Lecture 3 animal cell types
- 1. Lecture 3 - Introduction to Animal Cell Biotechnology continued
- 2. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture Primary Cultures cells taken directly from animal tissue are added directly to medium, establishing a primary culture often established from embryonic tissue → easily dispersed, superior growth potential tissues are broken up mechanically (scissors or forceps) fragmented tissures are treated with proteolytic enzymes such as trypsin or collagenase (10-20 minutes)
- 3. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture
- 4. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture How to grow/select specific cells? selective overgrowth of a particular cell type controlling media composition gradient centrifugation
- 5. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture
- 6. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Types Fibroblasts spindle-shaped, often striated, form parallel lines as they attach to substratum/substrate → in vivo – wrap around collagen (fibrous protein) → in vitro – glass
- 7. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Types Epithelial cover organs and line cavities (i.e. skin) cobblestone morphology, form monolayer anchorage dependent, need solid substratum
- 8. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Types Muscle cells follows a series of differentiation steps from precursor cells (myoblasts), leading to cell fusion, form multinucleate complex mature cells don‟t grow well, but are used to study cell differentiation cells are removed from animal embryo, subsequent changes are monitored and studied
- 9. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Types Neuron transmit electrical impulses can grow embryonic neurons, not adult addition of nerve growth factors cause the formation of outgrowths called neurites
- 10. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Types Lymphocytes large nuclei found in vivo in blood (liquid suspension) can grow in suspension in liquid medium in lab
- 11. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – What‟s Normal „Normal‟ mammalian cells have the following properties: a diploid chromosome number (46 chromosomes for human cells) anchorage dependence a finite lifespan nonmalignant (non-cancerous) density inhibition
- 12. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – What‟s Not Transformed cell characteristics – a review infinite growth potential loss of anchorage-dependence aneuploidy (chromosome fragmentation) high capacity for growth in simple growth medium, without the need for growth factors called an “established” or “continuous” cell line
- 13. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Anchorage Dependence Example of Anchorage Dependence
- 14. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Anchorage Dependence cells need to attach to solid substratum before growth occurs combination of electrostatic attraction and van der Waal‟s forces divalent cations (Ca2+) and basic proteins form layer between cells and substratum mediated by a range of nonspecific proteins which form a layer on the substratum prior to cell attachment
- 15. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Anchorage Dependence substratum may be negatively or positively charged alkali treatment (25 mM NaOH + 0.1 M EDTA) for borosilicate glass to induce a negative charge: Si-O-Si → Si-O- sulfuric acid treatment or high-voltage electrical treatment for polystyrene plastic to induce a negative charge
- 16. Fig. 3.5 Modification of polystyrene to obtain a charged surface
- 17. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Anchorage Dependence to induce a positive charge on the substratum: → DEAE dextran → polylysine → polyarginine → polyhistidine → polyornithine → polyacrylamide
- 18. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture Passaging - establishing Secondary → Tertiary Cultures growth of cells prolonged by inoculating some of the cells into fresh medium „cell line‟ refers to cell population that continues to grow through passaging or subculturing genetic alteration may occur during the first few passages as cells adapt to a new chemical environment
- 19. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture subculture within a day or two of maximum cell density must detach anchorage-dependent from growth surface → trypsinization → EDTA in Ca 2+ - and Mg 2+ -free solution
- 20. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture Oh No! Contamination! bacteria and fungi are main sources of contamination culture contamination observed by: → drop in pH → turbidity of medium → may observe granules between mammalian cells contamination by mycoplasma could be a huge problem
- 21. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture Mycoplasma contamination commonly associated with mammalian cells penicillin and streptomycin is ineffective, due to lack of mycoplasma cell wall slow growing, may affect cellular growth rate, morphology, viability and metabolism 0.2-2 μm, infect cytoplasm of mammalian cells high requirement for arginine, causes a rapid increase in culture pH should test every 3-6 months for mycoplasma contamination
- 22. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture must maintain aseptic techniques throughout process of establishing primary culture → animal cells‟ doubling time ~24 hours → bacteria ~15-20 minutes include antibiotics in growth media dissection instruments must be sterile all working surfaces should be wiped with 70% alcohol
- 23. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation differentiation is the process by which non- specialized cells become specialized, with characteristic phenotypes differentiated cells tend to lose ability to grow in culture → undifferentiated stem cells continue to grow → nerve, muscle cells grow poorly differentiated tumor cells retain the phenotypic characteristics of normal cells but also grow quickly
- 24. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation
- 25. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation Maintaining Cultures of Differentiated Cells use of hormones, growth factors, Ca 2+ chemical agents (i.e. DMSO) cell to cell interactions (with high cell density) interaction with the growth surface
- 26. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation Embryonic stem cells have the following characteristics: pluripotent propagates indefinitely in a non-differentiated state associated with specific cell markers normal diploid karyotype high activity of telomerase forms a teratoma in immunocompromised mice
- 27. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation Adult stem cells undifferentiated cells found amongst differentiated cells in tissue or organs differentiate along a more limited pathway than embryonic stem cells associated with cell replacement or repair of tissue damage may be induced into cell types belonging to other tissue (known as „transdifferentiation‟ or „plasticity‟)
- 28. Lecture 3 Animal Cell Biotechnology Characteristics of Cells in Culture – Cell Differentiation
- 29. Cell culture collections The American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. web-site: www.atcc.org The European Collection of Animal Cell Culture (ECACC), Public Health Laboratory Service (PHLS), Centre for Applied Microbiology Research (CAMR), Porton Down, Salisbury SP4 OJG, U.K. web-site: www.ecacc.org
- 30. . Common cell lines obtainable from culture collections Cell line Origin Cell type Comment BHK Baby hamster kidney fibroblast Cells are anchorage-dependent but can be induced into suspension; used for vaccine production CHO Chinese hamster ovary epithelial Cells will attach to a surface if available but will also grow in suspension; used extensively for genetic engineering. HeLa Human cervical carcinoma epithelial Fast-growing human cancer cell isolated in the 1950s L Mouse connective tissue fibroblast Many culture techniques developed from the 1950s were based on this tumour cell line. L6 Rat skeletal muscle myoblast Can be used to demonstrate the differentiation of a muscle cell. MDCK (Madin Darby) canine kidney epithelial Anchorage-dependent cells with good growth characteristics; used for veterinary vaccine production. MRC-5 Human embryonic lung fibroblast Finite life-span, 'normal' cells; used for human vaccine production. MPC-11 Mouse myeloma lymphoblast Derived from a mouse tumour; secretes immunoglobulin. Namalwa Human lymphoma lymphoblast Derived from cells from a human suffering from Burkitt's lymphoma; used for alpha-interferon production. NB41A3 Mouse neuroblastoma neuronal Tumour cells with good growth rate. Cells have nerve cell characteristics including a response to nerve growth factor. 3T3 Mouse connective tissue fibroblast Vigorous growth in suspension; Cells used widely in the development of cell culture techniques. WI-38 Human embryonic lung fibroblast Finite life-span, 'normal' cells; used for human vaccine production. Vero African green monkey kidney fibroblast An established cell line capable of continuous growth but with many 'normal' diploid characteristics; used for human vaccine production.
- 31. CHO cell line Chinese hamster ovary anchorage-dependent, or grown in suspension high capacity for amplification and expression of recombinant genes glycosylation of proteins