawed tissues to study syncytialization, while validating the importance of HtrA4 in the process which was shown previously in cell models. These represent significant new knowledge to the field of placental biology.
Project description:Cell-laden microgels have been used as tissue building blocks to create three-dimensional (3D) tissues and organs. However, traditional assembly methods can not be used to fabricate functional tissue constructs with biomechanical and structural complexity. In this study, we present directed assembly of cell-laden dual-crosslinkable alginate microgels comprised of oxidized and methacrylated alginate (OMA). Cell-laden OMA microgels can be directly assembled into well-defined 3D shapes and structures under low-level ultraviolet light. Stem cell-laden OMA microgels can be successfully cryopreserved for long-term storage and on-demand applications, and the recovered encapsulated cells maintained equivalent viability and functionality to the freshly processed stem cells. Finally, we have successfully demonstrated that cell-laden microgels can be assembled into complicated 3D tissue structures via freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. This highly innovative bottom-up strategy using FRESH 3D bioprinting of cell-laden OMA microgels, which are cryopreservable, provides a powerful and highly scalable tool for fabrication of customized and biomimetic 3D tissue constructs.
Project description:The cryopreservation of cancer tissues to generate frozen libraries is a common practice used worldwide for storing patient samples for later applications. However, frozen samples stored by existing methods cannot be used for initiating living cell cultures, such as patient-derived tumor organoids (PDOs), which offer great potential for personalized treatment. To overcome this challenge, we developed a novel procedure for culturing PDOs using frozen live tumor tissues. We show that tumor specimens stored using this technique maintain their viability and can be successfully used to generate organoids even after long-term freezing, with an impressive success rate of 95.2 %. Importantly, we found that the structural features, tumor marker expression, and drug responses of organoids derived from frozen tissues are similar to those derived from fresh tissues. Moreover, organoids derived from frozen tissues can be routinely passaged and frozen, making them ideal for high-throughput drug screening at any time. Notably, cryopreserved tumor tissues can also be utilized in air-liquid interface (ALI) culture. This method allows for preserving the original tumor microenvironment, making it an invaluable resource for conducting tests on antitumor drug responses, including immune checkpoint inhibitors (ICIs). This innovation has the potential to enable the identification of potentially effective drugs for patients and facilitate the development of novel therapeutic drugs. Thus, we have established protocols for the long-term cryopreservation of cancer tissues to maintain their viability and microenvironment, which are useful for personalized therapy.
Project description:Developing precise species-specific in vitro models that closely resemble in vivo intestinal tissues is essential for advancing our understanding of gastrointestinal physiology and associated diseases. This is especially crucial in examining host-pathogen interactions, particularly in bovines, a known reservoir for microbes and pathogens posing substantial public health threats. This research investigated the viability of producing bovine rectal organoids from cryopreserved tissues. We compared two cryopreservation methods with a traditional technique using fresh tissues, evaluating their effectiveness through growth rates, long-term viability, and comprehensive structural, cellular, and genetic analyses. These assessments utilized phase-contrast imaging, immunofluorescence imaging, and RT-qPCR assays. Additionally, the study developed a sophisticated method for forming a functional epithelial barrier from organoid-derived bovine rectal monolayers, incorporating a wide range of epithelial cells. This methodology employed transepithelial electrical resistance (TEER), parallel artificial membrane permeability assay (Papp), confocal microscopy, and advanced imaging techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings decisively show that bovine rectal organoids can be effectively generated from cryopreserved biopsy tissues. Moreover, we formulated a robust and optimized protocol for creating functional rectal monolayers from these organoids. This significant progress is particularly relevant given the susceptibility of the bovine rectum to various enteric pathogens of public health concern, marking a vital step forward in veterinary and biomedical research. The creation of accurate species specific in vitro models that faithfully mimic in vivo intestinal tissues is critical for enhancing our understanding of gut physiology and related pathologies. This is particularly relevant in studying the interactions between hosts and microbes or pathogens with significant public health risks where bovine can be the major reservoir.
Project description:Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates.We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence.There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring.
Project description:Background Insulinoma is the most common pancreatic neuroendocrine tumour in dogs and humans. The understanding of driving factors and critical survival genes in insulinomas is limited and overall survival is poor for canine and human malignant insulinoma. This study aimed to use single-cell RNA-sequencing to conduct a multispecies analysis of insulinoma cell lines to understand their single-cell transcriptomic landscape. Secondly, the impact of freeze-thawing on the pancreatic beta single-cell transcriptome was investigated, to determine whether cryoarchiving of primary insulinoma samples may be feasible in future studies. Methods Single-cell transcriptomic analysis was performed using fresh and cryopreserved multispecies insulinoma cell lines (canINS, CM, INS-1 and MIN6). R and Seurat were used to perform cell clustering and specific cluster marker genes were identified by the FindMarkers function. Metascape was used to identify statistically enriched pathways for specific cell clusters. Differentially expressed genes between fresh and cryopreserved single-cell transcriptome profiles, were defined as genes with a log2 fold change >0.25 and a Bonferroni-adjusted P<0.05, based on the Wilcoxon rank sum test. Results Based on the specific cell line single-cell transcriptome profiles, five or six cell clusters were constructed per cell line. All cell lines expressed neuroendocrine markers and additionally INS-1 and MIN6 displayed a gene signature indicative of mature/functional pancreatic islet/beta-cells. DEPTOR, BICC1, GHR, CCNB2, CENPA, LMO4, VANGL1, and L1CAM were identified as cross-species conserved insulinoma cluster marker genes. Little effect was found of cryopreservation and thawing on overall gene expression at the single-cell level in insulinoma cell lines: only 6 and 29 genes had a log2 fold change > 1 in cryopreserved versus fresh canINS and CM, respectively. Conclusions canINS, CM, INS-1 and MIN6 are all principally relevant as insulinoma models and the demonstrated differences in their single-cell transcriptomic profiles could aid researchers in selecting the appropriate cell lines for specific study objectives. Cross-species conserved insulinoma cluster marker genes were identified that harbour oncogenes and their involvement in insulinoma tumourigenesis should be investigated in future studies. The good comparability between cryopreserved and fresh insulinoma cells allows for inclusion of cryopreserved insulinoma patient samples in future studies, which allows for reduced assay-based variability.
Project description:Ballan wrasse (Labrus bergylta) is used as a cleaner fish in Norwegian aquaculture to control sea lice. Thus, knowledge on the digestive physiology and nutrient requirements, as well as the ontogeny of the immune system is important. In this study, two different diets were tested; diet 1 was used as control diet consisting of artemia and rotifers cultivated and enriched in the in-house facility at IMR, Austevoll, Bergen (Norway). Diet 2 consists in plankton which has successfully been used before in cod larvae generating more robust individuals. Sampling was done according to the standard length of the larvae rather than age (dph). Sampling point 0 (BW0) (4 mm), sampling point 2 (BW2) (4,5 mm), sampling point 3 (BW3) (5,7 to 6 mm), sampling 4 (BW4) (7 to 7,5 mm), sampling point 5 (BW5) (10 to 10,5 mm), sampling point 6 (BW6) (16 to 16,5 mm), and sampling point 7 (BW7) (25 to 30 mm). Ontogeny of lymphoid organs and mucosal associated lymphoid tissues in ballan wrasse and the effect of different diets were investigated.