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[6] Induction and production of human interferon with human leukemic cells
[6]
PRODUCTION OF HUMAN INTERFERON
[6] I n d u c t i o n
45
and Production of Human Interferon with Human Leukemic Cells
B y ROBERT D . HERSHBERG, E I L E E N G . GUSCIORA, P H I L I P C . F A M I L L E T T I , SARA RUB1NSTEIN, C Y N T H I A A . ROSE, a n d SIDNEY PESTKA
Patients with chronic myelogenous leukemia (CML) and elevated white cell counts afford a large number of leukocytes for production of interferon. A single patient with CML can yield more than 70 liters of primary suspension culture for interferon production. In comparison, many units of normal whole blood must be processed for a single liter of the same primary suspension culture. 1 Leukocytes can be removed selectively from red cells and plasma from patients by leukapheresis? The cell suspension collected by leukapheresis consists mainly of leukocytes and some erythrocytes, which can be removed by selective lysis with ammonium oxalate) Procedures for establishment of primary leukocyte cultures established by Strander and Cantell have been adapted for largescale use with leukemic leukocytes. 4 Primary leukocyte cultures are established in a synthetic medium with casein instead of the serum requirement and are induced with Newcastle disease virus to produce interferon .4--6 Procedures involved in the establishment of the primary culture are performed with aseptic techniques. Equipment is steam-sterilized, heattreated at 230 ° or rinsed with denatured ethyl alcohol (3A) depending on applicability. Whenever possible, procedures are performed in a laminarflow hood. For more detailed discussions of tissue culture procedures, see this series, Vol. 58. Medium for Cell Suspension. The medium used for the primary cell suspension consists of 10% (v/v) casein suspension, 10 mM N-l-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma Chemical Co.), and 100 units of penicillin per milliliter and 100/~g of streptomycin per milliliter (Gibco No. 600-5140) in Eagle's minimum essential medium (Gibco No. 410-110) supplemented with 2.2 g of sodium bicarbonate per liter. A. A. Waldman, R. S. Miller, P. C. Familletti, S. Rubinstein, and S. Pestka, this volume
[5]. 2 K. R. r H. s K. E.
B., McCredie, E. J. Freireich, J. P. Hester, and C. Vallejos, Transfusion 14, 357 (1974). H. Aster, H. E. Cooper, and D. L. Singer, J. Lab. Clin. Med. 63, 161 (1964). Strander and K. Cantell, Ann. Med. Exp. Biol. Fenn. 44, 265 (1966). Cantell and D. R. Tovell, Appl. Microbiol. 22, 625 (1971). F. Wheelock, J. Bacteriol. 92, 1415 (1966).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181978-7
46
INDUCTION AND PRODUCTION OF INTERFERONS
[6]
Casein Suspension. To make the casein suspension, 300 g of nonfat dry milk (Carnation Co.) in a 6-liter Erlenmeyer flask are dissolved in 3 liters of deionized distilled water and autoclaved for 12-15 min. The resultant solution is off white in color. As much as 30 liters of autoclaved milk are combined in a carboy after passage through several layers of cheesecloth to remove insoluble material generated by autoclaving. Penicillin-streptomycin solution is added to produce a final concentration of 300 units of penicillin and 300 gg of streptomycin per milliliter. The milk is centrifuged in an Electronucleonics continuous-flow preparative ultracentrifuge in an RK rotor at 35,000 rpm and a flow rate of 75 ml/min. The supernatant is recentrifuged in a J- 1 rotor at 55,000 rpm with a flow rate of 75 ml/min. The centrifugation process is performed on successive days. The pellet from the first centrifugation (approximately 2.5 kg) is scraped from the rotor, weighed, and homogenized in 6 liters of minimal essential medium containing sufficient penicillin and streptomycin to produce per milliliter approximately 300 units and 300/zg, respectively, of penicillin and streptomycin in the final suspension volume. The homogenate is stirred overnight at 4 °. The pellet from the second centrifugation (approximately 250 g) is homogenized and combined with the first homogenate. The combined homogenate is brought to a volume in liters 10 times the combined weight in kilograms of the pellets. Homogenizations are performed with an Ultra Turrax machine (Tekmar Co., Cleveland, Ohio) with a No. G-450 generator. The medium is apportioned into l-liter bottles and stored at 4 ° until use. This casein suspension is stable for approximately 3 months. Establishment of Primary L e u k o c y t e Suspension Culture
Purification of Leukocytes. Cells are obtained in blood collection bags after leukapheresis of chronic myelogenous leukemia patients. Leukocytes are decanted from the bags and centrifuged at 1500 rpm for 14 min in a Sorvall H6000 rotor. The supernatant is removed by aspiration and discarded. The pellet is resuspended in an equal volume of I% (w/v) ammonium oxalate by gently swirling. The leukocyte suspension is then decanted from any red cell pellet that may adhere to the bottom of the bottle. The suspension is further diluted twofold with 1% ammonium oxalate, incubated at 37 ° for 8 min to lyse contaminating red cells, and centrifuged at 1500 rpm for 14 min in a Sorvall H6000 rotor. The red supernatant in addition to the very light-colored and buoyant layer above the pellet is removed by aspiration. The cell pellet is resuspended in minimum essential medium. If cell lysis occurs during the above processes, the cell suspension is filtered through one layer of cheesecloth. A 0.10-ml aliquot is removed and serially diluted 10-fold three times in 0.4% (w/v) trypan
[6]
PRODUCTION
OF HUMAN
INTERFERON
47
blue. The 1000-fold diluted cell suspension is counted in a Neuberger hemacytometer. The cell suspension is then diluted into suspension culture medium to produce l0 T cells/ml. Stirred suspension cultures are contained in carboys with Teflon blade stirrers turned at approximately 30 rpm by overhead motors. Sterility is maintained within the culture vessel by a sterile delrin bell covering the shaft opening for the stirrer. Induction oflnterferon. The cell suspension is incubated with stirring for 30-40 min at 37 °. Sufficient stock Newcastle disease virus is added to produce 15 hemagglutination units/mlY The culture is stirred for 5 min to mix the virus followed by a period of I hr ofunstirred incubation. The cell suspension is then stirred at 37 ° overnight. Isolation and Concentration of lnterferon. After incubation overnight, a sample of the culture medium is centrifuged at 100,000 g for 45 min and assayed for interferon activity. The cell suspension is siphoned into l-liter centrifuge bottles and centrifuged at 4500 rpm in a Sorvall H6000 rotor for 10 min to sediment the cells. The clarified culture broth is carefully decanted and cooled in an ice bath. Approximately 25 ml of 1 N HC1 per liter is added dropwise with stirring to the clarified culture broth to reduce the pH to 4.0. The acidification of the culture broth causes the precipitation of casein, which settles by gravity over a period of 2 hr at 4 °. Gravity sedimentation of casein results in a heavy precipitate (in approximately 10% of the total volume) at the bottom of the vessel. Clarification of the supernatant is achieved by pumping the supernatant through a 3-/xm polypropylene filter (Pall-Trincor Co., Cortland, New York, No. SLK 7002 BP). The heavy precipitate is centrifuged in a Sorvall H6000 rotor for 20 min at 4500 rpm for a total recovery of the acidified culture broth. The filtrate and supernatant are combined. Interferon is precipitated by dropwise addition with stirring of 31 ml of 50% (w/v) trichloroacetic acid per liter of acidified culture broth. The mixture is allowed to stand overnight at 4 ° while the precipitate containing interferon settles to the bottom of the vessel. A quantitative recovery by gravity sedimentation is promoted by wiping the walls of the vessel with a plastic rod to cause precipitate adhering to the wall to settle. The supernatant is removed with a siphon, and the precipitate is recovered in approximately one-tenth of the culture volume. The suspension is poured into a smaller vessel and allowed to resettle. Successive gravity sedimentations result in a greater than 90% recovery of the precipitate that contains interferon and a reduction in volume by 20-fold or more. The supernatant is decanted and discarded. The remaining suspension is centrifuged in a Sor7 p. C. Familletti, this volume [43].
48
I N D U C T I O N AND P R O D U C T I O N O F I N T E R F E R O N S
[7]
vail H6000 rotor at 4500 rpm for 20 min. The pellet is dissolved in a minimum volume of 0.1 N NaHCOz (approximately 1/100 of the original culture volume). The solution is centrifuged to remove insoluble material. The pellet obtained is extracted with 10-20 ml of 0.1 N NaHCOn for recovery of residual interferon. The extract is centrifuged at 8000 rpm in a Sorvall SS-34 rotor. The supernatant is added to the first supernatant. The 0.1 N NaHCOa solution can be frozen and stored for several months until use.
Concluding Comments Titers of interferon from leukemic leukocytes can be as high as 200,000 units/ml. However, this titer is quite variable. For 101 preparations, the average titer has been 12,500 units/ml. A quantitative recovery of interferon activity is obtained for the procedures outlined. Acknowledgments We thank Drs. Jeane Hester and Jordan Guttermanfor supplyingus withleukemiccells obtained by leukapheresis; and PhyllisBegin and April Durett for shippingthe cells to us.
[7] I n d u c t i o n
and Production of Interferon with Porcine, Bovine, and Equine Leukocytes
By WILLIAM A. CARTER and
FREDERICK H.
JOHNSON,
JR.
The discovery that interferon derived from a lower animal source, porcine leukocytes, has high biological activity in human cell cultures 1 raises new interest in animal interferon for both its scientific and potential clinical value. All other current procedures for production of interferon for use in humans, as described in this volume, involve human cells because of an apparent species specificity. Based on the present annual slaughter of pigs in the United States alone, this one source would greatly exceed the available supplies of interferon from all human cell sources, both current ones as well as those immediately projected. ~ W. A. Carter, L. R. Davis, Jr., K. C. Chadha, and F. H. Johnson, Jr., Mol. Pharmacol. 15, 685 (1979). 2 W. A. Carter, Pharmacol. Ther. 7, 245 (1979).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1819/8-7
PRODUCTION OF HUMAN INTERFERON
[6] I n d u c t i o n
45
and Production of Human Interferon with Human Leukemic Cells
B y ROBERT D . HERSHBERG, E I L E E N G . GUSCIORA, P H I L I P C . F A M I L L E T T I , SARA RUB1NSTEIN, C Y N T H I A A . ROSE, a n d SIDNEY PESTKA
Patients with chronic myelogenous leukemia (CML) and elevated white cell counts afford a large number of leukocytes for production of interferon. A single patient with CML can yield more than 70 liters of primary suspension culture for interferon production. In comparison, many units of normal whole blood must be processed for a single liter of the same primary suspension culture. 1 Leukocytes can be removed selectively from red cells and plasma from patients by leukapheresis? The cell suspension collected by leukapheresis consists mainly of leukocytes and some erythrocytes, which can be removed by selective lysis with ammonium oxalate) Procedures for establishment of primary leukocyte cultures established by Strander and Cantell have been adapted for largescale use with leukemic leukocytes. 4 Primary leukocyte cultures are established in a synthetic medium with casein instead of the serum requirement and are induced with Newcastle disease virus to produce interferon .4--6 Procedures involved in the establishment of the primary culture are performed with aseptic techniques. Equipment is steam-sterilized, heattreated at 230 ° or rinsed with denatured ethyl alcohol (3A) depending on applicability. Whenever possible, procedures are performed in a laminarflow hood. For more detailed discussions of tissue culture procedures, see this series, Vol. 58. Medium for Cell Suspension. The medium used for the primary cell suspension consists of 10% (v/v) casein suspension, 10 mM N-l-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma Chemical Co.), and 100 units of penicillin per milliliter and 100/~g of streptomycin per milliliter (Gibco No. 600-5140) in Eagle's minimum essential medium (Gibco No. 410-110) supplemented with 2.2 g of sodium bicarbonate per liter. A. A. Waldman, R. S. Miller, P. C. Familletti, S. Rubinstein, and S. Pestka, this volume
[5]. 2 K. R. r H. s K. E.
B., McCredie, E. J. Freireich, J. P. Hester, and C. Vallejos, Transfusion 14, 357 (1974). H. Aster, H. E. Cooper, and D. L. Singer, J. Lab. Clin. Med. 63, 161 (1964). Strander and K. Cantell, Ann. Med. Exp. Biol. Fenn. 44, 265 (1966). Cantell and D. R. Tovell, Appl. Microbiol. 22, 625 (1971). F. Wheelock, J. Bacteriol. 92, 1415 (1966).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181978-7
46
INDUCTION AND PRODUCTION OF INTERFERONS
[6]
Casein Suspension. To make the casein suspension, 300 g of nonfat dry milk (Carnation Co.) in a 6-liter Erlenmeyer flask are dissolved in 3 liters of deionized distilled water and autoclaved for 12-15 min. The resultant solution is off white in color. As much as 30 liters of autoclaved milk are combined in a carboy after passage through several layers of cheesecloth to remove insoluble material generated by autoclaving. Penicillin-streptomycin solution is added to produce a final concentration of 300 units of penicillin and 300 gg of streptomycin per milliliter. The milk is centrifuged in an Electronucleonics continuous-flow preparative ultracentrifuge in an RK rotor at 35,000 rpm and a flow rate of 75 ml/min. The supernatant is recentrifuged in a J- 1 rotor at 55,000 rpm with a flow rate of 75 ml/min. The centrifugation process is performed on successive days. The pellet from the first centrifugation (approximately 2.5 kg) is scraped from the rotor, weighed, and homogenized in 6 liters of minimal essential medium containing sufficient penicillin and streptomycin to produce per milliliter approximately 300 units and 300/zg, respectively, of penicillin and streptomycin in the final suspension volume. The homogenate is stirred overnight at 4 °. The pellet from the second centrifugation (approximately 250 g) is homogenized and combined with the first homogenate. The combined homogenate is brought to a volume in liters 10 times the combined weight in kilograms of the pellets. Homogenizations are performed with an Ultra Turrax machine (Tekmar Co., Cleveland, Ohio) with a No. G-450 generator. The medium is apportioned into l-liter bottles and stored at 4 ° until use. This casein suspension is stable for approximately 3 months. Establishment of Primary L e u k o c y t e Suspension Culture
Purification of Leukocytes. Cells are obtained in blood collection bags after leukapheresis of chronic myelogenous leukemia patients. Leukocytes are decanted from the bags and centrifuged at 1500 rpm for 14 min in a Sorvall H6000 rotor. The supernatant is removed by aspiration and discarded. The pellet is resuspended in an equal volume of I% (w/v) ammonium oxalate by gently swirling. The leukocyte suspension is then decanted from any red cell pellet that may adhere to the bottom of the bottle. The suspension is further diluted twofold with 1% ammonium oxalate, incubated at 37 ° for 8 min to lyse contaminating red cells, and centrifuged at 1500 rpm for 14 min in a Sorvall H6000 rotor. The red supernatant in addition to the very light-colored and buoyant layer above the pellet is removed by aspiration. The cell pellet is resuspended in minimum essential medium. If cell lysis occurs during the above processes, the cell suspension is filtered through one layer of cheesecloth. A 0.10-ml aliquot is removed and serially diluted 10-fold three times in 0.4% (w/v) trypan
[6]
PRODUCTION
OF HUMAN
INTERFERON
47
blue. The 1000-fold diluted cell suspension is counted in a Neuberger hemacytometer. The cell suspension is then diluted into suspension culture medium to produce l0 T cells/ml. Stirred suspension cultures are contained in carboys with Teflon blade stirrers turned at approximately 30 rpm by overhead motors. Sterility is maintained within the culture vessel by a sterile delrin bell covering the shaft opening for the stirrer. Induction oflnterferon. The cell suspension is incubated with stirring for 30-40 min at 37 °. Sufficient stock Newcastle disease virus is added to produce 15 hemagglutination units/mlY The culture is stirred for 5 min to mix the virus followed by a period of I hr ofunstirred incubation. The cell suspension is then stirred at 37 ° overnight. Isolation and Concentration of lnterferon. After incubation overnight, a sample of the culture medium is centrifuged at 100,000 g for 45 min and assayed for interferon activity. The cell suspension is siphoned into l-liter centrifuge bottles and centrifuged at 4500 rpm in a Sorvall H6000 rotor for 10 min to sediment the cells. The clarified culture broth is carefully decanted and cooled in an ice bath. Approximately 25 ml of 1 N HC1 per liter is added dropwise with stirring to the clarified culture broth to reduce the pH to 4.0. The acidification of the culture broth causes the precipitation of casein, which settles by gravity over a period of 2 hr at 4 °. Gravity sedimentation of casein results in a heavy precipitate (in approximately 10% of the total volume) at the bottom of the vessel. Clarification of the supernatant is achieved by pumping the supernatant through a 3-/xm polypropylene filter (Pall-Trincor Co., Cortland, New York, No. SLK 7002 BP). The heavy precipitate is centrifuged in a Sorvall H6000 rotor for 20 min at 4500 rpm for a total recovery of the acidified culture broth. The filtrate and supernatant are combined. Interferon is precipitated by dropwise addition with stirring of 31 ml of 50% (w/v) trichloroacetic acid per liter of acidified culture broth. The mixture is allowed to stand overnight at 4 ° while the precipitate containing interferon settles to the bottom of the vessel. A quantitative recovery by gravity sedimentation is promoted by wiping the walls of the vessel with a plastic rod to cause precipitate adhering to the wall to settle. The supernatant is removed with a siphon, and the precipitate is recovered in approximately one-tenth of the culture volume. The suspension is poured into a smaller vessel and allowed to resettle. Successive gravity sedimentations result in a greater than 90% recovery of the precipitate that contains interferon and a reduction in volume by 20-fold or more. The supernatant is decanted and discarded. The remaining suspension is centrifuged in a Sor7 p. C. Familletti, this volume [43].
48
I N D U C T I O N AND P R O D U C T I O N O F I N T E R F E R O N S
[7]
vail H6000 rotor at 4500 rpm for 20 min. The pellet is dissolved in a minimum volume of 0.1 N NaHCOz (approximately 1/100 of the original culture volume). The solution is centrifuged to remove insoluble material. The pellet obtained is extracted with 10-20 ml of 0.1 N NaHCOn for recovery of residual interferon. The extract is centrifuged at 8000 rpm in a Sorvall SS-34 rotor. The supernatant is added to the first supernatant. The 0.1 N NaHCOa solution can be frozen and stored for several months until use.
Concluding Comments Titers of interferon from leukemic leukocytes can be as high as 200,000 units/ml. However, this titer is quite variable. For 101 preparations, the average titer has been 12,500 units/ml. A quantitative recovery of interferon activity is obtained for the procedures outlined. Acknowledgments We thank Drs. Jeane Hester and Jordan Guttermanfor supplyingus withleukemiccells obtained by leukapheresis; and PhyllisBegin and April Durett for shippingthe cells to us.
[7] I n d u c t i o n
and Production of Interferon with Porcine, Bovine, and Equine Leukocytes
By WILLIAM A. CARTER and
FREDERICK H.
JOHNSON,
JR.
The discovery that interferon derived from a lower animal source, porcine leukocytes, has high biological activity in human cell cultures 1 raises new interest in animal interferon for both its scientific and potential clinical value. All other current procedures for production of interferon for use in humans, as described in this volume, involve human cells because of an apparent species specificity. Based on the present annual slaughter of pigs in the United States alone, this one source would greatly exceed the available supplies of interferon from all human cell sources, both current ones as well as those immediately projected. ~ W. A. Carter, L. R. Davis, Jr., K. C. Chadha, and F. H. Johnson, Jr., Mol. Pharmacol. 15, 685 (1979). 2 W. A. Carter, Pharmacol. Ther. 7, 245 (1979).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1819/8-7