Cell Technology

• American Type Culture Collection
• Rosewood Park Memorial Institute
• Hank's Balanced Salt Solution
• Fetal Bovine Serum
• Phosphate Buffered Saline
• Dulbecco's Modification of Eagle's Medium

• 89% DMEM
• 10% FBS
• 1% Glutamine

• 1) Control of environment
• 2) Better use of reagents
• 3) Other issues (Ethical, moral and legal)

• 1) Expertise needed
• 2) Quantity and cost (of equipment)

• 1) The nature of phase on/in which the cell grows (Adherent or suspension)
• 2) Medium
• 3) Constitution of gas phase
• 4) Incubation temperature

• 1) Cells secrete extracellular matrix (ECM)
• 2) Matrix adheres to charged substrate
• 3) Cell then binds to the matrix through specific receptors

• Cadherins
• Interactions between homologous cells
• Calcium dependent

• Integrins
• Interactions between cell and substrate
• Binds to matrix molecules via RGD motif

• Transmembrane proteoglycans
• Interactions between cell and substrate
• DOES NOT bind to matrix molecules via RGD motif

Glycolysis and Citric Acid Cycle

• pH
• CO2 and Bicarbonate
• Buffering
• Oxygen
• Temperature

• Amino Acids
• Vitamins
• Salts
• Glucose
• Antibiotics
• Serum

The concentration of AA influences the cell survival and growth rate of the cells

• Essential AA: Isoleucine, leucine, lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine
• Cysteine
• Tyrosine
• Glutamine

Fat soluble (A,D,K,E) and water soluble (Choline, folic acid)

• Ca2+ --> Cell adherence (cadherins)
• Na+,K+,Cl- --> Regulates membrane potential
• SO42-, PO43-, HCO3- --> Regulates intracellular charge

Glycolysis

• Advantages
• Reduces contamination

• Disadvatages
• - Antibiotic resistant strains of organisms may arise
• - Hides low level contaminants that may appear when antibiotics is removed
• - Encourages poor aseptic techniques
• - Antimetabolic effects

• Growth factors (eg. Platelet derived growth factor PDGF, Fibroblast growth factors FGF, Epidermal growth factor EGF)
• Adhesion factors (eg. Fibronectin)
• Proteins (Major componet in serum. Carriers for other molecules)
• Hormones (eg. Insulin and Hydrocortisone- cell attachment and proliferation)
• Lipids (eg. Oleic acid) BOUND TO PROTEINS
• Minerals (eg. Iron, copper, zinc) BOUND TO PROTEINS

Filtration using a 0.22um filter.

• 1) Inject animal with substance of interest (usually mixed with adjuvant)
• 2) After 1 month, a booster is given
• 3) Two weeks later, blood is drawn and serum is harvested

The second antigenic challenge is:

• More immediate in onset
• Of a higher titer
• Higly specific for that antigen
• Persists longer

Polyclonal antiserum contains antibodies that bind to an antigen, but at different binding sites or affinities. This is because they have differenct hypervariable regions (where they bind to the antigen).

B cells that are primed to proliferate turn to plasma cells which make antibodies. Cytokines drive differentiation and proliferation of B cells to become plasma cells.

Monoclonal antibodies come from the same B cell. They bind to the same antigen at the same site and at the same antigen binding site. (They are all the same!)

• A hybridoma is a cell line that produces monoclonal antibodies and is immortal.
• (Basically Spleen cells+Myeloma Cells)

• PEG - Polyethylene glycol
• fuses myeloma and spleen cells
• HAT - Hypoxanthine aminopterin thymidine
• Blocks a pathway in myeloma cells so they die. Myeloma cells don't have the enzyme HGPRT while spleen cell does, but spleen cell dies within 2 days.

Balb/c mice and rat.

The cells must not synthesise immunoglobulin, must die in selective HAT media, and must be immortal.

Purified proteins, glycoproteins, carbohydrates, recombinant proteins, whole cells.

Adjuvant is use to augment the immune response. A common adjuvant to use is Freund's Complete Adjuvant (FCA).

Thioguanine.

Myeloma cells 1x10^7 and Spleen cells 5x10^7

1:5

Use ELISA

Human Anti-mouse Antibodies

Research (analysing cell surface markers and Intra cellular markers) and Clinical Applications (Diagnostic and Therapeutic)

Chimeric antibodies (human constant, mouse variable) and humanised antibodies (Human antibody with mouse hypervariable regions)

Bispecific antibodies are antibodies that are attached to antibodies

Bifunctional antibodies are antibodies that are attached to other molecules (eg. toxin)

• Avoid genomic and prototypical changes
• Avoid cross contamination by other cell lines
• Incubator failure
• Maintaining cell lines which are not for immediate use
• For distribution

• Slow - Water move out of cell
• Fast - Water will crystalise and damage cells

• Step 1: Harvest cells
• Step 2: Place cells in freezing media (growth media + 10% FBS + 10% DMSO or glycerol)
• Step 3: Place in vials
• Step 4: In -70C
• Step 5: In -196C

• Operator technique
• Environment
• Use and maintenance of laminar-flow hood
• Humid Incubators
• Cold stores
• Sterile materials
• Imported cell lines and biopsies

• Eye
• Microscope
• Gramstaining on culture plates

• DNA fluorescent staining (Hoechst)
• PCR
• ELISA
• Immunostaining

• Sudden change in pH
• Cloudiness in the media
• Granules, ovoid particles and filamentous mycelia seen under microscope

• Immunostaining
• ELISA
• PCR

• Wash cultures several times in high concentration antibiotics
• Grow the cultures for 3 subcultures in media with antibiotics
• Further cultures for a further 3 subcultures without antibiotics
• Check to ensure contamination is eliminated

If its for mycoplasma, use Mycoplasma Removal Agent (MRA)

Use a Hemocytometer or a Electronic Counter

(Count 4 big squares and take the average)

%viability = live cells/live and dead cells x100%

Viable cell concentration (cells/mL) = average number of life cells x dilution factor x 10^4

Total number of cells = viable concentration of cells x volume

• Primary cell culture are cells that are first obtained from the source.
• Sources are mouse embryos, human biopsies, and chicken embryos.

• Dissection (Migration) (Chop tissues to fine pieces and then seed in flask)
• Mechanical Force (Sieves, syringe+needle or vigorous pipetting)
• Enzymatic Disaggregation (Trypsin, Pronase, Collagenase and Dispase)

• Species differences
• Lineage differences
• Culture conditions

Transfect with Telomerase, oncogenes, interactions with SV40 genome/whole virus

Passage number - The number of times a culture has been subcultured

Generation (Doubling) number - Number of doublings a culture has undergone