cell culture- Van Casteren Flashcards
1
Q
Advantages and disadvantages of culturing cells
A
Advantages:
- faster
- simplifies cellular environment
- greater control over experimental factors
- less expensive
- reduction of animal use
Disadvantages:
- artificial/not very representative of in-vivo conditions
- is isolation healthy for neurons?
2
Q
what is biosafety hood?
A
- laminar flow hood;
- a space where airflow is uniform
- culture is protected from contamination (but the user is usually isn’t protected)
- air is filtered via HEPA filter
- 2 types: horizontal and vertical hoods (vertical protects both the culture and the user)
3
Q
what are the most important components of a culture medium?
A
- source of glucose
- Buffer (keeps pH between 7.2-7.4)
- medium often bicarbonate
- pH indicator (usually phenol red)
- serum (not always, for better control the medium should be serum free and supplied with N2/B27)
- supply of AA, vitamins and minerals
4
Q
what cells are used for culture?
A
- immortalised cell lines
- primary cell/tissue culture (slice/explant/dissected culture)
- stem cell culture (neural SC, embryonic SC, iPSC)
5
Q
immortalised cell culture
A
- Tumorous cells/ artificially manipulated cells
- Well characterised
- Genetically homogenous
- Easy to culture (very robust and survive in many types of media)
- do not require a line animal
- Allow to create cells tat express gene of interest easily
- Limitations: not normal cells; lead to genetic drift after several passages (thus need to be split on a regular basis–> best between 70-80% confluence)
6
Q
types of immortalised cells
A
- HELA cells (epithelial, cancer cells)
- COS7 (monkey kidney cells)
- HEK293 (human embryonic kidney cells)
- MDCK (dog kidney epithelial cells)
- PC12 (most human like)
7
Q
Primary cell culture (what are they, advantages/disadv….)
A
- tissue directly animal
- 3 types: slice, explant, dissociated culture)
- relevant cell type can be cultured (and also the circuitry if slices are used)
- good for comparing WT and transgenic subjects
limitations: limited life time; health depends on the health of the animal; called are more heterogenous
8
Q
types of slice culture
A
- Acute slice culture
- organotypic slices
9
Q
what is explant culture
A
- Fragments of tissue
- often cultured with growth factors
- culture do not preserve the orientation/organisation of the brain
10
Q
Dissociated cell culture
A
- individual cells grown on coverslips
- come from specific brain regions that are dissected and digested (mechanically/enzymatically)
- closely resemble cells of the brain
- can survive for a long time and mature if growth factors are added
- limitations: lacking natural organisation of networks; can only be used for some cell types
11
Q
stem cell culture
A
- similar to immortalised cell culture
- primary cells collected from living organism (blood/skin/urine cells..)
- cells are cultured with growth factors and special media (in which they are reprogrammed)
- limitations: not optimised to mimic in-vivo (quite new); chromosomal abnormalities; maturation of the cells
- types: embryonic SC, neural SC and iPSC
12
Q
how can one manipulate cell culture?
A
- Transfection/infection (genetic modification)
- Co-culture systems (banker/autaptic culture)
- pharmacological manip.
- Antibody studies
13
Q
what is a banker culture?
A
Banker culture, is when cells are cultured together with astrocytes:
- astrocytes (P1-2) are plated on inserts and left to grow
- -> 1 week later: neurons plated on coverslip and added to astrocytes
- -> grown for another 2 weeks–> mature neurons (neurons in the bottom and astrocytes on top)
14
Q
reverse banker culture
A
same as banker culture but with neurons on top and astrocytes in the bottom
15
Q
Autaptic culture
A
- single neuron grown on an astrocyte island
- a glass coverslip treated with a stamp that creates collagen/PDL coated islands
- -> astrocytes only attach to coated islands–>neurons can be plated
Advantages:
- easy to access
- it is possible to switch between solutions (fast drug application)
- no risk of unspecific spillover or accumulation of drugs
- good for: synapse formation and testing mutations in synaptic signalling
16
Q
example of autaptic culture
A
autoimmune encephalitis:
- antibodies from a patient were cloned, purified and sequenced
- -> cultured with HEK cells (?) –> growth of antibodies
- -> result: antibodies lead to internalisation or inactivation of NMDAR, by binding 2 receptors at a time, building clusters and thus blocking receptor activity–> death of receptors
17
Q
name all 4 transfection techniques
A
- electrical transfection
- chemical transfection
- virus based transfection
- physical transfection
18
Q
types of electrical transfection
A
- Electroporation- changes properties plasma membrane (permeability) by exposing cells to a single, strong voltage pulse–> charged materials (e.g. plasmids) can enter the cell
- Nucleofection- Modified electroporation, series of voltages pulses
- Single cell electroporation- Modification of electroporation, single cell, electroporated by patch pipette.
19
Q
types of chemical transfection
A
- Ca2+ -phosphate co-precipitation- formation of DNA crystals with the Ca2+ ions in phosphate buffer–> precipitate onto cells and taken up via endocytosis
- Lipofectamine- cationic lipid molecules form unilamellar liposome that interact w. neg. charged nucleic acids (NA) –> NA complex fusion with plasma membrane
20
Q
types of virus based transfection
A
- Adenoviruses- used in vitro and in vivo; no genome integration –> gentle to the cell and effective
- Adeno- associated virus (AAV)- allows specific transduction of various cell types; AAV can’t replicate more than once–> non pathogenic
21
Q
Types of physical transfection
A
- Microinjection- nucleic acids injected into cytoplasm or nucleus with fine glass capillaries
- Biolistics- based on the injection of DNA-coated gold particles by using motive force e.g. helium
pressure
22
Q
Types of contamination
A
- bacterial - easy to spot
- Fungal - easy to spot- branch like fibres on medium
- yeast - hard to get rid of; brown sturdy structures on medium
- Mycoplasma- hard to spot- only possible w. DAPI staining
- viral- most difficult to detect, often comes from animal derived materials used for culture
- chemical- any non living contaminant; originate from reagents/water used for media/buffer
23
Q
how can one avoid contamination?
A
- work in laminar hood
- sterile pipettes
- disinfection (of EVERYTHING)
- often: used of antibiotics on medium
