Lecture 3 animal cell types

Lecture 3 - Introduction to Animal Cell Biotechnology continued

Lecture 3 Animal Cell BiotechnologyCharacteristics 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)



selective overgrowth of a particular cell type

controlling media composition

gradient centrifugation

How to grow/select specific cells?


Lecture 3 Animal Cell BiotechnologyCharacteristics 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

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


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


TypesNeuron transmit electrical impulses can grow embryonic neurons, not adult addition of nerve growth factors cause the

formation of outgrowths called neurites


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

Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture –

What’s Normal

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:

Lecture 3 Animal Cell BiotechnologyCharacteristics 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

Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture – Anchorage

DependenceExample of Anchorage Dependence


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



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

Modification of polystyrene to obtain a charged surfaceFig. 3.5



to induce a positive charge on the substratum:→ DEAE dextran→ polylysine→ polyarginine→ polyhistidine→ polyornithine→ polyacrylamide


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 by mycoplasma could be a huge problem

Oh No! Contamination!


Characteristics of Cells in CultureMycoplasma 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


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


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





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

Maintaining Cultures of Differentiated Cells



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

Embryonic stem cells have the following characteristics:



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’)

Adult stem cells



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

http://www.atcc.org/

.Common cell lines obtainable from culture collectionsCell 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.

CHO cell line

Chinese hamster ovary anchorage-dependent, or

grown in suspension high capacity for

amplification and expression of recombinant genes

glycosylation of proteins

Technology

Lecture 3 animal cell types