cell culture techniques

Question 1

outline the timeline of cell culture history.

Answer 1

1882: Sidney Ringer develops solutions of salt to maintain frog heart.
1885 :Wilhelm Roux cultures embryonic chick tissue
1940-50:
Development of cell culture techniques for growing viruses
1951: George Otto Gey propagates HeLa cells from Henrietta Lacks.
1951: Jonas Salk and his team grow polio virus in monkey kidney cells.
1954: Fnders Weller and Robbins receive Nobel prize.

Question 2

what is cell culture and why do scientists use it?

Answer 2

• lab method (in vitro) by which cells are grown under controlled conditions outside their natural environment.
• study how cells function.
• study how diseases develop
• test new treatments without endangering patients

Question 3

What are advantages of cell culture?

Answer 3

1. control the physiochemical environment (pH, temp, osmolarity) and physiological conditions (levels of hormones and nutrients)
2. control micro-environment of the cells
3. cells can easily be characterised by cytological or immune -staining techniques and visualised using imaging techniques.
4. cells can be stored in liquid nitrogen for long periods(cryopreservation)
5. cells can be easily quantified.
6. Reduces use of animals
7. Cheaper to maintain

Question 4

Why is it important cells can duplicate themselves?

Answer 4

• large quantity of cell culture needed so scientists can repeat the experiments over and compare results with other scientists
• so important to have population that duplicates themselves.

Question 5

What is so special about HeLa cells?

Answer 5

• normal human cells have built in mechanisms that only lets them divide a certain number of times before apoptosis.
• Cancer cells ignore these signals and divide rapidly so we can generate large cell culture to study diseases BUT they can not survive Invitro (outside the human body , in labs)
• HeLa cells were the only cells that survived in labs (immortal)
• HeLa cells took up the polio virus and replicated, enabling Jonas Salk test a vaccine for polio.

Question 6

What are two types of cells in culture?

Answer 6

1. primary tissue cells

2. Immortalised cell lines.

Question 7

What are characteristics of primary tissue cells?

Answer 7

• cells derived directly from tissue/patient, good for personalised medicine
• finite lifespan (~6/7 divisions)
• cells divide/differentiate
• cells carry out normal functions.

Question 8

Outline method of isolation for primary tissue cells.

Answer 8

1. cells allowed to migrate out of an explant.

2. Mechanical( mincing, sieving, pipetting) or enzymatic dissociation ( trypsin p, collagenases, proteases)

Question 9

Define explant

Answer 9

-tissue which has been transferred from an animal/plant to a nutrient medium.

Question 10

what are methods of mechanical dissociation?

Answer 10

1. minicing
2. sieving
3. pipetting

Question 11

What are

enzymatic agents used for dissociation?

Answer 11

1. trypsin
2. collagenase
3. hyaluronidase
4. protease
5. DNAase.

Question 12

Give an example where mechanical and enzymatic dissociation occurs.

Answer 12

• Magnetic immuno-purification of CD31+ Placental endothelial cells:
  1. mincing = cutting into small uniform pieces.
  2. dispase, trypsin, collagenase

Question 13

What is an exception for dissociation of cells?

Answer 13

-Haemopoietic cells (blood cells) do not need to be disaggregated because they are already found as individual cells circulating in blood.

Question 14

What are methods of isolation for hematopoietic cells?

Answer 14

1. density centrifugation (sediment in test tube according to molecular weight)
2. immunopurification
3. Fluorescence activated cell sorter(FACS)
   => all filtering methods

Question 15

What are the layers of in density centrifugation?

Answer 15

1. plasma
2. PMBCs
   3.Density gradient medium
3. Granulocytes, Erythrocytes
   => 1-4 , smallest to largest molecular weight.

Question 16

What are examples of non - hematopoietic primary cells?

Answer 16

• liver, endothelial cells, muscle, skin, nerves, fibroblast, prostate.

Question 17

What are examples of haematopoietic cells?

Answer 17

• Stem, progenitor cells , T and B cells, monocyte, osteoblasts , dendritic cells, neutrophils, erythrocytes, megakaryocytes and platelets.

Question 18

What are disadvantages of primary cells?

Answer 18

• inter-patient variation
• limited number (small amount at high cost)
• finite lifespan and hard to maintain
• Difficult molecular manipulation
• phenotypic instability
• variable contamination

Question 19

What are characteristics of immortalised cell lines?

Answer 19

• immortalised cell lines are prolonged so scientists can study cells.
• less limited number of cell divisions
• phenotypically stable, defined population
• limitless availability(
• easy to grow
• good reproducibility
• good model for basic science

Question 20

What is the method to produce cell lines?

Answer 20

1. isolated from cancerous tissue (eg. HeLa)

2. immortalisation of healthy primary cultures (usually through genetic manipulation)

Question 21

How do you produce immortal cells through genetic manipulation?

Answer 21

• target processes that regulate cellular growth and ageing
• p53, pRb involved in apoptosis so you genetically modify these/make them inactive, to prevent apoptosis = immortal cells line.
• telomerase mantains telomeres and prevents them from shortening so it needs to be activated = immortal cell line(tumour cells basically)

Question 22

What genes and protein are involved in genetic manipulation?

Answer 22

1. p53 = supports DNA repair by arresting cell cycle.
2. pRb = tumour surpressor product of retinoblastoma susceptibility gene.
3. Telomerase = reverse transcriptase which helps, maintains telomeres and prevents them from shortening, elongates telomere repeat sequences.

Question 23

Why do we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to turn primary cell line (finite) to immortalised cell line (infinite) ?

Answer 23

• tumour suppressor genes suppress the tumour gene preventing uncontrolled division leading to finite number of cells but to make a cell line (infinite cells) we need to inhibit tumour suppressor.
  E6 (viral oncoprotein) targets tP53 for degradation and E7 binds to pRb inactivating it.
• telomeres shorten over time with age and this leads to senescence and apoptosis (finite life span) telomerase sythesises telomere cells repeats reversing the loss of DNA in each round of replication ( infinite in cell lines)
  => the telomerase gene introduced into target primary cell

SV40’s T - antigen is responsible for viral DNA replication and it can cause increased growth of viral DNA by interacting with p53 and pRb (function of these proteins is intact)

• some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for immortalisation.

Question 24

What is 3D culture?

Answer 24

• artificially created environment in which cells are permitted to grow or interact with their surroundings in all 3D.

Question 25

what are advantages and disadvantages of 2D cultures?

Answer 25

Advantage: 
- simple, well established 
-affordable
Disadvantage: 
-forced apical -basal polarity 
-high stiffness
-limited communication with other cells
-no diffusion of gradients 
- results not relevant to human physiology

Question 26

What are advantages and disadvantage of 3D cell cultures?

Answer 26

Advantage: 
- adhesion in all 3D 
-no forced polarity 
-variable stiffness 
-diffusion gradients of nutrients and waste products 
-more relevant to human physiology
Disadvantage: 
- more complex 
-more expensive

Question 27

What are 2 main types of 3D cultures?

Answer 27

1. Spheroids (generated from cell lines)
   -cellular aggregate composed of 1 or more cell types
   -undergo differentiation or self organisation.
   2.Organoids
   (generated from primary tissue)
   - derived from either PSCs, neonatal tissue stem cells or adult progenitors in which cells
   spontanously self-organise into properly differentiated functional cell types.

Question 28

Define transfection

Answer 28

• process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab.
• eg. plasmid, a SRISPR/Cas9 complex
• transfection has to happen in 2D, bc in 3D some organelles may not be reached.

Question 29

What are chemical methods of transfection?

Answer 29

-lipofection , calcium phosphate, cationic polymer, DEAE-Dextran, Magnet -mediated transfection, activated dendrimers.

Question 30

What are physical methods of transfection?

Answer 30

• electoporation, nucleofection, microinjection, biolistic particle delivery, laserfection
• VIRAL transduction(goes against definition of transfection)

Question 31

What properties of liposomes and cell membrane enable lipofection?

Answer 31

-liposomes have a positive charge and membrane is negatively charged so thats why there is an attraction.

Question 32

outline method of lipofection.

Answer 32

1. interaction with the cell membrane
2. taken up by endocytosis
3. release from the endosome
4. transport to the nucleus
   5 . entry to the nucleus inefficient and may need mitosis

Question 33

outline electroporation as a method of transfection.

Answer 33

1. high electric field forms pores which then reseal

2. rate of pore sealing is dependent on temperature

Question 34

Outline nucleofection as a method of transfection.

Answer 34

• combination of electroporation and lipofection
• increased efficiency particularly of non- dividing cells
• technology is protected under patent
• Different solution and protocols are used for each cell type.

Question 35

Outline viral infection/transduction as a method of cell transfection.

Answer 35

• exploits the mechanism of viral infection
• high transfection efficiency
• retrovirus, adenovirus but most commonly lentivirus are used.
• target cells need to express the viral receptor to work
• there are safety aspects to consider.