Microfluidic Devices and Methods Technology Package

P05144: Methods of Finding, Selecting and Studying Cells in Heterogeneous Co-Cultures

Overview: Current methods for isolating and culturing primary (stem/progenitor) cells remain unsatisfactory because most primary cells die when transferred to culture, and the remaining cells usually develop mutations rendering them unfit for an in vivo environment.

Invention Summary: UW-Madison researchers have developed a method of co-culturing heterogeneous primary cells. The cells are cultured in a very small, convection-free space, such as a microchannel, so they behave more as they would in vivo. Because there is no fluid flow, all movement of components in the environment is by diffusion. The culture contains at least one growth-promoting cell and at least one cell capable of proliferating.

Applications:

• Drug screening
• Isolation, purification and/or identification of stem or progenitor cells
• Study of autocrine and paracrine regulation

Key Benefits:

• Provides the ability to precisely direct and evaluate physical, chemical and biological interactions between cells and other factors in a controlled environment
• Physically constrains the diffusion of soluble factors, allowing cells to more closely imitate the in vivo environment
• Cells can come from a single source or from multiple sources
• Heterogeneous cells can be in cell-to-cell contact or spaced apart
• Uniquely enables the study of stem cells by avoiding problems associated with stem cell assays in standard tissue culture
• Avoids need for costly and time-consuming transplantation of cultured cells into a host to determine whether proliferation is occurring in a culture
• Molecular gradients of test agents or compounds can be established in the microenvironment
• May be used to determine the effect of a cancer treatment on proliferative capacity of an affected tissue

P05284: Microfluidic System for High Throughput Screening

Overview: Most high throughput screening (HTS) for new chemicals is done in microtiter well plates. Reagents for HTS can be very expensive, making miniaturization of the plates desirable. Microfluidics is an alternative to miniaturization, but previous attempts to develop a microtiter plate with microfluidic channels have required expensive and complicated pumping equipment and have not been robot-friendly.

Invention Summary: UW-Madison researchers have developed a microtiter plate that uses a simple and inexpensive microfluidics channel system in place of wells for standard high throughput screening using commercially available liquid-handling robots. This technology uses a passive pumping system that eliminates the need for external pumping equipment.

Each microtiter plate includes several microchannels with openings on either end. The input end of each channel consists of a port with multiple pores for receiving drops of fluid. After a liquid-dispensing instrument deposits a drop in the port, the passive pumping system draws it into the channel. To pump liquid through the system, each microchannel is filled with fluid, and a pressure gradient is generated so that fluid flows through the channel toward the output. Assays involving channel networks with multiple ports have also been demonstrated using this system, suggesting that it facilitates high throughput execution of many novel microfluidic cell-based assays as well.

Applications:

• High throughput screening of new chemicals

• Provides a direct, drop-in replacement for standard microtiter plates
• Enables fast, automated washing with simplified robotics
• Easily manufactured through injection molding
• Does not require expensive pumping equipment
• Reduces evaporation - only a small fraction of the liquid surface is exposed to the atmosphere

P06168: Device for Improved Cell Staining and Imaging

Overview: Preparing cover slips for optical cell imaging is labor intensive and current methods are limited to one treatment per slip. Imaging multiple cover slips is also cumbersome, due to the time needed to remove and replace each cover slip on the microscope.

Invention Summary: A UW-Madison researcher has developed a multichannel microfluidic device that increases the efficiency of sample preparation for cell imaging. The microfluidic device includes a cartridge with a patterned inlet port to contain reagent droplets and avoid cross-channel contamination. The lower surface of the cartridge includes several recesses that define channels for containing cells. Different reagents can be added to each channel. The upper surface of a cover slip is placed against the lower surface of the cartridge, and the cartridge is mechanically clamped to the cover slip to ensure no leakage during an assay. After staining is complete, the cover slip can be removed and traditionally mounted to a glass slide for standard imaging protocols.

Applications:

• Optical cell imaging

• Increases efficiency
• Multiple treatments can be performed on one cover slip in the time required to process one standard cover slip.
• Moving from one treatment to another does not require removing and replacing the cover slip.
• Saves money by using fewer reagents
• Saves time because cells are in well defined and predictable locations
• A range of channel dimensions is available.
• Fiduciary marks allow autofocusing, either in the channel structure with the channel in place or on the cover slip.
• A funnel-shaped outlet port design facilitates robust and easy mating of a pipette to the port.
• Designed for use with many types of microscopes, including high-resolution confocal microscopes

P06373: Simple Biological Method and Device for Detecting a Toxin or Other Chemical

Overview: Most schemes for detecting toxins or other chemicals are based on complex electronic, photonic and/or electrochemical methods, or on more elegant biomolecular methods, such as ELISA (Enzyme-Linked Immunosorbent Assay). Because these methods are generally expensive and complicated, a need remains for practical, cost-effective biosensors.

Invention Summary: UW-Madison researchers have developed a simple biological sensing method that can be used to detect toxins or other chemical compounds. The technology uses the elastic instability of swelling hydrogels to act as a trigger when contacted by a designated stimulus, such as a particular chemical.

Two different types of hydrogels are bonded together by a sensitive material, such as a degradable adhesive material specific to a certain enzyme or chemical. The hydrogels swell at different rates, causing them to bend. Because the adhesive restricts the motion of the hydrogels, an elastic force is generated. When the adhesive material contacts the target chemical, the adhesive degrades, releasing the elastic instability and causing the two hydrogels to separate with an explosive motion that is detectable by the naked eye.

Applications:

• Creation of simple dipstick sensors to detect toxins, enzymes or chemicals

Key Benefits:

• Does not require external power sources
• Sensors may be portable or disposable.
• Highly sensitive and selective
• Rapid response time
• Generates few false alarms
• Simple to use
• Easy to manufacture
• Signal is easily observable.

P07025: Population-Averaged Method to Quantify Cell Motility and Migration

Overview: Cell motility and migration are involved in a range of biological processes, from embryo development to cancer metastasis. Although many methods have been developed to study cell movement, current techniques are labor intensive and provide limited quantitative information.

Because most migrating cells rely on a chemical gradient to orient their movements, flow-based microfluidic systems have been designed to generate well defined gradients to study the migration of blood, tumor and neural cells; however, these systems rely on video microscopy for readout, which limits throughput and requires expensive software for data analysis. In addition, the flow may wash away cell signaling components.

Invention Summary: UW-Madison researchers have developed an alternative, microtechnology-based method for conducting cell mobility assays. This technique combines microfluidic gradient generation with micro-patterning to simplify the extraction of important migratory information. Rather than tracking individual cells, it uses parameters from the cell population as a whole.

A population of cells is labeled (e.g., with fluorescent dye) and patterned within a microchannel network so the cells are uniformly dispersed along the channel in the form of a generally rectangular strip. A predetermined medium that includes a migration- or motility-promoting signal is patterned along one sidewall of the channel. Then a first image of the population is obtained. After a predetermined time period, a second image is obtained and compared to the first. Simple mathematical processing of the images yields quantitative measurements that can be used to calculate the average directional migration and motility of the cell population.

Applications:

• Analyzing cell motility and migration

• Allows the user to simply and easily determine quantitative motility and directional migration data for a cell population
• Multiple populations of cells can be compared.
• Data is averaged across the population, improving sensitivity.
• Compatible with robotic micropipetting stations
• Amenable to high throughput operation
• Requires only phase contrast or fluorescence microscopy for readout

P07030: Method for Controlling Communication Between Multiple Access Ports in a Microfluidic Device

Overview: Microfluidic devices have been proven useful for exploring a variety of biological questions, such as how cell-derived soluble signaling affects cellular processes. Passive pumping is a common method used in microfluidics to provide fluid flow, requiring neither active components nor physical connections. Instead, fluid flow is driven solely through the fluid tension forces found at the microfluidic channel inlet and outlet ports. This approach allows fluid to be transferred to and from microfluidic channels in large arrays that can be addressed via generic liquid handling instruments.

Over the course of an experiment it can be beneficial to reconfigure a microfluidic network at some stage or to connect different microfluidic channels. However, current techniques and systems lack practical means for altering the fluid communication schemes of microfluidic networks on the fly. A more elegant method is needed for altering microfluidic systems so that modification of experimental parameters can take place within the duration of an experiment without the need for additional external equipment.

Invention Summary: UW-Madison researchers have developed a method and structure for controlling fluid communication between components in a passive pumping-based microfluidic system. The microfluidic network is designed so that a small drop of fluid can cover two nearby ports, promoting communication between the fluid-coupled channels.

In its simplest form, the communication device contains one input port and one output port, connected by a fluid-filled channel. If drops of fluid are pipetted onto the ports, the fluid will flow in the direction of the larger drop. The flexibility in the design provides a wide range of configurability and dynamic connectivity for executing changes to experimental setups on cue.

Furthermore, an intricate design can create a form of binary code in the presence or absence of drops on the ports. Microfluidic systems can have a complex network of channels and input/output ports that serve as logic gates, analogous to electronic circuit components. This multifaceted design allows limitless modifications throughout the duration of an experiment without adding external equipment. 

Business Opportunity:

• A 2006 Frost and Sullivan report indicates that there is an obvious need for improvement in cell-based assay technology, which is expected to contribute to growth in the microfluidics market.
• Cell assays represented 12% of the U. S. microfluidics market in 2005, corresponding to a market size of approximately $10M.

Applications:

• Cell-based assays, including cell-to-cell communication, cell migration and immunocytochemistry.

Key Benefits:

• Can be operated automatically
• Cheap and easy to manufacture
• Enables delivery of soluble cell signals to multiple cell culture environments without cell contamination
• Reduces the amount of costly reagents and other chemical solutions needed for an experiment
• Provides a more portable system due to reduced need for additional external equipment
• Allows microfluidics to perform more complex functions
• Allows time-dependent control of systems due to adjustable resistance

Stage of Development: Various cellular studies have been carried out utilizing this tunable fluid communication technique amongst a network of microfluidic devices.

P08041: Coupling Microfluidic Devices Yields Physiologically Relevant Micro-Environment for Cellular Assays

Overview: Cellular interactions and signaling in multicellular organisms involve complex pathways that can be difficult to study when using macroscale in vitro culture systems, in part because traditional macroscale systems do not adequately mimic the in vivo cellular environment. For example, concentration gradients play a key role in mediating biological activity in vivo, but the large volumes in macroscale systems make it impractical to control concentration gradients to coordinate the growth, differentiation and metabolism of cells. In addition, traditional macroscale systems necessitate the use of large amounts of expensive reagents.

Investigating signaling pathways on a smaller, more physiologically relevant scale is crucial to understanding the fundamental roles of particular biochemical processes. New approaches utilize microfluidic devices as they offer microscale dimensions that mimic necessary in vivo conditions and offer the ability to regulate the experimental micro-environment. Further improvements to current methods and in vitro culture system designs are necessary to pave the way for developing new drugs that target signaling pathways.

Invention Summary: UW-Madison researchers have developed an improved device to create a controllable, physiologically relevant micro-environment for studying cellular interactions and pathways. This device provides a means for coupling two discrete microfluidic channels using only fluid contact. Two microchannels, each having one inlet and one outlet, can be coupled by combining fluid droplets on the outlet port and inlet port of the respective microchannel. This fluid contact method allows channels with two isolated environments to initiate the transfer of signaling molecules by means of diffusion or flow, thus allowing controllable physiological communication between cells. Cells can be exposed to a variety of cellular signals without cell contamination by simply breaking fluid contact with the current microchannel and forming a new fluidic coupling with a new microchannel. This new approach can be particularly useful in the co-culture of cells, where cell contamination can be prevalent and result in skewed data.

The controllable micro-environment implemented by this device also provides improved parameters for cellular assays and is well suited for high throughput screening. The reduction of culture dimensions in this microfluidic system results in a more physiologically relevant cellular micro-environment due to certain physical phenomena and interactions that become more dominant. The scale of microfluidic systems offer more precise control over parameters that affect the cellular micro-environment, including fluid shear stress, diffusion of soluble factors and patterning of cells and extracellular matrix (ECM). These parameters can influence cell development and signaling pathways. For example, shear forces can modulate stem cell differentiation pathways and/or apoptotic activity.

In addition, the fluid reduction that results from using a microscale, rather than a macroscale, culture system allows minimal use of expensive reagents. Less reagent use also increases the repeatability and reliability of assays and reduces the amount of time necessary to move cells and reagents in and out of channels.

Applications:

• More robust cellular assays including co-culture of cells; soluble factor delivery; and cell capture, identification and sorting
• Adaptable for high throughput screening

Key Benefits:

• Reduces costs by using smaller amounts of expensive reagents
• Minimizing reagent use results in more efficient, repeatable and reliable experiments.
• Provides precise control of cellular micro-environment parameters, such as fluid shear stress, diffusion of soluble factors and patterning of cells and extracellular matrix (ECM)
• Suitable for ordinary laboratory purposes and very large-scale applications

P110347: Improved Self-Loading Microfluidic Device for Determining Effective Antibiotic Dose and Other Chemical and Biological Assays

Overview: The minimum inhibitory concentration (MIC) of a compound is the lowest dose that prevents the growth of a cell during a set time interval. Because of the increase in antibiotic-resistant bacteria and the slowdown in discovery of new antibiotics, determination of the MIC or susceptibility of antibiotics against microbes has become very important for increasing the longevity of clinical antibiotics. Determining the MIC of antibiotics against bacteria generally is performed using diffusion or dilution methods; however, diffusion-based assays are slow and insensitive and dilution-based assays, although accurate, are very slow and labor intensive. Microfluidic technology addresses some of these drawbacks, but also brings its own disadvantages. Alternative microfluidic devices and methods of using these devices to determine the MIC and susceptibility of antibiotics and for identifying bacterial strains are needed.

Invention Summary: UW–Madison researchers have developed a portable, self-loading microfluidic device and method for determining therapeutically effective amounts of agents, MICs and toxicity levels. It can be used to identify bacterial strains and for performing chemical and biological assays. The device comprises a porous organic polymer, a reaction well, an inlet port, a vacuum well, a main channel and a side channel. 

Applications:

• Determining MIC and antibiotic susceptibility
• Identification of pathogenic strains of bacteria and other microbes
• Various chemical and biological assays

Key Benefits:

• Reduces the number of steps for determining MICs and antibiotic susceptibility
• Reduces the cost and time of the assay
• Improves sensitivity/resolution
• Does not require external instruments

Stage of Development: MIC, susceptibility and bacterial identification measurements have been obtained using this microfluidic device.

Included IP:

• P08041US (PAT)
• P07030US (PAT)
• P07025US (PAT)
• P06373US (PAT)
• P06168US (PAT)
• P05284US (PAT)
• P05144US (PAT)
• P110347US01 (PAT)
• P110347US02 (DIV)