Patent Application
20190291112
The present disclosure generally relates to a microfluidic system for generating droplets and methods for making and using the same, and in particular, to a microfluidic system and methods useful for, e.g., drug screening of cells, especially cancer cells, and especially primary tumor cells.
Cancer is one of the most lethal diseases, which threatens millions of people worldwide, accounting for approximately 13% of all deaths globally. Although all clinically approved drugs and drug combinations have been tested in vitro using cultured cells, in vivo using animal models and in clinical trials, there is no guarantee that a particular treatment will successfully treat a patient's cancer, due to insufficient knowledge of cancer etiology, diversity of cancer types and properties.
Understanding the heterogeneous drug responses from individual cancer cells within a population of cancer cells can be critical to understanding cancer etiology and cancer diversity. However, most of the current in vitro drug screening platforms provide drug responses from a bulk population of cancer cells, and tend to overlook the heterogeneity of drug responses within the bulk population of cells. Hence, there is a need to develop a drug screening platform that provides drug response from a bulk population of cancer cells, as well as from individual cells within the bulk population of cancer cells.
In vivo drug screening using animal models is generally more reliable yet more expensive than in vitro drug screening using cultured cells. Most primary tumors contain multiple subclones and have genetic heterogeneity. However, the majority of cancer cell lines are propagated through hundreds of passages and as a result a single clone dominates the culture and the genetic heterogeneity of the primary tumor is lost. Primary tumor cells, directly obtained from an animal tumor tissue, closely resemble the parental tumor tissue and have similar biological responses to an in vivo situation. Thus, in vitro drug screening using primary tumor cells directly obtained from tumor tissue could better predict treatment outcome.
However, the major hurdle to drug screening on primary tumor cells is low sample input. For example, while leukemia patients provide over 10 million cancer cells from 2 ml of patient blood, mammary tumors of 2×2 cm can only result in less than 1 million cells in sum after dissociation.
Hence, there is need to develop a reliable and inexpensive drug screening method that requires low sample input.
Provided herein is a microfluidic chip that enables, e.g. quick sample loading, robust droplet formation, and/or automatic droplet investigation with a small sample input size. The sample includes but is not limited to cancer cells, such as cancer cell lines, primary tumor cells, secondary tumor cells, cancer stem cells, or circulating tumor cells. Other cell types for other disease models, e.g. metabolic diseases, respiratory diseases, and infectious diseases, are also within the contemplation of the present disclosure.
In a first aspect, a microfluidic system of the present disclosure is capable of loading sample more quickly and requires as short as 5 minutes even with manual loading, whereas conventional methods usually require 1-1.5 hours with manual loading and still require 10-30 min even with automatic loading. The quick sample loading method of the microfluidic system of the present disclosure allows high throughput screening and is beneficial for special samples that can only survive for a short period of time, such as primary tumor cells.
In a second aspect, a microfluidic system of the present disclosure is capable of generating robust droplets and preventing droplet escape during overnight incubation at 37° C. A robust droplet refers to a droplet with minimal loss of sample, no droplet coalescence, e.g. merging of two or more droplets, and/or no cross-contamination between droplets, e.g. mixing of the sample from one droplet with the sample from the other droplet, which are the prerequisites for reliable drug screening of the samples enclosed within the droplets. In existing droplet microfluidic systems, the formed droplets tend to escape from the droplet-forming wells during overnight incubation at 37° C., which causes undesired loss of sample and alteration of screening conditions that can ultimately affect drug screen outcomes. Therefore, the special configuration of the microfluidic system of the present disclosure significantly improves the reliability of the drug screening.
In a third aspect, a microfluidic system of the present disclosure is capable of filling the droplet-forming well fully to maximize channel space usage and save cost.
In a fourth aspect, a drug screening method of the present disclosure is capable of providing efficient evaluation of drug susceptibility of cancers with as few as 16,000 cells obtained from primary cancer sample obtained from a patient for each treatment condition within 24 h. Moreover, the sample input size can be potentially reduced to 100 cells per drug dose based on the configuration of the microfluidic system of the present disclosure. In addition, the cost of the drug screening method of the present disclosure is as low as HKD 0.20 per chip, making it pragmatically affordable for all cancer patients.
In certain embodiments, the present disclosure relates to microfluidic chip 100 for generating a plurality of droplets from a loading fluid, comprising at least one droplet-forming channel 200, each of the at least one droplet-forming channel 200 comprising: plural droplet-forming units serially connected together; an inlet 201 for receiving the loading fluid and providing the loading fluid to the plural droplet-forming units; and an outlet 202 for discharging the loading fluid remained after passing through the plural droplet-forming units; wherein: an individual droplet-forming unit 209 comprising an inflow channel 203, a neck channel 204, a droplet-forming well 205, a restricted flow port element 206, and an outflow channel 207 all of which are sequentially arranged along a flow direction of the loading fluid; the inflow channel 203 is configured to accept the loading fluid and is in fluid communication with the neck channel 204, the neck channel 204 is in fluid communication with the droplet-forming well 205 for delivering a first portion of the loading fluid from the inflow channel 203 to the droplet-forming well 205, and is configured to have a cross-sectional width that is smaller than a cross-sectional width of the droplet-forming well 205 to prevent droplet escape from the droplet-forming well 205; the restricted flow port element 206 is configured to generate a restricted flow to facilitate droplet formation in the droplet-forming well 205; and wherein: the individual droplet-forming unit 209 further comprises a bypass channel 208; the bypass channel 208 is located around the droplet-forming well 205, and is configured to deliver a second portion of the loading fluid from the inflow channel 203 to the outflow channel 207.
in certain embodiments, the neck channel 204 and the bypass channel 208 have a cross-sectional width ratio of the bypass channel to the neck channel, the cross-sectional width ratio being selected such that the first portion of the loading fluid fills the droplet-forming well 205 before the second portion of the loading fluid fills the bypass channel 208.
In certain embodiments, the cross-sectional width ratio of the bypass channel to the neck channel is approximately 0.2 to approximately 1.0.
In certain embodiments, the cross-sectional width ratio of the bypass channel to the neck channel is approximately 075.
In certain embodiments, the neck channel 204 has a cross-sectional width of approximately 50-150 μm.
In certain embodiments, the droplet-forming well 205 has a cross-sectional width of approximately 100-500 μm.
In certain embodiments, the restricted flow port element 206 is a restriction channel having a cross-sectional width of approximately 5-20 μm.
The present disclosure also relates to a mold comprising complementary features to a microfluidic chip 100, the microfluidic chip 100 comprising: at least one droplet-forming channel 200, each of the at least one droplet-forming channel 200 comprising: plural droplet-forming units serially connected together; an inlet 201 for receiving the loading fluid and providing the loading fluid to the plural droplet-forming units; and an outlet 202 for discharging the loading fluid remained after passing through the plural droplet-forming units; wherein: an individual droplet-forming unit 209 comprising an inflow channel 203, a neck channel 204, a droplet-forming well 205, a restricted flow port element 206, and an outflow channel 207 all of which are sequentially arranged along a flow direction of the loading fluid; the inflow channel 203 is configured to accept the loading fluid and is in fluid communication with the neck channel 204, the neck channel 204 is in fluid communication with the droplet-forming well 205 for delivering a first portion of the loading fluid from the inflow channel 203 to the droplet-forming well 205, and is configured to have a cross-sectional width that is smaller than a cross-sectional width of the droplet-forming well 205 to prevent droplet escape from the droplet-forming well 205; the restricted flow port element 206 is configured to generate a restricted flow to facilitate droplet formation in the droplet-forming well 205; and wherein: the individual droplet-forming unit 209 further comprises a bypass channel 208; the bypass channel 208 is located around the droplet-forming well 205, and is configured to deliver a second portion of the loading fluid from the inflow channel 203 to the outflow channel 207.
In certain embodiments, the material for the mold is selected from the group consisting of crystalline silicon, amorphous silicon, glass, quartz, and metals.
In certain embodiments, the neck channel and the bypass channel of the mold have a cross-sectional width ratio of the bypass channel to the neck channel, the cross-sectional width ratio being selected such that the first portion of the loading fluid fills the droplet-forming well before the second portion of the loading fluid fills the bypass channel.
In certain embodiments, the cross-sectional width ratio of the bypass channel to the neck channel of the mold is approximately 0.75.
The present disclosure also relates to a method for drug screening, wherein the method comprising steps of:
In certain embodiments, the carrier fluid comprises an oil and a surfactant.
in certain embodiments, the carrier fluid is a perfluorinated trialkyl amine oil supplemented with approximately 1-5% fluorosurfactant.
in certain embodiments, the sample fluid comprises cells, a drug, a cell culture medium, an additive, a dead cell indicator, and/or a metabolic indicator.
In certain embodiments, the cells are cancer cells selected from the group consisting of cancer cell lines, primary tumor cells, secondary tumor cells, cancer stem cells, and circulating tumor cells.
In certain embodiments, the cell culture medium comprises fetal bovine serum at a concentration of 1%-20% (v/v).
In certain embodiments, the dead cell indicator is selected from the group consisting of ethidium homodimer 1, Alamar Blue, SYTOX Green nucleic acid stain, and propidium iodide; and the metabolic indicator is selected from the group consisting of Calcein AM, C12-resazurin, SYTO 10 dye, and SYBR 14 nucleic acid stain.
In certain embodiments, the methyl cellulose has a percentage of 0.5%-3% (m/v) in the sample fluid.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.
The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features.
Other aspects and advantages of the invention will be apparent to those skilled in the art from a review of the ensuing description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The above and other objects and features of the present disclosure will become apparent from the following description of the various embodiments described herein, when taken in conjunction with the accompanying drawings, in which:
For the purposes of the present disclosure, cancer cells are described in connection with the microfluidic system and the methods using thereof described herein only as exemplary embodiments. It should be appreciated that the uses of the system and methods are not limited to cancer cells, but also other cell types for different disease models, or other biological samples, e.g. bacteria.
Additionally, to assist in the description of the structural configuration, words such as length, width, height, depth, upper, lower, top, bottom, transverse, longitudinal, horizontal and the like are used. Unless their contextual usage indicates otherwise, these words are to be understood herein as having no structural, functional or operational significance and as merely reflecting the arbitrarily chosen orientation.
The term “channel” or “well” as used herein is to be interpreted in a broad sense. Thus, it is not intended to be restricted to elongated configurations where the transverse dimension or longitudinal dimension greatly exceeds the diameter or cross-sectional dimension. Rather, such term is meant to comprise cavities or tunnels of any desired shape or configuration through which fluids, such as liquids and gases, may be directed. Such a fluid cavity may, for example, comprise a flow-through cell where fluid is to be continually passed or, alternatively, a chamber for holding a specified, discrete amount of fluid for a specified amount of time. “Channels” or “wells” may be filled with or may contain internal structures comprising, for example, valves, filters, or equivalent components and materials. A microfluidic channel can have a cross-sectional dimension in the range between about 1.0 μm and about 500 μm, between about 25 μm and about 200 μm or between about 50 μm and about 150 μm.
The term “transverse dimension” as used herein refers to the dimension of a plane that is parallel to the plane defined by the top or bottom surface of a channel or well and is parallel to the flow direction. The term “longitudinal dimension” as used herein refers to the dimension of a plane that is perpendicular to the plane defined by the top or bottom surface of a channel or well and is parallel to the flow direction. The term “cross-sectional dimension” as used herein refers to the dimension of a plane that is perpendicular to both the transverse plane and the longitudinal plane, and is also perpendicular to the flow direction.
The term “cross-sectional width” or “width” as used herein refers to the dimension that is perpendicular to the longitudinal dimension while parallel to the transverse dimension. The term “length” as used herein refers to the dimension that is parallel to the transverse dimension and longitudinal dimension while perpendicular to the cross-sectional dimension. The term “height” or “depth” as used herein refers to the dimension that is perpendicular to the transverse dimension while parallel to the longitudinal dimension and the cross-sectional dimension.
The term “microfluidic” as used herein is to be understood, without any restriction thereto, to refer to structures or devices through which fluid, such as liquids and gases, is capable of being passed or directed, wherein one or more of the dimensions is less than about 500 μm.
The term “in fluid communication” throughout the present disclosure, unless the context indicates otherwise, does not indicate a fluid must flow from one of the two components in fluid communication directly to the other. There can be one or more other components, such as devices, valves, ports, ducts, tubings, etc. between the two components.
As used herein the term “and/or” includes any and all combinations of one or more of the associated listed items.
The term “serially connected” used herein is not limited to the case where subjects, e.g. droplet-forming units, are directly connected, and thus the term may refer to the case where any other members are disposed between the subjects.
The term “sequentially arranged” as used herein refers to, for example, “A and B are sequentially arranged” representing the elements A and B arranged in the order described above, and the other elements, e.g. C, may be interposed between A and B, for example, A, C, and B may be provided in the order described above.
The term “element” as used herein is intended to include meanings of other like-terms such as “component” and so forth.
The term “complementary features” as used herein refers to a set of features that are complementary to another set of features so that when the two sets of features are merged together there is minimal space left therebetween, :For example, a set of concave features are complementary features to a set of convex features of the same dimensions with the set of concave features.
The term “sample input size” as used herein refers to the volume of sample fluid and/or the total number of cells within a sample fluid.
As used herein, the term “prevent” or “preventing” refers to any method to partially or completely preclude, avert, obviate, forestall, stop, hinder or delay the consequence or phenomenon following the term “prevent” or “preventing” from happening. The term “prevent” or “preventing” does not mean that the method is necessarily absolute, but rather effective for providing some degree of prevention or amelioration of consequence or phenomenon following the term “prevent” or “preventing”.
The inflow channel 203 of the droplet-forming unit 209 is configured to accept the loading fluid from the inlet 201, and is in fluid communication with the neck channel 204, as exemplified in
The loading fluid comprises a sample fluid and a carrier fluid in distinct layers separated by an interface. In certain embodiments, the loading fluid has a carrier fluid, a sample fluid A, the carrier fluid, and a sample fluid B, all of which are sequentially arranged in a loading chamber along the direction of ejecting the loading fluid. When the loading fluid is ejected from the loading chamber and is infused into the droplet-forming channel 200 through the inlet 201, the sample fluid B will first reach the inlet 201, followed by the carrier fluid, followed by the sample fluid A, and then followed by the carrier fluid. The loading chamber can hold two or more sample fluids, which are segregated by a carrier fluid between two adjacent sample fluids. As such, the droplet formed in a droplet-forming well 205 comprises a shell formed by the carrier fluid encompassing a sample fluid therewithin, as exemplified in
The carrier fluid can be a mixture comprising an oil and a surfactant, which facilitates the formation of droplets in the droplet-forming wells 205, segregates two distinct sample fluids in the loading chamber, prevents the coalescence of two droplets of two distinct sample fluids, and helps maintain constant drug concentrations within the droplets.
The neck channel 204 is in fluid communication with the droplet-forming well 205 for delivering a first portion of the loading fluid from the inflow channel 203 to the droplet-forming well 205, as exemplified in
The incorporation of the neck channel 204 into a droplet-generating microfluidic chip 100 and the configuration of the neck channel 204 with respect to the droplet-forming well 205 can significantly improve the robustness and/or stability of a droplet formed within the droplet-forming well 205, restrict crosstalk with subsequent flow during sample loading and prevent droplet escape during overnight incubation at 37° C. In particular,
Furthermore, the fidelity of the observed loaded volume as compared to the preset loading volume allows the adjustability of the number of screening conditions, since the total number of screening conditions can be calculated by dividing the total number of droplet-forming wells 205 on a droplet-forming channel 200 by the number of the occupied wells, which can be predicted based on the preset loading volume of a sample fluid, as shown in
The restricted flow port element 206 is in fluid communication with both the droplet-forming well 205 and the bypass channel 208, and is configured to generate a restricted flow to facilitate droplet formation in the droplet-forming well 205, as exemplified in
The bypass channel 208 is located around the droplet-forming well 205, and is configured to deliver a second portion of the loading fluid from the inflow channel 203 to the outflow channel. In certain embodiments, the bypass channel 208 of a droplet-forming unit 209 starts from the intersection between the inflow channel 203 and the neck channel 204, and ends at the intersection between the restricted flow port element 206 and the outflow channel 207, as illustrated in
In certain embodiments, the microfluidic chip 100 can be prepared by a) providing a mold comprising complementary features to the microfluidic chip 100 with features described above; b) contacting the mold with a polymer liquid and then solidifying the polymer liquid to form a mold covered by a layer of the solidified polymer; c) detaching the solidified polymer from the mold to obtain the microfluidic chip 100. The mold can be made from a material selected from a group consisting of crystalline silicon, amorphous silicon, glass, quartz, and metals. Other materials having similar properties are also within the contemplation of the present disclosure. In certain embodiments, the mold is a silicon wafer having complementary features to the microfluidic chip 100 as described above. The polymer used to prepare the microfluidic chip 100 includes, but not limited to, poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate), polyethylene, polyetheretherketone, polyurethane, polypropylene, polyimide, polystyrene, hydrogel, polycarbonate, and combinations thereof. Other polymers having similar properties are also within the contemplation of the present disclosure. In certain embodiments, the polymer used to fabricate the microfluidic chip 100 is PDMS, because of its ease of fabrication, transparency and biocompatibility.
Microfluidic Chip Design and Fabrication and Assembly of a Microfluidic System
Soft photolithography by photomask (Shenzhen Newway, China) was used to fabricate SU-8 negative photoepoxy (Microchem, USA) on silicon wafer (Harbin Tebo Technology, China) following standard procedures to make the patterned wafers. The patterned wafers used in this study were determined to be 62-78 μm in height using KLA-Tencor AlphaStep D-600 Stylus Profiler (KLA-Tencor, USA).
Polydimethylsiloxane (Dow Corning, USA) at 1:7 ratio (w/w) base to curing agent ratio (w/w) was poured onto the patterned wafers, baked in an oven at 65° C. for 25 min, and peeled off to generate PDMS slabs. Lastly, the PDMS slabs were plasma bound to 2.4×2.4 cm No. 1.5 square glass coverslips using Harrick Plasma PDG-002 Expanded Plasma Cleaner (Harrick Plasma, USA) to generate the ready-to-use microfluidic systems after baking at 65° C. overnight.
In certain embodiments, the restricted flow port element 206 of the microfluidic chip 100 has a transverse dimension of approximately 15 μm×150 μm, the droplet-forming well 205 of the microfluidic chip 100 has a transverse dimension of approximately 300 μm×1150 μm, and the neck channel 204 of the microfluidic chip 100 has a transverse dimension of approximately 100 μm×225 μm. Other suitable dimensions are also within the contemplation of the present disclosure.
A complete drug screening assay or method for drug screening will now be described with reference to the microfluidic system of the present disclosure, as shown in
Preparation of Loading Fluid
In certain embodiments, the loading fluid is prepared by sequentially withdrawing a carrier fluid followed by a sample fluid, segregated by a carrier fluid before withdrawing another sample fluid into a loading chamber. In certain embodiments, the loading chamber can be a tubing. In other embodiments, the loading chamber can be a tubing that is connected on one end with a pressure-asserting device, e.g. a syringe pump, and connected to the inlet of the microfluidic system on the other end. Any other loading chambers that are compatible with the microfluidic system are also within the contemplation of the present disclosure.
In certain embodiments, the sample fluid can be a mixture comprising cells, a drug, a cell culture medium, an additive, a dead cell indicator, and/or a metabolic indicator. As such, drug concentration can be freely adjusted during premixing with cells before loading on chip. In certain embodiments, the cells are cancer cells. In certain embodiments, the cancer cells can be cancer cell lines, primary tumor cells, secondary tumor cells, cancer stern cells, or circulating tumor cells. In certain embodiments, the cancer cell line can be Jurkat E6.1 cells, MDA-MB-231 cells, or NAS1604C that is derived and amplified from the primary tumor cell NAS1604 in the present disclosure. In certain embodiments, the primary tumor cells can be obtained from primary tumors from human patients. In certain embodiments, the primary tumor is nasopharyngeal tumor, colon carcinoma, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovary cancer, pancreas cancer, brain cancer, head cancer, neck cancer, lymphoma, leukemia, or brain cancer. Any other solid tumors are also within the contemplation of the present disclosure. The secondary tumor cells are cells obtained from secondary tumors, and secondary tumors are cancers that have spread and/or metastasized from the place where it first started to another part of the body. For example, cancer cells may spread from the breast (primary cancer) to form new tumors in the lung (secondary tumor). Cancer stem cells are cancer cells found within tumors or hematological cancers that possess characteristics associated with normal stem cells, specifically ability to give rise to all cell types found in a particular cancer sample.
In certain embodiments, the suspended cancer cell line is Jurkat E6.1 cells, derived from human acute T cell leukemia. Other suspended cancer cell lines are also within the contemplation of the present disclosure. In certain embodiments, the adherent cancer cell line is MDA-MB-231 cells, derived from human metastatic breast adenocarcinoma. Other adherent cancer cell lines are also within the contemplation of the present disclosure. In certain embodiments, the primary tumor cells can be dissociated from primary nasopharyngeal tumors from human patients. Other primary tumor cells are also within the contemplation of the present disclosure.
The cell culture medium can be any conventional culture medium suitable for a particular cell line. In certain embodiments, the cell culture medium contains v/v 1%-20% Fetal Bovine Serum (FBS). In certain embodiments, the volume to volume (v/v) percentage of FBS is 2%-19%, 3%-18%, 4%-17%, 5%-16%, 6%-15%, 7%-14%, 8%-13%, 9%-12%, or 10-11%. In certain embodiments, the volume to volume percentage of FBS is approximately 5%. FBS possesses emulsification properties that affect droplet formation and stability, and approximately 5% (v/v) FBS resulted in optimal droplet formation and stability.
In certain embodiments, the additive in the sample fluid prevents the clustering of adherent cell lines and improves the reliability of the automatic cell viability investigation. In certain embodiments, the additive is methyl cellulose, Pluronic® F-68, and Matrigel®. In certain embodiments, the additive is 0.5%-3% (m/v) methyl cellulose. In certain embodiments, the additive is 0.6%-2.5% (m/v), 0.7%-2.0% (m/v), 0.8%-1.9% (m/v), 0.9%-1.8% (m/v), 1.0%-1.7% (m/v) (m/v), 1.1%-1.6% (m/v), 1.2%-1.5% (m/v), or 1.3%-1.4% (m/v) methyl cellulose. In certain embodiments, the additive is methyl cellulose of approximately 1.0% (m/v). Any other additives and any other mass to volume percentages of the additives that prevent the clustering of adherent cell lines while do not affect droplet formation are also within the contemplation of the present disclosure.
In certain embodiments, the dead cell indicator can be ethidium homodimer 1, Alamar Blue, SYTOX Green nucleic acid stain, or propidium iodide. In certain embodiments, the metabolic indicator can be Calcein AM, C12-resazurin, SYTO 10 dye, or SYBR 14 nucleic acid stain. In certain embodiments, the dead cell indicator is 2 μM ethidium homodimer 1. Any other dead cell indicators and/or metabolic indicators that do not affect cell viability at the time frame of drug screening are also within the contemplation of the present disclosure.
In certain embodiments, the carrier fluid can be a mixture comprising an oil and a surfactant or a detergent. The carrier fluid facilitates the formation of droplets in the droplet-forming wells 205, segregates two distinct sample fluids in the loading chamber, prevents the coalescence of two droplets of two distinct sample fluids, and helps maintain constant drug concentrations within the droplets. The oil can be any oil that is substantially inert under the screening conditions and substantially immiscible with water. The selection of the appropriate oil is well within the skill of a person of ordinary skill in the art. In certain embodiments, the oil is a perfluorinated alkane, a perfluorinated trialkyl amine, and/or a mixture thereof. In certain embodiments, the oil is a C6-C12, C6-C10, or C8-C12 perfluorinated alkane. In certain embodiments, the oil is C6F14, C7F16, C8F18, C9F20, C10F22, C11F24, C12F26 or a mixture thereof. In certain embodiments, the oil is C10F22, i.e. Fluorinert® FC-3283 oil. In certain embodiments, the oil is C6F10, i.e. Fluorinert® FC-72 oil. In certain embodiments, the oil is a perfluorinated trialkyl amine. In certain embodiments, the oil is (CmFm+2)(CnFn+2)(CpFp+2)N, wherein each of m, n, and p is independently a whole number selected from 1-12. In certain embodiments, each of m, n, and p is independently a whole number selected from 1-6. In certain embodiments, the oil is perfluorinated tripentyl amine, i.e. Fluorinert® FC-70 oil. In certain embodiments, the oil is bisnonafluorobutyl trifluoromethyl amine, i.e. Fluorinert® FC-40 oil.
In certain embodiments, the carrier fluid is a mixture comprising an oil supplemented with approximately 1-5% (w/v) fluorosurfactant. In certain embodiment, the carrier fluid is a mixture comprising an oil supplemented with approximately 2% (w/v) fluorosurfactant. A fluorosurfactant is a synthetic organofluorine chemical compounds that have multiple fluorine atoms, which can be polyfluorinated or fluorocarbon-based perfluorinated). A fluorosurfactant has a fluorinated “tail” and a hydrophilic “head”, such as perfluorooctanesulfonic acid and pertluorooctanoic acid. In certain embodiments, the carrier fluid is a mixture comprising Fluorinert® FC-40 oil supplemented with approximately 2% 008-Fluorosurfactant (Ran Biotechnologies, USA). The selection of the appropriate fluorosurfactant is well within the skill of a person of ordinary skill in the art. The surfactant or detergent is added to prevent the cross-contamination between two adjacent droplets containing two different samples (
In certain embodiments, the microfluidic system is assembled by attaching the microfluidic chip with a transparent substrate that is suitable for microscopic observation. The attachment between the microfluidic chip and the transparent substrate can be achieved by any existing methods, such as plasma treatment of the substrate. In certain embodiments, the transparent substrate can be a glass slide, a glass coverslip, or a glass-bottom dish. In certain embodiments, the transparent substrate is a glass coverslip. Any other transparent substrates, e.g. plastic substrates, that are suitable for cell culture, droplet formation, and can be used for microscopic observation are also within the contemplation of the present disclosure.
In certain embodiments, the inlet and outlet of the microfluidic system are connected with tubings that are further connected with a loading chamber. Any other intermediate elements that connect the microfluidic chip and the loading chamber are also within the contemplation of the present disclosure.
Flushing the Droplet-Forming Channel with a Carrier Fluid
In certain embodiments, the microfluidic system is flushed with the carrier fluid to remove any impurities within the newly assembled microfluidic system and to form a layer of oil phase at the wall of the droplet-forming channels. In certain embodiments, the microfluidic system is flushed with the oil phase from the outlet to the inlet. In certain embodiments, the microfluidic system is flushed with the oil phase at 500 μL/h by syringe pump.
Infusing the Droplet-Forming Channel with a Loading Fluid
After the microfluidic system is flushed with the oil phase, the loading fluid is infused to the microfluidic system to form droplets of the samples that have been pre-loaded in the loading chamber. In certain embodiments, the loading fluid is infused from the inlet, in certain embodiments, the infusing speed of the loading fluid is 10-60 μL/h. In certain embodiments, the infusing speed of the loading fluid is 15-55 μL/h, 20-50 μL/h, 25-45 μL/h, 30-40 μL/h, or 35 μL/h. In certain embodiments, the infusing speed of the loading fluid is 25 μL/h. Any other infusing speeds that do not affect the droplet formation and stability are also within the contemplation of the present disclosure.
In certain embodiments, the infusing of a loading fluid can be achieved by an autosampler or a syringe pump.
The inlet and outlet are sealed after the loading fluid is infused into the microfluidic chip, which is then placed in a humidified environment for further incubation.
Imaging and Automatic investigation of Cell Viability
In certain embodiments, the microfluidic system containing droplets of a plurality of samples treated with different drugs at different concentrations is placed under a microscope system for acquisition of images of the each sample under each condition within the droplets. In certain embodiments, one sample under one condition is enclosed in a serial array of consecutive droplets.
In certain embodiments, cell viability is carried out by counting the number of live cells and dead cells within a droplet either by labeling dead cells using a dead cell indicator, e.g. ethidium homodimer 1, Alamar Blue, SYTOX Green nucleic acid stain, or propidium iodide, or by labeling live cells using a metabolic indicator, e.g. Calcein AM, C12-resazurin, SYTO 10 dye, or SYBR 14 nucleic acid stain. In certain embodiments, the investigation of cell viability can be manual or automated as shown in
In certain embodiments, the microfluidic system can be further coupled with RNA sequencing library preparation for single cells for single-cell next generation sequencing (NGS). In certain embodiments, the microfluidic system can be further coupled with fluorescence activated cell sorting (FACS). Other applications that could help elucidate the molecular background of the investigated cancer to be coupled with the microfluidic system are also within the contemplation of the present disclosure.
Below are examples of using the microfluidic system assembled using the droplet-forming microfluidic chip 100 of the present disclosure in a drug screening assay. In certain embodiments, the microfluidic system can be used for monitoring responses of cancer cells or primary cells at different drug concentrations, or to efficiently and accurately determine drug efficacy based on a small sample input size. It should be understood, however, that the description is only for illustrative not limiting purpose. The microfluidic system of the present disclosure can be used for many other purposes.
Cancer Cell Lines and Cell Culture
Jurkat E6.1 cells (ATCC® TIB-152™) and MDA-MB-231 cells (ATCC® HTB-26™) were used as models for suspended and adherent cancer cell lines respectively. Jurkat cell line was derived from human acute T cell leukemia, whereas MDA-MB-231 cell line was derived from human metastatic breast adenocarcinoma.
Jurkat cells were cultured in Advanced RPMI 1640 medium (Life Technologies, USA) supplemented with 5% fetal bovine serum (FBS) (Gemini, USA), 100 U/mL Penicillin-Streptomycin (Life Technologies, USA), 2 mM L-glutamine (Life Technologies, USA), and 10 mM HEPES pH7.4 (Life Technologies, USA).
MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle Medium (Life Technologies, USA) supplemented with 5% FBS, 100 U/mL Penicillin-Streptomycin and 2 mM L-glutamine.
All cells were cultured in humidified incubator at 37° C. supplemented with 5% CO2.
Primary Tumor and Tumor Dissociation
All human studies were conducted with the approval of the Panel on Research Ethics of University of Macau and the Research Ethics Committee of Kiang Wu Hospital, according to the Materials Transfer Agreement between University of Macau and Kiang Wu Hospital. Informed consent for sampling and publication without identifiable information was obtained from all participating patients. All patient sample names were double encoded by the university and the hospital, respectively, to remove any trace of patient identity during sample collection, transfer, processing and analysis. Primary tumors were obtained from surgery conducted at Kiang Wu Hospital immediately after tumor resection. Tumor tissue was dissociated as previously described. Briefly, tumor tissue was first cut into small pieces by a scalpel, then transferred to a 50 mL conical tube containing 5 mL Digestion Buffer I (DMEM/F12 medium containing 5% FBS, 5 μg/mL insulin, 500 ng/mL hydrocortisone, 10 ng/mL epidermal growth factor (EGF), 20 ng/mL cholera toxin, 300 U/mL collagenase III and 100 U/mL hyaluronidase), and digested for no more than 12 h with shaking at 100 rpm in humidified incubator at 37° C. supplemented with 5% CO2. After spinning down at 400 g at ambient temperature for 2 min, the cells were resuspended with 2 mL Digestion Buffer II (DMEM/F12 medium containing 5 mg/mL dispase II and 0.1 mg/mL deoxyribonuclease I), followed by digestion at ambient temperature for 5 min. The cells were then washed with 10 mL HBSS (Life Technologies, USA). 2 mL RBC lysis buffer (eBioscience, USA) was used to lyse red blood cells at ambient temperature for 3 min; this step was repeated until the solution becomes translucent. 12 mL HBSS (Life Technologies, USA) was finally added to stop the lysis. Dissociated cells were extracted by centrifugation of the filtrate through a 40 μm strainer (Falcon, USA). Lastly, the cells were resuspended in StemMACS iPS-Brew XF medium (Miltenyl Biotec, USA) and used for drug screening on chip.
On Chip Drug Screening Assay
All drugs used in this study were listed in Table 1, bortezomib and vorinostat were chosen as target drugs for leukemia, i.e. Jurkat cells, whereas cisplatin and epirubicin were chosen as target drugs for breast cancer, i.e. MDA-MB-231 cells. Another consideration of the chosen drugs was diverse therapeutic targets.
#This is not microfluidic-based assay.
On chip drug screening was performed using the PDMS-based microfluidic chip as described above. A 500 μL glass syringe (Hamilton, USA) and polytetrafluoroethylene (PTFE) tubings with appropriate bore (Cole Parmer, USA) was used to connect between the syringe pump (Harvard Apparatus PHD Ultra Syringe Pump, USA) and the microfluidic chip. Fluorinert® FC-40 (Sigma-Aldrich, USA) supplemented with 2% 008-Fluorosurfactant (Ran Biotechnologies, USA) was used as oil phase; relevant cell culture medium, supplemented with 1% (w/v) methyl cellulose (Sigma-Aldrich, USA) was used as aqueous phase. Cells treated with 0.1% dimethyl sulfoxide (DMSO) were used as negative control.
Briefly, cells at final concentrations of 1-2×106 cells per mL were aliquoted in 0.2 mL PCR tubes, then mixed with corresponding drugs and 2 μM ethidium homodimer 1 (Life Technologies, USA) by manual pipetting. Next, 100-200 nt, cell-drug mixtures were loaded into the tubing, consecutively segregated by oil phase at withdrawal rate of 200 μL/h by syringe pump. After loading all mixtures, the tubing was inserted into the microfluidic chip, which was back-flushed with oil phase at 500 μL/h by syringe pump. After that, the mixtures were infused at 25 μL/h by syringe pump. Finally, the inlet and outlet tubings were cut and sealed with Vaseline (Vaseline, USA). The chips were placed in 150 mm cell culture dish (Coming, USA) containing wet paper towels, and transferred to humidified incubator at 37° C. supplemented with 5% CO2 for 16-24 h incubation. Brightfield and red fluorescence images (Ex. 531/40 nm, Em. 593/40 nm) were taken under 10× magnification (Life Technologies EVOS FL Imaging System, USA).
Microtiter Plate Drug Screening Assay
Microtiter plate drug screening assays were carried out on 96-well clear round flat-bottom plates (Corning, USA) or 384-well white square flat-bottom plates (Corning, USA).
First, 5.0×105 or 1.0×105 cells were seeded per well for 96-well and 384-well plates respectively. Drugs were diluted with Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies, USA), and subsequently added to achieve final drug concentrations as indicated on the graphs. Afterwards, the plates were transferred to humidified incubator at 37° C. supplemented with 5% CO2 for 16-24 h incubation. Finally, Alamar Blue assay was used to measure cell viability. Fluorescence intensity (Ex. 560 nm, Em. 590 nm) using auto-cutoff was measured from bottom on plate reader (Molecular Devices SpectraMax MS Plate Reader, USA). Cells treated with 0.1% dimethyl sulfoxide (DMSO) were used as negative control, while no cells were added to blank control. All experiments were performed in triplicate for 96-well plates and in quadruplicates for 384-well relates.
Image Processing for on Chip Data Analysis
For on chip assays, brightfield and red fluorescence images were initially processed by ImageJ v.1.50i, Cell counting was either performed manually or by Matlab v.R201.5a based on the workflow shown on
Cell Viability Calculation
For on chip assays, the number of cells was counted in brightfield and red fluorescence images from each well respectively. Cell viability was calculated as follows:
where Total cells and Dead cells referred to the total number of cells counted from brightfield and red fluorescence images, respectively.
For normalized cell viability, mean cell viability of all sample wells were normalized to mean cell viability of all negative control wells.
where Sample and DMSO control represented mean cell viability of sample and negative control wells respectively.
For microliter plate assays, average relative fluorescence signal measured by plate reader from 6 reads of each well was used as raw data point. Cell viability was calculated as follows:
where Sample represented raw data points of each sample well, whereas DMSO control and Blank represented average raw data points of all DMSO control and Blank wells, respectively.
Bar graphs and line plots were drawn by GraphPad Prism 5.1. Scatter plots were drawn by R v.3.3.2 using custom scripts. Figures were prepared by assembling images, graphs and plots using Adobe® Illustrator® CS6 v.16.0.0.
Improvement of Microfluidic Chip Design and Validation
In this example, the microfluidic chip is fabricated from PDMS, and contains 2 droplet-forming channels 200, wherein each droplet-forming channel 200 has 6 rows and each row contains 8 droplet-forming units 209, wherein each droplet-forming unit 209 has a droplet-forming well 205, an inflow channel 203, neck channel 204, a restriction channel 206, an outflow channel 207, and a bypass channel 208, and two adjacent droplet-forming units 209 are connected by the outflow channel of the preceding unit and the inflow channel of the succeeding unit (
Within the droplet-forming unit 209, the neck channel 204 is designed to work as a droplet back flow restriction (
The robustness of the droplet formation was tested by checking the preservation of the loaded sample volume in the droplet-forming wells. The loading fluid was loaded with a syringe pump, and a good correlation between the observed volume or observed loaded volume and the preset volume or theoretical loading volume as preset on the syringe pump was observed (
The reliability of the droplet-forming microfluidic chip in droplet formation and separation was also tested using food dyes of different colors. In this experiment, the food dyes were dissolved in water, representing the sample fluid or aqueous phase; the carrier fluid or oil phase comprised Fluorinert® FC-40 oil supplemented with 2% 008-Fluorosurfactant. The sample loading workflow was illustrated in
Taken together, these results proved that the microfluidic chip is applicable for multi-drug conditions screening on a single channel with high flexibility based on sample input size and throughput requirements.
Drug Screening Platform Setup and Optimization
The microfluidic chip validated in Example 1 was used for cell-based drug screening. The following criteria were considered: (1) the drug screening platform should maintain cell viability under investigated conditions; (2) the method is robust and well-controlled; (3) the readout is accurate and reproducible; and (4) the system is versatile for different cell culture systems.
Firstly, we optimized the carrier fluid or oil phase to assure cell viability on chip for a minimum of 7 days for cancer cell lines. Commercial oils including Fluorinert® series oils, e.g. fluorocarbon oils, silicone oil, and mineral oil in combination with different surfactants (either mixed with oil phase or aqueous phase) at various concentrations have been tested. Among these, Fluorinert® FC-40 supplemented with 2% 008-Fluorosurfactant by Ran Biotechnologies provided optimal properties in terms of viscosity, volatility and droplet stability. Fluorosurfactant was added to prevent droplets from coalescence and cross-contamination of droplet contents when they touched each other (
Although droplet shrinkage due to evaporation after overnight incubation might affect drug concentration, it was assumed that all droplets on the same chip had equal evaporation rate, so results should be comparable. This problem would be addressed in future design by continuous perfusion. On the other hand, the Droplet Generation Oil for Probes by Bio-Rad was also good for droplet generation, but its high volatility rendered it suboptimal for long-term cell culture on PDMS-based chips. Other oils and surfactant combinations were not used due to adverse properties: (1) the investigated oils in absence of surfactant provided poor droplet generation efficiency on our chip; (2) mineral oil had high viscosity that hindered its loading on chip; (3) surfactants like Triton X-100 and sodium dodecyl sulfate (SDS) compromised cell viability at all tested concentrations; and (4) the Pluronic® series surfactants did not alter droplet generation efficacy, nor enhanced adherent cells to remain in suspension.
For the aqueous phase or the sample fluid, applying optimal cell culture medium for cell survival is critical. Next comes the consideration of droplet generation. In this study, three culture medium recipes, namely Dulbecco's Modified Eagle Medium (DMEM), Advanced RPMI 1640 and StemMACS iPS-Brew XF medium, have been tested to be feasible for drug screening on chip. Other medium recipes should also be feasible because mammalian cells, in general, require similar ionic strength, which is a major consideration factor for chip performance. Fetal bovine serum (FBS) frequently used in mammalian cell culture possesses emulsification properties that affect droplet formation and stability. In this study, it was found that 5% FBS was optimal, but empirical testing is recommended due to potential variance between products. Alternatively, additives should be added with caution. In our trial experiments, cell culture additives like 1% methyl cellulose, 0.1% Plutonic® F-68, and 8mg/mL Matrigel® did not affect droplet formation (data not shown).
Secondly, optimal cell density on the chip was tested. Optimal cell density was considered based on two reasons: (1) sufficient cell population for statistical analysis of drug susceptibility, and (2) optimization of droplet cell density to avoid overcrowding. Overcrowded droplets led to cell aggregates, resulting in poor cell shredding during image processing and hence collapse of intelligent solution. By loading gradient concentrations of cells on chip, results manifested that there was positive correlation between the average number of cells per well and the concentration of cells before loading on chip (
Thirdly, different cell viability indicator dyes were tested for staining efficiency, indicator reliability and cytotoxicity on chip. Since the sample fluid was premixed before loading to the chip, we tested for different cell viability indicator dyes and variant concentrations to ensure that the indicator dye had no impact on cell viability, at least within the time frame of the drug susceptibility test. Eventually, ethidium homodimer 1 staining was adopted to indicate dead cells. Ethidium homodimer 1 is a cell-impermeable, high affinity nucleic acid stain emitting strong red fluorescence after binding to DNA. It gave strong red fluorescence after cells were treated with drug but not DMSO) (
Lastly, we tried to make our drug screening platform versatile for different kinds of cancers. Because cells were suspended in droplets during drug treatment, adherent cells formed aggregates in the absence of physical support (
Drug Screening of Suspended and Adherent Cancer Cell Lines
Next, we performed drug screening experiment on Jurkat cells and MDA-MB-231 cells, as models for suspended and adherent cells, respectively. We tested these two cell lines against four anti-cancer drugs, namely bortezomib, epirubicin, cisplatin and vorinostat (Table 1). Comparison of the chip screen results of both cell lines with those obtained from conventional plate reader assays using 96-well and 384-well microtiter plates manifested qualitative assessment of drug efficacy regardless of drug screening platform (
In this experiment, ranking of drugs under all three assays using 96-well plate (
Alternatively, MDA-MB-231 cells demonstrated less consistency in drug ranking among assays using 96-well plate (
Close inspection of the dose response curves between different screening platforms prevailed overall smaller amplitude in the chip screen curves as compared to the plate reader assay curves (
In conclusion, our chip assay provided qualitative drug potency assessment in MDA-MB-231 cells. It provided drug ranking assessment of Jurkat cells against the four anti-cancer drugs used in this study, which was comparable to that obtained from 96-well and 384-well plate reader assays.
Drug Screening of Primary Tumor Dissociated Cells from Human Patients
Lastly, we screened seven primary nasopharyngeal tumors from human patients using our microfluidic chip assay. The tumors were collected by surgical resection from human cancer patients. The seven tumors varied in size from 0.2 cm to 0.5 cm in diameter and yielded variable cell numbers for use in this study from 0.5-1.0×105 cells using identical tumor dissociation technique. Given the limited number of cells obtained from human primary tumors, parallel drug screening on 96-well or 384-well plates could be not be attained due to their requirement for higher cell numbers (Table 2). Consequently, all tumor samples were screened on chip against two anti-cancer drugs, namely bortezomib and cisplatin, and DMSO control for up to 5 conditions. Cells were treated for a total of 16-24 h and cell viability was measured by ethidium homodimer 1 staining.
Mock treatment of the seven human primary tumor cells showed diverse cellular response after 16-24 h treatment with DMSO alone (
Next, we compared the chip screen data of all seven human primary nasopharyngeal tumor samples against two anti-cancer drugs, namely bortezomib and cisplatin, respectively (
Drug susceptibility between the primary tumor cell NAS1604 and its derived cell line NAS1604C was compared (
Collectively, these data reflected that: (1) primary tumor cells had diverse susceptibility towards different drugs, thus supporting the need for personalized cancer therapy; and (2) primary tumor cells before and after in vitro amplification (primary tumor cells are re-named as primary tumor cell lines after in vitro amplification in the present disclosure) might prevail similar drug susceptibility, while their morphological cell type might be different.
In order to exemplify the capability of our microfluidic chip, one nasopharyngeal tumor sample was shown in details as an example (
Further investigation into drug response among individual droplets showed stark contrast before and after overnight incubation (
In conclusion, our data demonstrated that mean cell viability could be used to reveal the percentage of cells that responded to the investigated drug(s), applied to qualitative drug potency assessment and drug ranking. On the other hand, the range of droplet cell viability suggested the conformity of cellular drug response towards the investigated drug(s).
Discussion
In this study, we used a centimeter-sized PDMS-based droplet microfluidic chip to provide efficient evaluation of drug susceptibility of cancers. Our data indicated that our system could be used to screen as few as 16,000 cells obtained from primary cancer for each treatment condition within 2.4 h after tumor resection from cancer patients. Rapid screening for effective therapy is virtuous, especially for fast-growing cancers from the pancreas (20.8%), lung (32.1%), brain (40.1%) and oesophagus (41.9%), which kills patients within one year after diagnosis. Our current assay provided evidence for rapid drug potency assessment within 2.4 h. This would allow clinical doctors to determine their patient's therapeutic regime within 2 days. Furthermore, the cost of our chip was merely HKD0.20 per chip (Table 2), making it pragmatically affordable for all cancer patients. Hence, our technology provided unprecedented opportunity for rapid evidence-based decision making for personalized cancer therapy.
Our microfluidic chip was designed so that the loading of samples can be performed by injecting a sequence of samples segregated by a carrier fluid, i.e. oil phase, through an inlet of the droplet-forming channel, which allows high throughput screening that otherwise cannot be realized by existing microfluidic devices using pipette loading methods. More importantly, cell lines were prone to survive and proliferate on chip, whereas primary tumor cells died quickly under mock treatment after 16-2.4 h treatment (
Ethidium homodimer 1 staining was used to measure single cell viability in this assay. Our data supported for observation of differential drug response in cancer cell lines and primary tumor cells from human cancer patients. On the population scale, mean cell viability obtained on chip could be applied to assessing drug potency and ranking drugs in a drug panel. Additionally, the conformity of drug response could be implied from the range of droplet cell viability.
In conclusion, our microfluidic chip assay provides a powerful tool for rapid, low-input drug screening of primary cancers. Adaptation of the assay to suspended and adherent cancer cell lines suggests of its application in potentially all types of cancers. It provides us the opportunity to observe and quantify cellular drug response on the single cell level, whereas population analysis is achievable by statistical analysis of multiple droplets.
Although the invention has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by the claims which follow.
The present application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 62/645,816, entitled DRUG SCREENING OF CANCER CELL LINES AND HUMAN PRIMARY TUMORS USING DROPLET MICROFLUIDICS, which was filed on Mar. 21, 2018, and is hereby incorporated by reference in its entity.
| Number | Date | Country | |
|---|---|---|---|
| 62645816 | Mar 2018 | US |
