Tracing blastomere fate choices of early embryos in single cell culture

Blastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in ≥50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro

Isolation of equine endothelial cells and life cell angiogenesis assay

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis

Label-free enrichment of adrenal cortical progenitor cells using inertial microfluidics.

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner

Isolation and primary cultures of human intrahepatic bile ductular epithelium

A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments. © 1988 Tissue Culture Association, Inc

Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).España, Junta de Andalucía grant P07-CVI-02792 and group BIO-284España, MINECO y Fondo Social Europeo grant BFU2013-41712-

Preparation of Neurospora crassa mitochondria

The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewher