Animal cell culture Flashcards
Why culture cells?
- Production of monoclonal antibodies and proteins
- Viral vaccine production
- Drug activity investigations
- Cell therapies
- Clinical investigations
Name some representative cell lines
CHO – Chinese hamster ovary cells
3T3 – mouse fibroblasts, MEFs - mouse embryonic fibroblasts
MDCK - Madin-Darby Canine Kidney Epithelial Cells
Vero – ‘Verda Reno’ – kidney epithelial cells from an African Green Monkey
HEK293 – human embryonic kidney cells
HeLa – immortalised cell line from a young woman named Henrietta Lacks that was suffering from cervical cancer
Many more
Explain the properties of CHO
- Epithelial cells from the ovaries of the chinese hamster.
- The original CHO cell line was created in the late 1950s by Theodore Puck. CHO cells were initially selected for radiation studies because of their low chromosome number (2n=22).
- Multiple CHO cell lines were derived from the original cell line (e.g. CHO-K1, CHO-DXB11, CHO-pro3, CHO-DG44).
- CHO-K1 is a continuous line, with short doubling times (15 h), highly adaptable, can be cultured as either adherent or suspension cells → used extensively in Biotechnology
Other animal cell lines of importance: MDCK cells
The development of FLUCELVAX (US) / Optaflu (EU) (Novartis) which is the first mammalian-cell based vaccine against the Influenza virus (2012)
Other animal cell lines of importance: Vero cells
Host cells for virus production because they are interferon-deficient and widely used for vaccine production (e.g. rabies virus vaccine and polio virus vaccine); FDA-approved for vaccine production
Other animal cell lines of importance: HeLa cells
Were the first human ‘immortal’ cells grown in the lab and were the first human cells successfully cloned → extensively used for research purposes (e.g. cancer, AIDS, gene mapping)
State some historical facts about cell culture
1885: Roux maintained embryonic chick cells in a saline culture
1907: Harrison cultivated frog nerve cells in a lymph clot and observed the growth of nerve fibres in vitro for several weeks →The FATHER of CELL CULTURE
1911: Lewis and Lewis - first liquid media consisting of sea water, serum, embryo extract, salts and peptones
1913: Carrel introduced strict aseptic techniques for longer cell culture periods
1916: Rous and Jones - proteolytic enzyme trypsin for the subculture of adherent cells
1923: Carrel and Baker developed ‘Carrel’ or T-flask as the first specifically designed cell culture vessel. They employed microscopic evaluation of cells in culture.
1940s: The use of antibiotics such as penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture
1952: Gey established the first human continuous cell line from cervical cancer (HeLa)
1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used, chemically defined medium → EMEM (Eagle’s Minimum Essential Medium)
1978: Sato - the basis of serum-free media from cocktails of hormones and growth factors
Initiation of cell culture:
Explain the process of explant culture
- Tissue removal (biopsy)
- Transfer to a glass / culture vessel
- Add culture medium until submerged
- Transfer to a controlled environment (37ºC, 5% CO2, 100%RH)
- After a few days in culture, the cells will move from the tissue onto the culture vessel substrate
- Cells will begin to grow and divide (i.e. proliferate)
What are the steps in the process of enzymatic dissociation?
- Remove tissue, then mince or chop into smaller pieces
- Add proteolytic enzyme to digest
- Cells are released from the tissue
- Single cells transferred to culture vessels
- Cells will grow and divide
Describe the morphology of fibroblastic cells
- Bipolar or multipolar
- Elongated
- Require attachment
Describe the morphology of epithelial-like cells
- Polygonal with more regular dimensions
- Grow attached in discrete patches
Describe the morphology of lymphoblast-like cells
Spherical usually grown in suspension
Compare Anchorage dependent vs independent cell lines
- Most cell lines derived from normal tissues are anchorage-dependent (grow only on suitable substrate) (e.g. tissue cells)
- Suspension cells are anchorage-independent (e.g. blood cells)
- Transformed cell lines can either grow as monolayer or as suspension (E.g. CHO – both)
What is cell adhesion critical for?
For adherent cell survival and growth
Describe adherent cell culture
- Seed cells in culture dishes/flasks
- Provide nutrients, growth factors
- Cells grow to cover culture surface
- Once confluence is reached, growth slows down and eventually ceases (contact inhibition)
- Subculture is now required
Describe suspension cell culture
Free-floating in medium, no requirement for an attachment substrate;
E.g. blood cells
When reaching confluency, cells clump together and the medium appears turbid → subculture
Why is subculturing cells necessary?
- Is necessary to keep cells in a healthy and growing state
- When the available substrate surface is completely covered with cells (i.e. confluent), cell growth slows down and then ceases
- Cell subculture (i.e. passage) needs to be done when about 80-90% confluency is reached in order to maintain cells in a proliferative state
Describe subculturing for adherent cells
For adherent cells, cell passage is done by using an enzyme (e.g. trypsin) in combination with a ion chelator (EDTA) to break the cell-cell and cell-substrate bonds made by the Cell adhesion molecules (CAMs) found the cell membrane
Describe subculturing suspension cells
Subculturing suspension cells is done very easily by removing a part of the cell suspension and replacing it with fresh medium
What are the general rules of subculture?
- Use actively growing cells in log phase of growth.
- Keep exposure to trypsin at a minimum.
- Handle the cells gently.
- Optimal feeding regime & sub-culturing.
- Low concentration of cells to initiate subculture of rapidly growing cells & higher concentration for slower growing cells.
What are the properties of senecent cells?
Larger in size
Increased enzymatic activity for SA-β-GAL
Upregulation of pro-survival pathways to resist apoptosis and unique secretome
The growth cycle is divided into which phases
Growth cycle is divided into phases:
- Lag phase (cell adaptation): a drop in cell number as a result of adaptation to culture conditions;
- Logarithmic (growth) phase: an exponential increase in cell number;
- Stationary phase (plateau): there is an equal number of cells dividing and cells dying;
- Death phase: the number of cell dying is greater than the number of cells dividing.
Compare primary vs cell lines
Primary cells:
- Derived directly from the excised tissue
- Heterogeneous, still represent parent cell types
- Closest phenotype to in vivo
- Finite life span (Example: bone marrow derived MSCs: up to 10 passages)
- Macrophages and Neurons do not divide in vitro, so have to be used as primary cultures
Cell lines:
- Subculturing of primary cells leads to generation of cell lines
- May be established only if cells are capable of proliferation
- Limited life span, might become senescent (old)
- Can be anchorage-dependent or independent
What basic equipment is required for cell culture?
- Laminar flow safety cabinet: to provide protection to the user and maintain an aseptic environment
- Incubator: to provide a suitable environment (Temperature 37°C; 5% CO2; 99% rH; pH ~ 7.2-7.4; oxygen)
- Fridge / freezer: to store liquid medium at 4°C, enzymes & media components (e.g. glutamine & serum) at -20°C or -80°C
- Centrifuge: to concentrate cells
- Microscope: to visualise cells
Images of cell contaminants
Describe microplasms
- Found in cultures at high concentrations (up to 108 organisms per mL of medium)
- No visible effects or turbidity
- Ubiquitous, but unseen organisms
State planar culture vessels
- T-flasks
- Multilayer plates
- Cell factories
- Roller bottles
- Well plates
Describe scalable culture systems
- Suspension cells: free floating
- Adherent cells: with the help of microcarriers to act as attachment substrates
Scalable culture systems - What are the different bioreactors?
- Aiflift bioreactor
- Hollow fibre bioreactor
- Stirred tank ioreactor
- Packed bed bioreactor
Describe manual cell counting
- Haemocytometer and Trypan Blue staining
- Trypan blue is a negatively charged dye which only stains cells with a compromised cell membrane, hence indicating cell death. Dead cells are stained in blue.
Describe automated cell counting
NUCLEOCOUNTER (CHEMOMETEC)
- uses either multiple chamber slides or Via-1 cassettes
- staining with acridine orange (live cells) and DAPI (damaged membranes)
Acridine orange is an organic compound. It is used as a nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic attractions respectively.
What are the disadvantages of automated cell counting?
- Relies on software estimations
- Assumes homogeneity
What are the indirect methods of monitoring cell growth and viability?
- Luminescent ATP monitoring
- Fluorescent live/dead staining
- Fluorescent proliferation assays
- Presto Blue
- Alamar Blue
- Colorimetric proliferation assays
- XTT
- MTT
What are the characteristics of live/Dead fluorescent staining?
- CALCEIN – AM: is virtually non-fluorescent and once it enters the live cells, under the effect of intracellular enzymes, is converted to the polyanionic dye CALCEIN that is well retained within live cells, producing an intense uniform green fluorescence.
- ETHIDIUM HOMODIMER: enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells.
