Cell Culture and Chromosome Staining

Question 1

What are the four main statges of cell culture?

Answer 1

1. Setup
2. culturing
3. harvesting (hypotonic treatment, fixation)
4. chromosome preparation (spreading, ageing, chromosome banding / chromosome staining).

Question 2

What are the three main aplications of cell culture in genomics laboratories?

Answer 2

1. Postnatal diagnosis: Peripheral blood (e.g. high resolution banding), blood for breakage studies (Fanconi anaemia FA; ataxia telangiectasia AT; Bloom syndrome BS), skin biopsy
2. Prenatal diagnosis: AF Amniotic fluid, CVS chorionic villus sampling, PUB percutaneous umbilical blood, POC product of conception, tissue biopsy
3. Oncology: Bone marrow, neoplastic blood (unstimulated), pleural effusion, ascites fluid, cerebrospinal fluid, and tissue biopsy (lymph node, solid tumour)

Question 3

What are the four main types of culture methods utilised by genomic laboratories?

Answer 3

1. Direct preparation (neoplastic blood, bone marrow, lymphatic cells from lymph nodes after physical mincing)
2. Single cell suspension (blood, bone marrow, lymphatic cells)
3. Monolayer (cells grow attached to surface of cell culture flask)
4. in situ culture (AF, CVS on cover slips).

Question 4

What is meant by ‘single cell suspension’?

Answer 4

• In cell culture, cells remain only viable if grown in suspension (blood, bone marrow, and lymph node cells) or thin monolayers (e.g. AF, CVS, solid tumors, skin biopsies).
• Blood requires no processing for single cell suspension cultures
• Tissue separation methods to yield single cells: dissecting, e.g. chorionic villi followed by enzyme treatment (Collagenase, trypsin:EDTA) or mincing of tissue, e.g. lymph nodes, soft non-fibrous tumors.

Question 5

What is meant by ‘in situ’ culture?

Answer 5

• In situ method provides information about the colony / clonal origin of cells, important to distinguish true mosaicism from pseudo-mosaicism.
• Open system: 37oC, pH: 7.2-7.4, 5% CO2 / incubator.
• Or closed system for short-term cultures: lids of tubes are tightly closed, no gas exchange, kept at 37oC.

Question 6

What are the key ingredients within the media and supplements used for cell culture?

Answer 6

1. L-Glutamine (essential amino acid not made by the cell)
2. serum (foetal calf serum, 10-20%)
3. antibiotics
4. buffers - maintain proper pH
5. growth factors (e.g. epithelial growth factor supplemented to stimulate growth of epithelial tumours)
6. mitogens.
7. salts and energy source e.g. glucose

Question 7

What mitogens are commonly used in the cell culture process?

Answer 7

1. PHA: Phytohemagglutinin, from red kidney beans, stimulates T lymphocytes to undergo blast transformation at 37oC. This allows the chromosomal analysis of peripheral and umbilical blood. Cell division starts 48hrs after addition, with additional waves of division in 24hr intervals.
2. LPS: Lipopolysaccharide; this is a polyclonal B-cell activator which can stimulate division of B-cell lymphocytes; isolated from E. coli, often used to stimulate bone marrow cultures of patients with chronic lymphoproliferative disorders.
3. PWM: Pokeweed; isolated from a toxic plant, can stimulate both T- and B-lymphocytes; may be used for hematologic cultures.
4. TPA: 12-O-Tetradecanoylphorbol-13-acetate, a B-cell mitogen.

Question 8

What is ‘Cell cycle synchronization’ and why is this important?

Answer 8

• Cell cycle synchronization aims to achieve chromosome preparations with long chromosomes needed for high resolution banding
• Can be achieved by adding chemicals which block cells in S phase of the cell cycle
• The cells can then be released from the block to proceed through rest of cell cycle in synchrony.

Question 9

What key chemicals are used in cell synchronisation?

Answer 9

1. S-phase block: FuDR or excess thymidine
2. Release: ‘Washing’ of cultures

Question 10

After synchronisation, what factors are important when Harvesting cultured cells?

Answer 10

• Good quality chromosome banding relies on harvesting chromosomes in mitosis.
• This is usually achieved by treating cells with tubulin inhibitors, such as Colcemid to depolymerize the mitotic spindle and so arrest the cell at this stage.
• Excessively long incubations with Colcemid result in over-condensed chromosomes that band poorly.
• Adding: ethidium bromide, actinomycin D and/or BrdU can help counteract this effect.

Question 11

What is the typical culture conditions for a routine postnatal blood sample?

Answer 11

Usually 72 hours synchronised in the majority of cases.

Question 12

How can culture conditions be modified for a urgent postnatal blood samples?

What drawbacks could this bring to the results?

Answer 12

• 48hr-unsynchronised for urgent cases e.g. new born, foetal bloods (on-going pregnancy),
• Resolution will be sufficient to identify aneuploidies and very large structural rearrangements, but not subtle changes e.g. microdeletions.

Question 13

How can culture conditions be modified for haemato-oncology samples e.g. Bone Marrow?

Answer 13

• BM contains actively dividing cells and can be harvested directly or cultured for 12- 48hrs.
• Direct = no culture, straight to fixation for slide making
• Longer cultures are not advised as the abnormal cells might be lost or diluted by normal precursor cells

Question 14

How are prenatal and tissue sampels cultured?

Answer 14

Chorionic villus samples, Amniotic fluid and solid tissue: Longer kept, monitored cultures.

Should be left 5-7 days undisturbed.

When colonies start to form, medium is added.

Can be sub-cultured before harvest.

Can take upto 10-14 days total.

Question 15

Can you draw a schematic of the culture/slide making process?

Answer 15

Question 16

What is the purpose of chromosome ‘banding’?

Answer 16

• ‘Banding’ is a technique used to produce a visible karyotype by staining condensed chromosomes.
• Staining is necessary for visualisation of chromosome structure (if more than chr number is required) and useful for identifying genetic diseases caused by structural and sub-chromosomal abnormalities.
• Different stains reveal different aspects of base composition, heterochromatin density, gene density and timing of replication

Question 17

What are the most commonly used chromosome banding techniques?

Answer 17

1. G banding
2. R banding
3. Q banding
4. C banding
5. NOR staining

Question 18

Describe the process of G-banding

Answer 18

1. The metaphase chromosomes are first treated with trypsin (selectively digests GC rich regions)
2. They are then stained with Giemsa stain.
3. Heterochromatic regions are stained more darkly in G-banding (tend to be AT- rich and gene-poor)
4. Euchromatic regions are stained lightly
5. Typically produces ~450 bands per haploid set

Question 19

How is R-banding different to G-banding?

Why would R-banding be selected over G-banding?

Answer 19

• R bands are produced by treatments that selectively denature AT rich DNA
• Thus, R banding patterns are the reverse (R) of G banding
• R- bands are Giemsa-ve
• Tend to be CG- rich and gene-rich
• Good for evaluation of pale G-banded regions
• Good for evaluation of telomeres as most are euchromatic and thus pale on G-banding

Question 20

How is C-banding different to G-banding?

Why would C-banding be selected?

Answer 20

• C banding used to identify the heterochromatic segments
• Constitutive heterochromatin in human chromosomes may vary in size or position in the chromosome.
• The bands at the centromeres are readily observed and can be used to identify a suspected dicentric chromosome vs a normal variant (heteromorphism).
• Can be used to detects most common chromosomal variant in humans (0.6% population), the inv(9)(p11q13).

Question 21

How is NOR-staining different to G-banding?

Why would NOR staining be selected?

Answer 21

• NORs (nucleolar organizing region) are located in the stalk region of chromosomes 13, 14, 15, 21 and 22.
• They code for the 18s and 28s subunits of the ribosomes and are responsible for the formation of the nucleolus in interphase cells.
• After silver staining satellites and stalks of acrocentics stain dark whilst all other areas are pale thus NOR staining is used to investigate variations in the p arms of acrocentric chromosomes.
• The NOR can be translocated to another chromosome (usually the terminal region) often without phenotypic effect.
• Major application: assess whether or not a marker is derived from an acrocentric chromosome or to investigate translocations involving acrocentric chromosomes
• FISH can be used as alternative and isn’t limited to just active NORs.

Question 22

What is ‘solid staining’ and when should this be used?

Answer 22

• Chromosomes are stained directly (i.e. no trypsin treatment to generate banding) with Giemsa
• Readily shows fragile sites e.g. fragile X
• Some fragile sites are common in the population thus are classified into rare, intermediate and common fragile sites
• Also be used to assess chromosome morphology for Roberts syndrome or Mosaic Variegated Aneuploidy referrals. It is the best way to visualise sister chromatid separation.

Question 23

What is Early replicating DNA and what are the general properties?

Answer 23

• Replication timing refers to the order in which segments of DNA along the length of a chromosome are replicated.
• Early replication takes place in the interior of the nucleus in the first stages of S-phase
• Light G bands
• Dark R bands
• relatively GC rich, euchromatin

Question 24

What is Late replicating DNA and what are the general properties?

Answer 24

• Replication timing refers to the order in which segments of DNA along the length of a chromosome are replicated.
• Early replication takes place in the chromatin around the periphery of the nucleus in the later stages of S-phase.
• Dark G bands
• Light R bands
• AT rich, heterochromatin
• condense early in mitosis and meiosis

Question 25

What is ‘Differential replication staining’?

Answer 25

• Differential replication staining enables the identification of ‘late replicating’ vs ‘early replicating’ DNA.
• Achived by adding an analogue of thymidine (BrdU) that gets incorporated into DNA at different stages of the cell-cycle.
• BrdU containing DNA is not stainined by Giemsa.
• B-pulse: When BrdU is added for the last part of one cell cycle, chromosome regions replicating early will stain dark
• T-pulse: When BrdU is added for the first part of one cell cycle, chromosome regions replicating late will stain dark
• When BrdU is present for two full cell cycles, one chromatid contains DNA with BrdU substituted into both chains.

Question 26

What are the applications of ‘Differential replication staining’?

Answer 26

1. This technique is used for indicating the late replicating (inactivated) X chromosome: late replicating X will incorporate BrdU and therefore appear pale.
2. Also used to assess X-autosome translocations: inactivation will spread from late replicating X into autosome and therefore affects the phenotype.
3. When BrdU is present for two full cell cycles, the resulting difference in staining is the basis of the detection of sister chromatid exchanges.

Question 27

How are cell culture conditions modified in order to study ‘chromosome breakage disorders’?

Answer 27

Chromosomal instability syndromes may demonstrate an elevated spontaneous breakage rate in standard blood cultures.

But each can also be cultured in the presence of specific clastogens - a material which is capable of causing chromosomal breaks.

The number of genome-wide breaks can then be quantified and used to assess the likelihood of a given breakage disorder.

Question 28

Why are long-term cultures required for prenatal specimens?

Answer 28

1. Cytogenetic studies on prenatal samples are required to check for major rearrangements and aneuploidies outside of the main trisomies
2. Cultures are also used to confirm any positive trisomy QF-PCR results
3. Long term cultures need to provide sufficient material for analysis, potential FISH studies, DNA studies and possibly biochemical tests.

Question 29

How long does it take to complete culture for prenatal specimens?

Answer 29

• Most samples are cultured for approximately 7-10 days
• even if sparse after 10 days harvesting is usually done in order to give time to report within the 14 day deadline.
• The first 5-7 days of culture the cells are left in the media in which they are first added and not checked.
• After this a ‘first-look’ is made to count the number of patches/colonies and asses the extent of growth
• If harvesting is not required the media is changes and the culture left for another 5-7 days.

Question 30

It may be necessary to ‘modify’ the culture prior to harvesting of a prenatal specimen.

What procedures can be performed during the culturing process to improve the quality of outcome?

Answer 30

1. Supplement: Instead of discarding the old media, it is added to a new culture tube and allowed to grow.
2. Spead: If cultures grow unevenly with a big colony in one lump not ideal for metaphases as only the cells at the edge of colony divide.
3. Sub-culture: if a tube has massively overgrown cells trypsinised and split across two tubes.