Facultad de Medicina

Asignatura BIOLOGIA MEDICA

P12-Práctica/sem–cell culture.
Laboratorio
Materiales
Métodos
Cultivos primarios
Líneas celulares.
HeLa cells

Seguridad
Criopreservación
Cuantificacion de los cultivos

Este obra está bajo una Licencia Creative Commons

Atribución-NoComercial-SinDerivadas 3.0 Unported.
 1878 Claude Bernard
proposed the possibility of maintaining physiological systems of
the organism even after the death of organism, in a living system.
1885 Roux
maintained the embryonic chick cells in saline culture.
1897 Loeb
survival of cells isolated from blood and connective tissue in plasma and serum
1903
Observation and detailed study of in vitro cell division of salamander leucocytes.
1907 Ross Harrison
cultivated the nerve cells of frog by the hanging drop method.

HANGING
DROP
METHOD

1910 Barrows
cultivated the chicken embryo cells using cells of plasma clots
1911 Lewis
The very first liquid media for supporting the monolayer.
1912 Alexis Carrel
Cell culture technique by using liquid media, and chick embryo extract.
The liquid culture media provided all essential growth nutrients
The use of trypsin for isolation of cells from was vital in this technique.

History of animal cell culture – Science of Healthy

Cell Culture Aseptic Techniques
Aseptic techniques ensure that all cell culture procedures are performed to a standard
that will prevent contamination from bacteria, fungi, mycoplasma and cross
contamination with other cell lines.
All work is done
under a tissue
culture hood. A
movable glass panel
or sash covers the
face area of the
tissue culture hood
and acts as a
physical barrier that
helps to maintain a
particulate-free
environment and
laminar air flow.
In case the sash
stop is overriden an
alarm will sound.
Cell Culture Microbiological Safety Cabinets
A microbiological safety cabinet is probably the most important piece of equipment for
cell culture since, when operated correctly, it will provide a clean working environment
for the product, whilst protecting the operator from aerosols.

In these cabinets operator and/or product protection is provided through the use of
HEPA (high efficiency particulate air) filters. The level of containment provided varies
according to the class of cabinet used.

http://www.sigmaaldrich.com/technical-documents/protocols/biology/design-and-equipment.html
Cell Culture Aseptic Techniques
Tissue culture hood.

http://sligoschneider.wordpress.com/2012/07/19/7-17-12-changing-cell-culture-media/
http://www.sigmaaldrich.com/life-science/cell-culture.html

http://www.sigmaaldrich.com/video/life-science/microbiological-safety-cabinet-
and-its-use.html
Cell Culture
The Micropipette
Micropipettes are used for accurately transferring small
volumes of liquid in the milliliter and microliter range.

We will cover three basic sizes of these handy little

instruments.
The P - 1000 accurately delivers 200 to 1000 microliters.
The P- 200 accurately delivers 30 to 200 microliters.
The P-20 accurately delivers 2 to 20 microliters.

The liquid is drawn into, and dispensed from a disposable

pipette tip
The disposable tip is changed between liquid transfers.
Pre-rinse every new pipette tip with the liquid to be pipetted.

video
https://www.youtube.com/watch?v=TFXX8yCWjMo
http://www.sigmaaldrich.com/life-science/cell-culture.html

http://www.sigmaaldrich.com/life-science/cell-culture/learning-
center/cell-culture-technical-videos.html
Cell Culture
Centrifuges
A small bench-top centrifuge with controlled braking is sufficient for most purposes.
Cells sediment satisfactorily at 80 – 150 x g. Higher gravitational forces may cause
damage and promote agglutination of the cell pellet.

Heraeus™ Pico™ and Fresco™ Microcentrifuges

Accelerate routine sample preparation processes with the high

speed and generous capacity of Thermo Scientific™ Heraeus™
Pico™ and Fresco™ Microcentrifuges. Safe and convenient, the
Heraeus Pico and Fresco 17 are ideal for routine laboratory work
while the Heraeus Pico and Fresco 21 are perfect for high-end
research applications, reducing spin times up to 20%.
Microcentrifuges are available in two speed options, with ventilated
and refrigerated models. - See more at:
http://www.thermoscientific.com/en/product/heraeus-pico-fresco-
microcentrifuges.html#sthash.iNmcGXSS.dpuf

Heraeus™ Labofuge™ 200 Centrifuge

Meet the needs of medical practices, small laboratories and the most
rigorous lab or clinical environment with the maintenance-free Thermo
Scientific™ Heraeus™ Labofuge™ 200 Centrifuge. This compact clinical
centrifuge comes standard with a 12-place fixed-angle rotor and a full set
of adapters. - See more at:
http://www.thermoscientific.com/en/product/heraeus-labofuge-200-
centrifuge.html#sthash.oA5YNvDY.dpuf
Cell Culture
Incubators
Cell cultures require a strictly controlled environment in which to grow. Specialist
incubators are used routinely to provide the correct growth conditions, such as
temperature, degree of humidity and CO2 levels in a controlled and stable manner.
Generally, they can be set to run at temperatures in the range of 28°C (for insect cell lines)
to 37°C (for mammalian cell lines) and set to provide CO2 at the required level (e.g. 5-
10%). Some incubators also have the facility to control the O2 levels
Cell Culture
Plasticware and Consumables
Almost every type of cell culture vessel, together with support consumables such as
tubes and pipettes, are commercially available as single use, sterile packs.
Suppliers include Sigma-Aldrich, Nunc, Greiner, Bibby Sterilin and Corning.

The use of such plasticware is more cost effective than recycling glassware, enables a
higher level of quality assurance and removes the need for validation of cleaning and
sterilisation procedures. Plastic tissue culture flasks are usually treated to provide a
hydrophilic surface to facilitate attachment of anchorage dependent cells.

T flasks Cell Culture Flasks

Cell Culture
Plasticware and Consumables
 Cell Culture Plasticware

http://www.weiku.com/products/9748777/Cell_Culture_Flask.html
Cell Culture
Culture Media

Basic Constituents of Media

Inorganic salts
Carbohydrates
Amino Acids
Vitamins
Fatty acids and lipids
Proteins and peptides
Serum
Trace Elements

Fetal bovine serum

cell culture medium

http://www.sigmaaldrich.com/technical-documents/protocols/biology/the-cell-environment.html
Cell Culture
Culture Media
Buffering Systems
Most cells require pH in the range 7.2-7.4
and close control of pH is essential for
optimum culture. Fibroblasts prefer a
higher pH (7.4-7.7) whereas, continuous
transformed cell lines require more acid
conditions pH (7.0-7.4).

Regulation of pH is achieved by (i) a

“natural” buffering system where gaseous
CO2 balances with the CO3/HCO3 content
of the culture medium and (ii) chemical
buffering using a zwitterion called HEPES.
Cultures using natural bicarbonate /CO2
buffering systems need to be maintained in
an atmosphere of 5-10% CO2 in air
usually supplied in a CO2 incubator.

Most commercial culture media include

phenol red as a pH indicator. Usually the
culture medium should be changed or
replenished if the colour turns yellow (acid)
or purple (alkali).

http://www.sigmaaldrich.com/technical-documents/protocols/biology/the-cell-environment.html
Cell Culture
Cell lines
New cell lines should only be acquired from a specialist, reputable culture collection
such as ECACC. Moreover, if a laboratory believes it already has a certain cell line in
its liquid nitrogen store, the identity and purity of such a cell line should be
questioned in the absence of a well recorded culture history and recent test data.

The European Collection of Cell Cultures

(ECACC) is one of the world’s largest
Biological Resource Centres supplying a
diverse range of authenticated cell lines.

http://www.hpacultures.org.uk/products/celllines/index.jsp

When a Culture Collection acquires a new cell line it will characterise the cell line
using techniques such as isoenzyme analysis and DNA profiling so that the
identity of the cell line subsequently can be verifi ed. The Collection will then
establish a hierarchy of Master and Working cell banks, cryopreserved in liquid
nitrogen, that are demonstrated free from microbial contamination including
mycoplasma.
Cell Culture
Cell lines
http://www.hpacultures.org.uk/products/celllines/index.jsp
HeLa cell culture
The woman from which the HeLa cells are
derived was named Henrietta Lacks, and she
was a wife and mother of five when she passed
away at John Hopkins University at the age of
thirty-one
A sample of one of her tumors was sent in 1951
to George and Maragret Gey who had been
seeking a line of human cells that would survive
indefinitely outside the body for research
purposes. The tumor cells they received
multiplied like nothing they had ever seen
before and soon the cells, dubbed HeLa in a
truncated form of Lacks' name, were being
shipped to their colleagues stationed around the
world.
The cells later became a laboratory standard
http://www.lifesciencesfoundation.org/events-item-239.html

The Immortal Life

of Henrietta Lacks

Scientists have
grown some 20 tons
of her cells and there
are almost 11,000
patents involving
HeLa cells.
HeLa cell culture
Subculture Routine: Split sub-confluent cultures (70-80%) 1:3 to 1:10 i.e. seeding at
1.3x10,000 cells/cm² using 0.25% trypsin/EDTA; 5% CO2; 37°C.
Culture Medium: EMEM (EBSS) + 2mM Glutamine + 1% Non Essential Amino Acids
(NEAA) + 10% Foetal Bovine Serum (FBS).

Phase Contrast differential interference contrast (DIC)

HeLa cells can easily invade other cultures during routine lab transfer procedures and had
contaminated many other cell lines used in research by the late 1960s. As a result,
numerous research papers that were published based on a variety of cultured cell types
have subsequently been proven to be HeLa cells

http://www.microscopyu.com/staticgallery/dicphasecontrast/heladic.html
HeLa cell line
DNA Profile
Tandemly repeated DNA sequences are common in the human STR-PCR
genome and show considerable variability among individuals. Short tandem repeat
Such polymorphisms have become useful tools for DNA (STR) analysis is a
analysis and typing, and are currently employed for human molecular biology
identification, paternity testing, and genetic mapping. method used to
compare specific loci
Short tandem repeats (STRs) contain tandemly repeated DNA on DNA from two or
sequences ranging in length from about two to seven base more samples.
pairs.
Of the STR loci used in identity testing, most have A short tandem repeat
tetranucleotide repeats and allele sizes between 100 and 350 is a microsatellite,
base pairs. STR alleles are defined and named by the number consisting of a unit of
of repeated sequences they contain, and as more samples and two to thirteen
populations are being examined, the number of reported alleles nucleotides repeated
is increasing. hundreds of times in a
row on the DNA strand.
The locus CSF1PO (proto-oncogene for CSF-1 receptor
gene) is included in the thirteen core loci used by the FBI for STR analysis
the Combined DNA Index System (CODIS) database. Reported measures the exact
alleles of this STR locus contain 6 to 15 repeats of the number of repeating
tetranucleotide AGAT units

Margolis-Nunno H, Brenner L, Cascardi J, Kobilinsky L. A new allele of the short

tandem repeat (STR) locus, CSF1PO. J Forensic Sci 2001;46(6):1480–1483.
HeLa cell line
DNA Profile
Detection of porcine X and Y chromosome–specific sequences by using
PCR of the amelogenin gene
The gene for amelogenin can be used in sex determination of samples from unknown
human origin through the Polymerase Chain Reaction (PCR).
Using primers specific for intron 1 of the gene, the gene sequence for the intron can be
amplified. The X chromosome gene, AMELX, gives rise to a 106 bp amplification product
(amplicon) and the Y chromosome gene, AMELY, a 112 bp amplicon.nHence, the AMELX
contains a 6 bp deletion in the intron 1.

Therefore, when the amplicons are run on an agarose gel, samples from male sources
(XY) will show two bands on an agarose gel (one for the 106 bp fragment and one for the
112 bp fragment), whereas females (XX) will show only one band. Thus, this process
allows for sex determination of unknown samples
Kashyap VK, Sahoo S, Sitalaximi T, Trivedi R. (2006). "Deletions in the Y-derived
amelogenin gene fragment in the Indian population". BMC Med Genet. 7: 37.

Gel electrophoresis showing amplicons

of the amelogenin (AMEL) gene
fragments. Upper band, amplicons
derived from AMELX; lower band,
amplicons derived from AMELY.
Molecular weight markers: ΦX174
HincII digest.

S. Sembon, M. Iwamoto, M. Hashimoto, T. Oishi, D. Fuchimoto, S. Suzuki, S. Yazaki, A. Onishi, Porcine androgenetic
embryos develop to fetal stage in recipient mothers, Theriogenology, Volume 78, Issue 1, 1 July 2012, Pages 225-231,
Safety Aspects of Cell Culture
The main aim of risk assessment is to prevent injury and avoid harm to
individuals and the environment. In many countries the performance of risk
assessment is a legal requirement. There are European Community directives
covering Health and Safety at work.
Visit the European Agency for Safety and Health at Work website
(www.europe.osha.eu.int) for information on legislation and standards.

Risk assessments must be undertaken prior to starting any activity.

The assessment consists of two elements:
Identifying and evaluating the risks.
Defining ways of avoiding or minimising the risk.
For animal cell culture the level of risk is dependent upon the cell line to be used and is based
on whether the cell line is likely to cause harm to humans.
Non human/non primate continuous cell lines and some well characterised
Low risk human continuous lines.
Medium risk Poorly characterised mammalian cell lines.
Primary cells derived from human/primate tissue or blood.
Cell lines with endogenous pathogens (the precise categorisation is dependent
upon the pathogen) – refer to ACDP guidelines, for details†.
High risk
Cell lines used following experimental infection where the categorisation is
dependent upon the infecting agent – refer to ACDP guidelines, for details.

†Advisory Committee on Dangerous Pathogens (ACDP) (1995) Categorisation of Biological Agents According to Hazard
and Categories of Containment, 4th edition, HSE books, Sudbury, UK. ‘Second supplement (Edition 2000)
An update to the Approved List of Biological agents was issued in 2004, available at:
http://www.hse.gov.uk/pubns/misc208.pdf

http://www.sigmaaldrich.com/technical-documents/protocols/biology/safety-aspects-of.html
Cryopreservation of Cell Lines
There has been a large amount of developmental work
undertaken to ensure successful cryopreservation and cell lines can be
resuscitation of a wide variety of cell lines of different cryopreserved in a
cell types. suspended state for
The basic principle of successful cryopreservation and indefinite periods
resuscitation is a slow freeze and quick thaw. provided a
Although the precise requirement may vary with temperature of less
different cell lines as a general guide cells should be than -135°C is
cooled at a rate of –1°C to –3°C per minute and thawed maintained.
quickly by incubation in a 37°C water bath for 3-5
minutes

1 Cultures should be healthy with a viability of >90% and no signs of

microbial contamination.
2 Cultures should be in log phase of growth (this can be achieved by using
pre-confluent cultures i.e. cultures that are below their maximum cell density
and by changing the culture medium 24 hours before freezing).
3 A high concentration of serum/protein (>20%) should be used. In many
cases serum is used at 90%.
4 Use a cryoprotectant such as dimethyl sulphoxide (DMSO) or glycerol to
help protect the cells from rupture by the formation of ice crystals. The most
commonly used cryoprotectant is DMSO at a final concentration of 10%,

http://www.sigmaaldrich.com/technical-documents/protocols/biology/cryopreservation-and.html
Subculture of Adherent Cell Lines
Primary Cultures
Primary cultures are derived directly from excised, normal animal tissue
and cultures either as an explant culture or following dissociation into a
single cell suspension by enzyme digestion. Such cultures are initially
heterogeneous but later become dominated by fibroblasts.

Continuous Cultures
Continuous cultures are comprised of a single cell type that can be serially
propagated in culture either for a limited number of cell divisions
(approximately thirty) or otherwise indefinitely. Cell lines of a finite life are
usually diploid and maintain some degree of differentiation.
the cell lines are categorized into two types:
a) Finite cell Lines - The cell lines which have a limited life span and go through a limited
number of cell generations (usually 20-80 population doublings) are known as Finite cell
lines. These cell lines exhibit the property of contact inhibition, density limitation and
anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours.
b) Continuous Cell Lines - Cell lines transformed under in vitro culture conditions give rise
to continuous cell lines. The cell lines show the property of ploidy (aneupliody or
heteroploidy), absence of contact inhibition and anchorage dependence. They grow in
monolayer or suspension form. The growth rate is rapid and doubling time is 12-24 hours.

Monolayer cultures - When the bottom of the culture vessel is covered with
a continuous layer of cells, usually one cell in thickness, they are referred to
as monolayer cultures.
Suspension cultures - Majority of continuous cell lines grow as monolayers.
Some of the cells which are non-adhesive e.g. cells of leukemia or certain
cells which can be mechanically kept in suspension, can be propagated in
suspension.
Commonly used cell lines

http://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-types-culture.html
Phases of Cell Growth

http://www.roche-applied-science.com/sis/cellanalysis/index.jsp?id=cell_050400
Features of cell culture with evolution of a cell line

http://www.biotechnology4u.com/animal_biotechnology_types_cell_cultures.html
Cell Quantification

Number Live Cells Counted

Viable Cell Count =
Number of large corner Squares x 10,000 x Dilution
(live cells per millilitre)
counted
Cell Quantification Haemocytometer

Neubauer ruling
http://www.swtafe.vic.edu.au/toolbox/lab_ops/laboratory/studynotes/SNHaemo.htm
Cell Culture

http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook.html
BIOL
P12 PRACT/SEM [plan trabajo]

visitar todos los enlaces marcados [7]

discusión y aclaración de conceptos
autoevaluación

1. Tipos de cámaras de flujo laminar

2. Parámetros de incubación en estufas de cultivo (porqué se pone CO2)
3. Tipos de medios de cultivo (y ejemplos)
4. Como se controla el pH y comose detecta
5. The European Collection of Cell Cultures (qué es, cómo se gestiona)
6. Origen y características de las células HeLa
7. Qué son Short tandem repeats (STRs) y para que sirve medirlos en líneas celulares
8. Cuáles son los cultivos celulares considerados de alto riesgo
9. Cuatro reglas para conseguir la criopreservación de cultivos.
10. Parámetros de criopreservación (bancos de tejidos)
11. Clasificaciones de los tipos de cultivo cellular
12. Fases de crecimiento de un cultivo
13. Etapas de una línea cellular hasta que deja de multiplicarse in vitro (porqué?)
14. Como se hace un recuento para averiguar la densidad cellular de un cultivo
15. Como se averigua el porcentaje de células muertas en un cultivo