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WO2016183513A1 - Enceinte de culture cellulaire conditionnée et transportable - Google Patents

Enceinte de culture cellulaire conditionnée et transportable Download PDF

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Publication number
WO2016183513A1
WO2016183513A1 PCT/US2016/032520 US2016032520W WO2016183513A1 WO 2016183513 A1 WO2016183513 A1 WO 2016183513A1 US 2016032520 W US2016032520 W US 2016032520W WO 2016183513 A1 WO2016183513 A1 WO 2016183513A1
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WIPO (PCT)
Prior art keywords
chamber
enclosure
cell culture
transportable
chambers
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Ceased
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PCT/US2016/032520
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English (en)
Inventor
Randy YERDEN
Alicia Henn
Steven BURROWS
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BioSpherix Ltd
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BioSpherix Ltd
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Publication date
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Publication of WO2016183513A1 publication Critical patent/WO2016183513A1/fr
Anticipated expiration legal-status Critical
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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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • 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
    • C12M23/44—Multiple separable units; Modules
    • 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
    • C12M23/54—Constructional details, e.g. recesses, hinges hand portable
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
    • C12M37/02—Filters

Definitions

  • This invention pertains to apparatus useful for growing cells in vitro in highly controlled environmental conditions.
  • Cells in particular stem cells or other mammalian tissue cells, have to be grown in highly controlled environmental conditions. Such cells normally exist in vivo, and to cultivate mammalian cells in vitro requires careful control of factors such as temperature, gases (oxygen and carbon dioxide levels), nutrients, pH of the growth medium, and other factors. Maintaining highly sterile conditions is also critical.
  • WO 201 1/022325 discloses a modular clean room system including biosafety units, and methods of making, and sealing biosafety units.
  • the units comprise at least one controlled air, sealable, sterilizable cleanroom and associated air handling units that provide redundant air to the cleanroom with at least Class 100,000 air purity.
  • this system does not solve the problem of cell culture apparatus being generally open to the environment within the room.
  • US6062976A discloses an apparatus and method for reducing or minimizing the particulates and other contaminants which may be brought into a restricted or clean room area.
  • XVIVOTM system a barrier isolator that provides a modular set of closed incubators and closed hoods, all integrated as co-chambers and sub-chambers. These are relatively small, self- contained units, which greatly reduces the volume of space that must be kept sterile and otherwise conditioned, such as maintaining specific temperature and specialized gas levels.
  • XVIVOTM system chambers can be rapidly assembled in an ordinary laboratory room or surgical suite that requires no specialized air conditioning within the room. The sterile, temperature-controlled, and gas-controlled atmosphere is limited to the XVIVOTM system chambers, not an entire room.
  • C-CHAMBERTM hypoxia chamber for cell culture.
  • This chamber can control the atmosphere around cell cultures in an external incubator.
  • barrier isolators although they are difficult to handle as they can be large and heavy. Their non-slip feet make sliding them between modules in the barrier isolator extremely difficult, so each movement requires lifting in the isolator. They are also more complex, with fans and gas sensors, and are difficult to surface disinfect with disinfectant-soaked gauze.
  • a transportable conditioned cell culture enclosure is disclosed herein.
  • the transportable conditioned cell culture enclosures can be conditioned with the sterile, warmed, gas- controlled isolator atmosphere and used to transport cultures to and house cell cultures within external incubators.
  • This device and the process used with it can help extend the risk reduction of a barrier isolator to the unlimited and flexible incubation capacity of external incubators for industrial cell production. While this device was developed with massively parallel patient-specific batch (scale-out) production in mind, it could also be used to scale-up cultures for larger single batches.
  • the transportable conditioned cell culture enclosure may be an environmentally secure portable enclosure adapted to contain one or more passive culture vessels.
  • the enclosure may have an environmentally sealing door adapted to fit in an isolation apparatus, wherein the door is only opened within the isolation apparatus.
  • a barrier-isolator cell culture growth apparatus having a plurality of interconnected chambers with controlled environmental conditions within, with at least one chamber having an exit port to the external environment, at least one buffer chamber and at least one work chamber.
  • Each chamber has an environmentally sealing door at the interface with any other chamber. Any chamber except a chamber with an exit port is maintained as a highly sterile environment isolated from the external environment in which the plurality of interconnected chambers resides.
  • the temperature and gases within each chamber can be precisely controlled.
  • a transportable conditioned cell culture enclosure with an environmentally sealing door is adapted to fit within a buffer chamber and configured so the door of the transportable conditioned cell culture enclosure only opens into the processing chamber.
  • HEPA air filtration apparatus need only maintain low particulate counts for the air in the chamber, not in an entire room. Also, since operators only work through glove-box gloves, or with a robotic arm, the need for full body sterile garb and air showers is avoided.
  • the transportable conditioned cell culture enclosure passive culture vessels as described herein solve the significant operational problem of minimal storage space in this system, by providing a highly efficient method of transporting materials in an environmentally secure manner to other apparatus such as incubators or refrigeration units.
  • FIG. 1 A is a perspective view of an embodiment of the transportable
  • Fig. 1 B is a perspective view of an embodiment of the tray 20.
  • Fig. 1 C is a perspective view of the transportable enclosure and tray together.
  • Fig. 1 D is a side elevation view of an embodiment (12) of the transportable conditioned cell culture enclosure.
  • Fig. 1 E is a perspective view of embodiment 12 of the transportable
  • Fig. 1 F is a perspective view of embodiment 14 of the transportable
  • Fig. 1 G is a cut-away perspective view of embodiment 14 of the transportable conditioned cell culture enclosure.
  • Fig. 2A shows the transportable chamber being transported manually on the tray, with an operator using the handles.
  • Fig. 2B shows the transportable chamber in a buffer chamber with the door open into the processing chamber.
  • Fig. 2C shows a series of transportable chambers stacked into a temperature controlled incubator.
  • FIG. 3 shows a barrier-isolator cell culture growth apparatus illustrating several modular chambers interconnected.
  • FIG. 4 shows schematics of the work flow of the transportable enclosure inside a barrier-isolator cell culture growth apparatus.
  • FIG. 4A shows a schematic of a barrier-isolator cell culture growth apparatus, depicting two zones, one with culture enclosures sealed, and the other where culture enclosures are open.
  • Fig. 4B shows the throughput of transportable conditioned cell culture enclosures schematically.
  • Fig. 4C shows the throughput of transportable conditioned cell culture enclosures schematically.
  • Fig. 4D shows the throughput of transportable conditioned cell culture enclosures schematically.
  • Fig. 4E shows the throughput of transportable conditioned cell culture enclosures schematically.
  • Fig. 5A shows cell growth kinetics of K562 cells in the transportable chambers compared culture inside the isolator.
  • Fig. 5B shows cell viability of K562 cells in the transportable chambers compared culture inside the isolator.
  • Fig. 5C shows stable variability of K562 cells grown in several transportable chambers.
  • Fig. 5D shows the number of cell divisions in three separate transportable chambers was comparable to cells grown in the isolator.
  • Fig. 5E shows that variability in cumulative cell numbers did not increase over time when grown in several transportable chambers.
  • Fig. 5F shows that cell viability was comparable between cultures maintained in the transportable chamber and cells maintained full-time in the barrier isolator.
  • Fig. 5G shows a plot of the atmosphere in an inventive chamber over time with various filter and door arrangements.
  • FIG. 6 shows schematically how environmental monitoring of bioburden showed no contamination of processing chamber air or floor was performed.
  • chamber refers to the interior compartments of the barrier isolator system.
  • enclosure refers generally to box-like enclosures designed to contain passive culture vessels.
  • vessel refers to various embodiments of vessels that directly contain culture media and cell cultures.
  • a transportable conditioned cell culture enclosure is provided.
  • the transportable conditioned cell culture enclosure may be an enclosure adapted to contain one or more passive cell culture vessels in an
  • a "passive culture” is a cell culture in which the cells and the medium rests in a vessel on an incubator shelf with no active gas exchange or mechanical agitation. A passive culture only gets gas exchange with the incubator through passive gas diffusion, such as through a loose lid, vents or membranes in the vessel. There is no mechanical agitation of the medium or vessel, such as stirring, swirling, shaking, rocking, with or without the bubbling of piped gases through the medium, to increase gas exchange rates or to stir the contents of the culture.
  • a cell culture growth apparatus having a plurality of interconnected chambers that serve as a barrier isolator 400 (Fig. 3) with controlled environmental conditions within each chamber.
  • at least one chamber has an exit port to the external environment
  • at least one chamber is a buffer chamber and at least one chamber is a processing chamber.
  • Each chamber may have an environmentally sealing door at the interface with any other chamber. Any chamber except a chamber with an exit port may be maintained as a highly sterile environment isolated from the external environment in which the plurality of interconnected chambers resides.
  • the temperature and gases within each chamber can be precisely controlled.
  • a transportable conditioned cell culture enclosure with an environmentally sealing door may be adapted to fit within a buffer chamber and configured so the door of the transportable conditioned cell culture enclosure only opens into the processing chamber.
  • the transportable conditioned cell culture enclosure can be used to extend the risk reduction of a barrier isolator to external incubators for increased cell culture incubation capacity.
  • the barrier isolator plurality of interconnected chambers is modular and adapted to rapid assembly and disassembly with uniform fittings.
  • the plurality of interconnected chambers has an air handling apparatus that maintains air particulates to ISO 8 (ISO 14644-1 ), also termed class 100,000 (FED-STD-209E) or better.
  • the air particulates may be ISO 7, 6, 5, 4, 3, 2, 1 or better.
  • the apparatus used to achieve this low particulate count is a HEPA filter.
  • all gases introduced into the barrier isolator chambers are from high purity compressed gas tanks, or liquid nitrogen. Other air filtration and conditioning systems may also be employed.
  • at least a small positive pressure of conditioned gases is maintained in the barrier isolator chambers to minimize any back pressure causing external air to enter into the barrier isolator.
  • a HEPA filter is integrated into a wall or door of the transportable conditioned cell culture enclosure.
  • a HEPA filter is integrated into one or both doors at each end of the enclosure.
  • the gas contents of at least one chamber are precisely measured and controlled. In most cases, this refers to the level of oxygen and carbon dioxide within the chamber, but other gases may be relevant, such as nitrogen, nitrous oxide, carbon monoxide, or other reactive or inert gases.
  • the chamber where the gas contents is likely to be most critical is the processing chamber, where the culture vessels are opened and sealed, and where other manipulations are performed, such as addition of nutrients and growth medium, or harvesting and isolation of cells following incubation.
  • the temperature of at least one chamber is precisely monitored and controlled.
  • the processing chamber has a window permitting an operator to view the interior.
  • one or more of the chambers has integrated gloves permitting an operator to manually perform mechanical operations in the interior of the chamber.
  • a robotic arm is installed within at least one chamber for performing mechanical operations in the interior of the chamber.
  • a robotic arm is installed that spans at least two adjacent chambers, wherein the arm can perform mechanical operations in the interior of either chamber or move contents from one chamber to another or both.
  • the amount of light within the interior of any chamber is precisely controlled.
  • At least one chamber is a disinfectant chamber situated between a chamber having an exit port and a processing chamber.
  • At least two buffer chambers are present between the processing chamber and any other chamber.
  • the door on the transportable conditioned cell culture enclosures is generally illustrated herein as having magnetic strips that form a sufficient hermetic seal yet are easy to open within a processing chamber.
  • other door sealing embodiments are possible, such as mechanical latches and rubber seals.
  • the door may also have a hinge.
  • the transportable conditioned cell culture enclosure shown in Fig. 1A. as 10, has a box 100, a door 110.
  • a handle 112 on the door facilitates opening and closing of the door.
  • Magnetic strips 150 form a hermetic seal.
  • the chamber 10 may rest in tray 20, shown in Fig. 1 B.
  • Tray 20 has a platform 200 adapted to hold chamber 10, and handle sections 210 that allow an operator or robot to pick up the tray and move it efficiently.
  • the chamber and tray combination is shown in Fig. 1 C.
  • Tray 20 may be adapted to easily slide on skids or small wheels on a smooth surface, such as the floor of the barrier isolator, allowing the tray and its contents to be easily moved from one location to another without the need to pick up the tray.
  • a door on an end of the enclosure is a HEPA air filter in a frame and sized to fit snugly on an end of the enclosure. This is shown as 14 in Figs. 1 F and 1 G.
  • the HEPA filter frame 164 supports HEPA filter 162 within door 114.
  • Door 114 may include a hermetic seal, for example with magnetic strips 150 or latches and gaskets that make an airtight seal when latched.
  • the latches may be lever operated-type latches.
  • HEPA filter frame-doors 114 are used at each end of the enclosure.
  • the HEPA filter frame-door 114 is used instead of any other type of door for the inventive enclosure.
  • the transportable conditioned cell culture enclosure may have one HEPA filter door 114, and one simple door 110.
  • a transportable conditioned cell culture enclosure may have an air filter integral with the enclosure. This is illustrated as enclosure 12 in Figs. 1 D and 1 E. Enclosure 12 has a HEPA filter 160 shown here opposite the door panel 110. Enclosure 12 may be transported using a tray 20.
  • the transportable conditioned cell culture enclosures 10 and 12 are designed to be used with a barrier isolator system 400, such as shown in Figs. 3 and 4.
  • Fig. 3 shows an embodiment of an installed system, with an entry/exit port chamber (laminar flow hood), two buffer chambers 410, a disinfectant chamber 430, and two processing chambers 420, shown with integral gloves 450 for performing manipulations within the processing chambers.
  • the processing chambers and disinfectant chambers are gloveboxes.
  • the operators (not shown) would stand or sit with their arms inside the gloves.
  • base support cabinets 460 are also shown.
  • FIG. 4 shows several schematics of barrier isolator systems illustrating the work flow of how the inventive transportable conditioned cell culture enclosure 10 might be used.
  • the transportable conditioned cell culture enclosure 10 (marked TC-4) enters the system through the laminar flow hood entry and exit port.
  • a laminar flow hood may have a strong positive pressure of filtered air or compressed gases and may have a door, so that when the door is opened, outside air, which is potentially contaminated, will not enter the hood.
  • the transportable conditioned cell culture enclosure 10 is moved to a buffer chamber, where the buffer chamber environment is subject to air exchange by iterative log-dilution (for example, dilution to 1/10 th of initial oxygen concentration with nitrogen, then re-charge with oxygen to initial level) with filtered gas to remove airborne particles and minimize external atmosphere exposure to future steps.
  • the buffer chambers may be pumped with a vacuum pump to evacuate the internal atmosphere before fresh gases are introduced.
  • the buffer chamber may have hermetic doors on two sides, allowing the enclosures such as 10 to be inserted into the interior of the buffer chamber on a first side and removed from the buffer through a second door on a different side of the buffer chamber.
  • the buffer chamber doors are sealed, and the interior of the buffer chamber may be subject to the dilution and gas replacement operation as described in this paragraph.
  • the chambers 10 are moved to the next processing station, which may be the disinfection chamber or the processing chamber.
  • this operation may also be used in reverse when enclosures such as 10 or 12 are being moved out of the barrier isolator.
  • the enclosures 10 or 12 are being moved out, there is no need to dilute the buffer chamber atmosphere, since the enclosure will be moved outside of the barrier isolator.
  • a transportable conditioned cell culture enclosure 10 enters through a laminar flow hood.
  • TC4 Handling Zone two zones in the barrier isolator apparatus are clearly depicted.
  • the first is marked “TC4 Handling Zone,” and in all chambers in this region, the culture vessels are sealed.
  • This zone includes the laminar flow hood entry/exit port, the first set of buffer chambers, and the disinfectant chamber.
  • the transportable conditioned cell culture enclosures remain sealed.
  • the second zone is the "Cell Handling Zone” and in this zone, the culture vessels are opened and manipulations to the cells or culture vessels are performed.
  • the transportable conditioned cell culture enclosures are opened to the sterile and highly conditioned environment within the processing and other operational chambers.
  • the barrier isolator system (such as the XVIVOTM system) is modular and other chambers and work configurations can be added.
  • Fig. 4A several additional processing chambers are depicted, such as a chambers containing incubators, a centrifuge, and a microscope.
  • Fig. 4B depicts two transportable conditioned cell culture enclosures moving through the barrier isolator.
  • One transportable conditioned cell culture enclosure (TC4- A) is being conditioned in a buffer chamber 2, and the other (TC4-B) is in the laminar flow hood section waiting to move into the barrier isolator.
  • Fig. 4C depicts the transportable conditioned cell culture enclosure TC4-A parked in buffer chamber 4 and open to the processing chamber.
  • Transportable conditioned cell culture enclosure TC4-B is in buffer chamber 2 and being moved to the disinfection chamber.
  • a third transportable conditioned cell culture enclosure, TC4-C is in the laminar flow hood waiting to be moved into the barrier isolator.
  • Fig. 4D depicts TC4-A being loaded with a passive culture vessel, and TC4-B in buffer chamber 1 ready to be opened into the processing chamber. Enclosure TC4-C is in buffer chamber 2 being conditioned for the disinfection chamber.
  • Fig. 4E depicts enclosure TC4A in buffer chamber 1 ready to be moved out of the barrier isolator through the laminar flow hood.
  • TC4-B is being actively worked on in the processing chamber.
  • TC4-C is being moved to the disinfectant chamber.
  • material can be safely and efficiently moved in and out of highly conditioned sterile zones without contaminating the cell culture vessels being transported or the highly conditioned sterile zones within the barrier isolator.
  • various disinfecting operations may be performed in the disinfection chamber. This may include exposure to UV light and wiping and cleaning the exterior of the chamber 10 with disinfectant-soaked wipes, such as SporKlenz- soaked (a disinfectant) gauze. This cleaning operation may include all surfaces of the chamber 10 and all surfaces of the tray 20.
  • disinfectant-soaked wipes such as SporKlenz- soaked (a disinfectant) gauze.
  • This cleaning operation may include all surfaces of the chamber 10 and all surfaces of the tray 20.
  • the box 10 is then moved into a second buffer chamber (marked chamber 4 in Fig. 4) and an additional log-dilution air exchange is performed.
  • the buffer chamber is then equilibrated with filtered gas content to match the processing chamber.
  • the buffer chamber door is then opened into the processing chamber.
  • the transportable conditioned cell culture enclosure 10 is then ready to be opened to the processing chamber for access to cultures and equilibration with isolator atmosphere.
  • the box 10 remains in the buffer chamber and only the door 112 of box 10 is removed. This exposes the interior of enclosure 100 to the environment of the processing chamber.
  • the operator can remove the contents of the transportable conditioned cell culture enclosure 10, which will typically be cell culture vessels.
  • Various embodiments of cell culture vessels are known in the art, such as flasks, well plates, and bags.
  • flasks containing a culture medium are illustrated.
  • Some operations that may be performed in the processing chamber include, but are not limited to, adding or removing contents of the cell culture vessels, harvesting cells from the cultures, and testing media for cell growth. Pipetting and other liquid transfer operations may be performed. Nutrients, chemical reagents, buffer solutions, etc., may be added. Chemical measurements, such as pH measurements, colorimetric measurements, standard molecular biology techniques such as agar gel
  • Additional modules of the isolator system may be ganged onto the processing chamber.
  • a chamber with a microscope may be added, or a chamber with a centrifuge.
  • Other chambers may have bioreactors for growing specialized tissue types.
  • two or more buffer chambers are provided between the processing chamber and the disinfection chamber. In an embodiment, two or more buffer chambers are provided between the disinfection chamber and the entry/exit port.
  • the use of a plurality of buffer chambers greatly increases the throughput possible in the barrier isolator system. For example, an operator can work with one transportable conditioned cell culture enclosure 10 while a second chamber 10 is being equilibrated for ingress or egress to the processing chamber.
  • all materials including chemical reagents, mechanical or electrical equipment, samples, harvested products, etc., must pass through the buffer chambers to enter or exit the barrier isolator once the isolator is set up and operational.
  • FIG. 2A The transportable conditioned cell culture enclosure 10 shown in Fig. 2A, being held by a person, with the accompanying transport tray 20. Chamber 10 is depicted as containing cell cultures in flasks.
  • the transportable conditioned cell culture enclosure 10 can be manually disinfected with a disinfectant wipe inside the barrier isolator to reduce the risk of contamination of the processing chamber.
  • Fig. 2B the transportable conditioned cell culture enclosure 10 is shown within a buffer chamber 410, with the hatch 412 open. Enclosure 10 is shown resting on tray 20 with handles 210 visible. In this embodiment, the hatch swings up to open the buffer chamber to allow the transportable conditioned cell culture enclosure and other equipment and supplies to be moved in and out of the highly controlled environment in the interior of the barrier isolator apparatus.
  • FIG. 2C multiple transportable conditioned cell culture enclosures 10 are housed in an exterior incubator 510, shown with the door 512 (partially cut away) open.
  • a plurality of such incubators can be employed to expand incubation space at relatively low cost.
  • the transportable enclosure remains outside of the critical space for open cell processing at all times (the cell handling zone depicted in Fig. 4A), minimizing the risk of microbial contamination from the external environment. All manipulations of cell cultures occur in a "safe zone", in modules further separated from the room environment than the modules in which the transportable chamber is handled. All cell cultures are always enclosed in the transportable chamber when outside of this "safe zone” for cell handling. In an embodiment, the transportable enclosure never enters the cell handling "safe zone", but remains parked in the buffer chamber during cell handling. [0081] Table 1 shows experiments showing that the processing chamber was not contaminated by the transportable conditioned cell culture enclosure 10 when used as disclosed herein.
  • the transportable conditioned cell culture enclosure 10 can be modified to have the following features:
  • Fig. 5A shows K562 cells grown in the isolator (in triplicate) in 20% O2 or in TC4 chambers conditioned in the isolator during routine cell culture manipulations and housed in external incubators had indistinguishable cumulative cell numbers (p>0.5, paired T-test, Day 25).
  • Fig. 5B shows that viability was greater than 98% at all times as measured by trypan blue exclusion.
  • Cell cultures in separate TC4 chambers did not vary in cell growth characteristics increasingly over time (Fig. 5C), as would be expected if critical cell growth parameters varied between chambers.
  • Fig. 5D shows the number of cell divisions in three separate CTCs was comparable to cells grown in the isolator and cells grown in open conditions. This data was generated using a prototype TC4 using twice-weekly cell counts the number of cell divisions at each time point. Cumulative cell divisions were quite similar between different growth chambers, isolator, and open (flask on exterior incubator shelf, subcultured in laminar flow hood) cultured cells.
  • Fig. 5E shows that variability in cumulative cell numbers did not increase over time. If critical cell parameters varied between the three separate TC4 chambers and the cultures maintained in the barrier isolator system, then we would expect increasing differences between the cultures as they grew over time. However, the standard deviation in cumulative cell numbers did not increase.
  • Fig. 5F shows that cell viability was comparable between cultures maintained in the TC4 and cells maintained full-time in the barrier isolator. Viability as assessed by trypan blue exclusion dipped slightly when there was a change in FBS lots in use at day 7, but was greater than 90% in all closed system culture.
  • Fig. 5G shows the atmosphere in an inventive chamber over time with various filter and door arrangements.
  • the atmosphere in the incubator is line 300.
  • the conditioned atmosphere is line 310.
  • An oxygen probe was sealed into a test TC4 chamber through a small hole in the top surface.
  • the chamber was equilibrated in the barrier isolator to 5% oxygen to assess equilibration with incubation air (-20% oxygen).
  • the test chamber was assayed three times in each configuration; with no doors, with two covers in place over the filters, with one filter uncovered, and with two filters (for example embodiment is a perspective view of embodiment 14 of the transportable conditioned cell culture enclosure, as shown in Figs. 1 F and 1 G) uncovered.
  • Interior oxygen levels were recorded every fifteen minutes. With filter doors uncovered on each end, the chamber equilibrated rapidly, reaching maximal oxygen in about 30 min. The mean of three trials is shown for each configuration. This experiment demonstrates rapid equilibration of the chamber with HEPA-filtered incubator atmosphere.
  • FIG. 6 shows that environmental monitoring of bioburden showed no contamination of processing chamber air or floor. While no microbial contamination of cell cultures was evident over 4 weeks, we monitored air in the unit during each cell processing session. An air sampler was used to draw air onto a bacterial growth touchplate. In addition, after processing each TCC culture, touchplates were used to monitor three places on the chamber floor. Less than one CFU was found on each plate after incubation.

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Abstract

La présente invention concerne une enceinte de culture cellulaire conditionnée et transportable qui est prévue pour contenir des récipients de culture cellulaire passive dans une enceinte portable sûre pour l'environnement. Le récipient peut comporter une porte hermétique à l'environnement adaptée pour s'intégrer dans un appareil d'isolation, la porte étant seulement ouverte à l'intérieur de l'appareil d'isolation. Dans un mode de réalisation, l'enceinte de culture cellulaire conditionnée et transportable comprend un filtre HEPA d'un seul tenant. L'invention concerne également une appareil de croissance de culture cellulaire de type barrière-isolateur comprenant une pluralité de chambres interconnectées dans lesquelles règnent des conditions environnementales régulées, avec au moins une chambre comprenant un orifice de sortie sur l'environnement extérieur, au moins une chambre tampon et au moins une chambre de traitement. La température et les gaz à l'intérieur de chaque chambre peuvent être régulés avec précision. Le récipient de culture passive de l'enceinte de culture cellulaire conditionnée et transportable est adapté pour se loger à l'intérieur de la chambre tampon et configuré de sorte que la porte de l'enceinte de culture cellulaire conditionnée et transportable s'ouvre uniquement dans la chambre de traitement.
PCT/US2016/032520 2015-05-13 2016-05-13 Enceinte de culture cellulaire conditionnée et transportable Ceased WO2016183513A1 (fr)

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EP3199614A4 (fr) * 2014-09-26 2018-06-06 Public University Corporation Yokohama City University Système de culture cellulaire
EP3736091A4 (fr) * 2018-01-05 2021-04-28 JGC Japan Corporation Système d'isolateur
IT202000024310A1 (it) * 2020-10-15 2022-04-15 Bioair S P A Sistema di trasferimento di campioni biologici per sistemi chiusi
CN114774235A (zh) * 2022-04-12 2022-07-22 温州维科生物实验设备有限公司 生物培养系统及方法
CN115627228A (zh) * 2021-05-31 2023-01-20 北京永泰生物制品有限公司 密闭培养系统

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US20070281351A1 (en) * 2006-06-02 2007-12-06 Toyoshige Kobayashi Culture system, culture apparatus, culture vessel box, and air cleaning method
US20110183411A1 (en) * 2010-01-22 2011-07-28 Highres Biosolutions Self-sterilizing automated incubator

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US5730777A (en) * 1993-07-16 1998-03-24 Peter Mosborg Peterson Method and apparatus for performing operations
WO1998052629A2 (fr) * 1997-05-20 1998-11-26 Zymequest, Inc. Appareil et procede de traitement de cellules biologiques
US20070281351A1 (en) * 2006-06-02 2007-12-06 Toyoshige Kobayashi Culture system, culture apparatus, culture vessel box, and air cleaning method
US20110183411A1 (en) * 2010-01-22 2011-07-28 Highres Biosolutions Self-sterilizing automated incubator

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3199614A4 (fr) * 2014-09-26 2018-06-06 Public University Corporation Yokohama City University Système de culture cellulaire
US10647955B2 (en) 2014-09-26 2020-05-12 Public University Corporation Yokohama City University Cell culture system
EP3736091A4 (fr) * 2018-01-05 2021-04-28 JGC Japan Corporation Système d'isolateur
EP3964289A1 (fr) * 2018-01-05 2022-03-09 JGC Japan Corporation Système d'isolateur
IT202000024310A1 (it) * 2020-10-15 2022-04-15 Bioair S P A Sistema di trasferimento di campioni biologici per sistemi chiusi
EP3985093A1 (fr) 2020-10-15 2022-04-20 Bioair S.p.A Système de transfert d'échantillons biologiques
CN115627228A (zh) * 2021-05-31 2023-01-20 北京永泰生物制品有限公司 密闭培养系统
CN114774235A (zh) * 2022-04-12 2022-07-22 温州维科生物实验设备有限公司 生物培养系统及方法

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