Download animal cell and tissue culture

ANIMAL CELL AND TISSUE CULTURE
Principles
Primary cells, cell lines
• and immortalized cells
Media for cell cultivation
Cell growth and metabolism
Cell characterization and preservation
Animal Cell Growth
Growth:
Increase of cell numbers during time in culture
Characteristic Growth Curve (mostly anchorage-dependent cells)
Lag Phase upon seeding
Exponential Growth Phase
Plateau Phase (saturation
cell density)
Cells
become
confluent,
occupying all available growth
surface;
Contact inhibition: inhibition of
cell motility, membrane ruffling…
Limited proliferation can also be
due to nutrient, growth factor
depletion
and
metabolite
accumulation (limiting factor for
suspension cells)
Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson
Prentice Hall Bioengineering, 2004
Cell Growth Terminology
Growth parameters:
Viable cell concentration, nv
Fraction of viable cells, fv
Specific growth rate, µ
dn = increase in cell number
dt = time interval
n = cell number
Doubling time, Dt
(i.e. time for a population to double
in number)
Population doubling time, PD
(i.e. total number of population doublings
of a cell line since its initiation in vitro)
Specific death rate, kd
1  dn 
µ=  
n  dt 
Dt =
ln 2
µ
n
log( )
n0
PD = ∏
log 2
Animal Cell Metabolism
Metabolism
•
•
•
•
Sources of carbon and
energy:
glucose
and
glutamine
(and anabolic precursors)
By-product metabolites:
lactate and ammonia
Source: Ratledge, C, Kristiansen, B, Basic Biotechnology, Cambridge University Press, 2001
Nutrient Metabolism
•Limited supply of glucose can be
compensated by an increased
consumption of glutamine and viceversa
“Flexibility”
” of
animal cells
Glutamine is also a nitrogen source for
animal cells, so its limitation can lead
to an increased consumption of other
amino acids to compensate lower
nitrogen intake.
Metabolic Pathways
Glucose Metabolism:
Source: www.uic.edu/classes
It is mainly metabolized via the glycolytic route to pyruvate;
Metabolic Pathways
Glucose Metabolism:
Pyruvate is mainly reduced to lactate;
Source: www.uic.edu/classes
Lactate is excreted and accumulates in the culture medium;
Metabolic Pathways
Glucose Metabolism:
Source: www.uic.edu/classes
Alternatively, pyruvate may be converted to acetyl-CoA which enters
the tricarboxylic acid cycle (TCA cycle);
.
Source: www.uic.edu/classes
Metabolic Pathways
Source: Wikipedia
Metabolic Pathways
Metabolic Pathways
Glucose Metabolism:
4 – 8 % of consumed glucose goes via the pentose phosphate
pathway (PPP) which supplies ribose-5-phosphate (R-5-P) for the
synthesis of nucleotides, as well as reducing equivalents (NADPH) for
biosynthesis.
Metabolic Pathways
Glutamine/Glutamate Metabolism:
Metabolic Pathways
Glutamine Metabolism:
It can be catabolized in a number of ways, all beginning with deamination of
glutamine that yields glutamate and NH4+;
A smaller amount is metabolized via transamidation reactions in which the
amide group is transferred to a precursor metabolite which is used in biosynthesis
of purines, pyrimidines and aminosugar moities incorporated into glycan structures;
The second step is the conversion of glutamate to α-ketoglutarate
via glutamate dehydrogenase
yielding another NH4+
via transamination with pyruvate or
oxaloacetate as amino-group acceptors
yielding alanine or aspartate. Alanine, in
particular, is easily excreted from the cell
and accumulates in the culture medium.
Both glucose-derived and glutamine-derived carbon enter the TCA cycle, as
acetyl-CoA and α-ketoglutarate, respectively.
Inhibition by Metabolic By-products
Lactate (20-60 mM)
Inhibition of growth by the decrease in pH that follows
from lactate excretion to the culture medium.
Metabolic
By-Products
NH3 / NH4+ (1-5 mM)
Inhibition includes interference with electrochemical
gradients, changes in intracellular pH, apoptosis and a
futile cycle of NH3 / NH4+ which increases the demand
for maintenance energy. Also disturbs the glycosylation
pattern of the product.
Alanine (not believed to harm the cells)
Cell Characterization in Tissue Culture
Detection of Biochemical Components
Many biochemical assays exploit the use of highly specific antibodies which are
combined with enzymatic or fluorescent labels:
o Enzymatic staining: color change in a substrate and production of immunohistochemical
stains;
o Fluorescent staining: immunofluorescent labeling
Non-antibody-based techniques:
• Expression of reporter fluorescent proteins: gene for the protein (e.g. GFP) is transferred to a
cell and expressed continuoulsy with a protein of interest; the fusion protein can be tracked
dynamically in live cells;
• Expression of b-lactamase: genetic transfer of the gene to the cells; the enzyme cleaves a
fluorescent substrate and produces a color change in proportion to the gene expression;
In vivo Imaging
Noninvasive: ultrasound, planar x-rays, fluoroscopy, computer-assisted tumoghraphy,
magnetic resonance imaging and nuclear medicine scans, which uses radioisotipes to
image organ function;
Invasive: miniaturized fiberoptic probes which can be guided through a small incision or
through a vessel.
Measurement of Cell Characteristics
Cell Morphology
Morphology: form and structure of cells
Qualitative assessment of the purity, general health and density of cell cultures;
Cells can be described as being:
• Fibroblastic (elongated and branched)
• Epithelial-like (rounded and cobblestonelike patterns)
• Lymphoblast-like (rounded and in suspension)
Influenced by the density (confluence) of the culture;
Unhealthy cells may show an increase in cytoplasmic granules (dark) and vacuoles
(clear) or detach from the substrate.
Cell number
Hemocytometer: chamber of fixed volume with grid lines that can be observed under the
microscope to count cells in suspension (also adherent cells after harvesting)
Source: www.wisc.edu
Source: www.uvm.edu
Cell Counting
Source: Domingos Henrique, Cell and Tissue Engineering Classes, IST, 2008-2009
Measurement of Cell Characteristics
Cell number (cont.)
Coulter counter: electronic particle counter for single-cell suspension
Cell aggregation can limit the accuracy of this technique.
Indirect methods: require to sacrifice the culture in order to measure the number of cells
(so-called destructive assays):
• DNA content
• Protein content
• Metabolic activity (ex. MTT, MTS, …)
Require calibration to convert DNA or protein content, or metabolic activity to cell
number.
Measurement of Cell Characteristics
Cell Viability
Viability Criteria
Methods
Ability to attach and divide upon plating at
very low density
Clonogenic Assays
Plasma-membrane integrity
Trypan blue stain
Polar nuclear stain
Fluorescent cytoplasmic stains
Lactate dehydrogenase release
Metabolic activity
Mitochondrial potential stain
Presence of DNA
Nonpolar nuclear stain
Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson Prentice Hall Bioengineering, 2004
Viable cell fraction is assessed by light or fluorescent microscopy or flow
cytometry analysis.
Measurement of Cell Characteristics
Cell Function: metabolism, protein synthesis, signal transduction…
Function Studied
Method
Protein synthesis and secretion
Incorporation of 3H-leucine, 35S-methionine and
3H-proline
Protein-specific assays (ELISA)
DNA synthesis
Incorporation of 3H-thymidine, 3H-deoxycytidine
and BrdU
Metabolic activity
Rate of consumption of
glucose/glutamine/aminoacids/oxygen and rate of
production of lactate/ammonia
Signal transduction
Intracellular calcium or pH dyes
Microphysiometer (changes in extracellular pH)
Biochemical assays on cell extracts
Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson Prentice Hall Bioengineering, 2004
Measurement of Tissue Characteristics
Tissue Properties: combination of both cells and extracellular matrix
(ECM)
General appearance: size, shape, color, weight…(microscopy, enzymatic tissue
disruption…);
Cellular component: results from a dynamic equilibrium between various
cellular-fate processes (cell death, cell migration);
ECM component: also a result of a dynamic equilibrium process (microscopy,
radioisotope labeling, immunostaining…);
Function: (ex. Contractility in cardiac tissue, insulin-producing function by β-islet
cells in pancreas)
Mechanical measurements: viscoelasticity, contractility, mechanical loading,…
Physical properties: optical, acoustic, conductive, thermal…
Cryopreservation
Cryopreservation: storage of the cells by freezing
Why?
the ability to recover a well-characterized cell state for tissue-engineered
medical products;
the prevention of genotypic drift due to genetic instability;
storage prior to senescence;
prevention of transformation;
control of phenotypic instability;
a back-up in case of contamination;
storage for characterization and pathogen testing.
Cryopreservation
Cryopreservation (cont.)
How?
Cells are usually removed from the substrate by enzymatic or mechanical
disruption and suspended in a solution (typically, culture medium) that contains a
cryoprotectant.
Cryoprotectant: chemical additive such as dimethyl sulfoxide (DMSO) or glycerol
that is used to prevent ice formation inside the cell, which is a major cause of cell
damage.
Cells are cooled and stored in a liquid nitrogen tank at -196ºC;
Cooling rate is a critical parameter that affects subsequent cell survival
(e.g. -1ºC per minute)
Contaminants
Contamination: can be suspected by a macroscopic change in the
culture (cloudiness, observable colonies, change in pH…)
5 major types of microbes
Bacteria: distinguished by their shape (rods, cocci or spheres) and observable
motility;
Yeast: round, refractile particles that increase in number over time;
Fungi: observed as a combination of thin filamentous extensions and clusters of
spores;
Mycoplasma and viral detection: particularly difficult to observe; mycoplasma
and some viruses pass through the sterile filters used for cell culture research (0.2
µm pore diameter); unexpected behavior of cultured cells: morphology, growth
rate…; confirmed by specific assays (e.g. PCR).
Prevention by using sterile culture techniques and antibiotics