Biology of cultured cells

1. Biology of cultured cells

2. The Culture Environment
• Changes of Cell’s
microenvironment needed that
favour the spreading, migration, and
proliferation of unspecialized
progenitor cells rather than the
expression of differentiated
functions.
• Cell – cell interaction and cell –
matrix interaction are reduced
because the cells lack the
heterogeneity and three dimensional
architecture found in vivo
• Many hormonal and nutritional
stimuli are absent

3. The Culture Environment
Influences of environment on the culture is expressed
via 5 routes:
 The nature of substrate
 Degree of contact with other cells
 Physicochemical and physiological constitution of
medium
 The constitution of gas phase
 The incubation temperature

4. Cell adhesion
• Most cell from solid tissue grow as adherence
monolayer.
• They need to attach and spread out on the
substrate before start to proliferate.
• Glass-slight negative charge
• Plastic- treated with an electric ion discharge
• Cell adhesion mediated by specific sell surface
receptor for molecules in the extracellular
matrix

5. Cell Adhesion molecule
• Three major classes of transmembrane proteins:
• CAMs(Ca2+ independent) and cadherin (Ca2+ dependent)
involved in interaction between homologous cells.
• Intergrin – Receptor for matrix molecules such as fibronectin,
entactin, laminin, and collagen, which bind to them via a specific
motif usually containing arginine-glycine-aspartic acid (RGD)
• Transmembrane proteoglycans-interact with matrix constituent
such as other proteoclycans or collagen, but not via RGD motif.

7. Intercellular junctions
• Some adhesion molecules are arranged in Plasma membrane.
• Others are organized into intercellular junctions.
• There are different junctions namely
• Desmosomes and adherens junctions which hold epithelial cells
together
• Tight junctions which seal the space between cells e.g., between
secretory cells in a duct, between endothelial cells in a blood
vessel
• Gap junctions which allow ions, nutrientsand small signaling
molecules to pass between cells in contact.

8. • Desmosomes are usually distributed throughout the
area of plasma membranes in contact and they are
often associated with tight junctions.
• As epithelial cell differentiate in confluent culture
they form an increasing number of desmosomes and
if some morphological organization occurs, can
form junctional complexes - difficult to
disaggregate.
• As many of the adhesion molecule within this these
junctions depend on Ca2+ ions, chelating agent as
EDTA is often in trypsin added before
disaggregation.

9. Extracellular matrix
• Intercellular spaces in tissues are filled with extracellular
matrix(ECM)
• The constituent of ECM depends on the cell types. E.g.,
Fibrocytes secrete Type I collagen and fibronectin into the
matrix; Epithelial cells produce laminin.
• If Adjacent cell types are different – boundary of dermis
( fibrocytes) and epidermis (epithelial Keratinocytes) will
contribute to the composition of ECM produce a basal lamina.

10. • The constituent of ECM depends on the cell types.
• The complexity of ECM is a significant component in the
phenotypic expression of the cells attached to it.
• ECM control its composition and, in turn the composition of
the ECM regulates the cell phenotype.
• Cultured cell line are allowed to generate their own ECM, but
primary culture and propagation of some specialized cells, and
the induction of their differentiation, may require exogenous
provision of ECM.

11. • Two components of interaction of ECM:
• Adhesion-to allow attachment and spreading that are
necessary for cell proliferation.
• Specific interaction-reminiscent of the interaction of
an epithelial cell with basement membrane, with
other ECM constituents or with adjecent tissue
• The use of ECM constituent can be highly beneficial
in enhancing cell survival, proliferation, or
differentiation but unless recombinant molecules are
used

12. Cytoskeleton
• Cell adhesion molecules are attached to element of
cytoskeleton.
• The attachment of integrins to actin microfilaments via
linker proteins is associated with reciprocal signaling
between cell surface and the nucleus.
• Cadherin can also link to the actin cytoskeleton in
adherens junctions, mediating changes in cell shape.

13. • Demosomes, which also employ cadherins,
link to the intermediate filament via an
intracellular plaque.
• Intermediate filament are specific to cell
lineages and can be used to characterized
them.
• The microtubules are the 3rd component of
cytoskeleton – Function: Cell motility and
intracellular trafficking of micro-organelles

14. Cell motility
• Culture cells are capable of movement on a substrate.
• The most motile are fibroblast at a low cell density when
cell are not in contact
• The least motile are dense epithelial monolayers.

15. CELL PROLIFERATION
• Cell cycle is made up of four phases
• M, G1, S & G2
• M phase – Chromatin condenses into chromosomes &
2 chromatids of the chromosome segregate to each
daughter cell
• Gap 1 (G 1) – cell either progresses toward DNA
synthesis & another division cycle or exits the cell
cycle reversibly to commit to differentiation.

16. • G1 – cell is susceptible to control of cell cycle progression at a
number of restriction points, which determine whether the cell
will re-enter the cycle, withdraw from it, or withdraw and
differentiate.
• S (DNA synthesis) – DNA replicates.
• G2 – cell prepares for reentry into mitosis.
• Checkpoints at the beginning of DNA synthesis and in G 2
determines the integrity of the DNA & will halt the cell cycle
to allow DNA repair or entry into apoptosis if repair is
impossible.
• Apoptosis or programmed cell death is a regulated
physiological process whereby a cell can be removed from a
population
• It can be marked by DNA fragmentation, nuclear blebbing and
cell shrinkage.

18. Control Of Cell Proliferation
• Environment Regulates Entry Into Cell Cycle
• External Growth Factors Promote Cell Proliferation
– PDGF, EGF, FGF (+ve)
– TGF-β (-ve)
– Interact with surface receptors
• High Density Inhibits Proliferation (Contact
Inhibition)
• Inside The Cell Both Positive and Negative Factors
– Positive, cyclins, Growth Factor Receptor
Activation
– Negative, p53, Rb, Checkpoints

19. Proliferation vs Differentiation
• Proliferation Does NOT Promote Differentiation
• Differentiation Often Requires
– High density
– Cell-Cell Interaction
– Cell-Matrix Interaction
– Differentiation Factors
• The Above Conditions Can Be Antagonistic To
Proliferation

21. Tissue Retains Function Longer
• 3-D Tissue Retains Its Properties Longer But Can Not
Be Propagated
• To Overcome This Limitation
– Cells Are Cultured On Matrices
– Matrigel Is Commercially Available
• Not Perfect But Promising
– Heterotypic Cultures Are Promising
– Pathological Behavior Can Be Studied

22. Dedifferentiation
• Inability To Express In Vivo Phenotype Is Attributed
To Dedifferentiation
• Dedifferentiation Occurs may be:
– Wrong lineage of cells is selected in vitro
– Undifferentiated cells dominate differentiated cells
of reduced proliferative capacity
– Absence of appropriate inducers, hormones, matrix

23. Deadaptation vs Dedifferentiation
• Deadaptation-enviroment suppresses phenotype,
reversible
• Synthesis of specific products of specialized function
under regulatory control by hormones and can be
reinduced if the correct condition can be recreated.
• Dedifferentiation
• Specialized properties of the cell are lost - conversion
to primitive phenotype, irreversible.
• For example, a hepatocyte would lose its characteristic
enzymes and could not store glycogen because of
reversion or conversion to a precursor cell.

24. Evolution Of Cell Lines
• Approximately 10 Passages
• Senescence Follows
– Thought To Be Due To Telomeres
– Every Division Telomeres Shorten
– Germ, Stem Cells Use Telomerase
• Transformation Is Needed If Division Will
Continue

25. • Approximately 10 Passages
• Senescence Follows
–Thought To Be Due To Telomeres
–Every Division Telomeres Shorten
–Germ, Stem Cells Use Telomerase
• Transformation Is Needed If Division Will
Continue

26. Continuous Cell Lines
• Finite Cell Lines Can Change To Continuous
• Often p53 Mutation or Deletion Occurs
• Overexpression Of Telomerase
• Transformation vs Immortalization
– Transformation-additional changes in growth
characteristics
– Immortalization-infinite lifespan
• Aneuploidy Is A Characteristic Of Cont. Cell Lines
– In between diploid and tetraploid
– Heteroploidy is also observed
• Most Cells Never Become Continuous Cell Lines