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Regenerative Medicine > Animal Cell Cultures > Flashcards

Animal Cell Cultures Flashcards

1
Q

What’s a cell culture?

A

Processe by which a prokaryotic, eukaryotic or plant cells are grown under controlled conditions

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2
Q

What’s tissue culture?

A

Term for removal of cells, tissue or organs from an animal and their placement in an artificial growth environment

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3
Q

Give 5 examples of products from cell culturing

A

Produce monoclonal antibodies and proteins

Viral vaccine production

Drug activity inverstigation

Cell therapy

Clinical investigation

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4
Q

Name 3 out of the 7 representative cell lines. (Bonus name all 7)

A

Cho - Chinese hamster ovary cells
3T3 - mouse fibroblasts
MEFs - mouse embryonic fibroblasts
MDCK - Madin-darby canine kidney epithelial cells
Vero - Verda Reno- kidney epithelial cells from an African green monkey
HK293- human embryonic cells
HeLa - immortalised cell line from a young women named Henrietta lacks suffered form cervical cancer

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5
Q

Explain what the CHO cell line is

A

Epithelial cells from ovaries of Chinese hamster

Created late 1950

Initially selected fro radiation studies due to low chromosome number 2n=22

Multiple cell lines from CHO developed from original - CHO-k1 , CHO-DXB11

CHO-K1 continuous line with short budding time 15s - can be cultured as either adherent or suspension cells -> used a lot in biotechnology

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6
Q

Explain the MDCK animal cel lines

A

development of flucelvax/ optaflu which is first mammalian cell based vaccine against influenza virus

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7
Q

Explain what vero cell line are and what they’re used for

A

Host cells fro virus production as they’re interferon-deficient

Widely used for vaccine production - fda approved

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8
Q

Explain HeLa cell lines and they’re use

A

First immortal human cells grown in lab

First human cells successfully clones

Used for research in cancer aids and gene mapping

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9
Q

How do you initiate a cell culture?

A

1) ExplaiCulture
- tissue removal (biopsy)
- transfer to glass/culture vessel
- add culture to medium till submerged
- transfer to controlled environment (37 deg, 5% co2 , 100% RH
- after few days cells move from tissue onto culture vessel substrate
- cells will begin to divide and grow(proliferation)

2) Enzymatic Dissociation
- remove tissue , mince into small pieces
- add proteolytic enzymes to digest
- cells released from tissue
- single cells transferred to culture vessels
- cells grow and divide

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10
Q

What are the 3 cell morphology + explain what they look like

A

Fibroblast- bipolar or multipolar, elongated, require attachment

Epithelial-like - polygonal with more regular dimensions, grow attached in discrete patches

Lymphoblast-like - spherically usually grown in suspension

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11
Q

What cell lines are anchorage dependant?

A

Most cell lines derived from normal tissue are anchorage dependant (grow only in suitable substrate)(tissue cells)

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12
Q

What type of cells are anchorage independent?

A

Suspension cells (blood cells)

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13
Q

How do transformed cel line grow?

A

As mono layer or as suspensions

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14
Q

Why is cell adhesion important?

A

Critical for adherent cell survival and grown
Cell adhesion molecules(CAMs)

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15
Q

How do you initiate an adherent cell culture?

A

1) seed cells in culture dish
2) provide nutrients, growth factors
3) cells grow to cover culture Durga e
4) once confluence reached growth slows down and eventually stops (contact inhibition)
5) subculture is required now

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16
Q

What are suspension cells and how do they work?

A

Free-floating I’m medium, no requirements for an attached substrate (blood cells)

When confluence reached, cells clump together and medium appears turbid => subculture then needed

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17
Q

What is confluence?

A

When adherent cells cover the adherent surface of culture dish

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18
Q

What is turbidity?

A

Cloudy, suspended matter

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19
Q

What is subculturing cells?

A
  • necessary to keep cells in healthy growing state
  • when confluence reaches and cells stop dividing subcultures needed (passage)
  • when 80-90% confluence reached subculture needed to maintain proliferating state
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20
Q

How is cell passage or subculturing done with adherent cells?

A

Using an enzyme( trypsin) combined with ion chelator (ETDA) to break cell-cell and cell-substrate bonds made by cell adhesion molecules bound in the cell membrane.

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21
Q

How are suspension cells subcultured?

A

Removing part of the cell suspension and replacing it with fresh medium

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22
Q

What are the rules for subculturing?

A
  • Use actively growing cells in log phase
  • keep exposure to trypsin at a minimum
  • handle cells gently
  • must maintain optimal feeding regime and subculturing
  • use low concentrations of cells to initiate subculture of rapidly growing cells and higher concentrations of slower growing cells
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23
Q

What is the limit of the amount of subculturing that can be done to normal cells ?

A

Ability to be split/continue to divide is limited

Normal cells limited number of times to be subcultured normally between 50-100 passages

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24
Q

What’s cellular senescence?

A

Phenomenon by which cells arrest their proliferation (hayflick limit)

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25
Q

What are the properties of a senescent cell?

A
  • Large in size
  • increase enzymatic activity for SA-beta-GAL
  • up regulation of pro survival pathways to resist apoptosis and unique secretome
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26
Q

Explain the phases of the growth cycle of cells

A

1) lag phase (cell adaptation) - a drop in cell number as a result of adaptation to culture conditions

2) logarithmic phase (growth) - exponential increase in cell number

3) stationary phase (plateau) - equal number of cells dividing and dying

4) death phase - number of cells dying greater than cells dividing g

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27
Q

When is subculture carried out in the growth cycle?

A

Exponential phase

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28
Q

explain the properties of primary cells

A
  • derived directly from excised tissue
  • heterogenous, still represent parent cell types
    Closest phenomenon to in Vigo
  • finite life span ( example - bone marrow derived from MSCs up to 1 passages)
  • macrophages and neurons do not divide in vitro so have to be used as primary cultures
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29
Q

Explain the properties of cell lines?

A
  • Subculturing of primary cells leads to generation of cell lines
  • may be established only if cells can proliferate
  • limited life spans - might become senescent (old)
  • can be anchorage dependant or independent
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30
Q

What are continuos/transformed cell lines?

A

Cell lines that can be propagated indefinitely due to transformation (Timor cells, chemical treatments)

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31
Q

What’s a disadvantage if continuous/transformed cell lines

A

Retain very little of original invivo characteristics

32
Q

What are the characteristics of continuous cell lines?

A

Smaller, more rounded, less adherent with higher nucleus-cytoplasm ratio

Fast growth, aneuploid chromosome number (loss or duplication)

Reduce serum and anchorage dependence, grow more in suspension

Ability to grow up to higher cell densities

Different phenotypes from donor tissue

Stop expressing tissue specific genes

33
Q

What are the parameter that need to be considered in terms of good culture conditions?

A

Solid phase - substrate or phase

Liquid phase - constitution of medium

Gaseous phase

Temperature

Aseptic environment

34
Q

What is medium formulation dependant on?

A

Medium formulation is cell type dependant

35
Q

Name 5 types of basal medium

A

DMEM (dulbeccos modified eagles medium)
EMEM (eagles minimum essential medium)
MEM(minimum essential medium)
RPMI1640
HAM F12

36
Q

What does EMEM contain?

A

Balanced salt solution

Non essential amino acids

Glucose

Sodium bicarbonate(for ph control in a co2 atmosphere , can be replaced by HEPES which doesn’t require co2)

Sodium pyruvate (provides more atp)

37
Q

What does DMEM contain?

A

EMEM with iron and phenol red(ph indicator pink at ph 7.2, yellow when acidic and purple when alkaline

38
Q

What are the two type of EMEMS?

A

Low glucose (1g/L)

High glucose (4.5g/L)

39
Q

Excluding EMEM and DMEM what other factors need to be incorporated when formulating a culture medium?

A

Further supplementation - serum - needed for most cell cultures, growth factor, glutamine, additional amino acids

Sterilised used by filtration through a 0.2 micron filter

Antibiotic supplementation possible for prevention of bacterial contamination

Stored refrigerated at 4c

40
Q

What basic equipment is needed for creating cell cultures and what are they used for?

A

Laminar flow safe cabinet’s - provides protection, aseptic environment

Incubator - provides a suitable environment (temp=37.5 , 5% co2 , 99% rH, 7.2-7.4 pH)

Fridge/freezer - store liquid medium at 4c , enzymes and media components (glutamine and serum) -20 to -80 c

Centrifuge - concentrate cells

Microscope - visualise cells

41
Q

What causes chemical contaminants?

A

Caused by Endotoxins, plasticisers, metal ions or traces of disinfectants

Hard to detect

42
Q

What are the signs and causes of biological contamination?

A

Visual signs of effect on culture

Signs - turbid medium, abnormally high PH, cell lysis, graining cellular appearance, vacuolisation, poor attachment

Mycoplasm, yeast, bacteria, fungus or cross contaminants

43
Q

What are mycoplasma in terms or cell contamination?

A

A bacteria

Found in culture at high concentrations (up to 10^8 organisms per ml of medium)

No visible effects or turbidity

Ubiquitous, but unseen organisms

44
Q

Name 5 planar culture vessels

A

T-flask

Multiple layer plate

Cell factories

Roller bottles

Well plates

45
Q

Name 4 bioreactors

A

Airlift bioreactor

Hollow fibre bioreactor

Stirred tank bioreactor

Packed bed bioreactor

46
Q

What 2 methods can be used for manual cell counting?

A

Haemocytometers

Ty pan blue staining

47
Q

How does trypan staining work?

A

Negatively charged due only stains cells with compromised cell membrane

Indicates cell death (stained blue)

48
Q

What’s are 2 disadvantages of trypan staining?

A

Tedious to perform

Low statistical resolution

49
Q

What can be used to automatically count cells?

A

Nucleocounter (chemimetec)
- uses either multiple chamber slides or via 1 cassettes

50
Q

What is used to stain cells when using a nucleocounter?

A

Acridine orange (live cells)

DAPI ( damaged membranes

51
Q

What is acridine orange?

A

Organic compound

Used as a nucleus acid-selective fluorescent cationic dye

Useful for cell cycle determination

Being cell permeable it interacts with DNA and RNA by intercalation or electrostatic attractions respectively

52
Q

What are 2 disadvantages of automated cell counting?

A

Relied on software estimation

Assumes homogeneity

53
Q

What can be used to monitor cell growth and viability?

A

Indirect methods
- luminescent ayo monitoring
- fluorescent live/dead staining
- fluorescent proliferation assays
- presto blue
- Almar blue
Colorimetric proliferation assays
- XTT
- MTT

54
Q

What is used in love/dead fluorescent staining?

A

Calcein- am

Ethidium homodimer

55
Q

How does calcein work?

A

Non fluorescent

When it enters live cells due to intercellular enzymes it’s converted to polyanionic dye

We’ll retained calcein in cells produces bright green uniform fluorescence

56
Q

How does ethidium homodimer work?

A

Enters cells with damaged membranes

Undergoes 40 fold enhancement of fluorescence upon binding to nucleic acids

Producing bright red fluorescence I’m dead cells

57
Q

What are the properties of stem cells that make them so useful in cell based therapies?

A

Self renewal - replicates or undergoes numerous cycles of cell division

Potency - differentiates into specialised cells

58
Q

Give 4 applications of stem cells

A

Cell therapy

I’m vitro drug testing

Models for normal growth and birth defects

Cells as complex delivery agents

59
Q

What is totipotent?

A

Cell division to produce all differentiated specialised cells and cells needed during early embryo development (amnion, placenta)

60
Q

What’s a zygote?

A

Formed when sperm joins an egg

61
Q

What’s a morula?

A

Solid ball of approx 4 undifferentiated (non specialised) cells

62
Q

What’s a blastocyst?

A

Very early embryo development

Approx 50-100 cells

63
Q

What’s pluripotent?

A

Cells divide to produce all differentiated cells but not cells needed during early embryo development (embryonic stem cells, induced pluripotent cells)

64
Q

What’s multipotent?

A

Cells can divide into somatic cells of certain lineages (mesenchymal stem cells)

65
Q

Give the 2 types of stem cells when determined by maturity/potency and give examples

A

Pluripotent cells - embryonic stem cells

Multipotent cells - adult stem cells
- haematopoietic stem cells
- mesenchymal stem cells

66
Q

Give the 3 types of stem cells defined by their source

A

Autologous - donor and recipient are same individuals

Allogeneic - donor and recipient are different individuals

Xenogeneic - donor and recipient belong to different species

67
Q

What are the culture requirements for embryonic stem cells (ESCs)

A

Adherent cells

Can grow as single cells or colonies

Feeder layers (MEFs) or protein coatings (matrigel, laminin)

Chemically defined optimised media - expensive

daily medium changes

When I’m suspension culture they form embryonic bodies -> differentiation

68
Q

Give 2 disadvantages of embryonic stem cells

A

Ethical issues

Safety risks - formation of teratomas

69
Q

What are induced pluripotent stem cells?

A

Somatic cells reprogrammed

No ethical issues

Same properties as ESCs

Pluripotent

More stable

Same culture requirements as ESCs

Unlikely to result in immune rejection as they match donor identically

Following isolation somatic cells are cultured in vitro and transduced with expression vectors encoding transcription factors associated with pluripotency

70
Q

What are haemotopoietic stem cells and their properties

A

Are suspension cells

Give rise to all blood cell types

Found in bone marrow

Commercially available optimised media to keep them in an undifferentiated state

71
Q

What are mesenchymal stem (stromal) cells and it’s properties?

A

Adherent-dependant cells

Can be isolated from many sources (bone marrow, fatty tissue, umbilical cord, blood)

Has ability to differentiate and limited self renewal as they become senescent after a small number of passages

Don’t require coatings

Cultured in both serum based and serum free, xeno free media

72
Q

Name the properties of hESCs

A

Differentiation Capacity - pluripotent

Self renewal - theoretically indefinite

Isolation - difficult

Source - allogenic

Ethics issues - destruction of embryo

Safety - concern if tumour formation

Rejection - likely

73
Q

Name the properties of adult stem cells

A

Differentiation capacity - multipotent

Self renewal - limited

Solution - routine (depends if source)

Source - autologous and allogenic

Ethical issue - none

Rejection - unlikely

74
Q

Name the properties of ips stem cells

A

Differentiation capacity - pluripotent

Self renewal - theoretical indefinite

Isolation - routine

Source - autologous (allogenic possible but unlikely)

Ethical issues - none

Safety - use of viruses and concerns of tumours

Rejection - unlikely

75
Q

What factors need to be considered when bioprocessing stem cells?

A

Quality is a must to minimise safety risks

Identity - to contain intended cellular and non cellular components

Potency - to possess the inherent or induced biologically functions

Purity - to not contain undesired components

Safety - not contaminated with microbes or adventitious agents and if appropriate does not have tumorigenic potential

Regenerative Medicine (8 decks)

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