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Techniques in Neuroscience > Fundamentals of tissue culture > Flashcards

Fundamentals of tissue culture Flashcards

1
Q

What is a tissue culture?

A

Cultivation of eukaryotic tissues outside of the organism

- in a growth media with the necessary nutrients

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2
Q

Which nutrients are required in a tissue culture to function in a physiologically normal manner?

A
  • Inorganic salts

- pH (potential of hydrogen, acid/basic)

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3
Q

What is a ‘cell culture’?

A

Culturing of dissociated cells rather than pieces of tissue (tissue culture)

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4
Q

Why is tissue culture useful?

A

> Model system for studying basic processes of cell biology

> Clinical applications

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5
Q

How did Kohler and Milstein (1975) clinically apply tissue culture?

A

Generation of monoclonal antibodies

- production of vaccines as a result of the development of hybridoma cells

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6
Q

How did Steptoe and Edwards (1977) clinically apply tissue culture?

A

In vitro fertilisation

- through techniques developed for culture of early embryo

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7
Q

Who achieved short term maintenance of tissue outside an organism in 1885?

A

Wilhelm Roux:

  • short term maintenance of neural folds from early chick embryos in a saline solution
  • BUT media did not support long term growth
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8
Q

Who achieved the first maintenance of animal cells in a substrate supporting growth AND long-term survival in 1907?
How?

A

Ross Granville Harrison:

  • removed small section of frog embryos and embedded them in blood clots on underside of coverslips to allow microscopic evaluation
  • good aseptic technique

-> he observed the outgrowth of nerve cells over several weeks

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9
Q

Who achieved the first long-term cell cultures in 1911?

A

Carrel and Burrows:

- generated the first ‘cell line’ from embryonic chicken heart

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10
Q

How can cell lines grow indefinitely in culture?

A

Through genetic mutations and chromosomal abnormalities

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11
Q

Which problems rose from media based upon blood products?

Why?

A

Problems with reproducibility of results

- due to its poorly defined nature

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12
Q

Who made the first defined liquid media to overcome the problems of blood-based media in 1911?
How?

A

Margaret Reed Lewis and Warren H. Lewis:

  • cultivation of tissues from chick embryos in simple defined liquid media
  • solutions of NaCI, CaCI2, KC1 and NaHCO3
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13
Q

In what kind of serum are cell types typically grown with nowadays?

A

Media containing serum

- e.g. human fibroblasts

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14
Q

Who first dissociated tissues into individual cells for culture in 1916?
How?

A

Francis Peyton Rous and F.S. Jones:

  • enzymatic dissociation
  • use of proteolytic enzyme trypsin
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15
Q

How do most cell types - with the exception of blood cells - grow?

A

Attached to an extracellular matrix (ECM)

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16
Q

What is an extracellular matrix (ECM) composed of?

A

A complex mixture of polysaccharides and proteins

- e.g. collagens and laminin

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17
Q

How do tissue cultures support attachment and normal functioning of many types of adherent cells?

A

> Tissue culture vessels are coated with purified or unpurified components of the ECM

> Binding of cell adhesion molecules to components of the ECM needs to be disrupted to detach cells without causing cell death

> Use of trypsin allows the passaging (re-plating) of cells grown attached to a substrate
- essential for dividing cells to grow and occupy all space in a cell culture vessel

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18
Q

What happens to dividing cells when they no longer have room to grow in a cell culture vessel?

A

They undergo contact inhibition:

  • stops the cells from dividing further
  • can alter characteristics of the examined cells
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19
Q

What is the current use of trypsin?

A

Still used to enzymatically dissociate tissues into single cells

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20
Q

What is the limitation of trypsin?

A

For the passaging of cells, trypsin can cause a degree of cell death

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21
Q

What are the alternative methods to trypsin?

What do they tend to result in?

A

> Gentler enzymes such as Accutase
Non-enzymatic methods such as EDTA solutions (ethylenediamine tetraacetic acid)

=> reduced cell death

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22
Q

How do non-enzymatic methods work for the passaging of cells?

A

They chelate ions (e.g. Ca2+) that are essential for the function of cell adhesion molecules

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23
Q

What are chelating agents?

A

Organic compounds capable of linking together metal ions to form complex ring-like structures called chelates

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24
Q

What is the passaging of cells?

A

Passaging, or subculturing of cells:

  • common procedure wherein cells from a given culture are divided into new cultures
  • and fed with fresh media to facilitate further expansion
25
Q

What are the 4 steps to freeze and thaw cells?

A
  1. Cells are detached using same methods used for passaging cells; and re-suspended in a solution with cyroprotectant (e.g. DMSO - dimethyl sulfoxide)
  2. Cells are frozen down in -80°C freezer
    - at rate of 1°C/minute using a vessel filled with isopropanol
  3. Cells are transferred to a liquid nitrogen vessel to be stored indefinitely
  4. Cells are revived by rapidly thawing in a 37°C water-bath
    - minimises ice crystal formation before replacing in growth media
26
Q

What is the point of using a cryoprotectant in the solution where detached cells are re-suspended in?

A

Reduces formation of ice crystals, which would cause cell death

27
Q

What are the problems created from Ross Granville Harrison’s tissue culture method - ‘hanging drop technique’?

A

> Cells in the blood clots were difficult to view under the microscope
Are unable to grow to a larger size

28
Q

How did Alexis Carrel and Lillian E. Baker address in 1923 the problems of Harrison’s ‘hanging drop technique’?

A

Developed new vessel for tissue culture: the Carrel Flask
- angled neck to prevent airborne particles from settling into the flask when open

  • allows for sterilisation with a flame, further reducing the risk of airborne contaminants infecting the culture
29
Q

How are most modern tissue culture vessels made?

A
  • Made of plastic
  • sterile
  • intended for single use

-> reduces the risk of microbial contamination AND cross-contamination

30
Q

In what is most tissue culture performed nowadays?

A

In plates or flasks, which come in varying sizes

  • plates range from single dishes up to 15cm diameter, to plates with 384 wells
  • flasks typically range in size from 25 to 175cmsq, often with vented lids
31
Q

What is the function of vented lids in flasks?

A

To prevent airborne particles entering, whilst allowing the free exchange of gasses

32
Q

What was the effect of the widespread use of the antibiotics, penicillin and streptomycin in the 1940s onwards?

A

Reduced the problem of microbial contamination of cultures

33
Q

What are antibiotics, penicillin and streptomycin ineffective to?

A

Against a certain common strains of bacteria

- e.g. mycoplasma

34
Q

What are mycoplasma?

A

> Very small (<1 micron in length)

> Can be a significant problem in long-term culture

35
Q

How to fight against mycoplasma?

A

> Some antibiotics are effective against mycoplasmas
- e.g. Citoprofloxacin

> Best practice is to prevent contamination by employing an aseptic technique

36
Q

What do biological safety cabinets represent historically?

A

One of the most important developments in improving aseptic techniques

37
Q

For what purpose were class I biological cabinets developed in 1909?

A

Preparation of tuberculin for mycobacterium tuberculosis

38
Q

What are the characteristics of class I biological cabinets?

A

> Protect both the user and the environment from the sample, from hazardous microbes

> Does not protect the sample from airborne particles

39
Q

For what purpose were class II biological cabinets developed in the 1960s?

A

Manipulation of biological materials (e.g. eukaryotic cells)

- to subsequently be grown in culture for extended periods of time without microbial contamination

40
Q

What are the characteristics of class II biological cabinets?

A

> Protect sample from outside contamination

> Relies on continuous uniform flow of clean filtered air travelling down over sample

41
Q 
What are class II biological cabinets used for?
Why?
A

Most routine tissue culture

- they’re very effective at reducing microbial contamination from airborne particles when manipulating the sample

42
Q

For what purpose were class III biological cabinets developed?

A

For samples requiring greater level of confinement than class I or II cabinets

43
Q

What are the characteristics of class III biological cabinets?

A

Completely encloses the sample, which can only be accessed through the gloves integrated into the cabinet

44
Q

What are the characteristics of tissue culture incubators?

A

Maintain critical parameters to allow optimal growth and survival:
- constant levels of temperature, humidity, CO2 and O2

  • specific incubators can reduce oxygen levels with displacement by nitrogen, for cell types that grow better under low oxygen conditions
45
Q

What is the atmosphere required for most media buffers in use for tissue culture?

A

Atmosphere of 5% CO2 to maintain a physiological pH

46
Q

Who derived the first strains of human fibroblasts (WI-38) in 1961?

A

Leonard Hayflick and Paul Moorhead

47
Q

What is the distinction between primary cells, cell lines and cell strains made by Leonard Hayflick and Paul Moorhead in 1961?

A

> Primary cells are derived from normal tissue and grown without passaging

> Cell strains are derived from primary cells which have limited capacity for growth and division, but retain a normal karyotype

> Cell lines have the capacity to grow indefinitely, AND have abnormal karyotypes invariably

48
Q

What is a karyotype?

A

The size, shape, and number of chromosomes in a cell

49
Q

Who developed immortal human cell lines in 1951?

How?

A

George Otto Gey

  • cultured cells from Henrietta Lacks, who had cervical cancer
  • cells derived from the cervical tumour could grow and divide indefinitely

=> HeLa cells

50
Q

What was, and is the use of HeLa cells?

A

> Aided the development of the first Polio vaccine

> Still being used for research today

51
Q

What did Martin Evans achieve regarding embryonic stem cells in 1982?

A

Established cultures of cells derived from mouse blastocysts

- can in principle generate any cell type of the body in a cell culture dish

52
Q

Who first generated human embryonic stem cells from human blastocysts in 1998?
How?

A

Jamie Thomson:
- allowed for the generation of inaccessible cell types (e.g. neurons) in large numbers for the first time

  • ethical issues: requires destruction of human embryos
53
Q

Who developed induced pluripotent stem cells (iPSCs) in 2006-2007?

A

Sinya Yamanaka:

  • directly generated ES cells by directly manipulating fibroblasts in a ‘reprogramming’ process
  • produced iPSCs
  • used to study genetic diseases and inaccessible cell types
  • no ethical issues as those surrounding embryonic stem cells
54
Q

What are the 4 Yamanaka factors of pluripotency, highly expressed in embryonic stem cells?

A
  • Oct3/4
  • Sox2
  • Klf4
  • c-Myc
55
Q

What are neural ‘rosettes’?

What are they composed of?

A

> Considered as 2D-cell culture model of cortical neural tube development

> Composed of radial glial cells, which in turn generate intermediate progenitors, which in turn generate neurons

56
Q

What is the prospect of using iPSCs?

A

Personalised regenerative medicine

57
Q

What is the process of regenerative medicine using iPSCs?

A

Human biopsy -> somatic cell -> reprogramming into iPSCs

  • differentiation: neurons, thymus epithelial cells, hematopoietic cells, B-cells
  • transplantation
58
Q

What is the major advantage of regenerative medicine through iPSCs?

A

Transplanted cells will be genetically identical to the biopsied individual
- eliminating the risk of rejection or the use of immunosuppressive drugs

Techniques in Neuroscience (6 decks)

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