Cell Storage Flashcards
Why preserve cells?
Banking
- consistent starting material
- no need for continuous culture - saves cost/time and minimising risk of phenotypic and genotypes changes
- avoid loss by contamination
- to avoid aging and transformation of finite cell lines
Uncouple
- expansion and manipulation of cells
- cell characterisation
Transport
- to end user
What are the challenges of cells storage?
Short shelf life’s
Storage method => determines effect on product quality
Contamination
What are the three main methods of cell storage and their properties?
1) cryopreservation
- medium to long term
- mammalian cells, micro organisms and plan cells
2) Desiccation
- medium to long term
- micro-organisms, some mammalian cells
3) hypothermia
- short term
- microorganisms, some mammalian cells
What is cryopreservation?
Cryopreservation is the use of very low temps to preserve structurally intact living cells
Preservation of cells below 0*c
Long term storage - in liquid nitrogen in the vapour phase at -197*c (any temperature driven reactions are prevented
What happens during cryopreservation?
From room temp to 0*c
- cellular metabolism slows down, rapidly disrupting active transport and ionic pumping
- usually this distraction does not result in cellular damage if the culture medium is osmotically balanced
0 to -20*c
- ice crystals from in the extra cellular environment => water moves out of cells and into partially frozen extracellular medium (cellular dehydration and shrinkage)
While many cell cultures are successfully stored at -70c to -90c for month or even years biological time is not stopped only slowed and cellular damage or changes will accumulate.
Below -30*c
- the glass transition point below which liquid water does not exist and diffusion is insignificant
What happens to cells when they freeze and when the thaw?
When cells shrink and dehydrate during freezing and swell during thawing
The cells sufferfrom sever osmotic stress and or ice crystal damage during the freezing and thawing processes
What’s osmotic tolerance limit (OTL)?
The maximum concentration of solutes an organism or cell can tolerate without being damaged or dying
Cell membranes can only swell so much before damage occurs
Damage can be observed through changes in mass transport
Knowing the OTL, damage caused by solution effect (osmotic pressure) can be reduced
What are the effects of slow and fast cooling in cells with and without cryo protectant?
Without cryprotectant
Slow cooling
- cellular death from dehydration effects
Fast cooling
- cellular death from internal crystal damage
With cryoprotectant
Slow cooling
- cells dehydrate but survive
Fast cooling
- cellular death from internal ice crystal damage
Explain what rapid cooling and slow cooling are and how cells may be able to survive freezing and thawing l
Rapid cooling - intracellular ice crystals form before complete cellular dehydration - damage and cell death during recovery
Slow cooling - free intracellular water is pulled osmotically from the cells resulting in complete dehydration and shrinkage - can lead to cell death
If cooling rate is slow enough to prevent intracellular ice formation but fast enough to avoid serious dehydration effect the cells may survive the freezing and thawing process
This survival zone is easily observed in many bacteria and other prokaryotes but most eukaryote cells it it is non existent or very difficult to find without using cryoprotective agents
How can cell damage be minimised when cryofreezing?
1) using cryoprotectuve compound prior to freezing for storage
2) controlling the transient cooling and waning rates during preservation (mr freeze box)
What is a cryoprotectant and how does it work, give a disadvantage
CPAs protect cells by preventing damage caused by slow freezing (dehydration and shrinkage). The damaged caused by formation of internal ice crystals as a result of fast freezing is minimised by freezing rate and not CPAs
Alter the kinetics of the cells ( membrane mass transport )
May be harmful to cells by causing toxicity or volume changes
What are the properties and make-up of penetrating and non penetrating CPA?
Penetrating (low Mw)
- eg DMSO 10% (10% v/v to 90% FBS)
- eh glycerol
- replace intracellular water, reduce cell shrinkage
- reduce freezing point of intracellular water
Non-penetrating (high Mw)
- eg polyethyleneglycol (PEG), sugars, starch, polyvinylpyrrolidone
- typically used in conjunction with penetrating CPAs
Name 2 CPAs, their concentration and an issue each of them may face
DMSO most often used at a final conc of 5 - 15% (v/v) (high purity grades)
- some cell lines are adversely affected by prolonged contact with DMSO. This can be reduced or eliminated by adding DMSO to the cel suspension at 4*c and removing it immediately upon thawing
Glycerol is generally used at a final concentration of between 5ams 20% (v/v) although less toxic than DMSO, glycerol frequently causes osmotic problems especially after thawing
Glycerol is always added at room temp or above and removed slowly by dilution
Why might high serum concentrations be used?
To help cells survive freezing
Replacing standard media-cryo-protectant mixtures with 95% serum and 5% DMSO may be superior for some overly sensitive cell lines
How does DMSO effect cells and patients ?
DMSO found in most freezing solutions
Effects on cells:
- Cytotoxicat temperatures below 0*c
- mechanism of action is poorly understood
- has been reported to induce cell differentiation
In patients:
- can cause adverse reactions if infused into patients
Give an alternative to DMSO
Trehalose
Explain the properties of the storage vessels in regards to storage size, types of vessel and it’s design
Material of choice for storage vessel is critical ( resistant to extreme temps)
The sealing system or cap design is critical to maintain the integrity of the vessel during storage in liquid nitrogen
Two types of vessels in different sizes (1 to 5 ml) although smaller sizes are preferred for cryogenic storage
1) heat sealable glass ampules
2) plastic (polypropylene) screw capped vials (internal or external) preferred choice and safe
Explain why a liquid nitrogen freezer is used
Liquid nitrogen freezers permit storage either in the vapour phase above the liquid at temperatures between -140 and -180 or submerged in the liquid at temps below -196
Why is vapour phase storage used?
Greatly reduces the possibility of leaky vials or ampules exploding during removal
explain desiccation and give 2 examples of it in terms of cell storage
Desiccation is the removal of moisture from something
Based on inherent capacity of some cells to survive almost complete dehydration
1) lyophilisation(freeze drying) - removes moisture by sublimation of ice into water vapour and requires the use of lyoprotectants (trehalose)
2) vacuum desiccation
- medium to long term storage
- would enable transport at ambient temperatures - impacts on cost
Explain lyophilisation(freeze drying)
The removable of frozen solvents by sublimation under a vacuum and unfrozen by desorption
What can lyophilisation preserve?
Small molecules (drugs)
Proteins(enzymes antibodies)
Food(coffee milk)
Plasma components and hormones
Whole cells(bacteria viruses)
What are the stages of lyophilisation?
Freezing (solidification) 5*c to -50 : 0h- 10h
Vacuum region start, temp inc to 10 : 10h-40h
Primary drying (ice sublimation) 10h-40h
End vacuum at 40h in
Start secondary drying temp inc to 20 deg :40h to 50h
What are concerns involving desiccation?
Impact on cell function
Lack of long term studies
- including impact of variation in storage temp/humidity
Will it be widely applicable?
What is hypothermia in terms of cell storage?
Also called cell pausing
Cells preserved at above 0c
Short term preservation
Has been used for organ transport prior to transplantation
Enables transport at ambient temps or 4c
Tested on a range of cell types including : heoatocytes MSCs red blood cells neurons renal cells endothelial cells hescs
How is hypothermia carried out in terms of cell storage?
Encapsulating cells in hydrogels for preservation
Formation of desperate capsules or by supplementation the medium with different stimuli responsive polymers/hydrogels ( alginate, methyl-cellulose )
How are cells transported to clinics and what are its issues?
Transport either frozen or refrigerated
Effects of prod quality unknown could change due to factors:
- delays and shelf life
- vibration and hypothermic storage
- fluctuations in temp
What happens when it reaches the clinic?
- does the clinic have the correct trained personnel or available equipment
What is the typical process chain for autologous cell therapies?
Collection of cells from patients
Or apheresis collection (only needed components of for example blood given)
Manufacturing cells produced for cell therapies
Therapy is returned to the patient
Give an example of autologous cell therapy
Provenge (dendreon)
Autologous cell therapy that uses patients own dendritic cells to target prostate cancer
Apheresis collection from patients day 1
Product manufactured on days 2-3
The patient is infused on day 3-4
What’s a limitation concerning provenge?
Provenge has a very short shelf life of about 18h in sealed container once opened shelf life reduced to 2h so product must be administered to patient in very short time frame
explain a logistical complexity of an autologous therapy?
Timor cell collection
- a kit is needed specialise
- packaged
- goes to receipt inventory
- stored and monitored and transported
Apheresis collection
- specialised kit needed and produced
- cells collected
- packaged
- sent to manufacturing facility
Manufacturing of cells
- processes and stored ready for distribution
Therapy to patient
- transit to clinic for therapy
- distribution to many locations
- storage monitoring
- receipt and inventory
- transit
- product acquisition
- clinical investigation
Administration kit production
What are the challenges concerning logistical complexity?
- Higher than usual performance standard
- ultra complex timings
- quality beyond compliance
Need specialist prover which is limited
Need specialised limited shipping technologies
What are the two strategies which are determined by manufacturing strategy?
Hub and spoke - centralised manufacturing
- Centralised hub carries out production and oversight
- manufacturing code may be cheaper
- point to pony logistics of final product challenging
- hub is single point of failure of network in event of fire flood natural disaster etc
Hub and node - decentralised manufacturing
- Centralised hub may produce materials carry out some oversight and back ends production logistics
- manufacturing cost more expensive
- logistics for final product simpler
- network less dependant and more resilient
