Cell Culture - Introduction (PDF)

Introduction to cell culture Dr. Bilal Malik [email protected] ©Dr. Bilal Malik2024, [email protected] Learning Outcomes What is a cell culture Origin of cell lines Types of Tissue culture- Primary, sec Stages of Cell culture (from...

Introduction to cell culture Dr. Bilal Malik [email protected] ©Dr. Bilal Malik2024, [email protected] Learning Outcomes What is a cell culture Origin of cell lines Types of Tissue culture- Primary, sec Stages of Cell culture (from patient to the dish) Types of cell cultures- monolayer, 3D and organoids Cell culture models vs animal models- an open question? Ethics governing cell culture Application of Cell culture techniques (in medicine, biotech and Pharma) What is a cell culture Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established.  Medullary plate of an embryonic chicken- for 13 days Development of Cell culture ©Dr. Bilal Malik2024, [email protected] Cell culture vs Tissue culture Cell culture” refers to the maintenance in vitro of disaggregated cells, while “organ culture” refers to a culture of a non-disaggregated tissue. The term “tissue culture” encompasses both terms Common terminology used in Cell culture Langdon, S. P. (2004) Basic principles of cancer cell culture, Methods Mol Med 88, 3–15 ©Dr. Bilal Malik2024, [email protected] Types of Cell culture - 1. By Origin I. Tissue specificity (Breast cancer, Kidneys, Neurons) 2. By adhesion I. Monolayer II. Suspended 3. By differentiation I. Differentiated II. Stem Cells Types of Cell culture - Fibroblasts Adipocytes Neurons What other cell lines can you think of? Types of Cell culture -Adhesion Adherent Suspension What do you think will their requirement? Types of Cell culture - Adhesion ©Dr. Bilal Malik2024, [email protected] Types of Cell culture -Differentiation Which one would you use and when? Cell Differentiation ©Dr. Bilal Malik2024, [email protected] Two types of differentiated cells Human Embryonic stem cells (hES) Induced Pluripotent cells (iPSCs) Also read about -Stem cells Embryonic Stel Cells ©Dr. Bilal Malik2024, [email protected] Induced Pluripotent cells ©Dr. Bilal Malik2024, [email protected] Types of Cell culture - How do they compare Low Clinical relevance High Clinical relevance Advantages Disadvantages Types of cell cultures- 2D, 3D doi: https://doi.org/10.1101/2021.03.18.436024 Types of cell cultures- organoids Organoids are simple tissue-engineered cell-based in vitro models. Derived from pluripotent or tissue- resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. Used to create diseases to get a ‘real-life- like’ scenario ©Dr. Bilal Malik2024, [email protected] Types of cell cultures- organoids From Nature Reviews 01 December 2022 https://doi.org/10.1038/s43586-022-00174-y Components of Organoid cultures Have you heard of Bioprinting? From Nature Reviews 01 December 2022 https://doi.org/10.1038/s43586-022-00174-y  Primary vs Secondary cell culture ©Dr. Bilal Malik2024, [email protected]  Primary vs Secondary cell culture Primary cell cultures are derived from tissues or organs of animals. These represent the organ from which they were derived from closely. They provide the most accurate representation of physiology and biochemistry or the primary organ Good examples- Epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells They have a finite life span ©Dr. Bilal Malik2024, [email protected]  Primary vs Secondary cell culture Why can’t we use primary cells for all research? Primary cell culture They take longer to grow and are very sensitive to changes in the microenvironment they grow in. Vary greatly in their characteristics depending on health and genetic state of the donor –so will not give consistent results. The time and cost associated with obtaining them, stabilising and growing them in the lab is too high compared to the chances of successful growth and benefits they provide. ©Dr. Bilal Malik2024, [email protected]  Primary vs Secondary cell culture Scalability Primary cell cultures can not be scaled up which is one of the biggest limitations for their use in the labs ©Dr. Bilal Malik2024, [email protected]  So what is the alternative? ©Dr. Bilal Malik2024, [email protected]  Secondary cell culture –cell lines Secondary cell culture Initially derived from a donor animal/ organ Immortalised artificially or naturally (by infection with a virus) Faster growth Well Established Conditions and Protocols Lowe Cost ©Dr. Bilal Malik2024, [email protected] Primary vs Secondary cell cultures Where would you use immortalised cells in research? ©Dr. Bilal Malik2024, [email protected] Stages of Cell culture Stages of primary Cell culture Aseptic conditions are maintained to work with cell culture models 1. Tissue sampling 2. Isolation of cells 3. Washing and Purification 4. Seeding and Expansion 5. Cryopreservation (Cold Storage) Stages of secondary Cell culture What are the requirements for cell culture? 1. Aseptic environment 2. Culture media 3. Incubators- CO2, temp and Humidity control 4. Storage facility (liquid nitrogen?) What to watch out for? 1. Contamination 2. Loss of cell characteristics (mutations etc) Bacterial Contamination of 293 cells  Yeast Contamination of 293 cells ©Dr. Bilal Malik2024, [email protected] Contaminants Bacteria Yeasts Fungi (mould) Other mammalian cells! How do you think you can avoid these? How reliable are cell lines? Different responses of Primary and sec cells Primary cells and cell lines show variability in responses to anticancer drug camptothecin, so data acquired through cell lines cannot easily be replicated in an in vivo model. ©Dr. Bilal Malik2024, [email protected] Cell line authentication 1. Immunocytochemistry and Immunofluorescence 2. RT-PCR 3. Confocal Microscopy 4. Karyotyping 5. Next Gen Sequencing 6. Flow Cytometry 7. Western Blot 8. Immunohistochemistry For more details https://doi.org/10.3389/fcell.2021.711381 Cell line authentication What do you think was the need for this ? Cell Volume 29, Issue 12, 1 December 2022, Pages 1685-1702.e22 ©Dr. Bilal Malik2024, [email protected] ©Dr. Bilal Malik2024, [email protected] Break! Cell culture vs animal models What are your thoughts? Cell culture vs animal models - Context dependent When should you use or avoid use of animals What can/can’t you do with the cell culture models Cell culture vs animal models - Context dependent When should you use or avoid use of animals What can/can’t you do with the cell culture models Cell culture vs animal models I use Drosophila as a model animal Why do we use animal models- other advantages (Drosophila) Shorter life cycle Inexpensive No animal welfare regulations Simple genetics Highly advances genetic tools Small size, can be grown in small containers in large numbers Highly understood genetics Fully sequenced genome Cell culture vs animal models - Context dependent Number of animals used in German laboratories in 2015. Data were extracted from the annual report of the Federal Ministry of Food and Agriculture. Regulations for use of animals or animal derived materials Animals (Scientific Procedures) Act 1986 (the Act). Post-mortem sector (Code B) Anatomy sector (Code C)) Public display sector(Code D) Research sector Code E: Research Research standards and guidance Regulations for use of animals or animal derived materials ©Dr. Bilal Malik2024, [email protected] Regulations for use of animals or animal derived materials Cell lines Cell lines – is licensing or ethical approval required? -Cell lines Cell lines are not relevant material under the Human Tissue Act (although primary cell cultures are). Storage of cell lines for research does not require a HTA licence. However, cell lines are subject to the The Human Tissue (Quality and Safety for Human Application) Regulations 2007 (The Quality and Safety [Q&S] Regulations), which deal with the storage and use of human tissue for human application/therapeutic use. Research using cell lines does not require ethical review either under the Human Tissue Act or NHS research governance systems. https://www.hra.nhs.uk/ ©Dr. Bilal Malik2024, [email protected] Application of tissue culture technology Any thoughts? Primary Cell culture applications ©Dr. Bilal Malik2024, [email protected] Primary Cell culture applications ©Dr. Bilal Malik2024, [email protected]  Further Reading Chapter 45 Cell culture and cell analysis CellCultureBasicsEU.pdf (vanderbilt.edu) ©Dr. Bilal Malik2024, [email protected] Further Reading Advancements in 3D Cell Culture Systems for Personalizing Anti-Cancer Therapies https://doi.org/10.3389/fonc.2021.782766 ©Dr. Bilal Malik2024, [email protected] Further Reading Organoids Zhao, Z., Chen, X., Dowbaj, A.M. et al. Organoids. Nat Rev Methods Primers 2, 94 (2022). https://doi.org/10.1038/s43586-022-00174-y ©Dr. Bilal Malik2024, [email protected]