Title of Invention

CASSETTE CONTAINING GROWTH MEDIUM

Abstract The invention features devices for capturing and culturing cells (e.g., microorganisms cells containing microorganisms, or cells from eukaryotic cell cultures) and methods of using these devices. One device is a cassette containing growth media that may be employed in an automated rapid enumeration system. The cassette has, for example, been enhanced with features for controlling surface flatness, optical imaging, controlled Dehydration of semi solid nutrient media, controlled air and particle exchange, and automated handling. Another device of the invention is a filtration funnel that may used to concentrate cells in a sample onto a membrane.
Full Text

PATENT ATTORNEY DOCKET NO. 50050/007WO2
CASSETTE CONTAINING GROWTH MEDIUM
BACKGROUND OF THE INVENTION
The invention relates to the fields of cell culture and detection.
In many industries, particularly the food, beverage, healthcare, electronic, and pharmaceutical industries, it is essential to rapidly analyze samples for the degree of contamination by microorganisms, such as bacteria, yeasts, or molds.
One microbial culture technique, called microbial enumeration or colony counting, quantifies the number of microbial cells in a sample. The microbial enumeration method, which is based on in situ microbial replication, generally yields one visually detectable "colony" for each microbial cell in the sample. Thus, counting the visible colonies allows microbiologists to determine the number of microbial cells in a sample accurately. To Performa microbial enumeration, bacterial cells can be dispersed on the surface of nutrient agar in Petri dishes ("agar plates") and incubated under conditions that permit in sits bacterial replication. Microbial enumeration is simple, ultra-sensitive, inexpensive, and quantitative but is also slow. The long time required results in increased costs in healthcare and in manufacturing. More rapid enumeration methods have been developed but, while shortening the time required, they have sacrificed one or more of the critical advantages of microbial culture.
There is a need for additional culturing devices and methods for microbial enumeration.
SUMMARY OF THE INVENTION
The invention provides a cell culture device - referred to as a cassette -that includes a housing that contains growth medium for microorganisms, wherein at least a portion of said housing is substantially non-radioactive. The device may further include a detachable, scalable lid having an optically clear

window disposed to allow imaging of the growth medium. This lid may be the substantially non-radiative portion of the housing. Preferably, the window and housing are substantially non-radiative, substantially non-reflective, or both. The housing may also include openings that provide for a tortuous passage of gases from the growth medium to the ambient environment, e.g., so that the housing has a gas exchange cross-sectional area of The cassette of the invention may also be provided in a kit for detecting microorganisms together with a membrane adapted to be placed over the growth medium such that microorganisms deposited on the membrane receive nourishment from the medium. The kit may further include a filtration vessel that allows the membrane to collect microorganisms from a sample passed through the membrane. In one embodiment, prior to being placed over the medium, the membrane remains substantially planar during the passing of the sample through the membrane whereby microorganisms are deposited on the membrane. Preferably, the membrane is substantially non-radiative or black or both.
The cassettes and kits of the invention may be used in any method for growth, assay, or maintenance of microbes, including enumeration, detection, diagnosis, or therapeutic response.
By a "non-radiative" object is meant an object that does not emit light, e,g., by fluorescence, phosphorescence, or luminescence.
By a "non-reflective" object is meant an object that reflects less than 25%, 10%, 5%, 1%, or 0.1% of the light used to image the object.
Other features and advantages will be apparent from the following description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA is a schematic depiction of the imaged side of a cassette of the invention. Figure IB is a schematic depiction of a funnel that may be employed in conjunction with a cassette of the invention.
Figure 2 is a schematic depiction of the imaged side of a cassette and a cross-section of the cassette. The elements of the cassette that allow for detection of auto-fluorescence from microbial colonies are indicated.
Figure 3 A is a schematic depiction of various views of a cassette showing the air management channels. Figures 3B-3C are cross-sectional views of the cassette showing an inlet for air.
Figure 4A is a schematic depiction of growth medium disposed in a cassette. Figures 4B-4C are various schematic views of the cassette containing growth media.
Figure 5 A is a schematic depiction of a removable lid against which growth media is cast. Figure 5B is a schematic depiction of the forces acting on the growth media.
Figure 6 is a schematic depiction of the removal of the lid from the cassette yielding raised and proud growth media surface.
Figure 7 is a schematic depiction of the cross-sectional views of a cassette illustrating a masking feature that covers the edges of a membrane disposed on top of growth media.
Figure 8 is a schematic depiction of the sealing of an optical window to the cassette.
Figures 9A-9B are schematic depictions illustrating how design elements of the cassette can be used to aid automatic loading of the cassette. Figures 9C-9D are schematic depictions of a cassette carrier and a plurality of cassettes loaded in the carrier that allow for automatic manipulation of multiple cassettes. Figures 9E-9H are photographs of a cassette carrier and a latch for locking the carrier in place.
Figures lOA-old are schematic depictions of automated gripping and release of a cassette.

Figure 11 is a schematic depiction of a through hole in the mask in the cassette that provides a fluorescent fiducially mark.
Figure 12 is a schematic depiction of a funnel.
Figures 13A-13B are various views of a funnel.
Figure 14A is a schematic depiction of a manifold adaptor for use with a funnel. Figure 14B is an exploded view of the components of a funnel. Figure 14C is a schematic view of a component of a funnel having indentations for removing a membrane.
The figures are not necessarily to scale
DETAILED DESCRIPTION OF THE INVENTION
The invention features devices for capturing and culturing cells (e.g., microorganisms, cells containing microorganisms, or cells from eukaryotic cell cultures) and methods of using these devices. One device is a cassette containing growth media (also referred to as nutrient media) that may be employed in an automated rapid enumeration system such as the Growth Direct™ system, e.g., as described in U.S. Publication No. 2003/0082516, which is hereby incorporated by reference. Another device is a filtration funnel that may used to concentrate cells in a sample onto a membrane. An exemplary cassette and a filtration funnel assembly are shown in Figures lA-IB.
The Growth Cassette '^^ is an advanced device that has, for example, been enhanced with features for controlling surface flatness, optical imaging, controlled dehydration of semi solid nutrient media, controlled air and particle exchange, and automated handling. These attributes enable, e.g., automated imaging of microbial growth. Any solid or semi-solid growth media can be employed in the present cassette. Examples include Sabouraud dextrose agar (SDA), R2A agar, cryptic soy agar (TSA), and plate count agar (PCA).
In various embodiments, the cassette facilitates automated imaging of auto fluorescent microbial microcolonies containing fewer than 500 cells, for example, by employing materials with fluorescence properties commensurate

with such detection* An exemplary material is black K-Resin® (styrene-butadiene-copolymer; Chevron Phillips). The cassette may also employ a transparent lid that has fluorescence properties commensurate with detection of autofluorescent microbial microcolonies. An exemplary material for the lid is Zeonor® 1060R (polycycloolefin resin; Zeon Chemicals LP). Glass may also be employed. A porous membrane may also be employed that has fluorescence properties commensurate with detection of autofluorescent microbial microcolonies. Membranes may be manufactured from materials including cellulose, cellulose acetate, polystyrene, polyethylene, polycarbonate, polyethylene terephthalate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, nylon, and silicone copolymer. The choice of membrane depends, in part, on the type of cell to be cultured (e.g., microorganisms that grow attached to a surface (anchorage-dependent), microorganisms that grow in suspension (anchorage-independent), or microorganisms that grow as attached to a surface or in suspension), degree of permeability, and rate of transfer of fluids and gases. An exemplary membrane is a black mixed cellulose ester membrane (Sartorius AG). Portions of the cassette that will not be imaged may be made of any suitable material, e.g., acrylonitrile-butadiene-styrene or styrene-acrylonitrile.
Preferably, a cassette has minimal air exchange with the ambient environment (e.g., air exchange cross sectional area of The cassette may also be designed for introducing nutrient media through a face other than the face that is proximal and parallel to the growth surface, i.e., from the side. Such a cassette may include a port on the edge of the cassette. The media in a cassette may also be sealed prior to use to minimize dehydration. Such cassettes have media encapsulated on the top, bottom, and sides. A flat media surface desirable for planar imaging may be achieved through the design of a removable pouring lid and method for pouring

lid retention post sterilization, e.g., by including glass beads to prevent relaxation of the plastic and detachment from nutrient medium. Preferably, the media is proud with respect to the material of the cassette. This configuration allows for the retention of flatness as the media dries during use. Typically, media is highly fluorescent, and the invention allows for the use of a porous membrane to cover the media to reduce background emission. The cassette may also feature a mask, i.e., a rim, that covers the edges of the membrane, and any exposed media, during use.
Preferably, a cassette is capable of being stacked in a carrier, e.g., designed to transfer and introduce a group of cassettes to an automated imaging instrument. Such automated handling of a cassette may include transport, interfacing between the cassette and carrier, positioning for automated handling, and capability for robotic transfer. The cassette may also be designed to allow for reproducible mechanical positioning, i.e., repeatedly being able to return the same cassette to same location for automated imaging.
A cassette may also include design features that facilitate alignment of multiple images. Imaging fiducial marks include a through hole aperture over fluorescent plastic or media. Imaging fiducial marks also include printed or embossed fluorescent material on cassette. Other fiducial marks are known in the art.
The funnel of the invention may be employed to capture on a membrane, e.g., by size-based filtration, microorganisms present in a sample, which may be liquid or air. Capture is achieved by introducing the medium, e.g., a liquid or gas sample, to be filtered in the funnel and creating a pressure difference to cause the medium to flow through the membrane. The membrane may then be contacted with a growth medium in a cassette, e.g., by manual transfer from the funnel assembly. Microorganisms on the membrane may then grow into colonies and be imaged, e.g., according to the methods of U.S. Publication No. 2003/0082516. Other methods for transferring the membrane or otherwise placing samples on the cassette are known in the art. Preferably, the filter includes a flat surface, e.g., sintered plastic beads from Pore, to support the

membrane during filtration to reduce or prevent marks on the membrane. Concomitant with this feature, the filter may include access areas to allow for manipulation of the membrane without damage to the imaged area. The filter may also prevent media from passing through the edge of the membrane, e.g., to prevent microorganisms from depositing on the edges of the membrane that may be covered by the mask described above.
Cassettes with or without funnels may be employed in any method where microbial growth is desired, including detection, enumeration, diagnosis, and therapeutic response. Exemplary fields of use include testing liquid, air, or surface samples for microbial bioburden; testing industrial samples, sterile pharmaceutical product samples, non-sterile pharmaceutical product samples for microbial bioburden; and testing samples for anaerobic microbial bioburden. The cassettes are compatible with imaging autofluorescent microcolonies under anaerobic conditions and may include components for actively removing molecular oxygen (e.g., a gas-pack or equivalent). Any microbe, including bacteria, cyan bacteria, protozoa, fungi, mammalian cells, plant cells, or other eukaryotic cell, may be employed in conjunction with the cassette and funnel described herein.
The invention will now be further described with respect to certain preferred embodiments.
Cassette facilities auto imaging of autofluorescent microbial microcolonies
Figure 2 shows elements of cassette materials that allow auto imaging of autofluorescent microbial microcolonies, e.g., that facilitates auto imaging of autofluorescent microbial microcolonies containing fewer than 500 cells. As is shown in the figure, the top surface is manufactured from (or coated with) a material that has low autofluorescence and reflection. The cassette includes an

optical window to allow for imaging of microbes on a membrane disposed to cover growth media. The membrane is also not fluorescent.
Nutrient media controlled drying profile, air management
To achieve a flat nutrient media surface for imaging over maniple days, we have invented a minimum air exchange passage system, whereby the microorganisms are delivered sufficient oxygen for growth promotion, but the air exchange is greatly limited compared to a standard Petri dish. The flow of the air and its impingement upon the media surface is managed to provide uniform drying of the nutrient media. Figures 3A-3C show elements of the air management system. Figure 3 A illustrates how two halves of a cassette may be mated to produce a tortuous air path. Figures 3B-3C show a close-up view of an air hole and its spatial relation to the media.
Sealable nutrient media cassette, fellable via a port on the edge of the cassette
To retain and encapsulate the nutrient media within the cassette so that it is contacted with the circumferential side walls and the back or bottom surface as well as a third surface formed by the removable pouring lid, we have invented a side fill cassette that is fully sealed post filling. The side filled cassette allows the media to be encapsulated over 100% of its surface area (top, bottom, and sides). The sealed cassette provides for greater shelf life and prevents media drying while in storage. To prevent trapped air bubbles from accumulating in the media, we may utilize a split overfill chamber. Figures 4A-4C show elements of the side filled, sealable cassette.
Flat media surface for imaging, achieved thru design of removable pouring lid and design method for pouring lid retention post sterilization.
To achieve a flat media surface for imaging, we have designed a removable pouring lid with a radial lip to seal to the cassette. The profile of the lid's underside surface may be flat or contoured such that the radial retention

loads counteract and create a flat surface. A specially designed top surface, gate, and process controls allow the material to be fed into the mold to prevent any disturbance or visible blush to the underside of the lid, i.e., the surface of the lid is smooth. Glass beads may be embedded into the resin to counteract the creep effects of plastic under load and the creep effects induced through common sterilization methods, such as gamma irradiation. The combined effect of these attributes is a pouring lid that allows for rapid impingement of the molten liquid media during filling without being displaced or bowing or warping.
Figures 5A-5B show elements of the flat media surface for imaging, achieved thru design of removable pouring lid and design method for pouring lid retention post sterilization.
Design method of maintaining flat media surface for imaging over multiple days by means of proud nutrient media
To allow the media the opportunity to shrink as it dries in a controlled flat manner, we have designed a feature to have a portion of the media proud compared to the cassette side walls. This feature prevents any interaction and restriction to the edge of the media that would occur if it were in contact and subject to resistance of movement.
Figure 6 shows elements of the design method of maintaining flat media surface for imaging over multiple days by means of proud nutrient media.
Design to block fluorescence from the nutrient media
The cassette specifically uses a low fluorescent, e.g., flat black, membrane to cover the highly fluorescent nutrient media agar. To cover the edge effects where the membrane, e.g., mixed cellulose ester, may not completely cover the media or where absorption and drying create a bright fluorescent edge to the membrane, we have designed a mask to cover this region. To prevent the mask from reflecting light back to the imager, the mold

surfaces of the mask are treated to form a vapor honed non reflective surface. The mask may be an integral component of the cassette lid.
To prevent microorganisms from being deposited in this masked zone, we may seal the membrane during filtration, such that the growth area formed is kept within the imaged area over the duration of the test as the media receeds. This design approach of controlled growth zone and controlled masking zone may also be used to negate user membrane placement error.
Figure 7 shows elements of the mask to block fluorescence from the nutrient media.
Enhanced tortuous air path
Traditional Petri dishes and Road plates provide for a controlled growth environment by means of a tortuous air passage. We have enhanced this significantly to allow for rapid movement and flipping associated with automatic robotic handling and plate processing. We have designed multiple convolutions and entrapment zones in the cassette's tortuous air management system. Figures 3A-3C show elements of the enhanced tortuous air path.
Method to retain and seal optical window
To retain and seal the optical window, e.g., fabricated in glass or plastic, we have designed an integral circumferential seal. Flexure of the retention element is achieved by deflecting into an open channel. This open channel design also allows the single piece molded design to be extracted from the mold tool. Figure 8 shows elements of the design method to retain and seal optical window.
Cassette transport design, carrier interface, and positioning for automated handling.
A desirable characteristic of the cassette is that it be capable of being repeatedly placed in the Growth system, or other automatic imaging system. Also a carrier is desirably provided to transport cassettes from sample

site to the imager. To achieve this result, we have designed a unique interface between the cassette and a cassette carrier - two half moon truncated cones. Oilier truncated cone on each side of the cassette serves to key the cassette to the carrier device. A cassette may be rotated into position or deposited from the top. A large chamfer surface negates the miss installation of the cassette. The mating rod like features on the carrier engage the half moon feature, preventing a cassette from falling out in normal use. These features in conjunction with the carrier ensure the cassette is always properly placed for automatic robotic processing.
Figures 9A-9H shows elements of the cassette transport design and cassette-carrier interface.
Method to align cassette to axis of automatic processing machine, Growth Direct. ™
Automated cassette or plate handling may require that the system zero out or minimize cassette placement error relative to the robotic grip, or the system must know where the cassette is in space. We have designed a zero draft tapered wedge slot interface to the robotic system. Two blade like features on the automated robotic grip need only find any entry point into the slot, one in the act of expanding the grip outward cams the cassette to the line of action of the gripper and squares up the cassette.
Figures lOA-lOD show elements of the method to align a cassette to an axis.
Imaging fiducial through hole aperture over fluorescent plastic
Our system may employ the ability to know where the same cassette is placed relative to the imager, every time that same cassette is cycled through for imaging. To achieve this result, a through hole in the mask may be utilized to create a stepped, fluorescent mark in an image by having the upper most plastic, i.e., mask with through hole, be of lower natural fluorescence than the plastic structure behind, in this case the cassette base. With the brighter plastic

behind, it illuminates the through hole creating a reproducible fiducial for accurate image placement.
An alternate means of providing the back fluorescence is to have the fiducial hole located over the nutrient media as the media inherently has a high degree of fluorescence.
Figure 11 shows elements of the imaging fiducial mark through hole aperture over fluorescent plastic.
Filtration assembly internal seal design and use of sintered porous plastic
Any seal or noncontiguous stepped feature which contacts a membrane inherently leaves a mark on the membrane when it is released or removed. Many commercial funnel assemblies seal the membrane directly between two rigid plastic elements, leaving a circular ring indentation at that point. To prevent such a mark or indentation from appearing within our image zone, (image zone defined as the wetted area of the membrane on which microorganisms will grow), we have solved the problem as shown in Figure 12. An inner seal traps the membrane to a porous plastic pad, e.g., made from sintered plastic beads, such as those available from Porex. The porous pad has specific properties and a smooth surface to prevent marring, wrinkling, or other perturbations to the membrane, which may interfere with imaging. Vacuum induces movement of air and liquid containing the organisms to pass through this porous barrier, leaving the microorganisms trapped on the membrane surface. An outer seal ensures vacuum is generated and flow occurs in the desired area.
Figure 12 shows elements of the internal seal design and use of sintered porous plastic.
Filtration assembly, dual mode manifold interface in a single device.
The filtration assembly may contain a molded plastic base referred to as the manifold adapter, unlike current products which are designed for a specific

device. For example, either they fit a traditional rubber stopper (used in common bio safety cabinet manifold system) or they fit a common pump head system such as that produced by Millipore. The filtration assembly shown below has a dual interface. The center nozzle spout fits common stoppers and the lid of the base manifold adapter conforms to most pump head (tulip) assemblies.
Figures 13A-13B show elements of the dual mode manifold interface.
Filtration assembly, ease of manual membrane removal by means of an integral pocket for grasping edge of membrane.
A common problem with current membrane filtration assemblies is the difficultly of grasping the membrane for manual transfer to the media. We have solved this problem through the use of integral pockets and raised castle protrusions that position the edge of the membrane for easy grasping with tweezers or forceps. This feature allows desirable membrane imaging as it works to keep the grip indentation from manual transfer out of the imaging zone.
Figures 14A-14C shows elements of the integral pocket that allows forceps to access the edge of the membrane for ease of retrieval.
Other Embodiments
All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims. What is claimed is:











CLAIMS
L A cell-culturing device comprising a housing that contains growth medium for microorganisms, wherein at least a portion of said housing is substantially non-radiative.
2. The device of claim 1, further comprising a detachable, scalable lid comprising an optically clear window disposed to allow imaging of said growth medium.
3. The device of claim 2, wherein said window is substantially non-radiative.
4. The device of claim 2, wherein said window is substantially non-reflective,
5. The device of claim 1, wherein said housing includes openings that provide for a tortuous passage of gases from said growth medium to the ambient environment.
6. The device of claim 5, wherein the housing has a gas exchange cross-sectional area of 7. The device of claim 4, wherein said housing and window are both substantially non-radiative and non-reflective.
8. The device of claim 1, further comprising a removable lid in conformal contact with said growth medium.
9. A kit for detecting microorganisms, comprising the device of claim 1, together with a filtration vessel comprising a membrane that collects microorganisms from a sample passed through the membrane.

10. The kit of claim 9, wherein said membrane is adapted to be placed over
the growth medium of the device of claim 1 such that microorganisms
present in the sample and deposited on the membrane receive
nourishment from said medium.
11. The kit of claim 9, wherein, prior to being placed over the medium, the
membrane remains substantially planar during the passing of the sample
through the membrane whereby microorganisms are deposited on the
membrane.
12. The kit of claim 8, wherein the membrane is substantially non-radiative.
13. The kit of claim 11, wherein the membrane is black.
14. A kit for detecting microorganisms, comprising the device of claim 1,
together with a membrane adapted to be placed over the growth medium
such that microorganisms deposited on the membrane receive
nourishment from said medium.
15. The kit of claim 14, wherein the membrane is substantially non-
radiative.
16. The kit of claim 14, wherein the membrane is black.


Documents:

1450-CHENP-2008 CORRESPONDENCE OTHERS 22-03-2013.pdf

1450-CHENP-2008 FORM-13 22-03-2013.pdf

1450-CHENP-2008 FORM-6 22-03-2013.pdf

1450-CHENP-2008 OTHER DOCUMENTS 22-03-2013.pdf

1450-CHENP-2008 POWER OF ATTORNEY 22-03-2013.pdf

1450-CHENP-2008 AMENDED CLAIMS 13-02-2014.pdf

1450-CHENP-2008 AMENDED CLAIMS 28-04-2014.pdf

1450-CHENP-2008 AMENDED PAGES OF SPECIFICATION 13-02-2014.pdf

1450-CHENP-2008 CORRESPONDENCE OTHERS 29-05-2013.pdf

1450-CHENP-2008 CORRESPONDENCE OTHERS 03-09-2013.pdf

1450-CHENP-2008 CORRESPONDENCE OTHERS 11-04-2014.pdf

1450-CHENP-2008 EXAMINATION REPORT REPLY RECEIVED 13-02-2014.pdf

1450-CHENP-2008 EXAMINATION REPORT REPLY RECEIVED 28-04-2014.pdf

1450-CHENP-2008 FORM-3 03-09-2013.pdf

1450-CHENP-2008 FORM-3 13-02-2014.pdf

1450-CHENP-2008 FORM-3 28-04-2014.pdf

1450-CHENP-2008 FORM-5 13-02-2014.pdf

1450-CHENP-2008 OTHER PATENT DOCUMENT 03-09-2013.pdf

1450-CHENP-2008 PCT NOTIFY 13-02-2014.pdf

1450-CHENP-2008 POWER OF ATTORNEY 11-04-2014.pdf

1450-CHENP-2008 FORM-18 27-08-2009.pdf

1450-chenp-2008-abstract.pdf

1450-chenp-2008-claims.pdf

1450-chenp-2008-correspondnece-others.pdf

1450-chenp-2008-description(complete).pdf

1450-chenp-2008-drawings.pdf

1450-chenp-2008-form 1.pdf

1450-chenp-2008-form 3.pdf

1450-chenp-2008-form 5.pdf

1450-chenp-2008-pct.pdf


Patent Number 260509
Indian Patent Application Number 1450/CHENP/2008
PG Journal Number 19/2014
Publication Date 09-May-2014
Grant Date 02-May-2014
Date of Filing 25-Mar-2008
Name of Patentee RAPID MICRO BIOSYSTEMS, INC.
Applicant Address ONE OAK PARK DRIVE, 2ND FLOOR, BEDFORD MA 01730
Inventors:
# Inventor's Name Inventor's Address
1 STRAUS, DON 19 BUCKINGHAM STREET CAMBRIDGE, MA 02138
2 MACE, CHAD AMHERST, NEW HAMPSHIRE
3 BROWNE, DOUGLAS GROTON, MASSACHUSETTS
PCT International Classification Number C12Q1/04
PCT International Application Number PCT/US06/37410
PCT International Filing date 2006-09-26
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/720,683 2005-09-26 U.S.A.