[go: up one dir, main page]

WO2008000038A1 - Dispositif de culture cellulaire - Google Patents

Dispositif de culture cellulaire Download PDF

Info

Publication number
WO2008000038A1
WO2008000038A1 PCT/AU2007/000905 AU2007000905W WO2008000038A1 WO 2008000038 A1 WO2008000038 A1 WO 2008000038A1 AU 2007000905 W AU2007000905 W AU 2007000905W WO 2008000038 A1 WO2008000038 A1 WO 2008000038A1
Authority
WO
WIPO (PCT)
Prior art keywords
culture
cell
wheel
cell culture
cavity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2007/000905
Other languages
English (en)
Inventor
Jason Paul Hayes
John Edward Mccormack
Matthias Schuenemann
Matthew Daniel Solomon
David Sean O'brien
Jason William Spittle
Jeremy Thompson
Andrew Hinsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
William A Cook Australia Pty Ltd
Original Assignee
William A Cook Australia Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2006903511A external-priority patent/AU2006903511A0/en
Application filed by William A Cook Australia Pty Ltd filed Critical William A Cook Australia Pty Ltd
Publication of WO2008000038A1 publication Critical patent/WO2008000038A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • 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
    • C12M23/10—Petri dish
    • 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
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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

Definitions

  • the present invention relates to a device for culturing, such as culturing cells or tissues. More specifically the invention relates to a microfluidic device for culturing, such as culturing cells or tissues.
  • the invention will be discussed in relation to it application for cell culture but its application is not so limited and can also relate to cell manipulation, cry ⁇ preservation and tissue culture.
  • the invention has particular application to mammalian cell culture including mammalian cell replication and/ or reproduction.
  • the invention is used for culturing embryos for in vitro fertilization (IVF).
  • Microfluidics is the technology used to design, model, manufacture and mass- produce microsystems that handle fluids - gases, vapours or liquids in volumes that can be as small as nano or pico litres. Active and passive microstructures control the flow and mixing of the fluids to produce physical, chemical, biochemical and microbiological reactions in a rapid, cost-effective manner. Microfluidics have a range of applications, including the culture of cells and the automation of highly manual laboratory processes, such as in vitro fertilization (IVF) onto a single substrate.
  • IVF in vitro fertilization
  • Culturing of distinct cells, or distinct populations of cells such as embryos, in a single culture device provides certain difficulties. It has been demonstrated that the exchange of certain autocrine, paracrine and endocrine molecules improves the success rate of culturing cells, and a fluidic communication between distinct cells is considered beneficial to the cells in a culture environment. However, benefits also exist in the separation of cells and the provision of a unique cell distinct from other cells in the culture environment. benefits of the exchange of autocrine, paracrine and endocrine molecules, and the maintenance of distinct cells. However, the provision of physical barriers has required the use of large fluid volumes, and the exchange of media between the physical barriers often results in the cells being dislodged from their desired location.
  • Physical barriers also often result in dead volumes and trapped gas bubbles that hamper fluid transport. Furthermore, physical barriers may also increase the time required to exchange culture media during the culturing process, and where no fluidic communication exists between distinct cells, the exchange of media can be laborious as well as exposing the cells to mechanical, thermal and chemical stress.
  • cell cultures usually need to be covered with cell culture oil. Additionally, the manual introduction, handling and removal of cells such as embryos is prone to airborne contamination, involuntary dislocation of embryos and operator-introduced handling errors.
  • the present invention seeks to overcome, or at least minimise, the problems associated with the prior art.
  • the invention will be discussed in relation to it application for cell culture but its application is not so limited and can also relate to cell manipulation, cryopreservation and tissue culture.
  • the present invention provides a device for use in manipulating or culturing a cell or tissue, the device comprising at least one culture chamber in a culture wheel, the culture wheel being in a culture cavity, the culture wheel including a vertical axis and the culture wheel being moveable around the vertical axis, the dimensions of the culture wheel and the culture cavity being selected such that a gap between the cell culture wheel and the culture cavity is smaller than the diameter of the cell or tissue to be cultured such that the cell or tissue is maintained within the culture chamber.
  • the device comprising at least one culture chamber in a culture wheel, the culture wheel being in a culture cavity, the culture wheel including a vertical axis and the culture wheel being moveable around the vertical axis, the dimensions of the culture wheel and the culture cavity being selected such that a gap between the cell culture wheel and the culture cavity is smaller than the diameter of the cell or tissue to be cultured such that the cell or tissue is maintained within the culture chamber.
  • the culture chambers are in fluid connection via the gap between the culture wheel and the culture cavity.
  • the culture cavity is connected to a loading port, a unloading port and a culture medium feed port.
  • the loading port and unloading port can include holding port, whereby a vertical fluid connection with a funnel section smaller than the diameter can hold the firm with back pressure for the purpose of manipulation of eh cell.
  • the culture medium feed port mates with a device, such as a liquid introduction device.
  • the culture cavity is connected to a culture medium aspiration port and the culture medium aspiration port mates with a device, such as a liquid introduction device.
  • the culture wheel can be rotated around its vertical axis, such as to relocate a selected culture chamber opposite the loading channel entry into the culture cavity and the culture wheel can be rotated around its vertical axis, such as to relocate a selected culture chamber opposite the unloading channel exit from the culture cavity.
  • the culture wheel can be rotated around a horizontal axis. This means that the cell could be lifted out of the fluid and held in the culture cavity for the purpose of manipulation i.e. sperm injection.
  • the rotation of the culture wheel can be controlled by a magnetic field from outside the culture device or by a mechanical connection between said culture wheel and a thumb wheel.
  • the culture wheel can be rotated by a fluid force such as the fluid flow to the device.
  • the culture wheel can be rotated by electrical means such as a stepper motor.
  • the invention comprises a method of culturing a cell, the method comprising providing culture media in the at least one culture chamber of a device as described above and incubating the device.
  • Figure IA shows a schematic perspective view of a device in accordance with the invention.
  • Figure IB shows a schematic explosion view of a device in accordance with the invention.
  • Figure 1C shows a schematic plan view of a device in accordance with the invention.
  • Figure 2A shows a schematic perspective view of the embryo loading station.
  • Figure 2B shows a schematic explosion view of the embryo loading station.
  • Figure 3A shows a schematic perspective view of a cell culture device in accordance with the invention.
  • Figure 3B shows a schematic plan view of an embodiment of the cell culture device (upper fluidic disc removed).
  • Figure 3C shows a schematic plan view of another embodiment of the cell culture device with separate cell loading/ unloading and culture media exchange channels (upper fluidic disc removed).
  • Figure 4A shows a horizontal cross-sectional view of the cell culture cavity area during cell loading.
  • Figure 4B shows a horizontal cross-sectional view of the cell culture cavity area during cell incubation.
  • Figure 4C shows a horizontal cross-sectional view of the cell culture cavity area during cell unloading.
  • Figure 5A shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 5B shows a schematic plan view of the device of figure 5A.
  • Figure 5C shows a schematic explosion view of the device of figure 5A.
  • Figure 5D shows a schematic cross-section through AA' of the device of figure 5B.
  • Figure 5E shows an expanded detail view of the cell loading channel entry into the cell culture cavity and a cell unloading channel exit from the cell culture cavity of figure 5D.
  • Figure 6A shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 6B shows a schematic plan view of the device of figure 6A.
  • Figure 6C shows a schematic explosion view of the device of figure 6A.
  • Figure 6D shows a schematic cross-section through AA' of the device of figure 6B.
  • Figure 6E shows an expanded detail view of the cell loading channel entry into the cell culture cavity and a cell unloading channel exit from the cell culture cavity of figure 6D.
  • Figure 7 A shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 7B shows a schematic plan view of the device of figure 7 A.
  • Figure 7C shows a schematic explosion view of the device of figure 7 A.
  • Figure 7D shows a schematic cross-section through AA' of the device of figure 7B.
  • Figure 7E shows an expanded detail view of the culture medium inlet into the cell culture cavity of figure 7D.
  • Figure 7F shows an expanded detail view of the of the cell culture cavity area of figure 7B, indicating the flow path for culture medium.
  • Figure 8A shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 8B shows a schematic plan view of the device of figure 8A.
  • Figure 8C shows a schematic explosion view of the device of figure 8 A.
  • Figure 8D shows a schematic cross-section through AA' of the device of figure 8B.
  • Figure 8E shows an expanded detail view of the culture medium inlet into the cell culture cavity of figure 8D.
  • Figure 8F shows an expanded detail view of the of the cell culture cavity area of figure 8B, indicating the flow path for culture medium.
  • Figure 9A shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 9B shows a schematic plan view of the device of figure 9A.
  • Figure 9C shows a schematic explosion view of the device of figure 9A.
  • Figure 9D shows a schematic cross-section through AA' of the device of figure 9B.
  • Figure 9E shows an expanded detail view of the culture medium inlet into the cell culture cavity of figure 9D.
  • Figure 9F shows an expanded detail view of the of the cell culture cavity area of figure 9B, indicating the flow path for culture medium.
  • Figure 1OA shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure 1OB shows a schematic plan view of the device of figure 1OA.
  • Figure 1OC shows a schematic explosion view of the device of figure 1OA.
  • Figure 1OD shows a schematic cross-section through AA' of the device of figure 1OB.
  • Figure 1OE shows an expanded detail view of the culture medium inlet into the cell culture cavity and the culture medium outlet from the cell culture cavity of figure 10D.
  • Figure HA shows a schematic perspective view of an embodiment of a cell culture device in accordance with the invention.
  • Figure HB shows a schematic plan view of the device of figure HA.
  • Figure HC shows a schematic explosion view of the device of figure 11 A.
  • Figure HD shows a schematic cross-section through AA' of the device of figure HB.
  • Figure HE shows an expanded detail view of the culture medium inlet into the cell culture cavity and the culture medium outlet from the cell culture cavity of figure HD.
  • Figure HF shows an expanded detail view of the of the cell culture cavity area of figure HB.
  • Figure 12 A-D show planar and cross-sectional views of different embodiments of the cell culture wheel.
  • Figure 13A shows a planar view of the cell culture wheel with latching dimples.
  • Figure 13B shows a cross-section through AA' of the device of figure 13 A, depicting an arrested cell culture wheel in absence of a magnetic field.
  • Figure 13C shows a cross-section through AA' of the device of figure 13A, depicting an released cell culture wheel in presence of a magnetic field.
  • Figure 14A shows a schematic perspective view of an embodiment of a device in accordance with the invention.
  • Figure 14B shows a schematic plan view of the device of figure 14A.
  • Figure 14C shows a schematic explosion view of the device of figure 14A.
  • Figure 14D shows a schematic cross-section through AA' of the device of figure 14B.
  • the preferred embodiments of the device of the present invention provide co- culturing of a distinct cell, such as a cell that may give rise to an embryo, so that a common culture media can be used.
  • the present device also provides a physical barrier to inhibit co-mingling of cells.
  • the device also provides for protection of the cells from airborne contamination.
  • the present device also provides for individual loading and unloading of selected cells.
  • the present device also provides for the changing of culture media to be effected gradually so as to reduce the shock from a sudden change in culture conditions.
  • Figures IA to 1C and 2A and 2B show various views of a cell culturing device according to one embodiment of the present invention.
  • the device 01 may be inserted into a loading station 02 for cell loading and unloading and for cell manipulation.
  • the loading station 02 provides for a controlled rotation of the cell culture wheel around a vertical axis.
  • the loading station 02 comprises a loading station base 03, a thumb wheel 05 and one or more magnets 04.
  • the magnets 04 are mounted on the thumb wheel 05.
  • the thumb wheel 05 is assembled into the loading station base 03 such that the thumb wheel can rotate freely around its vertical axis.
  • FIGS 3A to 3 C show the cell culture wheel in more detail.
  • the device consists of a cell culture wheel 07 with one or more cell culture chambers 08 around its circumference.
  • the cell culture wheel 07 is assembled into a cell culture cavity 06.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the cell loading port 10 is connected to a fluid reservoir 16 via a cell barrier 17.
  • the cell culture cavity 06 is also connected to the cell unloading port 13 via a cell unloading channel 14.
  • the cell unloading port 13 may be connected to an aspiration port 18 via a cell barrier 17.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20, and to the culture medium aspiration port 22 via a culture medium aspiration channel 23.
  • the diameter of the cell culture wheel 07 is about 3 mm to about 12 mm. More preferably, the diameter of the cell culture wheel 07 is about 3 mm to about 7 mm.
  • the difference between the inner diameter of the cell culture cavity 06 and the outer diameter of the cell culture wheel 07 forms an aperture or gap which enables fluid communication, but restricts embryo movement into the gap between cell culture cavity 06 and cell culture wheel 07.
  • the difference between the inner diameter of the cell culture cavity 06 and the outer diameter of the cell culture wheel 07 is about 40 ⁇ m to about 60 ⁇ m.
  • the height of the cell culture wheel 07 is about 250 ⁇ m to about 1000 ⁇ m. More preferably, the height of the cell culture wheel 07 is about 500 ⁇ m.
  • the difference between the height of the cell culture cavity 06 and the height of the cell culture wheel 07 forms an aperture which enables fluid communication, but restricts embryo movement into the gap between the cell culture cavity 06 and the cell culture wheel 07.
  • the difference between the height of the cell culture cavity 06 and the height of the cell culture wheel 07 is about 40 ⁇ m to about 60 ⁇ m.
  • the cell culture wheel 07 comprises multiple fluidly connected cell culture chambers 08, which are arranged along its circumference.
  • the cell culture wheel 07 comprises between 2 and 40 cell culture chambers 08. More preferably, the cell culture wheel 07 comprises 20 cell culture chambers 08.
  • Each cell culture chamber 08 is of a cylindrical shape and has a diameter at its base of about 250 ⁇ m to about 1000 ⁇ m. More preferably, the cell culture chamber 08 has a diameter at its base of about 300 ⁇ m to about 500 ⁇ m.
  • the volume of the cell culture chamber 08 is about 0.025 ⁇ l to about 1 ⁇ l. More preferably, the volume of the cell culture chamber 08 is about 0.1 ⁇ l.
  • Each cell culture chamber 08 fully opens to the top and bottom planes of the cell culture wheel 07.
  • Each cell culture chamber 08 also opens to the circumferential plane of the cell culture wheel 07.
  • this opening has a rectangular shape and a width of about 125 ⁇ m to about 250 ⁇ m. More preferably, said opening has a width of about 150 ⁇ m.
  • the device of the present invention may be used for culturing a single cell 09 per cell culture chamber 08, or a population of cells in each cell culture chamber 08.
  • a single cell may be cultured in the cell culture chamber 08.
  • the cell culture chamber 08 functions to restrict movement of a cell and retain the cell in the cell culture chamber 08.
  • the cell culture chamber 08 also maintains a unique address for cells, facilitates cell communication via autocrine, paracrine and endocrine molecule exchange, and facilitates media exchange.
  • the cell culture wheel 07 has two or more latching dimples 30 at one of its planar faces (see Figure 13A), and the cell culture cavity 06 has corresponding latching grooves 31 in its opposing face.
  • the latching dimples 30 are placed on the top plane of said cell culture wheel 07, and for cell culture wheels 07 made from a material with a higher specific density than any of the cell culture media, the latching dimples 30 are placed on the bottom plane of the cell culture wheel 07.
  • the latching dimples 30 in the cell culture wheel 07 engage with the latching grooves 31 in the cell culture cavity 06, thus preventing undesirable spontaneous rotation of the cell culture wheel 07 during transport, storage and incubation (see Figure 13B).
  • the latching dimples 30 in the cell culture wheel 07 disengage from the latching grooves 31 in the cell culture cavity 06, and the cell culture wheel 07 is free to be rotated around its vertical axis (see Figure 13C).
  • the cell culture wheel 07 is labelled. Labelling of the cell culture chamber
  • the labels are readable for both left- and right-handed operation of the device, and/ or for reading from above or below.
  • Figures 5 A to 5E show how the cell culture device can be assembled according to one embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the cell culture cavity 06 is connected to the cell unloading port 13 via a cell unloading channel 14.
  • the upper fluidic disc 26 closes the cell culture cavity 06 and provides access to the cell loading port 10 and the cell unloading port 13.
  • Figures 6A to 6E show how the cell culture device can be assembled according to another embodiment of the present invention. In this embodiment the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port 10 and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell unloading channel exit 15 is set above the cell culture wheel 07 such as to avoid accidental unloading of cells during cell loading.
  • Figures 7 A to 7F show how the cell culture device can be assembled according to another embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port 10 and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20 and a culture medium inlet 21, and to the culture medium aspiration port 22 via a culture medium outlet 24 and a culture medium aspiration channel 23.
  • Culture medium inlet 21 and culture medium outlet 24 fan out to a single wide shallow aperture with a depth smaller than cell diameter, such as to prevent cell escape during culture medium exchange.
  • Figures 8A to 8F show how the cell culture device can be assembled according to another embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20 and a culture medium inlet 21, and to the culture medium aspiration port 22 via a culture medium outlet 24 and a culture medium aspiration channel 23.
  • Culture medium inlet 21 and culture medium outlet 24 fan out to a single wide aperture having the same height as the cell culture cavity 06.
  • the cell culture cavity 06 is separated from the culture medium inlet 21 and culture medium outlet 24 by a cell barrier 25, realized by a filter structure with a pore size smaller than cell diameter, such as to prevent cell escape during culture medium exchange.
  • Figures 9A to 9F show how the cell culture device can be assembled according to another embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port via a cell loading channel.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port 10 and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20 and a culture medium inlet 21, and to the culture medium aspiration port 22 via a culture medium outlet 24 and a culture medium aspiration channel 23.
  • the culture medium inlet 21 and culture medium outlet 24 fan out to a wide opening connected with the cell culture cavity 06 through a cell barrier 25.
  • the cell barrier 25 is realized by a multitude of narrow apertures with a width smaller than cell diameter, such as to prevent cell escape during culture medium exchange.
  • Figures 1OA to 1OF show how the cell culture device can be assembled according to another embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port 10 via a cell loading channel 11.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port 10 and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20 and a culture medium inlet 21, and to the culture medium aspiration port 22 via a culture medium outlet 24 and a culture medium aspiration channel 23.
  • Culture medium inlet 21 and culture medium outlet 24 are realised as single apertures placed on opposite sides of the cell culture cavity 06 concentrically "with the rotational axis of the cell culture wheel 07, respectively. No additional cell barrier is necessary to prevent cell escape during culture medium exchange.
  • the culture medium feed channel 20 is capped with an upper cover lid 28, and the culture medium aspiration channel 23 is capped with a lower cover lid 29.
  • Figures 11 A to HF show how the cell culture device can be assembled according to another embodiment of the present invention.
  • the device is assembled by placing the cell culture wheel 07 into a cell culture cavity 06 in a lower fluidic disc 27.
  • the cell culture cavity 06 is connected to the cell loading port via a cell loading channel 11.
  • the upper fluidic disc 26 closes the cell culture cavity 06, provides access to the cell loading port and connects the cell culture cavity 06 with the cell unloading port 13 via a cell unloading channel exit 15 and a cell unloading channel 14.
  • the cell culture cavity 06 is connected to the culture medium feed port 19 via a culture medium feed channel 20 and a culture medium inlet 21, and to the culture medium aspiration port 22 via a culture medium outlet 24 and a culture medium aspiration channel 23.
  • the culture medium inlet 21 consists of a multitude of apertures arranged circular around the rotational axis of the cell culture "wheel 07.
  • the culture medium outlet 24 consists of a single wide aperture, placed concentrically with the rotational axis of the cell culture wheel 07 on the opposite face of the cell culture cavity 06. No additional cell barrier is necessary to prevent cell escape during culture medium exchange.
  • the culture medium feed channel 20 is capped with an upper cover lid 28, and the culture medium aspiration channel 23 is capped with a lower cover lid 29.
  • Figures 14A to 14D show how the cell culture device can be assembled according to a variation of the present invention. In this embodiment the device is assembled by placing a cell culture wheel 07 with a direct mechanically coupled thumb wheel 39 onto a lower fluidic disc 27.
  • the cell culture cavity 06 is formed in the upper fluidic disc 26, and connected to the cell loading 10 and unloading 11 ports via cell loading 11 and unloading 14 channels, respectively.
  • the upper fluidic disc 26 features a cutout 41, enabling mechanical operation of the thumb wheel 39. Fluidic sealing of the cell culture cavity 06 is provided by a seal-ring 40, such as an o-ring.
  • the upper fluidic disc 26 is capped with an upper cover lid 28.
  • the upper fluidic disc 26, lower fluidic disc 27, upper cover lid 28 and lower cover lid 29 are manufactured from materials selected from the group comprising cyclic olefin copolymer (COC), polycarbonate (PC), polystyrene (PS), polymethyl-methacrylate (PMMA), polyethyleneterephthalate (PET), polyimide (PI), polyetherimide (PEI), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), cellulose acetate (CA), cellulose acetate butyrate (CAB), high density polyethylene (HDPE), low density polyethylene (LDPE), polyamide (PA), polybutylene terephtalate (PBT), poly ether ether ketone (PEEK), polyethylene terephtalate glycol (PETG), polymethylpentene (PMP), polyoxide methylene (POM), polypropylene (COC), cyclic olefin copolymer (COC
  • the cell culture wheel 07 is manufactured from materials selected from the group comprising soft magnetic materials with high permeability such as iron (Fe), nickel (Ni), cobalt(Co), iron boron (FeB) based alloys, iron cobalt (FeCo) based alloys, iron nickel (FeNi) based alloys, iron phosphorous (FeP) based alloys or combinations thereof.
  • the soft magnetic materials used to manufacture the cell culture wheel 07 are coated on one or all surfaces with a barrier layer 37, such as Parylene, in order to render the bulk material non-cytotoxic (see Figure 12A).
  • the cell culture wheel 07 is manufactured from polymeric materials 38 selected from the group comprising cyclic olefin copolymer (COC), polycarbonate (PC), polystyrene (PS), polymethylmethacrylate (PMMA), polyethyleneterephthalate (PET), polyimide (PI), polyetherimide (PEI), polydimethylsiloxane (PDMS), acrylonitrile butadiene styrene (ABS), cellulose acetate (CA), cellulose acetate butyrate (CAB), high density polyethylene (HDPE), low density polyethylene (LDPE), polyamide (PA), polybutylene terephtalate (PBT), polyether ether ketone (PEEK), polyethylene terephtalate glycol (PETG), polymethylpentene (PMP), polyoxide methylene (POM), polypropylene (PP), polysulfone (PSU), polytetrafluoroethylene (PTFE), polyvinylch
  • One or all parts of the device may be coated on one or all surfaces with a barrier layer, such as Parylene, in order to render the bulk material non-cytotoxic, to lower the water absorption of the bulk material, to lower the water vapour absorption of the bulk material, and to protect the bulk material from potential harmful interaction with culture medium or culture oil.
  • a barrier layer such as Parylene
  • the device 01 is comprised of separate parts, such as upper fluidic disc 26, lower fluidic disc 27, upper cover lid 28 and lower cover lid 29, the parts may be connected using an adhesive, or by other bonding methods such as thermal diffusion bonding, ultrasonic bonding, solvent bonding, microwave welding and laser welding.
  • the hydrophilicity of the microfluidic surfaces may be improved by surface treatment techniques such as plasma polymerisation, UV treatment, saponification, polyethylene oxide grafting, surface texturing or electrowetting may be applied.
  • the cell culture device 01 is placed into the loading station 02.
  • a defined volume of a cell cleavage medium is injected into the fluid reservoir 16 or the cell loading port 10.
  • the cell culture medium flows, preferably by capillary flow or by applied pressure difference, via the cell loading channel 11 to the cell culture cavity 06, and subsequently fills the cell culture cavity 06, the cell culture chambers 08, the cell unloading channel 14 and the cell unloading port 13.
  • Embryos 09 are manually pipetted into the cell loading port 10 by applying a negative relative pressure to the aspiration port 18 (either by manually using a pipette or a syringe or by applying a tube connected to a pump), a pressure gradient from fluid reservoir 16 to aspiration port 18 is generated, which results in a flow of culture medium along that gradient (see Figure 3B).
  • Embryos 09 follow the flow into the cell loading channel 11 towards the cell culture cavity 06 and the cell culture wheel 07.
  • the first cell culture chamber 08 of the cell culture wheel 07 is aligned with the cell loading channel 11. Due to the fluidic design of the cell culture cavity 06, the generated flow passes through the cell culture chamber 08.
  • the first embryo 09 is transported by the flow into the cell culture chamber 08.
  • the cell culture wheel 07 is rotated so that the next empty cell culture chamber 08 is aligned with the cell loading channel 11.
  • the next embryo 09 is loaded into the now accessible cell culture chamber 08 (see Figure 4A). This process is repeated until all embryos 09 are sitting in their respective cell culture chambers 08 (see Figure 4B).
  • the device 01 is then removed from the loading station 02 and incubated at the required temperature (typically 37.8 0 C) for the required time (typically 48 to 72 hrs).
  • the device 01 is then returned to the loading station 02.
  • a defined volume of a blastocyst medium to promote the development of early embryos to blastocysts is applied to the culture medium feed port 19.
  • a set amount of fluid is then extracted from the culture medium aspiration port 22, either by wicking, pipetting or pumping, thus gradually replacing the cell cleavage medium in the cell culture cavity 06 and cell culture chamber 08 with the blastocyst medium.
  • the process may be repeated until all the cell cleavage medium is either completely removed or diluted to the required volume, for example 5% of the total volume of cell culture medium.
  • the device 01 is then removed from the loading station 02 and incubated again at the required temperature (typically 37.8 0 C) for the required time (typically 48 to 72 hrs).
  • the cell culture device 01 is then returned to the loading station 02.
  • the cell culture wheel 07 is rotated into a position where the cell culture chamber 08 of the embryo 09 to be extracted is aligned with the cell unloading channel 14.
  • a pressure gradient from cell loading port 10 to cell unloading port 13 is generated, which results in a flow of culture medium along that gradient.
  • the embryo 09 follows the flow from the cell culture chamber 08 into the cell unloading channel 14 towards the cell unloading port 13.
  • the embryo is extracted from the unloading port 13 by for example pipetting.
  • the cell culture wheel 07 is then rotated to the next position, and the process is repeated until all desired embryos are unloaded.
  • the culture medium feed port 19 is connected to an external culture medium reservoir or to another culture medium source via a flexible tube or another type of fluidic connection
  • the culture medium aspiration port 22 is also connected to a waste reservoir via a flexible tube or another type of fluidic connection.
  • a fluidic pump is connected to either the culture medium feed line or to the culture medium aspiration line.
  • the device with the attached tubes is then placed into an incubator and incubated at the required temperature (typically 37.8°C) for the required time (typically 48 to 120 hrs).
  • culture medium is slowly pumped through the cell culture cavity 06 and the cell culture chambers 08, either continually or in defined intervals for defined periods of time, in order to feed nutrients to the cells 09 and to remove waste products from the cells 09.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un dispositif (1) de culture ou de manipulation cellulaire ou tissulaire. Le dispositif de l'invention a au moins une chambre de culture (8) dans une roue de culture (7). La roue de culture est située dans une cavité de culture (6) du dispositif et peut tourner autour d'un axe vertical entre un orifice de chargement (10), un orifice de déchargement (13) et un orifice d'alimentation en milieu de culture (19). Selon l'invention, les dimensions de la roue de culture et de la cavité de culture sont sélectionnées de façon qu'un espace entre la roue de culture et la cavité de culture soit inférieur au diamètre de la cellule dont on fait la culture de sorte que la cellule est maintenue à l'intérieur de la chambre de culture.
PCT/AU2007/000905 2006-06-30 2007-06-29 Dispositif de culture cellulaire Ceased WO2008000038A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2006903511 2006-06-30
AU2006903511A AU2006903511A0 (en) 2006-06-30 Cell culture device

Publications (1)

Publication Number Publication Date
WO2008000038A1 true WO2008000038A1 (fr) 2008-01-03

Family

ID=38845047

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2007/000905 Ceased WO2008000038A1 (fr) 2006-06-30 2007-06-29 Dispositif de culture cellulaire

Country Status (1)

Country Link
WO (1) WO2008000038A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002102969A2 (fr) * 2001-06-18 2002-12-27 Pig Improvement Company Uk Limited Systeme
US6653124B1 (en) * 2000-11-10 2003-11-25 Cytoplex Biosciences Inc. Array-based microenvironment for cell culturing, cell monitoring and drug-target validation
WO2004020573A1 (fr) * 2002-08-27 2004-03-11 Vanderbilt University Bioreacteurs comprenant un groupement de chambres et une conduite d'alimentation commune
US6875605B1 (en) * 2002-08-21 2005-04-05 Florida State University Research Foundation, Inc. Modular cell culture bioreactor and associated methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6653124B1 (en) * 2000-11-10 2003-11-25 Cytoplex Biosciences Inc. Array-based microenvironment for cell culturing, cell monitoring and drug-target validation
WO2002102969A2 (fr) * 2001-06-18 2002-12-27 Pig Improvement Company Uk Limited Systeme
US6875605B1 (en) * 2002-08-21 2005-04-05 Florida State University Research Foundation, Inc. Modular cell culture bioreactor and associated methods
WO2004020573A1 (fr) * 2002-08-27 2004-03-11 Vanderbilt University Bioreacteurs comprenant un groupement de chambres et une conduite d'alimentation commune

Similar Documents

Publication Publication Date Title
US11498072B2 (en) Microfluidic device for storage and well-defined arrangement of droplets
WO2006089354A1 (fr) Dispositif de culture
US6811752B2 (en) Device having microchambers and microfluidics
US11061043B2 (en) Loading station
US8865464B2 (en) Microfluidic cell culture device
US9757728B2 (en) Microfluidic aliquoting for single-cell isolation
AU2006304634B2 (en) Microfluidic cell culture device and method for using same
US20040029266A1 (en) Cell and tissue culture device
EP3173787A1 (fr) Système de réseau de culture de cellules pour épreuves automatisées et procédés de fonctionnement et de fabrication de celui-ci
US20110130310A1 (en) Microbioreactor and microtiter plate comprising a plurality of microbioreactors
US20100323447A1 (en) Microfluidic cell culture device and method for using same
US20070217964A1 (en) Microreactor with auxiliary fluid motion control
US20070015276A1 (en) Fluid transfer device
US20070036690A1 (en) Inlet channel volume in a reactor
WO2011135339A2 (fr) Réacteur
WO2008000038A1 (fr) Dispositif de culture cellulaire
US20240263233A1 (en) Systems for Processing a Cellular Sample, Multi-Microwell Array-Flowcell Cartridges for Use Therein, and Methods of Use Thereof
AU2006216110A1 (en) Culture device

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07719142

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07719142

Country of ref document: EP

Kind code of ref document: A1