Sera radiosterilization: studies and applications

Pergamon

0969~806X(94)EOO58-Q

Radiat. Phvs. Chem. Vol. 45, No. 2, pp. 275-281, 1995 Copyright 0 1994 El&e-r Science Ltd Printed in Great Britain.All rightsreserved 0969-806X195 $7.00 + 0.00

SERA RADIOSTERILIZATION: APPLICATIONS

STUDIES

AND

JORGE H. LOMBARDO, CRISTINA FERNANDEZ DEGIORGI, DANIEL QUATTRINI, SEVERINO MICHELIN

and

EDUARDO E. SMOLKO

Comisi6n National de Energia At6mica (CNEA), Av. Libertador 8250, 1429 Buenos Aires, Argentina (Received I7 May 1993; accepted 25 January 1994) Abstract-The

application of ionizing radiation for sterilization of animal serum increased rapidly over the last few years, due to the fact that most radiation-resistant living organisms (fungi, bacteria, viruses, etc.) can be readily inactivated without damage to the serum. The quality of sera sterilized by ionizing radiation has been investigated. Radiosterilization was carried out in the frozen state at doses between that serum proteins did not 25 and 50 kGy. The source of gamma radiation was %o. To demonstrate suffer alterations during the process of irradiation, SDS-PAGE, electrofocusing and non equilibrium pH gradient electrophoresis of the sera were carried out before and after irradiation. The biological efficiency of the irradiated sera was demonstrated by growth curves of several cell lines in cell cultures supplemented with it. The results demonstrated that radiosterilization is a simple and effective method for sera.

quality and sterility controls (about 200 1 of sera).

INTRODUCTION

The use of animal cells for tissue culture requires a system to reproduce, as much as possible, in vivo conditions. These are defined by physico-chemical factors that affect cellular metabolism such as pH, temperature, osmotic pressure, electrolytes, essential and non-essential metabolites, hormones and specific factors. Serum is one of the main components of cellular culture media and it is also the principal mycoplasmal or viral contamination bacterial, source. Therefore, it is essential for sera to be free of these microorganisms. Application of ionizing radiation for sterilization of medical and biological products is increasing rapidly all over the world (McLaughin and 1973) because most radiation-resistant Halm, living organisms, such as fungi, bacteria and viruses, can be inactivated without excessive damage to the material. Inactivation of fungi, bacteria and viruses by gamma radiation have been described (Sullivan et al., 1973; Lombard0 and Smolko, 1990). Irradiation of biochemical drugs and reagents is more advantageous in the dry or frozen state, as most of the products irradiated in solution lose their biological activity (Nordheim er al., 1985). The purpose of the present paper is to describe an alternative procedure for sera sterilization using ionizing radiation in the frozen state. Usually, sera sterilization is carried out by filtration. Radiation effects on proteins could be related to changes in either physico-chemical properties or specific biological functions of proteins. Accordingly, we studied the modification of the protein properties and performed

in 15 fetal serum lots

MATERIALS AND METHODS

Sera collection

Blood was collected by cardiac puncture avoiding hemolysis. After 1 h at room temperature, blood was kept for 24 h at 4°C for coagulation. Clots were disrupted and serum was filtrated by glass wool and centrifuged at 3000g for 30min at 4°C. After this, serum was dispensed into glass flasks and stored at - 20°C until irradiation. Radiation

source

Sera in glass flasks were frozen at -78°C and irradiated with a gamma radiation @%o source of 18.5 PBq (5 x lo5 Ci) at the Industrial Irradiation Installation of CNEA. Irradiation was carried out at doses of 25, 32 and 50 kGy. Dosimetry was performed with a modified Fricke dosimeter (Swallow, 1960). Cells Baby hamster kidney [BHK 21(C-13)], 3T3 (Swiss mouse embryos), Madin Darby Bovine Kidney (MDBK), P3X63-Ag 8.653 myeloma, fetal bovine testis (FTB) were used throughout the study. 3T3 cells were grown in Dulbecco’s modified EagleMEM, P3X63-Ag 8.653 cells in RPMI-1640 (Gibco) and other cell lines in Glasgow-MEM (Gibco). Chemical

analysis and microbiological

tests

Protein, pH, osmolality and hemoglobin of sera were assayed. Measurements were performed before 275

276

JOHGEH. LOMBAKLX~ er ul

and after irradiation. Hemoglobin was tested to verify correct collection and processing procedures (Drabkin and Austin, 1935). Total protein was determined by the Lowry method (Lowry c’t ul., 1951). The presence of bacteria and fungi were investigated by standard techniques. For bacterial controls, Nutrient Broth (Difco) and Thioglycollate Broth (Difco) were used, in aerobic and anaerobic assay. Sabouraud Dextrose Agar (Difco) was used for fungal controls. For mycoplasmal detection in irradiated serum culture mycoplasma method was used (Freundt 1983). Briefly, 5 ml irradiated serum were added into 50 ml broth medium and incubated for 72 h at 36 C. After this, they were subcultured in agar plates for 5 days and stained (Dienes, 1939). Stained plates were examined by phase contrast microscopy ( x 20). In order to investigate if the doses used to irradiate serum inactivate mycoplasmas, the mycoplasmas were added to serum, irradiated and viability assayed. M,IYY&~~K~ or& was used in this study. The mycoplasmas were inoculated into 50 ml of broth medium and incubated at 36 C for 72 h. After two subcultures, three aliquots of 5 ml were handled as follows: one was irradiated. the second was diluted with fetal bovine serum and then irradiated. and the third was kept as control. After treatment, the three samples wcrc inoculated in 50 ml of broth medium, incubated at 36 C for 72 h. subculturcd in agar plates for 5 days and stained as described above. For viral testing, Bovine Hcrpcsvirus I (BHV-I) and Foot and Mouth Disease virus (FMDV) were propagated in MDBK and BHK cells respectively. Bovine cells cultures wcrc passaged three times in growth medium containing 15% irradiated sera. Cytopathogenic effects were observed through hcmatoxilin and eosin staining tests. Control cultures were grown with control serum (FBS Gibco). In other viral test, FMDV strain “A” and BHV-I strain Los Angeles wcrc added to serum at a final concentration of IO’ UFP:‘ml (viral inoculum) and then irradiated. BHV-I wax titrated according to Talens and Zcc (1976), with modifications. Briefly 0.1 ml of the viral inoculum was added in duplicate onto MDBK monolayer. Following adsorption for 45 min at 37 C, the monolayers were overlaid with 0.2% karaya gum in MEM without serum. Plaques were counted three days post inoculation. The same assay was pcrl’ormcd for FM DV using BHK 21 (C-13) as host cells. For BVD and PI3 viral tests, fetal bovine testis cells (FTB) (virus free) were used. One milliliter of strum was adsorbed for an hour onto FTB cells in 25 cm’ flasks. Then, serum was rcmo\ed and Glasgow-MEM without serum was added. Cultures from three successive blind passages were examined before the sample being considered negative. All passages were assayed for BVD virus hy the direct fluorescent antibody technique (Rossi 1’1 trl.. 1980). For P13, hemoadsorption assay (Cunningham. 1966) in the

3th. cellular passage was performed. Briefly, FBT cells were washed with Phosphate Buffered Saline (PBS) pH 7.4. After this, freshly obtained and washed guinea pig red blood cells were prepared at a concentration of 0.4% in PBS and IO0 11I of this suspension added to a monolayer of the FBT cells. The monolayers were incubated 30 min at room temperature and washed three times with PBS buffer. The presence of red blood cells adhered onto the cell monolayer was observed by optical microscopy ( x 40).

Cells were propagated through 7 subcultures, using media supplemented with 15% irradiated or control serum (FBS Gibco). Cells were examined throughout the subcultures for evidence of morphological changes or cytotoxic effects. On the other hand, 5 x IO” MDBK, BHK 2l(C-13) and 3T3 cultures cells were inoculated in Petri dishes (Falcon Tissue Dish) and kept in culture medium supplemented with 10% irradiated or control fetal bovine serum (FBS Gibco). Total cell number was counted every 24 h: each data was the result of the avcragc from triplicate dishes. Cultures wcrc controlled up to the end of the logarithmic growth phase.

Cells used to determine cloning efficiency assays were P3X63-Ag 8.653. Cells were grown in RPMI1640 medium supplemented with 5 and 10% irradiated or control serum (FBS Gibco). Cells were inoculated at concentration of I and 5 ccll:‘well in a 96-well plates with 200/11 of growth medium and incubated for I5 days at 37 C and 5% CO, in a humidified incubator. Then. the plates were observed microscopically. Percentage of cloning efficiency was formulated as number of positive wells:total numbct of wells inoculated x 100.

Protein synthesis was studied in MDBK cells by “S-methionine incorporation in the presence of irradiatcd or control sera. Cells were propagated through 7 subcultures, using culture medium supplcmcntcd with 5 and 10% irradiated and reference serum. Cells wcrc inoculated into 24-well plates. Monolayers were washed once with mcthionine-free GlasgowMEM and “S-mcthioninc (ICN Biochcmlcala Inc.) (100 ji Ci in 400 1~1 of mcthioninc-free GlasgowMEM) wcrc added to each well. After I? h at 37 C. the medium was rcmovcd and the cells washed 3 times with cold PBS. The cells hcrc removed bvith tripsinc and counted in a Ncubauer chamber. Cells w’crc lysed with 0.4 ml of cold NP40-DOC buffer [Tris-HCI 50 mM pH 7.4. NaCI IO mM, M&l, I.5 mM. sodium deoxicholate 0.5%. Nonidet P40 I”‘,,, phcnyl methyl sulphonyl fluoride (PMSF) 0.5 mM]. After cell lysis. the cell extract was ccntril’ugcd at HOOOg for 20 min

Sera radiosterilization

plaque. Proteins were visualized by staining with Coomassie blue before drying. lsoelectrofocusing (IEF) was carried out as described by O’Farrell (1975) with some modifications. Gels were 4% acrylamide, 0.2% bis acrylamide, 2% NP-40, ampholithes (pH 3310) and 9.5 M urea. The upper and lower electrode solutions were 0.2 M NaOH and 0.1 M H,PO, respectively; gel thickness was 0.75 mm. The gel was prerun without sample for 15 min at 200 V, 30 min at 300 V and 30 min at 400 V. After sample application, the run was carried out for 12 h at 400 V and 1 h at 800 V. Gels were stained with Coomassie blue. Non-equilibrium pH gradient electrophoresis (NEPHGE) was carried out according to O’Farrell et al. (1977), gel conditions were the same described for isoelectrofocusing. The upper and lower electrode solutions were 0.1 M H,PO, (anode) and 0.2 M NaOH (cathode), respectively. The gel was prerun for 35 min at 200 V and 35 min at 300 V. The run was performed at 400 V for 5 h (2000 V. h).

at 4°C and the supernatant frozen at -20’C for the analysis of cellular proteins. Five microliters of supernatant were precipitated with 2 ml of 10% trichloroacetic acid, left 10 min in an ice bath, filtered through Millipore-type membranes and washed with 5% trichloroacetic acid. Filters were dried, transferred to vials containing 5 ml of Bray’s scintillation fluid and counted in a Model 4530 Tricarb Liquid Scintillation System (Packard Instruments Co., Downers Grove, IL). For the analysis of cellular proteins, cell extracts, were adjusted to the sample buffer conditions of the SDS-PAGE Electrophoresis described by Laemmli (1970). SDS-PAGE was performed on 12% polyacrylamide slab gels. For fluorography (Bonner and Laskey, 1974) the gel was impregnated overnight with 22.2% w/v 2,5-diphenyl oxazole (PPO) in Dimethyl Sulfoxide (DMSO), washed with methanol : acetic acid : water 50 : IO : 40, dried and placed in contact with X-ray film (AGFA) at - 70 ‘C. Serum protein analysi.5 Electrophoresis on cellulose acetate was carried out using cellulose acetate strips (Cellogel Chemetron). Samples were run at 1.5 mA for each strip. The electrophoresis buffer was sodium diethyl barbiturate 1.04%, barbituric acid 0.134%. Proteins were visualized by staining the strips with 0.5% amido black in methanol : acetic acid : water 45 : 10: 45. Quantitative determinations of proteins were done by spectrodensitometry. SDS-PAGE was carried out in 7.5% polyacrylamide slab gels of 0.75 mm thickness by using a 18cm vertical electrophoresis apparatus, at 20 mA per

tests

Microbiological tests were performed for sera irradiated at 25, 32 and 50 kGy. Chemical tests were performed only for sera irradiated at 25 kGy. Fifteen serum lots were assayed for pH, osmolality, protein and hemoglobin contents before and after irradiation. The results showed no meaningful differences applying the Fisher statistical test (Table 1). Furthermore, these serum lots did not show bacterial or fungal contamination, after incubation in specific

BHK 21

8 _

0

RESULTS

Chemical and microbiological

P

12

10 -

217

i

2

4

6

0

2

4

6

0

Days

+

Unirradiated

Fig. I. Growth

curves

+ of BHK-2I(C-l3),

MDBK

Irradiated

and 3T3 cells.

2

4

6

rncdla for IO days. Also mycoplasmas \vcrc not dctccted in scra after 5 days incubation. On the other hand. mqcoplasmas alone or diluted with serum and irradiated. did not Ihrm colonies visible though the microscope. In contrast. colonies in plates inoculated with .&I. wrrlr without irradiation were observed by phase contrabt microscopy ( x 10). Thcsc colonies had a dcnsc ccntcr with ;I less dense pcriphcry. When the Dicnes stain was applied many blue densely staining colonies were observed. Cl topathogenic ctfccts such as round-like cells, polyxryocitc formation, cytoplasniic nuclear inclusion bodies. or cytolitic effect were not ohscrved in viral tats. Even though FMDV strain “A” 01. BHV-I \ iral paI-ticlcs wcrc added in 5onic cxpcrimcnts to the scra. they were not dctcctcd after irradiation. This is in food agreement with prebious studies performed with other viruses (Stilli\an PI t/l.. 1971: Lombardo and Smolko. 1990). BVD virus WI> not detectable by imlnLlnofluorcscence in any of the three passages of scr;i assayed. Also PI3 was not dctcctcd by hemoadsorption assays.

After 7 subcultures using media supplemented with cells wcrc microirradiated or control serum. scopically examined. For the 25 and 32 kGy doses. mot-phologic~il characteristics and growth patterns \+crc cquivalcnt to those of the rcfcrcncc. This suggested that radiosteriliration dd not C;ILIW cytotoxical alterations. Results obtained from the SO kGq dose were not that clear. Taking into account these results. it was dccidcd to use scra irradiated Mith 25 kGy for subscqucnt cxpcl-iments. Growth curves for MDBK. BHK 2l(C-13) and 3T3 ccllula~- lines Mere pcrtbrmcd in the presence of

irradiated

diffcrcnccs cloning

or

unirradiatcd

were obscrvcd efficiency

assays

w-a.

No

signilicative

(Fig. I ). The results for arc shown in Table 4. Cells

groMn with irradiated serum grew slighty raster than those grown with rcfcrence serum. but this result is not statistically significant. In order to demonstrate that there was not dcgradation

of

strum

proteins

during

irradiation.

WC

SDSPAGE. elcctrolhcusing and non-equilibrated pH gradicnt elcctrophorcsis. The electrophorctic patterns in ccllulosc acetate u’crc used for spectrophotometric analq~is. Irradiated and unirradiatcd serum were comp~tred. Total proteins and different fraction values xc shown in Table 2. Thcrc were no significant diffcrcnccs bct\\ccn data ofirradiatcd or unirradiatcd set-urn. Results for SDS-PAGE. elcctrofocusing and non-ccluilibr~ltcd pH gradient clcctrophoresis are shown in Figs 7, 3 and 4. Polypeptidc profiles were identical for both scra. Table 3 show “S-mcthionine incorporatlon on MDBK cells. Some reduction in incorporation with 5% strum was observed, but differences were not significativc between irradiated or unirradiated sera. Fipurc 5 shows separation of cellular polypcptides by SDS-PAGE gel electrophorcsis. At least 46 polypcptides could be ~rcsolved. Once more, no differences wcrc obxrvcd in the pattel-ii of cellular proteins grwn in the presence of irradiated or unirradiatcd scrx. carried

out clcctrophorcsis

on ccll~~losc acetate,

I)IS(‘LSSION loniring biomedical ;I Ions

radiation products

air

time. The present

sterilization

could

;I sterilizing

I~I used for

method

has been satisfactorily study

be applied

reports to serum

with good results. The irradiation during this work consist of a ““Co

that

radio-

steriliration facilities used source with a

279

Sera radiosterilization

_ Table 3 [“S]Methionine uptake on MDBK cells grown in Irradiated and unirradiated sera Activity (cpm/cell)

Treatment MDBK-5% MDBK-IO% MDBK-5% MDBK-IO%

c

+

2

I

4

3

Fig. 3. Isoelectrofocusing (IEF) of unirradiated I-2) and irradiated sera (lanes 34).

sera (lanes

energy of 1.33 MeV. In these conditions, in the irradiated no radioactivity is induced sample. Irradiation was performed on frozen sera because in a liquid diluted system. an indirect effect is produced by diffusion of free radicals when the medium absorbs radiation energy. They are mainly derived from radiolysis (i.e. H’, OH’, HO,, and HzO,). Nucleic acid and proteins are affected by these radicals (Scholes et al., 1960) and if compounds with -SH groups are present, they exert a protective action (Michael et ul.. 1983). In a solid material or a frozen liquid, the crystalline lattice will not allow free diffusion of free radicals. Therefore, there will be less interaction among them and the total effect will be minimized. maximal

6.9 7.7 6.7 7.4

irradiated sera irradiated sera unirradiated sera unirradiated sera

k + f f

0.7 0.8 0.7 0.8

Another kind of free radicals are produced in frozen or dry substratum. For example, when frozen viruses are irradiated in the presence of phosphate ions, damage is high due to the long-term radiomimetic products produced by these ions (Ginoza, 1963). There is a linear relationship between damage and the logarithm of the phosphate ion concentrations. At a given dose, radiation effects can change if exogenous conditions such as temperature, presence of O2 or dose rate are modified. Shape and size of the target, physical conditions, pH, etc., can also change these effects. Regarding the sterilization of contaminating microorganisms, radioresistance is a function of the water present in the cytoplasm, the DNA size and structure, and the repairing system (Hall, 1978). The biological and physico-chemical assays carried out during the present work demonstrated that irradiated serum is not significantly altered, if irradiation is performed on frozen sera. Tissue culture testing procedures as cellular maintenance, growth curves and cloning efficiency assays, indicate that radiosterilization did not cause cytotoxical alteration, when a 25 kGy dose was used. Also, 32 kGy did not produce modifications of the sera, but 25 kGy was chosen for the assays, so as not to apply a dose higher than that necessary for sterilization. Bacterial or fungal contamination was not observed in all sera irradiated with 25 kGy. This is particularly important because sterility is the primary condition required for sera that will be used for tissue culture. Mycoplasmas are another critical problem.

+

1

2

FIN. 4. Non equilibrium

3 pH gradient

4

5

6

7

electrophoresis (NEPHGE) irradiated sera (lanes 6-10).

8

9

of unirradiated

10 sera (lanes

l-5) and

‘X0

Viruses arc simpler biological structures; they have the highest radiorcsistancc. which may vary according to the salt concentration. organic materials in the substratum. irradiation temperature and hydration. Because during irradiation viruses are mainly in a complex mcdium. the ctfccrs ofcncrgy absorption will bc strongly influcnccd by the composiGon of the medium (Sullivan cut trl.. 1971). The D,,, value is the dose that \vill rcducc a given population h) ;I lilctor of IO. D,,, for viruses is considcrablq higher in frorcn samples (Sullivan c’t (I/.. 197 I). Sullivan c’t trl. (I 973) have rcportcd a D,,, of 6 kGy for Coxsackiebirus B-2 in foodstutrs or in other similar protective substratum and Lombardo and Smolko. (1990) have reported u D,,, around 5 kGy for FMDV. HSV. and Rauscher leukemia virus inactivatcd by gamma radiation. Adequate radiation doses found in this work were 25 kGy. These doses. were from 4 to 5 times higher than the D,,, previously found for the mentioned viruses. Radiation did not af’fect “S-mcthioninc incorporation into cellular proteins. so WC can infer that

Sera radiosterilization Scholes G., Ward J. F. and Weiss J. (1960) J. Mol. Biol. 2, 379. Simic M. G. (1978) Radiation chemistry of amino acids and peptides in aqueous solutions. J. Agric. Chem. 26, 6. Sullivan R., Fassolitis A. C., Larkin E. P.. Read R. B. Jr and Peeler J. T. (1971) Inactivation of thirty viruses by gamma radiation. Appl. Microbial. 22, 6 I,

281

Sullivan R., Fassolitis A., Larkin E. P., Read R. B. and Peeler J. T. (1973) Gamma radiation inactivation of Coxsakievirus B-2. Appt. Microbial. 26, 14. Swallow A. J. (1960) Radiation Chemistry of OrganicCompound, p. 42. Pergamon Press, Oxford. Talens L. T. and Zee Y. C. (1976) Purification and bouyant density of infectious bovine rhinotracheitis virus (39159). Proc. Sot. Exp. Biol. Med. 151, 132.