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Regenerative Medicine > Animal Cell Cultures > Flashcards

Animal Cell Cultures Flashcards

(75 cards)

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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
What are the properties of a senescent cell?
- Large in size - increase enzymatic activity for SA-beta-GAL - up regulation of pro survival pathways to resist apoptosis and unique secretome
26
Explain the phases of the growth cycle of cells
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
27
When is subculture carried out in the growth cycle?
Exponential phase
28
explain the properties of primary cells
- 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
29
Explain the properties of cell lines?
- 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
30
What are continuos/transformed cell lines?
Cell lines that can be propagated indefinitely due to transformation (Timor cells, chemical treatments)
31
What’s a disadvantage if continuous/transformed cell lines
Retain very little of original invivo characteristics
32
What are the characteristics of continuous cell lines?
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
What are the parameter that need to be considered in terms of good culture conditions?
Solid phase - substrate or phase Liquid phase - constitution of medium Gaseous phase Temperature Aseptic environment
34
What is medium formulation dependant on?
Medium formulation is cell type dependant
35
Name 5 types of basal medium
DMEM (dulbeccos modified eagles medium) EMEM (eagles minimum essential medium) MEM(minimum essential medium) RPMI1640 HAM F12
36
What does EMEM contain?
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
What does DMEM contain?
EMEM with iron and phenol red(ph indicator pink at ph 7.2, yellow when acidic and purple when alkaline
38
What are the two type of EMEMS?
Low glucose (1g/L) High glucose (4.5g/L)
39
Excluding EMEM and DMEM what other factors need to be incorporated when formulating a culture medium?
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
What basic equipment is needed for creating cell cultures and what are they used for?
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
What causes chemical contaminants?
Caused by Endotoxins, plasticisers, metal ions or traces of disinfectants Hard to detect
42
What are the signs and causes of biological contamination?
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
What are mycoplasma in terms or cell contamination?
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
Name 5 planar culture vessels
T-flask Multiple layer plate Cell factories Roller bottles Well plates
45
Name 4 bioreactors
Airlift bioreactor Hollow fibre bioreactor Stirred tank bioreactor Packed bed bioreactor
46
What 2 methods can be used for manual cell counting?
Haemocytometers Ty pan blue staining
47
How does trypan staining work?
Negatively charged due only stains cells with compromised cell membrane Indicates cell death (stained blue)
48
What’s are 2 disadvantages of trypan staining?
Tedious to perform Low statistical resolution
49
What can be used to automatically count cells?
Nucleocounter (chemimetec) - uses either multiple chamber slides or via 1 cassettes
50
What is used to stain cells when using a nucleocounter?
Acridine orange (live cells) DAPI ( damaged membranes
51
What is acridine orange?
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
What are 2 disadvantages of automated cell counting?
Relied on software estimation Assumes homogeneity
53
What can be used to monitor cell growth and viability?
Indirect methods - luminescent ayo monitoring - fluorescent live/dead staining - fluorescent proliferation assays - presto blue - Almar blue Colorimetric proliferation assays - XTT - MTT
54
What is used in love/dead fluorescent staining?
Calcein- am Ethidium homodimer
55
How does calcein work?
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
How does ethidium homodimer work?
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
What are the properties of stem cells that make them so useful in cell based therapies?
Self renewal - replicates or undergoes numerous cycles of cell division Potency - differentiates into specialised cells
58
Give 4 applications of stem cells
Cell therapy I’m vitro drug testing Models for normal growth and birth defects Cells as complex delivery agents
59
What is totipotent?
Cell division to produce all differentiated specialised cells and cells needed during early embryo development (amnion, placenta)
60
What’s a zygote?
Formed when sperm joins an egg
61
What’s a morula?
Solid ball of approx 4 undifferentiated (non specialised) cells
62
What’s a blastocyst?
Very early embryo development Approx 50-100 cells
63
What’s pluripotent?
Cells divide to produce all differentiated cells but not cells needed during early embryo development (embryonic stem cells, induced pluripotent cells)
64
What’s multipotent?
Cells can divide into somatic cells of certain lineages (mesenchymal stem cells)
65
Give the 2 types of stem cells when determined by maturity/potency and give examples
Pluripotent cells - embryonic stem cells Multipotent cells - adult stem cells - haematopoietic stem cells - mesenchymal stem cells
66
Give the 3 types of stem cells defined by their source
Autologous - donor and recipient are same individuals Allogeneic - donor and recipient are different individuals Xenogeneic - donor and recipient belong to different species
67
What are the culture requirements for embryonic stem cells (ESCs)
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
Give 2 disadvantages of embryonic stem cells
Ethical issues Safety risks - formation of teratomas
69
What are induced pluripotent stem cells?
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
What are haemotopoietic stem cells and their properties
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
What are mesenchymal stem (stromal) cells and it’s properties?
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
Name the properties of hESCs
Differentiation Capacity - pluripotent Self renewal - theoretically indefinite Isolation - difficult Source - allogenic Ethics issues - destruction of embryo Safety - concern if tumour formation Rejection - likely
73
Name the properties of adult stem cells
Differentiation capacity - multipotent Self renewal - limited Solution - routine (depends if source) Source - autologous and allogenic Ethical issue - none Rejection - unlikely
74
Name the properties of ips stem cells
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
What factors need to be considered when bioprocessing stem cells?
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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