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  • 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
  • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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  • 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
  • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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  • C12M41/14—Incubators; Climatic chambers
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  • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
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  • 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
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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  • 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
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/48—Automatic or computerized control
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
• • G—PHYSICS
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2200/06—Fluid handling related problems
  • B01L2200/0689—Sealing
• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2300/046—Function or devices integrated in the closure
  • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0829—Multi-well plates; Microtitration plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

• the invention in various embodiments relates to assays, systems, and/or devices for culturing cells or other biologic material in controlled environments and that are applicable to related fields generally using microfluidic systems.
• Particular embodiments involve configurations that can be used with various standard automated handling systems, with active or passive loading and perfusion of medium and to provide high-throughput multi-assay automated systems for culturing, viewing, and analyzing cell growth, invasion, movement, chemotaxis or other properties.
• the invention relates in specific embodiments to heat control systems for microfluidic culture plates and other automated systems for culture plates.
• Microfiuidic cell culture is an important technology for applications in drug screening, tissue culturing, toxicity screening, and biologic research and can provide improved biological function, higher- quality cell-based data, reduced reagent consumption, and lower cost.
• High quality molecular and cellular sample preparations are important for various clinical, research, and other applications.
• In vitro samples that closely represent their in vivo characteristics can potentially benefit a wide range of molecular and cellular applications.
• Handling, characterization, culturing, and visualization of cells or other biologically or chemically active materials has become increasingly valued in the fields of drug discovery, disease diagnoses and analysis, and a variety of other therapeutic and experimental work.
• Cytoplex, Inc. 6,653,124 "Array-based microenvironment for cell culturing, cell monitoring and drug-target validation.”
• the present invention involves various components, systems, and methods related to improved microfiuidic cell culture devices and systems, in particular systems for the culturing and/or analysis and/or viewing of cells under controlled temperature and gas parameters.
• the invention involves novel microfiuidic cell culture devices, systems and methods that have advantages over previously proposed culture systems providing in some embodiments, more convenient and unobtrusive heating and gas control and also providing automatic handling.
• the invention involves novel structures and methods for integrating heating and gas control with multiple microfiuidic cell culture units in various multi cell culture unit systems, such as to a microtiter well plate structure including various standard well plate formats (e.g., a 96-well SBS culture plate, or other plate formats, including plates having 6, 12, 24, 96, 384 or 1536 sample wells, as well as open bottom standard well plates, as well as plates with specific areas for cell culture and/or viewing.
• standard well plate formats e.g., a 96-well SBS culture plate, or other plate formats, including plates having 6, 12, 24, 96, 384 or 1536 sample wells, as well as open bottom standard well plates, as well as plates with specific areas for cell culture and/or viewing.
• design features include providing a cell culture device in a convenient format that allows for the elimination of tubing and connectors to the plates themselves and provides temperature control and monitoring mostly or entirely contained in a removable manifold that fits onto the plates with a gas seal, thereby providing the ability to maintain long-term cell culture with temperature and/or gas control in a culture platform that maintains the ability to easily observe cells and that is easily removable from the culture plate.
• a system of the invention can be used with a variety of cell culture units on culture plates, such as those described in the above referenced patent applications, including cell culture units for detennining cellular invasion, culture units with gel culture media, and a variety of other culture units as described herein or in incorporated related applications.
• Living cells generally require careful control of their physical environment, including nutrient and gas exchange, temperature regulation, and protection from stress.
• Advanced micro-scale cell culture methods such as described in above referenced patent applications enable structural control (microfabrication), and perfusion control (microfluidics).
• the present invention is directed to an additional area of this field, referred to at times herein as micro-incubation, to provide control of temperature and gas atmosphere for use in micro-scale cell culture systems in a way that is unobtrusive to observational equipment and that further is easily attached and removed from a culture plate in specific embodiments.
• the invention provides a miniaturization of the traditional cell culture incubator concept to perform dynamic, continuous temperature and gas regulation directly to a microfiuidic chamber.
• this is possible through the creation of a micro-incubator chamber of about 5 mL volume in contact with the microfiuidic cell culture chambers (1 ul).
• the temperature and gas content of the micro-incubator quickly transfers to the cell culture chamber (by conduction and diffusion).
• the micro-incubator is maintained by a novel manifold design as described herein. Manifolds according to specific embodiments of the invention can be controlled by various systems and software.
• Full microscope enclosures surround the entire microscope except for some heat generating or heat sensitive components such as the camera and illumination sources.
• the air within the enclosure is circulated and maintained at the desired temperature and gas environment of the sample.
• Stagetop incubators surround only a small volume intended to house one or more Petri dishes, slides or other culture platform. These provide local temperature regulation and enable limited gas environment control. They are convenient, but do not provide the same level of control as a microscope enclosure, as the stagetop incubator must mimic the mechanics of an enclosure, but in a smaller size and adapt to the microscope. For example, uniform temperature control in a stagetop incubator is limited by the heat sink of the stage itself, and cutouts to provide for the light path for optical clarity further reduce uniformity. In addition, the complexity of the design of stagetop incubators make them expensive.
• Perfusion chambers generally consist of an assembly that encloses a flowing liquid volume, with heating elements either directly through the walls of the chamber or immediately upstream of the inlet flow path.
• the design of the control elements need to be carefully considered, as issues such as heat/mass transfer may make proper maintenance of a steady state condition difficult.
• flow chambers are infrequently used in live cell imaging due to the myriad difficulties of adapting them for typical uses.
• the present invention by contrast, integrates temperature, flow, and gas control directly to a microfluidic culture plate via the use of a manifold that seals to the microfluidic plate. While manifolds similar in some aspects are discussed in some of the above referenced applications to control perfusion on microfluidic plates, the previous designs did not incorporate all of the features described herein as such incorporation was difficult due to the compact nature of the manifold and culture plate.
• the present manifold design includes novel temperature or gas "microincubator" compartments created by operation of the manifold. These compartments have been demonstrated to provide novel and critical advantages for the proper integration for live cell imaging needs.
• the present invention enables direct cell culture on a microscope stage without the use of external environment chambers such as enclosures or stage -top heaters.
• the "microincubator" concept according to specific embodiments of the present invention provides precise control, long-term cell culture, and ease of dynamic changes of conditions that has not been available in this context in other designs.
• microfluidic structures and culture units and systems and methods of various configurations as described herein can also be deployed independently of any well-plate, such as in various integrated lab-on-a-chip systems that are not configured to be used in conjunction with well plates or various other microfluidic devices or systems.
• any combination of elements described herein should be understood to include every sub-combination of any subset of those elements and also any sub-combination of any subset of those elements combined with any other element described herein or as would be understood to a practitioner of skill in the art.
• the present invention is described in terms of the important independent embodiments of multi-component devices or systems. This should not be taken to limit various novel aspects of the invention, which, using the teachings provided herein, can be applied to a number of other situations.
• the present invention is described in terms of a number of specific example embodiments including specific parameters related to dimensions of structures, pressures or volumes of liquids, temperatures, electrical values, durations of time, and the like. Except where so provided in the attached claims, these parameters are provided as examples and do not limit the invention, which encompasses other devices or systems with different dimensions.
• FIG. 1 shows a side view of an example micro-incubator manifold according to specific embodiments of the invention shown in place in a micro-incubator system with a well plate and microscope viewer.
• FIG. 2 illustrates one example of a top plane view of a manifold with a heat controller according to specific embodiments of the invention.
• FIG. 3 illustrates one example of a topside view of a manifold with a heat controller sealed to a well-plate and mounted in a microscope viewer according to specific embodiments of the invention.
• FIG. 4 illustrates one example of a culture plate with four culture units placed onto a 96-well standard SBS plate.
• This example shows four culture units (corresponding to rows labeled A-D) with six inlet wells (labeled A1-D6), four microfluidic culture areas placed under the large viewing oval, and two outlet wells (7-8).
• A-D inlet wells
• A1-D6 inlet wells
• 7-8 microfluidic culture areas placed under the large viewing oval
• FIG. 4 illustrates one example of a culture plate with four culture units placed onto a 96-well standard SBS plate.
• This example shows four culture units (corresponding to rows labeled A-D) with six inlet wells (labeled A1-D6), four microfluidic culture areas placed under the large viewing oval, and two outlet wells (7-8).
• A1-D6 inlet wells
• 7-8 two outlet wells
• FIG. 5A-D are schematic drawings of an example implementation of a manifold with a heat controller from various views according to specific embodiments of the invention.
• FIG. 6(A) illustrates the bottom portion of a heat exchange module according to specific embodiments of the invention.
• the bottom portion attaches to the pneumatic portion of the manifold.
• (B) illustrates the top portion of a heat exchange module according to specific embodiments of the invention.
• FIG. 7 is a schematic side view of the pneumatic portions of a manifold sealed to a culture plate and showing the gas in lines connecting to an environment control volume according to specific embodiments of the invention.
• FIG. 8 is a schematic showing how a manifold interfaces to a microfluidic plate according to specific embodiments wherein a positive seal is created by applying a vacuum to the cavity surrounding all the wells.
• the heating unit is not shown in this figure.
• FIG. 9 illustrates a manifold with additional gas line and a heated objective lens according to an earlier manifold design.
• FIG. lOA-C shows a top view, side view, and plan view of a schematic of an example pneumatic manifold according to earlier designs.
• the eight tubing lines to the right are for compressed air, and each is configured to provide pressure to a column of cell inlet wells in a microfluidic array.
• the left-most line in the figure is for vacuum and connects to an outer vacuum ring around the manifold.
• This basic manifold design is modified using the teachings herein to produce the heated manifold.
• FIG. 11 illustrates an example microfluidic perfusion system (ONIXTM), microincubator controller and manifold (MIC) according to specific embodiments of the invention.
• ONIXTM microfluidic perfusion system
• MIC manifold
• FIG. 12 illustrates an example microfluidic perfusion system (ONIXTM), microincubator controller and manifold (MIC) and computer control system according to specific embodiments of the invention.
• ONIXTM microfluidic perfusion system
• MIC manifold
• FIG. 14 illustrates one alternative of plate and culture unit design with an example culture unit filled with blue dye with the image taken from top according to specific embodiments of the invention.
• FIG. 15 is a screenshot showing integration of the ONIX microfluidic perfusion system with an open-source microscopy application.
• FIG. 16 illustrates a microincubation system integrated with a microscope system for cell analysis.
• the dimensions of the manifold in specific embodiments allow it to sit on a standard well plate stage, with a transparent optical path that does not interfere with light microscopy. This allows time-lapsed imaging of cells cultured in the micro-incubator.
• FIG. 17 is a block diagram showing a representative example logic device in which various aspects of the present invention may be embodied.
• FIG. 18 is a block diagram showing an automated piston driven system according to specific embodiments of the invention.
• FIG. 19 (Table 1) illustrates an example of diseases, conditions, or states that can be evaluated or for which drugs or other therapies can be tested according to specific embodiments of the present invention.
• FIG. 20A-D are schematics of an example implementation of electronic control circuits for a manifold according to specific embodiments of the invention.
• a “particle” refers to biological cells, such as mammalian or bacterial cells, viral particles, or liposomal or other particles that may be subject to assay in accordance with the invention. Such particles have minimum dimensions between about 50-100 nm, and may be as large as 20 microns or more. When used to describe a cell assay in accordance with the invention, the terms “particles” and “cells” may be used interchangeably.
• a “microchannel” or “channel” or “flow channel” generally refers to a micron-scale channel used for fluidically connecting various components of systems and devices according to specific embodiments of the invention.
• a microchannel typically has a rectangular, e.g., square, or rounded cross-section, with side and depth dimensions in a preferred embodiment of between 10 and 500 microns, and 10 and 500 microns, respectively. Fluids flowing in the microchannels may exhibit microfluidic behavior.
• microchannel and “channel” are used interchangeably.
• Flow channel generally denotes channels designed for passage of media, reagents, or other fluids or gels and in some embodiments cells.
• Cell culture channel generally denotes a portion of a cell culture structure that cells are designed to flow through and also remain during cell culture (though the cells may be localized into a particular culture area of the culture channel in some embodiments).
• Air channel generally denotes a roughly micron-scale channel used for allowing gases, such as air, oxygen enriched mixtures, etc., to pass in proximity to flow channels or culture areas.
• Perfusion channel is sometimes used to indicate a flow channel and any perfusion passages or structures that allow media to perfuse to the culture area.
• a "perfusion barrier” refers to a combination of solid structures and perfusion passages that generally separate a flow channel from a cell culture area or chamber.
• the perfusion passages are generally smaller than the microchannel height and/or width (for example, on the order of 5-50% or on the order of about 10%) and are designed to keep cells, other culture items, and in some embodiments gels, from migrating into the flow channels, while allowing some fluidic flow that is generally of a much higher fluidic resistance than the fluid flow in the flow channels.
• the perfusion barrier has a perfusion passage that is 4 microns high and that otherwise runs most of the length of the microchannel.
• a perfusion barrier has many perfusion passages that are about as high as the microfluidic channel, but about 4 microns wide.
• a "microfluidics device” refers to a device having various station or wells connected by micron- scale microchannels in which fluids will exhibit microfluidic behavior in their flow through the channels.
• a "microwell array” refers to an array of two or more microwells formed on a substrate.
• a “device” is a term widely used in the art and encompasses a broad range of meaning.
• “device” may signify simply a substrate with features such as channels, chambers and ports.
• the “device” may further comprise a substrate enclosing said features, or other layers having microfluidic features that operate in concert or independently.
• the “device” may comprise a fully functional substrate mated with an object that facilitates interaction between the external world and the microfluidic features of the substrate.
• an object may variously be termed a holder, enclosure, housing, or similar term, as discussed below.
• microfluidic systems provide a powerful tool to conduct biological experiments. Recently, elastomer-based microfluidics has especially gained popularity because of its optical transparency, gas permeability and simple fabrication methods.
• the present invention involves integrated microfluidics used for various culture and assay applications and systems for providing heating control and automating various handling of culture plates. Advantages of specific embodiments include use of a standard-sized microtiter plate format, tubing free plates, and easy and effective mating of plates with a manifold to provide gas recirculation and heating control.
• a novel cell loading system uses a pneumatic manifold and pneumatic pressure to place cells in the micro culture area.
• microfluidic cell culture and analysis can be fully automated using other automated equipment that exists for handling standard titer plates.
• heating and gas circulation elements are incorporated into the manifold to provide a micro- incubator system.
• a gravity driven flow culture configuration utilizes the medium level difference between the inlet and outlet well as well as engineering the fluidic resistances to achieve the desirable flow rate in nL/min regime can be used to "passively" flow culture medium for long periods of time (e.g., up to 4 days) without the use of bulky external pumps or tubes in an environment such as an incubator to control temperature and then the heat controlled manifold, as provided herein, can be used for control of the cell culture during observation.
• a custom pneumatic flow controller can be attached to the gas and electric connectors in the manifold and thereby used to load the cells into the culture regions, to switch between different exposure solutions, and to control the temperature of the culture region.
• a digital software interface can be used to allow a user to program specific inputs (pulses, ramps, etc.) over time to expose the cells to complex functions during time-lapse imaging while maintaining or varying temperature and gas exposure as desired.
• one or more micro culture areas are connected to a medium or reagent channel via a grid of fluidic passages (or diffusion inlets or conduits), wherein the grid comprises a plurality of intersecting high fluidic resistance perfusion passages.
• passages in the grid are about 1 to 4 ⁇ in height, 25 to 50 ⁇ in length and 5 to 10 ⁇ in width, the grid allowing for more even diffusion between medium or reagent channels and the culture area and allowing for easier manufacturing and more even diffusion.
• the high fluidic resistance ratio between the microchamber and the perfusion/diffusion passages or grid offers many advantages for cell culture such as: (1) size exclusion of cells; (2) localization of cells inside a microchamber; (3) promoting a uniform fluidic environment for cell growth; (4) ability to configure arrays of microchambers or culture areas; (4) ease of fabrication, and (5) manipulation of reagents without an extensive valve network. Examples were illustrated wherein a grid-like perfusion barrier can be much shorter than the culture area or can be near to or at the same height, according to specific embodiments of the invention and further wherein various configurations for culture devices were illustrated.
• FIG. 1 shows a side view of an example micro-incubator manifold according to specific embodiments of the invention shown in place in a micro-incubator system with a well plate and microscope viewer.
• FIG. 2 illustrates one example of a top plane view of a manifold with a heat controller according to specific embodiments of the invention.
• FIG. 3 illustrates one example of a topside view of a manifold with a heat controller sealed to a well-plate and mounted in a microscope viewer according to specific embodiments of the invention.
• FIG. 4 illustrates one example of a culture plate with four culture units placed onto a 96-well standard SBS plate. This example shows four culture units (corresponding to rows labeled A-D) with six inlet wells (labeled A1-D6), four microfluidic culture areas placed under the large viewing oval, and two outlet wells (7-8).
• a Microlncubator Manifold can interface to a variety of microfluidic plates and provides one or more of cell-loading, perfusion, temperature and gas environment control.
• a convective heat exchanger adds or removes heat to the gas mixture using a Peltier thermoelectric device.
• the cells are kept warm through conducted heat from the warm gas mixture and the desired gas concentration diffuses in the media surrounding the cells through a gas permeable membrane on the microfluidic plate.
• a sealed gas chamber is formed from the heat exchanger, under the manifold and into the area above the culture microfluidics. Gas introduced into the area above the microfluidics is circulated by a fan or other gas circulatory means in the heat exchanger, thereby providing control of both the gas environment and the temperature above the culture area using the manifold.
• the microincubator manifold includes both a gas input and a recirculating fan to control the gas composition.
• controlled and pressurized gases enter the manifold through the gas lines 5.
• the pneumatic portion of the manifold is shown for convenience in this example in two pieces, top piece 10a and bottom piece 10b, which comprises a gasket for tightly fitting to plate 20, the plate containing a number of wells 22.
• the pneumatic operation of the manifold and fitting to the well plate is as described in herein referenced patent applications.
• the manifold also includes heat exchange module 40, with internal heat exchange fins 42 and a transparent cover plate (e.g., glass or acrylic) 44. When the manifold is placed over plate 10, the open region above the culture areas is connected with the heating element to create a gas circulation region 30.
• the well plate can be any standard or custom well plate as described herein. It will be understood from the teachings provided herein that different configurations of manifold 40 are constructed to accommodate different well plate designs.
• the gas and heating controls and elements are entirely contained in the manifold and the manifold can mate with any number of differently configured micro- well plates, including micro well plates that have no specific modifications to allow them to receive a heat source.
• two fans are used in the heat exchange element one to circulate the sealed gas in the gas area and one to interact with the heat exchanger.
• FIG. 5A-D are schematic drawings of an example implementation of a manifold with a heat controller from various views according to specific embodiments of the invention.
• the pneumatic portions of the manifold operate similarly to previously disclosed designs.
• the heat exchange module is described in more detail below.
• a heat exchange module in one example embodiment controls the temperature within the chamber by the cycling of air at desired temperature.
• temperature control is provided by a thermoelectric Peltier module, which are well known devices that convert an electric current into a temperature gradient and can also be used as a temperature controller that either heats or cools. While other heating sources can be used in a manifold of the invention, a Peltier module is a presently preferred mechanism as it can be fully incorporated into the heat exchange module and the manifold.
• a heat exchange module in an example implementation has three main outer pieces: (1) a metal top enclosure, (2) a plastic bottom enclosure and (3) a manifold with an oval cutout that allows air to flow into the cell culturing chamber of the plate and cycle back out.
• FIG. 6(A) illustrates the bottom portion of a heat exchange module according to specific embodiments of the invention.
• the bottom portion attaches to the pneumatic portion of the manifold.
• (B) illustrates the top portion of a heat exchange module according to specific embodiments of the invention.
• the plastic enclosure of the bottom portion is attached to the top of the manifold (or example by screws or glue or other means).
• the bottom of this enclosure has a cut out of two 2 mm deep flow paths. These paths merge into one over the imaging chamber. When they merge, the depth of the path rises to 3 mm.
• Around the outskirt of the flow path is an o-ring that prevents the exchange of air between the flow path and the environment.
• the size of the flow paths must be wide enough to allow a desirable amount flow to circulate into the cell culture chamber. This decreases the difference in temperature in the plate cell culturing region and the Heat Exchange Module (at the location of the temperature probe).
• a vertical extrusion sits above the paths. It contains 3 chambers. Above one flow path is a heat sink and above the other sits a fan. The third chamber connects to another chamber in the metal top enclosure to make room for a PCB board that connects the wires of the Peltier module, a small temperature probe, a relay, connections to the microincubator controller and 2 thermal cutoffs (one for each heat sink).
• the temperature probe is routed from the electronics chamber through a screw hole on the fan and into the top of its flow path to measure the temperature of air cycling through.
• the plastic enclosure also has a cut out from the top that forms a frame for a piece of 2mm glass. This piece of glass sits right above the cell culturing chamber in order to create optimal condition for microscope imaging without disrupting heating.
• the metal top enclosure (FIG. 6B) has fin extrusion features that allow it to act as a heat sink for the other side of the Peltier module.
• it optionally contains a chamber for a second fan to speed up the cooling process as well as room for a thermal cutoff that connects to the electronics chamber of the plastic bottom enclosure.
• thermal grease is applied to the top of the Peltier module in order to attach it to the metal top enclosure.
• the bottom of the plastic enclosure is securely fastened to the top enclosure, for example using screws or glue.
• FIG. 7 is a schematic side view of the pneumatic portions of a manifold sealed to a culture plate and showing the gas in lines connecting to an environment control volume according to specific embodiments of the invention.
• FIG. 8 is a schematic showing how a manifold interfaces to a microfluidic plate according to specific embodiments wherein a positive seal is created by applying a vacuum to the cavity surrounding all the wells.
• the heating unit is not shown in this figure.
• FIG. lOA-C shows a top view, side view, and plan view of a schematic of an example pneumatic manifold according to earlier designs.
• the eight tubing lines to the right are for compressed air, and each is configured to provide pressure to a column of cell inlet wells in a microfluidic array.
• the left-most line in the figure is for vacuum and connects to an outer vacuum ring around the manifold.
• the manifold is placed on top of a standard well plate.
• a rubber gasket lies between the plate and manifold, with holes matching the manifold (not shown).
• the vacuum line creates a vacuum in the cavities between the wells, holding the plate and manifold together.
• Pressure is applied to the wells to drive liquid into the microfluidic channels (not shown).
• a typical pressure of 1 psi is used; therefore the vacuum strength is sufficient to maintain an air-tight seal.
• each column is connected to a single pressure line. Columns above the cell imaging regions are skipped.
• Pressurized cell loading has been found to be particularly effective in preparing cultures of aggregating cells (e.g., solid tumor, liver, muscle, etc.). Pressurized cell loading also allows structures with elongated culture regions to be effectively loaded. Use of a pressurized manifold for cell loading and passive flow for perfusion operations allows the invention to utilize a fairly simple two inlet design, without the need for additional inlet wells and/or valves as used in other designs.
• the plate manifold optionally also included an additional "gas line" that is used to bathe the cells in the microfluidic device with a specified gas environment (for example, 5% C0 2 ).
• a specified gas environment for example, 5% C0 2
• Other examples include oxygen and nitrogen control, but any gaseous mixture can be sent to the cells.
• the gas flows through the manifold into the sealed wells above the cell culture area and holes in the microfluidic device enable the gas to flow into specified microfluidic air channels, as described above.
• the gas permeable device layer (PDMS) allows the gas to diffuse into the culture medium prior to exposing the cells. By continuously flowing the gas through the microfluidic plate, a stable gas environment is maintained. This provides an optional means for controlling the gas environment to placing the microfluidic plate into an incubator.
• FIG. 12 illustrates an example microfluidic perfusion system (ONIXTM), microincubator controller and manifold (MIC) and computer control system according to specific embodiments of the invention.
• ONIXTM microfluidic perfusion system
• MIC manifold
• FIG. 14 illustrates one alternative of plate and culture unit design with an example culture unit filled with blue dye with the image taken from top according to specific embodiments of the invention.
• any culture units in any configuration of culture plates can be used with a correctly dimensioned manifold according to specific embodiments of the invention. This includes open top units, invasion units, liver mimetic units, gel units, etc as described in above referenced applications.
• Cell assay can be performed directly on the microfluidic cell culture using standard optically based reagent kits (e.g. fluorescence, absorbance, luminescence, etc.). For example a cell viability assay utilizing conversion of a substrate to a fluorescent molecule by live cells has been demonstrated (CellTiter Blue reagent by Promega Corporation). The reagent is dispensed into the flow inlet reservoir and exposed to the cells via gravity perfusion over a period of time (e.g., 21 hours). For faster introduction of a reagent or other fluid, the new fluid can be added to the flow inlet reservoir followed by aspiration of the cell inlet reservoir.
• standard optically based reagent kits e.g. fluorescence, absorbance, luminescence, etc.
• a cell viability assay utilizing conversion of a substrate to a fluorescent molecule by live cells has been demonstrated (CellTiter Blue reagent by Promega Corporation).
• the reagent is dispensed into the flow inlet reservoir
• Data can be collected directly on the cells/liquid in the microfluidic plate, such as placing the plate into a standard fluorescence plate reader (e.g., Biotek Instruments Synergy 2 model).
• the substrate may diffuse into the outlet medium, and therefore be easily detected in the cell inlet reservoir.
• the plate can be placed on a scanning microscope or high content system.
• an automated Olympus 1X71 inverted microscope station can be used to capture viability of cultured liver cells with a 20X objective lens.
• By repeatedly filling/aspirating the wells cells can be maintained for long periods of time with minimal effort (e.g. compared to standard "bioreactors" which require extensive sterile preparation of large fluid reservoirs that cannot be easily swapped out during operation).
• Integrated systems for the collection and analysis of cellular and other data as well as for the compilation, storage and access of the databases of the invention typically include a digital computer with software including an instruction set for sequence searching and/or analysis, and, optionally, one or more of high-throughput sample control software, image analysis software, collected data interpretation software, a robotic control armature for transferring solutions from a source to a destination (such as a detection device) operably linked to the digital computer, an input device (e.g., a computer keyboard) for entering subject data to the digital computer, or to control analysis operations or high throughput sample transfer by the robotic control armature.
• the integrated system further comprises valves, concentration gradients, fluidic multiplexors and/or other microfluidic structures for interfacing to a microchamber as described.
• software for providing the data and/or statistical analysis can be constructed by one of skill using a standard programming language such as Visual Basic, Fortran, Basic, Java, or the like.
• a standard programming language such as Visual Basic, Fortran, Basic, Java, or the like.
• Such software can also be constructed utilizing a variety of statistical programming languages, toolkits, or libraries.
• FIG. 17 shows an information appliance (or digital device) 700 that may be understood as a logical apparatus that can read instructions from media 717 and/or network port 719, which can optionally be connected to server 720 having fixed media 722. Apparatus 700 can thereafter use those instructions to direct server or client logic, as understood in the art, to embody aspects of the invention.
• One type of logical apparatus that may embody the invention is a computer system as illustrated in 700, containing CPU 707, optional input devices 709 and 711, disk drives 715 and optional monitor 705.
• Fixed media 717, or fixed media 722 over port 719 may be used to program such a system and may represent a disk- type optical or magnetic media, magnetic tape, solid state dynamic or static memory, etc.
• the invention may be embodied in whole or in part as software recorded on this fixed media.
• Communication port 719 may also be used to initially receive instructions that are used to program such a system and may represent any type of communication connection.
• FIG. 18 is a block diagram showing an automated piston driven system according to specific embodiments of the invention.
• an auto-sealer somewhat similar to a plate reader commonly used in biotechnology with the main difference is the design of the system component to allow automated handling of the microfluidic plates.
• the positive seal between manifold and microfluidic plate is still accomplished by applying vacuum to the interstitial areas, but the necessary initial downward force is applied mechanically.
• the area above the manifold is clear to allow access by an automated liquid hander such as the Tecan EVO. Vacuum and pressure sensors as well as plate presence and carriage position sensors allow for intelligent software based error handling.
• 18B is an image sequence showing how the automatic sealing device accepts and seals the manifold to a microfluidic plate.
• a single pneumatic linear actuator e.g., a piston
• This single operation allows more precise control of the manifold and plate and holds the plate in place during operation of the pneumatic manifold.

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