Animal Cell Cultures Flashcards
What’s a cell culture?
Processe by which a prokaryotic, eukaryotic or plant cells are grown under controlled conditions
What’s tissue culture?
Term for removal of cells, tissue or organs from an animal and their placement in an artificial growth environment
Give 5 examples of products from cell culturing
Produce monoclonal antibodies and proteins
Viral vaccine production
Drug activity inverstigation
Cell therapy
Clinical investigation
Name 3 out of the 7 representative cell lines. (Bonus name all 7)
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
Explain what the CHO cell line is
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
Explain the MDCK animal cel lines
development of flucelvax/ optaflu which is first mammalian cell based vaccine against influenza virus
Explain what vero cell line are and what they’re used for
Host cells fro virus production as they’re interferon-deficient
Widely used for vaccine production - fda approved
Explain HeLa cell lines and they’re use
First immortal human cells grown in lab
First human cells successfully clones
Used for research in cancer aids and gene mapping
How do you initiate a cell culture?
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
What are the 3 cell morphology + explain what they look like
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
What cell lines are anchorage dependant?
Most cell lines derived from normal tissue are anchorage dependant (grow only in suitable substrate)(tissue cells)
What type of cells are anchorage independent?
Suspension cells (blood cells)
How do transformed cel line grow?
As mono layer or as suspensions
Why is cell adhesion important?
Critical for adherent cell survival and grown
Cell adhesion molecules(CAMs)
How do you initiate an adherent cell culture?
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
What are suspension cells and how do they work?
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
What is confluence?
When adherent cells cover the adherent surface of culture dish
What is turbidity?
Cloudy, suspended matter
What is subculturing cells?
- 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
How is cell passage or subculturing done with adherent cells?
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.
How are suspension cells subcultured?
Removing part of the cell suspension and replacing it with fresh medium
What are the rules for subculturing?
- 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
What is the limit of the amount of subculturing that can be done to normal cells ?
Ability to be split/continue to divide is limited
Normal cells limited number of times to be subcultured normally between 50-100 passages
What’s cellular senescence?
Phenomenon by which cells arrest their proliferation (hayflick limit)
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
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
When is subculture carried out in the growth cycle?
Exponential phase
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
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
What are continuos/transformed cell lines?
Cell lines that can be propagated indefinitely due to transformation (Timor cells, chemical treatments)
What’s a disadvantage if continuous/transformed cell lines
Retain very little of original invivo characteristics
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
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
What is medium formulation dependant on?
Medium formulation is cell type dependant
Name 5 types of basal medium
DMEM (dulbeccos modified eagles medium)
EMEM (eagles minimum essential medium)
MEM(minimum essential medium)
RPMI1640
HAM F12
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)
What does DMEM contain?
EMEM with iron and phenol red(ph indicator pink at ph 7.2, yellow when acidic and purple when alkaline
What are the two type of EMEMS?
Low glucose (1g/L)
High glucose (4.5g/L)
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
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
What causes chemical contaminants?
Caused by Endotoxins, plasticisers, metal ions or traces of disinfectants
Hard to detect
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
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
Name 5 planar culture vessels
T-flask
Multiple layer plate
Cell factories
Roller bottles
Well plates
Name 4 bioreactors
Airlift bioreactor
Hollow fibre bioreactor
Stirred tank bioreactor
Packed bed bioreactor
What 2 methods can be used for manual cell counting?
Haemocytometers
Ty pan blue staining
How does trypan staining work?
Negatively charged due only stains cells with compromised cell membrane
Indicates cell death (stained blue)
What’s are 2 disadvantages of trypan staining?
Tedious to perform
Low statistical resolution
What can be used to automatically count cells?
Nucleocounter (chemimetec)
- uses either multiple chamber slides or via 1 cassettes
What is used to stain cells when using a nucleocounter?
Acridine orange (live cells)
DAPI ( damaged membranes
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
What are 2 disadvantages of automated cell counting?
Relied on software estimation
Assumes homogeneity
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
What is used in love/dead fluorescent staining?
Calcein- am
Ethidium homodimer
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
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
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
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
What is totipotent?
Cell division to produce all differentiated specialised cells and cells needed during early embryo development (amnion, placenta)
What’s a zygote?
Formed when sperm joins an egg
What’s a morula?
Solid ball of approx 4 undifferentiated (non specialised) cells
What’s a blastocyst?
Very early embryo development
Approx 50-100 cells
What’s pluripotent?
Cells divide to produce all differentiated cells but not cells needed during early embryo development (embryonic stem cells, induced pluripotent cells)
What’s multipotent?
Cells can divide into somatic cells of certain lineages (mesenchymal stem cells)
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
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
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
Give 2 disadvantages of embryonic stem cells
Ethical issues
Safety risks - formation of teratomas
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
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
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
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
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
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
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
