Cell Culture Techniques – Basic Principles 2024/2025 PDF

20/09/2024 Cell Culture Techniques – Basic Principles Fernanda Malhão 2024/2025 Cellular and Molecular Biology Techniques and Master Degree in Marine Sciences...

20/09/2024 Cell Culture Techniques – Basic Principles Fernanda Malhão 2024/2025 Cellular and Molecular Biology Techniques and Master Degree in Marine Sciences Applications in Marine Sciences What is Cell Culture? Cell culture refers to the removal of cells from an organism and their subsequent growth in a favorable artificial environment Fish Rat Organisms: Mollusc Insect Rabbit Plant Human Monkey Others… 1  20/09/2024 Applications Cell Culture Regulatory Industry Research testing (synthesize) purposes - Basic research – model system - Proteins (monoclonal antibodies, - Toxicity testing insulin, hormones,…) - Drug screening and development - Vaccines (polio, hepatitis B, rabies …) - Disease research - Replacement of tissue or organs - Genetic engineering - Cellular Agriculture - Gene therapy -… -… Examples of applications - Toxicology 2  20/09/2024 Examples of applications 3  20/09/2024 Examples of applications - Aquaculture Examples of applications 4  20/09/2024 How are cells obtained? Tissue/organ Tissue isolation isolation Primary culture Organ Organotipic Explant Cell Culture Culture Culture Adapted from Culture of Animal Cells - A Manual of Basic Technique 5th ed - I. Freshney (Wiley, 2005) Cell culture – types Cell Culture Primary Culture Cell Line Obtained from original tissue/organ Continuous- Immortalised Finite (limite number of (infinite number of cell cell divisions) divisions) Derived from tumors Senescence transformed spontaneously or transformed e.g virus 5  20/09/2024 Primary culture from fish Hepatocytes isolation Procedure: - Anaesthetize the animal; - Open the ventral region and liver exposure; - Portal vein cannulation; - In situ perfusion of the liver with buffers and enzymatic solutions; - Disperse cells in buffer; - Cell washing; - Determination of cell number and viability; - Use for cell suspension assays or primary cultures of hepatocytes. Other examples: Gills, kidney, … https://youtu.be/oOHUXk-KBVs https://youtu.be/ai84eQ5Mktg Cell Immortalization Spontaneous mutation Virus infection Expression of Telomerase Reverse Transcriptase (TERT) Protein https://www.creative-bioarray.com/support/immortalized-cell-culture-guide.htm 6  20/09/2024 Cell Growth Finite Cell Line Primary culture and Cell Line Animal Plant Tissue Enzimatic digestion Primary culture First subculture Cell line (limited or unlimited life span) 7  20/09/2024 Primary versus Cell lines Primary Culture Cell lines Derived directly from a tissue Derived from primary cultures Advantages: (tumors or transformed) Retain many of the differentiated Advantages: characteristics of the cell in vivo Can be propagated finite or (high specificity) indefinitely Greater ability to biotransformation More homogenous (genotypic and (retain enzymatic activity) phenotypic uniformity) Disadvantages: Unlimited availability Initially heterogeneous High growth capacity The preparation is labour intensive Free of pathogens Each assay is needs new isolation Consistent/Reproducible results Limited period of time (few days) Disadvantages: May harbor pathogens that were Retained a few original present in the tissues characteristics of the cell in vivo Type of growth Adherent or Suspension culture monolayer culture Growth attached to a solid or semi-solid substrate specifically treated to allow cell adhesion and spreading (e.g., tissue- Cells grow in suspension in the culture medium culture treated surfaces) Nonadhesive cells (e.g.,hematopoietic) Growth in monolayer Growth is limited by concentration of cells in the Growth is limited by surface area (when medium confluent, requires enzymatic or Does not require enzymatic or mechanical mechanical dissociation) dissociation Requires agitation for adequate gas exchange 8  20/09/2024 Cell Culture Morphology Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate (e.g., CHO – Chinese Hamster Ovary Cells) Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate (e.g., HeLa – tumour cell derived from the carcinoma of human cervix; RTL-W1 – cell line isolated from liver of an adult rainbow trout). Lymphoblast-like cells are spherical in shape and usually grown in suspension (e.g., HL60 – Human Leukemia – derived cells) **Cell morphology can vary with the cell density Henrietta Lacks – The story beyind cell culture On February 8, 1951, George Gey of Johns Hopkins University isolated some cells from a cervical cancer biopsy and placed them into a petri dish with some medium 9  20/09/2024 Outputs Molecular Biology (gene expression, protein expression) Immunocytochemistry CELL CULTURE Flow Cytometry Microscopy Spectrophotometr Cytogenetic y 19 1961 10  20/09/2024 Examples of applications - Fish cell line-based assays have been proposed as alternatives to fish in aquatic toxicity testing - As tools to explore toxicity pathways at the molecular and cellular levels and to predict internal concentrations in fish by using toxicokinetic models - Recommended its use for acute fish toxicity testing “The first permanent cell line from fish was developed from the gonads of Rainbow trout, Salmo gairdneri (RTG2)” in 1962 Sources of cell lines Cell Banks ATCC (American Tissue Culture Cell Collection) ECACC (European Collection of Cell Culture) DSMZ (German collections of microorganisms and cell cultures) Others…. Research collaborations 11  20/09/2024 http://www.lgcstandards- atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Animal.aspx More than 10.000 cell lines in Cellosaurus database http://www.lgcstandards-atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Human.aspx 12  20/09/2024 “The first permanent cell line from fish was developed from the gonads of Rainbow trout, Salmo gairdneri (RTG2)” in 1962 http://www.lgcstandards-atcc.org/Products/Cells%20and%20Microorganisms/Cell%20Lines/Animal/Fish.aspx?geo_country=pt 13  20/09/2024 Lab Conditions Equipment and facilities Biosafety “Laboratory biosafety describes the containment principles, technologies and practices that are implemented to prevent the unintentional exposure to pathogens and toxins, or their accidental release” (WHO – World Health Organization) 14  20/09/2024 Biosafety Level Most cell culture labs should be at least BSL-2, but depend upon the cell line used and the type of work conducted Human cells 15  20/09/2024 Biosafety Level 2 - Design  Lockable doors – restrict acess  Easily cleaned surfaces  Surfaces resistant to chemicals  BSCs installed properly (away from doors and windows)  Decontamination of laboratory waste  Others… 16  20/09/2024 Equipments Laminar flow hood - provides an aseptic working area Incubators Incubator with humidified atmosphere of 5% CO2 and temperature control (human cells 37°C; fish cells 18°C, normally do not require CO2) 17  20/09/2024 Inverted Microscope - Light source and condenser on the top - Objectives are below the stage pointing up - Observation of cells at the bottom (culture flasks or plates) Other equipment Water bath Sterilizer (i.e., autoclave) Centrifuge 18  20/09/2024 Other equipment pH meter Refrigerator Liquid nitrogen (N2) -80ºC Freezer freezer or cryostorage container Culture Conditions Materials Reagents Skilled human Sterile Technique resources 19  20/09/2024 Material Sterile Tubes, Syringes, Filters Sterile pipettes Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multiwell plates) Reagents - Culture Medium (DMEM, MEM, RPMI, L15…..) - Fetal Bovine Serum (FBS) – 5-20% - Antibiotic (e.g., penicillin and streptomycin) - Antimicotic (e.g., amphotericin B) - Buffers (e.g., PBS) - Trypsin 20  20/09/2024 Reagents Culture medium provide: Necessary nutrients Example: Growth factors Stabilize the pH of the culture Osmotic pressure of the culture Main components of culture medium: Vitamins Carbon source (glucose) Amino acids Inorganic Salts Trace elements (iron, zinc) Indicators for pH change (phenol red) pH Indicator – Phenol red Acidic Optimal pH 7.2 -7.4 Basic 21  20/09/2024 Reagents Fetal Bovine Serum - FBS Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells Cell Culture Contamination Contamination is the most common problem encountered in cell culture laboratories, sometimes with very serious consequences Could affect: - Cell growth and function - Morphology and differentiation state - Gene expression 22  20/09/2024 Cell Culture Contamination Chemical Contamination - Impurities in media, serum, and water (endotoxins, plasticizers, and detergents) Bacteria Cross contamination Yeast with other cell lines Biological Contamination Mycoplasma Virus Cell Culture Contamination Fungi Yeast 23  20/09/2024 Cell Culture Contamination Mycoplasma: - Simple bacteria that lack a cell wall - Minimal size (typically less than one micrometer) - Very difficult to detect only when they achieve extremely high densities and cause severe damage in the cell culture (altering the behavior and metabolism) Cell Culture Contamination Cell line cross-contamination is an endemic problem “…15–20% of cell lines in current use are estimated to not be what they are thought to be.” Freshney RI. Database of misidentified cell lines. Int J Cancer 2010; 126: 302 Contamination cost: - Time - Money - Reputation 24  20/09/2024 Cell Culture Contamination How to detect cell contamination? - Turbid culture media; - Color change of the medium; - Loss of cell adhesion; - Change in the growth rates; - Multi-nucleated cells; - Vacuolization; - Cell lysis - Change in the cell line behaviour Discard the infected cell lines immediately Cross Contamination Figure 3 Normal human fibroblasts (orbital shaped) cross-contaminated with HeLa (rounded cell colonies) Nims RW, et al., In Vitro Cell Dev Biol Anim. 2010;46(10):811–819. 25  20/09/2024 Cell Culture Contamination Macro and microscopic observation (medium color; contaminations; cell morphology; confluence) Bian, X., et al. A Combination of Species Identification and STR Profiling Identifies Cross-contaminated Cells from 482 Human Tumor Cell Lines. Sci Rep 7, 9774 (2017) 26  20/09/2024 Cell Authentication – Types of tests The aim is to compare the test cell line with other samples from the same donor or against a database. - Karyotyping - Isoenzyme analysis - STR (Short Tandem Repeat) analysis - SNP (Single-nucleotide polymorphism) analysis Fazekas, J. et al., Oncotarget. 2017 May 23; 8(21): 35076–35087 When should I authenticate? - If creating new cell lines from patients - For in-house (noncommercially available) cell lines - Prior to starting new experiments - Every three to six months during active growth as a routine quality control check - When receiving a cell line from a nonrepository source - If you have unexpected experimental results or changes in the cells morphology/growth rate - Prior to publication - Prior to submission of grant proposals https://www.biocompare.com/Editorial-Articles/350110-STR-Profiling-for-Cell-Line-Authentication/ 27  20/09/2024 Aseptic technique - Aim of the aseptic technique is keep the cells free from contamination by microorganisms Main aspects of aseptic technique: - Sterile work area (UV light, ethanol 70%) - Sterile reagents and media - Good personal hygiene - Sterile handling Respect the rules of the lab Some recomendations Cleaning the lab and equipment Maintaining good aseptic technique Wash hands; wear lab coats and gloves; avoid touching door handles, phones, calculators, etc Use materials and reagents certified for cell culture Work with only one cell type at once (do not share material and reagents) Dedicate media, reagents and suplies to a single cell line Keep individual record of all cell culture work 28  20/09/2024 Maintainence of Cell Cultures Change medium (maintain the growth conditions) Subcultivation (allow the propagation) % of Confluency Determination of cell density Viability test Freezing/thawing cells Subculturing - Allow the propagation of the cell line (transfer of cells from a previous culture into fresh growth medium) Confluent Subculture Subculture Sparse 29  20/09/2024 Subculturing In Vitro Age of a Cell Culture Passage number - indicates the number of times the cell line has been sub-cultured. Although there are no specific guidelines regarding the optimal passage range, common practice is to not use cells after 20 to 30 passages The in vitro age of cell culture is particularly useful to know for cell lines with a finite lifespan or unstable characteristics that change over time in continuous culture Subculturing Confluent Monolayer Cell density Cell viability enzymatic and/or Counting mechanic dissociation Plating New flasks with lower density Cell scrapers 30  20/09/2024 Cryopreservation X reduce the risk of ice crystal formation, which can damage cells and cause cell death Mr Frosty box slower cooling rate 1ºC/ min, -80ºC Fast Thawing Cryovials containing frozen cells Cryopreservation -80°C Some months Cell Storage liquid phase (-196ºC) liquid nitrogen Gas phase (-156ºC) 31  20/09/2024 Cryopreservation Cryopreservation Materials Needed Cryovials Boxes Liquid Nitrogen Containers 32  20/09/2024 Cryopreservation Why cryopreservating cells? - Cell lines in continuous culture are prone to genetic drift - Finite cell lines are fated for senescence - Cell cultures are susceptible to microbial contamination - May occurs equipment failure Because a cell line is a valuable resource and its replacement is expensive and time consuming, it is important that they are preserved for long-term storage. Cell Culture - Advantages Cell culture is a powerful tool - High number of tests in reduced time and space - Cost effective and easy to use when compared with animal experiments - Ethical concerns – Replace and Reduce the use of animals 33  20/09/2024 Cell Culture - Advantages Cell culture is a powerful tool - Unlimited supply of material - Consistency and reproducibility of the results - Specific monitoring of:  physico-chemical (e.g. temperature, pH, osmotic pressure, O2 and CO2 tension)  physiological conditions (e.g., hormone and nutrient concentrations) Cell Culture - Disadvantages - Material cost - Skilled human resources - Genetic instability - Loss of specific cell functions (cells desdifferentiation) - Difficulty in extrapolating results - Unexpected contaminations with microorganisms or even cross- contamination with other cell types 34  20/09/2024 Co-Culture models – 2D - System where two or more different cell types are grown together in the same environment. - Used to study the interactions between different cell types, closely mimicking the complex cellular environments found in tissues and organs. - Co-culturing is valuable for understanding cellular communication, signaling, and the influence of one cell type on another. Co-Culture models – 2D https://www.stemcell.com/how-to-co-culture-hpsc-derived-forebrain-neurons-and-astrocytes.html 35  20/09/2024 Co-Culture models – 2D Zhu et al., 2018. International Journal of Molecular Sciences, 19(8):2284 3D cell culture and other advanced techniques Fernanda Malhão 2024/2025 Cellular and Molecular Biology Techniques and Master Degree in Marine Sciences Applications in Marine Sciences 36  20/09/2024 Tumor microenvironment http://www.biotek.com/resources/articles/3d-cell-culture-review-of-current-techniques.html Cell Culture – 3D Carvalho et al., 2016. Carbohydrate polymers 37  20/09/2024 Cell Culture – 2D vs 3D 2D 3D Cell-to-cell contact limited Cell-to-cell contact Cell mostly in contact with the Cells interact with extracelular surface of the plastic matrix (ECM) No gradients present Diffusion gradient of drugs, No microenvironment exist oxygen, nutrientes, and waste Co-culture of multiple cell mimics microenvironment Shows resistance to anticancer drug as in vivo tumor Cell Culture – 2D and 3D 38  20/09/2024 3D Cell Culture 39 20/09/2024 40 20/09/2024 41  20/09/2024 Cell Culture – Organs-on-chip Possible applications: - Research on basic fish physiology - Refinement of fish feed in aquaculture - Predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. Cell Culture – Organs-on-chip  Organ-on-a-chip is a microfluidic cell culture technology that mimics the physiological functions, architecture, and microenvironment of human organs on a miniature scale  It consists of small, transparent chips, typically made from polydimethylsiloxane (PDMS)  The microchannels and chambers lined with living cells, allowing the researchers to study organ function, disease mechanisms, and drug effects in a controlled, human-relevant environment https://wyss.harvard.edu/technology/human-organs-on-chips/ 42  20/09/2024 Cell Culture – Organs-on-chip The chips can be used instead of animals in disease research, drug testing, and toxicity testing and have been shown to replicate human physiology, diseases, and drug responses more accurately than crude animal experiments do. https://wyss.harvard.edu/technology/human-organs-on-chips/ 43  20/09/2024 https://www.thermofisher.com/pt/en/home/g lobal/forms/cell-culture-basics/cell-culture- basics-virtual-lab.html Thank you for your attention Fernanda Malhão [email protected] 44