since the hybridoma technology was established for 
 preparation and production of monoclonal antibody ( mab ) in 1975 
 , a large variety and quantity of antibodies have been 
 produced and widely used in biological researches , medical 
 diagnoses , and therapies .
the production of mab from ascites by 
 culturing hybridomas in the peritoneum of a living mouse makes a 
 high yield of antibody ; however , it is not only restricted by 
 animal protection laws , but also involved in some problems of the 
 antibody purity and practicability in human therapies .
many 
 efforts have been made to improve the production of mab at high 
 titer in consideration of the economical effectivity . aside from 
 conventional t - flask culture ,
a variety of methods of hybridoma 
 cell culture in vitro have been developed , such as roller bottles 
 ( or spinner flasks , one - mouthed ) , hollow fibre bioreactors , 
 dialysis tubing , and macroporous microcarrier beads [ 2 , 
 3 , 4 , 
 5 , 6 ] . 

the process to raise mab with both high yield and 
 quality is still highly interesting , and processing of the 
  resultant hybridoma cell culture in vitro needs further 
 investigating and improving .
rotated cell culture system ( rccs ) by simulating 
 microgravity was developed on the basis of clinostat equipment 
 ( us patent ) .
it was first used by 
 plant physiologists more than 100 years ago to study 
 gravitational biological effects on plants .
recently , it 
 has been used to study mammalian cell growth , differentiation , and 
 morphogenesis in response to alterations in gravitational 
 conditions [ 10 , 11 , 
 12 , 13 ] . 

it was strongly recommended 
 that clinostat experiments should first be investigated before a 
 biology object was proposed for a space - flight experiment 
 . in this cultural system , when the radius and the 
 revolution speed of a rotating bottle are on a certain scale , the 
 centrifuge force is therefore closely equal to the gravity on 
 cells . under this condition , it allows the cells to grow in three 
 dimensions , which could be , in some sense , simulated to the 
 parent tissue condition in vivo , such as some cultures for liver 
 cells and cartilage cells . applying rccs ,
various cell lines were 
 successfully cultured , including breast and prostate cancer cells 
 that were difficult to culture under the conventional culture 
 conditions .
a new type of dmrb ( double - mouthed rolling bottle ) , 
 derived from conventional rotated bottle , has been designed 
 ( chinese patent ) .
it is operated conveniently with a 
 better gas - exchanging efficiency and a lower cost .
furthermore , a 
 large scale of mab can be produced by laying more dmrbs on the rollers of the 
 rolling cell culture system ( chinese patent ) . in this 
 paper , hybridoma cell growth and mab productivity cultured in 
 dmrbs and t - flasks  

 reagents and cell culture apparatus . 
 dmem cell culture medium with a low glucose concentration ,
fetal 
 calf serum ( fcs ) , and trypsin were from sigma chem co ( st louis , 
 mo , usa ) .
microcarriers beads ( cytodex 3 ) were from pharmacia 
 biotech ( uppsala , sweden ) .
the volume of the bottle is 50100  ml and the 
 diameter is 45  cm . during cell cultivation
, the two mouths 
 of the bottle were covered with a piece of foil or a lid and the 
 bottle was placed on the rolled - spindles of the cell culture system , by the curved 
 surface .
the rolling cell culture system generally 
 includes rolling machine with rolled - spindles and a 
 special electric device to control revolution speed .
the dmrb was 
 allowed to rotate in the direction either clockwise or 
 anticlockwise .
the bottles and rolling machine were installed in 
 the humidified incubator containing 5% co2 at 37c . 

the 
 cured surface of the bottle touches on the rolling spindles 
 horizontally and rotates at a suitable revolution speed .
the hybridoma cell line ( 3e8 ) was established resulting from 
 fusion of murine p3-x63 ag 8.653 myeloma cells with mouse balb / c 
 b - lymphocytes immunized against human chorionic gonadotropin 
 ( hcg ) as described . 

the dmem medium was supplemented 
 with 50  m gentamycin , at ph 7.4 ( adjusted with 7.4% 
 nahco3 when necessary ) .
viable cells were seeded at a density 
 of 1.52  10/ml for both t - flasks ( 50  ml , 
 25  cm containing 10  ml of medium ) and dmrbs ( also 
 containing 10  ml of medium ) .
cells were allowed to grow in 
 dmem medium with 1% , 5% , and 10% fcs , respectively . for 
 rotating dmrb culture ,
three bottles , at least , were not rotated as static control . to enhance productivity by increasing the surface area to a given 
 medium volume
, the hybridoma cells were cultured in the presence 
 of microcarriers . in the experiment
silicone was dissolved in 5% chloroform , then the 
 silicochloroform solution was poured into glassware and poured 
 out when the wall was immersed equably . the glassware was dried 
 and autoclaved prior to use .
dry cytodex microcarriers 
 ( 800  cm/50  ml ) were hydrated with calcium- and 
 magnesium - free phosphate - buffered saline ( ca- and 
 mg - free pbs ) in a siliconized spinner flask on a shaker 
 overnight .
the microcarriers were washed twice with ca- 
 and mg - free pbs and then autoclaved for 15  minutes at 
 115c , 15  psi .
residual ca- and mg - free 
 pbs was removed and the microcarriers were washed and resuspended 
 in warm media before use . usually , 100  ml medium contained 
 0.3  g of microcarriers . 

cell concentrations were determined by counting a cell suspension 
 diluted 1:1 ( v / v ) with 0.2% trypan blue by a hemacytometer . 

visual analysis of the cultures revealed an admixture of live 
 ( trypan - blue negative ) and dead ( trypan - blue positive ) cells 
 under each experimental condition . 

antibody levels in the supernatants of hybridoma cultures were 
 determined by an enzyme - linked immunoassay ( elisa ) . 

maxi - sorp f96 plates were coated with hcg ( 20  g / ml in 
 pbs , 100  l / well ) and incubated at 4c 
 overnight , and the wells were blocked with pbs containing 1% bsa 
 for 2  hours at 37c ( 200  l / well ) .
then the 
 wells were washed three times with pbs containing 0.5% triton 
 x-100 ( 200  l / well ) .
the samples , negative control 
 ( medium ) and positive control ( ascites of the mice with 
 hybridomas ) , were added ( 50  l / well ) and incubated for 
 1  hour at 37c .
the wells were then washed for three 
 times with pbs containing 0.5% triton x-100 and 1% bsa 
 ( 100  l / well ) , and incubated for 1  hour with 
 alkaline - phosphatase - conjugated goat anti - mouse igg at 

they were washed three times with pbs containing 
 0.5% triton x-100 and twice with double   distilled water 
 ( 200  l / well ) .
then 3,3,5,5-tetramethylbenzidine ( tmb , in 
 pb buffer ,   ph 6.0 ) chromogen - substrate was added 
 ( 100  l / well ) and incubated in dark .
the chromogenic 
 reaction was stopped after 1530 minutes with 0.5  m 
 h2so4 and the absorbance of each well was detected at 
 492  nm in a spectrophotometer ( bio - rad model 3550 microplate 
 reader jp41 ) .
microcarrier beads ( 0.3  g/100  ml medium ) were added in 
 the media containing fcs ( 1% , 5% , and 10% ) in both dmrb and 
 t - flask ( figure 2b ) . in static culture ,
cells can 
 perch on the microcarriers and occupy the most surface of the 
 beads ( 90%95% ) , growing in a three - dimensional manner in dmrb 
 ( figure 2a ) . for t - flask , however , 40%50% of 
 hybridoma cells perch and grow on the beads under the same 
 conditions .
the higher cell - perching ratio onto the microcarriers 
 in dmrb may be resulted from a better ventilation of the 
  two opposite necks , which are helpful to gas exchanging .
the dmem medium was supplemented 
 with 50  m gentamycin , at ph 7.4 ( adjusted with 7.4% 
 nahco3 when necessary ) .
viable cells were seeded at a density 
 of 1.52  10/ml for both t - flasks ( 50  ml , 
 25  cm containing 10  ml of medium ) and dmrbs 
 ( containing 10  ml of medium ) .
microcarrier beads 
 ( 0.3  g/100  ml medium ) were added to the medium and cells 
 were allowed to grow in dmem medium ( 5% co2 , at 37c ) 
 with 1% , 5% , and 10% fcs , respectively .
cells 
 growing in t - flask were as control ( bar = 0.1  mm ) . for 
 rotating dmrb culture ( supplemented with 10% fcs ) , cells were 
 under a revolution speed of 2 turns / min for 72 hours .
for each 
 experiment , three bottles , at least , were as a group . under a rotating condition ( 210  rpm ) ,
the 
 hybridoma cells on the beads are subjected to an increase in 
 shearing force .
the faster the revolution , the higher the shearing force 
 and the more cells shorn off the beads .
only 1%3% cells can 
 perch on the beads when revolution speed gets to 10  rpm .
this 
  is to say , the number of cells perching on the beads is directly related to the 
 revolution of dmrb . therefore , it is important to use a suitable 
 revolution speed to have a low shearing force on cells . on the 
 basis of our experiments ,
a suitable revolution speed should be 
 about 2  rpm , under which cell perching ratio can keep at 
 40%50% ( figure 2c ) .
figure 3 shows total ( left ) and viable ( right ) 
 numbers of hybridoma cells cultured in both dmrbs and t - flasks .
total ( left ) and viable ( right ) numbers of 
 hybridoma cells cultured in both dmrbs and t - flasks in medium 
 containing 10% ( a ) , 5% ( b ) , and 1% 
 ( c ) fcs , respectively .
the black curve represents cell 
 number in dmrbs and the red curve represents cell number in 
 t - flasks .
the maximum densities of cells cultured in rotating dmrbs are 
 observably higher than those in t - flasks , in all the media 
 containing 1% , 5% , and 10% fcs .
the total number of cells 
 reaches approximately 1.5  10/ml around 72  hours 
 while cells are cultured in the medium containing 10   fcs in 
 rotating dmrbs .
however , it is approximately 1.0  10/ml 
 while cells are cultured in t - flasks .
experiments also 
 show that cells densities in rotating dmrbs are higher than those in static dmrbs ( data 
 not shown ) , suggesting that the clinonational rotation plays an 
 important role in a rotating dmrb culture .
aliquots were daily taken from the culturing media to measure the 
 mab concentration during the culture .
changes in mab 
 concentrations in the medium supplemented with 5% fcs are shown 
 in figure 4 . as indicated ,
both accumulated ( a ) and daily ( b ) mab 
 production of cells cultured in rotating dmrbs are 
  distinguishably higher than those of cells cultured in 
 t - flasks .
interestingly , for dmrbs , the highest production rate 
 is achieved on the first day .
the first three - day mab production 
 accounts for over 95% of the total yield during the five - day 
 culture .
it suggests that the antibody should be harvested in 
 72  hours , to have a high yield of antibody , saving time and 
 materials .
statistically , the final production of a rotating dmrb 
 is approximately 42.5% higher than that of a t - flask .
this 
 result indicates that rotation plays a significant role , not only 
 in cell growth , but also in mab production .
the mab concentrations are measured in the medium 
 supplemented with 5% fcs in both dmrbs and t - flasks .
cell growth and mab production in dmrb groups and t - flask groups 
 within 72  hours are shown in table 1 . 

increase in production rate of mab is highly correlated with the 
 viable cell number .
it indicates that the increasing mab 
 production is largely resulted from an increase of the viable 
 cell number . in other words ,
a further possibility for 
 enhancing mab productivity is considered with the increase in the 
 surface area by the microcarriers on which cells grow and 
 perch .
consequently , the cell density per culture volume may be 
 improved by means of the microcarriers [ 20 , 
 21 ] .
for our 
 experiments , cytodex 3 is used as the microcarriers , which are 
 macroporous matrix based on polyethylene and weighted by silica . 

these microcarriers , alkali and acid resistant , are steamily 
 sterilizable at 121c ( 1  bar ) and are not swollen up 
 on hydration .
cytoline-3 microcarriers provide both an external 
 surface and an interior space with pore size between 10 and 
 400  m , which can be populated also by cells grown in 
 suspension and used in stirred cultures .
however , cells 
 inhabiting inside the pores could not be observed during the 
 culture .
comparison 
 of dmrb groups with t - flask group ;  
 p<.1 , 
  
 p<.05 .
cell growth density , mab production , and reasonable cost are 
 important criteria to evaluate alternative methods to 
 conventional t - flask .
the method using dmrb to culture hybridoma 
 cells has some significant advantages : ( 1 ) to produce higher mab 
 at a low cost , ( 2 ) easily to handle in lab , ( 3 ) conveniently to process and investigate the 
 optimal conditions for different mab , and ( 4 ) readily to 
 produce a larger - scale mab when necessary .
furthermore , the 
 method makes it possible to produce mab as a putative process on 
 an industrial scale , and to have obvious advantages , such as a 
 high yield and a low cost .
microcarrier beads ( 0.3  g/100  ml medium ) were added in 
 the media containing fcs ( 1% , 5% , and 10% ) in both dmrb and 
 t - flask ( figure 2b ) . in static culture ,
cells can 
 perch on the microcarriers and occupy the most surface of the 
 beads ( 90%95% ) , growing in a three - dimensional manner in dmrb 
 ( figure 2a ) . for t - flask , however , 40%50% of 
 hybridoma cells perch and grow on the beads under the same 
 conditions .
the higher cell - perching ratio onto the microcarriers 
 in dmrb may be resulted from a better ventilation of the 
  two opposite necks , which are helpful to gas exchanging .
the dmem medium was supplemented 
 with 50  m gentamycin , at ph 7.4 ( adjusted with 7.4% 
 nahco3 when necessary ) .
viable cells were seeded at a density 
 of 1.52  10/ml for both t - flasks ( 50  ml , 
 25  cm containing 10  ml of medium ) and dmrbs 
 ( containing 10  ml of medium ) .
microcarrier beads 
 ( 0.3  g/100  ml medium ) were added to the medium and cells 
 were allowed to grow in dmem medium ( 5% co2 , at 37c ) 
 with 1% , 5% , and 10% fcs , respectively .
cells 
 growing in t - flask were as control ( bar = 0.1  mm ) . for 
 rotating dmrb culture ( supplemented with 10% fcs ) , cells were 
 under a revolution speed of 2 turns / min for 72 hours .
for each 
 experiment , three bottles , at least , were as a group . under a rotating condition ( 210  rpm ) ,
the 
 hybridoma cells on the beads are subjected to an increase in 
 shearing force .
the faster the revolution , the higher the shearing force 
 and the more cells shorn off the beads .
only 1%3% cells can 
 perch on the beads when revolution speed gets to 10  rpm .
this 
  is to say , the number of cells perching on the beads is directly related to the 
 revolution of dmrb . therefore , it is important to use a suitable 
 revolution speed to have a low shearing force on cells . on the 
 basis of our experiments ,
a suitable revolution speed should be 
 about 2  rpm , under which cell perching ratio can keep at 
 40%50% ( figure 2c ) .
figure 3 shows total ( left ) and viable ( right ) 
 numbers of hybridoma cells cultured in both dmrbs and t - flasks .
total ( left ) and viable ( right ) numbers of 
 hybridoma cells cultured in both dmrbs and t - flasks in medium 
 containing 10% ( a ) , 5% ( b ) , and 1% 
 ( c ) fcs , respectively .
the black curve represents cell 
 number in dmrbs and the red curve represents cell number in 
 t - flasks .
. the maximum densities of cells cultured in rotating dmrbs are 
 observably higher than those in t - flasks , in all the media 
 containing 1% , 5% , and 10% fcs .
the total number of cells 
 reaches approximately 1.5  10/ml around 72  hours 
 while cells are cultured in the medium containing 10   fcs in 
 rotating dmrbs .
however , it is approximately 1.0  10/ml 
 while cells are cultured in t - flasks .
experiments also 
 show that cells densities in rotating dmrbs are higher than those in static dmrbs ( data 
 not shown ) , suggesting that the clinonational rotation plays an 
 important role in a rotating dmrb culture .
aliquots were daily taken from the culturing media to measure the 
 mab concentration during the culture .
changes in mab 
 concentrations in the medium supplemented with 5% fcs are shown 
 in figure 4 . as indicated ,
both accumulated ( a ) and daily ( b ) mab 
 production of cells cultured in rotating dmrbs are 
  distinguishably higher than those of cells cultured in 
 t - flasks .
interestingly , for dmrbs , the highest production rate 
 is achieved on the first day .
the first three - day mab production 
 accounts for over 95% of the total yield during the five - day 
 culture .
it suggests that the antibody should be harvested in 
 72  hours , to have a high yield of antibody , saving time and 
 materials .
statistically , the final production of a rotating dmrb 
 is approximately 42.5% higher than that of a t - flask .
this 
 result indicates that rotation plays a significant role , not only 
 in cell growth , but also in mab production .
the mab concentrations are measured in the medium 
 supplemented with 5% fcs in both dmrbs and t - flasks .
cell growth and mab production in dmrb groups and t - flask groups 
 within 72  hours are shown in table 1 . 

increase in production rate of mab is highly correlated with the 
 viable cell number .
it indicates that the increasing mab 
 production is largely resulted from an increase of the viable 
 cell number . in other words ,
the synthesis rate of mab increased 
 proportionally to the cell number . a further possibility for 
 enhancing mab productivity is considered with the increase in the 
 surface area by the microcarriers on which cells grow and 
 perch .
consequently , the cell density per culture volume may be 
 improved by means of the microcarriers [ 20 , 
 21 ] . for our 
 experiments ,
cytodex 3 is used as the microcarriers , which are 
 macroporous matrix based on polyethylene and weighted by silica . 

these microcarriers , alkali and acid resistant , are steamily 
 sterilizable at 121c ( 1  bar ) and are not swollen up 
 on hydration .
cytoline-3 microcarriers provide both an external 
 surface and an interior space with pore size between 10 and 
 400  m , which can be populated also by cells grown in 
 suspension and used in stirred cultures .
however , cells 
 inhabiting inside the pores could not be observed during the 
 culture .
comparison 
 of dmrb groups with t - flask group ;  
 p<.1 , 
  
 p<.05 . cell growth density , mab production , and reasonable cost are 
 important criteria to evaluate alternative methods to 
 conventional t - flask .
the method using dmrb to culture hybridoma 
 cells has some significant advantages : ( 1 ) to produce higher mab 
 at a low cost , ( 2 ) easily to handle in lab , ( 3 ) conveniently to process and investigate the 
 optimal conditions for different mab , and ( 4 ) readily to 
 produce a larger - scale mab when necessary .
furthermore , the 
 method makes it possible to produce mab as a putative process on 
 an industrial scale , and to have obvious advantages , such as a 
 high yield and a low cost .
 cell growth rate and production of monoclonal antibody ( mab ) of 
 hybridoma cells producing anti - human chorionic gonadotropin ( hcg ) 
 mab have been used as investigation criteria in double - mouthed 
 rolling bottle ( dmrb ) . compared with t - flask cell culture , both 
 of the cell number and mab production increased by approximately 
 42.5% when 
 the medium was supplemented with 5% fetal calf serum 
 ( fcs ) and dmrb rotated at 2 turns per minute . 
 yield of mab was 
 experimentally related to the number of viable cells . 
 
 interestingly , mab yield was four times as high as that cultured 
 in t - flask in the first 24  hours , and about 75% yield of 
 total mab was secreted by 48  hours during the culture . 
 it 
 appears that the promoted cell growth and mab yield are resulted 
 from the three - dimensional growth of hybridoma cells under a 
 suitably revolving condition . 