Cryopreservation of human red blood cells

a technology of red blood cells and cryopreservation, which is applied in the field of cryopreservation of human red blood cells to achieve the effect of reducing the level of hemolysis

Inactive Publication Date: 2006-06-15
LIFECELL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] In one embodiment for preserving human red blood cells the method includes drawing a volume of packed red blood cells in ADSOL, a currently licensed additive solution sold by Baxter Travenol, using standard blood banking protocols. The ADSOL is removed by centriftigation, and a volume of preservation solution containing the desired cryoprotectants and any additional biochemical reagents is added. The red blood samples containing cryoprotectants are submerged in liquid nitrogen for freezing and immediately transferred to a −80° C. freezer for extended storage. Biochemical stabilization may be targeted to maintain metabolic components, antioxidant potential, intracellular ionic distribution, membrane fluidity and integrity of cytoskeletal structure. Additionally, specific second messenger pathways, such as the cyclooxygenase, lipoxygenase, hexose monophosphate and phosphorylation pathways, may be manipulated directly by biochemical reagents or indirectly through regulation of specific messenger molecules, including c-AMP, c-GMP, intracellular calcium, inositol triphosphate, and diacylglycerol. In one preferred embodiment the biochemical reagents are present in a concentration so as to prevent the hemolysis of the red blood cells during the freeze/thaw cycle and even more prefera

Problems solved by technology

Stabilization with these reagents renders the cells partially resistant

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0036] The following example demonstrates the damage induced during the freeze / thaw cycle when the glycerol concentration is decreased. A significant percentage of the hemolytic damage is shown to be reversed by the addition of a preservative solution containing adenine, glutamine, sodium chloride, mannitol, and dextrose, with the essential additive being dextrose, and final concentrations of 5 mM pentoxifylline and 25 μg / ml flurbiprofen in DMSO as a carrier.

[0037] Specifically, 50 ml of packed red blood cells was added to a PVC freezing bag with a maximum volume of 150 ml. Isotonic saline or preservative solution containing the additional biochemical reagents pentoxifylline and flurbiprofen was mixed with the red blood cells. A volume of Glycerolyte 57 (Fenwall) was added slowly until the final concentration of glycerol was achieved. The total volume of all additions was 50 ml, making the final concentration of the red blood cell unit 100 ml. The PVC bags were frozen at −80° C. fo...

example 2

[0039] The following example further demonstrates the damage induced during the freeze / thaw cycle when the glycerol concentration is decreased, after red blood cells are frozen and stored at −80° C. Fifty ml of packed red blood cells were added to a PVC freezing bag. Isotonic saline or preservative solution containing the additional biochemical reagents of interest was mixed with the red blood cells. The samples were treated as follows prior to freezing at −80° C.:

[0040] 1. 40% Glycerol

[0041] 2. 20% Glycerol

[0042] 3. Nicotinamide

[0043] 4. Nikethamide, Nifedipine

[0044] 5. Nikethamide, Nifedipine, Pentoxifylline, Flurbiprofen

[0045] 6. Nicotinamide Nikethamide, Nifedipine, Pentoxifylline, Flurbiprofen

[0046] A volume of Glycerolyte 57 (Fenwall) was added slowly until the final concentration of glycerol was achieved. The total volume of all additions was 50 ml, making the final volume of the red blood cell unit 100 ml. The PVC bags were frozen at −80° C. for 2-7 days and thawed by...

example 3

[0048] The following experiment demonstrates the ability of biochemical addition to provide protection against damage during cryopreservation. The model used is a reduced glycerol cryopreservation system. 10% (w / v) glycerol is added to the unit prior to freezing at −80° C. A significant level of hemolysis occurs so that protection by the added reagent may be distinguished.

[0049] A single unit of packed red blood cells in ADSOL was obtained using standard blood banking protocols. The ADSOL was removed by centrifugation and a volume of preservation solution was added until the resulting hematocrit was about 50%. From this red blood cell unit 50 ml of red cells was placed into each of 3 PVC freezing bags having a maximum volume of 150 ml. An equal volume (50 ml) was added to each of the bags composed of the above preservation solution and the experimental conditions. The final composition of the first bag was 10% (w / v) glycerol. The second contained 10% (w / v) glycerol and 2.5% DMSO an...

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Abstract

A red blood cell storage composition includes a composition of red blood cells and biochemistry altering reagents, the biochemistry altering reagents being present at a concentration so as to reduce the percent hemolysis of the red blood cells during the freeze-thaw cycle below that of the percent hemolysis of the red blood cells in the absence the biochemistry altering reagents. The red blood cell storage composition preferably includes reagents selected from: modifiers of glycolytic/metabolic components, modifiers of antioxidant potential, effectors of intracellular ionic distribution, modifiers of membrane fluidity, modifiers of cytoskeletal structure, effectors of the cyclooxygenase second messenger pathway, effectors of the lipoxygenase second messenger pathway, effectors of the hexose monophosphate second messenger pathway, effectors of the phosphorylation second messenger pathway, modifiers of specific messenger molecules, and combinations thereof.

Description

[0001] Priority of provisional application Ser. No. 60 / 086,836 Filed: May 26, 1998 is claimed. BACKGROUND OF THE INVENTION [0002] Blood is composed of plasma and cellular constituents and plays a prominent role in several key physiological systems of the human body, including immunology, hemostasis, and tissue oxygenation. Movement of the respiratory gases oxygen (O2) and carbon dioxide (CO2) is the chief function of erythrocytes or red blood cells. This facilitated movement Of O2 and CO2 is carried out by hemoglobin, an iron-containing, multi-subunit protein contained within red blood cells. The necessity for encapsulating hemoglobin within the red blood cell is twofold. First, hemoglobin has very specific binding parameters to ensure delivery of these critical molecules to the proper sites. By regulating the concentration of cofactors and free ions, the red blood cell provides the proper environment for optimum uptake and release of O2. Secondly, free heme has toxicity effects on ...

Claims

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Application Information

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IPC IPC(8): A61K35/14A01N1/02A61K31/70A61K31/7048A61K31/522A61K31/7012
CPCA01N1/02A61K35/18A01N1/0226
Inventor LIVESEY, STEPHEN A.BURNETT, MICHAEL BRIANCONNOR, JEROMEWAGNER, CHRISTOPHER TODD
Owner LIFECELL
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