UNIT 12

Experimental models for cell biology

Model organisms Cell cultures

Under laboratory conditions (in vitro):

Under natural conditions (in vivo):

 Two conditions for any animal experimental model:
1- It should be easy to work with the model
2- We should already known the complete genome sequence

1-The Escherichia coli (E.Coli) bacteria: The simplest

procariotic model

- Grows easily under certain laboratory conditions

- divides every 20 minutes
- Genome: 4.6 million base pair – 4000 genes
- Nutrients mixture for optimal growing:
glucose, salts, amino acids, nucleic acid precursors and vitamins
- Nutrients mixture for limited growing:
glucose, salts and a source of nitrogen

What we have learnt from E.Colí?

- DNA replication
- The genetic code
- Gene expression
- Protein synthesis
- Amino acids and nucleic acid biosynthetic pathways
 Experimental models in cell biology
2- Yeasts, the simplest model for eukaryotic cells (Saccharomyces cerevisiae)

- More complex, but still grow easily under certain laboratory

conditions
- divides every 2 hours
- Genome: 12 million base pair – 6000 genes
- It contains nuclear membrane and subcellular organelles
- The DNA is organized in 16 linear chromosomes

From this model we learnt how an eucaryotic cell works:

- DNA replication, RNA processing, protein sorting and the regulation of cell división.

3- Caenorhabditis elegans (c.elegans) A multicellular nematode (a worm)

- It grows easily and can be genetically modified
- divides every 2 hours
- Genome: 100 million base pair – 19.000 genes
- Adult worms have 959 somatic cells
- It has been extensively studied under the microscope.
- It is very interesting model for animal development and cell
differentiation
 Experimental models in cell biology

4. Drosophila melanogaster
- Genome: 180 millions base pair - 14,000 genes
- Reproduction cycle: two weeks

Very useful organism for genetic experiments, the relationship between

genotype and phenotype has been extensively studied in this organism

5. The Zebrafish (26000 genes)

- They can be easily maintained in a laboratory
- They reproduce quite rapidly (every three to four months)
- The embrios develop outside of the mother and are transparent,
so, first steps in development can be easily observed
- Many mutations have been already identified.

A very interesting model to study the cardiac system.

 Experimental models in cell biology

6. The mouse

- Mice and men share about 97.5% of their genes (Around 30.000
codifying genes)
- Mice can be genetically modified (with technical difficulties)
- The generation of new mice with mutant genes

The mouse is a great model to study physiology

7. Animal Cell culture

- Isolated cells in culture can be manipulated under
controlled conditions.
- Signaling mechanisms controlling cell functions
Two types
1- Primary cell cultures:
Somatic terminal differentiated cells (muscle cells
and neurons)
2- Cell lines:
Embrionic stem cells or cells derived from tumors
 Experimental models in cell biology
Animal models
Cell cultures

Under laboratory conditions (in vitro):

1. Control of the physical-chemical

environment in which the culture is
Under natural conditions (in vivo): developed (pH, temperature, nutrients, gas
concentration…)
1. Uncontrolled variables 2. Cellular homogeneity of the culture
2. Cellular diversity 3. loss of homeostatic regulation systems
3. Homeostatic maintenance 4. Ability to repeat the experimentation in the
4. Non-reproducible results same or very similar conditions of analysis
5. Major cost of maintenance 5. Lower cost of experimentation

Cell culture is the technique of growing cells in the laboratory outside an organism. It allows for a steady supply of
cells to experiment on. Cells can be grown under different experimental conditions to mimic disease states in the
body. The differences in intracellular signaling can be observed to better understand the pathology and physiology of
the body.
 Type of cells

Fibroblast-like cells: bipolar or multipolar, elongated in shape and grow attached to

the plate (adherent)

Epithelial-like cells: polygonal, with regular and adherent dimensions forming

groups

Lymphoblast-like cells: spherical forms that grow in suspension (non-adherent)

 Cell culture media
Composition of culture media for mammalian cells (ex. Dulbecco's Modified Eagle's
Medium - DMEM)
 Experimental conditions
Culture media:

Incubation:
 Incubation temperature: 37ºC
 Incubation atmosphere: 5% CO2, to allow an exchange with the
bicarbonate of the medium and thus regulate a pH=7
 Humidity: 95%
Flask and p35 culture plates
Cell line subcultures

Cell line maintenance

 Cell culture media
There are many different cell cultures:
 Cell culture contamination
Culture media are normally complemented with antibiotics such as
Streptomycin, Penicillin y Amphotericin
Biological agents that can contaminate cell cultures are:

Bacteria such as E.coli Yeast

Mycoplasma
Adherent cells vs suspensión cells

Adherent cells

Cells in suspension
 GROWTH CURVE

Phenol red
pH

Day 1 Neutral
Day 2

Day 3
Acid

Adaptation Glucose and Glucolisis

Acidic environment
amino acid
consumption

Accumulation of metabolites
HeLa Cells (70% confluence)
 Introduction to engineering replacement tissues and regenerative medicine

Tissue engineering: Three components – scaffolds, cells, and biochemical signals

1. Scaffolds - Biomaterials serve as scaffolds for regenerative medicine to:

- Deliver cells
- Provide biological signals and physical support to the
transplanted cells
- Mobilize endogenous cells to repair tissues

The goal of a biomaterial is to mimic and modulate native tissue microenvironments, to replace the lost form
and to recover the relationships between structure–function -> understand cell–material interaction

2. The cellular component of the system is the only living

portion that secretes and builds the tissue.

Endogenous cells
a) from the surrounding environment that migrate into a scaffold
b) cells recruited from distant sites: bone marrow-derived stem cells.

Exogenous cells that can be delivered to a patient

a) include autologous cells, which are expanded in culture or prepared
* There are some examples of cellular
immediately after extraction
self-organization strategies that do not
b) allogeneic cells harvested from healthy donors.
use materials, such as cell sheets and
organoids
3. Biological cues - can be incorporated into the scaffold to
enhance cellular function and tissue production: Scaffold
functionalization

Approaches to engineering replacement tissues:

in vitro cultivation and in vivo development:

a) In vitro: static cultures or complex bioreactors -> to produce living tissues for transplantation that
are ready for their desired biological function

- biomaterials -> for cell attachment and growth can incorporated biological or physical cues to
encourage tissue formation. Depending on the maturity of in vitro cultivated ‘tissue’, the
biomaterial scaffold may or may not be present when the construct is implanted

b) In vivo: the body can be used as an incubator for tissue development

- Biomaterials, cells or biological cues can be implanted alone or in combination to locally

stimulate tissue growth. In vivo incubation and development provide innumerable biochemical
and biophysical cues to support tissue growth

as biomaterials become more sophisticated with respect to biological cues, the potential for efficiently
inducing in vivo tissue development and directing repair without adding cultured cells increases