U.S. patent number 3,847,749 [Application Number 05/377,806] was granted by the patent office on 1974-11-12 for apparatus and method for rapid harvesting of roller culture supernatant fluid.
This patent grant is currently assigned to Duke University, Inc.. Invention is credited to Frank E. Kozoman, Ralph E. Smith.
United States Patent |
3,847,749 |
Smith , et al. |
November 12, 1974 |
APPARATUS AND METHOD FOR RAPID HARVESTING OF ROLLER CULTURE
SUPERNATANT FLUID
Abstract
A conventional perfusion tissue culture apparatus using roller
bottles is modified such that the supernatant fluids may be removed
automatically and at frequent intervals from the roller culture
bottles. The modified apparatus is programmed such that the bottles
are periodically stopped in their rotative movement, are tilted,
are drained of their respective supernatant fluids, are replenished
with a predetermined amount of fresh medium, are then lowered and
rotation is resumed until the fluid removing cycle is repeated.
Inventors: |
Smith; Ralph E. (Durham,
NC), Kozoman; Frank E. (Durham, NC) |
Assignee: |
Duke University, Inc. (Durham,
NC)
|
Family
ID: |
23490589 |
Appl.
No.: |
05/377,806 |
Filed: |
July 9, 1973 |
Current U.S.
Class: |
435/286.5;
435/286.7; 435/298.2; 435/308.1; 428/375; 428/520; 428/483 |
Current CPC
Class: |
C12M
23/50 (20130101); C12M 29/00 (20130101); C12M
27/12 (20130101); C12M 33/00 (20130101); Y10T
428/31928 (20150401); Y10T 428/31797 (20150401); Y10T
428/2933 (20150115) |
Current International
Class: |
C12M
3/04 (20060101); C12b 001/00 () |
Field of
Search: |
;195/127,143 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Claims
What is claimed is:
1. In a roller tissue culture apparatus of the type having a
plurality of parallel rollers, means to drive at least some of the
rollers while others of the rollers are idled, a plurality of
culture bottles adapted to be placed on the rollers and to be
turned by the driven rollers and to in turn rotate the idled
rollers, said bottles each having a swivel cap adapted to mount
influent, effluent, and gas inlet-exhaust pipes, having a nutrient
supply, piping connecting said nutrient supply to respective bottle
influent pipes and with valve means to control the flow of said
nutrient to said influent pipes whereby to control the flow of
nutrient to said bottles, the improvement comprising:
a. tilting means provided below each of said bottles and operable
to engage, gradually raise and tilt each of said bottles
simultaneously in some predetermined angular amount while at least
a portion of each of said bottles remains on its respective said
rollers, being adapted to hold each of said bottles in said tilted
position for some predetermined time and then to lower said bottles
back to their normal position on said rollers;
b. electrical operator means connected to said tilting means for
operating said tilting means and energizable in one mode to cause
said tilting means to gradually raise and tilt said bottles, in
another mode to cause said tilting means to hold said bottles
tilted and in a third mode to cause said tilting means to gradually
lower said bottles and restore said bottles to their normal
position on said rollers;
c. a swivel cap like piping arrangement in each said bottle
including an influent pipe connected to receive nutrient from said
supply and to discharge the same through a section of said influent
pipe within the respective said bottle, a gas inlet-exhaust pipe
having one end communicating with the interior of said bottle and
an opposite end communicating with the environment external of the
respective said bottle and having an effluent pipe with one end
terminating proximate the forward end of the respective bottle and
adjacent the interior surface thereof;
d. a plurality of effluent piping means connected to said
respective said swivel cap effluent pipes at one end and to
suitable chilled supernatant storage container means at the
opposite end;
e. pumping means connected to and operative on said effluent drain
pipes to pump fluids from said bottles to said storage container
means; and
f. electrical timing control means connected to said operator
means, said pumping means, said nutrient supply valve means and
said roller drive means whereby said bottles once filled with a
predetermined amount of said nutrient may be caused to be rotated
for a predetermined time, be stopped, be tilted, be held in a
tilted position, be drained of the supernatant fluids in said
bottles, be replenished with said nutrient, be gradually lowered to
the normal position on said rollers and then again be rotated on
said rollers, to repeat the cycle on some predetermined sequence
whereby the supernatant fluid and its contents in said bottles may
be automatically harvested in said storage container means
according to said sequence.
2. In a roller tissue culture apparatus as claimed in claim 1
wherein said tilting means includes a shaft extending below and
perpendicular to the axis of said bottles and on said shaft a
plurality of yokes adapted to be rotated by said shaft to engage,
tilt and lower said bottles.
3. In a roller tissue culture apparatus as claimed in claim 2
wherein said operator means includes a reversible drive motor
connected to said shaft.
4. In a roller tissue culture apparatus as claimed in claim 3
wherein said swivel cap influent pipe includes an upturned section
within and near the mouth of each said respective bottle and said
swivel cap effluent pipe includes a section turned sharply down
within and near the mouth of the respective said bottle and
terminating proximate the interior surface thereof.
Description
The invention described herein was made in the course of work under
a grant or award from the Department of Health, Education and
Welfare.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates broadly to tissue culture and more
specifically to apparatus and methods used in perfusion tissue
culture wherein the cells are grown in bottles which rotate during
the growth of the cells and are therefore referred to as roller
bottles. Perfusion culture refers to the periodic replacement of
nutrient solution to prevent cell starvation and waste product
accumulation.
2. Description of the Prior Art
The technique of growing cells in a medium which is placed in a
bottle which is rotated during the growth of the cells has been
recognized. Also, commercial tissue culture apparatus using this
technique has been available in the marketplace. The perfusion
tissue culture technique has also been widely practiced. Typical
literature references which in turn cite a multitude of other
references include the following:
"Production and Purification of Large Amounts of Rous Sarcoma
Virus," Ralph E. Smith and Eugene H. Bernstein, Applied
Microbiology, Mar. 1973, pp. 346-353.
"Comparison of Immature (Rapid Harvest) and Mature Rous Sarcoma
Virus Particles," Kwok-Sing Cheung, Ralph E. Smith, Marie P. Stone,
and Wolfgang K. Joklik, Virology, Dec. 1972, pp. 851-864.
"Multi-Layer Perfusion Tissue Culture," R. Elsworth, Process
Biochemistry, Mar. 1970.
The commercial tissue culture roller bottle apparatus which has
thus far been available has not provided a satisfactory means or
method for removing the supernatant fluids automatically and at
frequent intervals to enable rapid harvesting of the cells. As an
example, the New Brunswick perfusion control apparatus which is
referred to in the above references has been provided with a
so-called perfusion swivel cap which allows fluids to be admitted
to and removed from the bottles while the bottles are rotated but
the conventional swivel cap arrangement does not allow for the
complete removal of the supernatant fluids. Further, this type of
conventional roller bottle tissue culture apparatus does not
provide an overall system and method for operation on a programmed
basis whereby the supernatant fluids from a plurality of roller
bottles may be automatically and periodically collected in chilled
flasks to arrest viral maturation, the medium automatically
replaced and the virus cells maintained at physiological
temperatures.
SUMMARY OF THE INVENTION
According to the method and apparatus of the invention the
plurality of roller bottles conventionally employed in perfusion
culture apparatus are mounted on and rotated by conventional bottle
roller apparatus. Through use of a timing control the bottle
rotating apparatus is periodically stopped and the bottles are
tilted slightly by means of a plurality of fork or yoke like arms
which are mounted on a common shaft turned by a reversible motor.
In one direction of movement of the fork or yoke arms, the rear of
each bottle is engaged by a fork arm which slides on the bottle
surface and gradually tilts the bottle in some predetermined amount
at which point the forks are brought to a rest with all of the
bottles in a tilted position. Each bottle connects through an
outlet tube to a suitable storage container. A pump is activated
after the forks bring the bottles to a full tilt and the
supernatant fluid in the bottles is then drained through the
respective outlet tubes and collected either in a common or
individual chilled storage flasks. To insure essentially complete
emptying of each bottle the conventional straight entrance end of
the outlet tube is given a relatively sharp bend within the swivel
cap so that when the bottle is tilted essentially all of the fluid
can be drained. The timing control now causes a predetermined
amount of fresh medium to be added through a commercially available
manifold-pinch valve arrangement and through inlet tubes passing
through the swivel cap of each respective bottle. The reversible
motor is then energized to cause the shaft on which the fork arms
are mounted to rotate in a direction to allow the bottles to return
to their normal horizontal position. The described cycle is
repeated whenever it is desired to drain the supernatant fluid and
add fresh medium.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a somewhat generalized schematic view of a roller tissue
culture system according to the invention.
FIG. 2 is a front view of a modified commercially available roller
tissue culture apparatus incorporating the invention.
FIG. 3 is a plan section view taken in the direction of line 3--3
of FIG. 2 and showing the yoke lifting arrangement.
FIG. 4 is a partial sectional, partial schematic view illustrating
the modified swivel cap arrangement used in the invention
apparatus.
FIG. 5 is a partial side elevation view of one bottle and one yoke
lifter showing how the bottle is tilted for draining purposes.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention apparatus has been arrived at by modifying a
conventional perfusion control apparatus as sold by New Brunswick
Scientific Co., Inc. of New Brunswick, New Jersey. Such an
apparatus is fully described in the company catalog entitled
"Perfusion Control Apparatus, Models PF-4 and PF-8" dated Oct. 22,
1971 and labeled "M1006- 5050." The operation and construction of
this apparatus is fully described in that catalog. Therefore, the
present description will go only into enough detail for those
skilled in the art to understand how the method and apparatus of
the invention are related to the conventional method and apparatus
and the mentioned New Brunswick apparatus will be used as a basis
of comparison.
Referring to FIG. 1, there is shown a schematic diagram of the
invention apparatus as well as the conventional apparatus with
which it most closely relates. The conventional perfusion control
apparatus includes five rollers, 21, 22, 23, 24 and 25. Rollers 22
and 24 are driven through belts 30 and 31 by a roller driven motor
32. Rollers 21, 23 and 25 are thus idler rollers and four bottles
are received as indicated in dashed lines in FIG. 1 between each
pair of rollers. When this roller driven motor 32 is energized,
each of the four bottles tends to rotate in a conventional
manner.
The nutrient is retained in a reservoir 40 which is connected to a
conventional manifold 41 through a supply tube 42 and each of the
four bottles are fed through individual supply tubes 43, 44, 45 and
46.
A pinch cock valve 50 controls the gravity flow between reservoir
40 and manifold 41. As best seen in FIG. 4, each bottle is provided
with a modified swivel cap 55. The modified swivel cap includes an
individual bottle inlet tube 56 which has a sharp upward bend 57 at
the fluid discharge end as compared to the normally straight
configuration, 57', shown in dashed lines. The manifold swivel cap
also includes a fluid drain line 58 whose entrance end 59 is bent
sharply downwardly as compared to the normally longer and straight
configuration 59'. The so called innoculation or gas exhaust line
is stuffed with a suitable cotton wadding or other air filtering
material 67. A thin metal bracket 65 is conventionally used on each
bottle and supports the front of the bottle during rotation as
shown.
The fluid drain line 58 from each respective bottle connects to
respective storage drain lines 70, 71, 72 and 73. Lines 70, 71, 72
and 73 in turn pass through a peristaltic pump 75 and then to a
suitable chilled supernatant storage container 76.
A horizontal shaft 80 extends below and perpendicular to and
rearwardly of the rollers 21, 22, 23, 24 and 25. Shaft 80 is in
turn connected through a suitable reversible motor gear drive 81
such that shaft 80 can be rotated back and forth upon suitable
energization of motor drive 81. Between each pair of rollers there
are mounted on shaft 80 lifting yokes 82, 83, 84 and 85. Referring
particularly to FIGS. 1, 3 and 5 it can be seen that when shaft 80
rotates in a clockwise direction, as seen in FIG. 5, each
respective yoke lifts the rear portion of each respective bottle
and causes the bottle itself to be tilted in some predetermined
amount, e.g. 5.degree.. Then when shaft 80 is rotated in a
counterclockwise direction as seen in FIG. 5 the bottle is lowered
back to a horizontal position. Motor drive 81 is purposely chosen
so that this movement is slow and gradual and the timing of motor
drive 81 is such that shaft 80 stops turning when the respective
bottles have reached a maximum tilt. Further, each yoke is formed
with its bottle engaging surface relatively smooth so that the yoke
tends to slide along the length of the bottle during the tilting
process. Since the rollers 21-25 are normally surfaced with a
friction material the bottles do not tend to slide axially of the
rollers 21-25. Also the previously mentioned brackets 65 tend to
maintain the bottles in their proper position. Thus, the bottles
can be tilted, drained, replenished with medium, and lowered back
many times without changing the relative position of the bottles on
the rollers.
Since those skilled in the art will readily see the general type of
timing controls called for by the invention, the same are only
generally represented in FIG. 1. In FIG. 2 the auxilliary timing
controls are also shown at 90 as they might appear in an actual
apparatus embodying the invention. Also in FIG. 2, since many of
the parts shown are conventional, only those parts of particular
interest to the invention have been identified by numbers since
those skilled in the art will readily see how the invention is
incorporated into a conventional apparatus.
Referring back to FIG. 1, the overall timing control generally
designated 95 includes a time control for the yoke drive motor
designated 96, a time control for the bottle roller drive motor
designated 97, a time control for the nutrient supply control valve
98 and a time control designated 99 for the peristaltic pump
designated 75. The necessary timing circuitry to coordinate,
interlock, etc. the individual timers is not shown, since those
skilled in the art will readily see a great variety of timing and
programming circuits suitable to the invention. In one embodiment,
eight timers (Industrial Timers, Inc., Parsippany, N.J.) were
required to allow the necessary operations. The main timer
regulated the length of time between medium/ladd cycles. Two timers
were provided: a 3-hour timer was used for intervals of from 5
minutes to 3 hours, and a 5-minute timer was used for intervals of
5 minutes or less. The minimum collection time was 30 seconds, in
which bottles were rotated for 12 seconds, and the balance of time
was required to complete the collection cycle. The bottle up timer
regulated the length of time that the motor driving the cam shaft
was in operation. This timer was regulated to stop the cams at
their maximum vertical elevation. The delay timer provided a 5
second delay to allow medium drainage to the front of the bottles
after they were tilted forward, and reversed the circuit to the
motor driving the cam device. The pump-on timer activated a Kraft
automatic pipettor (Model P300, Kraft Apparatus, Inc., Queens,
N.Y.).
In operation, medium was pumped into prechilled collection vessels
maintained either in an ice bath in the same room as the perfusion
apparatus, or in an adjacent cold room maintained at 4.degree.
Centigrade (a hold was cut in the wall separating the rooms). The
medium/add timer served the same function as in the unmodified
perfusion system, namely, to activate a solenoid, which released
pressure (provided by bottled nitrogen) on the automatic pinchcocks
and allowed the addition of fresh medium by gravity flow. The
amount of medium added to each bottle varied according to the
frequency of collection. For example, 10 ml of medium was added per
bottle during collection at 5 minute intervals, and 25 ml of medium
was added during collection at 2 hour intervals. During extended
perfusion cultures, the pH of the medium in the reservoir was
readjusted daily by bubbling with carbon dioxide passed through a
sterile cotton-plugged pipette. The bottle down timer lowered the
bottles, and the reset timer prepared the electronic circuitry for
the next cycle and started rotation of the apparatus of the
invention.
A prototype apparatus has performed satisfactorily during numerous
experiments for long-term collections of virus at frequent
intervals. For example, roller culture bottles containing chick
embryo fibroblasts transformed with Rous sarcoma virus (RSV) were
maintained on the apparatus for over 3 weeks, with collection of
supernatant fluid every 2 hours. The bottles were of contamination
during the entire period of collection, and no malfunction was
encountered. Cell growth was vigorous under the perfusion
conditions outlined. For example, a roller culture bottle
maintained by perfusion culture yielded more than twice the number
of cells obtained from a roller culture bottle maintained by daily
medium changes.
The device was designed to study virus particles recently budded
from the cell surface. During the course of an investigation
reported elsewhere it was found that ribonucleic acid (RNA)
purified from RSV harvested at intervals of 2 hours or shorter was
more uniform in size than the virus harvested at longer intervals
(12 to 24 hours). The manual harvesting of supernatant fluids every
2 hours is tedious, and can be done for only a portion of the day.
Since the modified perfusion allows automatic harvesting during the
entire day, including weekends, the apparatus is now proving useful
for obtaining large quantities of high quality RNA with relative
ease. The perfusion culture technique outlined could also be used
whenever sequential harvests of a virus, cell metabolite, or other
extracellular material is desired.
A number of advantages can be seen in the method and apparatus just
described. A principal advantage is that rapid harvesting can be
accomplished automatically and with essentially complete removal of
the supernatant fluids. The use of the peristaltic pump provides
positive removal of the supernatant fluids and allows the various
drain lines to be purged as required. Since the bottles are not
shifted axially and lifted, and since the tilting is accomplished
slowly and gradually, the cell growth is not substantially
disturbed during the tilting and draining cycles. A typical work
schedule according to the method of the invention would include the
following steps:
1. Fill the fresh nutrient reservoir;
2. Admit to the bottles a predetermined amount of fresh nutrient
and, if not already accomplished, simultaneously pre-chill the
storage container or containers;
3. Warm the bottle surroundings if not already done;
4. Start the bottle rollers;
5. Rotate the rollers for a predetermined time;
6. Stop the bottle rollers;
7. Tilt the bottles and hold tilted for a predetermined time;
8. Drain the supernatant fluids by positive pump action from the
bottles while they are tilted;
9. Replenish the bottles with a predetermined amount of fresh
nutrient while the bottles are tilted;
10. Lower bottles;
11. Resume rotation.
Those skilled in the art will readily appreciate that variations on
the above method of using the invention can be adopted. For
example, the fresh nutrient can be restored either with the bottles
in a tilted position or in a lowered position. It is preferred to
do this when tilted to avoid a sudden rush of nutrient and a
damaging action were the nutrient to suddenly flow as a wave from
one end of the bottle to the other. Also the bottle warming and
storage chilling steps can be accomplished at any suitable time.
Considering that the prior art method required manual operations
for all of the filling and removing steps indicated, it can be seen
that the researcher is given a new tool and an extremely valuable
apparatus and method for rapid harvesting.
* * * * *