Workshop Training Series Biomedical and Obesity Research Core Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules Introduction to Mammalian Cell Culture April 9, 2019 Yongjun Wang Ph.D. Director of Biomedical and Obesity Research Core Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules What Is Cell Culture Cell culture is the process by which cells are grown in controlled conditions outside of their native environment. Timeline: key milestone in cell cultures History of Cell Culture http://dx.doi.org/10.5772/66905 Primary vs Cell line Primary cells Cell lines Lifespan and division capacity Limited Indefinite Isolated in the lab or bought from Source Bought from commercial provider commercial provider Care and maintenance Complex and difficult Easy to maintain or proliferate Chromosomal aberration Minimal Several Retention of functional markers and Yes Not always signaling pathways Functional study, diagnosis, Drug development, vaccine and protein Application Gene therapy, et al. production, et al. Three Types of Cells Epithelial-like cells Fibroblast-like cells Lymphoblast-like cells Cell differentiation 3T3L1 cells C212 cells Cell Culture Vessels • Most adherent cells require attachment to proliferate • Polystyrene are treated to become hydrophilic and negatively charged once medium is added • Coating with basic synthetic polymers • Poly-L-lysine • Coating with matrix proteins • Collagen, laminin, gelatin, fibronectin Class II Biological Safety Cabinet The Class II Biological Safety Cabinet can be found in almost every tissue culture lab. It is designed to: 1. Protect you from being exposed to infectious aersoles that may be generated within the cabinet. 2. Protect your cultures from microbial contaminants that are ubiquitous in room air Cell Culture Incubator • Normal body temperature 37oC • Humidity must be maintained at saturating levels as evaporation can lead to changes in • Osmolarity • Volume of media and additives • Carbon dioxide (5%) • Buffering • Oxygen (20%) • Aerobic metabolism • Atmospheric 21% • Tissue levels between 1-7% Medium Formulation • Inorganic ions • Osmotic balance – cell volume • Trace Elements • Co-factors for biochemical pathways (Zn, Cu) • Amino Acids • Protein synthesis • Glutamine required at high concentrations • Vitamins • Metabolic co-enzymes for cell replication • Energy sources • Glucose • Phenol red (option) • pH indicator 6.8-8.2 (yellow to red) Serum provides the following • Basic nutrients • Hormones and growth factors • Attachment and spreading factors • Vitamins, minerals, lipids • Protease inhibitors • pH buffer Cell Culture initiation-Primary Cell https://www.creative-bioarray.com/support/primary-cell-culture-guide.htm Cell Culture initiation- Cell Lines From storage ATCC From other people Passaging or Sub-culture Adherent Cells 1. Remove medium 2. Wash with PBS 3. Trypsin-EDTA treatment 4. Cell dispersion and resuspension 5. Replenish with fresh medium 6. Count cells 7. Split cells (1:2-1:10 depending on growth rates of each cell lines) Suspension Cells 1. Cell collection 2. Centrifuge 3. Resuspend and split the cells Note: Label the vessels with cell line name, passage number, date, treatment and et al. Cell Harvesting and Storage Storage Cell Based Assay Cell Based Assay • Transfection and infection • Transcription • Protein expression • Reporter gene assay • Drug Treatment • Functional assay (Differentiation, senescence, migration, apoptosis, glycolysis, mitochondria respiration, and et al) Primary application of animal cell culture • Model Systems • Genetic Counseling and diagnosis • Drug Screening and Development • Genetic Engineering • Cell-Based Manufacturing • Gene Therapy Cell Culture Problems • Spotting (Bubbles) • Uneven Growth (Does not mix well) • Static Electricity (low attachment) • Meniscus Rings (low volume) • Uneven evaporation (96 well plates) • Contamination Sources of Contamination Type of contamination Visual recognition Detection methods Eradication Bacteria microscopically PCR, microbiological culture Discard culture methods Yeast microscopically bald PCR, microbiological culture Discard culture, , perhaps use an Anti-mycotic methods Fungi microscopically bald PCR, microbiological culture Discard culture, perhaps use an Anti-mycotic methods Mycoplasma only indirectly, PCR, microbiological culture Discard culture, special antibiotics for morphological changes methods mycoplasma of cells, slower growth Viruses very difficult, electron PCR, serological, cell-based assays Discard culture or set up a new safety protocol microscope only Prions impossible mostly too complicated, risk BSE-low risk reagents of animal/human origin mitigation by reagent tests (FCS, proteins) Chemical contamination impossible mostly too complicated, risk QA tests on reagents (plastic ware, reagents) mitigation by reagent tests Bacterial Contamination • Small black dots • Easy to mistake for cellular debris, especially at low levels • Multiply fast • Acidic pH, media • Cells die fast (overnight) Yeast Contamination • Small round bright dots • Multiply faster than mammalian cells but slower than bacteria • Visible in 2-3 days after contamination • Resistant to antibiotics like penicillin and streptomycin Other fungi contamination • Hyphae-branched, tubular, thread-like filaments. • Mycelium-consisting of a mass of branching, thread-like hyphae. • Multiply faster than mammalian cells but slower than bacteria • Resistant to antibiotics like penicillin and streptomycin Mycoplasma contamination • The incidence of mycoplasma contamination is high, being 15 to 85% • Does not generate the turbidity • Undetected for long periods of time • No cell wall and resistant to common antibiotics • Causes morphological changes and altered growth rates Note: About 11% of NCBI’s Gene Expression Omnibus (GEO) projects between 2012 and 2013 contain at least ≥ 100 reads / million reads mapping to mycoplasma’s small 0.6 Mb genome Contamination Prevention Cause Prevention Operator (hair, hands, breath, clothing) Wash hands thoroughly. Use a lab coat reserved exclusively for culture room. Dust from skin, hair, or clothing dropped or blown into the Tie back long hair or wear a cap. Face away from work when talking is culture necessary. Aerosols from talking, coughing, sneezing, etc. Avoid working with a cold or wear a mask. Nonsterile surfaces and equipment Clean the cabinet before and after using it. Do not leave extra tubes, pipettes Dirty cabinet inside the cabinet Do not pour liquids. Dispense by pipette, auto dispenser, or transfer device. Spillage on necks and outside of bottles and on work If pouring is unavoidable: (1)do so in one smooth movement, (2) discard the surface bottle that you pour from. Discard waste into a beaker with a funnel or use vacuum to draw off waste Splash-back from waste beaker whenever possible. Sedimentary dust or particles of skin settling on the culture Never pass hands over open vessels. Do not work over an open bottle or dish. or bottle Swab outside of vessels with 70% alcohol before use them in cabinet. Dirty vessels and inadequately glassware sterilization Monitor performance of the autoclave with a recording thermometer or sterility indicator. Contamination Prevention -Materials and Reagents Cause Prevention Instruments, pipettes Use individually wrapped, sterile, disposable supplies from a reputable supplier. Unreliable sterility Sterilize reusable items by dry heat before using them. Contact with a nonsterile surface or some other Do not grasp any part of an instrument or pipettes that will pass into a culture material vessel. Touching or holding pipettes too close to tips. Hold pipettes above graduations. Handle sterile pipettes by overwrap sleeve. Solutions Nonsterile reagents and media Filter or autoclave solutions before use. Inadequate sterilization procedures Test or culture sterilized solutions if contamination is suspected. Obtain media, buffers, sera only from reputable suppliers that certify quality and Poor quality control at commercial supplier source. Environment Clean out refrigerator and incubators regularly. Dirty storage or incubation conditions Use screw caps instead of stoppers. Swab bottles before placing in hood. Cover Dust and spores from incubator or refrigerator caps and necks of bottles with aluminum foil during storage or incubation. Use secondary containment for plates if necessary. Add disinfectant and change the water in the water bath regularly. Dirty water bath Clean and swab bottles with 70% alcohol before placing in hood Other Considerations • Cell confluence • Sensitivity to mechanical dispersal or enzymes (Trypsin concentration) • Cell-cell contact may be required for proliferation • Most primary cells require satisfactory adherence (special coating) • In a mixed primary culture, differences in growth rate may mean a loss of the cell type of interest – selection techniques • Limited life span of some cultures • Functional characteristic changes • Some cell are prone to spontaneous transformation Some cell are prone to spontaneous transformation Rules of Cell Culture Room • Do not forget to reserve the cell culture room on iLab before using it no matter how shot you use it. • The maximum reservation time is 4 hrs. for one use. Please plan your experiments ahead. • Reservation may be canceled if the users do not show up one or more hr after the start of the reservation. • Do not interrupt people when people are doing experiments. • If you have to use the cell culture room in the time reserved by other users. Please ask politely. Rules of Cell Culture Room • Please clean the cabinet after using it. Do not leave extra tubes, pipettes inside the cabinet. • Do not forget to turn off the vacuum. • Remove unused medium from refrigerator • Please check the water pan in the incubator Rules of Cell Culture Room • Please separate the trash in different trash cans/bags. • Regular (Gloves, wrapping plastic bags, et al) trash goes to the black trash can. • Serological pipettes go to one orange biohazard trash bag. • Other biohazard trash go to another orange bag. • The recycling container by the door is for the waste that can be recycled. .