EP0484519A1 - Procede de lyophilisation de cellules, de matieres analogues a des cellules et de plaquettes dans un melange de polymeres amphipathiques biocompatibles - Google Patents

Procede de lyophilisation de cellules, de matieres analogues a des cellules et de plaquettes dans un melange de polymeres amphipathiques biocompatibles

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Publication number
EP0484519A1
EP0484519A1 EP91912119A EP91912119A EP0484519A1 EP 0484519 A1 EP0484519 A1 EP 0484519A1 EP 91912119 A EP91912119 A EP 91912119A EP 91912119 A EP91912119 A EP 91912119A EP 0484519 A1 EP0484519 A1 EP 0484519A1
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European Patent Office
Prior art keywords
cells
medium according
medium
molecular weight
red blood
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP91912119A
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German (de)
English (en)
Other versions
EP0484519A4 (en
Inventor
Raymond P. Goodrich, Jr.
Johannes T. Derksen
Joseph Wyse
Samuel O. S. Coker
Leonor Directo
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Terumo BCT Inc
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Cobe Laboratories Inc
Cryopharm Corp
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Publication of EP0484519A1 publication Critical patent/EP0484519A1/fr
Publication of EP0484519A4 publication Critical patent/EP0484519A4/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes

Definitions

  • This invention relates to the general field of biochemistry and medical sciences, and specifically to processes for the preservation, storage and reconstitution of cells, particularly red blood cells and platelets, and cell-like materials (such as hemosomes) .
  • the oxygen is furnished from the lungs by an exchange-diffusion system brought about by a red, iron-containing protein called hemoglobin which comprises most of the total cell protein in a mature red cell.
  • hemoglobin combines with oxygen, oxyhemoglobin is formed and after oxygen is given up to the tissues, the oxyhemoglobin is reduced to deoxyhemoglobin.
  • the red cell membrane is composed of two major structural units, the membrane bilayer and a cytoskeleton.
  • a lipid bilayer and integral membrane proteins form the membrane bilayer, which has little structural strength and fragments readily by vesiculation.
  • the other major component, the membrane skeleton stabilizes the membrane bilayer and provides resistance to deformation.
  • the cytoskeleton is linked to the bilayer in the erythrocyte membrane, possibly by lipid-protein as well as protein-protein associations.
  • the hemoglobin, and other RBC components are contained within the red cell membrane.
  • bone marrow is active in the formation of new red blood cells. Once new erythrocytes enter the blood, these cells have an average lifetime of about 120 days. In an average person, about 0.83% of the erythrocytes are destroyed each day by phagocytosis, hemolysis or mechanical damage in the body, and the depleted cells are renewed from the bone marrow.
  • a wide variety of injuries and medical procedures require the transfusion of whole blood or a variety of blood components. Every patient does not require whole blood and, in fact, the presence of all of the blood components can cause medical problems. Separate blood fractions can be stored under those special conditions best suited to assure their biological activity at the time of transfusion.
  • erythrocytes are separated and stored by various methods. Such cells are storable in citrate- phosphate-dextrose at 4°C for up to five weeks, generally as a unit of packed erythrocytes having a volume of from 200 to 300 ml and a he atocrit value (expressed as corpuscular volume percent) of 70 to 90. Erythrocytes may also be treated with glycerol and then frozen at from -30° to -196°C and stored for up to seven years in a glycerol solution, but must be kept frozen at low temperatures in order to survive sufficiently for transfusion.
  • Freeze-drying of cells, since such cells could be stored at room temperature or an extended period of time and easily reconstituted for use. Freeze-dried cells (such as erythrocytes, platelets, or cell-like material, such as, hemosomes) could thus be easily stored for use in transfusions.
  • Freeze-dried cells such as erythrocytes, platelets, or cell-like material, such as, hemosomes
  • RBCs When RBCs have been lyophilized according to previous methods, for example in either an aqueous or phosphate- buffered saline (PBS) solution, the reconstituted cells are damaged to the extent that the cells are not capable of metabolizing, or the cell hemoglobin cannot carry oxygen or the cells lyse upon rehydration and are not useful for transfusion.
  • PBS phosphate- buffered saline
  • Glu araldehyde-fixed erythrocytes which have been lyophilized and reconstituted, have found use primarily in agglutination assays, in which only the preservation of certain cell surface antigens is desired. These fixed cells are metabolically non- viable and are unsuitable for use in transfusion medicine.
  • the process of the present invention allows for the lyophilization of red blood cells or platelets under conditions which are not deleterious to the structure and the biological activity of the cell, and which permits the reconstitution of the lyophilized red blood cells or platelets to form cells in which the biological activity found in freshly collected cells is preserved at useful levels.
  • the cells may be from in vitro cultures, peripheral blood cells, blood stem cells, or cell-like materials, such as liposomes, hemosomes or cell membrane ghosts. Furthermore, these may be mammalian cells, hybridoma cells, or any other type of cell.
  • the process comprises immersing a plurality of cells in an essentially isotonic aqueous solution containing a carbohydrate, and a mixture of at least two types of amphipathic polymers, freezing the solution, and drying the solution to yield freeze- dried cells which, when reconstituted, produce a significant percentage of intact and viable cells.
  • the process of the invention is preferably applied to red blood cells or platelets and allows for the lyophilization under conditions which maintain structure of the cell and the biological activity of the hemoglobin, and which permits the reconstitution of the lyophilized red blood cells or platelets to allow use on a therapeutic level.
  • the carbohydrate of the invention is biologically compatible with the cells, that is, non-toxic and non-disruptive to the cells, and is preferably one which permeates, or is capable of permeating, the membrane of the cells.
  • membrane-permeant carbohydrates apparently protect the intracellular components, to include the oxyhemoglobin, from freezing and drying damage.
  • the carbohydrate may be selected from the group consisting of monosaccharides, since disaccharides do not appear to permeate the membrane to any significant extent.
  • Monosaccharide pentoses and hexoses are preferred in concentrations of from about 7.0 to 37.5%, preferably about 23%.
  • Xylose, glucose, ribose, mannose and fructose are employed to particular advantage.
  • the use of a mixture of water soluble, biologically compatible amphipathic polymers in addition to the carbohydrate adds significantly to the percentage of biologically-active hemoglobin (in the case of red blood cells) which is retained in the cells and recovered after reconstitution of red blood cells after lyophilization. Retention of cell hemoglobin provides an easy assay for cell lysis or leakiness; use of polymers in the present invention appears to minimize loss of cell hemoglobin and therefore preserves cell integrity.
  • the polymers will preferably be amphipathic, meaning that there are hydrophilic and hydrophobic portions on a single molecule of the polymer.
  • the mixture of polymers may be present in the buffered lyophilization solution in total concentrations of from 0.7% (by weight) up to saturation.
  • each of the polymer types in the mixture has a molecular weight in the range of from about IK to about 600K (number average molecular weight) .
  • at least one of the types of polymers of the mixture will preferably have a molecular weight from about 5K to 40OK, and most preferably from 2OK to 360K.
  • one of the types of polymers of the mixture will preferably have a molecular weight in the range of about 100K to about 600K, most preferably in the range of about 100-500K.
  • each of the polymer types may be present in a concentration of from about .35% (by weight) up to its limit of solubility in the buffered lyophilization solution.
  • Polymers selected from the group consisting of polyvinylpyrrolidone (PVP) , polyvinylpyrrolidone derivatives, dextran, dextran derivatives, amino acid based polymers (j_.e. , proteins) and hydroxyethyl starch (HES) may be employed.
  • PVP polyvinylpyrrolidone
  • polyvinylpyrrolidone derivatives polyvinylpyrrolidone derivatives
  • dextran dextran derivatives
  • amino acid based polymers j_.e. , proteins
  • HES hydroxyethyl starch
  • Other amphipathic polymers may be used, such as poloxamers in any of their various forms.
  • a mixture of PVP molecular weight in the range of about 20K-360K
  • HES molecular weight in the range of about 100K-500K
  • the use of the carbohydrate-polymer solution in the lyophilization of red blood cells allows for the recovery of intact cells, a significant percentage of which contain biologically-active hemoglobin. While not intending to be bound by any theory, the amphipathic properties of the polymer allow them to bind to the cell membrane while protecting the membrane surface by extension of the hydrophilic portion into the aqueous environment. This may alleviate the damage to the cell membrane which causes other problems, such as cell aggregation.
  • the lyophilization buffer as well as the reconstitution buffer or washing buffer may further contain certain supplements which are particularly useful if the cells are cellular blood matter, including red cells, platelets, lymphocytes, stem cells; or other cell-like materials such as liposomes, hemosomes or membrane ghosts.
  • One class of supplements comprises antioxidants such as glutathione or alpha-tocopherol. It is believed that such antioxidants assist a cell in reducing oxidation damage (such as by cell membrane lipid peroxidation) which may otherwise occur during lyophilization or reconstitution.
  • a second class of supplements comprises chelating agents such as EDTA or desferrioxamine, which have the ability to scavenge free iron released from the degradation of cellular hemoglobin. The free iron or hemichromes are detrimental since they may in turn catalyze oxidative damage to cells.
  • a third class of supplements comprises amino acid based polymers (i.e.. peptides and proteins) , such as serum albumin which may act as a coating agent to coat the surface of the cells, thereby minimizing the formation of cell-cell aggregates.
  • preferred supplements include glutathione (GSH) preferably in a concentration of l- 60 M in the buffer (either lyophilization, reconstitution or wash buffer) ; alpha-tocopherol, preferably in the concentration of 1-3 mg/gm RBC; EDTA in a preferred concentration of 1-10 mM; desferrioxa ine in a concentration of 1-10 mM; and albumin in a concentration of 0.5-14% (w/v). Either human or bovine serum albumins are preferred.
  • the described solutions provide media which permit cells, particularly red blood cells, to be subjected to the stresses of freezing, water sublimation and reconstitution and to form freeze-dried cells which may be reconstituted to yield cells which are capable of functioning normally.
  • FIG. 1 is a graph of the methemoglobin half-life in samples of reconstituted lyophilized RBCs according to the invention and non-lyophilized RBCs.
  • FIG. 2 is a graph of the linear regression of methemoglobin over time in reconstituted lyophilized RBCs according to the invention and non-lyophilized RBCs.
  • the process of the invention provides media for the lyophilization of erythrocytes.
  • lyophilization is broadly defined as freezing a substance and then reducing the concentration of one of the solvents, namely water, by sublimation and desorption, to levels which will no longer support biological or chemical reactions.
  • the drying step is accomplished in a high vacuum.
  • the extent of drying (the amount of residual moisture) is of critical importance in the ability of cells to withstand long-term storage at room temperature.
  • cells may be lyophilized to a residual water content of less than 10%, preferably less than 5%, and most preferably to a water content of less than 3%.
  • the buffered lyophilization solution may contain, in addition to the monosaccharide and amphipathic polymer mixture, adjuvants, buffering agents, salts, cofactors, and the like.
  • a particularly preferred lyophilization buffer contains the following components:
  • the cells are mixed with lyophilization buffer at a hematocrit of 30%-40%.
  • the lyophilization buffer is as described above, with the polymer mixture used in each test set forth in Table 1. As a control, one run was performed using only 20% 24K PVP as the polymer.
  • the sample is then placed on a conventional pharmaceutical shelf freeze-dryer and the samples are then frozen on the refrigerated shelf, then vacuum is applied and the sample is allowed to dry until the sample is thoroughly dried as determined by a 58% weight loss.
  • the reconstitution buffer will contain a polymer as described above in connection with the lyophilization buffer (concentration preferably in the range of about 1-20 wt. %) which is amphipathic having a MW in the range of 1-600K, preferably 1- 360K.
  • a preferred reconstitution buffer is as follows:
  • reconstituted sample is prediluted with an equal volume of reconstitution buffer and agitated until thoroughly mixed.
  • the reconstituted and prediluted cells are centrifuged at room temperature.
  • the reconstituted sample is prediluted with an equal volume of reconstitution buffer and swirled until thoroughly mixed. At this point the cell suspension can be aseptically transferred to a sterile, enclosed cell washing system such as the COBE model 2991 cell washer.
  • the reconstituted and prediluted cells are centrifuged at room temperature to collect the cells.
  • the pellet is resuspended in wash buffer and centrifuged.
  • the wash buffer will preferably contain a polymer as described above in connection with the lyophilization buffer (concentration preferably in the range of about 1-20 wt/v %) which is amphipathic having a MW in the range of 1-600K, preferably 1- 360K.
  • the preferred wash buffer is as follows:
  • Another wash buffer is as follows:
  • An optional step involves a diluent buffer step to eliminate any fragile cells.
  • the pellet is resuspended in a diluent buffer at a 10-50 fold dilution and centrifuged.
  • the preferred diluent buffer is as follows:
  • Another diluent buffer is as follows:
  • the pellet is resuspended in the final solution, transfusion buffer, and centrifuged. This step is repeated once.
  • the transfusion buffer will preferably contain a polymer as described above in connection with the lyophilization buffer
  • the preferred transfusion buffer is as follows:
  • Another transfusion buffer is as follows:
  • NaCl solution is added to a sample of reconstituted cells. 5 ⁇ Ci of 51 Cr is added for every 0.1 ml of packed RBC pellet. The labelled pellet is incubated 15 min. at 37°C after which the labelling reaction is stopped by addition of 1 ul of ascorbic acid (50mg/ml in buffer) to every 0.1 ml of pellet. The pellet is then allowed to incubate another 5 min. at room temperature. The labelled sample is then washed 2 to 3 times in transfusion buffer. An aliquot of labelled cells is then transferred to 5 ml of autologous whole blood and the stability determined by the lysis of labelled cells at time points up to 24 hours.
  • the amount of free 51 Cr in the supernatant after centrifuging indicates the amount of cell lysis.
  • a 4-hour incubation is used, since lysis (if any) is complete by then.
  • Cell stability data show the stability and integrity of the lyophilized, constituted red blood cells.
  • the 51 Cr binds to the internal cell hemoglobin, and is released into the assay supernatant (therefore, lost) if the cells lyse.
  • retention of 51 Cr in the pellet measures cell integrity.
  • the high cell stability indicates sufficient cell preservation to be useful for diagnostic use, or for use in transfusion medicine.
  • EXAMPLE 1 Lyophilized reconstituted human red cells tested using the above procedures. Red cells were lyophilized using one polymer or a polymer mixture, and the whole blood stability of 51 Cr labeled reconstituted cells was studied. The reconstituted cells were processed using an automated cell washer as described in Example 2. The results are described as follows (Table I) : TABLE 1
  • This example illustrates use of an automated blood bank cell washer. Packed red blood cells are mixed in a container with lyophilization buffer at a hematocrit of 30%.
  • the lyophilization buffer is as described above, with the polymer mixture used containing 3% 360K PVP and 15% 500K HES.
  • the container is then placed in a standard shelf lyophilizer (Virtis SRC-15 Lyophilizer) and frozen.
  • the frozen sample is then placed under a vacuum of 10-30 mtorr.
  • the sample is allowed to dry, with a total weight loss of 58 ⁇ 2%.
  • the sample is returned to room temperature and the vacuum is removed.
  • the reconstituted sample is prediluted with an equal volume of reconstitution buffer and swirled until thoroughly mixed.
  • the reconstituted and prediluted cells are transferred to a COBE 2991 Blood Cell Washer, centrifuged at 3000 rp for 20 minutes, and repeated until all of the reconstitution buffer volume is added to the Cobe bag.
  • the cells are washed by the automatic protocol of the Cell Washer with the following solutions described in Example 1:
  • Wash buffer 500 ml, IX, 3000 rpm, 20 minutes.
  • Transfusion buffer 500 ml, 4X, 3000 rpm, 5 minutes.
  • This example shows the use of the automated cell washing equipment with the disclosed centrifugation conditions, to prepare reconstituted, washed human red cells.
  • Example 1 The procedure described in Example 1 was repeated with the substitution of 200K HES for 500K HES in a given HES/PVP polymer mixture in the lyophilization buffer. All other conditions were the same as those in Example 1. The results are described in Table 3. the use of 500K HES is marginally preferred over 200K HES in the polymer mixture.
  • Example l The procedure described in Example l was repeated with lyophilization buffers using 40% hematocrit mixtures with washed red blood cells.
  • the polymer composition used in these lyophilization buffers was 5:15% 24K PVP:500K HES.
  • the glucose concentration in the 40% lyophilization buffers is increased to 2.3 M (441.37 g/1). All other conditions were the same as those in Example 1.
  • the results are described as follows: TABLE 4
  • the 4-hr. whole blood stability was significantly increased using a polymer mixture as compared to using a single polymer.
  • EXAMPLE 5 The data shown in Table 5 indicate significant improvement in the osmotic stability, maximum cell deformability (DI max) , and cell density in cells lyophilized with the buffers modified with various supplements.
  • the osmotic stability assay was done with 51 Cr radiolabeled cells. Cell density was determined using discontinuous (step) density gradient centrifugation, which is a standard laboratory procedure. The method and equipment to measure the DI max is published in Mohandas, N. , Clark, M.R., Health, B.P. , Rossi, M. , Wolfe, L.C.,
  • osmotic stability in the cells treated with the supplements is at least about 75% of fresh cells.
  • osmotic stability is at least 60% of the stability of whole blood, and the
  • D ⁇ (max) is at least 50% of the D ⁇ (max) measured with fresh red cells.
  • MCV is the mean corpuscular volume in femtoliters.
  • MCH is the mean corpuscular hemoglobin in picograms.
  • MCHC is the mean corpuscular hemoglobin concentration as a w/v percent.
  • OxyHb is functional oxyhemoglobin measured as a percent recovery at the final stage (cells washed into transfusion buffer).
  • MetHb is oxidized methemoglobin (again % recovery at final step).
  • Hemichrome is a class of several forms of irreveisibly degraded hemoglobin (% recovery at final step).
  • DI (max) is a measure of the maximum defor ability (ellipticity) of red cells subjected to mechanical shear stress.
  • EDTA sodium ethylenediamine tetraacetate.
  • Albumin is serum albumin prepared from human plasma or bovine plasma.
  • Other antioxidants in addition to GSH include alpha-tocopherol used at 1-3 mg/gram of red cells.
  • Tables 6 and 7 one particular advantage of including albumin in the lyophilization buffer is shown (the experiment of Table 7 is the same as the 40 mM GSH + 14% albumin column in Table 5) in terms of a dramatic improvement in the cell density profile.
  • Table 6 and 7 show the fraction of lyophilized reconstituted human red cells that sediment above or below a solution (the density step gradient "cushion") of a known solution density. The percent of cells below the density cushion (i.e., having a cell density greater than the solution density) is indicated. The same percentage profile for normal human red cells as a control is also shown.
  • the lyophilization buffer was as described in Example 1, supplemented with GSH or GSH/albumin.
  • Blood was obtained from six healthy adult individuals with no history of either hemoglobmopathy or abnormal RBC metabolism. Blood was withdrawn from each donor into plastic transfer bags (Fenwal Laboratories, Deerfield, 111) containing 63mL of citrate phosphate dextrose-adenine (CPD-A) anticoagulant using conventional blood banking techniques. The blood units (500ml each) were centrifuged at 1500g for 5 minutes at room temperature (22C) to remove the buffy coat and plasma. The packed RBC were washed in isotonic dextrose saline according to standard washing procedures [11] using automatic cell washer (Model 2991, COBE, Lakewood, CO) .
  • CPD-A citrate phosphate dextrose-adenine
  • the washed and packed RBC (about 85% hematocrit) were resuspended to about 40% in lyophilization buffer as described in Example 2. (1800mOsmol, pH 7.4).
  • About 360g of the RBC suspension were transferred to plastic lyophilization bags and were placed in a conventional pharmaceutical shelf freeze-dryer (Cryopharm Corporation, Pasadena, CA) and then freeze-dried as described in Example 2.
  • the dried RBC were rehydrated and reconstituted in phosphate buffered rehydration buffers described in Example 2 (360mOsmol, pH 7.4) at 22C.
  • rehydration buffer 600g was added to the dried RBC and then agitated on a wrist action shaker (Burrel Corporation, Pittsburgh, PA) until the RBC were fully rehydrated.
  • additional 600g of rehydration buffer was added to the sample and then centrifuged at 1500g for 3 minutes. The supernatant was removed and the packed RBC were washed twice in wash buffers as described in Example 2 by centrifugation at 1500g, using COBE automatic cell washer. Reconstituted RBC were assayed for glycolytic enzyme activities and intermediates according to published methods.
  • Control blood samples were obtained from autologous donors at the time of reconstitution of lyophilized RBC.
  • Control RBC were treated similarly to reconstituted lyophilized RBC with respect to washing.
  • glycolytic enzyme activities of blood bank stored RBC were determined. See Tables 1 and 2.
  • Rate of Adenine Nucleotide Synthesis The rate of adenine nucleotide synthesis was measured by following the incorporation of carbon 14-labelled adenine into the adenine nucleotide pool in intact RBC according to the method described by Zerez et al. J. Lab. Clin. Med. 114, 43-50 (1989). Briefly, the RBC were incubated with carbon 14-labelled adenine ( 1 C) at 37C and at different times aliquots were removed, mixed with saline and immediately immersed in boiling water for 60 seconds. The mixture was chilled at 0 ⁇ C and then centrifuged to remove coagulated proteins.
  • the resultant supernatant contained 1 C-labelled adenine nucleotides along with an excess of 1 C-labelled adenine.
  • a modification of the method of Hershko [19] was used to separate Re ⁇ labelled adenine nucleotides from 14 C-adenine and radioactivity was counted in a liquid scintillation spectrometer (Model LS7500, Beckman instruments, Fullerton, CA) .
  • the rate of Methemoglobin Reduction The rate of methemoglobin (metHb) reduction in intact RBC was determined by using a published method. Zerez et al. Blood 76, 1008-1014 (1990). Briefly, to convert hemoglobin (Hb) to metHb, washed RBC were incubated for 10 minutes at 37C in a solution containing 0.1% (wt/v) NaN0 3 , 605mM Na 6 HP0 4 , pH 7.4 and 154mM NaCl at
  • Rates of ATP and lactate production were determined by the methods described by Beutler, Red Cell Metabolism: A Manual of Biochemical
  • Table 1 Summary of the activities of the glycolytic enzymes in hemolysates from rehydrated lyophilized and non-lvophilized RBC.
  • Data represent the mean ⁇ sd, for 6 samples. Data from blood bank stored RBC are included for comparison with rehydrated lyophilized RBC. Total number of blood bank samples analyzed was 3. Abbreviations: lyo, lyophilized; N-lyo, non- lyophilized; BB, Blood bank,; N-R, normal range; P, probability for comparison between lyophilized and non-lyophilized RBC; ND, not detected; NS, not significant.* Enzymes of Glycolytic Pathway; + Enzymes of the Pentose Phosphate Pathway.
  • the preferred useful reconstituted RBCs are characterized by hexokinase (HX) activity of at least 0.9 micromole/min/gram hemoglobin; diphosphoglyceromutase (DPGM) activity of at least
  • SUBSTITUTESHEET 3.0 micromole/min/gm hemoglobin; phosphofructokinase (PFK) activity of at least 8.0 micromole/min/gram hemoglobin; pyruvate kinase (PK) activity if at least 12.0 micromole/min/gm hemoglobin; glucose-6-phosphate dehydrogenase (G-6-PD) of at least 9.0 micromole/min/gm hemoglobin; 6-phosphogluconate dehydrogenase (6-PGD) of at least 7.0 micromole/min/gm hemoglobin; at least 0.5 micromole/min/gm hemoglobin each of transaldolase (TA) and transketolase (TK) ; and at least 6.0 micromole/min/gm hemoglobin of glutathione reductase.
  • PFK phosphofructokinase
  • PK pyruvate kinase
  • G-6-PD glucose-6-phosphate dehydrogen
  • SUBSTITUTESHEET Data represent the mean ⁇ S.D. for 6 samples. Normal values are included in the table for comparison with present data. Abbreviations: lyo, lyophilized; N- lyo, non-lyophilized; NV, normal values; P, probability for comparisons between lyophilized and non-lyophilized RBC.
  • the preferred useful reconstituted RBCs are characterized by at least 50 nmole/gm hemoglobin of glucose-6-phosphate (G6P) ; at least 100 nmole/gm hemoglobin of fructose-l,6-diphosphate (FDP) ; at least 2000 nmole/gm hemoglobin of 2,3- diphosphoglycerate (2,3-DPG); and at least 50 nmole/gm hemoglobin of pyruvate (pyr) .
  • G6P glucose-6-phosphate
  • FDP fructose-l,6-diphosphate
  • pyr 2,3- diphosphoglycerate
  • HX hexokinase
  • PFK phosphofructokinase
  • PK pyruvate kinase
  • the reconstituted lyophilized red cells retain the activity of diphosphoglyceromutase, which in human red cells shunts, 1,3-diphosphoglycerate (1,3-DPG), a glycolytic intermediate, to 2,3-DPG, which is a key allosteric effector of hemoglobin, and regulates the
  • the data shows steady-state levels of the metabolic intermediates to include levels of glucose-6- phosphate (G6P) , the product of hexokinase activity; fructose-l,6-diphosphate (FDP) , the product of phosphofructokinase activity; 2,3-DPG, the product of diphosphoglyceromutase activity; and pyruvate (pyr) , the product of pyruvate kinase (PK) activity.
  • G6P glucose-6- phosphate
  • FDP fructose-l,6-diphosphate
  • 2,3-DPG the product of diphosphoglyceromutase activity
  • pyr pyruvate kinase
  • the enzymes of the pentose phosphate shunt are functional; this pathway serves two vital functions in the red cell: it produces energy (ATP) and ribose-5-phosphate (R-5-P) used to make reduced glutathione as part of the cell's normal antioxidant defense system, and it produces 5-phosphoribosyl pyrophosphate (PRPP) , an intermediate used to make adenine nucleotides from exogenous adenine (exogenous adenine is imported into the cell from plasma, or in refrigerated stored cells from commercial storage solutions such as CPDA-1: citrate/phosphate/dextrose/ adenine) .
  • energy ATP
  • R-5-P ribose-5-phosphate
  • PRPP 5-phosphoribosyl pyrophosphate
  • NADH reduced nicotinamide adenine dinucleotide
  • NADPH reduced nicotinamide adenine dinucleotide phosphate

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Abstract

Procédé et milieu de lyophilisation de cellules (y compris de plaquettes) consistant à utiliser des solutions comprenant des hexoses et des pentoses monosaccharidiques, ainsi qu'un mélange d'au moins deux polymères amphipathiques biocompatibles.
EP19910912119 1990-05-25 1991-05-24 Process for lyophilizing cells, cell-like materials and platelets in a mixture of biocompatible amphipathic polymers Withdrawn EP0484519A4 (en)

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JP (1) JPH05501419A (fr)
AU (1) AU8064691A (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340592A (en) * 1988-05-18 1994-08-23 Cobe Laboratories, Inc. Lyophilization of erythrocytes
US5648206A (en) * 1988-08-26 1997-07-15 Cobe Laboratories, Inc. Lyophilization of cells

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5213814A (en) * 1989-04-10 1993-05-25 Cryopharm Corporation Lyophilized and reconstituted blood platelet compositions
CA2051092C (fr) * 1990-09-12 2002-07-23 Stephen A. Livesey Methode et appareillage pour la cryopreservation, la stabilisation a sec et la rehydratation de suspensions biologiques
US5336616A (en) * 1990-09-12 1994-08-09 Lifecell Corporation Method for processing and preserving collagen-based tissues for transplantation
US8067149B2 (en) 1990-09-12 2011-11-29 Lifecell Corporation Acellular dermal matrix and method of use thereof for grafting
US6114107A (en) * 1996-06-14 2000-09-05 Biostore New Zealand Limited Composition comprising raffinose, TMAO, sodium citrate and methods for the preservation of living tissues
US6361933B1 (en) 1996-06-14 2002-03-26 Biostore New Zealand Limited Solutions for the preservation of tissues
US6743575B2 (en) 1996-06-14 2004-06-01 Biostore New Zealand Ltd. Compositions and methods for the preservation of living tissues
US6037116A (en) * 1996-06-14 2000-03-14 Biostore New Zealand, Ltd. Compositions comprising betaine, sodium citrate and sodium chloride and methods for the preservation of biological materials
US6060233A (en) * 1996-06-14 2000-05-09 Biostore New Zealand, Ltd Methods for the lyophilization of platelets, platelet membranes or erythrocytes
US5962213A (en) * 1996-06-14 1999-10-05 Biostore New Zealand Limited Compositions and methods for the preservation of living tissues
US6040132A (en) * 1996-06-14 2000-03-21 Biostore New Zealand, Ltd. Methods for the lyophilization of living biological materials
US5827640A (en) * 1996-06-14 1998-10-27 Biostore New Zealand Limited Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose
CA2257497A1 (fr) * 1996-06-14 1997-12-18 Philippa M. Wiggins Compositions et procedes de conservation de tissus vivants
US5879875A (en) * 1996-06-14 1999-03-09 Biostore New Zealand Compositions and methods for the preservation of living tissues
EP1082006B1 (fr) * 1998-05-26 2006-02-01 Lifecell Corporation Cryoconservation amelioree d'erythrocytes humains
US6933326B1 (en) 1998-06-19 2005-08-23 Lifecell Coporation Particulate acellular tissue matrix
US7112576B1 (en) 1999-12-10 2006-09-26 Regents Of The University Of Minnesota Compositions and methods for cryopreservation of peripheral blood lymphocytes
JP2002226368A (ja) * 2001-02-02 2002-08-14 Fuji Chem Ind Co Ltd 赤血球の酸化的損傷抑制剤
GB0230152D0 (en) * 2002-12-24 2003-02-05 Sinvent As Product
AU2006294654B2 (en) 2005-09-26 2012-05-24 Lifecell Corporation Dry platelet composition
KR101410065B1 (ko) * 2011-12-09 2014-06-27 테고사이언스 (주) 세포의 유용물질을 상온에서 안정하게 보존하는 방법

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1014712A (en) * 1963-05-31 1965-12-31 Union Carbide Corp Improvements in and relating to liquid media in contact with animal cells
GB1057277A (en) * 1963-03-01 1967-02-01 Union Carbide Corp Improvements in and relating to preservation of cells
WO1990012582A1 (fr) * 1989-04-10 1990-11-01 Cryopharm Corporation Compositions cellulaires nouvelles de globules rouges reconstituees et lyophilisees
WO1991016060A1 (fr) * 1990-04-16 1991-10-31 Cryopharm Corporation Procede pour rendre inactifs des contaminants sanguins viraux et bacteriens
WO1991017655A1 (fr) * 1990-05-17 1991-11-28 Cryopharm Corporation Nouvelles compositions de cellules reconstituees et lyophilisees
WO1992014360A1 (fr) * 1991-02-15 1992-09-03 Cryopharm Corporation Procede de lyophilisation et de reconstitution de melanges de cellules non mammiferes non nuclees et de matiere sanguine

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3915794A (en) * 1973-02-09 1975-10-28 Rit Rech Ind Therapeut Stabilizing compositions for cell-free viruses and cell-free virus preparations containing them
DE2310964A1 (de) * 1973-03-02 1974-09-05 Schering Ag Antigen- bzw. antigen-proteinkonjugatbeschichtete erythrocytenpraeparation
US4963362A (en) * 1987-08-07 1990-10-16 Regents Of The University Of Minnesota Freeze-dried liposome mixture containing cyclosporin
IL90188A0 (en) * 1988-05-18 1989-12-15 Cryopharm Corp Process and medium for the lyophilization of erythrocytes
US5045446A (en) * 1988-08-26 1991-09-03 Cryopharm Corporation Lyophilization of cells
US4874690A (en) * 1988-08-26 1989-10-17 Cryopharm Corporation Lyophilization of red blood cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1057277A (en) * 1963-03-01 1967-02-01 Union Carbide Corp Improvements in and relating to preservation of cells
GB1014712A (en) * 1963-05-31 1965-12-31 Union Carbide Corp Improvements in and relating to liquid media in contact with animal cells
WO1990012582A1 (fr) * 1989-04-10 1990-11-01 Cryopharm Corporation Compositions cellulaires nouvelles de globules rouges reconstituees et lyophilisees
WO1991016060A1 (fr) * 1990-04-16 1991-10-31 Cryopharm Corporation Procede pour rendre inactifs des contaminants sanguins viraux et bacteriens
WO1991017655A1 (fr) * 1990-05-17 1991-11-28 Cryopharm Corporation Nouvelles compositions de cellules reconstituees et lyophilisees
WO1992014360A1 (fr) * 1991-02-15 1992-09-03 Cryopharm Corporation Procede de lyophilisation et de reconstitution de melanges de cellules non mammiferes non nuclees et de matiere sanguine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9118504A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340592A (en) * 1988-05-18 1994-08-23 Cobe Laboratories, Inc. Lyophilization of erythrocytes
US5648206A (en) * 1988-08-26 1997-07-15 Cobe Laboratories, Inc. Lyophilization of cells

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ZA913995B (en) 1992-03-25
CA2062941A1 (fr) 1991-11-26
WO1991018504A1 (fr) 1991-12-12
EP0484519A4 (en) 1993-12-29
AU8064691A (en) 1991-12-31
JPH05501419A (ja) 1993-03-18
IL98269A0 (en) 1992-06-21

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