Patent Application
20120216601
This specification relates to analysis of biological samples.
Neutrophil functioning is essential for our well-being and protection against many infectious agents from our close environment. Without neutrophils, we could only survive a few days the constant assault of bacteria and fungi in our normal environment. A tremendous selective pressure on neutrophils to function perfectly in a large number of conditions has made neutrophils one of the most efficient and remarkable cells in terms of migration speed and ability to reach distant targets. However, there are several conditions where neutrophil activity could produce more damage than benefits. While neutrophil activation is protective after minor trauma, hyper-active neutrophils after major injuries have systemic deleterious effects and can effectively damage several organs and tissues, even in the absence of infection. Many conditions like chronic inflammatory diseases, immune reactions post-organ transplantation, or severe forms of asthma can be exacerbated by active neutrophils. Other times, neutrophils become unresponsive, simultaneously with down-regulation of the immune system, leading to, or facilitating septic states. Despite tremendous advances in the understanding of signaling molecules and pathways acting inside neutrophils, our understanding of the changes in neutrophils during disease processes is limited, and consequently, or abilities to modulate the activity of neutrophils in health and disease, restricted to very few options.
Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Evidence indicates that, in addition to such post-burn inflammatory changes, the motility of neutrophils is also affected. Impairments in neutrophil function after burn injury have potential implications for the development of sepsis. Flow cytometry analysis of neutrophils has shown impairments in phagocytosis, bactericidal activity, phago-lysosomal activity, and the oxidative burst within two weeks post-injury. Neutrophils demonstrate impaired adhesion and complement receptor expression after burn injury, and these changes, in turn, correlate with an increased incidence of abscess formation in vitro.
However, prognostic and diagnostic studies of neutrophil motility have been difficult due to the cumbersome assays that have prohibited exploration of the significance of neutrophil motility after burn injury.
This specification describes microfluidic devices for analyzing one or more motile cells, e.g., neutrophil motility.
One example of the microfluidic devices described herein include a substrate in which a main channel is manufactured. Multiple side channels are formed in the substrate such that each side channel branches off the main channel. The side channels can be filled with a chemokine solution. After a motile cell, e.g., a neutrophil, is obtained from a blood sample, it is introduced into a side channel in a non-chemokine suspension, the neutrophil migrates through the channel due to a chemotaxis effect, i.e., a movement of a motile cell in response to a chemical stimulus. The motility of the neutrophil, as the neutrophil migrates through the side channel, can be measured. Such a microfluidic device can be used for prognostic and diagnostic studies of neutrophil motility, for example, in burn victims.
Various aspects of the invention are summarized as follows. In general, in a first aspect, the subject matter of the disclosure can be embodied in methods for manipulating cells in a chemo-attractant environment, in which the methods include filling a main channel and a side channel branching off of the main channel with a first fluid that includes at least one chemokine, and introducing a second fluid free of the at least one chemokine and that includes multiple cells into the main channel such that an individual cell of the multiple cells can enter and form a plug in the side channel such that a chemokine concentration gradient is formed in the side channel on opposite sides of the individual cell.
Implementations of the methods can include one or more of the following features and/or features of other aspects. For example, filling can include applying pressure at the main channel inlet that is sufficient to move the first fluid through the main channel and into the side channel. Applying pressure can include applying sufficient pressure to displace air in the side channel through a gas permeable substrate defining a portion of the side channel.
In some implementations, the methods further include filling the side channel with a gas that is soluble in the first fluid prior to filling the side channel with the first fluid.
In some instances, filling the side channel with the first fluid includes flowing the first fluid through the main channel past the side channel inlet, in which case the first fluid fills the side channel by capillary action.
In certain aspects, the subject matter of the present disclosure can be embodied in devices for analyzing migration of cells in a chemo-attractant concentration gradient, in which the devices each include: a main channel having a pair of side walls, a main channel inlet, and a main channel outlet, in which the main channel is sized to receive the cells; and a side channel that branches from the main channel, the side channel having an inlet formed in a side wall of the main channel and terminating in a closed end, in which the side channel is sized such that the cells can migrate from the main channel into and form a plug in the side channel and then move along the side channel in the present of a chemo-attractant concentration gradient established in the side channel.
Implementations of the devices can include one or more of the following features and/or features of other aspects. For example, the main channel and the side channel can be positioned on a substrate that includes a gas-permeable material.
In some implementations, the main channel has a cross-sectional area sufficient to allow the cells to flow through. In certain cases, the side channel has a cross-sectional area that is smaller than a cross-sectional area of the main channel. In some implementations, the side channel can be sized such that a cell forms a plug within the side channel. In certain implementations, the side channel is rectangular in cross-section, in which sides of the rectangular cross-section are about 3 μm and 6 μm.
In some implementations, the device further includes multiple side channels, each of which branches from the main channel and has a corresponding inlet formed on the side wall of the main channel. The side channels can branch in a direction that is perpendicular to an axis of the main channel between the main inlet and the main outlet.
In some cases, the side channel is a straight channel or in other implementations, the side channel can include a bend between the first end and the second end.
In certain implementations, the side channel can include a first portion that includes the inlet and a first end, and multiple second portions, each including a second end and terminating at a corresponding closed end, wherein each second end is connected to the first end.
In certain aspects, the subject matter of the present disclosure can be embodied in systems for analyzing target cells, in which each system includes a device having a main channel with a pair of sidewalls, a main channel inlet and a main channel outlet, and multiple side channels, each side channel having an inlet connected to a side wall of the main channel, in which each side channel terminates in a corresponding closed end, and cells are introduced through the main channel inlet and migrate through the multiple side channels. The system can also include a detection unit configured to detect a migration of the cells through the device.
In some implementations, the device includes a gas permeable substrate defining at least part of the side channels. In some other implementations, a cross-sectional area of each side channel is less than a cross-sectional area of cells that migrate through the side channel.
In certain implementations, the main channel has a rectangular cross-section and/or each side channel has a rectangular cross-section.
In some implementations, the main channel and the multiple side channels are microfluidic channels and the multiple side channels can be perpendicular to the main channel.
In some instances, a side channel is square in cross-section, and a side of the square cross-section is about 6 μm. In certain implementations, a side channel is sized such that the cell forms a plug in the side channel.
In additional aspects, the subject matter of the present disclosure is embodied in methods for analyzing target cells, in which the methods include: determining a first migration rate of control cells through a side channel having an inlet and terminating in a dead end, in which the control cells migrate through the side channel due to a chemokine concentration gradient, and the first migration rate includes a distance traveled by a control cell in the side channel per unit time; determining a second migration rate of target cells through the side channel, in which the target cells migrate through the side cells due to the chemokine concentration gradient, and the second migration rate includes a distance traveled by a target cell in the side channel per unit time; and analyzing the target cells by comparing the first migration rate and the second migration rate.
In some implementations of the new methods, determining the first migration rate includes filling the side channel with a first fluid that includes chemokines, introducing a second chemokine-free fluid that includes the control cell at the inlet establishing a chemokine concentration gradient in the side channel, in which the control cell migrates towards a region of high chemokine concentration in the side channel, and measuring the distance traveled by the control cell in the side channel per unit time.
In other aspects, the subject matter of the present disclosure is embodied in methods of modulating neutrophil motility within a patient, in which the methods include obtaining a first neutrophil sample from the patient, the first neutrophil sample including a first neutrophil, determining a first motility of the first neutrophil, and administering a medication that affects neutrophil motility to vary the first motility of the first neutrophil.
Implementations of the methods of modulating motile cell, e.g., neutrophil, motility can include one or more of the following features and/or features of other aspects. For example, administering the medication can include administering a quantity of the medication such that a motility of the neutrophil measured after the medication is administered is greater than (or less than) the first motility. In certain cases, determining the first motility of the first neutrophil includes introducing the first neutrophil to a device as described herein.
For example, the device can include a main channel having a main channel inlet and a main channel outlet connected by a side wall, and a side channel having an inlet connected to the side wall, in which each side channel terminates in a corresponding closed end, and the first neutrophil sample including the first neutrophil is introduced through the main channel, where prior to introducing the neutrophil in the device, a chemokine concentration gradient is established in the side channel, and where the first neutrophil migrates through the side channel due to the chemokine concentration gradient. Determining the first motility can further include measuring a motility of the first neutrophil through the side channel in the chemokine concentration gradient.
In some implementations, the first motility is the motility of the first neutrophil through the side channel.
In some instances, the methods further include obtaining a second neutrophil sample from the patient, the second neutrophil sample including a second neutrophil, in which prior to obtaining the second neutrophil sample, the medication that affects neutrophil motility has been administered to the patient; introducing the second neutrophil sample in the device; obtaining a second motility value of the second neutrophil sample in the side channel; and comparing the first and second motility values. The methods can further include varying the medication based on a result of the comparing.
Implementations of the subject matter described here can provide several advantages. For example, the microfluidic devices for measuring cell, e.g., neutrophil, motility are simple to operate and enable the measurement of neutrophil migration and persistence characteristics at single-cell resolution. The microfluidic devices described here enable the direct observation of moving cells. Consequently, the devices can be easily implemented in a clinical setting, and the prognostic potential of neutrophil motility in a chemokine environment can be explored. Because the neutrophils migrate under an effect of a chemokine gradient established within the microfluidic device, the requirement for expensive equipment to effect flow, for example, syringe pumps, can be decreased or eliminated. Further, the training required to educate an operator to use the microfluidic devices described herein can also be decreased.
The devices can utilize small volumes of blood, a practically important feature to avoid iatrogenic anemia by repeated blood draws from critically ill or pediatric patients. The devices can yield data in less than three hours, and their operation requires, in some embodiments, only the sequential injection of two solutions through the same port of the device, without syringe pumps or complicated priming. In addition, the devices can provide temporal information at the single-cell level, and while most of the motile cells, such as neutrophils, migrate at a steady speed along distinct channels of the devices, their migration speed can be measured to a high degree of precision. The mechanical confinement of neutrophils in the small channels in these devices can also facilitate persistent movement and can have practical consequences on the ease of quantification of motility.
Using the new devices, a reference set of values characteristic of motile cells, e.g., neutrophils, from healthy subjects can be easily obtained. Once a range of “normal values” has been established, these could be used as reference. The reference values are specific for combinations of side-channel size, extracellular matrix coating the channels, and chemokines. For example, the reference value for neutrophil migration in 3×6 μm channels coated with fibronectin and filled with 100 nM fMLP is 18±3 μm/minute. Similarly obtained migration speed values from burn patients and other patients can be compared with the reference values. Data collected in this manner can serve as an early indicator for concurrent infections. Complex relationships between impaired neutrophil motility and the clinical evolution of burn patients can be studied.
The devices can detect significant impairment of motile cell, e.g., neutrophil, migration speed as early as 24 hours after patient admission to the hospital. The ability to measure precisely the neutrophil migration speed independently of all the other parameters of chemotaxis can establish a strong correlation between the depression of migration speed at 72 hours after burn correlated and the total body surface area (TBSA) of burn injury. Overall, the microfluidic devices described herein can contribute to a better understanding of the context of neutrophil pathology associated with the burn injury and its contribution to the septic and other complications in burn patients.
In addition to their use for measuring motile cell, e.g., neutrophil, migration speed, the devices can also be used to measure directional persistence that represents a duration and a distance that a cell moves in one direction before stopping or turning. For example, cells that are motile, but have no directionality, may enter the side channels, but may move back and forth. Alternatively, or in addition, cells following chemo-attractant signals may enter the channels and move up the gradient without stopping or turning. Such directional persistence of cells can be measured and any bias can be designated with numbers that represent the persistence.
As used herein, “motility” means the ability of a motile cell to move, e.g., at a specific migration rate, at least under certain conditions. Motile cells include neutrophils and other immune cells such as granulocyte, monocytes, and lymphocytes, as well as certain cells that can move only under certain specific conditions, such as mast cell, fibroblasts, and endothelial cells (e.g., circulating endothelial cells (“CEC”)) or cells under pathological conditions, such as metastatic cancer cells (e.g., circulating tumor cells (“CTC”)). Sperm and many bacteria are also motile cells. A list of some examples of motile cells that can be monitored using the devices described herein is included in Table 1 below.
As used herein, a “chemokine” means a protein capable of inducing a chemotaxis effect in a cell.
As used herein, “chemotaxis” means a movement of a motile cell in response to a chemical stimulus.
As used herein, “chemo-attractant” means a chemical agent that induces a motile cell to migrate towards the chemical agent.
As used herein, “gas-permeable” means having openings that allow gas to pass through.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
The microfluidic devices described with reference to the following figures enable the investigation of the details of cell motility, such as neutrophil chemotaxis in burn patients. In particular, the no-flow microfluidic devices can be used to measure the directional migration speed of motile cells, such as neutrophils, in a chemoattractant gradient, with high throughput, and at single cell resolution. The devices were used to measure the migration speed of neutrophils in blood samples from healthy volunteers and to establish a set of reference values for healthy persons regardless of age and sex. Using the new devices, the impairment of cell motility, such as the impairment of neutrophil migration speed after burn injury, can be determined with a high degree of precision.
Results obtained using the devices showed that neutrophil motility is depressed as early as 24 hours after the burn injury and is inhibited the most at three to five days after injury. Further, the degree of neutrophil directional migration speed inhibition in burn patients correlates with the magnitude of burn trauma. The migration speed did not correlate with clinical parameters frequently used to monitor these patients, for example, absolute neutrophil numbers, age of circulating neutrophils, or the body temperature. An example of a microfluidic device that was used to obtain these results is described with reference to
The device 100 further includes side channels (110, 112, 114, 116, 118, 120). Each side channel, for example, side channel 110, branches from the main channel 105. Each side channel has an inlet formed in the side wall of the main channel and terminates in a closed end. The side channel is sized such that the cells, for example, the neutrophils, migrate through the side channel due to a chemokine concentration gradient established in the side channel. Methods to establish a chemokine concentration gradient in a side channel are described later.
The microfluidic device 100 can be manufactured using the following methods. Two layer of photoresist (SU8, Microchem, Newton, Mass.), the first one 3 μm thin and the second one 50 μm thick, can be patterned on a silicon wafer by sequentially employing two photolithography masks and can be processed using known methods. The wafer with patterned photoresist can be used as a mold to produce PDMS (Polydimethylsiloxane, Fisher Scientific, Fair Lawn, N.J.) parts, which can then be bonded irreversibly to standard glass slides (1×3 inches, Fisher).
Thus, the example microfluidic device 100, described above, includes a first glass substrate and a second PDMS substrate in which the main channel 100 and the multiple side channels (110, 112, 114, 116, 118, 120) can be formed. The inlet 125 and the outlet 130 can be formed either in the first glass substrate or the second PDMS substrate. In other implementations, both substrates can be PDMS substrates or other similar materials.
In general, the substrate in which the main channels and the side channels are formed should be selected to have the following characteristics. The substrate can be gas-permeable so that, when the side channel is filled with a fluid, air in the side channel can be displaced through the substrate as the fluid is pumped in. Alternatively, or in addition, the substrate can be selected so as to facilitate the filling of the side channel with a fluid by capillary action. Furthermore, the substrate can be transparent so as to facilitate image capture of neutrophil motility within the side channels. In some situations, the substrate can be selected such that its surfaces can be coated with agents, for example, proteins, glycoproteins, antibodies, or combinations of them. Such coating can prevent the absorption of soluble factors to the surfaces, and facilitate cell migration, capture of cells in the main channel from cell suspension, and the like.
When formed, the main channel 105 and the side channels (110, 112, 114, 116, 118, 120) are filled with air 135. As shown in
To do so, in some implementations, a syringe (for example, 1 mL syringe) can be filled with the chemokine solution 140 (for example, a solution of fMLP and fibronectin), and can be connected to the inlet 125 of the device. At this time, the outlet 130 can be blocked. By applying pressure to the syringe, the solution 140 can be flowed into the main channel 105. The pressure of the syringe can cause the chemokine solution 140 to displace the air in the side channels. In this manner, the air can be diffused out of the side channels through the PDMS. Once all the air has been displaced out of the side channels, both the main channel 105 and the side channels (110, 112, 114, 116, 118, 120) can be filled with the chemokine solution 140 (
In some implementations, after the chemokine solution 140 is flowed through the main channel 105, the inlet 125 and the outlet 130 can be closed. The chemokine solution 140 can wick into the side channels by capillary action. In some implementations, the microfluidic device 100 can be filled with a gas that dissolves in the chemokine solution 140 prior to flowing the chemokine solution 140 through the device 100. The gas can displace the air in both the main channel and the side channels. Subsequently, when the chemokine solution 140 is flowed through the main channel 105, the gas in the side channels will dissolve in the chemokine solution 140, and the chemokine solution 140 will flow into the side channels, for example, by capillary action.
In some implementations, the chemokine solution 140 can be diffusively loaded directly into the side channels. In some implementations, reservoirs filled with chemokine solution 140 can be connected to the ends of the side channels through micro-valves. The micro-valves can be active or passive, and can permit the transfer of the chemokine solution 140 from the reservoirs directly into the side channels. An example of a passive micro-valve configured to allow transfer of chemokine solution 140 from the reservoirs to the side channels is described with reference to
After filling the side channels and the main channel 105 with the chemokine solution 140, the main channel 105 can then be flushed with a wash buffer 145. In this manner, the chemokine solution from the main channel 105 can be removed from the microfluidic device 100 whereas the chemokine solution 140 in the side channels (110, 112, 114, 116, 118, 120) remains in the microfluidic device 100. The wash buffer can be the cell suspension itself.
Subsequently, a fluid suspension that is free of chemokines and includes the cells of interest (e.g., neutrophils) can be flowed into the main channel 105. The neutrophils can be obtained from blood samples drawn from healthy volunteer donors, for example, adults who are 18 years or older and who are not on any immuno-suppressants. For example, three 1 mL samples can be drawn from each volunteer with a time lapse of at least one week between draws.
In addition, neutrophils can be obtained from blood samples drawn from burn patients. For example, the blood samples can be drawn from burn patients who have sustained if burns cover at least 20% of their total body surface area. For example, a first 1 mL sample can be obtained within 72 hours after burn injury. Subsequently, two more 1 mL samples can be drawn at 48 hour intervals afterwards. Samples may not be drawn within 24 hours of an operative procedure.
Neutrophils can be isolated from blood samples using known techniques. For example, using sterile technique, neutrophils can be isolated from whole blood by density gradient separation using Polymorphprep (13.8% sodiuk diatrizoate and 8.0% polysaccharide, Axis-Shield, Rodelokka, Oslo, Norway), with centrifugation at 500 g for 40 minutes. To return the cells to an isotonic environment, the cells can be harvested and re-suspended in 10 mL of 0.5×PBS, then isolated by centrifugation at 400 g for 10 minutes. The neutrophils can be re-suspended in 50-100 μL of 1×PBS before loading into the microfluidic device 100. Samples can be processed within one hour after each blood draw and can be maintained at 37° C.
Once the neutrophils have been isolated from the blood and re-suspended in a fluid (for example, 1×PBS), the fluid suspension 150 that includes the neutrophils 155 can be infused into the device. To do so, the fluid suspension 150 can be introduced into the main channel 105, and the inlet 125 and the outlet 130 can be closed, for example, clamped. Notably, the fluid in which the neutrophils are suspended (for example, 1×PBS) is chemokine free. When the fluid suspension 150 is introduced into the main channel 105, the neutrophils 155 can settle, for example, at the bottom of the main channel 105 (
Because a side channel (for example, side channel 110) is filled with a chemokine solution and the main channel 105 is filled with a chemokine free solution, a chemokine concentration gradient is established within the microfluidic device 100. It is known that neutrophils experience a chemotactic effect in the presence of the chemokine concentration gradient, and consequently migrate from a region of lower chemokine concentration to a region of higher concentration. Consequently, the neutrophils 155 migrate through the side channels away from the main channel 105.
The microfluidic device 100 can be formed such that when several neutrophils 155 are introduced into the device, one neutrophil 155 migrates through each side channel. For example, the main channel 105 has a cross-section that is sufficient to allow the neutrophils 155 to be flowed through. Each side channel (110, 112, 114, 116, 118, 120) has a cross-section that is either as large as or smaller than a size of the neutrophil 155. Thus, when a neutrophil 155 enters, for example, side channel 110, the neutrophil 155 forms a plug within the side channel 110.
As a plug, the neutrophil 155 entraps the chemokine solution 145 in the region of the side channel 110 between the neutrophil 155 and the dead end of the side channel 110 (
By selecting the cross-section of the side channel 110 to be less than or equal to a size of the neutrophil 155, neutrophil migration in the absence of an active mechanism (for example, micro-pump, micro-valve, and the like) can be achieved. A photograph of an example of a microfluidic device 100 including a main channel 175 filled with neutrophils suspended in a chemokine-free solution is shown in
The system 600 further includes an imaging system 630 configured to capture images and/or video of the neutrophil migration. For example, the imaging system 630 is configured to perform time-lapse imaging, and can include a Zeiss Axiovert microscope operating at a 20× magnification. One example of an imaging system is the Nikon Biostation IM, which is a system for time-lapse imaging with heated and humidified environmental chamber. The humidified environmental chamber increases the observation duration, for example, 24 hours for observing the migration of cancer cells towards serum factors.
The system 600 further includes a computer system 635 that is operatively coupled to the imaging system 630. The computer system 635 can include a computer-readable storage medium (for example, a hard disk, a CD-ROM, and the like) that stores computer program instructions executable by data processing apparatus (for example, a computer system, a processor, and the like) to perform operations. The operations can include controlling the imaging system 630 to capture images of the migration of neutrophils 615 through the side channels. In addition, the computer system 635 can receive the captured images from the imaging system 630, and process the images to obtain a migration speed of a neutrophil in a channel.
Alternatively, or in addition, neutrophils can be manually tracked to obtain migration rates. For example, migration rates of neutrophils (for example, 50 neutrophils/sample) can be tracked, and the migration rates can be calculated using Image J (NIH) software executed by the computer system 635. In some situations, experiments to characterize the formation of gradients inside the device in the absence of neutrophils can be performed by replacing all or portions of the chemokine solution (for example, the fMLP) with fluorescein (Sigma) of comparable molecular weight, and analyzing the distribution and changes in fluorescence intensity from time-lapse imaging using the imaging system 630 and the computer system 635. Further, the computer system 635 is configured to execute computer software applications that perform statistical analysis of the data captured by the imaging system 630. For example, the computer system 635 is configured to execute the Shapiro-Wilk test to test the normality of the distribution of migration speed values for neutrophils in the same sample. The test indicates if the data is likely to be derived from a normally distributed population (p>0.05). The computer system 635 is further configured to perform multivariate analysis to determine correlations between neutrophil migration speed and clinical parameters.
The microfluidic devices described herein can be used to analyze different types of motile cells including neutrophils. Some of the different types of cells, components of a chemokine solution in which each cell experiences a chemotaxis effect, cell sizes, and corresponding side channel sizes, are shown in Table 1.
The devices can be single-use devices. In other situations, the devices can be re-used by lysing the neutrophils, for example, with distilled water, or some detergents, and removing the cell debris by persistent washing. In particular, if the side channels are connected to reservoirs through micro-valves, then the side channels can be washed by pumping suitable fluids into the side channels through the valves. Also, migration of control and target cells can be performed in the same device. Alternatively, control cell migration can be measured using a first device and target cell migration can be measured using a second, separate device.
In one implementation of process 900, a total of 23 blood samples were collected from 18 healthy volunteers aged 19-68 years (mean 40 years) (Table 2); 61% of volunteers were female, and 39% male. Volunteers had no past medical history with the exception of hypertension, hypothyroidism, and depression. The most common medications taken were oral contraceptives and antidepressants. No volunteer took immunosuppressant drugs.
Enterobacter,
Candida-
Average neutrophil migration speed for all volunteer samples was 18±5 μm/min (range 14 to 24 μm/min) (
Thus, the microfluidic device can be applied to the analysis of neutrophil motilities in burn patients. In some situations, neutrophils obtained from burn patients can be studied over a one week period, for example, at 48 hour intervals. To determine a long-term effect of the burn injury and treatment on the patients, the study period can be expanded to six months. In such situations, the neutrophil motility of the patients can be determined periodically. For example, for burn patients, neutrophil motility can be measured daily for the first two weeks and then weekly for the subsequent six months. For transplant patients, motility can be monitored on a monthly basis.
Different types of devices can be used at the different monitoring stages, thereby yielding different information. For example, monitoring the ability of T-helper lymphocytes during immuno-suppressive therapy would have the goal of identifying the minimal drug doses that keep the lymphocytes from moving too fast, and potentially preventing rejection. Monitoring the neutrophils would have the goal of identifying the maximal drug dose to identify the risk of too much immune suppression, which can increase the risk of infections.
Recovery for burn patients takes significantly longer than one week after the burn injury. Further, burn patients are susceptible to infections for life. Periodic studies of neutrophil motilities over a six month period can enable determining an effect of the treatment. In particular, neutrophil motility can be monitored to determine if there is a correlation between motility and infections. For example, if the neutrophil motility of the patient decreases over time, then it can be determined that the patient is at a higher risk of infection, and a treatment can be administered accordingly.
Similarly, neutrophil motility can be studied to determine if a correlation exists between motility speed and rate of wound healing. For example, if neutrophil motility increases over time and the patient's wounds are healing, then a rate of migration speed that indicates wound healing can be determined. In another burn patient, if the rate of migration speed does not match the rate previously determined as indicating wound healing, then a treatment can be administered to alter, i.e., increase the neutrophil migration speed.
Although neutrophil mobility is not the sole indicator of infections or healing in burn patients, it is a significant indicator. Because the microfluidic device described above enables easy determination of neutrophil mobility, a range of migration speeds that indicate wound healing or heightened risk of infection or both can be determined. Based on these determinations, treatments unique to patients can be tailored.
Further characterization of neutrophil chemotaxis using this microfluidic device may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions. For example, the device can be applied to analyze neutrophil motility in pediatric patients to identify patients who are at a higher risk for certain diseases. In transplantations, the device can be used to analyze neutrophil motilities to determine if there is a correlation between neutrophil motility under medication and the occurrence of complications, for example, infections and rejections. By determining a range of neutrophil motilities that correlate to low infection and at which immuno-suppressant functions are not suppressed, it may be possible to vary the quantities of immuno suppressant medication that is being administered to patients.
Neutrophil motility measured using this device can be one metric for identifying a treatment in patients suffering from fungal infections. Because such patients are critically ill, doctors often rely upon intuition to immediately start a course of treatment while waiting on test results that can take between 36 and 72 hours to obtain. Because neutrophil motility is believed to decrease due to fungal infections but not due to bacterial infections, motilities determined using this device can be used to determine that the infections are fungal, and then, as one metric in a selection of a course of treatment.
The metal patch 1415 prevents the layer 1430 from adhering to the substrate 1425. When the pressure in the chemokine solution channel 1420 is increased above a certain threshold, leakage will occur between the layer 1430 and the metal patch 1415. This will create an opening between the channel 1420 and the side channel 1410. Relieving the pressure will close the opening, stop the flow, and restore the sealing. In some implementations, the layer 1430 can be made from PDMS or any elastic material that is flexible under high pressure and retains its original shape upon relief of the pressure. The pressure required to open and close the micro-valve 1400 can depend on the side of the metal patch 1415 (for example, width and length) and the elasticity of the layer 1430. In addition, the pressure can also be affected by the stickiness of the layer 1430.
While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any disclosures or of what may be claimed, but rather as descriptions of features specific to particular implementations of particular disclosures. Certain features that are described in this specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Thus, particular implementations of the subject matter have been described. Other implementations are within the scope of the following claims.
The specification discloses methods, apparatus, and systems for analyzing neutrophil motility using microfluidic devices. Specifically, in some implementations, motility of a neutrophil in a chemokine concentration gradient is described. It will be appreciated that the microfluidic device and the techniques described herein can be used to detect motility of any type of cell in an environment in which the cell will migrate due to a concentration gradient. For example, the concentration gradient can be created using a light source such that the cell migrates from a region of low light intensity toward a region of high light intensity (or vice versa). In another example, the concentration gradient can be created using a magnetic source such that the cell migrates from a region of low magnetic field toward a region of high magnetic field. Thus, in general, the motility of cells that migrate in the presence of any type of concentration gradient can be studied by flowing a fluid including such cells into the microfluidic device having the main channel and the branching side channels. The universe of cells and organisms that can be tested in such a microfluidic device can include cells and organisms that have one or more cilia (for example, bacteria, sperm, and the like) that have sizes sufficient to be mechanically restricted within one of the branching side channels.
In some implementations, all the channels (including the side channel 1610 and the bifurcating channel 1615) can be filled with a first chemokine solution. A second chemokine solution can be introduced through the chemokine solution channel 1625 into the side channel 1610 through the micro-valve 1620. When the pressure in the chemokine solution channel 1625 is increased, the second chemokine solution flows into the side channel 1610 without washing the first chemokine solution through the bifurcating channel 1615. Cells in suspension can be introduced in the main channel, removing any excess of the second chemokine solution.
The cells migrate through the side channel 1610 in a first chemokine solution environment. When the cells reach the entrance of the bifurcating channel 1615, then the cells diffuse from the side channel 1610 into the bifurcating channel 1615. The geometry and arrangement of the side channel 1610 and the bifurcating channel 1615 can be selected to regulate the timing and temporal changes of the two chemokine solutions. In some scenarios, the first chemokine solution can be a drug. An array of bifurcating channels on both sides of each side channel can be used. By changing the ratios between the dimensions shown in
In some implementations, neutrophil chemotaxis analysis can be performed using whole-blood without separating the neutrophils from the blood. Such a system can offer several potential advantages such as, for example, less than five minutes from blood draw to moving neutrophils, minimal risks for activating/altering neutrophils during sample separation (due to absence of density-based separation media, anti-body capture, E-selecting capture, electrical or other physical fields), low blood volume (for example, 10 μL or less.
Alternatively, strictures can have a length between 4 and 50 μm and can be effective in blocking the entrance of red blood cells but allowing the passage of neutrophils. The strictures can be smaller or larger than those described here, and can be used to analyze the migration of other cells in blood, for example, lymphocytes, monocytes, natural killer lymphocytes, platelets and megacariocytes, epithelial cells, endothelilal cells, cancer cells, and the like.
Occasionally, few red blood cells pass through the stricture, either during whole blood loading or pushed forward by the moving neutrophils. These red blood cells appear not to interfere with the neutrophil chemotaxis. When many red blood cells clog the entrance of a channel, neutrophils can have a difficult time entering those channels.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/447,240, filed on Feb. 28, 2011, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61447240 | Feb 2011 | US |
