A METHOD OF AND APPARATUS FOR HARVESTING MAMMALIAN CELLS

Abstract: A technique for harvesting mammalian cells from an artificial substrate using ultrasonic energy in the range of 5 to 20 joules/sq. cm., at an intensity in the range of 30 to 150 mW/sq. cm, and at a frequency in the range of 20 to 60 kHz.

Description: -l- Description A Method Of And Apparatus For Harvesting Mammalian Cells Technical Field This invention relates to a technique of harvesting mammalian cells from substrates upon which they are being cultured. In particular, it relates to a technique of harvesting mammalian cells by means of ultrasound employed without chemical enhancement. Background Art Many types of mammalian cells must be anchored to a surface of a substrate if they are to reproduce. When th_ey are cultured, they are grown in glass or plastic vessels or on microcarrier beads, depending on the use for which the cultured cells are intended. When the growth is complete — that is, when the cells have formed a confluent monolayer on the surface of the substrate and ceased to proliferate - they must be recovered in a harvesting step. There are two classical ways of harvesting cells. One way is simply to scrape the cells off the substrate. That is time consuming if large numbers of substrates are involved and physically impossible for microcarrier beads. The other way to harvest the cells is to treat them with an enzyme (usually trypsin) which lyses the bonds attaching the cells to the substrate. This approach involves considerable rinsing, accurate timing, and so forth. It also introduces the problems of possible contamination of the harvest with trypsin and/or unexpected alteration of the cells or of their constituents by the trypsin. A harvesting method which avoids these disadvantages, and which is at the same time fast, reliable, and adaptable to mass production techniques would, accordingly, be very valuable. Others have used ultrasound to interfere with the reattachment of mammalian cells to substrates after they have been harvested from the substrates by other means. However, so far as it is known, the only effort to harvest cells by ultrasound was unsuccessful except when the ultrasound was enhanced chemically with enzymes or EDTA. However, as previously stated, the presence in the harvest of such chemicals is often objectionable. Accordingly, it would be highly desirable to have a technique by which mammalian.cells could be harvested solely by use of ultrasound without any chemical enhancement. Moreover, in addition to the question of efficacy, there is the question of safety. Any ultrasound technique used for harvesting mammalian cells must not only separate the cells from the substrate, it must do so without any significant damage to the cells. Siegel et al, "Cellular Attachment as a Sensitive Indicator of the Effects of Diagnostic Ultrasound Exposure on cultured Human Cells," 133 Radiology 175 (October 1979), is addressed to the question of possible damage to the cells, particularly fetal cells, by diagnostic ultrasound imaging. It discloses use of high frequency (2.25 MHz), low energy (0.1 to 0.4 joules/sq. cm.) ultrasound typical of diagnostic practice on cells very weakly attached to the substrate (45 minutes after seeding in the petri plates). A maximum of 50% of the cells were detached. Sanford, "A New Method for Dispersing Strongly Adhesive Cells in Tissue Culture," 10 In Vitro 281 (1974), reports that ultrasound accelerated detachment of cells from their substrates and broke up clumps of released cells when ultrasound was used in conjunction with trypsin and EDTA. However, Sanford specifically states that, in the absence of the chemicals, the ultrasound "did not remove cells from the flask surface or from each other." Disclosure of the Invention Quite unexpectedly. Applicants have discovered that mammalian cells in culture vessels can be harvested successfully by the use of ultrasound in particular energy and frequency ranges without the use of any chemical enhancement. The preferred energy range for this technique is 5 to 20 joules/sq. cm., the preferred ultrasonic intensity is 30 to 150 mW/sq. cm., and the preferred frequency range is 20 to 60 kHz. Preferably, the ultrasound is delivered to an external surface of a culture vessel containing the cells to be harvested. Brief Description of the Drawings FIGURE 1 is a perspective view of a first embodiment of apparatus according to the subject invention adapted to harvest cells from a roller bottle. FIGURE 2 is a transverse sectional view of the apparatus shown in FIGURE 1. OMPI FIGURE 3 is a perspective view of a culture flask. FIGURE 4 is a transverse sectional view of a second embodiment of apparatus according to the subject invention adapted to harvest cells from a culture flask. FIGURE 5 is a perspective view of a third embodiment of apparatus according to the subject invention adapted to harvest cells from microcarrier beads. Best Mode for Carrying Out the Invention The Method Applicants first grew normal human diploid fibroblast cells in a petri dish, then, using a pencil¬ like sonicator, stripped layers of the cells from the plate. Later, they successfully replated the cells. Applicants next cultured human fibroblast cells in a T-flask (a 25 cc vessel rather like a small, square sealed petri dish with a neck) . When the cells were firmly adherent and confluent, the flask was immersed in a water bath which itself was energized with ultrasound by a laboratory ultrasonic cleaner. Again the cells were all removed, and again the harvested cells multiplied enthusiastically when they were later replated. This showed the efficacy of ultrasound applied indirectly to the cells — i.e., through the walls of the T-flask. However, in practice the T-flask may be replaced by a conventional roller bottle or any other culture vessel. Applicants next grew human fibroblast cells on microcarrier beads. They put the loaded beads in a T- flask, and then they harvested the cells as above. Again, the harvested cells were viable. Next, Applicants cultured human fibroblast cells in a multiwelled vessel of the kind used for titers. By immersing the probe of a sonic transducer in a well in which cells were being grown and applying ultrasonic energy to the cells for various times and at various power settings and frequencies, they established the following parameters within which the ultrasound is effective for harvesting mammalian cells. Energy Frequency (joules/sq. cm.) (kHz) Results 100 20 No cells will replate 50 20-45 40% of the cells will not replate 5 to 20 20-45 Preferred range 2 45 No significant cell release in five minutes Additionally, Applicants' most effective experiments were conducted with an ultrasonic bath device that provides an ultrasonic intensity of approximately 30 to 40 mW/sq. cm.; it was much more difficult to obtain reproducible cell removal and survival at intensities of 2 to 5 W/sq. cm. At all energy levels, bubble formation and cavitation were associated with extensive cell death and failure to replate. Thus, the energy levels noted are effective only in the absence of cavitation. Generalizing from the foregoing data, our preferred range of exposure density is 5 to 20 joules/sq. cm., our preferred range of energy exposure is 30 to 150 mW/sq. cm., and our preferred frequency range is 20 to 60 kHz. In view of the publications of prior investigators indicating that direct application of ultrasound to cell cultures in liquid media causes death of the cells, it is our tentative hypothesis that rapid vibration of the substrate surface caused by application of the ultrasound to an external surface of the culture vessel shakes the cells loose. That is, we suspect that the harvesting is effected by the vibrations of the substrate surface caused by the ultrasound rather than by the ultrasound directly. A further refinement of the technique is particularly effective where the mammalian cells are grown on microcarrier beads. In this case, the microcarrier beads may be suspended in a nutrient liquid prior to harvesting, and, when the cells are • removed from the microcarrier beads by the ultrasound, they are released directly into the nutrient liquid. Once the cells have been released, the microcarrier beads may, of course, be strained from the nutrient liquid and the cells used for their intended purpose. Another possible refinement is "chirping" the frequency in each ultrasonic pulse from 30 to 60 kHz. "Chirping" would move the standing ultrasonic waves generated across the substrate surface and through the culture medium, resulting in a more uniform exposure of the cells to the ultrasonic energy applied. As for the duration of the ultrasonic radiation, it can range from tens of seconds to a few minutes. In practice, the setting of exposure density, energy intensity, and frequency determines the duration of ultrasonic irradiation used. The Apparatus A first embodiment of apparatus according to the subject invention adapted to harvest cells from roller bottles is shown in FIGURES 1 and 2. In this embodiment, a roller bottle 10, which is about 10 to 11 cm. in diameter and 20 to 25 cm. in length, is slowly rotated in an approximately 37°C water bath 12 by a drive roller 14. The roller bottle 10 is cradled by drive roller 14 and passive rollers 16. The roller bottle 10 contains a cell layer 18 (which may extend around the entire inner peripheral surface of the roller bottle 10) and a nutrient solution 20 which is in contact at any given instant only with a small portion of the cell layer 18. The drive roller 14 is powered by a motor 22 mounted on casing 24. An ultrasonic transducer 26 is mounted in the casing 24 in position to deliver ultrasonic energy to the exterior surface of the roller bottle 10. An on-off switch 28 is provided for the motor 22, and a timer switch 30 is provided for the ultrasonic transducer 26. A first continuously variable control switch 32 is provided to vary the power produced by the ultrasonic transducer 26 and a second continuously variable control switch 34 is provided to vary the rate of spin of the roller bottle 10 — that is, to vary the rate of spin of the drive roller 14 caused by the motor 22. Additionally, an on-off switch 36 is provided for a "chirper". That is, the ultrasonic transducer 26 can be used either to OMPI subject invention adapted to harvest cells from culture flasks is shown in FIGURES 3 and 4. In this embodiment, a culture flask 40, which is about 9 to 25 cm. long and which has a screw cap 42, is suspended in an approximately 37°C water bath 44 in a casing 46 by means of adjustable gripper brackets 48. The culture flask 40 contains a cell layer 50 covered by a nutrient solution 52. An ultrasonic transducer 54 is mounted on the casing 46 in position to deliver ultrasonic energy to the exterior surface of the culture flask 40. A timer switch (not shown) is provided for the ultrasonic transducer 54, a continuously variable control switch (not shown) is provided to vary the power produced by the ultrasonic transducer 54, and an on-off switch (not shown) is provided to vary the ultrasonic energy between continuous wave and chirping. A third embodiment of apparatus according to the subject invention adapted to harvest cells from microcarrier beads is shown in FIGURE 5. In this embodiment, a toroidially shaped ultrasonic transducer 60 surrounds a glass or plastic tube 62 which acts as a culture vessel through which an approximately 37°C nutrient liquid 64 flows in the direction of the arrow 66. Microcarrier beads 68 having cells 70 thereon are suspended in the nutrient liquid 64 upstream of the ultrasonic transducer 60. As the microcarrier beads 68 pass through the ultrasonic transducer 60, the cells 70 are harvested (i.e., separated) from the microcarrier beads 68. A power cable 72 connects the ultrasonic OMPI transducer 60 to a power supply/signal generator/amplifier 74. A first continuously variable control switch 76 is provided on the power supply/signal generator/amplifier 74 to vary the power produced by the ultrasonic transducer 60, and a second continuously variable control switch 78 is provided on the power supply/signal generator/amplifier 74 to vary the frequency of the ultrasonic transducer 60. Finally, an on-off switch 80 is provided to turn the ultrasonic transducer 60 on and off, and an on-off switch 82 is provided to vary the ultrasonic energy between continuous wave and chirping.

Claims: Clai s 1. A method of harvesting mammalian cells from an artificial substrate, said method comprising the step of exposing the cells to ultrasonic energy at a level which is sufficiently high to separate the cells from the substrate, but which is sufficiently low not to be harmful to the cells. 2. A method as recited in Claim 1 wherein the substrate is in a culture vessel and the cells are exposed to ultrasonic energy delivered to the external surface of the culture vessel. 3. A method as recited in Claim 2 wherein the culture vessel is a roller bottle. 4. A method as recited in Claim 2 wherein the culture vessel is a culture flask. 5. A method as recited in Claim 1 wherein the ultrasonic energy is in the range of 5 to 20 joules/sq. cm., the frequency is in the range of 20 to 60 kHz, and the ultrasonic intensity is in the range- of 30 to 150 mW/sq. cm. 6. A method as recited in Claim 1 wherein the artificial substrate is a microcarrier bead. 7. A method as recited in Claim 4 wherein the microcarrier bead is suspended in a liquid while being exposed to the ultrasound. 8. A method as recited in Claim 7 wherein the liquid is a nutrient liquid. 9. A method as recited in Claim 1 wherein the frequency in each ultrasonic pulse is chirped from 30 to 60 kHz. 10. Apparatus for harvesting mammalian cells from an artificial substrate, said apparatus comprising an ltrasonic transducer positioned to deliver ultrasonic energy to the cells at a level which is sufficiently high to separate the cells from the substrate, but which is sufficiently low not to be harmful to the cells. 11. Apparatus as recited in Claim 10: (a) and further comprising first means for mounting a culture vessel and (b) wherein the cells are exposed to ultrasonic energy delivered to the external surface of the culture vessel. 12. Apparatus as recited in Claim 11 wherein: (a) said apparatus further comprises a casing adapted to contain a controlled temperature bath; (b) said first means for mounting a culture vessel comprise a plurality of rollers mounted on said casing and positioned to cradle a roller bottle in position to be rotated about its central axis and to contact a controlled temperature bath in said casing; and (c) said apparatus further comprises second means for rotating at least one of said plurality of rollers. thereby rotating a roller bottle cradled by said plurality of rollers. 13. Apparatus as recited in Claim 12 and further comprising: (a) a timer switch for controlling the length of time that said ultrasonic transducer is on; (b) a control switch for varying the power produced by said ultrasonic transducer; (c) a control switch for varying the rate of spin of a roller bottle cradled by said rollers ; and (d) an on-off switch for said second means. 14. Apparatus as recited in Claim 13 and further comprising an on-off switch for varying the ultrasonic energy produced by said ultrasonic transducer between a continuous wave and a series of pulses in the form of chirps. 15. Apparatus as recited in Claim 11 wherein: (a) said apparatus further comprises a casing adapted to contain a controlled temperature bath and (b) said first means for mounting a culture vessel comprise means for suspending the culture vessel in position to contact a controlled temperature bath in said casing. 16. Apparatus as recited in Claim 15 wherein said second means comprises a pair of adjustable gripper OMPI brackets . 17. Apparatus as recited in Claim 10 wherein said ultrasonic transducer is shaped toroidially and surrounds a conduit adapted to contain a controlled temperature bath which flows through said conduit. 18. Apparatus as recited in Claim 17 and further comprising: (a) a control switch for varying the power produced by said ultrasonic transducer and (b) an on-off switch for said ultrasonic transducer. 19. Apparatus as recited in Claim 18 and further comprising an on-off switch for varying the ultrasonic energy produced by said ultrasonic transducer between a continuous wave and a series of pulses in the form of chirps. 20. Apparatus as recited in Claim 10 and further comprising a control switch for varying the frequency produced by said ultrasonic transducer. OMPI A

Inventors: FRITCHMAN KATHIE S, MELTON HEWLETT, RHEAD WILLIAM J, STOUT FERRIS M, FRITCHMAN, KATHIE, S., MELTON, HEWLETT, RHEAD, WILLIAM, J., STOUT, FERRIS, M.
Applicants: UNIV IOWA RES FOUND, UNIVERSITY OF IOWA RESEARCH FOUNDATION
Application year: 1984
Main classification: C12N13
IPC codes: C12M3, C12N1, C12N13, H01L31