B029079 - Biotechnology applied to eukaryotic cells, with lab

Italian

Cell systems: yeasts, insect cells, mammalian cell lines, stem cells, cancer cells. Maintenance of in vitro cultures. Two-dimensional and three-dimensional cell cultures. Maintenance and differentiation of stem cells. Protein expression in yeast, in insect cells, in mammalian cells. Genetic manipulation. Laboratory exercises.

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 up-to-date knowledge on the methods of culture and handling of yeasts, insect cells, mammalian cell lines, stem cells and tumour 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

The methods for evaluating the knowledge and skills acquired are: a) for the contents of the lessons, an oral exam. The maximum score obtainable is 28/30, which will be integrated by the points achieved in the laboratory activity (maximum 3). b) for the laboratory part, the drafting of a report with the description and interpretation of the results of the proposed experiments..

Lectures:
Introduction to different cell systems: yeasts, insect cells, human cell lines, stem cells, tumour 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 of cultures, protein expression by baculovirus infection. 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, reporter genes. 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, viewing of the instrument)
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.
Presentation by the students of an innovative scientific publication that represents an in-depth study of a topic covered in the class.