WO2009039433A1 - Système de culture analytique microfluidique - Google Patents

Système de culture analytique microfluidique Download PDF

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Publication number
WO2009039433A1
WO2009039433A1 PCT/US2008/077105 US2008077105W WO2009039433A1 WO 2009039433 A1 WO2009039433 A1 WO 2009039433A1 US 2008077105 W US2008077105 W US 2008077105W WO 2009039433 A1 WO2009039433 A1 WO 2009039433A1
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WO
WIPO (PCT)
Prior art keywords
microfluidic
cell
culture
culture system
micro
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PCT/US2008/077105
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English (en)
Inventor
Arthur Gershowitz
Nobuyuki Futai
Robert D. Powers
Enrico Picozza
Syed Zafar Razzacki
Original Assignee
Incept Biosystems Inc.
Crowley, Michael J.
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Application filed by Incept Biosystems Inc., Crowley, Michael J. filed Critical Incept Biosystems Inc.
Publication of WO2009039433A1 publication Critical patent/WO2009039433A1/fr

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    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • 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
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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
    • 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
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • 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
    • C12M41/48—Automatic or computerized control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles

Definitions

  • This document relates to an integrated microfluidic device and method for the cultivation and in situ monitoring of cells.
  • the document relates to in vitro culturing of any cell including prokaryotic and eukaryotic cells.
  • Such cells can be from a human or any other mammal.
  • a cell or cells can be oocytes, embryos, genetically modified cells derived from a multi-cellular organism, or portions or multiples thereof.
  • the in vitro culture i.e., growth of living cells in a controlled artificial environment
  • cells such as oocytes and embryos
  • a favorable environment can enable cells to thrive through various metabolic processes including fertilization, production, meiosis, growth, propagation, maintenance, and maturation.
  • fibroblast cells such as fibroblast cells, stem cells, cells from a whole pancreas, parotid gland, thyroid gland, parathyroid gland, prostate gland, lachrymal gland, cartilage, kidney, inner ear, liver, parathyroid gland, oral mucosa, sweat gland, hair follicle, adrenal cortex, urethra, bladder, bacteria, yeasts, fungi, and molds as well as genetically modified cells from multi-cellular organisms including plants, and mammals may also require similar conditions during cultivation.
  • cells such as fibroblast cells, stem cells, cells from a whole pancreas, parotid gland, thyroid gland, parathyroid gland, prostate gland, lachrymal gland, cartilage, kidney, inner ear, liver, parathyroid gland, oral mucosa, sweat gland, hair follicle, adrenal cortex, urethra, bladder, bacteria, yeasts, fungi, and molds as well as genetically modified cells from multi-cellular organisms including plants, and mammals may also require similar conditions during cultivation
  • a biocompatible, microfluidic cell chip having integrated microfluidic reservoirs, micro-channels, culture media sources, and sinks which allows simultaneous culturing and monitoring is described.
  • the microfluidic cell chip is part of a larger analytical microfluidic culture system that can sense and control environmental and cellular conditions and perform image analysis as described and can, for example, cultivate sensitive cells (e.g., oocytes, pre-implantation embryos, and the like) and mimic in vivo conditions to sustain such processes as fertilization, transformation, production, meiosis, growth, propagation, maintenance, maturation, and the like. Operations for cell culture may be automatically performed over an extended period of time thus minimizing perturbation to the culture environment, minimizing risk of contamination, and/or maximizing cell viability and utility.
  • One aspect of the analytical micro fluidic culture system is to provide a micro fluidic carrier and method that enables the monitoring of the mechanical functions of the micro fluidic carrier and to provide feedback on its operation.
  • Other aspects of the analytical micro fluidic culture system include providing equipment and analytical techniques that enable the monitoring of the constituents and by-products of the culture media and allowing for actively controlling the cell culture environment within the microfluidic carrier.
  • Yet another aspect is to provide equipment and techniques for interfacing the microfluidic carrier and analytical devices for the observation and imaging analysis of cells within an incubator.
  • one aspect of this document features a microfluidic culture system comprising (a) a microfluidic device comprising (i) a microfluidic chamber; (ii) a microfluidic channel fluidly connected to the microfluidic chamber; and (iii) an interface that is configured to accept a sensor; wherein the microfluidic device is configured to allow for manipulation by a tactile pin device; (b) a tactile pin device which applies pressure to alter the shape of at least a portion of the microfluidic device to control fluid flow within the microfluidic device; (c) a sensor that connects to the interface of the microfluidic device; and (d) a processing device adapted to receive information from the sensor and adapted to maintain or alter a culture condition based on the information.
  • the sensor can detect an analyte, a liquid level, a liquid flow rate, a temperature, a light intensity, a wavelength, a conductivity, a viscosity, or a combination thereof.
  • the sensor can be an imaging device.
  • the imaging device can be adapted to capture an image.
  • the culture condition can comprise a flow rate, a temperature, a volume, a culture medium, a cell location, a flow pattern, a gas content, a humidity, or an illumination level.
  • the processing device can be adapted to receive information from the sensor and can be adapted to maintain or alter two or more culture conditions based on the information.
  • the processing device can be adapted to alter the culture medium present within the microfluidic chamber gradually over time.
  • the processing device can be adapted to exchange the culture medium present within the microfluidic chamber with a second culture medium. The exchange can occur gradually over the course of more than one hour.
  • the processing device can be adapted to determine the viability of a cell or cells present within the microfluidic device based on the information.
  • this document features a microfluidic culture system comprising a fluidic channel, a cell chamber, and a lower, outer surface, wherein a surface of the fluidic channel comprises a flexible material such that the surface can be expanded into the fluidic channel to reduce or promote fluid movement within the fluidic channel, wherein the cell chamber comprises a bottom inner surface for supporting a cell, and wherein the distance between the lower, outer surface and the bottom inner surface is between 1 mm and 5 mm.
  • the distance can be between 1.5 mm and 4 mm.
  • a portion of the bottom inner surface can define a recess to segregate a single cell.
  • a portion of the bottom inner surface can define a recess to segregate an embryo.
  • the microfluidic culture system can comprise two or more cell chambers.
  • the microfluidic culture system can comprise a segregated cell chambers not fluidly connected to the fluidic channel.
  • this document features a microfluidic culture system comprising (a) a microfluidic device comprising (i) a microfluidic chamber; (ii) a microfluidic channel fluidly connected to the microfluidic chamber; and (iii) an interface that is configured to accept a sensor; wherein the microfluidic device is configured to allow for manipulation by a tactile pin device; (b) a tactile device which applies pressure to alter the shape of at least a portion of the microfluidic device to control fluid flow within the microfluidic device; (c) a sensor that connects to the interface of the microfluidic device; and (d) a processing device electronically coupled to the tactile device and the sensor; wherein contents contained in the microfluidic device are cultured in culture medium, the processing device receives information from the sensor relating to conditions inside the microfluidic device, and the processing devices uses the information to direct the tactile device to change fluid flow based on the information.
  • the sensor can detect an analyte.
  • the chamber can be a cell holding chamber, and the culture system can further comprise a viewing area proximate to a cell holding chamber. The viewing area can be configured to allow inspection of at least a portion of the interior of the cell holding chamber.
  • the culture system can further comprise an imaging device that is electronically coupled to the processing device and images at least a portion of the interior of the micro fluidic device.
  • the processing device can use information derived from images obtained from the imaging device to direct the tactile device to change fluid flow based on the information. A determination can be made of the viability of the contents of the micro fluidic device using the image derived information.
  • a micro fluidic culture system comprising (a) a microfluidic device comprising (i) a microfluidic chamber; (ii) a microfluidic channel fluidly connected to the microfluidic chamber; and (iii) a viewing area proximate to a cell holding chamber; wherein the viewing area is configured to allow for inspection of at least a portion of the interior of the microfluidic chamber; (b) a tactile device which applies pressure to alter the shape of at least a portion of the microfluidic device to control fluid flow within the microfluidic device; (c) an imaging system that can record an image of the portion of the interior of the microfluidic chamber through the viewing area; and (d) a processing device electronically coupled to the tactile device and the imaging system; wherein contents contained in the microfluidic device are cultured in culture medium, the processing device receives the image from the imaging system and uses information derived from the image to direct the tactile device to change fluid flow based on the information.
  • the microfluidic device can further comprise an interface configured to accept a sensor.
  • the microfluidic culture system can further comprise a sensor connected to the interface and electronically connected to the processing device.
  • the processing device can use information obtained from the sensors to direct the tactile device to change fluid flow based on the information.
  • a microfluidic culture system comprising a fluidic channel and an embryo chamber, wherein a surface of the fluidic channel comprises a flexible material such that the surface can be expanded into the fluidic channel to reduce or promote fluid movement within the fluidic channel, wherein the embryo chamber comprises a bottom surface comprising a material other than the flexible material.
  • the embryo chamber can comprise a frustropyramidal or frustroconical shape comprising the bottom surface.
  • the flexible material can comprise polyurethane, silicone, thermoplastic elastomers, rubber, or more rigid materials that can be deformed without fracture when sufficiently thin (e.g., polyester, polycarbonate, polyimide, PVC, or polyethylene).
  • the bottom surface can be an optically clear surface made from, for example, a thermoplastic, glass, thermoset, or silicone.
  • a micro fluidic culture system comprising a fluidic channel, an embryo chamber, and a lower, outer surface, wherein a surface of the fluidic channel comprises a flexible material such that the surface can be expanded into the fluidic channel to reduce or promote fluid movement within the fluidic channel, wherein the embryo chamber comprises a bottom inner surface for supporting an embryo, and wherein the distance between the lower, outer surface and the bottom inner surface is between 0.1 mm and 5 mm (e.g., between 1 mm and 5 mm).
  • the embryo chamber can comprise a frustropyramidal or frustroconical shape comprising the bottom inner surface. The distance can be between 1.5 mm and 4 mm.
  • FIG. 1 is a schematic representation of an exemplary micro fluidic device viewed from the top.
  • FIG. 2 is a schematic representation of an exemplary micro fluidic device viewed from the top.
  • FIG. 3 is a schematic representation of an exemplary micro fluidic device viewed from the side.
  • FIG. 4 is a schematic representation of an exemplary micro fluidic device viewed from the top.
  • FIG. 5 is a perspective view of an exemplary micro fluidic device.
  • FIG. 6 is a schematic representation of an exemplary micro fluidic device viewed from the bottom.
  • FIG. 7 is a schematic representation of a portion of an exemplary micro fluidic funnel viewed from the side.
  • FIG. 8 is an exploded view of an exemplary micro fluidic device viewed from the side.
  • the culture medium for cell cultivation generally contains, for example, water, salts, nutrients, essential amino acids, vitamins, hormones, proteins, and/or growth factors in pre-defined proportions and levels, as well as a buffer system establishing a defined pH-range, and a source of oxygen.
  • the buffer system can be a CO 2 /bicarbonate buffer system, where the bicarbonate can be incorporated into the aqueous culture medium at or before the start of the culturing period.
  • the buffer systems may be supplemented during the culture period, whereas the CO 2 (carbon dioxide) may be provided from the atmosphere surrounding the culture medium during the culture period.
  • the source of oxygen can be gaseous free oxygen (O 2 ) which may be supplied from the surrounding atmosphere to the culture medium.
  • the culture temperature should be kept within rather narrow limits in order to obtain optimum and/or successful results.
  • the handling of, for example, embryos intended for in vitro fertilization (IVF) and cell cultures for diagnostic analysis can be encumbered by the need to carefully manage the conditions of perfusion and incubation, and the need to quickly return the cultures to a controlled environment. Necessary repeated manipulation of the samples for analytical purposes can result in unwanted contamination, lost time, and increased costs associated with the repeated tasks.
  • the culture carriers may be disturbed each time the door of the incubator is opened during inspection or removal of a culture carrier (e.g., a Petri dish).
  • Sensors devices that can measure a measurable quantity, can be included in the control equipment may assist in restoring the parameters in the interior volume of the incubators to the pre-selected levels within a short period of time (e.g., thirty seconds), but within the culture carrier and media, the restoration of the environmental parameters to the pre-selected levels may take a much longer period of time (e.g., two minutes). Because the door of the incubator may be opened and closed many times during a working-day, the cumulative effect on the development, growth, and propagation of the cultured cells within the carriers may be significant and detrimental. The cumulative effect may result in decreased development and viability of cells in the culture.
  • Mammalian oocytes and pre-implantation embryos can be sensitive to environmental changes.
  • the in vitro production and propagation of such embryos may require frequent changes or supplementation of the culture media to maintain a constant environment, while certain types of cell cultures may require a changing environment.
  • These changing environments can be pre-selected to mimic in vivo conditions and may include changes in the concentrations of nutrients, growth factors, and vitamins, changes in pH, presence or absence of growth inhibitors, and the like.
  • the changing environment may also include a change in flow rate of fluid or a periodic fluctuation of fluid flow. Changing such factors is typically quite complex and cumbersome to perform manually by a technician.
  • Cell cultivation success rates may be reduced by limitations of current culturing methods. For example, some improvements in embryo culture methods have been obtained by simulating in vivo conditions and meeting the changing needs of an embryo.
  • Human and animal embryos can be cultured in controlled atmosphere incubators. Atypical culturing cycle is three days, followed by implantation into the female reproductive system.
  • Existing embryo culture systems can employ a single medium from the time of insemination until the transfer of the embryo.
  • a generally accepted method involves the placement of individual embryos within Petri dishes and submerging individual embryos, each in a drop of a growth-enhancing nutrient. In vivo, however, an embryo is bathed in constantly changing environments as it moves through the oviduct to the uterus.
  • microfluidic culture carriers can enable manipulation of, for example, nanoliter to microliter volumes of fluids and are useful for reducing reagent consumption and creating physiologic cell culture environments that better match the fluid-to-cell-volume ratios in vivo.
  • the devices can contain a plurality of interconnected micro-channels and reservoirs of very small sizes.
  • micro-channels can have width and height dimensions of 10 to 300 micrometers.
  • "Lab on a chip" devices can be constructed which contain most of the materials required to cultivate cells with the exception of, for example, external fluid supply and supply of electrical, magnetic, or pneumatic energy.
  • Micro fluidic systems can be made of materials such as polydimethylsiloxane (PDMS) because of its favorable mechanical properties, optical transparency, and bio-compatibility.
  • PDMS polydimethylsiloxane
  • Examples of micro fluidic cell culture carriers are disclosed in S. Takayama, et al., "Micro fluidic Cell Culture Device and Method for Using Same” published Patent Application No. 2007/0084706, and microfluidic cell culture carrier as disclosed in S. Takayama, et al., "Microfluidic Cell Culture Device” published Patent Application No. 2007/0090166, the contents of which are hereby incorporated by reference.
  • One goal in the design of cell cultivation equipment can be total process automation. Cell culture methods are still being performed in a step wise fashion even with microfluidic devices.
  • microfluidics techniques are well-suited to address process automation.
  • the changing of culture media may require no manual manipulation of the embryo.
  • the media can be gradually changed around the embryo, rather than subjecting it to sudden changes in environment.
  • the micro environment that can exist within a microfluidic device can simulate physiological conditions and may provide some beneficial influence on development.
  • the integration of a microfluidic channel for introduction of cells e.g., oocytes and sorted sperm
  • the oocyte can be directed to a secondary site within a microfluidic channel for cumulus removal, evaluation for fertilization, and/or embryo culture.
  • Sequential media can be provided to optimize embryo development with little or no cell manipulation. Miniaturization can allow the entire system to be small in size and to be implemented in a self-contained device. Decreased intervention by laboratory personnel can decrease gamete and/or embryo manipulation and provide for greater consistency of incubation conditions.
  • microfluidic cell culturing devices and their use, specific shapes, dimensions, and substrate materials of reservoirs and channels within a microfluidic device can provide specific advantages and benefits for cultivating cells and the like.
  • the culture media for supporting various cell types including embryos requires specific constituents and volumes.
  • the process of loading, positioning, placing, and/or segregating of cells within a micro fluidic chip as well as the accessibility of cells to observation, monitoring, manipulation, and retrieval from a micro fluidic chip also factors into the potential viability of the culture and success of harvesting.
  • Embryo culturing can involve the steps of visually recording the morphology of the embryos and attempting to determine the viability of each embryo based on its morphology and the position of certain features such as R's polar bodies and spindles. While these activities occur in culturing carriers including microfluidic devices, periodic visual inspections, note taking, and visual evaluation can be recorded to document the development of the embryos.
  • the visual monitoring of embryos during a culturing period can include a technician identifying and obtaining confirmation of specific embryos that may be more viable than the others and/or more likely to survive and further develop after implantation. This inspection can include removing the embryos from the incubator and transporting them to a bench top location for viewing through a microscope, or the like.
  • the selection of embryos to be implanted is made on the day of retrieval from the incubator. This is based on the then-existing morphological and physical characteristics of the embryos.
  • the selected embryos may be the only embryos that are incubated through the entire culturing period, and then implanted. Flawed embryos that are detected during the culturing period may be discarded.
  • one or more of the mentioned activities are labor intensive and subjective but necessary in the culturing process as presently performed in routine clinical practice.
  • a microfluidic system may integrate sensors, actuators, and controllers that enable monitoring and control of the culture media.
  • a system can measure and modify many different conditions of the culture medium, including, but not limited to, temperature, volume, pH, concentration, refractive index, color, turbidity, absorbance, fluorescence, conductivity, viscosity, density, fluid flow conditions, and the like.
  • the system can include quality control for the mechanical functions for the microfluidic cell culture carrier itself.
  • Such a system can enable optimal culturing conditions by monitoring and controlling many different mechanisms, including controlling the temperature, the humidity, the pH, the precipitant concentration, and the like to achieve enhanced efficiency and viability of cells during cultivation.
  • An analytical microfluidic culture system that includes a microfluidic device (e.g., a microfluidic chip) can be capable of enhancing efficiency and cell viability by mimicking in vivo conditions through controlling the cellular micro-environment within microfluidic channels, incorporating diagnostics on the carrier function and the culture media, permitting in situ optical inspection of cultures within the incubator with minimal disturbances of the culturing environment, and/or minimizing the number of steps necessary in the culturing process as presently performed in routine clinical practice.
  • a microfluidic device e.g., a microfluidic chip
  • an analytic microfluidic culture system contains a microfluidic device (e.g., a microfluidic chip) that is box-shaped with rounded corners and comprises microfluidic features such as one or more reservoirs for retaining one or more cells, one or more micro-channels for conducting fluid transport, one or more sources of fluid including culture media or reagents, and one or more receptacles (i.e. sinks) for collecting culture media and the like.
  • the fluid transport within micro- channels can be facilitated by using an active fluid transport system that comprises electronically activated and addressable tactile display pins to actively deform at least one wall of the micro-channels at specific locations along the channels thus creating one or more active valves capable of providing fluid transport.
  • the micro-channels can be arranged in a plurality of configurations to enable fluid transport between the reservoirs, the fluid sources, and the fluid sinks.
  • the reservoirs can have one or more inlets, one or more outlets, an interior, and/or a bottom support.
  • Also included in the active fluid transport system can be one or more fluid circulation components that are capable of non-turbulent delivery of culture medium to and/or from one or more of the reservoirs, sources, sinks, and the like through the micro-channels.
  • the walls of the reservoirs, channels, sources, and sinks can be constructed of a material (e.g., PDMS) that is optically transparent and permeable to external sources of oxygen or other gases suitable for the growth of cells.
  • the microfluidic device e.g., a microfluidic chip
  • the microfluidic device can comprise a substrate having at least one reservoir (e.g., funnel, chamber, and the like) and one or more micro-channels configured within the substrate.
  • the reservoir(s) can be used for retaining one or more cells, for segregating cells, and/or for holding culture media, reagents, waste fluid, and the like.
  • the micro-channels can comprise at least one rigid surface of a substrate and at least one deformable polymeric surface, preferably a bottom surface, capable of deformation as to substantially occlude fluid transport.
  • One or more locations within each micro-channel of the deformable surface can be configured to function as valves to enable active control of fluid movement within the microfluidic device.
  • At least one of the reservoirs of the microfluidic chip is used for retaining cells.
  • the reservoir has exemplary features including an interior surface, an interior upper portion, an interior lower portion, interior bottom surface, an interior space that substantially spans from the upper portion to the lower portion, and the interior space accessible through the upper portion.
  • the upper portion can be configured to permit the insertion and removal of one or more cells or the like from the reservoir, where insertion can be from the lower portion through the upper portion.
  • the reservoirs can each have one or more inlets and one or more outlets connected to one or more micro-channels.
  • the inlets and outlets positioned in the proximity of the bottom portion of the reservoirs are configured with dimensions relative to the size of a cell as to retain the cell mass within the reservoirs, preferably within the lower portion.
  • the reservoirs can also be configured with an interior space having the shape of a funnel whereby the interior dimension of the upper portion of the reservoir, for example its inner diameter, has a dimension of a defined value greater at the upper portion, where the value transitions linearly or geometrically to a smaller defined value of an inner diameter of a lower portion.
  • a reservoir can be configured to have a horizontal and or vertical cross-section with a defined shape such as square, rectangle, polygonal, truncated cone, and the like.
  • the inner dimension can be configured with dimensions to provide sufficient volumetric space or capacity for examination of embryo growth or manipulation of embryo during culture.
  • the microfluidic chip can comprise glass, quartz, silicon, plastics, and/or an optically transparent material.
  • the plastic can be a polymer or polymeric material of various polarities.
  • a surface may be polar or non-polar and/or modified to be hydrophilic.
  • the polymer may be selected from a group consisting of polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymers, polyamides, polydimethylsiloxanes, polyurethane, polysulfone, polytetrafluoroethylene, and elastomers.
  • the polymer may alternatively be selected from a family of polyacrylamides, such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA).
  • polyacrylamides such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA).
  • other polymers such as propylene glycol (PG), ethylene glycol (EG), and polyglycols including polypropylene glycols (PPG) and polyethylene glycols (PEG) may be used as the substrate.
  • other polymers such as propylene oxide (PO) and ethylene oxide (EO), and polyoxides including polypropylene oxides (PPO) and polyethylene oxides (PEO) may be used as the substrate.
  • Block copolymers of the polymers listed herein can also be used, including, for example, block copolymers of PPG and PEG and PAM and NIPAM and PDMS, and further including block copolymers such as polyacrylamide-block-N-isopropylacrylamide (PAM-NIPAM) and polydimethylsiloxane-block-polyethyleneglycol (PDMS-PEG).
  • PPG and PEG and PAM and NIPAM and PDMS block copolymers
  • block copolymers such as polyacrylamide-block-N-isopropylacrylamide (PAM-NIPAM) and polydimethylsiloxane-block-polyethyleneglycol (PDMS-PEG).
  • the inner surfaces of a reservoir and micro-channels are modified to provide hydrophilic surfaces.
  • the hydrophilic surfaces can enable rapid fluid priming of the micro-channels and reservoirs through enhanced wettability (i.e., modification of contact angle of a fluid with respect to a surface) as well as reducing adverse conditions including clogging, fouling, and the like.
  • the hydrophilic surfaces can provide a benign environment within the proximity of an inner surface of a reservoir (e.g., a funnel-shaped bottom surface used for retaining cells).
  • the surface can be modified using known methods including corona treatment, UV radiation treatment, gas-plasma treatment, surface coating, photochemical modification, attaching hydrophilic groups (e.g., OH, COOH, NH 2 , and the like), attaching PEG to the surface, using acid catalyzed hydrolysis and aminolysis on a surface, adding a surfactant, and the like.
  • hydrophilic groups e.g., OH, COOH, NH 2 , and the like
  • the reservoirs can be configured with characteristics and dimensions for the simple and efficient loading, placement, positioning, segregation, tracking, and retrieval of one or more cells.
  • the reservoirs can be configured having an inner bottom surface with one or more spatially defined circular dimple-shaped micro- depressions, each depression having an inner diameter of less than about 100 micrometers, such as about 70 micrometers, or about 50 micrometers, and a depth dimension of about 15 to 20 micrometers, and/or dimensions as to retain and secure at least a portion of a cell mass within the micro-depression.
  • Each depression can be arranged or positioned within the bottom whereby the micro-depressions are spaced based on a defined characteristic of the depression.
  • Exemplary arrangements can include a linear or geometric factor (e.g., a fraction, a multiple, and the like) of the radius of a micro-depression, a volumetric parameter, a shape, and the like.
  • each depression can be positioned relative to each other in a random fashion.
  • Each micro-depression can be configured with differing dimensions and different features (e.g., color, surface optical properties, surface roughness, distinguishing marks, and the like) as to allow the placement, isolation, and identification a specific cell, cell-type, cell mass, cell group, cell clump, cell family, oocyte, denuded mammalian zygote, and the like.
  • One or more of the reservoirs may be configured in combination within a microchip for retaining cells, holding cells, or for segregating embryos into different reservoirs or chambers.
  • the reservoirs can include an inner bottom surface accessible to visual observation and or optical analysis, described in detail below.
  • the bottom surface can have a wall thickness that is configured with specific dimensions to position one or more cells loaded in the proximity of the inner bottom surface as to enable optimal visual clarity and allowing simplification of manipulation or retrieval of one or more cells from an upper portion of the said reservoir.
  • the bottom surface can be configured with a dimensional thickness to provide visual observations within the proximity of a focal plane of an inverted microscope, preferably less than about 4 millimeters and greater than about 1.5 millimeters within the focal plane, such as about 2 millimeters within the focal plane.
  • the reservoirs can include an interior space that is configured with a total defined volumetric capacity to accommodate at least one fluid medium.
  • the fluid medium can include culture media, buffer, reagents, and the like.
  • an optimal volumetric capacity may be further defined as to enable optimal embryo development. It has been discovered that a culture medium of less than about 1 milliliter, such as about 500 microliters, or about 250 microliter, provides a capacity for the optimal flux of gases, nutrient, and/or waste in and out of the proximity of a cell, a cell group, an embryo, and the like.
  • the volume of culture medium can be static or actively transported in an out of the said reservoir using the micro-channels.
  • the optimal volume of culture medium may contain specific constituents in specific concentration and or proportion.
  • the optimal volume of a funnel can be measured and defined using one or more micro-channels in combination with one or more reservoirs within a microchip.
  • a funnel can function as a culture medium receptacle whereby a received volume is calculated using flow rate or vice versa.
  • the reservoir is configured as a funnel comprising an upper and a lower portion, having a circular cross-section, and connected to at least one channel, preferably two channels that function interchangeably as inlet and/or outlet channels.
  • Each channel comprises a proximal and distal portion with the proximal portion having a defined cross-sectional area that spans the majority of the length of the channel leading to the distal portion which furcates into two or more micro-channels, each micro-channel having individual cross-sectional areas of a smaller dimension relative to the cross-sectional area of the proximal portion.
  • the said channels are connected to the lower portion of the funnel via the micro-channels of the distal portions with each micro-channel providing an orifice at the wall of the funnel.
  • the micro-channels of the said channels can be arranged spatially along the circumference of the lower portion of the funnel as symmetrically opposing sets with orifices providing contiguous conduits for fluid flow to and from the interior of the funnel.
  • Each orifice can have a cross-sectional area that enables fluid transport and the like into and/or out of the funnel but negates the migration of, for example, a cell mass, a cell, an oocyte, and/or an embryo into a micro-channel thus retaining and securing a cell or an embryo within the funnel.
  • each said channel is configured with a distal portion having micro-channels with a total cross-sectional area that enables fluid transport into and out of the funnel with substantially the same total cross-sectional area as a single channel connected directly to the funnel having a cross-section area of the proximal portion.
  • Each channel may function synchronously or asynchronously, intermittently or periodically, or independently by active transport means (i.e., pressure, pin-flow valve actuation, and the like) to transport fluid into (i.e., ingress mode) and out (i.e., egress mode) of the funnel.
  • At least one channel connected to the funnel can function singly, pumping fluid into the funnel, while another functions as a redundant channel.
  • a micro fluidic device 100 comprises a funnel 101 with a circular cross-section area and interior 102 connected to channel 104 and channel 108.
  • the channel 104 and channel 108 comprise proximal portions 105 and 109 with entrances 107 and 111 as well as distal portions 106 and 110.
  • the entrances 107 and 111 are configured to be 300 micrometer in diameter.
  • each channel 104 and 108 are preferably spaced apart, parallel to each other, with a perpendicular length of 2400 micrometers between their longitudinal axes.
  • the channels 104 and 108 are preferably designed to be symmetric with respect to each other with a vertical diameter of the funnel 101 serving as the axis of symmetry.
  • the distal portions 106 and 110 of each channel comprise furcated micro-channels.
  • the distal portion 106 of channel 104 comprises three micro-channels.
  • the distal portion 110 of channel 108 comprises three micro-channels.
  • Each micro-channel preferably has a width of about 100 micrometers and a height of 30 about micrometers.
  • the funnel 101 is connected to each of the channels 104 and 108 by the micro-channels through a set of orifices.
  • the orifices provide fluid transport in and out of the funnel 101 and are spatially arranged along the circumference of a funnel wall 103 (see FIG. 3).
  • three micro-channels of the channel 104 are connected to the funnel wall 103 at three orifices.
  • three micro-channels of the channel 108 are connected to the funnel wall 103 at three orifices.
  • the cross-sectional area of each micro-channel is less the cross-sectional area of the proximal portions.
  • the micro-channels at the distal portion of each channel can be configured with one or more segments arranged at specific angles relative to each other as to enable the placement of orifices at spatially defined locations along the funnel wall 103.
  • an exemplary micro-channel of the distal portion 106 of the channel 104 has three segments, with two segments arranged at an angle with respect to each other. The angle can be 135 degrees for the exemplary micro-channel and the other micro- channels can be configured in a similar manner. Due to the symmetric nature of the device 100, the three micro-channels of the channel 108 can have similar angular arrangements.
  • one exemplary micro-channel from each channel 104 and 108 can be arranged having their orifices positioned symmetrically opposite each other, with their longitudinal axes coaxially aligned with a horizontal diameter of the cross-sectional area of the funnel 101.
  • Exemplary micro- channels from channel 104 can be arranged at an angle relative to the diameter of funnel defined by the opposing positions of orifices from channels 104 and 108. In some embodiments, the angle between the said diameter and the longitudinal axis of a segment of an exemplary micro-channel is 45 degrees.
  • the angle between the said diameter and the longitudinal axis of a segment of another exemplary micro- channel 114 is 45 degrees. Therefore, the sum of the two angles is 90 degrees for the channel 104, and similarly for the channel 108.
  • the micro-channel segments can be configured with other angles and depends on the number of orifices chosen to be connected to the funnel 101.
  • the device 100 is configured to pump a given volume of fluid using one or more of the micro-channels into (i.e. ingress mode) and out of (i.e. egress mode) of the funnel 101.
  • the orifices of the said micro-channels have cross-sectional areas of defined dimensions as to allow fluid transport into and out of the funnel 101 but retard embryos or cellular constituents placed within the funnel 101 from entering any said micro-channels.
  • Each of the channels 104 and 108 may be configured to furcate into two or more said micro-channels.
  • the total cross sectional areas of channels 104 and 108 are preferably the same as the total cross sectional areas of their respective orifices.
  • each of the channels 104 and 108 can function synchronously or asynchronously, intermittently or periodically, or independently by active transport means (e.g., pressure, pin-flow valve actuation, and the like) to transport fluid into and/or out of the funnel 101.
  • active transport means e.g., pressure, pin-flow valve actuation, and the like
  • At least one channel, for example the channel 104 connected to the funnel 101 is functioning singly, pumping fluid into the funnel 101, while the channel 108 functions as a redundant channel.
  • the arrangement serves to eliminate the potential of a cell or embryo to be transported by suction from the funnel 101 through the orifices and into the micro-channels during an egress mode.
  • active fluid transport e.g., provided by the fluid circulation components and/or controlled by the tactile display pins
  • flow rate can be determined by the accumulation of fluid volume within a sink.
  • the micro fluidic carrier contains interfaces to enable the measurement of one or more constituents of the culture media and/or one or more analytes that can be consumed or produced by a cell retained within the microfluidic carrier.
  • Exemplary analytes can include pH, K (potassium), oxygen, lactate, glucose, ascorbate, serotonin, dopamine, ammonina, glutamate, purine, calcium, sodium, potassium, NADH, protons, insulin, antibodies, antigens, receptors, NO (nitric oxide), and the like.
  • the active fluid transport system enables the regeneration of culture media.
  • Exemplary active fluid transport system functions can include delivering a culture medium to a reservoir, extracting product (e.g., removing waste material) from a reservoir, depositing material within one or more sinks, and/or replenishing nutrients to a culture medium through one or more micro-channels.
  • the active fluid transport system can be controlled to spatially or temporally vary a value, a concentration, and/or a gradient of one or more constituents of the cell culture media and/or one or more environmental parameters within the microfluidic culture carrier.
  • the microfluidic device can contain components such as micro-channels whose flow characteristics are to be actively varied. These components can be formed of compressible or distortable elastomeric materials.
  • substantially the entire microfluidic device can be constructed of a flexible elastomeric material such as an organopolysiloxane elastomer ("PDMS”), as described hereinafter.
  • PDMS organopolysiloxane elastomer
  • Polydimethylsiloxane (PDMS) is a rubbery, biologically compatible, microfabrication compatible, silicone elastomer that is low-cost, and generally non-toxic, non-flammable, thermally stable, chemically inert, optically clear, permeable to gases including oxygen and carbon dioxide and almost impermeable to water.
  • the device substrate may be constructed of hard, i.e., substantially non-elastic material at portions where active control is not desired, although such construction generally involves added construction complexity and expense.
  • the generally planar devices can comprise a rigid support of glass, silica, rigid plastic, metal, etc. on one side of the device to provide adequate support. In some alternative embodiments, actuation from both major surfaces may require that these supports be absent, or be positioned remote to the elastomeric device itself.
  • the surfaces of the micro-channel within the micro fluidic device can be modified to achieve particular flow characteristics and/or surface chemistry intended for particular set of experiments. Characteristics such as topographical and chemical patterning make PDMS an ideal candidate for use as a micro-channel substrate.
  • Pulsed plasma deposition of allylamine on hydrophobic PDMS creates cytophilic cell adhesion surfaces for securing cells within a reservoir. Attachment and growth of cultured cells can be manipulated using microcontact printing methods that permit the deposition of thin layers of various organic materials on PDMS with varying degree of organized architecture. Selective patterning of PDMS substrates may also be necessary to confine cell growth to particular regions within a reservoir or a micro-channel. Silanizing oxidized PDMS with an amino-terminated silane (aminopropyltriethoxysilate) can provide a reactive surface for a bifunctional cross- linker for protein attachment.
  • an amino-terminated silane aminopropyltriethoxysilate
  • Bio-fouling of channels can be minimized by grafting a poly (ethylene glycol) di-(triethoxy) silane onto an oxidized PDMS surface which causes the surface of PDMS to become permanently hydrophilic with reduced bio- fouling properties.
  • the culture system can include at least one active portion which alters the shape and/or volume of micro fluidic features, thus alerting the fluid flow capabilities of the device.
  • active portions include mixing portions, pumping portions, valving portions, flow portions, channel or reservoir selection portions, cell crushing portions, unclogging portions, and the like. These active portions can induce some change in, for example, the fluid flow, the fluid characteristics, the channel characteristics and/or the reservoir characteristics by exerting a pressure on the relevant portions of the device, thus altering the internal shape and/or volume of these features.
  • the active portions of the carrier can be actuated by pressure to close their respective channels or to restrict the cross-sectional area of the channels to accomplish the desired active control.
  • the microfluidic features e.g., sources, sinks, reservoirs, channels, and the like
  • the microfluidic features can be constructed in such a way that modest pressure from the exterior of the microfluidic carrier causes the microfluidic features to compress, causing local restriction, or total closure of the respective feature.
  • the walls within the plane of the carrier surrounding the feature can be elastomeric, along with the external surfaces (e.g., in a planar device, an outside major surface) such that a minor amount of pressure causes the external surface and/or the internal feature walls to distort, either reducing cross-sectional area at this point or completely closing the feature.
  • the pressure required to alter the shape of the active portion(s) of the carrier is supplied by an external tactile device such as are used in refreshable Braille displays.
  • the tactile actuator contacts the active portion of the device, and when energized, can extend and press upon the deformable elastomer, restricting or closing the feature in the active portion.
  • the pressure generated by the external tactile actuator can be measured and regulated based on the deformation resistance of one or more walls of the micro-channel of the micro fluidic carrier. The extent of pressure measured can provide feedback on the presence or absence of fluid within a micro-channel.
  • the rate of change of the presence or absence of fluid can be monitored to determine a fluid flow rate within the micro-channel.
  • the culture carrier comprises one or more interfaces for the integration of sensors and controllers.
  • sensors can include those designed to measure levels of glucose, lactate, sodium, potassium, pH, dissolved O 2 , dissolved carbon dioxide, and the like.
  • the microfluidic carrier can be designed with interfaces that allow modular "plug-in"s with functionalities that can be tailored to a particular application.
  • the functionalities can include the integration of microelectrode arrays for physiological studies, biosensors for detection and control of small molecules (e.g., oxygen, pH, glucose), biosensors for large molecules (e.g., immunosensors), non invasive spectro-photometric sensors, optical imaging systems, and the like.
  • Exemplary sensors can also include thermal detectors, electrical detectors, chemical detector, optical detectors, ion detectors, biological detectors, radioisotope detectors, electrochemical detectors, radiation detectors, acoustic detectors, magnetic detectors, capacitive detectors, pressure detectors, ultrasonic detectors, infrared detectors, microwave motion detectors, radar detectors, electric eyes, image sensors, and the like.
  • the interface can be located at a wall of a reservoir, at a wall of at least one micro-channel, and/or at a wall of a sink.
  • one or more of the analytical sensors can be introduced into the culture media located within a reservoir, a channel, and/or a sink of the microfluidic culture carrier.
  • the analytical sensors can sample an aliquot of culture media from a reservoir, channel, and/or sink for the purpose of conducting a measurement and providing a result.
  • Analytical parameters and constituents of the cell culture measured and monitored can include optical density, dissolved oxygen, temperature, humidity, pH, carbon dioxide, electrolytes, a cellular metabolite, proteins, organic compounds, and the like.
  • Exemplary sensors can include temperature sensors, humidity sensors, pH sensors, oxygen sensors, carbon dioxide sensors, and nutrient sensors.
  • One or more analytical results generated from information obtained from the sensors can be used to determine parameters of the cell culture media and/or the environmental condition within the microfluidic carrier. The results can be used with the active fluid transport and automated environmental control to control the culture environment and the culture medium.
  • the cell culture environment is under temperature control.
  • the microfluidic carrier can contain a heating element, such as a miniature resistive foil heater (e.g., a Minco HK5291) in contact with one surface of a reservoir.
  • the heating element can include lead wires that extend from the microfluidic carrier to a temperature controller coupled to a DC switching relay and a DC power supply that can be replaced with a battery for portability.
  • Exemplary power requirements for the heating element integrated with the microfluidic carrier are less than about 2 Watts, such as IW.
  • the culture media can be well circulated using the active fluid transport so that the temperature of the reservoir is essentially homogeneous throughout the microfluidics channels at any time.
  • the temperature of the culture media can be controlled and kept within a range of about ⁇ 1 0 C of a pre-selected level, such as within a range of about ⁇ 0.5 0 C, within about ⁇ 0.1 0 C, or within about ⁇ 0.05 0 C or less.
  • the temperature controller can be in the form of a proportional-integral-derivative (PID) controller that serves to maintain the temperature at a pre-selected level and automatically performs temperature control through an optimal set of constants (e.g., proportional, integral, differential, and the like) based on the thermal response of the system.
  • PID proportional-integral-derivative
  • a culture system can enable simultaneous optical viewing/imaging , fluidic control, electrical interfacing, and/or data acquisition, in an environment where the temperature, concentration, and/or perfusion of gases, nutrients or other relevant substances are controlled.
  • the cell culture environment within a microfluidic culture device can be actively controlled based on culture medium and cell parameters including physiological features and analytes produced by a cell.
  • the culture system can provide a method of selecting a culture environment parameter comprising steps of: culturing one or more cells in a plurality of reservoirs, wherein the micro fluidic device is operated under conditions in which the value of the culture environment parameter varies based on the measurement of cell parameters and analyte values (e.g., concentration, gradient, and the like); monitoring the cell features in each reservoir; and/or identifying one or more values of the culture environment parameter that results in optimum cell viability.
  • temperature can be monitored within the microfluidic culture device and actively controlled to replicate in vivo temperature for optimal cell growth.
  • the temperature of the media inside the microfluidic carrier environment is controlled at about 37 ⁇ .0.2 0 C.
  • In- situ quality control testing including priming condition and fluid flow rate may be performed automatically by the active fluid control device coupled to the microfluidic carrier.
  • One or more cells are introduced into a reservoir upon auto confirmation of the necessary optimal condition of the culture environment. The cells are kept within the culture for cultivation with minimal disturbances thereby reducing adverse events to cells, for example, the risk of contamination.
  • the culture system can enable the introduction of externally controlled temporally varying and/or spatially varying concentration gradients of transported culture media agents into the culture reservoir.
  • the environment of a cell culture can be actively changed by controlling the reservoir environment. For example, one or more programmable time-periodic injections of nutrients can be made, followed by periods of time where the contents of a reservoir are statically controlled.
  • the media can also be cycled in and out of the reservoir in some periodic or aperiodic fashion to break the temporal invariance of spatial gradients.
  • the ratio of fresh to used media can be varied to increase the amount of a chosen nutrient.
  • the determination of flow rate of the culture media can be determined by quantifying the accumulation or diminution of fluid volume within a source, a reservoir, or a sink.
  • the fluidic system may be easily adapted to address and individually control multiple culture reservoirs, sources, and sinks.
  • Reagents and culture media can be discretely applied to specific reservoirs and regions with micro-channels using the active portions of the carrier. This makes the microfluidic carrier ideal for time-dependent cellular analysis at dynamically controlled exchange rates. For example, extracellular and intercellular ion concentration can be controlled and altered within each reservoir.
  • the microfluidic carrier can contain interfaces that enable the sensing of, for example, the spent perfusate collected in one or more sinks.
  • the substances (e.g., analytes) released during cultivation can be analyzed using a chosen sensor for an analyte of interest (e.g., perfusion gases within the culture media).
  • the system can provide the capability of performing cell or embryo cultures in a specifically composed gas mixture atmosphere by monitoring and controlling the environment external to the carrier as to allow the perfusion of an optimal concentration of gas mixtures into a microfluidic cell carrier.
  • a gas mixture consisting of 5% carbon dioxide in air is used by many laboratories worldwide for maturation and fertilization of bovine embryos.
  • certain cultures require a gas mixture consisting of 5% oxygen, 5% carbon dioxide, and 90% nitrogen, while other cultures have to be performed in 2% carbon dioxide in air.
  • the percentage of oocytes developing to the blastocyte stage is significantly higher when the carbon dioxide level is set to 3.5%
  • the culture system can permit continuous and simultaneous monitoring of one or more cell culture parameters using, for example, a microscope charge coupled display (CCD) imaging system for image acquisition, visual observation, and/or spectro-photometric sensing.
  • CCD microscope charge coupled display
  • a modified microscope stage insert may be used to align and couple the microfluidic carrier to a microscope CCD imaging system.
  • the microfluidic carrier can be fabricated with one or more reservoirs with a surface having a matched index of refraction for receiving a portion of a microscope objective. This enables, for example, in situ cell culture viability, immuno- cytochemistry analysis, and the like.
  • the microscope CCD imaging system comprises an image capturing device.
  • the image capture device can include a microscope or other high magnification optical system having an objective lens, an X-Y stage plate adapted for holding the microfluidic carrier, a means for moving the plate to align the microfluidic carrier with the microscope objective and for moving the carrier in the direction to effect focusing, a CCD camera, a detection system with a light source for directing light into a reservoir of the microfluidic carrier, a means for directing light emitted from the cells to the CCD camera, and a computer means for receiving and processing digital data from the CCD camera.
  • Exemplary optical methods that may be used include bright field, dark field, phase contrast and/or interference contrast.
  • the computer means cam include a digital frame grabber for receiving the images from the camera, a display for user interaction and display of a cell, a digital storage media for data storage and archiving, and/or a means for control, acquisition, processing and display of results.
  • a still photo and/or video can be captured by the image capturing device and stored in an electronic format (e.g., bitmap, Graphics Interchange Format, JPEG file interchange format, TIFF, MPEG, and the like).
  • the image of the cell or cell culture can be captured by an analog camera and converted into an electronic form by a computer means.
  • the image capture device can include imaging systems, confocal or light microscopes, or cell screening systems known in the art. Each image may contain a single cellular structure or multiple cellular structures.
  • the culture system includes a method for determining characteristics of a cell using acquired images to perform classification analysis.
  • Acquired images can be processed using signal processing software (e.g., 2 and 3 dimensional) to extract features which can be used for cellular structure classification.
  • image features can be extracted by a texture analysis algorithm, and/or a border analysis algorithm.
  • the image analysis can be performed by a digital signal processor (DSP), application specific processor (ASP), other integrated circuits, and the like.
  • DSP digital signal processor
  • ASP application specific processor
  • characteristics of a cell can be determined thus enabling the classification of a given cellular structure.
  • an image of a cellular structure can be captured, a plurality of numerical image features can be derived from said image, and/or the cellular structure can be classified by comparison to a known cellular structure classification model.
  • Exemplary cellular structures can include types of cells, cellular organelles, cell cultures, and/or any discernible two- dimensional or three-dimensional biological or cellular structures.
  • a cellular structure can be single or multicellular, prokaryotic or eukaryotic, mammalian (e.g., mouse, rodent, primate, and/or human) or non-mammalian.
  • a cellular structure can be an egg cell, an embryo, a stem cell, a differentiated cell, a tumor cell, a colon cell, a breast cell, a lung cell, a prostate cell, a pancreatic cell, a kidney cell, an endometrial cell, a cervical cell, an ovarian cell, a thyroid cell, and the like.
  • the cellular structure can be an organelle such as a nucleus, cell membrane, cell wall, mitochondria, golgi, chromatin, DNA, cytoskeleton, ribosome, and/or endoplasmic reticulum.
  • an image is analyzed for the purpose of identifying image features contained within the image.
  • the structural textures of an embryo can be characterized, for example, as homogenous or heterogeneous, smooth, fine, and/or tight or loose.
  • Exemplary image features may be single or multi-cellular in nature and include cell texture, cell border, cell membrane, or any discernible two- dimensional or three-dimensional cellular structure.
  • image features include components of cell cytoplasm, cellular organelles, and/or portions thereof (e.g., nucleus, cell membrane, cell wall, mitochondria, golgi, chromatin, DNA, cytoskeleton, endoplasmic reticulum, and the like).
  • Image features can include biomolecular markers, expression pattern of biomarkers, recombinant proteins that emit a signal, molecular tags that emit a signal, and the like.
  • the emitted signal can be one or more electromagnetic signals within a detectable electromagnetic spectrum (e.g., bioluminescence, fluorescence, infrared, ultra-violet, and the like).
  • the culture system enables simultaneous imaging and acquisition of spectral data regarding absorption, transmittance, and/or reflectance of electromagnetic radiation of or from a cell culture.
  • classification of embryos based on the analysis of spectral data collected from the embryos using IR spectroscopy.
  • Spectroscopic analysis including IR spectroscopy, NIR spectroscopy, and Raman spectroscopy, permits identification of the chemical composition (e.g., surface chemistry, internal chemistry, and the like) of an embryo. It is known that the quality of an embryo can be related to the gross chemical composition of the embryo and/or its parts (e.g., the amount of water and storage compounds such as proteins, lipids, carbohydrates, and the like).
  • spectroscopic analysis can be used to classify one or more cells (e.g., embryos), obtaining an image or imaging is not limited to obtaining a visible image of cells, and may include acquiring spectral data from cells, or parts of cells, to identify the chemical composition.
  • a specific cell or embryo can be selected based on at least one physical feature and/or at least one analyte produced by the cell or embryo.
  • Exemplary steps include culturing at least one cell (e.g., each in an individual reservoir), measuring at least one characteristic feature of the cell, and selecting a cell based on at least one characteristic feature of the cell.
  • the analytic micro fluidic culture system includes methods for the non-invasive determination of various characteristics of a cell including the viability of a cell, the plasticity of a cell, the sternness of a cell, the rate of proliferation of a cell, the tumorigenic state of a cell, the rate of differentiation of a cell, the suitability for fertilization, the suitability of implantation, and the like.
  • the micro fluidic carrier can be constructed of materials and dimensions for use with a microscope charge coupled display (CCD) imaging system.
  • One or more walls of the reservoir of the micro fluidic carrier can be constructed of a material with an index of refraction matched to the objective of a microscope as to enable high optical clarity.
  • the CCD imaging system can be connected to a computer, and/or to a visual monitor, for use in recording and/or displaying images of a cell. For example, images of an embryo retained with a reservoir can be periodically captured and stored during the culturing period.
  • the microfluidic carrier can be constructed to facilitate multiple optical interfaces for imaging multiple views of an embryo. One interface, attached to a camera, can receive a first view of an embryo (e.g., the top view). An additional interface provides a first reflecting surface for receiving and reflecting a second view of the plant embryo (e.g., the side view) toward the camera. Using multiple camera interfaces, the microfluidic carrier can permit simultaneously imaging of both the first and second views of a cell or embryo.
  • Image features can include but are not limited to the physiological or morphological representations of a cellular border, a cell membrane, and cell texture of an embryo.
  • the image features may include a kinetic or spatio-temporal parameter relating to an embryo.
  • Kinetic or spatio-temporal parameters can include visual changes in the said image features over a given period of time. The changes in the said image features over a given period of time can be correlated to, for example, changes in the viability of the cellular structures, changes in the plasticity of the cellular structures, changes in the ability of an embryo to be implanted in vivo, changes in the state of fertilization of an embryo, the loss or gain of the viability of a cell.
  • the culture system includes an automated, noninvasive, quantitative method for determining the utility of one or more cells (e.g., an embryo) in culture.
  • the utility of a cell can include any physiological or phenotypic information associated with a cell, such as its viability, the plasticity/sternness of a cell, the rate of proliferation and/or differentiation of a cell, the suitability for fertilization an embryo, and/or the suitability of implantation into the uterus.
  • the utility of a cell may be obtained by classifying a cellular structure of the cell, and can include the steps of capturing an image of a cellular structure, deriving a plurality of image metrics from said image, and classifying the cellular structure according to classification models known in the art based on the derived image features.

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  • Analytical Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
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Abstract

L'invention concerne des puces microfluidiques pour cellules qui comportent des microcanaux et des chambres intégrés.
PCT/US2008/077105 2007-09-20 2008-09-19 Système de culture analytique microfluidique WO2009039433A1 (fr)

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EP2443224A1 (fr) * 2009-06-19 2012-04-25 University of Maryland, Baltimore County Détection non invasive de paramètres de bioprocédés
US8350820B2 (en) 2009-11-06 2013-01-08 Bose Corporation Touch-based user interface user operation accuracy enhancement
WO2013004644A1 (fr) * 2011-07-01 2013-01-10 Universiteit Twente Dispositif microfluidique doté de capteurs intégrés pour la culture de cellules
US8638306B2 (en) 2009-11-06 2014-01-28 Bose Corporation Touch-based user interface corner conductive pad
US8669949B2 (en) 2009-11-06 2014-03-11 Bose Corporation Touch-based user interface touch sensor power
US8686957B2 (en) 2009-11-06 2014-04-01 Bose Corporation Touch-based user interface conductive rings
US8692815B2 (en) 2009-11-06 2014-04-08 Bose Corporation Touch-based user interface user selection accuracy enhancement
US8736566B2 (en) 2009-11-06 2014-05-27 Bose Corporation Audio/visual device touch-based user interface
US9201584B2 (en) 2009-11-06 2015-12-01 Bose Corporation Audio/visual device user interface with tactile feedback
WO2016005971A1 (fr) * 2014-07-06 2016-01-14 Fertilesafe Ltd Dispositifs pour la culture d'un échantillon biologique dans un liquide de culture
WO2016131079A1 (fr) * 2015-02-17 2016-08-25 Genea Ip Holdings Pty Limited Procédé et appareil pour la culture dynamique d'un échantillon biologique
EP3239285A1 (fr) * 2016-04-27 2017-11-01 Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen Dispositif de determination et de surveillance de l'etat physiologique de cultures microbiennes dans chaque micro-bioreacteur d'une plaque de microtitration
CN111718853A (zh) * 2020-07-03 2020-09-29 中山大学 一种用于药物筛选的2d和3d一体化肿瘤器官培养芯片的制备方法
EP3907007A1 (fr) * 2020-05-08 2021-11-10 Technische Universität Wien Dispositif microfluidique
CN113789264A (zh) * 2021-08-09 2021-12-14 哈尔滨工业大学（深圳） 胚胎培养检测装置及胚胎培养检测方法
EP3943586A4 (fr) * 2019-03-19 2022-05-04 FUJIFILM Corporation Système de culture cellulaire et procédé de culture cellulaire
EP3943588A4 (fr) * 2019-03-19 2022-05-25 FUJIFILM Corporation Appareil de traitement d'informations, système de culture cellulaire, procédé de traitement d'informations et programme de traitement d'informations
EP4039791A1 (fr) * 2021-01-13 2022-08-10 Biothera Institut GmbH Appareil de commande d'un processus, ainsi que procédé de commande associé

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US20060166357A1 (en) * 2003-03-10 2006-07-27 The University Of Michigan Integrated microfludic control employing programmable tactile actuators
US20070161106A1 (en) * 2003-12-19 2007-07-12 Eric Jervis Cultured cell and method and apparatus for cell culture

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US20040115838A1 (en) * 2000-11-16 2004-06-17 Quake Stephen R. Apparatus and methods for conducting assays and high throughput screening
US20060166357A1 (en) * 2003-03-10 2006-07-27 The University Of Michigan Integrated microfludic control employing programmable tactile actuators
US20070161106A1 (en) * 2003-12-19 2007-07-12 Eric Jervis Cultured cell and method and apparatus for cell culture

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2443224A4 (fr) * 2009-06-19 2014-10-22 Univ Maryland Détection non invasive de paramètres de bioprocédés
EP2443224A1 (fr) * 2009-06-19 2012-04-25 University of Maryland, Baltimore County Détection non invasive de paramètres de bioprocédés
US8692815B2 (en) 2009-11-06 2014-04-08 Bose Corporation Touch-based user interface user selection accuracy enhancement
US8638306B2 (en) 2009-11-06 2014-01-28 Bose Corporation Touch-based user interface corner conductive pad
US8669949B2 (en) 2009-11-06 2014-03-11 Bose Corporation Touch-based user interface touch sensor power
US8686957B2 (en) 2009-11-06 2014-04-01 Bose Corporation Touch-based user interface conductive rings
US8736566B2 (en) 2009-11-06 2014-05-27 Bose Corporation Audio/visual device touch-based user interface
US9201584B2 (en) 2009-11-06 2015-12-01 Bose Corporation Audio/visual device user interface with tactile feedback
US8350820B2 (en) 2009-11-06 2013-01-08 Bose Corporation Touch-based user interface user operation accuracy enhancement
WO2013004644A1 (fr) * 2011-07-01 2013-01-10 Universiteit Twente Dispositif microfluidique doté de capteurs intégrés pour la culture de cellules
WO2016005971A1 (fr) * 2014-07-06 2016-01-14 Fertilesafe Ltd Dispositifs pour la culture d'un échantillon biologique dans un liquide de culture
JP2021072814A (ja) * 2015-02-17 2021-05-13 ジェネア アイピー ホールディングス ピーティーワイ リミテッド 生物学的試料を動的に培養するための方法および装置
WO2016131079A1 (fr) * 2015-02-17 2016-08-25 Genea Ip Holdings Pty Limited Procédé et appareil pour la culture dynamique d'un échantillon biologique
JP2018508234A (ja) * 2015-02-17 2018-03-29 ジェネア アイピー ホールディングス ピーティーワイ リミテッド 生物学的試料を動的に培養するための方法および装置
JP2022188054A (ja) * 2015-02-17 2022-12-20 ジェネア アイピー ホールディングス ピーティーワイ リミテッド 生物学的試料を動的に培養するための方法および装置
EP3239285A1 (fr) * 2016-04-27 2017-11-01 Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen Dispositif de determination et de surveillance de l'etat physiologique de cultures microbiennes dans chaque micro-bioreacteur d'une plaque de microtitration
EP3943586A4 (fr) * 2019-03-19 2022-05-04 FUJIFILM Corporation Système de culture cellulaire et procédé de culture cellulaire
EP3943588A4 (fr) * 2019-03-19 2022-05-25 FUJIFILM Corporation Appareil de traitement d'informations, système de culture cellulaire, procédé de traitement d'informations et programme de traitement d'informations
EP3907007A1 (fr) * 2020-05-08 2021-11-10 Technische Universität Wien Dispositif microfluidique
WO2021224328A1 (fr) 2020-05-08 2021-11-11 Technische Universität Wien Dispositif microfluidique
CN111718853B (zh) * 2020-07-03 2022-08-02 中山大学 一种用于药物筛选的2d和3d一体化肿瘤器官培养芯片的制备方法
CN111718853A (zh) * 2020-07-03 2020-09-29 中山大学 一种用于药物筛选的2d和3d一体化肿瘤器官培养芯片的制备方法
EP4039791A1 (fr) * 2021-01-13 2022-08-10 Biothera Institut GmbH Appareil de commande d'un processus, ainsi que procédé de commande associé
EP4039791B1 (fr) 2021-01-13 2023-08-30 Biothera Institut GmbH Appareil de commande d'un processus, ainsi que procédé de commande associé
CN113789264A (zh) * 2021-08-09 2021-12-14 哈尔滨工业大学（深圳） 胚胎培养检测装置及胚胎培养检测方法

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