Cell Storage

Question 1

Why preserve cells?

Answer 1

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

Question 2

What are the challenges of cells storage?

Answer 2

Short shelf life’s

Storage method => determines effect on product quality

Contamination

Question 3

What are the three main methods of cell storage and their properties?

Answer 3

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

Question 4

What is cryopreservation?

Answer 4

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

Question 5

What happens during cryopreservation?

Answer 5

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

Question 6

What happens to cells when they freeze and when the thaw?

Answer 6

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

Question 7

What’s osmotic tolerance limit (OTL)?

Answer 7

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

Question 8

What are the effects of slow and fast cooling in cells with and without cryo protectant?

Answer 8

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

Question 9

Explain what rapid cooling and slow cooling are and how cells may be able to survive freezing and thawing l

Answer 9

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

Question 10

How can cell damage be minimised when cryofreezing?

Answer 10

1) using cryoprotectuve compound prior to freezing for storage

2) controlling the transient cooling and waning rates during preservation (mr freeze box)

Question 11

What is a cryoprotectant and how does it work, give a disadvantage

Answer 11

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

Question 12

What are the properties and make-up of penetrating and non penetrating CPA?

Answer 12

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

Question 13

Name 2 CPAs, their concentration and an issue each of them may face

Answer 13

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

Question 14

Why might high serum concentrations be used?

Answer 14

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

Question 15

How does DMSO effect cells and patients ?

Answer 15

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

Question 16

Give an alternative to DMSO

Answer 16

Trehalose

Question 17

Explain the properties of the storage vessels in regards to storage size, types of vessel and it’s design

Answer 17

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

Question 18

Explain why a liquid nitrogen freezer is used

Answer 18

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

Question 19

Why is vapour phase storage used?

Answer 19

Greatly reduces the possibility of leaky vials or ampules exploding during removal

Question 20

explain desiccation and give 2 examples of it in terms of cell storage

Answer 20

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

Question 21

Explain lyophilisation(freeze drying)

Answer 21

The removable of frozen solvents by sublimation under a vacuum and unfrozen by desorption

Question 22

What can lyophilisation preserve?

Answer 22

Small molecules (drugs)
Proteins(enzymes antibodies)
Food(coffee milk)
Plasma components and hormones
Whole cells(bacteria viruses)

Question 23

What are the stages of lyophilisation?

Answer 23

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

Question 24

What are concerns involving desiccation?

Answer 24

Impact on cell function
Lack of long term studies
- including impact of variation in storage temp/humidity

Will it be widely applicable?

Question 25

What is hypothermia in terms of cell storage?

Answer 25

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

Question 26

How is hypothermia carried out in terms of cell storage?

Answer 26

Encapsulating cells in hydrogels for preservation

Formation of desperate capsules or by supplementation the medium with different stimuli responsive polymers/hydrogels ( alginate, methyl-cellulose )

Question 27

How are cells transported to clinics and what are its issues?

Answer 27

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

Question 28

What is the typical process chain for autologous cell therapies?

Answer 28

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

Question 29

Give an example of autologous cell therapy

Answer 29

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

Question 30

What’s a limitation concerning provenge?

Answer 30

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

Question 31

explain a logistical complexity of an autologous therapy?

Answer 31

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

Question 32

What are the challenges concerning logistical complexity?

Answer 32

• Higher than usual performance standard
• ultra complex timings
• quality beyond compliance

Need specialist prover which is limited
Need specialised limited shipping technologies

Question 33

What are the two strategies which are determined by manufacturing strategy?

Answer 33

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