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[22] High-efficiency entrapment of enzymes in resealed red cell ghosts by dialysis
[22]
ENTRAPMENT OF ENZYMES IN R B C OHOSTS
229
TABLE II EFFECT OF HEMATOCRIT ON ERYTHROCYTE-ENTRAPMENT PROCEDURES
Virus A
2
~X174
Amount added per ml 7.4 X 1.5 X 3.3 × 6.8 × 7.3 X 6.1 X 9.5 × 1.0 X 2.1 X
lOs 109 109 109 109 lOp 109 101° 10H 1.8 X 10 I°
Hematocrit (%) 7.2 25 32 67 80 17 10 80 80 10
Entrapment procedure
Equilibration (%)
Endocytosis
2.8 2.5 1.5 2.9 0.7 1.5 6.6 1.6 1.6 67
Dialysis
Dialysis
Entrapment of Drugs As an example of drug entrapment, glueantime (meglumine antimonate, N-methylglucosamine antimonate), an antimonial drug effectivein the treatment of leishmaniasis, was loaded into human erythroeytes by the preswell dilution procedure. High concentrations of this low-molecularweight drug (MW = 384) could not be used in hypotonic solutions because of osmotic considerations. However, 0.4 ml of erythroeytes loaded with 20 gmol/ml injected into hamsters infected with the parasites 17 days previously suppressed the disease and was much more effectivethan injection of the free drug itself.
[22] H i g h - E f f i c i e n c y E n t r a p m e n t o f E n z y m e s in R e s e a l e d R e d Cell G h o s t s b y Dialysis B y GEOROE L. DALE
The use of resealed erythrocytes as in vivo drug carriers was independently proposed by Ihler et a l l and Zimmermann 2 in 1973. Early work with resealed erythrocytes focused on their utilization in enzyme-replacement therapy for inborn errors of metabolismS; however, the proposed uses G. M. IMer, R. H. Glew, and F. W. Sehnure, Proc. Natl. Acad. Sci. U.S.A. 70, 2663 (1973). 2 U. Zimmermann, "Jahresbericht der Kernforschungsanlage," pp. 55-58. Nuclear Research Center, Julich, G.F.R., 1973.
METHODS IN ENZYMOIX~Y, VOL. 149
Copyright © 1987 by Academic Prem, Inc. All rights of reproductionin any form reserved.
230
CELLULARCARRIERS
[22]
for these cellular carriers have broadened to include flow-release depots for chemotherapeutic agents and processing centers for the enzymatic degradation of plasma metabolites. For reviews of clinical and laboratory applications of resealed erythrocytes, the reader is referred to recent reviews. 3,4 The utility of erythrocytes as drug carders was increased dramatically when the original dilution procedures for erythrocyte lysis and drug entrapment 1 were replaced by high-hematocrit dialysis procedures. 5-7 These dialysis methods substantially improved the incorporation etticiencies for the rare and valuable biochemicals which investigators wanted to entrap in erythrocytes. Several different procedures for dialysis loading of erythrocytes have been reported, but all take advantage of the same principle-that the semipermeable dialysis membrane maximizes the intracellular:extracellular volume ratio for macromolecules during lysis and resealing, yet also allows a free flow of small ions, which are required for lysis and resealing of the erythrocytes. It is this intracellular: extracellular volume ratio during the time that the erythrocyte membrane is permeable that determines the incorporation yield for entrapment: The following procedure is a generalized one for the dialysis loading of proteins into human erythrocytes. At the end of this article, each step or component of the loading procedure is examined to identify those variables that may or may not be modified to compensate for the features of various experimental systems. Blood is drawn into EDTA (1 mg/ml blood) and washed two times with at least l0 volumes of saline (0.15 M sodium chloride). Each centrifugation is at 1100 g for l0 min at 4°, and the buffy coat is removed along with the supernatant. The hematocrit of the washed, packed red cells is accurately determined with a microhematocrit centrifuge (IEC, Needham Heights, Massachusetts) run at full speed for 5 min. Normally, the hematocrit of the washed, packed red cells will be between 85 and 95%. Dialysis tubing (Spectra/Pot 4, 25 m m flat width; molecular weight cutoff 12,000, Spectrum Medical, Los Angeles, California) is hydrated in a large volume of water for 10 min and used without further preparation. In a typical experiment, 7 ml of washed, packed erythrocytes is mixed with phosphate-buffered saline (PBS) (0.15 M NaC1, 5 m M sodium phosphate, pH 7.4) containing the protein to be entrapped; the volume of PBS utilized is that amount which will lower the hematocrit of the red cells to 80%. For 3 G. M. Ihler, in "Drug Carders in Biology and Medicine" (G. Gregoriadis, ed.), p. 129. Academic Press, New York, 1979. 4 G. L. Dale, in "Drug Delivery Systems--Characteristics and Biomedical Applications" (R. L. Juliano, ed.), p. 237. Oxford Univ. Press, New York, 1980. s G. L. Dale, D. G. Villacorte, and E. Beutler, Biochem. Med. 18, 220 (1977). J. DeLoach and G. Ihler, Biochim. Biophys. Aeta 496, 136 (1977). 7 j. R. DeLoach, R. L. Harris, and G. M. Ihler, Anal. Biochem. 102, 220 (1980).
[22]
ENTRAPMENT OF ENZYMES IN R B C GHOSTS
231
example, 7 ml of red blood cell (RBC) solution with a hematocrit of 90% needs to be diluted with 0.875 ml of PBS solution to give a final hematocrit of 80%. Protein concentrations as high as l0 mg/ml have been used for entrapment without difficulty. This erythrocyte/protein mixture is then placed in the dialysis tubing, and the bag sealed with an air bubble included. The sealed dialysis bag should be rather rigid (a "tight sausage") with the air bubble representing at least 20% of the internal bag volume (see Fig. 1). This bubble is critical for the procedure in that, during lysis and resealing, it will traverse the length of the dialysis tubing and serve as a mixing agent. The sealed dialysis bag is then placed in a bottle containing at least 200 ml of lysis buffer (100 mM glycerol, 0.1 mM CaC12, 5 mM potassium phosphate, pH 7.4) at 0 °. The bottle is sealed, placed on a' mechanical rotator (Scientific Industries, Springfield, Massachusetts), and rotated in a vertical plane at 6-10 revolutions per min. If the dialysis bag is small in relation to the lysis bottle, then the dialysis bag will remain stationary as the bottle rotates and there will not be adequate mixing of the bag's contents. This problem can be eliminated by securing the dialysis bag to a glass rod as shown in Fig. l, thereby allowing the entire lysis bottle/dialysis bag apparatus to rotate effectively. 5 The lysis is done in a cold room at 4°; however, the reproducibility of the system is optimized if the rotating bottle is also submerged, at least partially, in a 0 ° ice bath (see Fig. 1 and the section on Lysis Temperature). The lysis continues for 120 min, after which the dialysis bag is changed to a
i c e - w a t e r bath FIG. 1. Schematic representation of apparatus for entrapping proteins into erythrocy~. The dialysis bag is tied tight to produce as rigid a bag as possible and contains an air bubble representing approximately 20% of the volume. If the dialysis bag is small relative to the bottle, it may be anchored to a glass rod as shown to ensure rotation of the bag while the bottle turns. The rotating platform is submerged to its axle in an ice-water bath to ensure that the lysis buffer in the bottle remains at 0 °.
232
CELLULAR CARRIERS
[22]
bottle containing at least 200 ml of room-temperature PBS; the bottle is rotated in a vertical plane at room temperature for 30 min to reseal the erythrocytes. The resealed erythrocytes are then washed two times with either PBS or saline at 1500 g for 15 min; each supernatant is saved for quantitation of the incorporation efficiency. With this procedure, utilizing an 80% hematocrit within the dialysis bag, the incorporation efficiency for nSI-labeled human serum albumin (HSA) is 64.4 _+ 1.8% (n = 9). The theoretical incorporation would be 80%, i.e., equal to the hematocrit. Variations in the entrapment yield will occur with different size proteins since the erythrocytes develop 200- to 500, A pores during lysis,s and some molecular sieving effects are seen. As mentioned earlier, the procedure outlined above is a generic one usable for many different applications. The following special notes refer to individual steps in the procedure and variations that are allowable while still achieving good entrapment yields. Erythrocytes EDTA, heparin, or acid-citrate dextrose may be used as the anticoagulant for blood drawing. Also, all of the data represented here were collected with freshly drawn human blood; the effects of using stored blood have not been studied. Erythrocytes from the following species have been used for drug entrapment: rabbit and ratg; bovine, equine, and porcine7; murine~°; and canine." If desired, erythrocytes may be totally freed of leukocytes by cellulose filtration ~2without affecting this entrapment procedure. The erythrocyte hematocrit present in the dialysis bag is a critical variable and without accurate determination of the hematocrit, as outlined above, results will vary from experiment to experiment.
Dialysis Tubing Dialysis tubing with different molecular weight cutoffs may be utilized when incorporating low-molecular-weight molecules into erythrocytes. For example, Spectra/Por 6, 1000-molecular-weight cutoff dialysis tubing has been used successfully for the incorporation ofbleomycin (MW 1200) into s p. Seeman,J. CellBiol. 32, 55 (1967). 9j. DeLoaeh,S. Peters,O. Pinkard,R. Glew,and G. Ihl©r,Biochim. Biophys. Acta 496, 507 (1977). ioM. B. Fiddler,L. D. S. Hudson,and R. J. Desnick,Biochem. J. 168, 141 (1977). 11A.R. Hubbard, U. Sprandel,and R. A. Chalmers,Biochem. Soc. Trans. 8, 578 (1980). 12E. Beutler,"Red Cell Metabolism-- A Manual of BiochemicalMethods,"2nd Ed. Grune & Stratton, New York, 1975.
[22]
ENTRAPMENT OF ENZYMES IN R B C GHOSTS
233
erythrocytes. ~3 The 1000-molecular-weight cutoff dialysis tubing has lysis and reseating kinetics that are indistinguishable from the standard 12,000molecular-weight cutoff dialysis tubing. The use of higher molecular weight cutoff tubing has not been investigated. The dialysis tubing may be treated with chelating agents to remove possible heavy metal contaminants or autoclaved to obtain sterility without affecting its performance. Lysis Buffers Variations in lysis buffers have not been examined systematically. DeLoach and Ihler 6 utilized water for lysis; Furusawa et al.14 used diluted PBS, and DeLoach et al. 7 used l0 m M phosphate, pH 7.0, with 2 m M glucose and 0.5 m M CaC12. Lysis T i m e Lysis time has been shown to be a function of the hematocrit present in the dialysis bag. 5 For example, 45 min gives total lysis with a 50% hematocrit; whereas 75 min is required with an 80% hematocrit. The difference presumably represents increased viscosity of the higher hematocrit and of the resulting lysed hemoglobin solution. For the generalized procedure outlined above utilizing an 80% hematocrit, a 120-rain lysis time provides a safety margin. No adverse effects of longer lysis times have been observed. Lysis T e m p e r a t u r e Repeated erythrocyte entrapment experiments in this laboratory gave frustrating variations in the total incorporation yield. The critical variable which caused this daily variation was lysis temperature. Initially, a cold room at approximately 4° was utilized for the lysis. However, when a 0 ° ice/H20 bath was included for improved temperature control (Fig. 1), the reproducibility of protein incorporation into resealed erythrocytes was greatly improved. Table I demonstrates this effect of accurately controlling lysis temperature with a 0 ° ice bath. In 12 resealing experiments using a cold room, the incorporation yield for entrapment was lower and the standard deviation larger than in 9 experiments using an ice bath (as shown in Fig. 1) to control temperature. ~3S. Bursteinand G. L. Dale, unpublishedobservations(1982). ~4M. Furnsawa, M. Yamalzumi,T. Nishimura,T. Uchida,and Y. Okada,Methods CellBiol. 14, 73 (1976).
234
CELLULAR CARRIERS
[22]
TABLE I EFFECT OF LYSIS TEMPERATURE ON ERYTHROCYTE ENTRAPMENT EFFICIENCYa
Lysistemperature
Incorporation of ~25I-labeledHSA (%)
Cold room (-4 °) Ice bath (0 °)
55.8 + 6.8 (n = 12) 64.4 + 1.8 (n = 9)
a All experiments were at 80% hematocrit with lysis for 120 min as described in the text. The differencesin incorporation are significantat p = 0.01. Resealing The time required for resealing has been shown to be relatively short, and anything greater than 15 rain is a d e q u a t e : The generalized protocol here is set so that the resealing buffer is a sufficient volume to ensure that the cold dialysis bag will not change the temperature o f the total system, thereby allowing resealing to occur at r o o m temperature ( 2 0 - 2 2 ° ) . Other investigators have performed resealing at 370. 7 The buffer for resealing has been varied from PBS, utilized here, to saline 6 to supplemented buffers. H For example, Hubbard et al. H have included several low-molecular-weight metabolites, such as glucose, adenine, and inosine, in the resealing solution, since these authors are interested in preparing red cells that will have a normal in vivo survival time and, therefore, wish to mimic as closely as possible physiological conditions within the resealed erythrocytes. Acknowledgment This is publication number 3739 from the Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, and was supported by Grant HL25552 and DK35220 from the National Institutes of Health. The author gratefully acknowledgesthe support of Dr. E. Beutler.
ENTRAPMENT OF ENZYMES IN R B C OHOSTS
229
TABLE II EFFECT OF HEMATOCRIT ON ERYTHROCYTE-ENTRAPMENT PROCEDURES
Virus A
2
~X174
Amount added per ml 7.4 X 1.5 X 3.3 × 6.8 × 7.3 X 6.1 X 9.5 × 1.0 X 2.1 X
lOs 109 109 109 109 lOp 109 101° 10H 1.8 X 10 I°
Hematocrit (%) 7.2 25 32 67 80 17 10 80 80 10
Entrapment procedure
Equilibration (%)
Endocytosis
2.8 2.5 1.5 2.9 0.7 1.5 6.6 1.6 1.6 67
Dialysis
Dialysis
Entrapment of Drugs As an example of drug entrapment, glueantime (meglumine antimonate, N-methylglucosamine antimonate), an antimonial drug effectivein the treatment of leishmaniasis, was loaded into human erythroeytes by the preswell dilution procedure. High concentrations of this low-molecularweight drug (MW = 384) could not be used in hypotonic solutions because of osmotic considerations. However, 0.4 ml of erythroeytes loaded with 20 gmol/ml injected into hamsters infected with the parasites 17 days previously suppressed the disease and was much more effectivethan injection of the free drug itself.
[22] H i g h - E f f i c i e n c y E n t r a p m e n t o f E n z y m e s in R e s e a l e d R e d Cell G h o s t s b y Dialysis B y GEOROE L. DALE
The use of resealed erythrocytes as in vivo drug carriers was independently proposed by Ihler et a l l and Zimmermann 2 in 1973. Early work with resealed erythrocytes focused on their utilization in enzyme-replacement therapy for inborn errors of metabolismS; however, the proposed uses G. M. IMer, R. H. Glew, and F. W. Sehnure, Proc. Natl. Acad. Sci. U.S.A. 70, 2663 (1973). 2 U. Zimmermann, "Jahresbericht der Kernforschungsanlage," pp. 55-58. Nuclear Research Center, Julich, G.F.R., 1973.
METHODS IN ENZYMOIX~Y, VOL. 149
Copyright © 1987 by Academic Prem, Inc. All rights of reproductionin any form reserved.
230
CELLULARCARRIERS
[22]
for these cellular carriers have broadened to include flow-release depots for chemotherapeutic agents and processing centers for the enzymatic degradation of plasma metabolites. For reviews of clinical and laboratory applications of resealed erythrocytes, the reader is referred to recent reviews. 3,4 The utility of erythrocytes as drug carders was increased dramatically when the original dilution procedures for erythrocyte lysis and drug entrapment 1 were replaced by high-hematocrit dialysis procedures. 5-7 These dialysis methods substantially improved the incorporation etticiencies for the rare and valuable biochemicals which investigators wanted to entrap in erythrocytes. Several different procedures for dialysis loading of erythrocytes have been reported, but all take advantage of the same principle-that the semipermeable dialysis membrane maximizes the intracellular:extracellular volume ratio for macromolecules during lysis and resealing, yet also allows a free flow of small ions, which are required for lysis and resealing of the erythrocytes. It is this intracellular: extracellular volume ratio during the time that the erythrocyte membrane is permeable that determines the incorporation yield for entrapment: The following procedure is a generalized one for the dialysis loading of proteins into human erythrocytes. At the end of this article, each step or component of the loading procedure is examined to identify those variables that may or may not be modified to compensate for the features of various experimental systems. Blood is drawn into EDTA (1 mg/ml blood) and washed two times with at least l0 volumes of saline (0.15 M sodium chloride). Each centrifugation is at 1100 g for l0 min at 4°, and the buffy coat is removed along with the supernatant. The hematocrit of the washed, packed red cells is accurately determined with a microhematocrit centrifuge (IEC, Needham Heights, Massachusetts) run at full speed for 5 min. Normally, the hematocrit of the washed, packed red cells will be between 85 and 95%. Dialysis tubing (Spectra/Pot 4, 25 m m flat width; molecular weight cutoff 12,000, Spectrum Medical, Los Angeles, California) is hydrated in a large volume of water for 10 min and used without further preparation. In a typical experiment, 7 ml of washed, packed erythrocytes is mixed with phosphate-buffered saline (PBS) (0.15 M NaC1, 5 m M sodium phosphate, pH 7.4) containing the protein to be entrapped; the volume of PBS utilized is that amount which will lower the hematocrit of the red cells to 80%. For 3 G. M. Ihler, in "Drug Carders in Biology and Medicine" (G. Gregoriadis, ed.), p. 129. Academic Press, New York, 1979. 4 G. L. Dale, in "Drug Delivery Systems--Characteristics and Biomedical Applications" (R. L. Juliano, ed.), p. 237. Oxford Univ. Press, New York, 1980. s G. L. Dale, D. G. Villacorte, and E. Beutler, Biochem. Med. 18, 220 (1977). J. DeLoach and G. Ihler, Biochim. Biophys. Aeta 496, 136 (1977). 7 j. R. DeLoach, R. L. Harris, and G. M. Ihler, Anal. Biochem. 102, 220 (1980).
[22]
ENTRAPMENT OF ENZYMES IN R B C GHOSTS
231
example, 7 ml of red blood cell (RBC) solution with a hematocrit of 90% needs to be diluted with 0.875 ml of PBS solution to give a final hematocrit of 80%. Protein concentrations as high as l0 mg/ml have been used for entrapment without difficulty. This erythrocyte/protein mixture is then placed in the dialysis tubing, and the bag sealed with an air bubble included. The sealed dialysis bag should be rather rigid (a "tight sausage") with the air bubble representing at least 20% of the internal bag volume (see Fig. 1). This bubble is critical for the procedure in that, during lysis and resealing, it will traverse the length of the dialysis tubing and serve as a mixing agent. The sealed dialysis bag is then placed in a bottle containing at least 200 ml of lysis buffer (100 mM glycerol, 0.1 mM CaC12, 5 mM potassium phosphate, pH 7.4) at 0 °. The bottle is sealed, placed on a' mechanical rotator (Scientific Industries, Springfield, Massachusetts), and rotated in a vertical plane at 6-10 revolutions per min. If the dialysis bag is small in relation to the lysis bottle, then the dialysis bag will remain stationary as the bottle rotates and there will not be adequate mixing of the bag's contents. This problem can be eliminated by securing the dialysis bag to a glass rod as shown in Fig. l, thereby allowing the entire lysis bottle/dialysis bag apparatus to rotate effectively. 5 The lysis is done in a cold room at 4°; however, the reproducibility of the system is optimized if the rotating bottle is also submerged, at least partially, in a 0 ° ice bath (see Fig. 1 and the section on Lysis Temperature). The lysis continues for 120 min, after which the dialysis bag is changed to a
i c e - w a t e r bath FIG. 1. Schematic representation of apparatus for entrapping proteins into erythrocy~. The dialysis bag is tied tight to produce as rigid a bag as possible and contains an air bubble representing approximately 20% of the volume. If the dialysis bag is small relative to the bottle, it may be anchored to a glass rod as shown to ensure rotation of the bag while the bottle turns. The rotating platform is submerged to its axle in an ice-water bath to ensure that the lysis buffer in the bottle remains at 0 °.
232
CELLULAR CARRIERS
[22]
bottle containing at least 200 ml of room-temperature PBS; the bottle is rotated in a vertical plane at room temperature for 30 min to reseal the erythrocytes. The resealed erythrocytes are then washed two times with either PBS or saline at 1500 g for 15 min; each supernatant is saved for quantitation of the incorporation efficiency. With this procedure, utilizing an 80% hematocrit within the dialysis bag, the incorporation efficiency for nSI-labeled human serum albumin (HSA) is 64.4 _+ 1.8% (n = 9). The theoretical incorporation would be 80%, i.e., equal to the hematocrit. Variations in the entrapment yield will occur with different size proteins since the erythrocytes develop 200- to 500, A pores during lysis,s and some molecular sieving effects are seen. As mentioned earlier, the procedure outlined above is a generic one usable for many different applications. The following special notes refer to individual steps in the procedure and variations that are allowable while still achieving good entrapment yields. Erythrocytes EDTA, heparin, or acid-citrate dextrose may be used as the anticoagulant for blood drawing. Also, all of the data represented here were collected with freshly drawn human blood; the effects of using stored blood have not been studied. Erythrocytes from the following species have been used for drug entrapment: rabbit and ratg; bovine, equine, and porcine7; murine~°; and canine." If desired, erythrocytes may be totally freed of leukocytes by cellulose filtration ~2without affecting this entrapment procedure. The erythrocyte hematocrit present in the dialysis bag is a critical variable and without accurate determination of the hematocrit, as outlined above, results will vary from experiment to experiment.
Dialysis Tubing Dialysis tubing with different molecular weight cutoffs may be utilized when incorporating low-molecular-weight molecules into erythrocytes. For example, Spectra/Por 6, 1000-molecular-weight cutoff dialysis tubing has been used successfully for the incorporation ofbleomycin (MW 1200) into s p. Seeman,J. CellBiol. 32, 55 (1967). 9j. DeLoaeh,S. Peters,O. Pinkard,R. Glew,and G. Ihl©r,Biochim. Biophys. Acta 496, 507 (1977). ioM. B. Fiddler,L. D. S. Hudson,and R. J. Desnick,Biochem. J. 168, 141 (1977). 11A.R. Hubbard, U. Sprandel,and R. A. Chalmers,Biochem. Soc. Trans. 8, 578 (1980). 12E. Beutler,"Red Cell Metabolism-- A Manual of BiochemicalMethods,"2nd Ed. Grune & Stratton, New York, 1975.
[22]
ENTRAPMENT OF ENZYMES IN R B C GHOSTS
233
erythrocytes. ~3 The 1000-molecular-weight cutoff dialysis tubing has lysis and reseating kinetics that are indistinguishable from the standard 12,000molecular-weight cutoff dialysis tubing. The use of higher molecular weight cutoff tubing has not been investigated. The dialysis tubing may be treated with chelating agents to remove possible heavy metal contaminants or autoclaved to obtain sterility without affecting its performance. Lysis Buffers Variations in lysis buffers have not been examined systematically. DeLoach and Ihler 6 utilized water for lysis; Furusawa et al.14 used diluted PBS, and DeLoach et al. 7 used l0 m M phosphate, pH 7.0, with 2 m M glucose and 0.5 m M CaC12. Lysis T i m e Lysis time has been shown to be a function of the hematocrit present in the dialysis bag. 5 For example, 45 min gives total lysis with a 50% hematocrit; whereas 75 min is required with an 80% hematocrit. The difference presumably represents increased viscosity of the higher hematocrit and of the resulting lysed hemoglobin solution. For the generalized procedure outlined above utilizing an 80% hematocrit, a 120-rain lysis time provides a safety margin. No adverse effects of longer lysis times have been observed. Lysis T e m p e r a t u r e Repeated erythrocyte entrapment experiments in this laboratory gave frustrating variations in the total incorporation yield. The critical variable which caused this daily variation was lysis temperature. Initially, a cold room at approximately 4° was utilized for the lysis. However, when a 0 ° ice/H20 bath was included for improved temperature control (Fig. 1), the reproducibility of protein incorporation into resealed erythrocytes was greatly improved. Table I demonstrates this effect of accurately controlling lysis temperature with a 0 ° ice bath. In 12 resealing experiments using a cold room, the incorporation yield for entrapment was lower and the standard deviation larger than in 9 experiments using an ice bath (as shown in Fig. 1) to control temperature. ~3S. Bursteinand G. L. Dale, unpublishedobservations(1982). ~4M. Furnsawa, M. Yamalzumi,T. Nishimura,T. Uchida,and Y. Okada,Methods CellBiol. 14, 73 (1976).
234
CELLULAR CARRIERS
[22]
TABLE I EFFECT OF LYSIS TEMPERATURE ON ERYTHROCYTE ENTRAPMENT EFFICIENCYa
Lysistemperature
Incorporation of ~25I-labeledHSA (%)
Cold room (-4 °) Ice bath (0 °)
55.8 + 6.8 (n = 12) 64.4 + 1.8 (n = 9)
a All experiments were at 80% hematocrit with lysis for 120 min as described in the text. The differencesin incorporation are significantat p = 0.01. Resealing The time required for resealing has been shown to be relatively short, and anything greater than 15 rain is a d e q u a t e : The generalized protocol here is set so that the resealing buffer is a sufficient volume to ensure that the cold dialysis bag will not change the temperature o f the total system, thereby allowing resealing to occur at r o o m temperature ( 2 0 - 2 2 ° ) . Other investigators have performed resealing at 370. 7 The buffer for resealing has been varied from PBS, utilized here, to saline 6 to supplemented buffers. H For example, Hubbard et al. H have included several low-molecular-weight metabolites, such as glucose, adenine, and inosine, in the resealing solution, since these authors are interested in preparing red cells that will have a normal in vivo survival time and, therefore, wish to mimic as closely as possible physiological conditions within the resealed erythrocytes. Acknowledgment This is publication number 3739 from the Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, and was supported by Grant HL25552 and DK35220 from the National Institutes of Health. The author gratefully acknowledgesthe support of Dr. E. Beutler.