WO2011089391A1 - Procédés de conservation de cellules de mammifère - Google Patents

Procédés de conservation de cellules de mammifère Download PDF

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Publication number
WO2011089391A1
WO2011089391A1 PCT/GB2011/000072 GB2011000072W WO2011089391A1 WO 2011089391 A1 WO2011089391 A1 WO 2011089391A1 GB 2011000072 W GB2011000072 W GB 2011000072W WO 2011089391 A1 WO2011089391 A1 WO 2011089391A1
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WIPO (PCT)
Prior art keywords
cells
mammalian cell
preservation
trehalose
mammalian
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PCT/GB2011/000072
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English (en)
Inventor
Nigel K. H. Slater
Andrew Lynch
Rongjun Chen
Original Assignee
Cambridge Enterprise Limited
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Publication date
Priority claimed from GBGB1000999.1A external-priority patent/GB201000999D0/en
Priority claimed from GBGB1016327.7A external-priority patent/GB201016327D0/en
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2011089391A1 publication Critical patent/WO2011089391A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Definitions

  • This invention relates to the preservation and storage of viable mammalian cells using non-permeating preservation agents, for example disaccharides , such as trehalose.
  • non-permeating preservation agents for example disaccharides , such as trehalose.
  • cryopreservation is currently the only technology that allows for reliable long-term stabilization of most cells. As cryopreservation is traditionally accomplished using high
  • Trehalose a non-reducing disaccharide of glucose, accumulates at high concentrations in a variety of freezing and dessication tolerant organisms across all kingdoms and has been applied to the
  • PGP Poly (L-lysine iso-phthalamide)
  • PRP Poly (L-lysine iso-phthalamide)
  • This invention relates to the finding that mammalian cells may be reversibly permeabilised to preservation agents using synthetic polymers. This polymer-mediated permeability allows cells to be loaded with high concentrations of preservation agents prior to preservation and may be useful in a range of cryopreservation and lyopreservation techniques .
  • An aspect of invention provides a method of increasing the amount of preservation agent in a mammalian cell comprising treating a mammalian cell with a membrane-permeating polymer and exposing the cell to the preservation agent.
  • the methods described herein may reduce the sensitivity of mammalian cells during preservation and/or increase the ability of the mammalian cells to survive preservation (e.g. increase cryosurvival ) relative to controls.
  • aspects of the invention provide a method of preparing a mammalian cell for preservation comprising treating the mammalian cell with a membrane-permeating polymer and exposing the mammalian cell to the preservation agent; and a method of preserving a mammalian cell comprising treating the mammalian cell with a membrane-permeating polymer, exposing the mammalian cell to a preservation agent and preserving the cell.
  • the mammalian cell may be treated with a membrane-permeating polymer and exposed to the preservation agent simultaneously.
  • the mammalian cell may be exposed to a solution which comprises both the preservation agent and the membrane- permeating polymer.
  • the mammalian cell may be treated with a membrane-permeating polymer and exposed to the preservation agent sequentially.
  • the mammalian cell may be exposed to a first solution which comprises the membrane-permeating polymer and then exposed to a second solution which comprises the preservation agent.
  • the membrane-permeating polymer in the first solution permeabilises the cell membrane and allows the influx of preservation agent from the second solution into the cell.
  • the mammalian cell membrane is generally impermeable to the
  • preservation agent i.e. the preservation agent is cell membrane impermeant and cannot diffuse across the cell membrane into the cell.
  • the membrane-permeating polymer Treatment of cells with the membrane-permeating polymer, for example by suspension in a solution comprising the polymer, permeabilises the cell membrane and allows extracellular preservation agent to diffuse into the mammalian cell from the external solution. The influx of preservation agent from the external solution into the cell increases the intracellular concentration of preservation agent and leads to the loading of the cell with preservation agent.
  • Cells loaded with preservation agent display increased viability following preservation, for example by cryopreservation, lyophilisation or desiccation techniques, relative to controls. Losses in viability associated with preservation are therefore reduced. In other words, the ability of cells to remain viable following preservation (i.e. the survivability or resistance of the cells to preservation techniques, such as freezing and drying) is increased when they are loaded with
  • preservation agent as described herein relative to cells loaded with preservation agent in the absence of membrane-permeating polymer.
  • the intracellular concentration of preservation agent in the mammalian cells may be increased by the methods described herein relative to control mammalian cells which are exposed to identical extracellular concentrations of preservation agent but are not permeabilised by membrane-permeating polymer. These cells display increased viability following preservation relative to the control mammalian cells.
  • Preservation agents are cell membrane-impermeant hydrophilic compounds which protect mammalian cells from damage caused by freezing or drying based preservation techniques. Treatment of mammalian cells with preservation agents may, for example, lead to increased viability following cryopreservation (cryoprotectants) , desiccation (desiccation protectants) or lyophilisation (lyoprotectants) i.e. a greater proportion of a mammalian cell population treated with preservation agents remain viable compared to a mammalian cell population not treated with preservation agents.
  • cryopreservation cryopreservation
  • desiccation desiccation protectants
  • lyophilisation lyoprotectants
  • Preservation agents include polyhydroxy compounds, for example mono-, di- and poly-saccarides such as glucose, sucrose, trehalose, mannitol, lactitol, palatinit, GPS (alpha-D-glucopyranosyl-l->6-sorbitol) , GPM (alpha-D-glucopyranosyl-l->6-mannitol) , maltooligosaccarides, and hydrogenated maltooligosaccarides.
  • Preferred preservation agents include trehalose.
  • a membrane-permeating polymer is a synthetic (i.e. non-naturally derived) polymer which forms a charged extended chain when the pH is above its Ka and a hydrophobic aggregation when the pH drops below its Ka.
  • Membrane-permeating polymers include amphipathic polymers with weakly ionizable carboxyl acid groups and hydrophobic side chains, such as co-polymers of: (a) a monomer selected from iso-phthalic acid and iso-phthaloyl chloride; and, (b) a monomer selected from 2,4- diaminopropionic acid; 2 , -diaminobutyric acid; ornithine; lysine ; or 2, 6-diaminopimelic acid.
  • suitable membrane-permeating polymers are disclosed in
  • the membrane-permeating polymer is a poly (lysine iso-phthalamide) ("PLP") polymer.
  • PLP poly (lysine iso-phthalamide)
  • a suitable membrane-permeating polymer, such as a PLP polymer, may have pendant groups substituted onto its carboxylic acid groups.
  • the pendant groups are hydrophobic.
  • a membrane-permeating polymer such as a PLP polymer
  • a PLP polymer substituted with phenylalanine may be employed.
  • the degree of grafting or substitution of the membrane-permeating polymer with the pendant group may be 10% or more, 20% or more, 30% or more, 32% or more, 40% or more, 46% or more, 50% or more, or 60% or more, 70% or more, 80% or more, 90% or more, or 100%, preferably about 50%.
  • a PLP polymer which is 30% or more substituted with phenylalanine may be employed.
  • the degree of substitution may be conveniently determined, for example by 1 H-NMR in d6-DMSO at room temperature.
  • a suitable membrane-permeating polymer may have a weight average molecular weight of 10,000 to 60,000, a number average molecular weight of 10,000 to 60,000, and a polydispersity of 1.25 to 3.0.
  • the physical properties of a membrane-permeating polymer may be determined using conventional techniques, such as chromatography, viscometry, osmometry, light or X-ray scattering, and sedimentation velocity.
  • membrane-permeating polymers such as PLP polymers
  • PLP polymers which are suitable for use in the present methods
  • membrane-permeating polymers suitable for use in the present methods are not associated with a payload.
  • the polymers are not bonding or complexed to any compound or moiety which is delivered to the interior of the mammalian cell through the interaction of the polymer and the cell.
  • the data set out herein shows that the membrane-permeating polymer does not associate with the preservation agent, such as trehalose i.e. the preservation agent which is introduced into the mammalian cell is not bonded or complexed to the membrane-permeating polymer.
  • the membrane-permeating polymer is not a poration agent and treatment with the polymer does not introduce pores into the cell membrane of the mammalian cell.
  • the mammalian cell may be exposed to a solution of membrane-permeating polymer of at least 10 pg mlT 1 , preferably at least 50 pg mL "1 and up to 1000 pg mlT 1 , preferably up to 500 pg mL "1 .
  • Mammalian cells which may be loaded with trehalose using the methods described herein may be nucleated or non-nucleated.
  • Suitable nucleated cells may include progenitor cells including stem cells, for example stem cells derived from umbilical cord blood or tissue, industrial and laboratory cell lines, such as HeLa, 3T3, HEK, BHK, COS, K562, SAOS-2, SH-SY5Y, and CHO cells, hybridoma cells, and cells isolated from mammalian tissue.
  • stem cells for example stem cells derived from umbilical cord blood or tissue, industrial and laboratory cell lines, such as HeLa, 3T3, HEK, BHK, COS, K562, SAOS-2, SH-SY5Y, and CHO cells, hybridoma cells, and cells isolated from mammalian tissue.
  • Suitable non-nucleated mammalian cells may include erythrocytes.
  • methods described herein may reduce the oxidation of haemoglobin in the erythrocytes during preservation and storage.
  • the mammalian cells are human cells.
  • a mammalian cell may contain no preservation agent or substantially no preservation agent before permeabilisation with membrane-permeating polymer and exposure to extracellular preservation agent. As described above, exposure to the preservation agent may occur either after or simultaneously with permeabilisation by the membrane-permeating polymer.
  • the mammalian cell may be exposed to a solution containing up 0.1 M or more, 0.2 or more, 0.3 M or more, 0.4 M or more or 0.5 M or more, of the preservation agent, such as trehalose. For example, the cell may be exposed to up 0.8 M, up 0.9 M or up 1.0 M of the preservation agent, such as trehalose.
  • the cell may be exposed to up 0.8 M, up 0.9 M or up 1.0 M of the preservation agent, such as trehalose.
  • the mammalian cell is preferably exposed to a concentration of preservation agent which is in excess of the concentration of membrane-permeating polymer.
  • concentration of preservation agent may be at least 100 fold, at least 1000 fold or at least 10000 fold greater than the concentration of membrane-permeating polymer .
  • the preservation agent is not covalently bonded to the membrane- permeating polymer, for example by covalent bond(s) and/or linking groups. Nor does the preservation agent form a complex with the membrane-permeating polymer or otherwise associate with it.
  • the intracellular concentration of preservation agent may be allowed to increase to 50 mM or more, 85 mM or more, 90 mM or more, 100 mM or more, 110 mM or more, 120 mM or more, 130 mM or more, 140 mM or more, 150 mM or more, 200 mM or more, or 250 mM or more, through exposure to the preservation agent to load the mammalian cell with preservation agent. Suitable methods for determining the intracellular
  • concentration of a preservation agent are known.
  • intracellular concentration of trehalose can be determined by the method described in Umbreit W et al., (1972) and in the examples below .
  • Polymer-mediated cell permeability is not sensitive to levels of metal ions, such as Zn 2+ .
  • Cells may be loaded with preservation agent as described above in the presence of greater than ⁇ Zn 2+ .
  • the mammalian cells may be exposed to preservation agent at a temperature of at least 29°C, preferably at least 34°C, and less than 43°C, preferably about 37°C.
  • the mammalian cells may be exposed to preservation agent for 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more or 9 hours or more .
  • the mammalian cells may be exposed to preservation agent at pH 7.8 or less, pH 7.6 or less, pH 7.4 or less, pH 7.3 or less, or pH 7.2 or less and at pH 5.0 or more, pH 5.5 or more, pH 6.0 or more or pH 6.5 or more.
  • the mammalian cells are exposed to trehalose at about pH 7.05.
  • Polymer-mediated permeabilisation and exposure to extracellular preservation agent as described herein may be used to load mammalian cells with a sufficient intracellular concentration of the
  • the preservation agent to increase the ability of the cells to survive desiccation and/or one or more cycles of freeze/thaw and remain viable.
  • the cells may be loaded with an intracellular concentration of preservation agent of 50 mM or more, 85 mM or more, 90 mM or more, 100 mM or more, 110 mM or more or 120 mM or more, 150 mM or more, 200 mM or more, or 250 mM or more.
  • the mammalian cell may be washed to remove the membrane-permeating polymer and reverse the permeability of the cell membrane. Any convenient washing method may be employed.
  • mammalian cells may be resuspended in a suitable washing solution, for example a buffer solution, such as phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the mammalian cell may be washed before or more preferably after preservation and storage.
  • Mammalian cells loaded with preservation agent as described herein may be stored for extended periods in a viable form. Any one of a variety of different techniques may be employed to preserve the loaded cells for storage. For example, the cells may be cryopreserved or dried, for example by vacuum-drying, air-drying, or freeze-drying .
  • mammalian cells loaded with preservation agent are preserved in the presence of extracellular preservation agent.
  • the cells may remain in the solution of preservation agent which was used to load the cells.
  • the cells may be removed from the loading solution, washed or otherwise treated, and then resuspended in a different solution of preservation agent for storage .
  • the concentration of preservation agent in the storage solution may be the same or different from the concentration in the loading solution.
  • the cells may be stored in an extracellular preservation agent concentration of 0.1 M or more, 0.2 M or more, 0.3 M or more, 0.4 M or more, 0.5 M or more, 0.6 M or more.
  • a method of preserving one or more mammalian cells as described herein may comprise;
  • a preservation agent such as trehalose
  • the preserved cells may be stored until required.
  • Mammalian cells loaded with preservation agent may be frozen and cryopreserved for storage. Any convenient freezing protocol may be employed and many suitable protocols are well-known in the art.
  • cells loaded with preservation agent may be suspended in a solution of the preservation agent and frozen to a cryopreservation temperature, for example -20°C or lower, -40°C or lower, -60°C or lower, or -80°C or lower.
  • the cells may be frozen in liquid nitrogen.
  • a container of loaded cells suspended in solution of preservation agent may be immersed in liquid nitrogen.
  • the frozen preservation agent-loaded cells may be stored for an indefinite period, until required.
  • the cells may be stored at a cryostorage temperature, for example -20°C or lower, -40°C or lower, - 60°C or lower or -80°C or lower.
  • the frozen cells may be thawed.
  • frozen cells may be thawed by incubation at up to 37°C in an air or water bath, or by exposure to air at room temperature.
  • the cells may be re-suspended in a culture medium to promote cell recovery.
  • the cells may be washed at this stage to remove the membrane-permeating polymer and reverse the permeability of the cell membrane.
  • a method of cryopreserving one or more mammalian cells as described herein may comprise;
  • a preservation agent such as trehalose
  • the mammalian cells may be thawed, optionally washed and re-suspended in culture medium, as described above.
  • Mammalian cells loaded with preservation agent may be freeze-dried for storage. Freeze-drying involves the removal of water from a frozen material by sublimation and desorption at reduced pressure. Suitable freeze-drying techniques are well known in the art * ⁇ ' 48 .
  • the loaded cells are frozen in a solution of the preservation agent as described above and then dried to lower the moisture content to a level suitable for freeze-dried storage.
  • the frozen cells may, for example, be dried by exposing them to a vacuum under conditions where water and other volatiles sublimate to produce freeze-dried cells with low water content.
  • Freeze-dried cells may be stored in a sealed container at room temperature, or more preferably at about 4°C, until required.
  • freeze-dried cells When required, the freeze-dried cells may be rehydrated. Typically, freeze-dried cells may be rehydrated by addition of an aqueous medium, such as a buffer or culture medium, at up to 37°C. Optionally, the cells may be washed at this stage to remove the membrane-permeating polymer and reverse the permeability of the cell membrane.
  • an aqueous medium such as a buffer or culture medium
  • a method of freeze-drying one or more mammalian cells as described herein may comprise;
  • the mammalian cells may be rehydrated as described above .
  • Mammalian cells loaded with preservation agent may be dried for storage.
  • the loaded cells may be dried in a solution of preservation agent to a level which permits dry storage. Drying may be carried out by any convenient technique. For example, vacuum- or air-drying may be employed. Typically cells may be dried by evaporation at reduced pressure and non-freezing temperatures.
  • the dried mammalian cells may be stored in a dried form.
  • the cells may be stored without refrigeration, for example at room or ambient temperature.
  • the cells may be rehydrated.
  • freeze-dried or dried cells may be rehydrated by re-suspension in an aqueous medium, such as a buffer or culture medium, at up to 37°C.
  • the cells may be washed at this stage to remove the membrane-permeating polymer and reverse the permeability of the cell membrane .
  • a method of dry storing a mammalian cell as described herein may comprise;
  • the mammalian cells may be rehydrated as described above . Because Zn 2+ is not required to reverse the mammalian cell permeability, mammalian cells may be thawed, rehydrated or otherwise recovered after storage, in the absence of Zn 2+ i.e. in a Zn 2+ free medium.
  • the viability of mammalian cells loaded with preservation agent as described herein following storage as described above may be increased relative to control cells i.e. cells not loaded with preservation agent by the methods described above.
  • the survival of mammalian cells loaded with preservation agent as described herein is increased following storage as described above relative to control cells .
  • Mammalian cells such as erythrocytes, which are loaded with
  • preservation agent using the methods described above display increased membrane integrity relative to control cells.
  • the loaded mammalian cells may release fewer intracellular components into the surrounding medium during preservation, storage and/or reconstitution (e.g. rehydration and/or thawing) relative to control cells.
  • erythrocytes loaded with preservation agent as described above may release less haemoglobin during preservation, storage and/or reconstitution relative to control erythrocytes (e.g. erythrocytes loaded with preservation agent using conventional methods) .
  • Loading a mammalian cell with .preservation agent using the methods described herein may therefore be useful in increasing the membrane integrity of the mammalian cell, for example relative to control cells not loaded with preservation agent by the methods described above.
  • Mammalian cells loaded with preservation agent using the methods described above may display decreased levels of cellular oxidation relative to control cells.
  • loaded mammalian cells may have decreased levels of oxidised intracellular or membrane proteins and/or lipids relative to control cells following storage.
  • erythrocytes that are loaded with preservation agent as described above may display decreased levels of oxidised haemoglobin relative to control erythrocytes, for example erythrocytes loaded with preservation agent by conventional methods.
  • Cellular oxidation levels in a preserved mammalian cell may therefore be decreased by loading the mammalian cell with preservation agent as described above.
  • Suitable control cells may include mammalian cells of the same type which have not been loaded with preservation agent using the methods described above e.g. cells loaded with preservation agent by
  • the mammalian cells may be used for any purpose.
  • preserved erythrocytes may be used for transfusion.
  • a method of preserving erythrocytes may comprise:
  • erythrocytes treated with a membrane-permeating polymer and exposing the cell to the preservation agent, as described above, to produce erythrocytes loaded with preservation agent.
  • Erythrocytes loaded with preservation agent may optionally be washed and then preserved, for example by drying, as described above.
  • the preserved erythrocytes may then be stored.
  • preserved erythrocytes may be stored without refrigeration, such as at room or ambient temperature.
  • the preserved erythrocytes may be reconstituted, for example by thawing and/or rehydration.
  • the reconstituted erythrocytes may, optionally be washed, and then admixed with isotonic buffers and other reagents for transfusion into an individual.
  • Mammalian cells in particular erythrocytes, or nucleated cells, such as progenitor cells or stem cells, may be formulated into a
  • composition by admixing with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients.
  • Suitable pharmaceutically acceptable excipients, carriers, buffers, preservatives, stabilisers, anti-oxidants are well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the mammalian cell. The precise nature of the carrier or other material will depend on the intended route of administration .
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included .
  • the pharmaceutical composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.
  • the pharmaceutical composition be administered to a patient, e.g. for treatment (which may include preventative treatment) or replacement of damaged or dysfunctional tissue.
  • a pharmaceutical composition comprising erythrocytes may be administered to a patient as a blood transfusion, for example for the replacement of lost or damaged blood.
  • Another aspect of the invention provides mammalian cells containing intracellular preservation agent which are produced by a method described above.
  • cells loaded by the present methods contain high intracellular concentrations of preservation agent.
  • mammalian cells may include erythrocytes containing lOOmM or more, preferably 150mM or more, or 200mM or more, or 250mM or more
  • the cells may have a membrane-permeating polymer associated with the cell membrane. In other embodiments, the cells may have been washed to remove the membrane-permeating polymer.
  • Suitable populations of cells may be in a dried or cryopreserved form.
  • Another aspect of the invention provides a mammalian cell containing at least 140 mM of an intracellular preservation agent.
  • a mammalian cell having an intracellular concentration of at least 140 mM of an intracellular preservation agent may for example be produced by loading the cell with the preservation agent as described herein.
  • the mammalian cell may be an erythrocyte containing 140 mM or more, preferably 150 mM or more, or 200 mM or more, or 250 mM or more of an intracellular preservation agent, such as trehalose.
  • the mammalian cell may be a preserved mammalian cell.
  • the cell may have been preserved by freezing and/or by drying.
  • the preserved cell may have been reconstituted for example by thawing and/or rehydration.
  • the mammalian cell may be an erythrocyte for use in transfusion.
  • mammalian cells containing intracellular preservation agent which are produced by a method described above and/or which contain at least 140 mM of an
  • intracellular preservation agent for use in the treatment of an individual .
  • erythrocytes containing intracellular preservation agent which are produced by a method described above and/or which contain at least 140 mM of an intracellular preservation agent may be used in the treatment of an individual, for example in a method of transfusion to replace lost or defective blood cells.
  • Mammalian cells such as erythrocytes, may be provided in a suitable container, such as a bag, sachet, capsule or vial, in which the contents are protected from the external environment.
  • a suitable container such as a bag, sachet, capsule or vial, in which the contents are protected from the external environment.
  • Another aspect of the invention provides a kit for preserving mammalian cells comprising membrane-permeating polymer and
  • the preservation agent and membrane-permeating polymer are described in more detail above and may be provided in separate containers, such as vials, in which the contents are protected from the external environment.
  • the kit may include instructions for use in a method of increasing the amount of preservation agent in a mammalian cell as described herein
  • a kit may include one or more other reagents required for the method, such as culture medium, buffer solutions etc.
  • a kit may also include one or more articles and/or reagents for performance of the method, such as tubes, vessels and other containers suitable for handling mammalian cells (such components generally being sterile) .
  • articles and/or reagents for performance of the method such as tubes, vessels and other containers suitable for handling mammalian cells (such components generally being sterile) .
  • Figure 1 shows the impact of polymer " pendant group on trehalose loading (blank) and haemolysis (stippled).
  • [Trehalose] 0.36 M;
  • Data were derived from four replicates. Error bars represent standard deviations. Data were analyzed for statistical significance using Student's t test for unequal variances.
  • Figure 2 shows the impact of polymer degree of substitution by phenylalanine on trehalose loading ( ⁇ ) and haemolysis ( ⁇ ) .
  • Figure 3 shows the impact of pH on trehalose loading ( ⁇ ) of
  • a PP-50 concentration of 75 pg mL "1 was utilized at 37°C with an incubation time of 9 hrs; data were derived from four replicates and error bars represent standard deviations.
  • pyrene was dissolved in an aqueous solution of PP-50 (25 pg mL "1 ) in 0.1 M buffer.
  • Figure 4 shows the impact of pH on PP-50 mediated trehalose loading (a) and hemolysis (b) of human erythrocytes and on PP-50-FITC
  • Figure 5 shows the trehalose loading (lower) and haemolysis (upper) of erythrocytes in 0.36 M trehalose solution (o)and with the addition of 50 pg mL "1 of polymer PP-50 ( ⁇ ) , 100 pg mL "1 PP-50 (A) , 150 pg mL “1 PP- 50 ( ⁇ ), and 300 pg mL "1 PP-50 ( ⁇ ) . Impact of polymer PP-50
  • Figure 8 shows the trehalose loading (upper) and haemolysis (lower) of erythrocytes at 29°C ( ⁇ ) , at 33 °C (A), at 35 °C ( ⁇ ) , and at 37 °C ( ⁇ ) .
  • [Trehalose] 0.36 M;
  • the central graph presents an Arrhenius plot of the loading data obtained during incubation for 9 hrs.
  • Figure 9 shows the osmotic fragility of erythrocytes before incubation (o) , after incubation in 100 ⁇ g mL "1 PP-50 alone ( ⁇ ) , after incubation in trehalose and 50 pg mL "1 of polymer PP-50 ( A ) , after incubation in trehalose and 100 ⁇ xq mL "1 of polymer PP-50 ( ⁇ ) , and after incubation in trehalose and 150 pg mL "1 of polymer PP-50 ( ⁇ ) .
  • Figure 10 shows the investigation of pore formation reversibility: (upper) trehalose loading and (lower) haemolysis of erythrocytes in 0.36 M trehalose solution after prior inducement of membrane
  • Figure 11 shows analysis of permeability pathways induced in human erythrocytes by polymer PP-50 ( ⁇ ) .
  • data were plotted as the logarithm of the relative permeability vs. the molecular hydrodynamic pore radius, to test the applicability of the Renkin pore model.
  • Sucrose has been taken as a reference solute.
  • the expected dependence for pores of different sizes were calculated according to the Renkin model and depicted as continuous functions [65] . Note that the experimental data does not match the model of diffusion through a pore (three pore sizes provided to make this clear) .
  • data were plotted as the logarithm of the relative permeability versus hydrodynamic volume to test for non-Stokesian diffusion [66]. Note that the logarithm of permeability decreases proportionally to molecular volume, indicating diffusion of a hydrophilic molecule through a hydrophobic membrane.
  • Solute designation A - sucrose; B - PEG 2000; C - PEG 4000; D - PEG 6000; E - PEG 8000; F - PEG 10000.
  • Figure 12 shows the correlation between topographic AFM micrographs of the surface of the erythrocyte membrane in the absence (a) and presence (b) of polymer PP-50 adsorption, AFM cross-sectional imaging along the white lines of these topography images in the absence (c) and presence (d) of PP-50 adsorption and a proposed mechanism of polymer mediated membrane disruption (e-g) based on the mechanism of action of amphipathic peptides [67, 71].
  • the native phospholipid bilayer (e) is initially disrupted through adsorption of the hydrophilic phase of PP-50 (large black circle) onto the polar head region of the bilayer, leading to the formation of a void in the hydrocarbon chain region (f ) .
  • This energetically unstable void is rapidly filled, leading to enhanced interaction with the hydrophobic phase of PP-50 (lower grey crescent) , disruption of the bilayer structure and localized membrane thinning (g) .
  • scale indicates membrane thickness only and is approximate.
  • Figure 13 shows a 1H-NMR spectra of neutralized PP-50 in d6-DMS0 at room temperature.
  • Figure 14 shows the repeat unit structure of poly (L-lysine iso- phthalamide) substituted by L-phenylalanine .
  • polymer PP-50 46.2% of carboxyl acid groups are grafted with L-phenylalanine.
  • Figure 15 shows the survival of K-562 cells after incubation for 9h at 37°C and pH 7.05 in cell culture media ("Media”; Dulbecco's Modified Eagle Medium), 0.2 M trehalose (“0.2”), 0.2 M trehalose + 200 pg mL "1 PP-50 (“0.2P"), 0.3 M trehalose (“0.3”), 0.3 M trehalose + 200 pg mL “1 (“0.3P”), 0.5 M trehalose (“0.5”), and 0.5 M trehalose + 200 pg mL “1 (“0.5P”) .
  • Figure 16 shows the cryosurvival of K-562 cells after incubation for 9h at 37°C and pH 7.05 in cell culture media ("Media"), 0.2 M trehalose (“0.2”), 0.2 M trehalose + 200 pg mL “1 PP-50 (“0.2P”), 0.3 M trehalose (“0.3”), 0.3 M trehalose + 200 yg mL “1 (“0.3P”), 0.5 M trehalose
  • Figure 17 shows the survival of SAOS-2 cells after incubation for 9h at 37°C and pH 7.05 in cell culture media ("Media”), cell culture media + 200 pg mL “1 PP-50 ("Media P") , 0.3 M trehalose (“0.3”), and 0.3 M trehalose + 200 pg mL “1 (“0.3P”).
  • Figure 18 shows the cryosurvival of SAOS-2 cells after incubation in cell culture media ("Media”) , cell culture media + 200 pg mL “1 PP-50 ("Media P") , 0.3 M trehalose (“0.3”), and 0.3 M trehalose + 200 pg mL “1 (“0.3P”).
  • Media cell culture media
  • Media P cell culture media + 200 pg mL "1 PP-50
  • Media P 0.3 M trehalose
  • 0.3P 0.3 M trehalose + 200 pg mL “1
  • Figure 19 shows the survival of SH-SY5Y cells after incubation in cell culture media ("Media”), 0.2 M trehalose (“0.2”), and 0.2 M trehalose + 200 pg mL “1 (“0.2P”). Incubation was for 9h at 37°C and pH 7.05.
  • Figure 20 shows the cryosurvival of SH-SY5Y cells after incubation for 9h at 37°C and pH 7.05 in cell culture medium ("Media"), 0.2 M trehalose (“0.2”), and 0.2 M trehalose + 200 pg mL “1 (“0.2P”) followed by freeze/thaw. Freezing was via immersion in liquid nitrogen; thawing was via immersion in a 37°C water bath. Data were analyzed for statistical significance using Student's t test for unequal variances.
  • Table 1 shows the compositions of poly (L-lysine iso-phthalamide) grafted with L-valine, L-leucine, and L-phenylalanine .
  • Table 2 shows the impact of cryopreservation protocol on survival (%) of erythrocytes suspended in PBS, erythrocytes suspended in
  • extracellular trehalose solution and erythrocytes loaded with 123 ⁇ 16 mM intracellular trehalose suspended in extracellular trehalose solution.
  • Iso-phthaloyl chloride, potassium carbonate, ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) , 4-dimethylaminopyridine (DMAP) , N,N- dimethylformamide (DMF) and triethylamine were purchased from Sigma- Aldrich (Dorset, UK) .
  • L-Valine methyl ester hydrochloride, L-leucine methyl ester hydrochloride and L-phenylalanine methyl ester hydrochloride were purchased from Alfa Aesar (Heysham, UK) .
  • Dulbecco's phosphate buffered saline (PBS); a-D-1, 1-a-D trehalose dihydrate, methanol (>99.8%, HPLC) ; anthrone; pyrene; and sulphuric acid (>95 ) were purchased from Sigma-Aldrich (Dorset, UK) .
  • Ovine erythrocytes (defibronated, sterile) were purchased from TCS
  • the biopolymers investigated have a backbone of poly (L-lysine iso- phthalamide) (PLP) grafted with L-valine (“PV'series) , L-leucine ("PL series) , or L-phenylalanine (“PP" series) .
  • the poly (L-lysine isophthalamide) backbone has a weight average molecular weight of 35,700, and a polydispersity of 1.99.
  • the specific polymers studied were PLP, PP-30, PP-50, PP-60, PP-75, PV-50, and PL-50.
  • Table 1 [20, 23] All polymers were synthesized in-house as has been previously reported [21, 22].
  • the grafted polymers were prepared from the coupling reaction between poly (L-lysine iso-phthalamide) and amino acid (L-valine, L-leucine and L-phenylalanine ) methyl ester
  • Polymers labelled with FITC were prepared by coupling FITC- NH- (CH2) 2-NH2, an amino derivative of FITC from the reaction with ethylene diamine using dibutyltin dilaurate as a catalyst, to the carboxylic acid groups of PP-50 using standard DCC/DMAP mediated coupling techniques [20] .
  • PBS contained 136.89 mM NaCl, 2.68 mM KC1, 1.47 mM ⁇ 2 ⁇ 0 ⁇ , and 8.10 mM Na 2 HP0 4 . (306.39 mOsm, pH 7.4) Higher concentrations of PBS were prepared by diluting a 10* strength PBS solution as necessary.
  • 0.1 M phosphate buffers in the pH range of 6.20-7.6 were prepared by mixing 0.2 M NaH 2 P0 3 and 0.2 M Na 2 HP0 3 aqueous solutions followed by two-fold dilution with deionised water. Final pH values were measured using a pH meter.
  • 0.1 citrate buffer at pH 5.8 was prepared similarly by mixing 0.2 M citric acid and 0.2 M trisodium citrate aqueous
  • Trehalose solutions of 0.19, 0.36, 0.52 M for incubation with RBC were made using 306.39 mOsm PBS and trehalose dihydrate ( Sigma-Aldrich; Dorset, UK) . These molar (mol solute / L solution) values correspond to 0.2, 0.4, and 0.6 molal, respectively.
  • a 0.7 trehalose solution was made using 300 mOsm PBS as solvent to 15% packed cell volume.
  • Haemolysis was evaluated by measuring haemoglobin absorbance at 541 nm using a Spectronic UV1 spectrophotometer (Thermo Fisher Scientific, USA). The Drabkin's method for hemoglobin assay was not employed because hemoglobin oxidation during trehalose loading was
  • Washed erythrocytes were used to create a calibration curve relating erythrocyte mlT 1 , counted using a haemocytometer
  • erythrocytes suspended in protective solution were stored as directed at 4°C and used within one week of the prescribed four week shelf life. Before use, erythrocytes were washed with PBS. Cells were centrifuged at 980g for 8 min and the supernatant discarded. Next, the erythrocytes were homogenously resuspended in 306.39 mOsm PBS, centrifuged at 980g for 8 min, and the supernatant discarded (first wash) . Two more PBS washes were performed before use. Trehalose loading
  • Freshly donated human erythrocytes were obtained from the United Kingdom National Blood Service and washed three times in 300 mOsm PBS prior to use. Washed erythrocytes were divided into 0.5 mL samples of approximately 40% packed cell volume (haematocrit) and centrifuged at 550g for 2 min. Their supernatants were discarded and 1 mL of
  • Resulting mixtures were homogenously resuspended and incubated in an air bath incubator (Binder UK; Wolverhampton, UK) of preset temperature .
  • erythrocyte samples were centrifuged and washed twice with 1.5 mL PBS buffer iso-osmotic to the incubation solution to remove extracellular trehalose.
  • cells were mixed with 5 mL 80% methanol and incubated at 85 °C in a water bath for 1 hour. Samples were then centrifuged at 980g for 2 min and their supernatants collected. This supernatant was evaporated in a vacuum oven
  • anthrone reaction was used based on Umbreit et al 1972 [24] . Briefly, 0.5 mL each of the trehalose solutions in deionized water were mixed with 5 mL of 66% sulphuric acid containing 0.05% w/v anthrone and incubated at 100 °C for 15 min. After cooling, sample absorbance was measured at 620 nm and compared to a trehalose concentration versus absorbance standard curve to evaluate trehalose concentration. Ovine mean corpuscular volume used for calculating intracellular trehalose concentration was measured using a Woodley abc haematology analyzer (Woodley Equipment Company Ltd.; Bolton, UK).
  • Negative controls ensured that all reported trehalose was truly intracellular.
  • the amount of residual extracellular trehalose after washing was measured by incubating red blood cells in trehalose for less than 1 minute (a negligible amount of time for trehalose loading) and then passing them through the trehalose measurement protocol.
  • the creation of a negative control of this kind was necessary for each concentration of trehalose used; all loading values are relative to their corresponding negative control values.
  • Erythrocytes were incubated in PBS of pH 7.05 along with 75 pg mL "1 PP-50 for 9 hrs at 37°C. Following incubation, cells were immediately washed twice and resuspended at room temperature in PBS of physiological osmolarity and pH. Washed cells were split into two groups and incubated in parallel at 37°C and pH 7.05 for 9 hrs: one group was incubated in 0.36 M trehalose alone and one group was incubated with 0.36 M trehalose and 75 pg mL "1 PP-50.
  • erythrocyte size and internal complexity were performed using a FACScan flow cytometer (Becton Dickinson; Oxford, UK) . Prior to analysis, erythrocytes were washed twice in PBS of physiological pH and osmolarity and diluted to approximately 3 * 10 7 erythrocytes per mL .
  • Erythrocytes were incubated at approximately 15% haematocrit in either 0.36 M trehalose or 0.36 M trehalose + 100 g mL "1 PP-50, each for 9 hrs at 37°C and pH 7.05.
  • the intracellular trehalose concentration achieved in the absence of polymer was insignificant ( ⁇ 2 mM) ; the intracellular concentration achieved in the presence of polymer PP-50 was 123 ⁇ 16 mM.
  • erythrocytes were washed twice in PBS buffer iso-osmotic to the incubation solution to reverse
  • Cyropreservation protocol 1 utilized relatively slow freezing and thawing; freezing was accomplished by placement of samples in a -80°C freezer (Sanyo; Loughborough, UK) as described by Farrugia et al [25] and thawing was accomplished by warming samples in air at room temperature as described by Chao et al [26] .
  • Cryopreservation protocol 2 utilized relatively slow freezing and relatively rapid thawing; freezing was accomplished by placement of samples in a -80°C freezer and thawing by warming samples in a 37°C water bath as described by Holovati et al [6].
  • Cryopreservation protocol 3 utilized relatively rapid freezing and thawing; freezing was accomplished by direct immersion of samples in liquid nitrogen as described by Pellerin-Mendes et al [27] and thawing was accomplished by warming samples in a 37°C water bath.
  • percent survival of erythrocytes after cryopreservation was calculated by subtracting percent haemolysis during freezing and thawing ("freeze-thaw”) from 100%. Percent increase in cryosurvival of cells loaded with trehalose relative to unloaded cells (all cells suspended in trehalose solution) was calculated using the following formula :
  • Nucleated mammalian cells (human immortalised myelogenous leukaemia line K562; human primary osteosarcoma cell line SAOS-2; and thrice- cloned human neuroblastoma cell line SH-SY5Y) were cultured and flasks full of cells retrieved. The cells were washed three times. Adherent cells were washed using PBS and trypsinated. Suspension cells were gently centrifuged (1000 RPM) and washed as for RBCs above.
  • Cells were then suspended in PBS pH 7.05 and add 0.2 mL of cell suspension solution (7. Ox 10 6 cells per mL with SAOS cells and SH-SY5Y cells and 10.5x 10 6 cells per mL in K562 cells in PBS solution) to 2 mL centrifuge tubes.
  • Cells were then frozen via immersion in liquid nitrogen in 2 mL centrifuge tubes as described above for RBCs. Cells were thawed in 37°C water bath for 15 min and resuspended via gentle shaking.
  • Cells were imaged at *63 magnification on a Leica DMI 6000 CS confocal unit ( etzlar, Germany). Prior to imaging, cells (15% packed cell volume) were incubated in extracellular trehalose (0.7 M) and PP-50- FITC (400 pg mL "1 ) for 9 h at 37°C and at pH 7.05 or 7.4. Cells adjusted to 15% packed cell volume were imaged immediately after incubation. The excitation and emission wavelengths were 492 nm and 525 nm, respectively. Determination of permeabilization mechanism
  • Permeability of molecules to the red blood cell membrane was assayed based on degree of hemolysis induced via colloidal osmotic lysis as previously described [39] . Permeability measurement took place after incubation of erythrocytes (108 erythrocytes mL “1 ) for 4 hours at 37°C and pH 7.05 in phosphate buffer (100 mM) containing PP-50 (200 g mL "1 ) plus sucrose, 1000 MW poly (ethylene glycol) ("PEG 1000”), PEG 2000, PEG 4000, PEG 6000, PEG 8000 or PEG 10,000 (each 10 mM) .
  • PEG 1000 poly (ethylene glycol)
  • hydrodynamic radius of sucrose is from the literature [77]; the hydrodynamic radius for PEG molecules was determined as previously [41] . Molecular volumes were determined as described by Bondi [47] .
  • erythrocyte sample aliquots (100 ih at 25% packed cell volume) were deposited evenly at the bottom of glass lyophilization vials (5 mL) and placed in a vented plastic box containing silica gel desiccant (2.5 g per sample). Extracellular trehalose, when present, was at a concentration of 0.7 M. The vented box was then placed in a six layer nylon-polyethylene FoodSaver vacuum sealer bag and put under vacuum (20 kPa abs) using a FoodSaver V2860 commercial vacuum food sealer (Rye, USA) . Samples under vacuum were stored in the dark at room temperature (22°C) for one week before rehydration.
  • Sample water content initially at 232 ⁇ 12 g H 2 0 per g dry weight, stabilized at 0.08 ⁇ 0.02 g H20 per g dry weight within 48 h. Sample water content was calculated as previously [12]. Rehydration of samples to pre desiccation volume was accomplished using room temperature PBS of osmolarity matching incubation osmolarity to minimize osmotic shock. Between rehydration and hemolysis assay, samples were gently agitated for 1 h, leading to homogenous cell resuspension .
  • oxidation of oxyhemoglobin to methemoglobin was found to be significant.
  • concentration of free oxyhemoglobin, deoxyhemoglobin and methemoglobin in samples was determined using spectrophotometry and the hemoglobin species' millimolar extinction coefficients as set forth by Benesch et al [80] . Percent hemoglobin oxidation was calculated by dividing sample methemoglobin
  • hemoglobin hemoglobin, oxyhemoglobin and deoxyhemoglobin
  • Percent loss in membrane integrity during desiccation and rehydration was calculated by dividing total hemoglobin concentration released during desiccation and rehydration by the total hemoglobin
  • Percent membrane integrity after desiccation and rehydration was calculated as 100% minus percent membrane integrity loss.
  • Cells were imaged using a Veeco Dimension 3100 Scanning Probe atomic force microscopy (AFM) unit (Cambridge, UK) . Scanning took place in air in tapping mode using a silicon tip of thickness 4 ⁇ 1 pm and length 125 ⁇ 10 pm with a force constant of 10-130 N/m and a resonance frequency of 24-497 kHz. Images were flattened using a first-order line fit. Prior to imaging, cells were immobilized on a polylysine coated microscope slide [81], cross linked in glutaraldehyde (1%) for 5 min [81], washed three times with PBS and then air dried [82].
  • AFM Veeco Dimension 3100 Scanning Probe atomic force microscopy
  • Polymer PP-50 yielded greater membrane permeability to trehalose than PL-50 by a factor of approximately 1.5 and greater membrane
  • Trehalose loading at pH 7.05 was investigated as a function of polymer concentration and used to investigate the impact of intracellular trehalose on erythrocyte desiccation tolerance. As shown in Fig. 6a, cellular uptake of trehalose increased dramatically from 46 ⁇ 6 mM to 175 ⁇ 5 mM upon introduction of 50 ⁇ ig mL '1 PP-50. As polymer
  • Hemoglobin oxidation decreased to 50 ⁇ 4% in cells loaded with 46 ⁇ 6 mM intracellular trehalose, consistent with the hemoglobin oxidation level of freeze-dried erythrocyte samples loaded with a comparable concentration of intracellular trehalose by hypertonic shock [64]. Oxidation was shown to decrease approximately linearly with further trehalose uptake down to 36 ⁇ 2% with 175 ⁇ 5 mM intracellular trehalose and down to 23 ⁇ 3% with 251 ⁇ 5 mM intracellular trehalose. A hemoglobin oxidation level of 66 ⁇ 1% was found in un-loaded human erythrocytes suspended in PBS. Impact of external trehalose concentration
  • Trehalose loading and haemolysis were shown to increase rapidly with temperature (Fig. 8a, b) . After nine hrs at 29°C, very little loading or haemolysis was experienced. At 37°C, trehalose loading increased to 123 ⁇ 16 mM and haemolysis reached 23 ⁇ 2 % . A particularly
  • trehalose imparts cellular osmoprotection to human erythrocytes and nucleated mammalian cells at low osmolarity [14, 32].
  • trehalose loaded into ovine erythrocytes via polymer-mediated membrane permeabilization provided osmoprotection up to an osmolarity of approximately 230 mOsm and increased osmotic sensitivity after this threshold (Fig. 9) .
  • erythrocytes indicating that osmotic sensitivity changes are likely due to intracellular trehalose rather than polymer-specific effects.
  • cryopreservation protocols In each of these protocols, intracellular trehalose delivered using PP-50 was shown to improve survival of erythrocytes after freeze-thaw relative to erythrocytes suspended in extracellular trehalose alone. Intracellular trehalose improved erythrocyte cryosurvival by 13.1 ⁇ 3.5 % using cryopreservation protocol 1 and by 26.6 ⁇ 6.2 % using protocol 2. The impact of intracellular trehalose on erythrocyte cryosurvival was most of intracellular trehalose delivered using PP-50 was shown to improve survival of erythrocytes after freeze-thaw relative to erythrocytes suspended in extracellular trehalose alone. Intracellular trehalose improved erythrocyte cryosurvival by 13.1 ⁇ 3.5 % using cryopreservation protocol 1 and by 26.6 ⁇ 6.2 % using protocol 2. The impact of intracellular trehalose on erythrocyte cryosurvival was most
  • cryopreservation protocol 3 82.6 ⁇ 3.4 % of erythrocytes with both extracellular and intracellular trehalose survived freeze-thaw while only 62.2 ⁇ 4.4 % of erythrocytes with extracellular trehalose alone survived; this is an increase in post-thaw viability of 32.8 ⁇ 9.0 %. Survival of erythrocytes suspended in PBS alone did not exceed 21 using any cryopreservation protocol.
  • Non-Stokesian diffusion is the mechanism for passive diffusion of hydrophilic molecules through the native phospholipid bilayer [38] .
  • non-Stokesian diffusion takes place via jumping of diffusing molecules into gaps between anchored solvent molecules. As incidence of gap formation decreases
  • amphipathic peptide magainin 2 acts as an antimicrobial agent by increasing microbe membrane permeability, eventually leading to cell lysis [67]. It has been shown that magainin 2 increases membrane permeability through the reversible integration of its hydrophilic moieties into the hydrophilic head region of the phospholipid bilayer and integration of its hydrophobic moieties into the membrane core [68].
  • Adsorption of the hydrophilic region of magainin onto the cell membrane head region creates void space in the hydrophobic core of the membrane because the volumetric ratio of hydrophilic to hydrophobic moieties of magainin does not match the ratio of membrane
  • phospholipid tails eliminate these voids by chain bends or increased trans-gauche isomerization (Fig. 12g) [69]. Because the total volume of the hydrophobic phospholipid tails is constant, the cell membrane becomes thinner in regions incorporating adsorbed peptide [67, 70]. Similar membrane-thinning behavior has been observed with other amphipathic peptides, including protegrins, alamethicin and, at some concentrations, melittin [72].
  • Synthetically derived PP-50 possesses similar amphipathic character to magainins, protegrins, alamethicin, melittin and other antimicrobial peptides. As presented in Fig. 12, it is proposed that PP-50
  • adsorption as demonstrated by confocal microscopy (Fig. 4c) and atomic force microscopy (AFM) (Fig. 12b), leads to thinning of the phospholipid bilayer according to the mechanism presented by Ludtke et al. [67] and Mecke et al. [71].
  • Cross-sectional AFM data showing depressions of the membrane around regions of polymer buildup at 50 nm resolution provides support for this hypothesis (Fig. 12 c,d). These depressions reach approximately 3 nm, 35-40% of the human erythrocyte bilayer thickness [73] . Because diffusive flux is proportional to membrane thickness [74], membrane thinning could contribute to the enhanced non-Stokesian diffusion demonstrated in trehalose loading (Figs.
  • hemolysis likely takes place when extensive bilayer thinning leads to cell membrane collapse.
  • Biomimetic polymers were utilized to reversibly increase the
  • permeability of the ovine erythrocyte membrane to trehalose yielding trehalose loading dependent on polymer type, polymer concentration, pH, external trehalose concentration, and incubation temperature.
  • Trehalose loading was shown to impart cellular osmoprotection up to an external osmolarity of 230 mOsm and impart increased osmotic sensitivity above this threshold. Both effects were approximately proportional to intracellular trehalose concentration.
  • the membrane permeabilization and desiccation protection of human erythrocytes achieved in this study allows for the employment of synthetic polymers for enhancement of non-Stokesian diffusion and the preservation of cells in the dry state.
  • the dramatic decrease in hemoglobin oxidation encountered in this study due to biopolymer- delivered intracellular trehalose is particularly significant to the realization of room-temperature supply chain for transfusion blood.
  • the number average molecular weight calculated based on weight percentage of amino acid grafts (wt%) and the n of poly (L-lysine iso-phthalamide) determined by GPC

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Abstract

Cette invention concerne des procédés qui permettent de rendre réversiblement perméables des cellules de mammifère à des agents de conservation, tels que le tréhalose, à l'aide de polymères synthétiques. Les polymères synthétiques appropriés comprennent les polymères de lysine et d'isophtalamide, en particulier les polymères de lysine et d'isophtalamide qui présentent un ou plusieurs acides aminés hydrophobes pendants, tels que la leucine ou la phénylalanine. La perméabilité à médiation par un polymère permet de charger les cellules de concentrations élevées d'agents conservateurs avant la conservation, par exemple la cryoconservation ou la lyoconservation, et ceci permet à une plus grande proportion de cellules de rester viables après la conservation.
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WO2022016148A3 (fr) * 2020-07-17 2022-02-17 The General Hospital Corporation Billes d'hydrogel d'absorption et de libération contrôlée d'agents cryoprotecteurs
US11297828B2 (en) 2016-03-14 2022-04-12 The Regents Of The University Of Michigan Surface tension mediated lyo-processing technique for preservation of biologics
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