US3871955A - Process for automated production of synchronous mammalian cells - Google Patents
Process for automated production of synchronous mammalian cells Download PDFInfo
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- US3871955A US3871955A US360047A US36004773A US3871955A US 3871955 A US3871955 A US 3871955A US 360047 A US360047 A US 360047A US 36004773 A US36004773 A US 36004773A US 3871955 A US3871955 A US 3871955A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/10—Rotating vessel
- C12M27/12—Roller bottles; Roller tubes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2517/00—Cells related to new breeds of animals
- C12N2517/04—Cells produced using nuclear transfer
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- the process for automated production of synchronous mammalian cells uses roller bottles containing cell medium which has been incubated for 30 minutes. Mammalian cells are then subcultured for about 24 hours in the roller bottles. Then, the roller bottles are speeded up sufficiently for a given period oftime to dislodge rounded mitotic cells in the medium. The detached cells in medium are pumped from the roller bottles and collected in a manifold, and then discharged into a growth flask.
- the process is then repeated at the desired time interval to suit the cell cycle time and the particular requirements of the investigator. All of these steps of the process are automatically carried out by suitable apparatus of a conventional type, without the prolonged presence of the investigator.
- an object of this invention to provide a process for automated production of synchronous mammalian cells which has no need for the presence of the investigator for prolonged periods of time.
- Another object of this invention is to provide a process for automated production of synchronous mammalian cells without the use of chemical treatment, such as inhibitors.
- a further object ofthis invention is to provide a process for automated production of synchronous mammalian cells in relatively high yields.
- a yet further object of this invention is to provide a process for automated production of biochemically balanced populations of synchronous mammalian cells, without the use of altered medium or lowered temperatures.
- Example 1 is an embodiment of the invention using Chinese hamster cells, known as cell line V 79.
- the medium for the cells is McCoys 5a medium supplemented with fetal calf serum and 100 mg/l neomycin.
- EXAMPLE I The entire cycle of the mammalian cell, Chinese hamster cell line V79, with a 12-hour generation time, is examined.
- a series of roller bottles is set up in a standard Bellco model 7400 roller apparatus modified to operate at increased revolutions by adding a one-way clutch'and a fractional horsepower bodine motor.
- the roller bottles are incubated'each with about 25 ml. of the medium for about 30 minutes at 37 C. Then, cells are subcultured,
- the roller apparatus is speeded up to a speed of about rpm for about 5 minutes, R thereby causing the dislodging of rounded mitotic cells, which are selectively detached in the medium in the roller bottles.
- the detached cells in the medium are collected through manifold M and discharged into a growth flask 0,, through valve V
- the first batch of detached cells is always discarded because it is contaminated with cells which have never attached or were loosely attached but were non-mitotic.
- the growth flasks are selected by the opening and closing of a series of valves V,,, V, through V the number of valves and flasks may be varied as desired.
- This first cycle may be called the purge cycle.
- the detached cells are kept at 37 C.
- the process is then repeated every hour for 12 hours automatically, so that detached cells will be pumped and collected through the manifold and valves selectively into growth flasks G through G by valves V through V
- Each of the growth flasks thus has a selected synchronous culture separated in the cell cycle from the others by 1 hour.
- the program is automatic and has no need for the presence of the investigator during this 12-hour period.
- the speed of the roller can be varied to suit the particular cell cycle time. Also, the slow speed of the roller may be set from 20 to minutes in l-minute increments.
- the fast turning of the roller may be varied as desired with respect to time of fast turning, prior to slow turning.
- Example 2 The process of Example 1 was repeated, except for a change in the slow and fast rotation of the rollers.
- the program controller was adjusted to provide a slow turning speed of about 0.5 rpm of the roller bottles for a period of 10 minutes. Thereafter, the fast turning speed of about 100 rpm for the roller bottles was set for 50 minutes, and the detached cells were discharged into the growth flasks.
- the quality of synchrony using the processes of Examples l and 2 was compared with that obtained by conventional manual selection of cells.
- the process of Example 1 produced about the same yield of cells per sample as by manual selection, but the process of Example 2 produced a yield of over three times the yield obtained by manual selection.
- Yields were tested by conventional methods of counting in a Coulter electronic cell counter and by centrifuging and fixing for cytological analysis.
- EXAMPLE 3 This Example used a normal diploid human cell line known as Wl-38, human fibroblasts. Wl-38 cells were brought into a synchronous state by repeated selection and automated detachment of mitotic cells from roller bottles according to the process of Example 1, except that these cells were subcultured at a density of 5 X cells per roller. Also, the program was set for a 24-hour period and used 24 growth flasks.
- Example 3 produced a relatively large yield of synchronous Wl-38 cells, about 0.5 X 10 cells per growth flask.
- a process for automated production of synchronous mammalian cells comprising:
- a process for automated production of synchronous mammalian cells comprising:
- a process for automated production of synchronous mammalian cells comprising:
- a process for automated production of synchronous mammalian cells comprising:
- a process for automated production of synchronous mammalian cells comprising:
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Abstract
A process for automatically producing relatively large, biochemically balanced populations of synchronous mammalian cells without requiring the presence of an investigator for prolonged periods of time, which includes subculturing the cells in a medium inside roller bottles, allowing growth of the cells, and then rotating the bottles at a speed sufficient to detach rounded mitotic cells and collecting the cells so detached.
Description
Unite States Patent [1 1 Klevecz Mar. 18, 1975 l l PROCESS FOR AUTOMATED PRODUCTION OF SYNCHRONOUS MAMMALIAN CELLS [76] Inventor: Robert R. Klevecz, 227 Bettyhill Dr., Duarte, Calif. 91010 221 Filed: May 14,1973
[21 Appl. No.: 360,047
[52] US. Cl. 195/l.8 [51] Int. Cl Cl2k 9/00 [58] Field of Search 195/127, 1.8
[56] References Cited UNITED STATES PATENTS 3 7l 1,379 l/l973 Adams l95/l27 OTHER PU BLICATIONS WillmerCells & Tissues in Culture, Vol. (1966), p. 91.
Primary Examiner-Sam Rosen Attorney, Agent, or Firm-John Joseph Hall ABSTRACT 9 Claims, 1 Drawing Figure PROCESS FOR AUTOMATED PRODUCTION OF SYNCHRONOUS MAMMALIAN CELLS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to the production of synchronous mammalian cells.
2. Prior Art Applicant is unaware of any prior art disclosing a process for automated production of synchronous mammalian cells without chemical treatment or lowered temperatures and which dispenses with the presence of the investigator for prolonged periods of time. Chemical inhibitors which align or synchronize cells only do so with respect to a single biochemical parameter and thus may detrimentally affect the resulting cell population. Alignment or synchronization methods with single inhibitors or a combination of inhibitors may alter normal cell metabolism and may introduce artifactual perturbations or obscure natural periodicities. Further, simple mitotic selection techniques usually result in low yields, besides requiring the investigators presence for long periods of time.
SUMMARY OF THE INVENTION The process for automated production of synchronous mammalian cells uses roller bottles containing cell medium which has been incubated for 30 minutes. Mammalian cells are then subcultured for about 24 hours in the roller bottles. Then, the roller bottles are speeded up sufficiently for a given period oftime to dislodge rounded mitotic cells in the medium. The detached cells in medium are pumped from the roller bottles and collected in a manifold, and then discharged into a growth flask.
The process is then repeated at the desired time interval to suit the cell cycle time and the particular requirements of the investigator. All of these steps of the process are automatically carried out by suitable apparatus of a conventional type, without the prolonged presence of the investigator.
It is, therefore, an object of this invention to provide a process for automated production of synchronous mammalian cells which has no need for the presence of the investigator for prolonged periods of time.
Another object of this invention is to provide a process for automated production of synchronous mammalian cells without the use of chemical treatment, such as inhibitors.
A further object ofthis invention is to provide a process for automated production of synchronous mammalian cells in relatively high yields.
A yet further object of this invention is to provide a process for automated production of biochemically balanced populations of synchronous mammalian cells, without the use of altered medium or lowered temperatures.
These and other objects will be more readily understood by reference to the following description and claims, taken in conjunction with the accompanying drawing illustrating a preferred sequence of operations of the process.
The following Example 1 is an embodiment of the invention using Chinese hamster cells, known as cell line V 79. The medium for the cells is McCoys 5a medium supplemented with fetal calf serum and 100 mg/l neomycin.
EXAMPLE I The entire cycle of the mammalian cell, Chinese hamster cell line V79, with a 12-hour generation time, is examined.
A series of roller bottles is set up in a standard Bellco model 7400 roller apparatus modified to operate at increased revolutions by adding a one-way clutch'and a fractional horsepower bodine motor. The roller bottles are incubated'each with about 25 ml. of the medium for about 30 minutes at 37 C. Then, cells are subcultured,
i.e., inoculated in the medium, at a density of 3 X 10 7 Then, the roller apparatus is speeded up to a speed of about rpm for about 5 minutes, R thereby causing the dislodging of rounded mitotic cells, which are selectively detached in the medium in the roller bottles.
The detached cells in the medium are collected through manifold M and discharged into a growth flask 0,, through valve V However, the first batch of detached cells is always discarded because it is contaminated with cells which have never attached or were loosely attached but were non-mitotic.
The growth flasks are selected by the opening and closing of a series of valves V,,, V, through V the number of valves and flasks may be varied as desired.
This first cycle may be called the purge cycle.
Next, a fresh 25 ml. charge of medium is then pumped into the roller bottles by pump P,- while valve V remains open to allow any medium and cells trapped in the line to drain into a waste receptacle. The slow roller speed is repeated for about 55 minutes, and then the fast speed for about. 5 minutes.
This time, the detached cells are pumped by pump P out of the roller bottles and collected in the manifold M, and, by suitable valve timing through valve V discharged into growth flask (3,.
Throughout the process, the detached cells are kept at 37 C.
The process is then repeated every hour for 12 hours automatically, so that detached cells will be pumped and collected through the manifold and valves selectively into growth flasks G through G by valves V through V Each of the growth flasks thus has a selected synchronous culture separated in the cell cycle from the others by 1 hour. The program is automatic and has no need for the presence of the investigator during this 12-hour period.
The speed of the roller can be varied to suit the particular cell cycle time. Also, the slow speed of the roller may be set from 20 to minutes in l-minute increments.
Similarly, the fast turning of the roller may be varied as desired with respect to time of fast turning, prior to slow turning.
EXAMPLE 2 The process of Example 1 was repeated, except for a change in the slow and fast rotation of the rollers.
The program controller was adjusted to provide a slow turning speed of about 0.5 rpm of the roller bottles for a period of 10 minutes. Thereafter, the fast turning speed of about 100 rpm for the roller bottles was set for 50 minutes, and the detached cells were discharged into the growth flasks.
The quality of synchrony using the processes of Examples l and 2 was compared with that obtained by conventional manual selection of cells. The process of Example 1 produced about the same yield of cells per sample as by manual selection, but the process of Example 2 produced a yield of over three times the yield obtained by manual selection.
Yields were tested by conventional methods of counting in a Coulter electronic cell counter and by centrifuging and fixing for cytological analysis.
I have found that the process produces successful results using human cells, as indicated in the following Example.
EXAMPLE 3 This Example used a normal diploid human cell line known as Wl-38, human fibroblasts. Wl-38 cells were brought into a synchronous state by repeated selection and automated detachment of mitotic cells from roller bottles according to the process of Example 1, except that these cells were subcultured at a density of 5 X cells per roller. Also, the program was set for a 24-hour period and used 24 growth flasks.
The process of Example 3 produced a relatively large yield of synchronous Wl-38 cells, about 0.5 X 10 cells per growth flask.
It is known that standard methods such as colcemidenhanced mitotic selection and S phase arrest have failed. Also, simple mitotic selection yielded only a vanishingly small population of cells.
Although I have described my invention in detail with reference to the accompanying drawing illustrating a preferred embodiment of the invention in conjunction with the specification and claims, it is understood that numerous changes in the details of construction and arrangement of parts and components may be made without departing from the spirit and scope of the invention as hereinafter claimed.
1 claim:
1. A process for automated production of synchronous mammalian cells, comprising:
adding a suitable medium to a seriesof roller bottles in a roller apparatus;
incubating the medium;
subculturing the medium with mammalian cells;
allowing growth of the cells for about 24 hours;
initiating turning of the roller bottles by automatic controllers at a speed sufficiently slow to prevent detaching said cells;
speeding up the turning of the roller bottles by automatic controllers to a speed sufficient to detach rounded mitotic cells;
removing the detached rounded mitotic cells from the roller bottles;
Collecting the detached rounded mitotic cells in a manifold; and
, discharging said cells into a growth flask through a valve.
2. A process according to claim 1 which the period of time for initiating turning of the roller bottles is about 55 minutes.
3. A process according to claim 1 in which the period of time for speeding up the turning of the bottles is about 5 minutes.
4. A process according to claim 1 in which the initiating turning of the rollers is about 0.5 rpm.
5. A process according to claim 1 in which the speeded up turning of the rollers is about rpm.
6. A process for automated production of synchronous mammalian cells, comprising:
adding a suitable medium to a series of roller bottles in a roller apparatus;
incubating the medium for about 30 minutes;
subculturing the medium with mammalian cells;
allowing growth of the cells for about 24 hours;
initiating turning of the roller bottles by automatic controllers for about 55 minutes at about 0.5 rpm;
speeding up the turning of the roller bottles by automatic controllers for about 5 minutes at about 100 rpm to detach rounded mitotic cells;
removing the detached rounded mitotic cells from the roller bottles;
collecting the detached rounded mitotic cells in a manifold; and
discharging said cells into a growth flask through a valve.
7. A process for automated production of synchronous mammalian cells, comprising:
adding a suitable medium to a series of roller bottles in roller. apparatus;
incubating the medium for about 30 minutes;
subculturing the medium with mammalian cells;
allowing grown of the cells for about 24 hours.
initiating turning of the roller bottles by automatic controllers for about 10 minutes at about 0.5 rpm;
speeding up the turning of the roller bottles by automatic controllers for about 50 minutes at about 100 rpm to detach rounded mitotic cells;
removing the detached rounded mitotic cells from the roller bottles;
collecting the detached rounded mitotic cells in a manifold; and
discharging said cells into a growth flask through a valve.
8. A process for automated production of synchronous mammalian cells, comprising:
subculturing mammalian cells in a suitable medium in a container rotatable around its axis;
allowing growth of said cells for about 24 hours;
causing relatively rapid rotation of said container by automatic controllers for a pre-determined period of time at a speed sufficient to detach rounded mitotic cells; and
collecting the detached rounded mitotic cells in a container.
9. A process for automated production of synchronous mammalian cells, comprising:
automatically pumping a charge of medium into a container rotatable around its axis;
subculturing mammalian cells in said medium for about 24 hours while the container is rotating at a relatively slow speed;
causing relatively rapid rotation of said container by collecting the detached rounded mitotic cells in a automatic controllers for a pre-determined period container by the automatic opening and closing of of time at a speed sufficient to detach rounded mia valve means. totic cells; and
Claims (9)
1. A PROCESS FOR AUTOMATED PRODUCTION OF SYNCHRONOUS MAMMALIAN CELLS, COMPRISING: ADDING A SUITABLE MEDIUM TO A SERIES OF ROLLER BOTTLES IN A ROLLER APPARATUS; INCUBATING THE MEDIUM; SUBCULTURING THE MEDIUM WITH MAMALIAN CELLS; ALLOWING GROWTH OF THE CELLS FOR ABOUT 24 HOURS; INITIATING TURNING OF THE ROLLER BOTTLES BY AUTOMATIC CONTROLLERS AT A SPEED SUFFICIENTLY SLOW TO PREVENT DETACHING SAID CELLS; SPEEDING UP THE TURNING OF THE ROLLER BOTTLES BY AUTOMATIC CONTROLLERS TO A SPEED SUFFICIENT TO DETACH ROUNDED MITOTIC CELLS; REMOVING THE DETACHED ROUNDED MITOTIC CELLS FROM THE ROLLER BOTTLES; COLLECTING THE DETACHED ROUNDED MITOTIC CELLS IN A MANIFOLD; AND DISCHARGING SAID CELLS INTO GROWTH FLASK THROUGH A VALVE.
2. A process according to claim 1 which the period of time for initiating turning of the roller bottles is about 55 minutes.
3. A process according to claim 1 in which the period of time for speeding up the turning of the bottles is about 5 minutes.
4. A process according to claim 1 in which the initiating turning of the rollers is about 0.5 rpm.
5. A process according to claim 1 in which the speeded up turning of the rollers is about 100 rpm.
6. A process for automated production of synchronous mammalian cells, comprising: adding a suitable medium to a series of roller bottles in a roller apparatus; incubating the medium for about 30 minutes; subculturing the medium with mammalian cells; allowing growth of the cells for about 24 hours; initiating turning of the roller bottles by automatic controllers for about 55 minutes at about 0.5 rpm; speeding up the turning of the roller bottles by automatic controllers for about 5 minutes at about 100 rpm to detach rounded mitotic cells; removing the detached rounded mitotic cells from the roller bottles; collecting the detached rounded mitotic cells in a manifold; and discharging said cells into a growth flask through a valve.
7. A process for automated production of synchronous mammalian cells, comprising: adding a suitable medium to a series of roller bottles in roller apparatus; incubating the medium for about 30 minutes; subculturing the medium with mammalian cells; allowing grown of the cells for about 24 hours. initiatIng turning of the roller bottles by automatic controllers for about 10 minutes at about 0.5 rpm; speeding up the turning of the roller bottles by automatic controllers for about 50 minutes at about 100 rpm to detach rounded mitotic cells; removing the detached rounded mitotic cells from the roller bottles; collecting the detached rounded mitotic cells in a manifold; and discharging said cells into a growth flask through a valve.
8. A process for automated production of synchronous mammalian cells, comprising: subculturing mammalian cells in a suitable medium in a container rotatable around its axis; allowing growth of said cells for about 24 hours; causing relatively rapid rotation of said container by automatic controllers for a pre-determined period of time at a speed sufficient to detach rounded mitotic cells; and collecting the detached rounded mitotic cells in a container.
9. A process for automated production of synchronous mammalian cells, comprising: automatically pumping a charge of medium into a container rotatable around its axis; subculturing mammalian cells in said medium for about 24 hours while the container is rotating at a relatively slow speed; causing relatively rapid rotation of said container by automatic controllers for a pre-determined period of time at a speed sufficient to detach rounded mitotic cells; and collecting the detached rounded mitotic cells in a container by the automatic opening and closing of a valve means.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US360047A US3871955A (en) | 1973-05-14 | 1973-05-14 | Process for automated production of synchronous mammalian cells |
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| Application Number | Priority Date | Filing Date | Title |
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| US360047A US3871955A (en) | 1973-05-14 | 1973-05-14 | Process for automated production of synchronous mammalian cells |
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| US3871955A true US3871955A (en) | 1975-03-18 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129742A1 (en) * | 2001-03-02 | 2003-07-10 | Helmstetter Charles E. | Bioreactor and methods for producing synchronous cells |
| WO2010118709A2 (en) | 2009-04-16 | 2010-10-21 | Univerzita Palackeho | Method of production of synchronized adherently growing cell lines and device for carrying out said method |
| CZ302682B6 (en) * | 2009-04-16 | 2011-08-31 | Univerzita Palackého | Production process of synchronized adherently growing cell lines and apparatus for making the same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3711379A (en) * | 1971-06-24 | 1973-01-16 | Cenco Medical Health Supply Co | Rotating flask culture apparatus |
-
1973
- 1973-05-14 US US360047A patent/US3871955A/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3711379A (en) * | 1971-06-24 | 1973-01-16 | Cenco Medical Health Supply Co | Rotating flask culture apparatus |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129742A1 (en) * | 2001-03-02 | 2003-07-10 | Helmstetter Charles E. | Bioreactor and methods for producing synchronous cells |
| US6867040B2 (en) | 2001-03-02 | 2005-03-15 | Charles E. Helmstetter | Bioreactor and methods for producing synchronous cells |
| WO2010118709A2 (en) | 2009-04-16 | 2010-10-21 | Univerzita Palackeho | Method of production of synchronized adherently growing cell lines and device for carrying out said method |
| CZ302682B6 (en) * | 2009-04-16 | 2011-08-31 | Univerzita Palackého | Production process of synchronized adherently growing cell lines and apparatus for making the same |
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