B029079 - Biotechnology applied to eukaryotic cells, with lab

italian

Cellular systems: yeasts, insect cells, mammalian cell lines, stem cells, tumor cells. Maintenance of in vitro cultures.

Material provided by the teacher and present on the e-learning platform
of the university (access of students enrolled in the course through the
own credentials)

The aim is to transmit updated information on the methods of culture and handling of yeasts, insect cells, mammalian cell lines, stem cells and tumor cells, in order to learn their main biotechnological fields of application

Good knowledge of the topics of general biology and molecular biology is required

Lectures and lab exercises

Oral examination

Lectures:
Introduction to different cell systems: yeasts, insect cells, human cell lines, stem cells, tumor cells. Maintenance of cultures in vitro, in suspension and in adhesion. Stem cells: maintenance, supports, co-cultures, growth factors, molecular mechanisms of differentiation, embryonic stem cells (ESC), somatic (ASC), induced pluripotent stem cells (iPSC). Cultures on three-dimensional supports, spheroids, organoids and their applications. Yeast: growth and maintenance conditions, genetic manipulation, expression of recombinant proteins, induction systems. Insect cells: characteristics and growth, genetic manipulation through baculovirus infection, protein expression. Mammalian cell lines: growth and maintenance, transfection techniques. Expression methods in mammalian cells: constitutive and inducible promoters, Tet-On / Tet-Off, expression of recombinant proteins, secretion, post-translational modifications. Methods for selecting stable cell lines, selection markers, fluorescent reporter. Genetic manipulation by use of transposons. Genome editing techniques: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 system.

Laboratories
Working with cells in adhesion. Detachment of cells with trypsin. Cell count in Burker's chamber. Methods for evaluating cell viability. Cells seeding for the determination of the IC50 value of a cytotoxic agent by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MMT) bromide assay. IC50 determination using GraphPad software.
Evaluation of cellular metabolism: dosage of lactate in cell culture supernatant. Measurement of oxygen consumption in whole cells. Clark electrode oxygraphy. Evaluation of mitochondrial function with Seahorse instrumentation (theoretical part, video viewing, instrument visit)
Preparation of a protein lysate starting from adhesion cells on the plate. Protein dosage with the Bradford method. Preparation of calibration curve and determination of protein concentration using Excel software.
Evaluation of cell migration. Wound repair essay. Migration on Boyden's chamber. Using ImageJ software for data analysis.
Virtual laboratory. Design of cloning and expression of a protein in eukaryotic cell systems. Presentation performed by the students.