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Equipment: laminar flow (biosafety) cabinet giving a sterile working area, CO2 incubator (37 degrees Celsius, 5 percent CO2, humidified), inverted microscope, centrifuge, autoclave, water bath and cryostorage in liquid nitrogen. Sterile technique: work in the cabinet, disinfect surfaces with 70 percent ethanol, use sterile disposable plastic and filtered pipettes, avoid talking over open dishes. Contamination (bacteria, fungi, mycoplasma) must be detected and excluded. Medium: a basal medium with glucose, amino acids, vitamins and salts, a bicarbonate buffer, phenol red as pH indicator, supplemented with serum (foetal bovine serum) and sometimes antibiotics. Adherent cells attach to plastic and are detached with trypsin; suspension cells are simply diluted. Cells are passaged before confluence and frozen with a cryoprotectant (DMSO).

Primary cells are taken directly from tissue: physiologically relevant but with a limited lifespan (senescence) and variability between donors. Cell lines are immortalised (from tumours or by genetic modification): unlimited growth and reproducibility, but they can differ from normal tissue and acquire changes over time (HeLa, HEK293, MCF-7). Three-dimensional culture (spheroids, hydrogels, organoids) reproduces tissue architecture, cell-matrix contacts and gradients much better than a 2D monolayer.

Yamanaka showed that four factors (Oct4, Sox2, Klf4, c-Myc) reprogram fibroblasts into induced pluripotent stem cells (iPSCs), which resemble embryonic stem cells: self-renewal and differentiation into any cell type. Uses: disease modelling (cells from patients differentiated into affected cell types), drug screening, and cell therapy. Advantages: no embryo destruction, patient-matched cells. Limitations: tumour risk (teratomas), genetic and epigenetic abnormalities, cost and variability between lines.

1. Why must cell culture be performed in a laminar flow cabinet? 2. What is the role of phenol red in the medium? 3. Compare primary cells and cell lines. 4. Which factors reprogram cells to iPSCs?

1. To maintain sterility and protect the cells from contamination. 2. It is a pH indicator (colour change with acidification). 3. Primary cells are physiologically relevant but have a limited lifespan; cell lines proliferate indefinitely but may differ from normal tissue. 4. Oct4, Sox2, Klf4 and c-Myc.

Polyclonal antibodies are a mixture recognising many epitopes; monoclonal antibodies recognise a single epitope and are uniform. Kohler and Milstein method: immunise a mouse with the antigen, fuse its spleen B cells with myeloma cells (with polyethylene glycol), select hybrids in HAT medium (unfused myeloma cells die because they lack HGPRT; unfused B cells die naturally), screen the clones for the desired antibody, expand and clone by limiting dilution. Humanisation: mouse antibodies cause immune reactions in patients (HAMA). Chimeric antibodies (mouse variable and human constant regions, suffix -ximab), humanised antibodies (only the hypervariable loops from mouse, suffix -zumab) and fully human antibodies (phage display or transgenic mice, suffix -umab) reduce this.

- Precipitation and immunodiffusion: soluble antigen and antibody form visible precipitates at equivalence. - Agglutination: antibodies cross-link particles (red cells, latex beads): blood grouping and pregnancy tests. - ELISA (enzyme-linked immunosorbent assay): antigen or antibody attached to a plate, detected by an enzyme-labelled antibody that converts a substrate to a coloured product. Formats: direct, indirect, sandwich (two antibodies binding two epitopes; most specific and sensitive) and competitive. - Radioimmunoassay and fluorescence or chemiluminescence assays use other labels.

Immunohistochemistry (IHC): antibodies on tissue sections show where a protein is (enzyme or fluorescent labels); used in diagnostic pathology (for example oestrogen receptor and HER2 in breast cancer). Western blot: proteins are separated by SDS-PAGE according to size, transferred to a membrane and detected with antibodies, giving presence and approximate size. Flow cytometry: cells in suspension pass one by one through a laser; forward scatter reflects size and side scatter reflects granularity, and fluorescent antibodies identify surface markers (for example CD4 and CD8 in lymphocyte counts). Flow can also sort cells (FACS).

1. Why are hybridoma cells grown in HAT medium? 2. What do the suffixes -ximab and -zumab mean? 3. Describe a sandwich ELISA. 4. What information does a western blot give? 5. What do forward and side scatter measure?

1. To select hybrids: unfused myeloma cells lack HGPRT and die, unfused B cells do not survive. 2. -ximab: chimeric antibody; -zumab: humanised antibody. 3. A capture antibody binds the antigen on the plate, and an enzyme-labelled detection antibody binds a second epitope and gives a coloured product. 4. The presence and molecular size of a specific protein. 5. Forward scatter: cell size; side scatter: granularity or complexity.

Reagents: template DNA, two primers flanking the target, thermostable DNA polymerase (Taq), dNTPs, buffer with magnesium. Cycle (25-40 repeats): denaturation (about 95 degrees Celsius, strands separate), annealing (50-65 degrees, primers bind) and extension (72 degrees, polymerase copies). The amount of product doubles each cycle. Controls: a negative control with no template detects contamination. RT-PCR (reverse transcription PCR): RNA is first converted to cDNA by reverse transcriptase, so gene expression or RNA viruses can be detected. Quantitative PCR (qPCR, real-time PCR): a fluorescent dye (SYBR Green) or probe (TaqMan) reports amplification each cycle; the cycle threshold (Ct) is lower when more starting template is present; used for gene expression (normalised to a reference gene) and viral load. Application: SARS-CoV-2 diagnosis by RT-qPCR.

Sanger sequencing: DNA synthesis with a small proportion of fluorescent dideoxynucleotides (ddNTPs) that terminate the chain; fragments of every length are separated by capillary electrophoresis and the fluorescence read out gives the sequence. Accurate, reads up to about 800 bases, used to confirm variants. Next-generation sequencing (NGS): library preparation (fragmenting DNA, adding adaptors), massively parallel sequencing (for example sequencing by synthesis on Illumina platforms), and bioinformatic analysis (alignment to a reference genome, variant calling). It allows whole-genome, exome and targeted panel sequencing, and RNA sequencing. Long-read technologies (nanopore, PacBio) read longer fragments.

A biobank collects, processes, stores and distributes human biological samples (tissue, blood, DNA) with associated clinical data. Requirements: informed consent, privacy protection, quality management, standardised sample handling and traceability, and governance of access. Biobanks support biomarker discovery, epidemiology and personalised medicine. The Marche Biobank project is a regional example.

1. List the three steps of a PCR cycle. 2. What is the difference between RT-PCR and qPCR? 3. What does a lower Ct value mean? 4. How does Sanger sequencing terminate chains? 5. What ethical requirements apply to biobanks?

1. Denaturation, annealing, extension. 2. RT-PCR includes reverse transcription of RNA to cDNA; qPCR measures amplification in real time by fluorescence. 3. More starting template. 4. Incorporation of dideoxynucleotides that lack the 3' hydroxyl. 5. Informed consent, privacy protection and governance of access.