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- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
The first atlas in many years giving researchers a good visual reference of the status of their cell lines. Given the increasing importance of well defined cellular models in particular in biomedical research this is a sorely needed resource for everyone performing cell culture.
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Seitenzahl: 340
Veröffentlichungsjahr: 2013
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Contents
Cover
Related Titles
Title Page
Copyright
Preface and Acknowledgments
Abbreviations
Chapter 1: Introduction
1.1 Introduction and Usage of This Book
1.2 General Remarks
Chapter 2: Basic Cell Culture Techniques
2.1 Safety Precautions for Frozen Cell Lines
2.2 Sterile Working
2.3 Handling Procedure for Cell Lines
2.4 Special Remarks on the Origin of the Cell Lines
2.5 Photographic Equipment
Chapter 3: List of Cell Lines and Human Primary Cells (in Alphabetical Order)
3.1 Human Cell Lines
3.2 Animal Cell Lines
3.3 Human Primary Cells
Chapter 4: Cell Lines and Human Primary Cells
4.1 Human Cell Lines
4.2 Animal Cell Lines
4.3 Human Primary Cells
Appendix A: Materials and Suppliers
Appendix B: Suppliers of Cell Culture Materials
Further Reading
Index
Related Titles
Freshney, R. I.
Culture of Animal Cells
A Manual of Basic Technique and Specialized Applications, 6th Edition
2010
ISBN: 978-0-470-52812-9
Freshney, R. I., Stacey, G. N., Auerbach, J. M.
Culture of Human Stem Cells
2007
ISBN: 978-0-470-05246-4
Cetrulo, C. L., Cetrulo, K., Cetrulo, C. L. (eds.)
Perinatal Stem Cells
2009
ISBN: 978-0-470-42084-3
Vunjak-Novakovic, G., Freshney, R. I. (eds.)
Culture of Cells for Tissue Engineering
2006
ISBN: 978-0-471-62935-1
Davey, M. R., Anthony, P.
Plant Cell Culture
Essential Methods
2010
ISBN: 978-0-470-68648-5
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Print ISBN: 978-3-527-32887-1
ePDF ISBN: 978-3-527-66993-6
ePub ISBN: 978-3-527-66994-3
mobi ISBN: 978-3-527-66995-0
oBook ISBN: 978-3-527-66992-9
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Preface and Acknowledgments
This comprehensive collection of photographs of various living cells and cell lines cultured in vitro represents the first of its kind.
Within the last decades the use of cells in culture has not only increased dramatically in basic research but also expanded into many industrial processes and techniques, for example, for the generation of antibodies and biopharmaceuticals.
In industrial processes, the cells used are tested thoroughly with the aid of many and diverse direct and indirect analytical methods. As such sophisticated and time consuming testing is not always possible in basic research laboratories, a fast first control check for their viability under the microscope would be done and no other control seemed to be necessary in the past and even in the present.
This cell culturing in T-flasks, in Petri-dishes or in multiwell-plates is a technique that can be deduced since more than 100 years without great improvements if you look just for the behavior of the seeded cells on the substratum and their image under the microscope: Either they are attached after one or two days (as normal cells derived from a body's tissue do) or they keep rounded up in suspension like blood cells do. Dying or dead cells do not attach to the substrate and they keep rounded up or even disintegrate into small-vesiculated membrane particles.
For many years the cell morphology was the main and nearly only characteristic for the viable cells in culture, taking advntage of the invention of the phase contrast microscopy in the 1930s. This kind of microscopy was almost the only technique for the observation of live cells in greater magnification and therefore indispensable for people who worked with cells in culture.
But even now, although modern analytic methods at the cell's molecular level are in use after the rapid developments within the last 30 years to look into cells, light microscopy is still the most important tool in the routine field for viewing cells in culture.
Working with live cells and cell lines and observing them as vital organisms still means using an inverted phase contrast microscope to control continuously not only the morphology but at the same time the proliferation of a cell under culture condition in the T-flask. Each cell type and each cell line has its own morphological features even though cells originating from the same tissue may differ from each other.
Although many photographs of cells and cell lines exist and various pictures from respective cell lines can be found, for example, in the World Wide Web, it may be a tedious and time consuming task to find them at the various websites and/or in numerous journals and other publications. In addition, the morphology of cultured cells varies from the onset of seeding until they become confluent and also from passage to passage. Density of cells causes striking changes of the morphology in vitro due to the availability of the substratum and their overgrowth. It is therefore very important to have a comparison of different densities of cultured cells in the flasks.
On the other hand, it must be emphasized that variations of the cell morphology during cultivation may derived from the use of different media, from the incubation conditions (seeding concentrations, CO2-concentrations, humidity, and temperature in the incubator, length of incubation time) and from the individual (!) treatment during passages and from laboratory to laboratory. Therefore, our pictures taken from the T-flaks at different times were made under certain and defined condition (media, temperature and CO2-concentration in the incubator, etc.) and these conditions are depicted within the text sides opposite to the pictures.
Our aim is to give a first impression of the individual cultivated cell line, but it must be emphasized again that our pictures of the cell morphology are derived from individual laboratory personnel. But nevertheless they may be representative for the respective cell line.
In our opinion, no 100% ideal picture of the respective cell exists. Our aim was to give an impression of an image of the cultured cells which comes closer to the truth than any other picture which may be found, for example, in the World Wide Web.
We want to introduce for the first time a comprehensive but limited number of living cell lines the photographs of which were taken during cultivation of the cells. This atlas may lead to a better control how these cultured cell lines may look alike under good cell culture practice (GCCP).
Our selection was certainly to some extent random. We could not introduce nearly all of the estimated 3500–4000 (?) cell lines listed in all scientific publications or in the catalogues of the cell banks. Our choice was to list the most used or most “popular” cell lines but certainly our choice may not find the consent of all people working with cell cultures. Proposals for introducing further cell lines are welcome.
Furthermore, it was not our aim to make “star pictures” for the “haute couture” of cells in culture, instead we made photographs under routine culture conditions with a “normal” microscopic equipment such as an inverted microscope equipped with a digital camera and a pdf-conversion program in the computer and/or printer. It was also not the aim to give pictures of contaminated or of sick cells in culture in all details. People, who had these kinds of problems may look further in the textbooks of cell and tissue culture.
Instead we recommend in the context of all cell culture practices to withdraw contaminated cell cultures immediately and not try to cure them with antibiotics.
In Chapter 2, the most basic cell culture techniques are described. For further reading we refer to very detailed and informative cell culture manuals such as “Culture of Animal cells” by R. Ian Freshney (6th ed. Wiley-Blackwell New York, 2010) or “Zell- und Gewebekultur” by Toni Lindl and G. Gstraunthaler (6th ed. Spektrum Verlag Heidelberg, 2008). Chapter 3 contains the list of all cell lines. Chapter 4 is divided into three subchapters, namely human cell lines originating from various tissues and animal cell lines originating from various animals and from various tissues. Also included are primary cells of human origin that are characterized by a finite life span. The photographs of the primary cells are courtesy of PromoCell GmbH, Heidelberg, Germany. We thank Dr. Hüttner for providing these highly informative photographs of these cells.
STR-analyses were performed using the cell lines of CLS Cell Lines Service GmbH in Eppelheim and are consistent with STR data published by ATCC (if available). All cell lines are listed alphabetically, and the search for one particular cell line should be an easy task. Each cell line comes with a short description and some basic information.
The authors would like to acknowledge Jessica Hirscher who has been busy with culturing the cell lines; Dagmar Lojewski for spending many hours to take the photographs and to arrange the best photographs at differing magnifications; Ute Fischer and Dott. Francesca Maggi Herbring for controlling the contamination status of the cell lines.
Eppelheim and Munich, April 2013
Rosemarie SteubingToni Lindl
Abbreviations
ACTHAdrenocorticotropic hormoneAMLAcute myeloid leukemiaANPAtrial natriuretic peptideAP-1Activator protein 1ArgArgeninATCCAmerican Type Culture CollectionATPaseAdenosintriphosphataseBBSBalanced salt solutionBCGBacille Calmette-GuérinbpBase pairBMP-6Bone morphogenetic protein°CDegree CelsiusC3b receptorComplement receptorCa2CalciumCCD cameraCharge-coupled device cameraCD2APCD2-associated proteinCEOChief Executive OfficerCFTRCystic fibrosis transmembrane conductance regulatorCLSCell Lines Service GmbHCM-1Cryomedium-1CM-5Cryomedium-5cm2Square centimeterCO2Carbon dioxideCSAColony stimulating activityCysCysteinDAPI4′,6-diamidino-2-phenylindoleDKFZDeutsches Krebsforschungszentrum (German Cancer Research Center)DMBAMetabolism of 7,12-dimethylbenzanthraeeneDMEMDulbecco's modified Eagle's mediumDMSODimethylsulfoxideDNADeoxyribonucleic acidEBNAEpstein-Barr nuclear antigenECACCEuropean Collection of Cell CulturesEDTAEthylendiamintetraacetateEEAErythroid-enhancing activityEGF-biotinEpidermal growth factor-biotinEREndoplasmic reticulumEUB -polymeraseEubacterial polymeraseFBSFoetal bovine serumFc receptorFragment crystallizablegGrammG418GeneticinG6PDGlucose-6-phosphate dehydrogenaseGABAGamma-aminobutyric acidG-CFSGranulocyte-colony-stimulating factorGenTSV §5Gentechnik-SicherheitsverordnungGLO-1Lactoylglutathione lyaseGM-CSFGranulocyte macrophage colony-stimulating factorhhourH-2d antigenHistoincompatibilityHAT sensitiveHypoxanthine/aminopterin/thymidine sensitiveHBsAgHepatitis B virus surface antigenHEPES-bufferN-2-hydroxyethylpiperazine-N-2′-ethanesulfonic acid bufferHisHistidinHIVHuman immunodeficiency virusHLAHuman leukocyte antigen systemHPV-16, HPV-18Human papillomavirus typeIGF IIInsulin-like growth factor IIIGFBPInsulin-like growth factor binding proteinsIFN-g-inducibleInterferon-gamma-inducibleIL-1, IL-6Interleukin 2, 6IST premixInsulin selenium transferrin complex premixKMG-2,KMG-5Konditioniertes medium growthLAVLymphadenopathy associated virusl-DOPA decarboxylasel-3,4-dihydroxyphenylalanine decarboxylaseLCMLymphocytic choriomeningitisLDVLactate dehydrogenase-elevating virusLtdLimitedLmx1bLIM homeobox transcription factor 1-betaLPS.LipopolysaccharideMAP-TestMitogen-activated protein testMEMMinimum essential mediumMg2+MagnesiaMHVMouse hepatitis virusminMinutemlMillilitermMMillimolarmRNAMessenger ribonucleic acidm-THPC-PEGMeta-tetra(hydroxyphenyl)chlorin-PEGMUC-1, MUC-2MucinMVMMinute virus of miceNaSodiumNa2CO3Sodium carbonateNaHCO3Sodium hydrogen carbonateNEAANonessential amino acidsNGFNerve growth factorNKNatural killerPAS positivePeriodic acid Schiff reactionPBSPhosphate buffered salinePCRPolymerase chain reactionPen/Strep-solutionPenicillin/Streptomycin- solutionPGM1Isozyme of phosphoglucomutasepHPotentia HydrogeniiPhePhenylalaninePPDPurified protein derivativepRBRetinoblastoma suppressorPTHParathyroid hormoneRCV/SDARat Corona Virus/Sialoda Cryoadenitis VirusRD114Endogenous retrovirusrpmRevolutions per minuteSCFStem cell factorSerSerinSMV provirusSoybean mosaic virusSTRShort tandem repeatSV40Simian Virus 40T75 flaskTissue 75 cm2 flaskTBE bufferTris, boracid, EDTA bufferTBSTTris-buffered saline containing 0.1% Triton X-100TNF alphaTumor necrosis factor alphaTPATissue plasminogen activatorTSHThyroid stimulating hormoneWT-1Wilms-Tumor-Protein1
Introduction
1.1 Introduction and Usage of This Book
To culture living cells in the laboratory and to keep them proliferating have become a revolutionary part in the Life Sciences. For more than 60 years now researchers are using permanent cell lines and in recent years the so-called primary cell lines. Within this time frame the number of these cell lines has increased tremendously since the first cell line (the mouse fibroblast cell L-929) has been established in 1943. When the first human cell line (HeLa) was introduced in 1952, a boom in the development of such cell lines started and continues until today.
During this development the increasing knowledge regarding the establishment of human and animal cell lines has influenced the culture of cell lines; however, the scientists suffered from various setbacks and problems which could not be reduced to cell's biology alone but rather to the cell culture practice. This started with the definition of the meaning of “cell line” which has not been defined as uniformly as it may be desirable for the biological scientific research.
Both cell lines mentioned above, L-929 and HeLa, have been cloned originally, it means these cell lines originate from one single cell. This basic principle of uniformity or clonality of cell lines has not been followed strictly within the last 50 years. Furthermore, the problem of cross-contamination, that is, the mixing of different cells with each other still poses a serious problem that is not overcome completely.
In the last couple of years a movement within the area of cell culture has established, which makes a point of a more stringent and careful maintenance of the cell lines regarding all the steps in cell culturing and the general handling of the cells. Strict rules of handling cell lines in particular were established (GCCP-Good Cell Culture Practice), and along with the application of these rules a reproducible and transparent work will be possible in the future.
This “Good Cell Culture Practice” should have been basic routine from the beginning, but 60 years ago cell culture work has not been as good resulting in mistakes not only during sterile handling of the cells. Also, the diagnostic instrumentation in the analysis of cells and cell lines in these early times of cell handling have not been present to be able to recognize any modification of a particular cell line on the molecular basis during cultivation such as a switch of the number of passages.
In the very beginning the analysis of vital cells was restricted to watching them in the microscope (without phase contrast at first); this represented the only possibility besides the analysis of the chromosomes. Still today, a relatively simple inverted microscope equipped with phase contrast and a digital camera is sufficient to visualize the viable cultures routinely. The distance between the light source and the object table should be large enough to be able to watch cells which are kept in large culture flasks such as roller bottles.
However, the microscope being equipped with the phase contrast is necessary to efficiently evaluate the morphology in vitro. A modern inverted microscope is fitted with an ocular tube and a second tube which is connected to a digital camera or a CCD camera together with a monitor.
Another useful tool for an inverted microscope is an object table with a coordinating device for exactly locating the cell colonies unambiguously. Special object clamps at the microscope table may facilitate working with the various culture flasks and petri dishes. Inverted microscopes equipped with a fluorescent device are available; however, it is recommended to purchase a conventional upright microscope with fluorescent device together with an inverted microscope to achieve maximum sensitivity and accuracy through the higher magnification and better light yield for maximal performance of the fluorescence technique.
The analysis of specific isozymes as diagnostic tools has been introduced for the first time in the 1960s and 1970s. Within the last decade the diagnostics of cells changed dramatically, at first DNA hybridization emerged to be followed by DNA-fingerprinting and today the DNA profiling in the characterization of cells has become almost routine testing.
1.2 General Remarks
All efforts to characterize human and animal cells and cell lines unequivocally rise and fall with the knowledge of the morphology of the cells. This oldest, most direct and simplest way to visualize and characterize the cells is based on the histology of the cells existing in the body of human beings and animals, how they arrange and appear.
It is important to distinguish between the situations “in vivo” and “in vitro”, which is evident and manifold; therefore simple extrapolation of cell pictures from a histological textbook can be misleading. Thus, observing the vital morphology by phase-contrast microscopy in routine cell culture life is highly recommended.
The environment and the development of the cells in vitro are not the same as they are in vivo, and these specific characteristics in vitro regarding the cellular morphology have to be taken into account and have to be observed and followed up intensely.
Normal epithelial cells cultured “ex vivo” as primary cells “in vitro” have almost all characteristics of epithelial cells; however, most cell lines may loose defined properties (of molecular kind) if they are transformed or transfected for example, which they may express in a different morphology under the microscope.
Culturing animal tissue cells on a chemically inert but charged material results in large differences to the situation “in vivo”, which poses a serious problem regarding this type of the morphological characterization. Culture of adherent cells results in the formation of a monolayer on the substrate. The image of a cell line, which can spread out on the bottom of the cell culture flask when seeded at low density may reflect best the morphological image of the cells in the “in vitro” environment.
If the optimum cell density “in vitro” is exceeded, the cells are being pushed together as soon as confluency is reached. At this stage formations and structures may arise that are less characteristic. It is evident that the morphology of the cells under the phase contrast microscope are studied best when the cells have not reached confluence yet; then, their origin can be defined as epithelial or fibroblastoid. However, as mentioned above, this conclusion is not always unambiguous.
An obvious discrimination between epithelial cells and fibroblasts in the microscope is as follows: cells are defined as being fibroblastoid if their length is more than twice their width. This structure is also called spindle-like. Epithelial cells in culture appear polygonal and plane. Furthermore, the characteristics of the division process of these two main cell types are differing. Following cytokinesis, the daughter cells of fibroblasts move away from each other and find their position on the substrate. Epithelial cells keep contact with their daughter cells via specific epithelial complexes such as tight junctions. Colonies of growing epithelial cells may arise.
Other environmental factors besides the substrate may play a major role in the formation of cellular morphology, such as the composition of the medium or the presence or absence of serum. The transformation of the cell line in question is an important criterion for the morphology. Diploid, that is, nontransformed cell lines, can be characterized much better than those whose status of ploidy differs from the original tissue.
In addition, the number of diploid cell lines is restricted, as almost all healthy tissue cells are subject to apoptosis. This means that the passage number is constrained, and therefore not many non-transformed lines exist which are useful for in vitro culturing compared to the majority of transformed cell lines. Therefore, the number of passages in the case of diploid, nontransformed cell lines is always required. A passage number of about 30–35 in human diploid fibroblasts, for example, MRC-5 or Wi-38, is sufficient to induce apoptosis. These apoptotic cells cease their proliferation and have to be substituted with cells of a lower passage number.
In this case the creation of a “Master Cell Bank” as a prohibitive strategy is very helpful, as nearly all healthy diploid cell lines possess a limited life span in vitro as well as in vivo. Regarding the maintenance in vitro, transformed cell lines can be cultured much easier than diploid cells but still this transformation process represents a dramatic change of the biology of the cell. This holds for the situation in vivo as well as in vitro. As transformed cells have been and are still widely used, a few remarks regarding the observation and analysis of the cellular morphology follow:
Our whole set of pictures represents viable cells cultured as monolayers or as suspension cells. The adherent cells attach to the respective surface or substrate, that is plasma-treated polystyrene with negative charges. No special treatments of the surface nor any other conditioning with, for example, collagen, extracellular matrices were used unless specified. No attempts were made to fix and/or to stain the cells and no three dimensional constructs were used for the pictures.
The pictures were made with a professional equipment (inverted microscope with phase contrast and a digital camera), no further retouch or improvements by digital processing were made. This guarantees that pictures taken in the laboratories of the readers may be comparable to our pictures without any manipulations or “improvements.”
Last but not the least, this book is not a textbook nor will give any detailed and special guidelines or protocols how to treat and process the respective cell lines in culture. Please refer to the many textbooks in this field and even the growing number of protocols and procedures of cell culturing appearing in the World Wide Web.
This book may be dedicated mainly to people with previous knowledge in cell culture techniques working in the laboratory.
2
Basic Cell Culture Techniques
2.1 Safety Precautions for Frozen Cell Lines
Protective gloves and clothing should be used and a facemask or safety goggles must be worn when storing in and/or removing from liquid nitrogen. The removal of a cryovial from liquid nitrogen can result in the explosion of the cryovial creating flying fragments.
2.2 Sterile Working
To assure a sterile working environment, all cell culture tasks should be performed within a class 2 safety laminar air flow cabinet.
2.3 Handling Procedure for Cell Lines
2.3.1 Frozen Cells
2.3.2 Receipt of Growing Adherent Cultures in T-flasks
The cell culture flask before shipping are completely filled with growth medium eventually with antibiotic/antimycotic solution to prevent loss of cells in transit and prevent from contamination. Remove all of the medium except for a small but sufficient volume to cover the inner surface of the flask. Incubate at 37 °C for 1 h. Then change to the desired incubation medium without antibiotics as recommended. (DMEM or RPMI-1640 or other incubation medium of your choice. Please check carefully the recommended CO2-concentration in the incubator.) But if you use routine antibiotics (e.g., pen/strep-solution) in the media, you can use your respective media without problems.
Sometimes the cultures are handled roughly in transit and some or even most of the cells may become detached and float in the culture medium. If this has occurred remove the entire contents of the flask after gently suspending the medium with a pipette and centrifuge at 200 × g for 10 min. Draw off the excess supernatant medium, resuspend the cells in 10 ml of the culture medium, and plate the entire cell suspension in a single flask of suitable size.
2.3.3 Receipt of Growing Suspension Cultures
The culture flask are completely filled up with growth medium for shipment. Remove the entire contents of the flask with a pipette into a centrifuge tube and centrifuge at 200 × g for 10 min. Resuspend the cell pellet as suggested under subculture procedure described in the cell lines descriptions with the respecitve incubation media.
2.3.4 Medium Replacement of Cells in Suspension
If medium is to be replaced with fresh cell culture medium, the flask containing the cells should be placed in an upright position to sediment the cells. After about 30–45 min, carefully remove an aliquot without removing cells, and replace it with the same amount of fresh medium.
If the cells do not sediment, transfer the cell suspension into sterile centrifuge tubes, centrifuge at 200 × g for 10 min, remove the spent medium and add an equal amount of fresh cell culture medium.
2.3.5 Subculture of Cells in Suspension
If the cells have reached the plateau phase, subculture them by preparing fresh flasks, label the flasks with the name of the cell line, passage number, the respective cell culture medium, and the date. Pipette an aliquot of fresh cell culture medium, add an aliquot of the dense cell suspension and resuspend the cells. Transfer the flasks into the incubator.
2.3.6 Subculture of Adherent Cells
If the cells cover about 85–90% of the substrate, subculture adherent cells using trypsin or alternative detaching enzymes. A split ratio of 1 : 2 to 1 : 16 is recommended, as described on the respective cell line information sheet.
Before trypsinization, wash the cell layer very carefully twice with balanced salt solution (BBS) without Ca 2+, Mg 2+ and without any serum. Thus, all remaining serum residues have been removed. If serum-free medium is used, one washing step using BBS is sufficient.
Trypsinization should be carried out according to general trypsinization protocols. It is advised to stop the trypsin activity using media containing serum, or using serum inhibitors, if serum-free media has been used.
Resuspend the cells carefully, centrifuge at 200 × g for 10 min, resuspend the cells in fresh medium and count the cells. Seed the cells at a concentration of 1 × 104 to 5 × 104 cellls/cm2 into new flasks or refer to the cell lines description.
If the trypsinization solution is free of EDTA, the centrifugation step can be omitted.
It is recommended to follow the instructions on the apropriate datasheet which contains details or routine maitenance including feeding and subculturing.
2.3.7 Subculture of Mixed Cell Lines (Adherent and Floating Cells)
Few cell lines grow as adherent as well as floating cells. In this case, collect the floating cells in sterile centrifuge tubes, detach the adherent cells according to the protocol described above for adherent cells, and combine both fractions. Following one centrifugation step at 300 × g for 5 min, resuspend the cells for cell counting, and dilute them in cell culture flasks as described.
2.3.8 Cell Counting
The counting of the cells can be performed using a Hemocytometer or using an electronic cell counter.
2.3.9 Cryopreservation of Cell Lines
To achieve best results, the cells to be frozen should be in the log-phase of the growth curve. Harvest these cells as usual.
Centrifuge the cell suspension at 200 × g for 10 min at room temperature and remove the supernatant. Wash once with fresh cell culture medium.
Resuspend the cell pellet using icecold cryomedia (see for composition the manufactor's catalogs or the textbooks), adjusted to a cell number of 2–4 × 106 cells/ml.
Quickly distribute the cell suspension into appropriate cryovials and close them tightly.
Do not allow the suspension to warm up to room temperature.
Place these cryovials containing the cells in a Cryo Freezing Container and cool down at a rate of 1 °C/min to at least −70 °C. At this point the frozen cryovials can be stored directly in liquid nitrogen or better in the gaseous phase of liquid nitrogen.
If you do not possess a Cryo Freezing Container, place the rack with the ampoules without covering in a freezer (−30 °C to −40 °C) for at least 60–120 min.
Immediately afterwards put the rack into an ultra freezer or into a container filled with dry ice (−72 °C – −80 °C) and keep the cryovials for at least 1 h.
Following this procedure, the cryovials can be stored in liquid nitrogen. To control the success of the freezing procedure, it is recommended to revitalize one cryovial 24 h after the cryovial had been placed into the liquid nitrogen. Thus, follow the general recommendations for thawing of cells.
2.3.10 Long Term Storage of Cells
It is not recommended to store cryopreserved cells on dry ice, as many biological processes are still going on at temperatures as low as the sublimation temperature of dry ice of about −78 °C. Biological activity substantially slows below the glass transition point of aqueous solutions of around −136 °C.
Therefore, the storage in the gas phase of liquid nitrogen at −196 °C is required for successful preservation of cells lines and primary cells.
2.3.11 Detection and Elimination of Contaminations
When cells are contaminated with bacteria, fungi, molds, and mycoplasm, they should be withdrawn and autoclaved, and the sterile routine should be examined step by step. Contaminations can be recognized in the microscope and by a sudden change in pH, which results in yellow medium. Fungi and yeast contamination appears at least within 3 days often without visible change of the media pH.
Mycolasma contamination cannot be recognized neither by eye nor in the microscope. Diagnosis of mycoplasma contamination can be carried out by staining fixed cells with DNA-specific fluorescent dyes (Hoechst 33258 or DAPI) or by polymerase-chain-recation (PCR). Direct culturing of mycoplasmas for diagnostic purposes in the cell culture laboratory is not recommended.
Although it is recommended to discard mycoplasma-infected cultures like those infected with bacteria and fungi, it was reported that some bactericidal agents (Tylosine, Minocyclin, Tiamulin and Ciprofloxacin and derivates there of) can be used to cure contaminated cells. But care should be taken that these infected and probably cured cell are monitored at least every three months (!) if reinfection occurs. Please consider the manufacture's recommendation for the appropriate concentration.
Viral contamination cannot be seen by visual inspection nor by phase contrast microsopy. Viral contamination can be part of the serum used, but there are no reliable methods for detecting or even eliminating viruses from cultures.
2.3.12 Cross-contaminations/Authentication
Cross-contamination is a very common problem in cell cultivation. The most prominent cell line HeLa, which has overgrown many slower growing cells. Other fast growing cell lines, like the T-24-line, have cross-contaminated at least three different cell lines.
Cross-contamination can be avoided, if good cell culture practice has been applied. However, authenticating the cell line(s) on a regular basis by standard STR analysis technique helps to avoid cross-contaminations.
2.4 Special Remarks on the Origin of the Cell Lines
The cell lines described in this book are deposited at ATCC (American Culture Tissue Collection), HPACC/ECACC (Health Protection Agency), DKFZ (German Research Cancer Institute), CLS Cell Lines Service GmbH and IAZ (Institut für Allgemeine Zellkultur).
2.5 Photographic Equipment
All photographs of the cell lines shown in this book were taken using the inverted microscope
3
List of Cell Lines and Human Primary Cells (in Alphabetical Order)
3.1 Human Cell Lines
5637769-PA-64 CLSA-204A-375A-427A-431A-498A-549A-673A-704AGSAsPC-1BeWoBT-20BT-474BT-549C-643Caco-2Caki-1Caki-2Calu-1CaLu-6Capan-1Capan-2CCRF-CEMCERV-186CERV-196CERV-215Chang-LiverCLS-54CLS-117CLS-354CLS-439CLS-54Colo-60HColo-94HColo-205Colo-320DMColo-680NColo-824DAN-GDMS-79DU-145ECV-304FAMPACGCTH-4HB-CLS-1HB-CLS-2HBL-52HEK-293HEL-299HeLaHeLa-S3Hep-2Hep-G2HGC-27HOSHRT-18 (HCT-8)HS1-CLSHS-683HS-695THS-729HSBHT-29HT-1080HuTu-80IGR-1IMR-32JARJurkat E6.1K-562Kasumi-1KATO-IIIKG-1AKHOS-240SKHOS-312HKHOS-NPLCLC-97TM1LnCaPLOVOLXF-289Ma-CLS-2MCF-7MDA-MB-231MDA-MB-436MDA-MB-468MEL-CLS-2MEL-CLS-3MEL-CLS-4MeWoMG-63MML-1MNNG-HOSMRC-5MSTO-211HMX-1NB-4NCI-H69NCI-H82NCI-H209NIH:Ovcar-3NIS-GOAW-42PA-CLS-52Panc-1PC-3PLC-PRF-5RC-124RCC-ERRCC-FG1RCC-FG2ZR-75-1RCC-LRRCC-MHRCC-OF1RCC-PRRCC-WKRDRD-ESRPMI 8226RT-4RT-112RT-112-D21SaOS-2SH-SY5YSk-BR-3Sk-LMS-1Sk-LU-1Sk-MEL-1Sk-MEL-2Sk-MEL-5Sk-MEL-28Sk-MES-1Sk-NEP-1Sk-N-LOSk-OV-3Sk-UT-1SW-480SW-579SW-684SW-872SW-948SW-1736T-47DT-84T-406TF-1THP-1TK-6U-87 MGU-118 MGU-251 MGU-937UM-SCC-14CWS-1Wi-38VA-13_2RAWS-1WS1-CLSWT-CLS1Y-793.2 Animal Cell Lines
3.2.1 Rat
AR42JAS-30-DBRL-3ADSL-6A-C1Zajdela HepatomaFRTL-5L-5222MH-3924NRK-49FO-342PC-12RBL-1Walker-2563.2.2 Mouse
3T3-Swiss Albino3T6-Swiss AlbinoC2C12CaD2CLS-103CLS-138Colon-26E11EL4.IL-2FS-C3HJ-774A.1KERA-308KERA-SP1KLN-205L-138L-929MCA-3DMeth-A-SarcomaMSC-P5NFS-60NIH-3T3P3X63Ag8.653P-19P388-D1PDVRAW-264.7RenCaSp2-O-AG14STOSVIWEHI-3bYAC-13.2.3 Hamster
BHK-213.2.4 Chicken
ECF-RMDCC-MSB13.2.5 Monkey
Cos-7CV-1VERO3.2.6 Pig
LLC-PK1PK-153.2.7 Opossum
OK3.2.8 Potoroo
PtK-1 (NBL-3)PtK-23.2.9 Bovine
BFA3.2.10 Dog
MDCK3.2.11 Insect
SF-93.3 Human Primary Cells
Airway Small EpitelialChondrocytesEndothelial Cells (Dermal Microvascular)Fibroblasts Dermal NormalHepatocytesHuman Follicle Dermal Papilla Cells (HFDPC) culture in phase contrastHuman Skeletal Muscle Cells (SkMC)Human Tracheal Smooth Muscle Cell (HTSMC) culture in phase contrastHuman Umbilical Vein Endothelial Cells (HUVEC)Keratinocytes Normal EpidermalMammary Epithelial CellsMelanocytes Epidermal NormalMelanocytes Epidermal in Melanocytes Growth MediumMesenchymal Stem Cells from Bone Marrow undifferentiated (Human)Muscle Cells Skeletal Human differentiatedMyocytesOsteoblastsPapillar Follicle Dermal CellsPericytes from the placenta proliferatingPreadipocytes undifferentiatedPreadipocytes after in vitro differentiation into AdipocytesSkeletal Muscle Cells undifferentiatedSmooth muscle cells (Artery Pulmonary)Tracheal Epithelial CellsUmbellical Vene Endothlial Cells, spheroidVascular Endothelial Growth Factor (VEGF)4
Cell Lines and Human Primary Cells
4.1 Human Cell Lines
5637, 100× Leica.
5637, 100× Leica.
5637, 400× Leica.
5637
Origin and General Characteristics
Organism:Homo sapiens (human)Ethnicity:CaucasianGender:MaleAge:68 yearsTissue:Bladder (urinary)Cell type:CarcinomaMorphology:EpithelialGrowth properties:MonolayerDescription:The 5637 cell line has been established from the primary bladder carcinoma (grade II) of a patient by Dr G. Cannon in 1974Culture Conditions and Handling
Culture medium:RPMI 1640 medium supplemented with 2 mM l-glutamine and 10% fetal bovine serumSubculture routine:Remove medium and rinse with 0.02 EDTA (versene). Add fresh 0.025% trypsin/0.02% EDTA solution at 37 °C until the cells detach. Add fresh medium, remove trypsin by centrifugation, aspirate, and dispense into new flasks. Subculture every six to eight daysSplit ratio:A ratio of 1 : 5 to 1 : 8 is recommendedFluid renewal:Two to three times weeklyBiosafety level:1Special Features of the Cell Line and Recommended Use
Karyotype:Phenotype Frequency Product: 0.0056DNA profile (STR):Amelogenin: X, Y; CSF1PO: 12, 13; D13S317: 9, 11; D16S539: 13; D18S51: 14; D21S11: 30, 31; D3S1358: 15, 18; D5S818: 9, 10; D7S820: 10, 11; D8S1179: 13; FGA: 24, 25; Penta D: 10, 13; Penta E: 10, 20; THO1: 6, 9.3; TPOX: 11; vWA: 17, 18Tumorigenic:In nude miceIsoenzymes:Me-2, 1; PGM3, 2; PGM1, 1; ES-D, 1; AK-1, 1; GLO-1, 1–2; G6PD, BProducts:IL-1, IL-6, G-CFS, GM-CSF, SCFATCC number:HTB-9CLS number:300105Further Reading
Fogh, J. et al.
