Lecture 3 animal cell types

Lecture 3 - Introduction to Animal Cell Biotechnology continued

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)

How to grow/select specific cells?

 selective overgrowth of a particular cell type
 controlling media composition
 gradient centrifugation

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


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


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

Neuron
 transmit electrical impulses
 can grow embryonic neurons, not adult
 addition of nerve growth factors cause the formation of
outgrowths called neurites


Lymphocytes
 large nuclei
 found in vivo in blood (liquid suspension)
 can grow in suspension in liquid medium in lab

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

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

Characteristics of Cells in Culture – Anchorage Dependence
Example 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

Fig. 3.5 Modification of polystyrene to obtain a charged surface

 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


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

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


 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

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

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

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

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‟)

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

. 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.

CHO cell line

 Chinese hamster ovary
 anchorage-dependent, or grown in
suspension
 high capacity for amplification and
expression of recombinant genes
 glycosylation of proteins

Lecture 3 animal cell types

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