Genetic Engineering & Biotechnology News

OCT15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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18 | OCTOBER 15, 2017 | | Genetic Engineering & Biotechnology News Profiling Large and Complex Cell Populations The ability to profile a large number of cells is becoming increasingly important for rare-cell detection and for the comprehen- sive classification of biological systems, ac- cording to Grace Zheng, Ph.D., senior scien- tist, 10x Genomics. "Because of the limited throughput with most scRNA-seq methods, our company devised a comprehensive, scal- able solution to quickly characterize and profile the transcriptome of hundreds to mil- lions of cells." Single cells, reverse transcription reagents, "gel beads" containing barcoded oligonucle- otides, and oil are combined on a micro- fluidic chip to form reaction vesicles called "Gel Beads in Emulsion," or GEMs, which are formed in parallel within the microflu- idic channels of the chip, allowing the user to process hundreds to tens of thousands of single cells in a single seven-minute Chromi- um ™ Controller run. Dr. Zheng reports, "Cells are loaded at a limiting dilution to maximize the number of GEMs containing a single cell, while en- suring a low doublet rate and maintaining a high cell recovery rate of up to ~65%. The speed, reproducibility, and high cell capture rates of the Chromium Solution facilitates profiling and discovery of precious and rare- cell populations." A challenge associated with the accumu- lation of such massive scRNA-seq datasets is their efficient processing. According to Dr. Zheng, such computational analysis has not been scalable beyond tens of thousands of cells, and has been a limiting factor to en- abling large-scale cell atlas efforts. "Our computational pipelines and visualization tools can now efficiently process million-cell datasets, and can be easily used by a non- computational biologist. These are the types of tools needed to enable large-scale cell at- las studies." As an example of the power of the tech- nology, the company profiled 1.3 million neurons from two embryonic murine brains. More than 100 single-cell libraries were completed in two days. Major neuronal and nonneuronal cell types were detected from different cortex layers, the hippocampus, and subventricular zones. Dr. Zheng notes, "We readily detected diverse and rare inter- neurons without the need to enrich by flow- cytometry sorting." Another important single-cell applica- tion is the characterization of paired T-cell receptor alpha and beta chains in tens of thousands of T cells. This application allows comprehensive immune repertoire profiling, for determining which functional subsets of T cells have undergone clonal expansion. Dr. Zheng believes this will be especially valuable in areas of infectious diseases and immuno-oncology. Single-Cell Nuclei Transcriptomes Cells of the central nervous system are particularly difficult to isolate as intact whole cells. Neurons are highly interconnected with axons and dendrites. To separate them by physical means often causes considerable damage. Further, proteolytic degradation of surface proteins to dissociate whole cells from tissue may stress the cell sufficiently enough to alter gene expression. Roger Lasken, Ph.D., director of single- cell genomics, J. Craig Venter Institute, and colleagues have developed an alternative approach that focuses on the nucleus and its stash of mRNA while eliminating tradi- tional harsh treatment utilizing whole cells. He reports, "RNA is made in the nucleus, processed, spliced, and exported to ribo- somes in the cytoplasm. We've made ex- tensive comparisons of nuclear and cellular transcriptomes and have demonstrated that nuclei can substitute for whole cells in most RNA-seq applications. For most genes, single-nuclei RNA-seq (snRNA-seq) pro- vides expression signatures that are very similar to those obtained from whole-cell controls." As an example, Dr. Lasken describes a study of memory carried out by collabora- tors using mouse hippocampus tissue. "They characterized the transcriptome from mouse neurons using snRNA-seq. The mice were initially raised in a very plain home cage, but then placed into a cage with a more enriched Single-Cell Genomics Continued from page 14 OMICS Cell cultures are a central part of life science research programs, and culture techniques are continuously evolving. Properly optimiz- ing workflows and utilizing the latest best practices are the keys to obtaining consistent results and advancing research. Cell Authentication Estimates suggest that as many as half of all cell lines are misidentified. Proper and regular authentication is the key to avoiding this pitfall. Planning future cell-based experiments: • Choose the cell line carefully, ensuring that it accurately reflects the biology of inter- est, can withstand the planned experimen- tal manipulations, and is able to generate reproducible results. • Obtain your cells from a trusted source with proper documentation of the cells' identify and quality controls. • Don't share or borrow cell lines informally. Insist on authentication and formal docu - mentation. Developing a new cell line: • Follow the ethical and legal requirements, including patient consent, pertaining to the acquisition of tissue for cell lines. • Keep detailed records about the line's origin, including: clinical information about the donor or patient, if applicable; cell type, source, lot numbers of all reagents used; images of the culture; and methods of genetic modification or reprogramming. • Preserve tissue samples for subsequent confirmation of origin. In the case of cells derived from a disease, also preserve a tissue sample for histopathology as well as a sample of normal tissue for comparison. Cells currently in use: • Check the International Cell Line Authen - tication Committee (ICLAC) registry of misidentified cell lines. • Validate cells using DNA genotyping, not phenotyping, at least once per year, ideally every six months. Comparison of short tandem repeats (STRs) or single nucleotide polymorphisms (SNPs) with available databases are the two most cost effective and acceptable genetic methods for intra-species comparison. Culture Preparation A successful culture requires proper sterile technique and the use of other best prac- tices. Best cell culture practices: • Proper training is essential. Generate standard operating procedures (SOPs) for all culture techniques and make sure everyone in the lab knows how to properly implement them. • Purchase high-quality reagents and sup - plies with documented quality control from reputable sources. Record lot numbers for later troubleshooting. • Perform regular sterilization, calibration, and servicing of all equipment, including pipettes, culture hoods, incubators, micro - scopes, and autoclaves. • Quarantine new cell lines until after authentication to safeguard against con- tamination. Sterile filtration: • The membrane choice depends on sample characteristics, composition, and volume. Both the solute and the dissolved molecules or particles of the sample being filtered must be chemically compatible with the filter membrane. • Sample agitation and adjusting the flow rate per unit area of filter surface can improve throughput and help prevent particulates from aggregating on the membrane surface. • Clarification and prefiltration is best ac - complished with a 0.45 µm pore size. • For fast filtration of most media and buff- ers use a polyethersulfone (PES) mem- brane with a 0.45 µm pore size. • For sterilizing media containing serum and other protein use a PES membrane with a 0.22 µm pore size. • For removal of mycoplasma use a PES membrane with a 0.10 µm pore size. • For very low protein binding and sterilizing solutions with high value biomolecules use a polyvinylidene difluoride (PVDF) mem - brane with a 0.2 µm pore size. Cell freezing: • Label stock vials completely and unam- biguously. • Freeze stock samples at the lowest pas- sage number possible. Cultivating Consistency: Tips and Tricks for Successful Cell Cultures MilliporeSigma and the Vibrant M are trademarks and SNAP i.d. and Muse are are registered trademarks of Merck KGaA, Darmstadt, Germany. 2017 - 06947 09/2017 Copyright © 2017 EMD Millipore Corporation. All Rights Reserved. ADVERTISEMENT FEATURE The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.

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