Lecture 3 animal cell types
-
Upload
sarah-aira-santos -
Category
Technology
-
view
2.634 -
download
3
description
Transcript of 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)
Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture
Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture
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
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
Lecture 3 Animal Cell BiotechnologyCharacteristics 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
Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture – Cell
TypesNeuron transmit electrical impulses can grow embryonic neurons, not adult addition of nerve growth factors cause the
formation of outgrowths called neurites
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
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
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
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
Modification of polystyrene to obtain a charged surfaceFig. 3.5
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
Lecture 3 Animal Cell BiotechnologyCharacteristics 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
Lecture 3 Animal Cell BiotechnologyCharacteristics 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
Lecture 3 Animal Cell BiotechnologyCharacteristics of Cells in Culture
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!
Lecture 3 Animal Cell Biotechnology
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
Lecture 3 Animal Cell BiotechnologyCharacteristics 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
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
Lecture 3 Animal Cell Biotechnology
Characteristics of Cells in Culture – Cell Differentiation
Lecture 3 Animal Cell Biotechnology
Characteristics of Cells in Culture – Cell Differentiation
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
Lecture 3 Animal Cell Biotechnology
Characteristics of Cells in Culture – Cell Differentiation
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:
Lecture 3 Animal Cell Biotechnology
Characteristics of Cells in Culture – Cell Differentiation
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
Lecture 3 Animal Cell Biotechnology
Characteristics of Cells in Culture – Cell Differentiation
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 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