Instrumentation In Medical Diagnostic, Laboratory And Blood Banking BIO62004 PDF

BIO62004 Instrumentation in Medical Diagnostic, Laboratory and Blood Banking Topic 2: Maintenance of Mammalian Cell Culture 2.2 (L5) : Aseptic Techniques in maintenance of cell culture in vitro ...

BIO62004 Instrumentation in Medical Diagnostic, Laboratory and Blood Banking Topic 2: Maintenance of Mammalian Cell Culture 2.2 (L5) : Aseptic Techniques in maintenance of cell culture in vitro  Learning objectives Students should be able to: 1. Discuss the principles of animal cell culture 2. Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media) 3. Demonstrate animal cell culture techniques 4. Relate animal cell culture to specialized applications 5. Disuses the principles of cryopreservation and cell banking  Protocols for animal cell culture Maintaining cell cultures Establishment and maintenance of animal cell cultures require standardized approaches for media preparation, feeding, and passaging (or subculturing) of the cells. Cultures should be examined regularly to check for signs of contamination and to determine if the culture needs feeding or passaging.  Freshney, 2005 Increase in cell number on a log scale plotted against days from subculture. a) Defines the lag, log (exponential), and plateau phases, and when culture should be fed and subcultured after the indicated seeding time.  Protocols for animal cell culture Passaging cells Many adherent cell cultures will cease proliferating once they become confluent (completely cover the surface of cell culture vessel) – trypsinization Suspension cells will exhaust their culture medium very quickly once the cell density becomes too high  Protocols for animal cell culture Trypsinization is a technique that uses the proteolytic enzyme trypsin to detach adherent cells from the surface of a cell culture vessel. http://www.sciencedirect.com/science/article/pii/S014296120 http://www.biochrom.de/en/products/enzymes/trypsin/ 5003832  Dissociating adherent cells Procedure Dissociation Agent Applications Shake-off Gentle shaking or rocking Loosely adherent cells, mitotic cells of culture vessel, or vigorous pipetting Scraping Cell scraper Cell lines sensitive to proteases; may damage some cells Enzymatic Trypsin Strongly adherent cells dissociation Trypsin + collagenase High density cultures, cultures that have formed multiple layers, especially fibroblasts Dispase Detaching epidermal cells as confluent, intact sheets from the surface of culture dishes without dissociating the cells  Subculture Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium Criteria for subculture 1. Density of culture 2. Exhaustion of medium 3. Time since last subculture 4. Requirements for other procedures http://www.biologydiscussion.com/cell/types-of-subculture-of-cell-2-types-with-diagram/10514  Cell banking For some cell cultures, especially valuable ones, it is common practice to maintain a frozen cell bank The storing process is usually done using small cryovials stored in liquid nitrogen using a technique called cryopreservation. http://cellbanking.net/Cell_Banking_FAQ.htm  Cryogenic storage Cell lines in continuous culture are likely to suffer from genetic instability as their passage number increases Essential to prepare working stocks of the cells and preserve them in cryogenic storage http://www.selectsurrogacyindia.com/ivf/cryopreserv ation_india.html  Cryopreservation Main advantages of cryopreservation – To reduce risk of microbial contamination – To reduce risk of cross contamination with other cell lines – To reduce cost – To maintain cell stocks at early passages – To reduce genetic drift and morphological change: continuous subculture of cell line may lead to genetic instability, dedifferentiation, loss of original phenotype and transformation into cancerous cell lines  Cryopreservation Basic principle Slow freeze and quick thaw Cells should be cooled at a rate of -1°C to -3°C per minute and thawed quickly by incubation at 37°C water bath for 3-5 minutes. Thermo Scientific™ Mr. Frosty™ Freezing Container  Cryopreservation Addition of cryoprotectant to the cells depresses the temperature at which intracellular ice is formed and allows cooling rates to be reduced for more efficient water loss  Cryopreservation When to cryopreserve cells? – Cultures are healthy with viability of >90% and have no microbial contamination – Cultures should be in the log phase of growth. Why?  Cryopreservation Freeze medium Either the growth medium supplemented with 30% (v/v) serum and 10% (v/v) cryoprotectant, or whole serum and 10% (v/v) cryoprotectant The choice of cryoprotectant will be determined by the cell type, but for the majority of cell lines dimethyl sulfoxide (DMSO) or glycerol can be used An alternative is polyvinyl pyrrolidone, a high-molecular weight polymer  Cryopreservation Success of the freezing process depends on: Proper handling and gentle harvesting of the cultures Use of appropriate cryoprotective agents A controlled rate of freezing Storage at cryogenic temperatures  Cell banks Several cell banks exist for the secure storage and distribution of validated cell lines Initial seed stock can be obtained from a reputable cell bank where the necessary characterization and quality control have been done Researchers can also submit their cultures to a cell bank, e.g. RIKEN, ATCC, and ECACC  Cell banking Two-tiered frozen cell bank: a master cell bank and a working cell bank. Working cell bank comprises cells from one of the master bank samples Master cell bank is used only when absolutely necessary.  Cell characterization Requirements of cell characterization: – Confirmation of the species of origin – Correlation with tissue of origin Identification of the lineage to which the cell belongs Position of the cells within that lineage (stem cell, precursor, differentiation status) – Confirmation of the absence of cross-contamination – Indication of whether the cell line is prone to genetic instability, transformation and phenotypic variation  Characterization Most laboratories will have, as an integral part of the research program, procedures in place for the characterization of new cultures. In case where cells are misidentified or cross- contaminated, DNA profiling can be used to confirm cell line identity  Cell characterization There are various assays for determining cell line identity and authenticating the cell lines used for production and drug discovery research – Isoenzyme analysis – Karyotyping – DNA barcoding – DNA analysis (DNA fingerprinting)  Cell characterization (Isoenzyme Analysis) Isoenzyme analysis assays are based on the presence of enzymes with similar or identical substrate specificity, but different molecular structures. The assay is based on the patterns of migration of isoenzymes present in cell lysates following electrophoresis using agarose gels. Isozyme patterns obtained are species-specific and therefore are used as quality control and cell authentication procedures to confirm species of origin of material. Specific banding patterns are analyzed for target enzymes including but not limited to: – Lactate dehydrogenase – Purine nucleoside phosphorylase – Glucose-6-phosphate dehydrogenase – Malate dehydrogenase  Cell characterization Isozyme analysis  Cell characterization (Karyotyping) Banded chromosome analysis Standard karyotyping analysis includes: – Metaphase nuclei harvest and chromosome count (to give the modal diploid chromosome number, frequency distribution and polyploidy levels) – Chromosomal abnormality quantification – Metaphases G-banding  Cell characterization (DNA barcoding) – Uses PCR amplification and DNA sequencing technology to visualize DNA polymorphism – Characterization of the mitochondrial cytochrome c oxidase I (COI) gene – Widely used to identify the species of cell cultures DNA barcoding  Cell characterization DNA Analysis (DNA fingerprinting, genotyping) DNA fingerprinting - DNA analysis is performed by interrogating hypervariable regions in the genomic DNA Minisatellites (repeat units of 10–100 bp) and microsatellites (repeat units of usually 1–4 bp) have proven particularly useful for cell identification  Cell characterization Comprehensive DNA analysis techniques includes: – RFLP (Restriction Fragment Length Polymorphism) identification of genomic DNA – Simple sequence repeats – AFLP (Amplified Fragment Length Polymorphism) detection – RAPD (Rapid amplification of polymorphic DNA) analysis – SNP (Single Nucleotide Polymorphism)  Cell characterization Other methods of cell characterization – Lineage or tissue markers: cell surface antigens, intermediate filament proteins, and differentiated products – Morphology/microscopy: morphological change – Enzyme activity: specialized functions in vivo are expressed in the activity of specific enzymes, e.g. urea cycle enzymes in the liver Many enzyme activities are lost in vitro and are no longer available as markers of tissue specificity  Contamination The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results. The simulated images below show an adherent 293 cell culture contaminated with E. coli. http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/biological- contamination/bacterial-contamination.html  Contamination Potential contamination routes: – Poor handling – Contaminated media, reagents, and equipment (e.g., pipets) – Microorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on the skin of the worker and in cultures coming from other laboratories.  Contamination Contamination of cell cultures is easily the most common problem encountered in cell culture laboratories – Cell culture contaminants can be divided into two main categories, chemical contaminants such as impurities in media, sera, and water, endotoxins, plasticizers, and detergents, and biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines. While it is impossible to eliminate contamination entirely, it is possible to reduce its frequency and seriousness by gaining a thorough understanding of their sources and by following good aseptic technique. This section provides an overview of major types of biological contamination. Types of microbial contamination – Bacteria, yeasts, molds, and mycoplasma  Bacterial contamination Bacteria are a large and ubiquitous group of unicellular microorganisms. Because of their ubiquity, size, and fast growth rates, bacteria, along with yeasts and molds, are the most commonly encountered biological contaminants in cell culture.  Bacterial contamination Bacterial contamination is easily detected by visual inspection of the culture within a few days of it becoming infected; infected cultures usually appear cloudy (i.e., turbid), sometimes with a thin film on the surface. Sudden drop in the pH of the culture medium is also frequently encountered. Under a low power microscope, the bacteria appear as tiny, moving granules between the cells, and observation under a high-power microscope can resolve the shapes of individual bacteria.  Bacterial contamination The simulated images above show an adherent 293 cell culture contaminated with E. coli. This phase contrast images of adherent 293 cells contaminated with E. coli. The spaces between the adherent cells show tiny, shimmering granules under low power microscopy, but the individual bacteria are not easily distinguishable (panel A). Further magnification of the area enclosed by the black square resolves the individual E. coli cells, which are typically rod-shaped and are about 2 μm long and 0.5 μm in diameter  Yeasts contamination Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in size from a few micrometers (typically) up to 40 micrometers (rarely). Cultures contaminated with yeasts become turbid, especially if the contamination is in an advanced stage. There is very little change in the pH of the culture contaminated by yeasts until the contamination becomes heavy Under microscopy, yeast appear as individual ovoid or spherical particles, that may bud off smaller particles.  Yeast contamination Phase contrast images of 293 cells in adherent culture that is contaminated with yeast. The contaminating yeast cells appear as ovoid particles, budding off smaller particles as they replicate  Fungi contamination Molds are eukaryotic microorganisms in the kingdom of Fungi that grow as multicellular filaments called hyphae. Similar to yeast, The pH of the culture remains stable in the initial stages of contamination, then rapidly increases as the culture become more heavily infected and becomes turbid.  Fungi contamination Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores. Spores of many mold species can survive extremely harsh and inhospitable environments in their dormant stage, only to become activated when they encounter suitable growth conditions.  Virus contamination Viruses are microscopic infectious agents that take over the host cells machinery to reproduce. Their extremely small size makes them very difficult to detect in culture, and to remove them from reagents used in cell culture laboratories. Because most viruses have very stringent requirements for their host, they usually do not adversely effect cell cultures from species other than their host.  Virus contamination However, using virally infected cell cultures can present a serious health hazard to the laboratory personnel, especially if human or primate cells are cultured in the laboratory. Viral infection of cell cultures can be detected by electron microscopy, immunostaining with a panel of antibodies, ELISA assays, or PCR with appropriate viral primers.  Mycoplasma contamination Mycoplasma are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. Mycoplasmal infections cannot be detected by naked eye; only noticed through signs of deterioration in the culture (omit) – Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection.  Mycoplasma contamination Some slow growing mycoplasma may persists in culture without causing cell death They can alter the behavior and metabolism of the host cells in the culture. – Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures.  Mycoplasma contamination The only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining (e.g., Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbiological assays.  Mycoplasma contamination Figure 2.4: Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels B and C). The cultures were tested using the MycoFluor™ Mycoplasma Detection Kit, following the kit protocols. In fixed cells, the MycoFluor™ reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that the culture is free from mycoplasma contamination (panel A). In fixed cells infected with mycoplasma, the MycoFluor™ reagent stains both the nuclei and the mycoplasma, but the intense relative fluorescence of the nuclei obscure the mycoplasma on or near the nuclei. However, the mycoplasma separated from the bright nuclei are readily visible (panel B). In live cells, the MycoFluor™ reagent does not have access to the nuclei, but readily stains the mycoplasma associated with the outside of cells (panel C). http://www.lifetechnologies.com/my/en/home/references/gibco-cell-culture-basics/biological-contamination/mycoplasma-contamination.html  Eradication of contamination The most reliable method of eliminating a microbial contamination is to – Discard the culture and the medium and reagents used with it, treating a culture may be unsuccessful or lead to the development of antibiotic resistant microorganisms  Cross contamination Cross-contamination of many cell lines with HeLa and other fast growing cell lines is a clearly-established problem with serious consequences. – it was realized that vigorous lines could contaminate and overgrow slower-growing cultures How to prevent cross contamination? Obtaining cell lines from reputable cell banks, periodically checking the characteristics of the cell lines, and practicing good aseptic technique are practices that will help you avoid cross-contamination.  Precautions to avoid cross contamination Do not handle more than one cell line at a time Do not share media or other reagents among different cell lines Do not share media or reagents with other people – or, if this is impractical, do not have culture vessels and medium bottles for more than one cell line open at one time, and never be tempted to use the same pipette or other device for different cell lines. Retain a tissue or blood sample from each donor and confirm the identity of each cell line by DNA profiling: – (a) when seed stocks are frozen – (b) before the cell line is used for experimental work or transplantation  Precautions to avoid cross contamination Keep a panel of photographs of each cell line, at low and high densities, above the microscope, and consult this regularly when examining cells during maintenance – This is particularly important if cells are handled over an extended period, and by more than one operator If continuous cell lines are in use in the laboratory, handle them after handling other, slower-growing, finite cell lines  Usage of antibiotics Antibiotics should not be used routinely in cell culture: why? – Continuous use encourages the development of antibiotic resistant strains – Allows low-level contamination to persist, which can develop into full-scale contamination once the antibiotic is removed from media – May hide mycoplasma infections and other cryptic contaminants. – Further, some antibiotics might cross react with the cells and interfere with the cellular processes under investigation. Antibiotics should only be used as a last resort and only for short term applications, and they should be removed from the culture as soon as possible.  References: Alison M. R., Vig P., Russo F., Bigger B. W., Amofah E., Themis M., et al. (2004). Hepatic stem cells: from inside and outside the liver? Cell Prolif. 37, 1–21. Chow, A. Y. (2010) Cell Cycle Control by Oncogenes and Tumor Suppressors: Driving the Transformation of Normal Cells into Cancerous Cells. Nature Education 3(9):7 Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons. Freshney, R.I. (2010) Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition, Wiley-Blackwell Kopnin, B. P.Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biokhimiya 65, 2–27 (2000). Kondo T, Raff M. (2000). Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science. 289(5485), 1754-1757. Takahashi K, Yamanaka S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663-676.  After studying this lecture, you should understand able to: 1. Discuss the principles of animal cell culture 2. Demonstrate animal cell culture techniques 3. Relate animal cell culture to specialized applications 4. Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media) 5. Disuses the principles of cryopreservation and cell banking