Cell Culture & Stem Cells PDF

Cell culture & stem cells By: Rehab Nady 1 Cell types Oocyte Hepatocytes Purkinje Cells from cerebellum Phagocytic c...

Cell culture & stem cells By: Rehab Nady 1 Cell types Oocyte Hepatocytes Purkinje Cells from cerebellum Phagocytic cell (cultured on Petri dish) 2  Cell culture ❑ Growing cells outside the organism. ❑ Removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. Why do we culture cells: 1) To be obtained in large quantities. 2) To study a single type of cell. 3) To study many different cellular activities, such as endocytosis, cell movement, cell division, and macromolecular synthesis. 4) Cells can differentiate in culture. 5) Cultured cells respond to treatment with drugs, hormones, growth factors, and other active substances. 3 Primary culture: cells are isolated directly from the tissue and proliferate under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). ▪ At this stage, the cells must be subcultured (i.e., passaged) by transferring them to a new vessel with a fresh growth medium to provide more room for continued growth. ▪ Cell dissociation is accomplished with enzymatic (trypsin or collagenases) or mechanical means. Secondary culture: cells derived from a previous culture. ▪ Normal cells can divide a limited number of times (typically 50 to 100) before they die; these cell lines are known as finite. 4 ▪ Primary cells can become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally (by a tumor virus that overcomes cell- cycle controls) induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line (ex., HeLa cell line has been growing worldwide in laboratories since 1951). ▪ Transformed cell lines have a higher growth rate, are continuous, and require less serum in media. Cryopreservation ❖ If a surplus of cells is available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (Liquid nitrogen has a boiling point of about −196°C) until they are needed. 5 6  Adherent Cell Culture Suspension Cell Culture Appropriate for most cell A few cell lines that are types, including primary nonadhesive (ex, hematopoietic) cultures Easier to passage, but requires daily cell counts and viability Requires periodic passaging determination to follow growth patterns Cells are dissociated Does not require enzymatic or enzymatically or mechanical dissociation mechanically Does not require tissue-culture Requires tissue-culture treated vessel, but requires treated vessel agitation (i.e., shaking or stirring) for adequate gas exchange 7 Morphology of Cells in Culture https://www.thermofisher.com/eg/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html 8  Culture Conditions * A medium that supplies the essential nutrients (amino acids, vitamins, inorganic salts, D-glucose) * Growth factors * Hormones * Gases (O2, CO2) * A regulated environment (pH, osmotic pressure, temperature) https://www.thermofisher.com/eg/en/home/life-science/cell-culture/mammalian-cell-culture/cell-culture-media.html 9 Co2 incubator 10 o Fetal bovine serum (FBS) is a common component of animal cell culture media. It is harvested from bovine fetuses taken from pregnant cows during slaughter. o FBS is a mixture of growth factors, proteins, trace elements, vitamins, and hormones, which are important for the growth and maintenance of cells in culture. 11 Because they are so rich in nutrients, tissue culture media are a habitat for the growth of microorganisms. To prevent bacteria from contaminating cell cultures, we must maintain sterile conditions within the working space. This is accomplished by using sterile gloves, sterilizing all supplies and instruments, adding low levels of antibiotics in the media, and conducting activities within a sterile hood. 12 13 Cell culture plates Cell culture flasks Cell culture vessels 14 15 16 17 Biological safety cabinets ❑ There are 3 main classes of Biological Safety Cabinets (BSCs) – they all protect the worker/environment from the cultures. Class II BSCs o The most common cabinets found in the lab and are used for mammalian cell culture. o Protect the worker, the environment, and the samples. o Air is HEPA-filtered as it is drawn into the cabinet and is also filtered upon exhaust. 18  Stem cell classification o Stem cells are unspecialized cells that can differentiate into any cell of an organism and can self-renew. o Stem cells exist in embryos (embryonic stem cells) and adult cells (adult or nonembryonic or somatic stem cells). o Totipotent stem cells can divide and differentiate into cells of the whole organism. o They have the highest differentiation potential and form both embryo and extra-embryonic structures (placenta). An example of a totipotent cell is a zygote. o Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures. o Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of the blastocyst. 19 https://stemcells.nih.gov/info/basics/stc-basics 20  o Multipotent stem cells have a narrower spectrum of differentiation than PSCs. An example is a hematopoietic stem cell, which can develop into several types of blood cells. o Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. o These cells can form one cell type, e.g. dermatocytes. 21 22  Induced Pluripotent Stem Cells (iPS cells) o It is now possible to produce pluripotent stem cells without the use of embryos. o Differentiated cells can be taken from an adult mouse or human tissue, grown in culture, and reprogrammed by artificially driving the expression of three or four transcription regulators, including Oct4, Sox2, Klf4, and Myc. o This treatment is sufficient to permanently convert fibroblasts into cells with practically all the properties of ESCs cells, including the ability to proliferate indefinitely and differentiate in diverse ways. 23 References 1) Alberts, B., Hopkin, K., Johnson, A. D., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2018). Essential cell biology: Fifth international student edition. WW Norton & Company. 2) https://bitesizebio.com/8998/biological-safety-cabinets-and- culture-hoods-know-the-difference/ 3) Pollard, T. D., Earnshaw, W. C., Lippincott-Schwartz, J., & Johnson, G. (2017). Cell biology E-book. Elsevier Health Sciences. 4) Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10, 68 (2019). https://doi.org/10.1186/s13287-019-1165-5 24 25