Micro-incubation systems for microfluidic cell culture and methods

Abstract

A micro-incubator manifold for improved microfluidic configurations and systems and methods of manufacture and operation for a manifold and automated microfluidic systems are disclosed. Various embodiments relate to assays, systems, and/or devices for culturing cells or other biologic material in controlled environments and 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. More specifically, specific embodiments relate to heat control systems for microfluidic culture plates and other automated systems for culture plates.

Claims

1. A method of culturing cells, comprising: disposing cells into a culture chamber, the culture chamber connected to a plurality of microfluidic channels, the culture chamber and microfluidic channels configured into a culture unit on a well plate; interfacing the well plate with a microincubator manifold, the microincubator manifold having a gasket for providing a removable air-tight seal to the well plate, and a heat exchange module, wherein the manifold seals to the well plate, thereby enclosing an incubation volume of gas, wherein the heat exchange module provides a controlled temperature above and in communication with the culture chamber; and observing and/or assaying the cells in the culture chamber, wherein the microincubator manifold comprises a transparent window in a region disposed above the culture chamber.

2. The method of claim 1, wherein the microincubator manifold interfaces with the well plate using a vacuum seal.

3. The method of claim 1, wherein the microincubator manifold further comprises one or more pneumatic connectors to control pressure to the plurality of microfluidic channels.

4. The method of claim 1, wherein the microincubator manifold further comprises one or more gas inlets.

5. The method of claim 1, wherein the microincubator manifold further comprises a fan to circulate gas in the incubation volume.

6. The method of claim 1, wherein the microincubator manifold further electrical connections for providing at least one of power, sensor and control connections.

7. The method of claim 1, further comprising releasing the microincubator manifold from the well plate.

8. The method of claim 7, further comprising: interfacing the microincubation manifold to a second well plate.

9. An automatic handling system for sealing a pneumatic manifold to a culture plate comprising: a pneumatic linear actuator cylinder; one or more position sensors; two arms for holding the manifold from the sides, without blocking a view through the top of the manifold; and a plate carriage and plate grippers for holding the culture plate; said system configured so that movement of the cylinder causes the plate carriage to move horizontally to a position under said manifold; and said system configured so that movement of the cylinder causes the manifold to descend onto the plate.

10. The system of claim 9, further wherein the cylinder is attached to the arms so that the cylinder provides both horizontal and vertical motion.

11. The system of claim 9, wherein a positive seal between the manifold and culture plate is accomplished by applying vacuum through the manifold to the interstitial areas of the culture plate while the necessary initial downward force is applied mechanically.

12. The system of claim 9, wherein an area above the plate remains clear prior to placement of the manifold to allow access by an automated liquid handler.

13. The system of claim 9, wherein an area above the plate remains clear during operation of the manifold to allow observation of culture areas during operation.

14. The system of claim 9, further comprising one or more vacuum and pressure sensors as well as plate presence and carriage position sensors to allow for intelligent software based control and error handling.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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.

(2) FIG. 2 illustrates one example of a top plane view of a manifold with a heat controller according to specific embodiments of the invention.

(3) 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.

(4) 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). This is an example only and placement and designation of the various wells and components will vary with different implementations.

(5) FIGS. 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.

(6) FIG. 6A 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. FIG. 6B illustrates the top portion of a heat exchange module according to specific embodiments of the invention.

(7) 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.

(8) 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.

(9) FIG. 9 illustrates a manifold with additional gas line and a heated objective lens according to an earlier manifold design.

(10) FIGS. 10A-C show a top view, side view, and plan view of a schematic of an example pneumatic manifold according to earlier designs. In this example, 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.

(11) FIG. 11 illustrates an example microfluidic perfusion system (ONIX), microincubator controller and manifold (MIC) according to specific embodiments of the invention.

(12) FIG. 12 illustrates an example microfluidic perfusion system (ONIX), microincubator controller and manifold (MIC) and computer control system according to specific embodiments of the invention.

(13) FIG. 13 shows NIH-3T3 mouse fibroblasts cultured using the microincubator system according to specific embodiments of the invention at t=0 (left) and after 15 hours (right) showing cell growth and viability. When no temperature or CO.sub.2 was controlled, the cells rapidly died within 2 hours.

(14) FIGS. 14A-B illustrate 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.

(15) FIG. 15 is a screenshot showing integration of the ONIX microfluidic perfusion system with an open-source microscopy application.

(16) 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.

(17) FIG. 17 is a block diagram showing a representative example logic device in which various aspects of the present invention may be embodied.

(18) FIG. 18A is a block diagram showing an automated piston driven system according to specific embodiments of the invention. FIG. 18B illustrates perspective views of the automated piston driven system of FIG. 18A in the ejected and sealed states.

(19) 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.

(20) FIGS. 20A-D are schematics of an example implementation of electronic control circuits for a manifold according to specific embodiments of the invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

(21) 1. Overview

(22) Definitions

(23) 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.

(24) 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. When used to refer to a microchannel within the microwell array device of the invention, the term 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. Culture channel or 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.

(25) 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. In one example embodiment, the perfusion barrier has a perfusion passage that is 4 microns high and that otherwise runs most of the length of the microchannel. In other embodiments, a perfusion barrier has many perfusion passages that are about as high as the microfluidic channel, but about 4 microns wide.

(26) 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.

(27) A microwell array refers to an array of two or more microwells formed on a substrate.

(28) A device is a term widely used in the art and encompasses a broad range of meaning. For example, at its most basic and least elaborated level, device may signify simply a substrate with features such as channels, chambers and ports. At increasing levels of elaboration, the device may further comprise a substrate enclosing said features, or other layers having microfluidic features that operate in concert or independently. At its most elaborated level, 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. Such an object may variously be termed a holder, enclosure, housing, or similar term, as discussed below. As used herein, the term device refers to any of these embodiments or levels of elaboration that the context may indicate.

(29) 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.

(30) According to further embodiments of the invention, as has been previously described, a novel cell loading system uses a pneumatic manifold and pneumatic pressure to place cells in the micro culture area. With the addition of this cell loading system, microfluidic cell culture and analysis can be fully automated using other automated equipment that exists for handling standard titer plates. In the present invention, heating and gas circulation elements are incorporated into the manifold to provide a micro-incubator system.

(31) In further embodiments, 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.

(32) In some embodiments, 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.

(33) 2. Microfluidic Culture System and Array

(34) The applications referenced above discussed a variety of different cell culture configurations and fabrication techniques. Portions of the operation of the cell culture areas and materials are useful as background to the present discussion. In some examples therein, 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. In one discussed example, passages in the grid are about 1 to 4 m in height, 25 to 50 m in length and 5 to 10 m 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 earlier application further discussed that the high fluidic resistance ratio between the microchamber and the perfusion/diffusion passages or grid (e.g., ratios in the range of about 10:1, 20:1 to 30:1) 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.

(35) 3. Pneumatic Manifold with Heat Control

(36) As discussed above, one difficulty in a number of culture systems is how to control the heating and temperature of the culture area while allowing for observation of the cellular processes. Previous solutions have relied on heating sources applied to the well-plate, for example, from the microscope viewer. (E.g., see FIG. 11), or containing the entire system in a controlled environment.

(37) The present invention provides an improved culture system by placing all or nearly all heat and gas controls in or attached to a typically removable manifold that in preferred embodiments is configured to be operational will not interfering with observational equipment. The invention will be more easily understood with reference to the specific examples illustrated, though these examples are intended to be illustrative and not limiting. 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).

(38) Thus, according to specific embodiments, a MicroIncubator 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.

(39) As can be seen from FIG. 1, when the manifold according to specific embodiments is in place over a culture 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.

(40) This design, according to specific embodiments of the invention, solves the problem of providing effective heating in a small space to deliver the controlled temperature to the cells themselves while also controlling the gas composition. According to specific embodiments of the invention, the microincubator manifold includes both a gas input and a recirculating fan to control the gas composition.

(41) In the example implementation shown in FIG. 1, 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. To show the device in context, microscope and lighting elements 102 and 104 are illustrated as they would generally be used in operation. 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.

(42) Recirculating on the Manifold.

(43) In this example design, 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 microwell plates that have no specific modifications to allow them to receive a heat source.

(44) According to specific embodiments, 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.

(45) FIGS. 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.

(46) Manifold Heat Exchange Module

(47) A heat exchange module in one example embodiment controls the temperature within the chamber by the cycling of air at desired temperature. In specific embodiments, 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.

(48) 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.

(49) Plastic Bottom Enclosure

(50) FIG. 6A 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. FIG. 6B illustrates the top portion of a heat exchange module according to specific embodiments of the invention. In a specific embodiment, 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).

(51) 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.

(52) The plastic enclosure also has a cut out from the top that forms a frame for a piece of 2 mm glass. This piece of glass sits right above the cell culturing chamber in order to create optimal condition for microscope imaging without disrupting heating.

(53) Metal Top Enclosure

(54) 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. In addition, 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.

(55) After the fan is placed inside the enclosure and the thermal cutoff attached to the smaller chamber, thermal grease is applied to the top of the Peltier module in order to attach it to the metal top enclosure. In a specific example, the bottom of the plastic enclosure is securely fastened to the top enclosure, for example using screws or glue.

(56) When temperature in the cell culturing region has to be raised, the Peltier module heats up the bottom heat sink by cooling the top heat sink. The bottom fan blows hot air across the bottom heat sink into the flow path beneath it. The heated air enters the cell culturing chamber as cooler air is circulated back to the fan. When temperature in the culturing region has to be lowered, the Peltier module cools the bottom heat sink by raising temperature on the top (up to ambient temperature).

(57) 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.

(58) 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.

(59) 4. Earlier Pneumatic Manifold

(60) While gravity or passive loading is effective for some microfluidic cell culture devices and desirable in some embodiments, a proprietary pneumatic manifold was previously described in the above referenced applications. This may be mated to the plate and pneumatic pressure is applied to the cell inlet area for cell loading and for culturing during invasion assays. FIGS. 10A-C show a top view, side view, and plan view of a schematic of an example pneumatic manifold according to earlier designs. In this example, 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. In one example there are 9 tubing lines to the pressure controller: 8 lines are for compressed air and 1 line is for vacuum (leftmost). In specific example embodiments, each column is connected to a single pressure line. Columns above the cell imaging regions are skipped.

(61) 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.

(62) While this manifold is effective for cell loading and some perfusion tasks, the manifold did not effectively provide for the recirculation of a gas over the culture area or for any heat control. As illustrated in the figure, heating was provided when necessary from the opposite side of the culture plate, for example form the vicinity of the microscope viewer.

(63) 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% CO.sub.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.

(64) FIG. 12 illustrates an example microfluidic perfusion system (ONIX), microincubator controller and manifold (MIC) and computer control system according to specific embodiments of the invention.

(65) As just one example, FIGS. 14A-B illustrate 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. However, 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.

(66) Cell Assay and/or Observation

(67) 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.

(68) 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). In some reactions, the substrate may diffuse into the outlet medium, and therefore be easily detected in the cell inlet reservoir. For cell imaging assays, the plate can be placed on a scanning microscope or high content system. For example, an automated Olympus IX71 inverted microscope station can be used to capture viability of cultured liver cells with a 20 objective lens.

(69) 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).

(70) Example Cell Culture

(71) Cells were cultured using the micro-incubation system to control temperature and gas atmosphere. In one example, human cancer cells (HT-1080, MCF-7, MDA-MB-231) were cultured at 37 C and 5% CO2 to monitor cell division over 24 hours. Additional cell types, including yeast, bacteria, primary cells, neurons, etc. have been successfully cultured using the microincubation system. As an example, FIG. 13 shows NIH-3T3 mouse fibroblasts cultured using the microincubator system according to specific embodiments of the invention at t=0 (left) and after 15 hours (right) showing cell growth and viability. When no temperature or CO2 was controlled, the cells rapidly died within 2 hours.

(72) Integrated Systems

(73) 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. Optionally, the integrated system further comprises valves, concentration gradients, fluidic multiplexors and/or other microfluidic structures for interfacing to a microchamber as described.

(74) Readily available computational hardware resources using standard operating systems can be employed and modified according to the teachings provided herein, e.g., a PC (Intel x86 or Pentium chip-compatible DOS, OS2, WINDOWS, WINDOWS NT, WINDOWS95, WINDOWS98, LINUX, or even Macintosh, Sun or PCs will suffice) for use in the integrated systems of the invention. Current art in software technology is adequate to allow implementation of the methods taught herein on a computer system. Thus, in specific embodiments, the present invention can comprise a set of logic instructions (either software, or hardware encoded instructions) for performing one or more of the methods as taught herein. For example, 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. Such software can also be constructed utilizing a variety of statistical programming languages, toolkits, or libraries.

(75) FIG. 7 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. In specific embodiments, 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.

(76) Various programming methods and algorithms, including genetic algorithms and neural networks, can be used to perform aspects of the data collection, correlation, and storage functions, as well as other desirable functions, as described herein. In addition, digital or analog systems such as digital or analog computer systems can control a variety of other functions such as the display and/or control of input and output files. Software for performing the electrical analysis methods of the invention are also included in the computer systems of the invention.

(77) Auto-sealer Automated System

(78) FIG. 18A is a block diagram showing an automated piston driven system according to specific embodiments of the invention. According to further specific embodiments, 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. In this implementation, 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. FIG. 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) provides horizontal and vertical motion. It has been found that this single operation allows more precise control of the manifold and plate and holds the plate in place during operation of the pneumatic manifold.

Other Embodiments

(79) Although the present invention has been described in terms of various specific embodiments, it is not intended that the invention be limited to these embodiments. Modification within the spirit of the invention will be apparent to those skilled in the art.

(80) It is understood that the examples and embodiments described herein are for illustrative purposes and that various modifications or changes in light thereof will be suggested by the teachings herein to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the claims.

(81) All publications, patents, and patent applications cited herein or filed with this submission, including any references filed as part of an Information Disclosure Statement, are incorporated by reference in their entirety.