Eb Drying Biological Material Mpangestu 34
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Transcript of Eb Drying Biological Material Mpangestu 34
Drying Biological Material
Mulyoto Pangestu, PhD1. Monash University Australia
2. Jenderal Soedirman University Indonesia
David Keilin, in his scholarly address in 1958 to The Royal Society reviewed several natural survival strategies including dehydration, cooling, lack of oxygen, and
increasing salt concentration,
and introduced term "Cryptobiosis" or now known as “Anhydrobiosis”.
Frontiers in Anhydrobiosis
Scientific observation on anhydrobisis has been started in early 18 century
• Leeuwenhoek (1702) discovered that certain ‘animalcules’ found in the sediments of roof gutters could be dried and then revived by the simple addition of water after being stored for his study in several months, – His finding as prompted considerable controversy,
but also much useful research.• Needham (1809) found similar effects with some
microscopic eelworms that infested wheat grains. These organisms, later identified as the larval stage of Anguillulina tritici, caused gall formation on the wheat, and in the dry state showed no sign of life or movement. Indeed, they were seen to crumble if touched while in this state, but were apparently restored to life if water was added
• Lazzaro Spallanzani (1803), Professor of Natural History at the University of Pavia, often credited with being the first to demonstrate survival of spermatozoa after cooling and rewarming, would not initially accept that organisms could undergo reversible desiccation. However, he eventually changed his mind and extended previous experiments by showing that dried rotifers could withstand heating up to 73C.
• Doy`ere (1842) demonstrated that, after thorough desiccation in a vacuum with sulfuric acid or calcium chloride, tardigrades and rotifers could withstand even higher temperatures, up to 125C.
Tardigrade
Artemia / sea-monkey
Artemia Merupakan kelompok udang-udangan dari phylum
Arthopoda, hidup di air asin dan toleran terhadap rentang
salinitas yang sangat luas, mulai dari nyaris tawar hingga
jenuh garam. Secara alamiah salinitas danau tempat mereka
hidup sangat bervariasi, tergantung pada jumlah hujan dan
penguapan yang terjadi. Apabila kadar garam kurang dari 6
% telur artemia akan tenggelam sehingga telur tidak bisa
menetas, hal ini biasanya terjadi apabila air tawar banyak
masuk ke dalam danau di musim penghujan. Sedangkan
apabilakadar garam lebih dari 25% telur akan tetap berada
dalam kondisi tersuspensi, sehingga dapat menetas dengan
normal.
Kista tertua artemia pernah ditemukan di sekitar Great Salt
Lake, diduga berusia sekitar lebih dari 10000 tahun
(berdasarkan metoda "carbon dating") dan masih bisa
menetas.
www.o-fish.com
www.o-fish.com
Siklus hidup artemia
Storage for Biological Materials
Frozen storageFrozen storage
• Advantages:– Has been proved by
many researchers– Produces live cells
and motile spermatozoa
• Disadvantages:– Requires access to liquid
nitrogen and appropriate storage tanks.
– Raises work safety issues– Accidental thawing is
detrimental– Possibility of cross
contamination– Limited storage capacity– COST
Frozen storage in liquid nitrogen is the standard protocol for storage of biological
material
Dry storageDry storage
• Significantly reduce the cost
• Allow unlimited storage capacity and save floor
space
• Circumvent all problems of cross contamination
• Allow genetic materials to be stored separately to
prevent use without consent.
• Significantly less likelihood of failure (eg.tanks)
• Eliminate all occupational health/building and
safety issues associated with liquid nitrogen
Dehydration is Destructive to living things.
But, when carried out in the Right Way it can
lead to long term preservation
• Why drying? Why drying? – Water is
highly reactive itself, may also contain remnants from the drug
manufacturing process including destructive enzymes such as proteases
– While in aqueous solution, these will be able
to degrade the product, but in the solid state produced by drying they are immobile
and harmless.
Freeze-dryingFreeze-drying
• Allows room temperature storage• Produces live and normal offspring
however, • Requires an expensive freeze-drying
machine and accessories or equipment
Freeze-drying is routinely used in food and pharmaceutical industries
Mouse sperm can be freeze-dried and stored at room temperature
(Wakayama and Yanagimachi, Nat. Biotech., 1998)
• Slow cooling– Extracellular ice formation from pure water in the
media– Increase osmolarity (as water turn into ice and left salt
behind)– Hypertonic effect– Slowly remove intracellular water– All pure water turn into ice– Cell completely dry (free of water)
Morphology, solubility and chemical integrity of the material can be maintained if water is
removed during frozen state.
From RT then slowly frozen
Pure water turn into ice and increase concentration of other molecules in remain suspension
Hypertonic effect, dehydration, CPA perme-abilises mem-branes
CPA replaces intracellular water
Cryoprotectant
Media (salt & water)
Ice
Intracellular components
• All extra cellular water (ice) sublimed/vacuumed out).
• Salts turn into crystal and
• Concentrated CPA but, may evaporate
• Dry specimen left behind.
Water removal
Vacuum to remove water
dry specimen
Vacuum pressure
Drying in the Right Way Important!!
to prevent damages and reduce destructive effects
Excipient (sugar)
Gen (?)
Water
Oxygen (?)
• Sugar molecules – produce stable solid products– form glassy matrix and preserve internal
structure– supporting tertiary structure
(polypeptide and protein)– preventing fro collapse when water
molecules are remove
• In the presence of sugar molecules, the process of drying becomes less damaging, and the solid product obtained is relatively stable.
• A small sugar called trehalose is found in many living things which have adapted to survive drying out.
Trehalose• has the ability to stabilize the cell during the drying
process making it possible to then rehydrate the cell and have it come back to full life.
• Trehalose is a naturally-occurring, glass-forming disaccharide (alpha-D-glucopyranosyl alpha-D-glucopyranoside), exploited by nature for millenia across a range of desiccation-resistant organisms.
• Found in the desert, the resurrection plant is a particular example, withstanding total drying for many years and coming back to life unharmed when re-hydrated.
• is an inert, non-reducing sugar, and does not interact with reactive molecules such as amino groups from peptides and proteins - preventing the degradation and aggregation which is common when drying with other excipients.
• is found in many of the foods we eat and is broken down to its glucose constituents by the human enzyme, trehalase.
Trehalose (Cont.)
Oxygen
• Oxidation/free radical damages– Membrane
– DNA
– Other cell components
– Impaired physiological system
Drying Mammalian (cells)
• No mammalian cells has been shown to synthesise or accumulate trehalose.– No cryotolerance– No anhydrobiosis– Can trehalose protect mammalian
cells against drying?
Incorporating trehalose into mammalian cells
• Genetic engineering using otsA and otsB genes (Guo et al, 2000)
• Reversible pores – genetically engineered mutant of Staphylococcus
aureus alpha-hemolysin to create pores in the cellular membrane. (Eroglu et al, 2000)
• Microinjection (Eroglu et al., 2002)
• Incubation (Guo et al., 2000; Wolkers et al., 2001)
• Electroporation (Shirakashi et al., 2002)
Methods &Reference
Cell type Treatments
Geneticengineering Guoet al., 2000
human embryonickidney cell line 293and human fibroblastcell line 12F
Cells were transfected with otsA and otsB from E. coliusing adenoviral vector. Air dry 72 h later by removingthe culture medium and storing cells at roomtemperature.Result: Viable and proliferate cells
IncubationPuhlev et al 2001
Human fibroblast Thermal shock 50mM T in DMEM followed byvacuum dry and storageResult: 60% viable day 1, 10% viable day 8.
IncubationMatsuo, 2001
Human cornealepithelial cells
0.2 M Trehalose in PBS followed by air drying for 30minResult: Viable cells after staining
IncubationWolkers et al.2001
Human platelets 52mM Trehalose in buffer at 37C followed by freezedrying.Result recovery >80% intact platelets.
IncubationGordon et al.,2001.
Human mesenchymalstem cells
50mM T; 3% Glycerol in hMSC medium 24h, vacuumstorage.Result: Up to 45% recovery of viable cells, butdecreased later.
Summary of reports on the use of trehalose in mammalian cell drying
Cell type Dryingmethod
Excipient Results References
Rat red bloodcells
Freeze drying PVP Recovered Meryman (1966)
Red bloodcells
Freeze drying Recover enzymaticfunction
Goodrich et al .(1992);Soewemimo-Coker
et al . (1993)
HES Recover enzymaticfunction
Rindler et al (1999a,b)
Fibroblast Air drying Trehalose Viable andproliferate cells
Guo et al (2000)
Cornealepithelial
Air drying Trehalose Viable cells Matsuo (2001)
Fibroblast Air drying Trehalose Viable cells Puhlev et al (2001)
Humanplatelets
Freeze drying Trehalose Recover enzymaticfunction
Wolkers et al .(2001b)
Humanmesenchymestem cells
Air dry andvacuum dry
Trehalose Recover viablecells but thenumber wasreduced by time
Gordon et al(2001)
Some attempts to preserve mammalian cells in the dry state.
Ucapan terima kasih
• Asian Development Bank
• Universitas Jenderal Soedirman
• Monash Institute Reproduction and Development
• National Institute of Health