cell culture- Van Casteren

Question 1

Advantages and disadvantages of culturing cells

Answer 1

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?

Question 2

what is biosafety hood?

Answer 2

• 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)

Question 3

what are the most important components of a culture medium?

Answer 3

• 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

Question 4

what cells are used for culture?

Answer 4

• immortalised cell lines
• primary cell/tissue culture (slice/explant/dissected culture)
• stem cell culture (neural SC, embryonic SC, iPSC)

Question 5

immortalised cell culture

Answer 5

• 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)

Question 6

types of immortalised cells

Answer 6

• HELA cells (epithelial, cancer cells)
• COS7 (monkey kidney cells)
• HEK293 (human embryonic kidney cells)
• MDCK (dog kidney epithelial cells)
• PC12 (most human like)

Question 7

Primary cell culture (what are they, advantages/disadv….)

Answer 7

• 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

Question 8

types of slice culture

Answer 8

• Acute slice culture

- organotypic slices

Question 9

what is explant culture

Answer 9

• Fragments of tissue
• often cultured with growth factors
• culture do not preserve the orientation/organisation of the brain

Question 10

Dissociated cell culture

Answer 10

• 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

Question 11

stem cell culture

Answer 11

• 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

Question 12

how can one manipulate cell culture?

Answer 12

• Transfection/infection (genetic modification)
• Co-culture systems (banker/autaptic culture)
• pharmacological manip.
• Antibody studies

Question 13

what is a banker culture?

Answer 13

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)

Question 14

reverse banker culture

Answer 14

same as banker culture but with neurons on top and astrocytes in the bottom

Question 15

Autaptic culture

Answer 15

• 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

Question 16

example of autaptic culture

Answer 16

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

Question 17

name all 4 transfection techniques

Answer 17

1. electrical transfection
2. chemical transfection
3. virus based transfection
4. physical transfection

Question 18

types of electrical transfection

Answer 18

1. Electroporation- changes properties plasma membrane (permeability) by exposing cells to a single, strong voltage pulse–> charged materials (e.g. plasmids) can enter the cell
2. Nucleofection- Modified electroporation, series of voltages pulses
3. Single cell electroporation- Modification of electroporation, single cell, electroporated by patch pipette.

Question 19

types of chemical transfection

Answer 19

1. Ca2+ -phosphate co-precipitation- formation of DNA crystals with the Ca2+ ions in phosphate buffer–> precipitate onto cells and taken up via endocytosis
2. Lipofectamine- cationic lipid molecules form unilamellar liposome that interact w. neg. charged nucleic acids (NA) –> NA complex fusion with plasma membrane

Question 20

types of virus based transfection

Answer 20

1. Adenoviruses- used in vitro and in vivo; no genome integration –> gentle to the cell and effective
2. Adeno- associated virus (AAV)- allows specific transduction of various cell types; AAV can’t replicate more than once–> non pathogenic

Question 21

Types of physical transfection

Answer 21

1. Microinjection- nucleic acids injected into cytoplasm or nucleus with fine glass capillaries
2. Biolistics- based on the injection of DNA-coated gold particles by using motive force e.g. helium
   pressure

Question 22

Types of contamination

Answer 22

1. bacterial - easy to spot
2. Fungal - easy to spot- branch like fibres on medium
3. yeast - hard to get rid of; brown sturdy structures on medium
4. Mycoplasma- hard to spot- only possible w. DAPI staining
5. viral- most difficult to detect, often comes from animal derived materials used for culture
6. chemical- any non living contaminant; originate from reagents/water used for media/buffer

Question 23

how can one avoid contamination?

Answer 23

• work in laminar hood
• sterile pipettes
• disinfection (of EVERYTHING)
• often: used of antibiotics on medium