Cell Culture Basics Handbook Includes transfection Gibco™ Education (Culture your career ThermoFisher [Link]/gibcoeducation SCIENTIFICInformation in this document is subject to change without notice, DISCLAIMER THERMO FISHER SCIENTIFIC AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR APARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, INNO EVENT SHALL LIFE ‘TECHNOLOGIES AND/OR TTS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, ©2016 Thermo Fisher Scientific Inc. All rights reserved. COL31122 0516Contents 1. Introduction Purpose of the Handbook . Introduction to Cell Culture What is cell culture? Finite vs, continuous call line . Culture conditions 2 Cryopreservation 2 Morphology of cells in culture 3 Applications of cell culture: 3 [Link] Culture Laboratory .. Safety Biosafoty lovels SDS 5 Safety equipment Personal protective equipment (PPE) Sate laboratory practices Cell Culture Equipment Basic equipment Expanded equipment Additional supplies 6 Cell Culture Laboratory Aseptic work area, 7 Cell culture hood 7 Cell culture hood layout 8 Incubator ° Storage 9 Cryogenic storage Cell counter 0 Aseptic Technique "1 Introduction coe : " Sterile work area Good personal hygiene... : " Stetle reagents and media 12 Sterle handling cee : 12 Coll Culture Basics | ii For educational purposes onlyAseptic Technique Checklist . +13 Biological Contamination .. Introduction Bacteria Yeasts Molds Viruses Mycoplasma Cross-contamination Using antibiotics coe : 7 3. Cell Culture Basics . . Cell Lines .. 18 Selecting the appropriate cal ine ‘Acquiring cell ines 18 Culture Environment . 19 Adherent vs, suspension culture . : 19 Media 20 pH coe : 21 co, 21 ‘Temperature 21 Cell Morphology « 22 Mammalian Cells 22 Variations in mammalian coll morphology 22 Morphology of 292 cells 23 Insect Cells 24 Morphology of $121 cells Morphology of $19 cells, 4, Cell Culture Methods Guidelines for Maintaining Cultured Cells . . = 26 What is subculture? 26 When to subculture? coe : ar Media recommendations for common cell lines 28 iv | Cell Cuture Basics For educational purposes onlyDissociating adherent cells TiypLE dissociation enzymos Subculturing Adherent Cells Materials needed Protocal for passaging adherent calls Notes on subcul Subculturing Suspension Cells Passaging suspension cultures Suspension culture vessels Materials needed Protocol for passaging suspension cells Notes on suiscutturing suspension insect cells Freezing Cells . Cryopreservation Guidelines for cryopreservation Freezing medium Materials needed Cryopreserving cultured calls Thawing Frozen Cells .. Guidelines for thawing Materials needed Thawing frozen cells 5. Transfection Basics Introduction to Transfection What is transfection? Terminology App tions ‘Types of Transfection .. Transient transfection Stable transfection Choosing a transfection strategy Gene Delivery Technologies Cationic lipid-mediated delivery 1ring adherent insect cells 36 For educational purposes onlyCalcium phosphate coprecipitation DEAE-doxtran-modiated delivery Delivery by other cationic polymers Viral deivery Electroporation ther physical delivery methods . Cationic Lipid-Mediated Transfection . . Mechanism c nic lipid transfection reagents Virus-Mediated Gene Transfer . Key properties of viral vectors, Common viral vectors Neon Transfection System . Selection of Stable Transfectants .. Selection antibiotics for eukaryotic cells Reporter Gene Assays . Transfection assays Gone regulation assays Common report genes RNAi and Noncoding RNA Research Glossary of common RNAI terms How RNAI works SIRNA analysis miRNA analysis Choosing an RNAi approach 6. Transfection Methods Factors Influencing Transfection Efficiency Cell type Cell health and viability Contiueney Media Serum Antibiotics 69 vi | CellCulture Basics For educational purposes only‘Type of molecule transfected Transfection method Selecting a Transfection Method (nonvit Continuous cel ines Primary colls and finite cutures Selecting a Viral DNA Delivery System . Expression in mammalian cells Expression in insect cells Guidelines for Plasmid DNA Transfection . . Vector considerations Quality of plasmid DNA Gene product and promoter Controls Optimization of Plasmid DNA Transfection . Considerations for calcium phosphate coprecipitation Considerations for cationic lipic-madiated delivery Considerations for electroporation Selection of Stable Transfectants .. Before starting Kill curve Selection workflow Selecting an RNAi Strategy SIRNA vs, vector approaches Nonvector siRNA technologies SIRNA transfection Vector-mediated RNAI Guidelines for RNA Transfection . IAI workflow Handling RNA Transfection efficiency Positive controls Negative controls Cotranstection SIRNA quality 78 79 80 82 = 83 83 83 83 For educational purposes onlySIRNA quantity Volume of transfection reagont Cell density Exposure to transfection agent/siRNA complexes Presence of serum during transfection Tins for a succossful siRNA experiment Optimization of siRNA Transfection . Factors affecting siRNA transfection efficiency Appendix Troubleshooting . Cell Culture and Transfection Pi Cel lines Media for mammalian cell cultu Media for insect cell culture Serum products for cell culture roducts . re Laboratory reagents for cell culture Antibiotics and antimycotics . Growth factors and purified proteins Accessory products for cell culture Transfection reagents Neon Transfection System RNA interference Additional Resources .. Mammalian and insect cell cultures Coll and tissue analysis: Transfection selection tool Safety data sheets. Cer ate of analysis Technical support References ... Notes -104 104 104 104 104 104 104 -105, = 108 vii | Cell Culture Basies For educational purposes only1. Introduction Purpose of the Handbook AN The Cell Culture Basics Companion Handbook is a supplement to the Cell Cutture Basics instructional videos available online at [Link]/celiculturebasics The handbook and videos are intended as an introduction to cell culture basics. The first four chapters of the handbook focus on cell culture, covering topics such as gatting {familar with the requiremants of a laboratory dedicated to call culture experiments, laboratory safety, asoptic technique, and microbial contamination of coll cultures, a ‘wall as providing basic methods for passaging, freezing, and thawing cultured cells. The sulssequent two chapters of the handbook focus on various transfection technologies and provide general guidelines for the selection of the appropriate transfection method, the transfection of cals with plasmid DNA, olgonuclectides, and RNA, as wollas culture preparation for in vitro and in vivo transfection and selection of the transfected cals, ‘The information and guidetnes presented in the handbook and the instructional videos focus on cell ines {finite or continuous) and omit experiments and techniques concerning primary cultures and stem cells, such as isolating and disaggregating tissues, reprogramming cells into pluripotent stem cells, or cifferentiting stem cells into various lineages, Note that while the basics of cell culture experiments share certain similarities, cel culture conditions vary widely for each call type. Deviating fiom the culture conaitions required for a particular cell tyoe can result in different phenotypes being exoressed; we therefore recommend that you familarize yourself with your cell line of interest, and closely folow the instructions provided with each product you are using in your experiments, Call Cuture Basics | 1 For educational ourposes onb,Introduction to Cell Culture What is cell culture? Finite vs. continuous. cell line Culture conditions Cryopreservation Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artical environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cuttvation, or they may be derived from a cell Ine or cell strain that has already been established, Primary culture Primary culture rofors to the stage of the culture after the cells arm isolated from the tissue and proliferated under the appropriate conditions until they occupy al of the available substrate (.¢., reach confluence). At this stage, the cells have to be subcultured {.. passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. Celine Ator the first subcuturo, the primary culture becomes known as a cell line. Cell ines dorived from primary cuftures havo a limited Ife span (.e, they are finite; see bolow), and as they are passaged, colls with the highest growth capacity predominate, resulting in a agree of genotypic andl phenotypic uniformity in the population Cell strain Ifa subpopulation of a cell line is positively selected ftom the cuture by cloning or some ‘other method, this cell Ine becomes a cell strain, A cell strain often acquires additional genetic changes subsequent to the ination of the parent line Normal cells usually divide only a limited number of times before losing thei abilty to pprolferate, which is a genetically determined event known as senescence; these call Ines are known as finite, However, some cell ines become immortal through a process called transformation, which can occur spontaneously or can ie chemically or vraly induced, When a finite osl ine undergoes transformation and acquires the abilty to divide indefinitely, t becomes a continuous cell line. ‘Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists ofa suitable vessel containing a substrate or medium that sugples the essential nutrients (amino acids, carbohydrates, vitamins, minerals) (growth factors, hormones, and gases (O,, CO,], and regulates the physiochemical milieu (PH, osmotic pressure, temperature). Mast cells are anchorage-dependent and must be cultured while attached to a sold or semisolid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture) Ifa surplus of cells are available from subculturing, they should be treated withthe appropriate protective agent (e.., DMSO or glycerol) and stored at temperatures below ~120°C (eryopreservation) unt they are needed. For more information on subcuturing and cryoprosarvng cols, rofe to the Guidelines for Maintaining Cultured Cells page 26. 2 | CallCuture Basics For educational purposes onby,Morphology of cells in culture Cals in culture can be divided into three basic cat appearance (.e., morphology) jories based on their shape and * Fibroblastic (or fbroblast-tke) cells are bipolar or mutipolar, have elongated shapes, and grow attached to a substrate, + Lymphoblastelike calls aro spherical in shape and usually grown in suspension without attaching to a surface Applications of cell culture Cell culture is one of the major tools used in celldlar and molecular biclogy, providing excelent model systems for studying the normal physiology and biochemistry of cells (e.g. metabolic stucies, aging), the effects of drugs and toxic compounds on the calls, and mutagenesis and carcinogenesis. tis also used in drug screening and development, and large-scale manufacturing of biological compounds (e.g, vaccines, therapeutic proteins) The major advantage of using call culture for any of these applications is the consistency and reproducibilly of resuts that can be oblained from using a batch of clonal cells. For educational purposes onby,2. Cell Culture Laboratory Safety Biosafety levels In addition to the safety risks common to most averydlay work places, such as electrical and fire hazards, a cell culture laboratory has a number of spectic hazards associated with handing and manipulating human or animal cols and tissues, as well as toxic, corrosive, ‘of mutagenic solvents and reagents. The most common of these hazards are accidental inoculations with syringe needles or other contaminated sharps, spils and splashes. conto skin and mucous membranes, ingestion through mouth oipetting, animal bites and scratches, and inhalation exposures to infectious asrosoks. ‘The fundamental objective of any biosafety program is to reduce or eliminate exposure of laboratory workors and the outside environment to potentially harmful biological agerts. ‘The most important element of safety in a cell culture laboratory is the strict adherence to standard microbiological practices and techniques. The regulations and recommendations for biosafety in the United States are contained in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH), and pubished by the US Department of Health and Human Services, The document defines four ascending fevels of containment, referred to as biosafety kvels 1 through 4, and describes the microbiological practices, safety equipment, and facilty safeguards for the corresponding level of risk associated with handling a particular agent, Biosafety Level 1 (BSL-1) BSL is the basic level of proteotion common to most research and clinical laboratories, and is appropriate for agents that are not known to cause disease in normal, healthy humans. safety Level 2 (BSL-2) BSL.2 is appropriate for moderate-tisk agents known to cause human disease of varying ‘severity by ingestion or through percutaneous or mucous membrane exposure. Most cell Colture labs should be at least BSL-2, but the exact reauirements depend upon the cell Ine used and the type of work conducted, Biosafety Level 3 (BSL-3) BSL js appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for agents that may cause serious and potential lethal infections. Biosafety Level 4 (BSL~4) BSL-4 is appropriate for exotic agents that pose a high individual isk of life-threatening disease by infectious aerosols and for which no treatment is available. These agents are restricted to high-containment laboratories, For mote information about the biosafety level guidetnes, refer to Biosafety in Microbiological and Biomadical Laboratoris, 5” Ealton, whichis availabe for downicacing at [Link]/od/ohs/biosfty/bmbiS/[Link] 4 | Coll Cuture Basics For educational purposes only,SDs Safety equipment Personal protective ‘equipment (PPE) Safe laboratory practices Safety Data Sheet (SDS) is a form containing information regarding the properties of a particular substance, inckicing physical data such as melting ooint, boling point, and flash point, as well as information on its toxicity, reactivity, heath effects, storage, cisposal, recommended protective equipment, and handling spils ‘SDSs for our products are available at [Link]/sds ‘Safety equipment in a cell culture laboratory includes primary barriers such as biosafety cabinets, enclosed containers, and other engineering controls dasigned to remove or minimize exposure to hazarcous materials, as well as personal protective equipment (PPE) that is often used in conjunction with the primary bariars. The biosafety cabinet {.2., callcuture hood) is the most important piece of equipment to provide containment of infectious splashes or aerosols gonorated by many microbiological procedures. For more information, soe Cell Culture Hood, page 7 PPE form an immodiate barrier botwoen the personnel and the hazardous agent, and they include items for personal protection such as gloves, laboratory coats and gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. They are often used in combination with biosafety cabinets and other devices that contain the agents, animals, or ‘materials being handled, We recommend that you consult your institution’: guidelnes for the appropriate use of PPE in your laboratory, The following reco mmenciations are simoly guidelines for safe laboratory practices, and they should nt be interpreted as a complete code of practice, Consult your insttution’s safety committee and follow local rules and regulations pertaining to laboratory safety. For mote information on standard microbiological practices and for specific biosafety level ‘Quidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5” Edition at ‘[Link]/od/ohs/biosfty/bmbIS/[Link] * Always wear appropriate PPE. Change gloves when contaminated, and dispose of Used gloves with other contaminated laboratory waste * Wash your hancis after working with potentially hazardous materials and before leaving the laboratory. * Do not eat, dink, smoke, handle contact lenses, apply cosmeties, or store food for human consumption in the laboratory. * Follow the institutional policies regarding safe handing of sharps (i, needles, scalpels, pipettes, and broken glassware) + Take care to minimize the creation of aerosols and splashes. * Decontaminate all work surfaces before and alter your experiments, and immeciately after any spill or splash of potentially infectious material with an aopropriate disinfectant. Clean laboratory equipment routinely, even ifit is not contain * Decontaminate all cultures, stocks, and other potentially infectious materials before disposal * Report any incidents that may result in exposure to infectious materials to appropriate personnel (e.g, laboratory supervisor, safoty officer For educational purposes onlyCell Culture Equipment The specific requirements of a cell culture laboratory dapend mainly on the type of research conducted; for example, the needs of mammalian cell culture laboratory ‘specializing in cancer research is quit citferent from that of an insect cell cuttue laboratory that focuses on protein expression. However, all cell culture laboratories have the common requirement of being ‘ree from pathogenic microorganisms (i.., asepsis), and share some of the same basic equipment that is essential for culturing cals. his section lists the equipment and supplies common to most cell culture laboratories, as wall as beneficial equipment that allows the work to be performed more effciantly or accurately, or permits a wider range of assays and analyses. Note that this st is not all inclusive; the requirements for any cell cuture laboratory depend on the type of work conducted Basic equipment — + Coll cuture hood! ie., laminar-flow hood or biosafety cabinet) + Incubator humid GO, incubator recommended) + Water bath + Centrituge + Rotrigerator and freezer 20°C) + Call counter (e.g. Invitrogen" Countess™ Il Automated Cell Counter o hemocytometer) * Inverted microscope * Liquid nitrogen (N,) freezer or cryostorage container * Sterilizer (ie, autoclave) Expanded equipment —* Aspiration pump (peristaltic or vacuum) + pH meter * Rollor racks {for scaling up monolayer cultures) * Confocal microscope * Flow cytometer * EG bioreactors * Coll cubes Additional supplies + Cell culture vessels (e.g, flasks, Pet dishes, roll bottles, mutiwel plates) + Pipettes and pipettors + Syringes and neces + Waste containers + Media, sera, and reagents + Cals 6 | Coll Cuture Basics For educational purposes only,Cell Culture Laboratory Aseptic work area Cell culture hood The major requirement of a call culture laboratory is the need to maintain an aseptic ‘work area that is restricted to cell cuture work. Although a separa tissue culture room is preferred, a designated cell culture area within a larger laboratory can stil be used for sterile handling, incubation, and storage of cal cultures, reagents, and media. The simplest ‘and most economical way to provide aseptic conditions is to use a cell culture hood (.°. biosafety cabinet. he coll cuture hood provides an aseptic work area while allowing the containment of infectious splashes or asrosols generated by many microbiological procedures. Three kinds of coll cuture hoods, designated as Class I, I, and Il, have boon developed to meat varying research and clinical needs. Classes of cell culture hoods lags I col cuture hoods offer significant levels of protection to laboratory personnel and 10 the environment when used with goad microbiological techniques, but they do not provide cultures protection from contamination, They are similar in design and air flow characteristics to chemical fume hoods. Class II coll culture hoods are designed for work iwolving BSL-1, 2, and 3 materials, and also provide an aseptic environment necessary for cell culture experiments, A Class I biosafety cabinet should be used for handling potentially hazarcious materials (0.9 primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents) Class Ill biosafety cabinets are gas-tight, andl they provide the highest attainable level of protection to personnel and the environment, A Class Ill biosafety cabinet is required for ‘work involving known human pathogens and other BSL-4 material, Air-flow characteristics of cell culture hoods ell culture hoods protect the working environment from dust and other airborne contaminants by maintaining a constant, unidirectional low of HEPA-filtered air over the work area. The flow can be horizontal, blowing parallel to the work surface, ort can be vertical. blowing from the top of the cabinet onto the work surface, Dspending on its design, a horizontal flow hood provides protection to the cultur ff the air flowing towarcis the use’) or to the user ff the aris drawn in through the front of the Cabinet by negative air pressure inside). Vertical flow hoods, on the other hand, provide significant protection to both the user and the cell culture. Clean benches Horizontal laminar flow or vertical laminar flow “clean benches" are not biosafety cabinets; these pieces of equipment cischarge HEPA-fitered air from the back of the cabinet across the work surface toward the user, and they may expose the user to potentialy hazardous materials. These devices only provide product protection. Clean benches can bs used for certain clean activities, such as the dust-tree assembly of sterile equipment or electronic devices, and they should never be used when handing cell culture materials or drug formulations, or when manipulating potentially infectious materials For mare information on the selection, instalation, and use of biosafety cabinets, refer to Biosafety in Microbiological and Biomedical Laboratorias, 5" Ealtion, which is available for downloading at [Link]/od/ohs/biosfty/bmbIS/[Link] For educational purposes only,Cell culture hood layout ‘A cell culture hood should be large enough to be used by one person at a time, be easily Cleanable inside and outside, have adequate lighting, and be comfortable to use without requiring awkward postions. Keep the work space in the cell culture hood clean and uncluttered, and keep everything in direct line of sight. Disinfect each item placed in the cell culture hood by spraying them with 70% ethanol and wiping clean, The arrangement of items within the cell culture hood usually adheres to the following right-handed convention, which can bbe modified to include adcitional tems used in specific applications, * Avwide, clear work space in the center with your cell culture vessels + Pipettor in the front right and glass pipettes in the let, where they can be reached easily + Reagents and media in the rear right to allow easy pipetting + Small container in the rear midalle to hokd liquid waste sha Wore Stace Figure 21. The basic layout ofa cell culture hood for right-handed workers. Let-handes workers may ‘sich the positions ofthe ters laid out on the work sface & | Coll Culture Basics For educational purposes only,Incubator Storage ‘The purpose of the incubator is to provide the appropriate environment for cell growth he incubator should be large enough, have forced-air crculation, and should have temperature control to within +0.2°, Stainless sleel incubators allow easy cleaning and provide corrosion protection, especially if humid air is required for incubation. Although the requirement for aseptic conditions in a cell cutture incubator is not as stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to avoid contamination of coll cultures. Types of incubators, hare are two basic types of incubators, cy incubators and humid CO, incubators. Dry incubators are more economical, but require the cell cultures to be incubated in sealed flasks to prevent evaporation. Placing a water dish in a dry incubator can provide some humility, but they do not allow prec'se control of atmospheric conditions in the incubator Humid CO, incubators are moro expensive, but allow superior control of cuture ‘conditions. They can be used to incubate colls cultured in Peti dishes or muttiwall plates, which requite a controlled atmosphere of high humidity and increased CO, tension, ‘A coll culture laboratory should have storage areas for iquids such as media and reagents, for chemicals such as drugs and antibiotics, for consumables such as disposable pipettes, culture vessels, and gloves, for glassware such as media bottles and glass pipettes, for specialized equipment, and for tissues and cell Glassware, plastics, and specialized equioment can be stored at ambient temperature (on shelves and in drawers; however, tis important to store all media, reagents, and chemicals according to the instructions on the label, ‘Some media, reagents, and chemicals are sensitive to ight; while their normal laboratory use uncier lighted coneitions is tolerated, they should le stored in the dark or wrapped in aluminum foll when not in use, Refrigerators For small cell cuture laboratories, a domestic reftigerator (oreferably one without an autodetrost freezer is an adequate and inexpensive piece of equipment for storing reagents and media at 2-8°C. For larger laboratoriss, a cold room restricted to cell culture is more appropriate, Make sure that the retigerator or the cold room is cleaned regulatly to ‘avoid contamination, Freezers Most cell culture reagents can be stored at ~8°C to ~20°C; therefore, an ultradeep ‘reezer (.2., a-80°C freezer) is optional for storing most reagents. A domestic freezer is a cheaper alternative to a laboratory freezer. While most reagents can withstand temperature ‘oscillations in an autodettost {.e., sel-hawing) treezer, some reagents such as ant and enzymes should be stored in a freezer thal does not autocletrost For educational purposes onlyCryogenic storage Cell counter Col lines in continuous culture are likely to suffer from ganetic instability as their passage number increases; therefore, itis essential to prepare working stocks of the cells and preserve them in cryogenic storage (for more information, see Freezing Cells, page 37). Do not store cells in -20"C or -80°C freezers, because their viabiliy, decreases when they are stored al these temperatures, There are two main types of liquid-ntrogen storage systems, vapor phase and liquid phase, which come as wide-necked or narrow-nackad storage containers. Vapor phase systems minimize the risk of explosion with cryostorage tubes, and are required for storing bichazardous materials, while the liquid phase sysioms usually have longer static holeling times, and are therefore more economical Narrow-necked containars have a slower nitrogen evaporation rate and are more ‘economical, but wide-necked containers allow easier access and have a larger storage capactty. A cell counter is essential for quantitative growth kinatics, andl a great advantage when ‘mote than two or three eal lines are cutured in the laboratory. The Countess ll Automated Cell Counter is a benchtop instrument designed to measure cell count and viabilty (ive, dead, and total cells) accurately and precisely in less than a minute per sample, using the standard trypan blue uptake technique, Using the same. ‘amount of sample that you currently use with the hemocytometer, the Countess II ‘Automated Cell Counter takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells. 410 | Coll Culture Basics For educational purposes onlyAseptic Technique Introduction Sterile work area Good personal hygiene ‘Successful cal culture depends heavily on keeping the calls free from contamination by ‘microorganisms such as bacteria, fungi, and viruses. Nonstetile supplies, mecla, and reagents, airborne particles laden with microorganisms, unclean incubators, and clrty work surfaces are all sources of biological contamination. ‘Aseptic technique, designed to provide a barrier between the microorganisms in the ‘environment and the sterile coll culture, depends upon a set of procedures to reduce the probabilty of contamination fram these sources. The elamants of aseptic technique are a storie work area, good personal hygiene, stele reagents and media, and storie handling ‘The simplast and most economical way to recluce contamination from airbome particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a coll culture hood, * The coll cuture hood shoul be properly set up, and be located in an area that is restricted to cell culture that is free from dtafts from doors, windows, and other ‘equipment, and with no through tratfc, * The work surface should be uncluttered and contain only tems required for a particular procedure; it should not be used as a storage area, * Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely * For routine cleaning, wine the work surface with 70% ethanol before and during work, especially ater any spillags * Using a Bunsen loumer for flaming is net necessary oF recommended in a cell culture hood, * Leave the coll culture hood running at all times, turning it off only when it will not be Used for extended periods of time, Wash your hands before and after working with cell cultures. In addition to protecting you from hazatclous materials, wearing personal protective equipment also reduces the probabilty of contamination from shed skin as well as dirt and dust from your clothes. " For educational purposes only,Sterile reagents and media Sterile handling Commercial reagents and media undergo strict quality contral to ensure their sterility, but they can become contaminated while handling. Follow the guidelines below for sterile handling to avoid contaminating them. Always steriize any reagents, media, or solutions prepared in the laboratory using the appropriate steriization procedure (@.g., autociave, stenle fier) Always wipe your hands and your work area with 70% ethanol + Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell cuture hood. * Avoid pouting media and reagents directly from bottles or flasks. * Use sterile glass or disposable plastic pipettes and a pipettor to work with Iquids, ‘and use each pipette only once to avold cross-contamination, Do not unwrap sterle pipettes unt they are to be used, Keep your pipettes at your work area, Always cap the bottles and flasks after use and seal mutiwell plates with tape or place thom in resealable bags to prevent microorganisms and airborne contaminants from gaining entry. * Never uncover a storie flask, bottle, Peti dish, etc. until the instant you are ready to use it and never leave it open to the ervironment. Return the cover as scon as you are fnishod, * Ifyou remove a cap or cover, and have to put it down on the work surface, place the ‘cap with opening facing down, * Use only sterile glassware and other equipment * Be careful not to talk, sing, or whistle when you are performing sterile procedures. * Porform your experiments as rapidly as possible to minimize contamination. 12 | Coll Culture Basics For educational purposes onlyAseptic Technique Checklist 5 a concise list of suggestions and to achieve a solid aseptic technique. For an in-depth review of aseptic Cutture of Animal Calls: A Manual of Basic Tec from drafts and thro atic? I culture hod in an area fr Is the work surface uneluttor your experiment? fin only ome raquired pe the work surface with 70% ethanol before work? ‘Avo you routinely clearing and sterilizing your incubators, refigarators, feezars, and ther laboratory equnment? Personal Hygiene he you wash your hands? ‘Ave you wearing personal protective eq shave long hai, st tod in the back? ‘Ae you using a pipator to work with iquids? Reagents and Media Have you sleriized any reagents, media, and solutions you have prepared in the laboratory using the appropriate procedure? before Did you wipe the outside of the placing them on your work surface? es, flasks, and plates with 70% ethar ‘Avo all your battles, asks, and other containors capped whan natin use’ ‘Ave all your plates stored in sterile resealable bags? 30 ay of your reagents look cloudy? ¢ particlas? Have a foul smell? Unusval ntaminated? Do they contain floating 7 Tyas, did you decontaminate and discard them? Handling ‘Ave you working slowly and deliberately, minaful of aseptic technique?” id you wipe the surfaces ofall the toms, including pipettor, bottles, a 0% ethanol before placing them inthe call cuture hood? ‘Ave you placing the caps or cavers face down on the work area? or str you using sterile glass pip alliquids? sable plastic pipettes to manipulate ‘Ave you using a sterile pipette only ones to avoid cross-contamination?” ‘Ave you careful nat to touch the pipate tp to anything nonster’ id you mop up any spillage immediately, and wipe the area with 70% athanol? 13 For educational purposes only,Biological Contamination Introduction Bacteria Contamination of cell cultures is easily the most common problem encountered in cell collure laboratories, sometimes with very serious consequences. Cell culture contaminants can be divided into two main categories, chemical contaminants such as imourites in media, sera, and water, including endotoxins, plasticizers, and detergents, and biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as wall as cross- contamination by other cell lines. While it is impossible to eliminate contamination entirely, itis 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, Bacteria are a large and ubiquitous group of unicellular microorganisms. They are typically ‘few micrometers in diameter, and can have a variety of shapes, ranging from spheres to rods and spirals, Because of ther ubiquity, sizo, and fast growth rates, bactora, along with yeasts anc molds, are the most commonly encountered biological contaminants in cell culture. Bacterial contamination is easily detected by visual inspection of the culture within a few days of it becoming infected; infected cutures usually anoear cloudy, sometimes with a thin flm on the surface. Sudden drops in the pH of the culture medium are also frequently encountered. Under a low-power microscope, the bacteria appear as tiny granules between the cells, and observation under a high-power microscope can resolve the shapes of individval bacteria, The simulated images below show an adherent 293 cell culture contaminated with E. col, B Figure 2.2. Simulated phase-contrast images of adherent 283 cells contaminated with E. col. The spaces Between the achoront calle snow try, shrmmerng granule under kw power miroscepy bul he iRdvival bacteria are nc easly cisingushable parel A) rurther magreation ofthe area enclosed by he black square resobes the indial Ecol calls, whch ae typically rod-shaped and are about? ym long ane ‘0 um In camer Each spol the bk sean panel A 100 un, 14 | Coll Culture Basics For educational purposes only,Yeasts Molds Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in size from a few micrometers (typical) up to 40 micrometers (arely). Like bacterial contamination, cultures contaminated with yeast become turbid, especially ifthe contamination isin an advanced stage. Thera is vary litle change in the pH of the culture contaminated by yeast untl the contamination becomes heavy, at which stage the pH Usually increases. Under microscopy, yeast appear as individual ovoid or spherical particles that may bud off smaller particles. The simulated image below shows adherent 293 cell culture 24 hours after plating that is infected with yeast. Figure 2.3. Simulated phase-contrastimages of93 cols in adherent culture thatis contaminated with yeast ‘Tho contaminating yeast cole aprosr as ovoWd paticls, budding 0" smaller paricies as trey ropfeao, ‘Molds are eukaryotic microorganisms in the kingclom of Fungi that grow as multicellular filaments called hyphae. A connected network of these multicellular flaments contain ‘genetically identical nuclei, and are referred to as a colony or mycelium. Similar to yeast Contamination, the pH of the culture remains stable in the intial stages of contamination, then rapidly increases as the culture becomes more heavily infected and becomes turbid. Under microscopy, the mycelia usually appear as thin, wisp-lke 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, 15 For educational purposes onlyViruses Mycoplasma Viruses are microscopic infectious agents that take over the host cell's 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 affect cell cultures from species other than their host. However, using virally infected cell cultures can present {a serious health hazard to the laboratory personnel, especially if human or primate calls are cultured in the laboratory. Viralnfection of cell cuturas can be detacted by alactron ‘microscopy, immunostaining with a panel of antibodies, ELISA assays, or PCR with appropriate viral primers. Mycoplasma ave simple bacteria that lack a call wall, and are considered the smallest sot-roplicating organism. Because oftheir oxtromoly smal size (typically lass than one micrometer), mycoplasma are very dificult to detect until they achieve extremely high donsitis and cause the cal cuture to deteriorate; until then, thore are often no visible signs of infection, Some slow-growing mycoplasma may persist in culture without causing coll death, but they can alter the behavior and metabolism of the host calls in the culture. CChronie mycoplasma infections might manifest themselves with decreased rates of call profferation, reduced saturation density, and agglutination in susponsion cultures; however, the only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining (e.g., Hoechst 23288), ELISA, POR, immunostaining, autoradiography, of microbiological assays. B Cc Figure 2.4. Photomicrographs of mycoplasma-tree cultured cells (pane! A) and coll infected with asia (panels B and C). The cuties were tsied ug the Ivirogen”™ MyooFIuor™ Mycoolasma Detection Ki flowing the at protocols. In fod cas, the Myco"luor™ reagent has access tothe cell race which ao inionslyslaned withthe reagent, but the absence of foresoon! exraraclear objects nsoales ‘that tho cutis ee rom myceplasma contarsnation panel. fixed cos infstoe vith mycoplasme, the ‘MycoFluor reagent stains both the nicki and the mycoplasma, but the intense relative fuorescence of the ructe Coscure the mycoplasma on or near the auc. However, the mycoplasma separated from the bight nucle ara ready vise panel In Ive cal, the MycoFluorreagant does not have access tothe nucle, but readily stan the mycoplasma associated withthe outside "cele panel C). The emission spectrum cf ho ‘conta inciec inthe kts degre tc have a nomagoneous ntonsty tha cbsol matches that of myopia Stained accorsng fo te Mycorluor mycaplatra sotcton protocal allowing the racearchars to wicerenint Between stared mycoplasma and otha forms of backoround luminescence, nluding vss, bactaria, anc ‘calla autofuorescence. The mages were obtaned wsing 366 rm exctation and a 100/1.8 Pan Neofuar™ ‘coctve lns coupled wih a 460 = 80 nm barcoass ther, 16 | Coll Culture Basics For educational purposes onlyCross-contamination Using antibiotics While not as common as microbial contamination, extensive cross-contamination of many call ines with HeLa and other fast-growing call ines isa clearly established problem with serious consequences. Oblaining cal lines from reputable cell banks, perocically checking the charactarstics of the call Ines, and practicing good aseptic technique are practices that will help you avoid cross-contamination. DNA fingerprinting, karyotype analysis, and isotype analysis can confirm the presence or absence of crass-contamination in your coll cultures. Antibiotics should never be used routinely in cell cuture, because their continuous Use encourages the develooment of antibiotic-resistant strains and allows low-level contamination to persist, which can develop inte full-scale contamination once the antibiotic is removed from media, and may hide mycoplasma infoctions and othor 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. I thoy are used in the long term, antiviotic-free cutures should be maintained in parallel as a control for cryptic infections. "7 For educational purposes only3. Cell Culture Basics Cell Lines This section provides information on the fundamentals of cel cuture, including the selection of the appropriate cell Ine for your experiments, media requirements for call couture, adherent vs. suspension culture, and morphologies of continuous cell ines available trom Thermo Fisher Sciantfic Note that the following information is an introduction to the basis of cel culture, ancl it Is intended as a starting point in your investigations. For more in-depth information, we recommend that you consutt published Iteralure and books, as wellas the manuals and product information sheets provided with the products you are using, Selecting the appropriate cell line Acquiring cell lines Consider the folowing criteria for selecting the appropriate col Ine for your experiments: * Species: Nonhuman and nonprimate cell ines usually have fewer biosafety restrictions, bbut ultimately your experiments will dictate whether to use species-specrfc cultures or rot. * Functional characteristics: What is the purpose of your experiments? For example, Iver and kidney-derived cell ines may be more suitable for toxicty testing © Finite or continuous: While choosing from finite cell ines may give you more options to express the correct functions, continuous cell nes are often easier to clone and maintain. * Normal or transformed: Transformed cell ines usually have an increased growth rate and higher plating efficiency, are continuous, andl require less serum in media, bout they have undergone a permanent change in their phenotype through a genetic, transformation * Growth conditions and characteristics: What are your requirements with respect to (growth rate, saturation density, cloning efficiency, and the abllty to grow in suspension? For exampk, to express a recombinant protein in high yields, you might want to choose a cellline with a fast growth rate and an abilty to grow in suspension, * Other criteria: Ifyou are using a finite cell ne, are there sufficient stacks available? Is the call ing well characterized, or do you have to perform the validation yourself? If you are using an abnormal cal ine, do you have an equivalent normal cell ne that you can Use as a control? Is the cell ne stable? Ifnot, how easy its to clone It and generate sufficient frozen stooks for your experiments? You may estabish your own culture from primary cells, or you may choose to buy established call cultures from commercial or nonprofit suppliers (.e.. cell banks). Reputable ‘supplies provide high-quality cel ines that are carefull tested for their integrity and to ensure that the culture is tree from contaminants. We advise against borrowing cultures from other laboratories because they cary a high risk of contamination. Regardless of their source, make sure that allew cell ines are tested for mycoplasma contamination before you bagin to use them. We otter a vaviety of primary cultures anc! established cell nes, reagents, media, sera, and ‘growth factors for your cell cutture exeariments. The Appendix section contains a lst of the more commonly used call ines available trom Thermo Fisher Scientific (see page 97). For mare information on our products, go to [Link] 18 | Coll Culture Basics For educational purposes only,Culture Environment One of the major advantages of cell culturs is the abiity to manipulate the physiochemical (.c., temperature, oH, osmotic pressure, O, and CO, tension) and the physiological environment (.c., hormone and nutrient concentrations) in which the cells propagate. With the exception of temperature, the culture environment is controled by the ‘growth mecia. While the physiological environment of the cuture is not as well defined as its physiochemical environment, a better understanding of the components of serum, the identification of the growth factors necessary for prolferation, and a better appreciation of the microenvironment of cells in culture (.¢., cel-cell interactions, cifusion of gases, actions wih the matrix) now allow the culture of certain fr Adherent vs. suspension culture There are two basic systems for growing calls in culture, as monolayers on an artical substrate (.0., adherent culture) or fo0-floating in the cutture mocium (suspension culture), Tho majority ofthe colls derived trom vertebrates, withthe exception of hematopoietic callines and a few others, are anchorage-clependent and have to be cultured on a suitable substrate that is specifically treated to alow cell adhesion and spreading (Le, tissue-culture treated), However, many cal ines can also be adapted for suspension cutture, Similarly, mast of the commercially available insect cell ines grow well in monolayer or suspension culture, Cells that are cutured in suspension can be maintained in culture flasks that are not tssue-culture treated, but as the culture volume- to-surface area is increased beyond which adequate gas exchange is hindered (usually 0.2-0.5 mLicm', the medium requites agitation. This agtation is usually achioved with a magnetic stiner or otating spinner Nasks. ‘Adherent Culture: Aapropriata for most cal tyoes, including primary cultures Roquies periodic passaging, but alows ‘easy visual inspection under inverted Calls are cssosiat Gibeo” Tiyp mechanically enzymaticaly | ass, vyosin) or 9 Growth is limited by surface area, which may Init product yolds Requires tissue-cutture treated vessel Used for cytology, harvesting products Continuously, and many research applications ‘Suspension Culture prate for ces adapted to su culture and a fow other call onadesive (e.g, hematopoietic Easior to passage, but requires dally col counts and vability determination to follow growth patterns; culture can be dikted to stimulate growth Does not require enzymatic o machanical dissociation Growth is Imited by concentration of ces in the medium, which allows aasy scale-up ‘Can be maintained n culture vessels that aro not tissue-culture trated, but requiras gation (Le., shaking or string) for adequate gas exchange Used for bulk production, bateh harvesting and many research applications For educational purposes only, 19Media The culture medium is the most important component of the culture environment, because it provides the necessary nutrients, growth factors, and hormones for call growth, as wall as regulating the pH and the osmotic pressure of the cullure. ‘Atthough intial cel cuture experiments were performed using natural media obtained from tissue extracts and ody fics, the need for standardization and media quailty, as well as an increased demand led to the development of chemically defined madia, The three basic classes of media are basal media, reduced-serum media, and serum-free media, which differ in their requirement for supplementation with serum. ‘Serum is vitally important as a source of growth and adhesion factors, hormones, lipids, land minerals for the culture of calls in basal media. In accion, serum also regulates call membrane permeability and serves as a carrer for lipids, enzymes, micronutrients, ‘and trace olomonts into the cal. However, using sorum in media has a numbor of disadvantages inclucing high cost, problams with standardization, specticity, and variabilty, and unwanted effects such as stimulation or inhibition of growth andlor collar function on cortain coll cultures, f the sorum is not obtained from reputable source, ‘contamination can also pose a serious threat to successful coll cuture experiments. Ou Gibco™ products, including sera, are tasted for contamination and guaranteed for their quality, safety, consistency, and regulatory complance, Basal media The majority of cell lines grow well in basal media, which contain amino acids, vitamins, inorganic salts, and a carbon soures such as gkicose, but these basal media formulations ‘must be further supplemented with serum, Reduced-serum media ‘Another strategy to recluce the undesired effects of serum in cell culture experiments is to Use reduced-serum media, Reduced-serum media are basal media formulations enriched with nutrients and animal-derved factors, which reduce the amount of serum that is needed. Serum-free media ‘Serum-free magia (SFM) circumvents issues with using animal sera by replacing the serum with appropriate nutritonal and hormonal formulations. Serum-free media formulations ‘exist for many primary cultures and cell ines, including recombinant protein-producing Ines of Chinese Hamster Ovary (CHO), various hyiaridoma cell ines, the insect lines S19 and S121 (Spodoptera frugiperda), and for cel ines that act as hosts for viral production, such as 283, VERO, MDCK, MDBK, and others. One of the major advantages of using serum-free media is the abil to make the medium selective for specific cell types by choosing the aporopriate combination of growth factors. The table below lsts the advantages and disadvantages of serum-free media ‘Advantages Disadvantages + roreased defnton ' Requirement for call type-specific mea * More consistent performance formulations + Nead for higher degree of reagent purty * Easior purifcation and downstream processing + Slower growth * Procise evaluation of cellular functions + Inreased productivity * Better control over physiological response + Enhanced detection of celular mediators 20 | Coll Culture Basics For educational purposes onlypH CO, Temperature We otfer a wide range of classical basal media, reduced-serum media, and serum-free media, as wellas sera, growth factors, suoplements, antibiotics, and reagents for your cell culture experiments. The Appendix section contains a ist of the more commonly used cell culture products available from Therma Fisher Scientific. For more information, go to [Link] Most normal mammalian cell lines grow well at pH 7.4, and there is very ltl variability ‘among different coll strains. However, some transformed cell nas have bean shown to (grow battor at sightly more acidic environments (oH 7.0-7.4), and some normal fisroblast Coll Ines prefer sightly more basic environments [oH 7.4-7.7). Insect cell lines such as ‘S19 and S121 grow optimally at pH 6.2. he growth medium controls the pH of the cuture and buffers the colls in culture against ‘changes in the pli. Usually, this buffering is achiaved by including an organic (e.g., HEPES) CO,--bicarbonate based buffer. Because the pH of the medium is dependent on the doleate balance of dissolved carbon dioxide (CO,) and bicarbonate (HCO, changes in the atmospheric CO, can ater the pH of the medium. Therefore, fis necessary to se exogenous CO, when using mecia bultfered with a CO,-bicarbonate-based buffer, ‘especially ifthe cell are cutured in open dishes or transformed cell ines are cultured high concentrations. While most researchers usually use &-7% CO, in alr, 4~10% CO, is common for most cell culture experiments, However, each medium has a recommended (CO, tension and bicarbonate concentration to achieve the correct pH and osmolalty; refer to the media manufacturer's instructions for more information, The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated, and to a lesser degree on the anatomical Variation in temperature (@.g., temperature of the skin may be lower than the temperature of skeletal muscle), Overheating is a more serious problem than undetheating for cell ccutures: therefore, often the temperature in the incubator is set slightly lower than the optimal temperature, + Most human and mammalian cell lines are maintained at 36-27°C for optimal ‘to * Insect cells are cultured at 27°C for optimal growth; they grow more slowly at lower temperatures and at temperatures between 27°C and 30°C, Above 20°C, the viability of insect cells decreases, and the cells do nol recover even aller they are returned to 27°C, * Avian cell lines require 38,5°C for maximum growth, Although these calls can also be maintained at 37°C, they will row more slowly. * Col lines derived from cold-blooded animals (o.g., amphibians, cold-water fish) tolerato a wide temperature range between 15°C and 26°C. Note that call culture conditions vary for each cell type. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. We therefore recommend that you familiarize yourself with your cal line of interest, andl closely follow the instructions provided with each product you are using in your experiments. 2 For educational purposes onlyCell Morphology Mammalian Cells Regularly examining the morphology of the cells in cullure (.¢, their shape andl appearance) is essential for successful coll culture experiments. In addition lo confirming the healthy status of your cels, inspec cells by eye and a microscope each time they are handled will low you to detect any signs of contamination early on and to contain it before it spreads to other cultures around the laboratory, Signs of deterioration of calls incluce granularity around the nuclous, detachment of the cells from the substrate, and cytoplasmic vacuolation. Signs of deterioration may be caused by a variety of reasons, including contamination of the culture, senescence of the coll Ine, or the presence of oxic substances in the medium, or thay may simply imply that the culture needs a medium change. Allowing the deterioration to progress too far will make it reversible. Variations in mammalian cell morphology Most mammalian cells in culture can be vided in to three basic categories based on their morphology. * Fibroblastic (or fbroblast-Ike) cells are bipolar or multipolar and have elongated shapes, Thay grow attached to a substrate, * Epithelialstike calls are polygonal in shape with more regular dimensions, and grow attachod to a substrate in disorete patches. + Lymphoblast-like calls are spherical in shape and are usually grown in suspension without attaching to a surface, In addition to the basic categories Isted above, certain cells display morphological characteristics specific o their specialized role in its host, * Neuronal cells exist in ctferent shapes and sizes, but they can roughly be alviced into two basic morphological categories, type with long axons used to move signals over long distances and type Il without axons. A typical neuron projects cellular extensions with many branches from the cell body, which is refered to as a dendritic tree. Neuronal ‘calls can be unipolar ot pseucounipolar with the denckite and axon emerging from the same process, bipolar with the axon and single dendrite on opposite endis of the soma {the central part ofthe cell containing the nucleus). or multipolar with more than two dendrites, 22 | Coll Culture Basics For educational purposes only,Morphology of 293 cells The 293 call lino is a permanent line established from primary embryonic human kidney, which was transformed with sheared human adenovirus type 5 DNA. The adenoviral ‘genes expressed in this cell line allow the cells to produce very high levels of recombinant proteins. We offer several variants of the 288 cell line, including thase adapted for high density suspension culture in serum-free media. For more information, visit the mammatian call culture pages on our website. he phase-contrast images below show the morphology of healthy 293 cells in adherent culture at 80% confluency (Figure 3.1) and in suspension cuture (Figure 3.2). Note that adherent mammalian cultures should be passaged when they are in the log phase, before they reach confluence (see When to subculture, page 27} Figure 3.1. Phaso-contrast images of healthy 288 coll in adheront culture, The cals wors plod at 3 soedng densiy of 9x "0" viable celsion? n Gbeo"™ 298 SM Imacum and gronn as a monolayer in & 37°C ncubator with a humilfed atmospnarec! 5% CO, in ak The Images were obtained using TOx and 20x ‘Objectives (panels A and B, respectively 4 days ater pang, Figure 2.2. Phase-contrast images of healthy 283F cells grown Is suspension. The culo vas statec ina shake faaiat a seeding densty 2° 2 x 10" vabl cell. ry Giboo 299 SFM I macsum and grown in 8 ‘87°C inebator witn a ruriciied atmosphere of 8% CO, in si. The cells were cikted 1:8, an the images. ‘were obtained using 10x and 20x objectwes ‘panels A and B, respectively 4 cays ater seeding For educational purposes onlyInsect Cells Morphology of Sf21 cells ‘S121 coll (PLB-S121-AB) are ovarian coll isolated fram Spodoptera frugiperca (all ammyworm). They are spherical in shape with unequal sizes, and have a somewhat (granular appearance. S121 cells can be thawed and used directly in suspension cuture for rapid expansion of call stocks, propagation of baculovitus stocks, and production of recombinant proteins. Because S121 calls attach frmly to surfaces, thay can be used as a monolayer for transfection or plaque assay applications. he images below show the morphology of healthy S21 insect cells in suspension culture [Figure 8.3) and in adherent cuture at confluency (Figure 2.4). Note that insect cells should bbe sulbcultured when they reach confluency (soe When to Subculture, page 27) Figure 3.3. Phaso-contrast images of healthy S121 insect cells grown in suspension. Tho culture was Started ina shake ask ata seeding consi of 3 10" vablecell/mL m Gibco'™ S001 SFM and mantained ih a 28°C, nonhumicied, ambient a-reguated incubstor. The images ver obtained using 10x and 20x objectives (panels A and [Link] 8 days ater seeding, Figure 2.4. Phase-contrast images of S121 insect cells grown as an adherent monolayer in Gitco™ 298 SFM Il medium, Tho coll wore pbc ata soocng density of 9 x 10" vlablecolefort a 7-25 Task ard grown as moralayors n a 28°C, nonhured, ambiontar-rogulated incubator. Tho agos wore ‘Sbiained using 10x and 20x cojectves pana A ane & respectively 7 Saye ater seecng, wnen the culture had reached confbancy 24 | Coll Culture Basics For educational purposes only,Morphology of Sf9 cells The S19 insect cell ine is a clonal isolate derived from the parental Spodoptera frugiperda cell ine IPLB-St-21-AE, and is a suitable host for expression of recombinant proteins ‘from baculovirus expression systems (e.g, Invitrogen" Bac-to-Bac™ and Bac-N-Blue™ Expression Systems). Although insect cels have been historically cultured in stationary systems ullzing I-lasks and serum-supplemented basal medium, insect cells are generally not anchorage dependent and can easily be maintained in suspension culture he images below show the morphology of heatthy S'9 insect cals in suspension and adherent cultures. Si9 cols attach firmly to surfaces, and their small, regular size makes them exceptional for the formation of monolayers and plaques. Figure 8.5, Phase-contrast images of healthy S19 insect cells grown in suspension. The cule was started i a shake flsk ata seeding densty of 8x 10" vale cellvm In SI-000 II SFM medium and was maintained ina 28°C, ronhuicifed. ambient 2r—egultas naunator. The sages were obtained using 10x and 20x objectives panels A and B, respectvaly) 8 days after seeing, Figure 8.6. Phase-contrast images of healthy S19 insect cells grown in adherent culture. Tasks ‘wore send! at 5» 10" viable calefow In 5-900 SFM and maraned n 828°C, nonhuridfed, ambent air-requlaiec incubator. The mages were obiained using 10x and 20x objectives (panels A and, respectively) 3 days ater sooding For educational purposes only4. Cell Culture Methods AN This section provides guidelines and general procedures for routine subculturing, thawing, and freezing of calls in culture. Note that cell cute conditions vary for each call type. The Cconeaquencas of deviating from the culture conditions required for a particular cell type ‘can range from the expression of aberrant phenotypes to a complete failure of the cell culture. We therefore recommend that you familiarize yourself with your call ine of interest, and closely foliow the instructions provided with each product you are using in your experiments, Guidelines for Maintaining Cultured Cells What is subculture? ‘Subcutturing, also roterrod to as passaging, is tho romoval of the medium and transfer Of cols rom a previous culture into fresh growtn medium, a procedure that enables the further propagation of the call ine or cal strain. The growth of cells in culture proceeds from the lag phase following seeding to the log phase, where the cells proliferate exponential. When the calls in adherent cultures ‘occupy all the available substrate and have no room let for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proiferation is greatly reduced or ceases entirely (see Figure 4,1, below), To keep the culture at an optimal density for continued cell growth and to stimulate further proliferation, the culture has to be divided and fresh medium supplied, 10° ‘Subculture 2 ze | g | 5 3 10 a ° | to Taman pea oO 2 4 6 8 10 Days Figure 4.1 Characteristic growth patter of cultured cells The sa-logaithmis po! shows he ost dersy ‘ets the te spent in cure, Cells in culture usualy praferate folowing a standars growth pattern, The ist ase of gow afte the cultures seeded isthe lag phase, whch is a period of show grow when tre cols are adapting tothe culture environment and preparing for fast growth. The ag phases flowed bythe log pase, ‘logarthnic" phase) a period wnee the cel rolfeateexporentialy a consume he nuvien’s fs the growth mectum, Wnon athe xourtn medlum s spent j..,on9 F move of ho rutionts i deplored oF when the cols occucy allt the avails suostrato, to cls entor ho statonary prasej.6., plateau phase), ‘hero the proleraton ie resuced or caases entire 26 | Coll Culture Basics For educational purposes only,When to subculture? The criteria for determining the need for subculture are similar in adherent and suspension cultures; however, there are some differences betweon mammalian and insect call lines. Cell density + Mammalian cells: Achorent cultures should be passaged when they are in the log pphase, before they reach confluency. Normal osls stop growing when they reach ‘confluency (eontact inhibition), and it takes thei longer to recover when reseed. Transformed cells can continue prolferating even after they reach confluency, but they usually deteriorate after about two douilings. Sinvlarly cals in suspension should be passaged when they are in log-phase growth before they teach confluency, When they reach confluency, cells in suspension clump together and the medium appears turbid when the cullue flask is swirled, * Insect cells: Insect cells should be subcultured when they are in the log phase, boefore they reach confluency. While tightly acherent insect cells can be passaged at confluency, which allows for easier detachment from the culture vessel insect cells that are repeatedly passaged at densities past contlienoy cisplay decreased douibing times, decreased viabilties, and a decreased abilty to attach, On the other hand, passaging insect cals in adherent culture before they reach confluency requites mote mechanical force to dislodge them from the monolayer. When repeatedly subcultured betore confluency, cells also display decreased doubling times and decreased viabilties, and are considered unhealthy, Exhaustion of medium ‘+ Mammalian cells: A crop in the pH of the growth medium usually indicates a buildup Of lactic acid, which is a by-product of cellular metabolism, Lactic acid can be toxic to the cells, and the decreased pH can be suboptimal for cell growth, The rate of change of pH is generally dependent on the cell concentration —cultures at a high cell concentration exhaust medium faster than cells at lower concentrations, You shoul suibculture your cels if you observe a rapid drop in pH (>0.1~0.2 pH units) with an Increase in cell concentration, * Insect cells: Insoct colls aro cultured in growth media that are usvaly more acidic than those used for mammalian cells, For example, TNM-FH and Grace's medium used for culturing S19 cells have a pH of 6,2, Unlike mammalian cell cutures, the pH rises gradually as the insect cals grow, but usually does not exceed pH 6.4, However, as with mammalian cells, the pH of the growth medium wil stat falling when insect cells reach higher densities, Subculture schedule Passaging your cells according to a stict schedule ensures reproducible behavior and allows you to monitor their health status. Vary the seeding density of your cultures until you achieve consistent growth rates and yields appropriate for your cal type from a given ‘seeding density. Deviations from the growth patterns thus established usually indicate that the cukure is unhealthy (e.g, deterioration, contamination) or a component of your culture system is nol functioning properly (e.g, temperature is not optimal, culture madium too cd). We strongly recommend that you keep a detailed eel eulture log, listing the feeding {and subculture schedules, types of media used, the dissociation procedure followed, split ratios, morphological observations, seeding concentrations, yields, and any antbiolic use. Itis best to perform experiments and other nonroutine procedures (0.9., changing type of media) accorcing to your subculture schedule. your experimental schadula doos not ft the routine subcuture schedule, make sure that you de not passage your cals while they 2 stil inthe lag period or when they have reached contuency and have ceased growing. 2 For educational purposes onlyMedia recommendations for common cell lines Mary continuous mammalian cell ines can be maintained on a relatively simple medium ‘such as MEM supplemented with serum, and a culture grown in MEM can probably be just as easily grown in OMEM or Mecium 199. However, when a specialized function is expressed, a more complex medium may be required. information for selecting the appropriate medium for a given cell type is usually available in published iterature, and ‘may also be obtained from the source of the calls or call banks. IF there is no information available on the appror the growth mi iate medium for your cell type, choose um and serum empirically or test several different mecia for best results cols. The conditions Iisted below can be used as a guideline when setting up anew ‘mammalian cell culture Insect cols are cultured in g Is, and it wth media that aro ide TNM-FH and Gr Mammalian Cell Culture Cell Line Colltype | Species Tissue Medium” 280 human | embryonic Kdrey 36 mouse | enbyo A549 ‘epithelial numan | tng carcinoma F-12K, 10% FES a9 mouse ctv tise DMEM, 10% FBS pth! mouse | ptutary tuner F=10, 18% horee serum, and 2 BALRISTS ‘robles mouse | embye BHK2 Aibrobles hamster | knay BHL-100 opto! ruman | breast MeCoy’s 8A, 10% FBS eT ‘blast ovne | ‘urbinate cate MEM, 10% FBS, and NEMA epithet numa adenocarcinoma chang ‘epithet human | Wer (CHO-KT epithelial hamster | ovary path cS F=12K, 10% FES eptheial ‘mouse | melanoma F-10, 15% horse sorum, and 2.5% FBS foro monkey | Kaney opto cat sehey ova foros monkey | kidney Dar epithelial cog osteosarcoma Daudi Iymohoblast | muman | stood tor a yrmohema patient | RPNI-T640, 10% FBS He pare 5 pituitary tumor F-10, 15% horse sou, and 2. * BME: Basal Medum Eagle; DMEM: Dulbecco's Mocltad Eagle Medium: FBS: Fetal Bovine Serum; GMEM: Glasgow Mima IMDM: Iscove's Modied Dulbecco’ Medivn: MEM: Minimum Essential Mediun; NEA: Nor-Essential Amino cds Salton, nil Machu 28 | Cell Culture Basios For educational purposes onlyMammalian Cell Culture, continued rmystoiest | muman | Taw tomenttvleuceria | guy ee ee ee EES ee * BME: Basal econ Eagle; MEM Dulbacco’s Mocked Eagle Medi FBS. Fatal Boyne IMDM: Iscove's Modfed Dulbecco’ Masi: MEM: Minimum Essential Maury; NEAA: Non-Essental Amino Acids Salton ‘TNM-FH: Tichoplusia ni Medum-Formulaton Hink je. Grace's hsect Medium, Supplemented) Serum GMEM: Glasgow Minimum Essential Mecium) For educational purposes only 29Dissociating adherent cells TrypLE dissociation ‘enzymes he first step in subculturing acherent cells is to detach them from the surface of the couture vessel by enzymatic or mechanical means. The table below lists the various cell dissociation procedures. Procedure Dissociation Agent ‘Applications Gant shaking or rocking of Shake-off couture vessal, or vigorous. Loosely adherent cals, mitotic beside cols soraping ll sraper Cal inas sonsitive to proteases; Sereping Gall scraper may damage some calls Trypsin Strongly adherent calls High-density cultures, cultures Trypsin and collagenase that have formed multiple layers, especially Fbroblasts Dotaching epidermal calls as Enzymatic confluent, intact sheets from the dissociation Discase swace of culture dishes withou dissociating the cels ‘Stongly adherent cells; dct substitute for trypsin; applications that woquio animal orgin=tres reagen's TiypLE dissociation enzyme Gibco" TrypLE™ Express and TiypLE™ Select enzymes are microblally produced cell dissociation enzymes with similar kinetics and cleavage specificities to trypsin. Although TiypLE enzymes can directly substitute for wypsin in dissociation procedures without a need for protocol changes, we recommend that you intially optimize the incubation time for dissociation for best results. Because TrypLE enzymes are recombinant fungal trypsin lke proteases, they are ideal for applications thal require animal origin~free reagents. The table below compares TrypLE Express and TrypLE Select enzymes to trypsin ‘TrypLE Express and TrypLE Select hs pgs erica a yr ‘Completely ree of animal- and human- Porcine- or bovine-derived detived camaonants Stable at oom temperature for at least six | Not stable at oom temperature Not inhibited by serum Inhibited by serum Doas not require trypsin nactivators Roguirs trypsin inactivators 80 | Coll Culture Basics For educational purposes onlySubculturing Adherent Cells Materials needed Protocol for passaging adherent cells he folowing protocol is a general procedure for subculturing adherent mammalian cells in cutture, Note that the procedure for passaging insect cals differs fram that {for mammalian cells on several crucial stens. For mare information, refer to Notes on ‘Subculturing Insect Cells, next page. For passaging your own cell line, we recommend that you closely follow the instructions provided with each product you are using in your experiments. The consequences of deviating from the culture conditions required for a particular cell ype ‘can range from the expression of aberrant phenotypes to a complete falure of the cell culture, Culture vessals containing your adherent calls * Tissue cullue-treated flasks, plates, or dishes * Complete growth medium, prewarmed to 27°C * Disposable, sterile 15 mL tubes * 37°C incubator with humisified atmosphere of 5% CO, * Balanced salt solution such as Dulbecco's Phosphate- fered Saline (DPBS), ccontaining no caleium, magnasium, or phenol rea * Dissociation reagent such as trypsin or TiypLE Exoress enzyme, without phenol red * Reagents and equipment to determine viable and total cell counts (@.g., Countess Automated Cell Counter) All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood, Remove and discard the spent cell cul ure medium from the cukure vessel Wash cells using @ belanced sat solution without calcium and magnesium (approximately 2mL per 10 om* cultute surface area). Gently add wash solution to the side of the vessel ‘opposite the altached call layer to avoid disturbing the cell layer, and rock the vessel back and forth several times. Note: The wash stop removes ary tracos of eerum, calcium, ancl magnesium that would inhibit the action ofthe cissocition reagent. Remove and discard the wash solution from the culture vessel ‘Add the prewarmed dissociation reagent such as typsin or Tryp the flask: use enough reagent to cover Gently rock the container to gf enzyme to the side of 1 coll layer (approximately 0.8 mL per 10 em’ t complete coverage of the cell layer Incubate the culture vessel at room temperature for approximately 2 minutes. Note that the ‘actual incubation time vavies with the cell line used, a For educational purposes only,Notes on subculturing adherent insect cells Observe the cells uncler the microscope for detachment. If calls are less than 90% detached, increase the incubation time a few more minutes, checking for dissociation every 80 seconds. You may also tap the vessel to expedite cell detachment, When 200% of the calls have detached, tit the vessel for a minimal length of time to allow the cells to drain, Add the equivalent of 2 volumes twice the volume used for the clscociation reagent) of prewarmed complete growth medium, Disperse the medium by pipetting over the call layer surface several times. ransfer the calls to a 15 mL conical tube and centrifuge them at 200 x g for 5-10 minutes. Note that the centrifuge speed and time vary based on the coll type. Resuspend the call pellet in a minimal volume of prewarmed complete growth medium and remove a sample for counting, Determine the total number of calls and percent viabilty using a hemocytometer, cell counter, and trypan blue exclusion, or the Countess Il Automated Coll Countor. If ecassary, add growth medium to the calls to achiave the desired cell concentration and recount the cals Dilute the call suspension to the seeding density recommended for the cell Ine, and pipet the appropriate volume into new cell culture vessels, and return the cells to the incubator. Note: If using culture flasks, loosen the caps before placing them in the incubator to allow proper gas exchange unless you are using vented flasks with gas-permeable caps. While the general procedure for suboulturing insect cals follows the same steps as ‘mammalian cells, some key requirements of these culture systems are diferent, For best resutts, always follow the instructions provided with each product you are using in your experiments, * Passage insect cells at log phase. However, if your insect cells are strongly adherent, you may passage them at confliency or slightly after when they are starting to pull away from the bottom of the flask. Gells will be easier to dislodge. * Densities lower than 20% confluency inhibit growth, The heathest cells are those taken from log-phase cultures, * CO, exchange is not recommended for insect call culture, * Maintain insect calls at 27°C in a nonhumidifed environment. Cells can be maintained at room temperature on the benchtop if protected from light or in a drawer. However, a 27°C controlled environment is recommended * Use media specifically formulated for insect cell growth, * Insect cells attach very tightly to substrates under serum-free conditions and require ditional effort to detach, To dislodge the calls, you may need to give the flask one uick shake using a wrist-snapping motion, To avoid contamination, always tighten the ‘cap before this procedure. Caution: We do not recommend shaking the flask vigorously, because it may resutt in damage to the cals. 982 | Coll Culture Basics For educational purposes onlySubculturing Suspension Cells Passaging suspension cultures, Suspension culture vessels The following protocols describe general procedures for subculturing mammalian cells in suspension culture. Note thal the procedure for passaging insect cols differs from that for mammalian cells on several crucial steps. For more information, refer to Notes on Subcutturing Insect Cells, page 36. For passaging your own callline, we recommend that you closely follow the instructions provided with each product you are using in your experiments. The consequences of deviating from the culture conditions requied for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cel culture. ‘Subcuturing suspension cals is somewhat less complicated than passaging adharent colls, Because the cals are already suspended in growth medium, there is no need to treat them enzymaticaly to detach them from the surface of the culture vessel, andl the whole process is fastor and less traumatic for the calls. Replacement of growth medium is not carried out in suspension cultures; instead, the colls aro maintainod by fooding them very 2-3 days untl they reach confluency. This can be dene by directly cluting the cells in the culture flask and continuing to expand them, or by withdrawing a portion of the cells trom the culture flask and diluting the remaining cells down to a seeding density appropriate for the call ine, Usually, the lag period following the passaging is shorter than that observed with adherent cultures, ‘Suspension outures can be maintained in stele culture flasks that are not tissue culture~ treated; however, spinner flasks (.¢., stirrer bottles) spectically designed for suspension cell culture allow for superior gas exchange and permit higher voimes of cells to be cultured, Roller bottles rotating on a rack may also be used to agitate suspension cultures. Spinner flasks have two basic designs: the medium is agitated (.e., stired) by a hanging ti-bar assembly or with a vertical impeller, The vertical impeller provides better aeration he total culture volume in a spinner flask should not exceed half ofthe indicated volume Of the spinner for proper aeration (e.g., a 00 mL spinner should never contain more than 2260 mL of culture) Verteal Iroetr 33 For educational purposes only,Materials needed —* Culture vessels containing your suspension calls + Shaker flasks without bates or spinner bottles (8e Suspension Culture Vessels, previous page) + Complete growth medium, prewarmed to 37°C * 37°C incubator with humistiod atmosphore of 5% CO, + Magnoti sti plate f using spinnor flask), roller rack (fusing rollr bottles), or shaking platform (fusing conventional culture flasks or Pati dishes) + Reagents and equipment to determine viable and total cell counts (e.g, Countess I Automated Cell Counter) Protocol for passaging suspension cells _Allsolutions and equipment that come in contact with the cells must be steril Always use proper sterile technique and work in a laminar flow hood. Subcuture colls vwhon they are in log-phase growth before they reach confluency, Wen they reach confluency, cells in suspension chimp together andi the meclum appears turbid wnen the culture flask is swirled. The maximum recommended cel density before passaging varios with coll lines: refer to the cell-speciic product insert or manual for deta Cells grown in shaker flasks The following protocol is a general procedure for passaging mammalian cells grown in suspension culture using shaker flasks in a shaking incubator, For detailed protocols, always refer to the cell-specific product insert, Note: Make sure that the shaker flask does not have baffles (.e., the indents at the bbottom of the flask designed to provide agitation), because they ruin the shaking rhythm, 1. When the cells are ready for passaging (.¢., log-phase growth before they reach ‘confluency), remove the flask fiom the shaking incubator, and take a small sample from the cukure flask using a sterile pipette. If cells have settled down before taking the sample, wi the flask to evenly cistribute the cells in the medium, 2. From the sample, determine the total number of cels end percent viabilty using the Countess Il Automated Cell Counter or a hemocytometer, cell counter, and trypan blus exclusion 2. Calculate the volume of media that you need to add to dilute the culture down to the recommenced seeding densly 4, Aseplically add the appropriate volume of prewarmed growlh medium into the culture flask. You may split the culture into multiple flasks if needed. 5. Loosen the cans of the culture flasks one full urn to alow for proper gas exchange (or use ‘a gas-permeable cap), and return the flasks to the shaking incubator. The shaking speed depends on the cell ine. Note: To minimize the accumulation of coll debris and metabolic waste by-oroducts in shaker cultures, gently centrifuge the cell suspension at 100 x g for 5~10 minutes, and resuspend the coll pellet in fresh growth medium once every three weeks (or as naedod) 94 | Coll Culture Basics For educational purposes onlyCells grown in spinner flasks he following protocol is a general procedure for passaging mammalian cals in suspension grown using spinner flasks. For detailed protocols, always refer to the cell-specific product insert. Note thal calls are sensitive to physical shearing. Ensure that impeller machanisms rotate {realy and do not contact vessel walls or the base, The top of the paddles should be slightly above the mecium to ensure adequate aaration of the cuture. Adjust the spinner ‘mechanism so that paddles clear the sides and the bottom of the vessel. The table below Its the minimum volumes of media needed for differant spinner flask sizes, Size of Spinner Fiask | Minimum Media Volume (00 mL 30 mL 260 mi 80m 500 mL 200 mL We do not recommend initiating a spinner cutture into a spinner flask larger than $00 mi \We suggest scaling up from smaller spinners that have already been established. When the cells are ready for passaging (Le., log-phase growth before they reach confluency), remove the flask from the shaking incubator, and take a small sample from the culture flask using a sterile pipette. If cells have settled down before taking the sample, wil the to flask evenly distribute the cells in the medium, . From the sample, determine the total numberof cells andl percent viabilly using the Couintess Il Automated Cell Counter or a hemocytometer, cell counter, and trypan blue exclusion Caloulate the volume of medium that you need to add to dilute the cuture down t recommenced seeding densty the Aseplically add the appropriate volme of prewarmed growth medium into the culture flask. You may split the culture into multiple flasks if needed. Loosen the side arm caps of the spinner flasks one full turn to allow for proper gas ‘exchange, and return the flasks to the incubator. The spinner speed depends on the cell Ine and the impeller type. Make sure that the spinner speed is kept within the recommenced values to avoid damage to the cells from shear stress. Note: To minimize the accumulation of cell debris and metabolic waste by-products in spinner cultures, gently centrifuge the cell suspension at 100 x g for 5=10 minutes, and resuspend the cell pallet in fresh growth medium once every three weeks (or as needed) For educational purposes onlyNotes on subculturing suspension insect cells \While the general procedure for subculturing insect calls folows the same steps as mammalian cells, some key requirements of these cullure systems are cifferent. For best results, always follow the instructions provided with the insect cell ines you are using in your experiments. * itis not necessary to change medium when you are culturing cols in suspension. Rogular subculturing requires the removal of cell suspension and the adaition of medium sufficient to dilute culture to the appropriate density (refer to the cell-specific product insert). Adding fresh medium is sufficient to replenish cel nutrients, * CO, exchange is not recommended for insect cell culture, * Maintain insect cells at 27°C in a nonhumidifed environment, Cells can be maintained at room temperature on the benchtop or in a drawer; however, a 27°C controled environment is recommended, * Use media specifically formulated for insect cell growth, * Uso a surfactant to decrease shearing. 0.1% Gisco™ Pluronic™ F-68 surfactant is recommended for spinner insect cultures, as it decreases coll membrane shearing due 10 impelor forces. Note: Sf-900 II SM and Express Five SFM already contain surfactants. * Cottain insect call ines may require adaptation to suspension culture. For more information, refer to the col/-ine specific product insort or manual 96 | Coll Culture Basics For educational purposes onlyFreezing Cells Cryopreservation Guidelines for cryopreservation Cal lines in continuous culture are prone to genetic drift, finite cell lines are fated for senescence, all cel cultures are susceptible to microbial contamination, and even the best-1un laboratones can experience equipment failure. Because an established cell ine is @ valuable resource and is replacement is expensive and time consuming, its vitally important that they are frozen down and preserved for long-term storage, ‘As soon as a small surplus of calls becomes avaiable from subculturing, they should be ‘rozen as a seed stock, protected, and not be macle available for general laboratory use. Working stocks can be prepared and replenished from frozen seed stocks. Ifthe seed stocks become depleted, cryopreserved working stocks can then serve as a source for proparing a fresh seed stock with a minimum increase in generation number from the initial freezing, he best method for eryopreserving cultured cells is storing them in liquid nitrogen in Complete medium in the presence of a cryoprotective agent such as dimethyisulfoxide (DMSO). Cryoprotective agents reduce the freezing point of the medium and also allow a slower cooling rato, greatly rocucing the risk af ice crystal formation, which can damage cols and cause call death, Note: OMSO is known to faciltate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards, posed by such materials, Dispose of the reagents in compliance with local regulations, Following the guidelnes below is essential for eryopreserving your cal lines for future use. ‘As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cell line for best results. * Freeze your cultured cells at a high concentration and at as low a passage number as possible, Make sure that the cells are at least 90% viable before freezing, Note that the ‘optimal freezing conditions depend on the cell line in use. * Freeze the oslls slowly by reducing the temperature at approximately 1°C per min Using a controllac-rate cryo- freezer oF a cryo-tieezing container (e.g., Theimo Solentfic™ Mr, Frosty" Freezing Container * Always use the recommencled freezing medium. The freezing medium should contain a eryoprotective agent such as DMSO or glycerol (soe What is Subculture?, page 26). * Store the frozen calls below 70°C; frozen calls begin to deteriorate above ~50°C. * Always use storie cryovials for storing frozen cells. Cryovials containing the frozen calls may be stored immersed in Iquid nitrogen or in the gas phase above the lauid nitrogen (see Safety Note, page 22) + Always wear PPE. * Allsolutions anc equipment that come in contact with the cells must be sterile, Always: Use proper steril technique and work in a lamina flow hood. 7 For educational purposes only,Safety note Bichazardous materials must be stored in the gas phase above the laud nitrogen. ‘Storing the sealed cryovials in the gas phase elminates the risk of exolosion. Ifyou are using liquid-phase storage, be aware of the explosion hazard with bath glass and plastic cryovials, and always wear a face shield or goggles. Freezing medium Always use the recommended freezing medium for cryopreserving your cells. The freezing ‘maclium should contain a cryoprotective agent such as DMSO or glycerol. You may also use a special formulated complete cryopreservation medium such as Giboo™ Recovery™ Cell Cuture Freezing Medium or Synth-s-Freeze"™ Cryopreservation Medium Recovery Call Cutture Freezing Medium is a ready-to-use complete cryopreservation ‘medium for mammalian coll cultures, containing an optimized ratio of fotal bovine sorum to bovine serum for improved cell viabilty and cell recovery after thawing ‘Synth-a-Froeze Cryopreservation Medium is a chemically detined, protoin-oo, storle cryopreservation medium containing 10% DMSO that is suitable for the cryopreservation ‘of many stam and primary coll types, with the exception of melanocytes. Materials needed —* Culture vessels containing cutured cells in log phase of growth * Complete growth medium * Cryoprotactive agent such as DMSO (use a bottle set aside for cel culture; open only ina laminar flow hood) or a freezing medium such as Synit-a-Freeze Cryopreservation Medium or Recovery Cell Culture Freezing Medium * Disposable, sterile 15 or $0 mL conical tubes * Reagents and equipment to determine viable and total cell counts (¢.g., Countess Automated Cell Counter) * Sterile cryogenic storage vials (Le, cryovials) * Controlied-rate freezing apparatus or isopropanol chamber * Liquid nitrogen storage container For freezing adherent cells, in addition to the above materials, you need: * Balanced salt solution such as Dulbecco's Phosphate-Buffered Saline (DPBS), containing no calelum, magnesium, or phenol red * Dissoolation reagent such as trypsin or TrypLE Express enzyme, without phenol red 988 | Coll Culture Basics For educational purposes only