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Cell and tissue culture
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Cell culture = propagation of cells outside the organism = in vitro
(Cells in the organism = in vivo) ADVANTAGES: cellular environment can be manipulated well defined cell type large quantities of cells can be obtained many cellular functions can be investigated animal experiments can be replaced living cells can be tested human cells can be tested DISADVANTAGES: does not mimic the complexity of the human body spontanous evolution
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TYPES OF CULTURED CELLS:
primary culture cell line finite continuous cell strain
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Primary culture Cells derived directly from tissues
A culture from the time of isolation until its first subculture First developed in 1907: axons grow in culture! spinal cord explant + lymphatic fluid 1 day
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cultured neuron extending processes
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Cell line Derived from primary culture via subcultivation (passage)
dissociate cells medium plate cells tissue explant cells dish Grow and divide while adhering (monolayer) to plastic dishes Require growth factor for growth Passaging: after physical or enzymatic dissociation of monolayer cultures the cells are reseeded in lower density
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Cell line Finite cell line
Finite lifespan A cell line that survives for a fixed number of population doublings, usually ~40–60 (Hayflick limit), before senescing and ceasing proliferation.
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Cell line Continuous cell line
Features: Immortal (indefinite lifespan, over 100 population doublings) Genetic abnormalities accumulate (transformed cells) Unlimited growth Loss of contact inhibition Loss of adherence dependency (suspension) injected in mice may form tumor Transformation can be spontanous or induced by viruses, carcinogens or radiation May derive: from finite cell line (in vitro transformation) – e.g. NIH3T3, CHO from tumors (in vivo transformation) – e.g. HeLa
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Cell strain A subpopulation of cell lines that have been purified by physical separation, selection or cloning Which has specific defined characteristics e.g. marker chromosome, virus resistance, antigen expression
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normal cells SEM transformed cells
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Cell cultures can be obtained e.g. from cell banks
where these cultures are kept frozen (-196 °C in liquid nitrogen)
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Density of the culture influences the cell shape
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Density of the culture influences the cell shape
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Requirements of cell cultures
Physical parameters Temperature O2/CO2. humidity Biological parameters ASEPTIC conditions Cell density Co-culture Metabolites Feeder cell layer Chemical parameters defined medium serum (growth factors, hormones) coat on culture surface (collagen, gelatine) pH osmolarity
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Laminar box (laminar flow cabinet)
is a carefully enclosed bench designed to prevent contamination of biological samples or any particle sensitive materials.
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CO2 Incubator The incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
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Plasticwares for cell culture
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Inverted Microscope Light source Objectives
in routine laboratories for live cell inspection
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R1 ES cells on fibroblast feeder layer
D3 ES (embryonic stem) cells on fibroblast feeder layer
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myeloma cells HeLa 53 MONOLAYER CELL SUSPENSION CELL CULTURE CULTURE
Adherent cells, they need surface for proliferation. These are normal cells except hematopoetic cells. Tumor cells may grow in monolayer, too. SUSPENSION CELL CULTURE Non-adherent, floating, generally hemopatopoetic or transformed, tumor cells, they do not need special surface for proliferation.
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Cell count in function of time
Mozog! sejtmorfológia változása a sejtdenzitás függvényében Confluent culture
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Cell count in function of time
3. Stationary phase /plateau without changing medium 2. log phase Cell count subculturing 1. lag phase time
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ENVIRONMENTAL INTERACTIONS
Infections (viruses, bacteria, parasites) Toxicology Immunology Carcinogenesis Biotransformation of xenobiotics GENETICS Transformation Cell fusion Cell cycle BIOCHEMISTRY DNA transcription RNA metabolism Protein synthesis Intermediate metabolism BIOTECHNOLOGY/ TISSUE ENGINEERING Cytokines/growth factors, hormones, antibody production Arteficial tissues CELL BIOLOGY Cell-cell and cell-matrix interactions Gene expression Cell proliferation Differentiation Cell migration, invasion
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(human cervical cancer)(MGG)
Slide 9. HeLa monolayer (human cervical cancer)(MGG) cytoplasm nucleus No contact inhibition Cell morphology changes with cell density!! 100% aneuploid.
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Use and perspectives of cell culture
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Pluripotent Pluripotent Unipotent
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iPS (induced pluripotent stem) cells
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Artificial human tissues and organs
Blood vessels - aorta Liver Bone Cartilage Skin Retina Requirements of tissue engineering
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Human epidermal epithelial cell cultures with
different density and different duration in culture. Cultured epidermal autograft (e.g. Epicel R)
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Therapeutic use of human epidermal epithelial cell culture.This photo
was taken 5 years after the transplantation.
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Generation of artificial teeth
or a scaffold printed in 3D scaffold may comprise compounds that are chemotactic, osteogenic, dentinogenic, amelogenic, or cementogenic. Nature Biotechnology 21, (2003)
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Diagnosis : Amniocentesis – genetic study of the embryo
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Ex vivo gene therapy In vivo gene therapy
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Ex vivo gene therapy in dentristry
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Facultative slides
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The immortal life of Henrietta Lacks
1951 Tissue were taken without her knowledge 1952 HeLa cells were used to develop polio vaccine 1955 Isolation of a single cell for cloning 1960 HeLa went to space before any astronaut 1984 HeLa was used to prove that HPV infection causes cancer 1986 Mechanism of HIV infection were studied 1989 Telomerase were described in HeLa 1993 Tuberculosis was studied 2013 Whole genome data of HeLa were published born Loretta Pleasant in 1920 George Otto Gey
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History of human embryonic stem cell research
1998 – James Thomson isolated cells from the inner cell mass of the early embryo and developed the first human embryonic stem cell lines Derivation of the H9 cell line. (A) Inner cell mass–derived cells attached to mouse embryonic fibroblast feeder layer after 8 days of culture, 24 hours before first dissociation. Scale bar, 100 μm. (B) H9 colony. Scale bar, 100 μm. (C) H9 cells. Scale bar, 50 μm. (D) Differentiated H9 cells, cultured for 5 days in the absence of mouse embryonic fibroblasts, but in the presence of human LIF (20 ng/ml; Sigma). Scale bar, 100 μm. Science 06 Nov 1998:
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Organ on a chip technique
Microfluidic chamber lined by living human cells Mimics the interconnectedness of organs within humans Fields of application: - disease modelling; - drug development; - personalized medicine Biologically inspired design of a human breathing lung-on-a-chip microdevice. (A) The microfabricated lung mimic device uses compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with ECM. The device recreates physiological breathing movements by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier. (B) During inhalation in the living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip), leading to distension of the alveoli and physical stretching of the alveolar-capillary interface. (C) Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-μm-thick PDMS membrane containing an array of through-holes with an effective diameter of 10 μm. Scale bar, 200 μm. (D) After permanent bonding, PDMS etchant is flowed through the side channels. Selective etching of the membrane layers in these channels produces two large side chambers to which vacuum is applied to cause mechanical stretching. Scale bar, 200 μm. (E) Images of an actual lung-on-a-chip microfluidic device viewed from above.
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A tissue-friendly scaffold covered with self-cells is
not immunogenic.
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The hydrogel embedded cells are printed in 3D
The hydrogel embedded cells are printed in 3D
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First artifical trachea
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