Lec # 15 Animal cell lines and culturing Shah Rukh Abbas, PhD 26.2. 2015

What is Cell culture ? Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.  The process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions But in practice it refers to the culturing of cells derived from animal cells/tissue

Why we need to culture animal cells? Cell is the Basic Unit for the Life Understanding functions and roles of various cell are crucial approach for modern biology

Cost effective than animal experiments With isolated cell culture, we can study/investigate desired cell using various techniques without interference of other cells/tissues/organ Microscopic Biochemical Molecular Biology Toxicology… Cost effective than animal experiments Avoid ethical problem in animal/human experiments Production of Protein/Antibody/Virus/Vaccine using cultured animal cell culture

History 1885 : medullary plate of an embryonic chickens (Wilhelm Roux) 1907 : Grow Frog nerve fiber in using hanging drop culture 1912 : Alexis Carrel culture chicken heart using chick embryo extract 1916 : Trypsinization and subculture of explants 1923 : Development of first cell culture flask 1925 : Subculture of fibroblastic cell lines 1940s : Discovery of Antibiotics -: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture. 1952: Cloned Tadpoles (Briggs and King) 1954 : Discovery of Contact Inhibition (Abercrombie) 1955: nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.(Eagle) DMEM Dulbecco’s Modified Eagle’s Medium 1961: isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture. A thickened plate of dorsal ectoderm that differentiates into the neural tube and neural crest

History 1965: first serum-free medium which was able to support the growth of some cells. (Ham) 1975: First hybridoma capable of secreting a monoclonal antibody (Milstein) 1978: development of serum-free media from cocktails of hormones and growth factors. (Sato) 1981 : Mouse Embryonic Stem Cell 1982: Human insulin : the first recombinant protein to be Licensed as a therapeutic agent. 1985: Human growth hormone produced from recombinant bacteria 1989 : Knockout mouse using Mouse ES Cell (Capecchi, Evans, Smithies) 1996 : The First mammal cloned from adult cells (Dolly, the sheep) 1998 : Human Embryonic Stem Cell (Thomson) 2006-2007 : First Induced Pluripotent Stem Cell (iPSC : Yamanaka and others) 2013 : First Human stem Cell generated from SCNT

because stage of differentiation) Mouse, mammals, Embryo Embryonated Eggs because stage of differentiation) Cell culture Grow in media -monolayer -suspension cells Finely cut tissue or explant explant Enzymic digestion Sterilize the site with 70% alcohol. Remove tissue aseptically. Transfer to the laboratory in transport medium If delay in transporting to lab, keep at 4°C for up to 72hour.

STAGES OF CULTURE Isolated tissue (disaggregation) Primary cell culture(limited lifespan after certain proliferations undergo senescence) Finite cell cultures Continous cell lines(immortalized cell line acquires ability to proliferate indefinitely by transformation)

Primary Cell Culture Cells taken directly from living tissue (e.g. biopsy material) and established for growth in vitro Undergone very few population doublings Proteolytic enzymes (trypsin and Collagenase) are commonly used to break the protein

Primary Cell Culture Pros more representative of the main functional component of the tissue Cons They are not well characterized, Have limited life span, Slow in proliferation

Established Cell Line After the first subculture, primary culture may be called secondary cultures Thereafter, if continued passage is possible, a cell line. Established or immortalised cell line : ability to prolierate indefinetely by Random Mutation Artificial Modification : expression of telomerase, insertion of cancer antigen Cell Line Organism Origin Tissue HeLa Human Cervical Cancer 293-T Embryonic Kidney A-549 Lung carcinoma ALC Murine Bone Marrow CHO Hamster Ovary HB54 Hybridoma

Growth Curve

‘Normal’ mammalian cells have the following properties: In the 1960’s, only “normal” mammalian cells could be used as the hosts for t he production of human vaccines, to ensure the safety of the products. A number of characteristics had to be met, as defined by Hayflick and Moorhead. ‘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 Indicates that no significant chromosomal damage has occurred. Cells require solid surface for attachment and growth. Growth occurs until a confluent monolayer of cells is formed. Typically 50 divisions, may be different for different cells. When injected into an immuno-compromised mouse the cells will not form a tumor. As cells grow on substratum, they cover surface with a single layer of cells, then stop growing, demonstrates regulatory control

Transformed cell characteristics 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

Example of Anchorage Dependence Cells need to attach to solid substratum before growth starts. Serum-derived glycoproteins (fibronectin) coat the surface of the substratum to aid in cell attachment. Conditioning factors are released by the cells and form a bond between the membrane-bound glycoproteins and the substratum. Divalent cations (Ca2+, Mg2+) may also assist in the interaction process.

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 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 When cells stop growing in culture, a new culture can be established by inoculating some of the cells into fresh medium.

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 Phenol red is used for pH indicator, culture turns yellow as pH drops.

HeLa The oldest and most commonly used human cell line 1951 : Derived from cervical cancer cell taken from Henrietta Lacks (1920-1951) Henrietta Lacks (circa 1945)

HeLa is famous for… Immotallised Cell Line : Due to mutation, it can evades normal cellular senescence and can keep undergo division Used to test the first polio vaccine / virus Culture More than 60,000 scientific articles has been published using HeLa Extensively Used for the Cancer Studies It has abnormal chromosome number : 82 4 copies of chromosome 12 3 copies of chromosome 6,8,7

How to grow/select specific cells? selective overgrowth of a particular cell type controlling media composition gradient centrifugation

Type of Cell by Morphology Fibroblast like spindle-shaped, often striated, form parallel lines as they attach to substratum/substrate → in vivo – wrap around collagen (fibrous protein) → in vitro – glass

Lymphoblast-like large nuclei found in vivo in blood (liquid suspension) can grow in suspension in liquid medium in lab

Epithelial-like cover organs and line cavities (i.e. skin) cobblestone morphology, form monolayer anchorage dependent, need solid substratum

Type of Cells Anchorage-independent cells: which can propagate in the suspension culture Blood Cells Cancer Cells Hybridoma Anchorage-dependent cells which can propagate as a monolayer attached to the cell culture vessel Will cease proliferating once they become confluent (completely cover the surface of cell culture vessel)

Suspension Culture Appropriate for cells adapted to suspension culture and a few other cell lines that are non-adhesive (e.g., hematopoietic) Used for bulk protein production, batch harvesting, and many research applications Can be maintained in culture vessels that are not tissue-culture treated, but requires agitation (i.e., shaking or stirring) for adequate gas exchange Easier to passage, but requires daily cell counts and viability determination to follow growth patterns; culture can be diluted to stimulate growth Spinner Flask

Adherent Cell Culture Adherent cell require surface to attach to grow Appropriate for most of cell types (including primary cultures) The majority of the cells derived from vertebrates are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading Growth is limited by surface area Cells are dissociated enzymatically or mechanically from surface

Utilization of Cell Culture Model System for Basic Science In Vitro Cell Toxicity / Screening Animal Cell Culture for Protein Productions Tissue/embryo Engineering