Lec # 15Animal cell lines and culturing

Shah Rukh Abbas, PhD26.2. 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

• 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.

• 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

History

•Mouse, mammals,•Embryo•Embryonated Eggs because stage of differentiation)

explant

Grow in media-monolayer-suspension cells

Cell culture

Finely cut

Finely cut tissue or 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

Cons • They are not well characterized, • Have limited life span,• Slow in proliferation

Pros • more representative of the main functional

component of the tissue

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 Human Embryonic Kidney

A-549 Human Lung carcinoma

ALC Murine Bone Marrow

CHO Hamster Ovary

HB54 Hybridoma Hybridoma

Growth Curve

• a diploid chromosome number (46 chromosomes for human cells)

• anchorage dependence

• a finite lifespan

• nonmalignant (non-cancerous)

• density inhibition

‘Normal’ mammalian cells have the following properties:

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

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

• 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

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 : 824 copies of chromosome 123 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)

Spinner Flask

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

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