Classifications

• • C—CHEMISTRY; METALLURGY
  • 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
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/12—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by pressure
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/08—Flask, bottle or test tube
• • C—CHEMISTRY; METALLURGY
  • 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
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/02—Separating microorganisms from their culture media
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
  • H01L31/00—Semiconductor devices sensitive to infrared radiation, light, electromagnetic radiation of shorter wavelength or corpuscular radiation and specially adapted either for the conversion of the energy of such radiation into electrical energy or for the control of electrical energy by such radiation; Processes or apparatus specially adapted for the manufacture or treatment thereof or of parts thereof; Details thereof
  • H01L31/18—Processes or apparatus specially adapted for the manufacture or treatment of these devices or of parts thereof
  • H01L31/184—Processes or apparatus specially adapted for the manufacture or treatment of these devices or of parts thereof the active layers comprising only AIIIBV compounds, e.g. GaAs, InP
  • H01L31/1844—Processes or apparatus specially adapted for the manufacture or treatment of these devices or of parts thereof the active layers comprising only AIIIBV compounds, e.g. GaAs, InP comprising ternary or quaternary compounds, e.g. Ga Al As, In Ga As P

Definitions

• This invention relates to a technique of harvesting mammalian cells from substrates upon which they are being cultured.
• it relates to a technique of harvesting mammalian cells by means of ultrasound employed without chemical enhancement.
• th_ey 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.
• 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.
• 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.
• the preferred frequency range is 20 to 60 kHz.
• the ultrasound is delivered to an external surface of a culture vessel containing the cells to be harvested.
• 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.
• 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.
• 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) .
• T-flask a 25 cc vessel rather like a small, square sealed petri dish with a neck
• the T-flask may be replaced by a conventional roller bottle or any other culture vessel.
• 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.
• 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.
• FIGURES 1 and 2 A first embodiment of apparatus according to the subject invention adapted to harvest cells from roller bottles is shown in FIGURES 1 and 2.
• 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.
• an on-off switch 36 is provided for a "chirper". That is, the ultrasonic transducer 26 can be used either to
• FIGURES 3 and 4 OMPI subject invention adapted to harvest cells from culture flasks is shown in FIGURES 3 and 4.
• 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.
• FIGURE 5 A third embodiment of apparatus according to the subject invention adapted to harvest cells from microcarrier beads is shown in FIGURE 5.
• 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.
• 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
• 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.
• an on-off switch 80 is provided to turn the ultrasonic transducer 60 on and off
• an on-off switch 82 is provided to vary the ultrasonic energy between continuous wave and chirping.

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• Engineering & Computer Science (AREA)
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• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Sustainable Development (AREA)
• Condensed Matter Physics & Semiconductors (AREA)
• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Electromagnetism (AREA)
• Microelectronics & Electronic Packaging (AREA)
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• 1984
  • 1984-08-13 WO PCT/US1984/001263 patent/WO1985001514A1/en unknown
  • 1984-08-13 EP EP84903224A patent/EP0158642A1/de not_active Withdrawn

Non-Patent Citations (1)

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