Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/16—Microfluidic devices; Capillary tubes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0681—Filter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/08—Regulating or influencing the flow resistance
  • B01L2400/084—Passive control of flow resistance
  • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles

Definitions

• the technical field generally relates to microfluidic devices that are used to digest tissue specimens or tissue samples into cellular suspensions.
• Biogrid device that was designed to mechanically cut neurospheres using sharp silicon knife- edges placed across the device cross-section. See Wallman et al., Biogrid - a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates, Lab Chip, 11(19), pp. 3241-8 (2011). While more effective, mechanical cutting in this fashion was harsh and only resulted in smaller aggregates, not single cells.
• a microfluidic device was disclosed that employed a network of branching channels to achieve highly efficient and rapid dissociation of cancer cell aggregates into viable single cells. See Qui et al., Microfluidic device for mechanical dissociation of cancer cell aggregates into single cells, Lab on a Chip, 15.1, 339-350 (2015).
• the inlet could not accommodate samples that were greater than 1 mm in size, requiring off-chip mincing and digestion of larger tissue specimens.
• full scale tissues have been employed in a single microfluidic application, namely, the culture and enzymatic digestion of rat liver biopsies, this device has a number of limitations. See Hattersley et al., Development of a microfluidic device for the maintenance and interrogation of viable tissue biopsies, Lab Chip, 8(11), pp. 1842-6 (2008). For example, this device just provided a means to incubate tissues with enzymes, and suffered from extremely low cell yields, even after prolonged digestion times. Summary
• a method of processing tissue using the microfluidic device includes placing the tissue within the sample chamber and then flowing a fluid containing a digestive enzyme into the inlet.
• the tissue that may be processed using the microfluidic device may include healthy or diseased tissue.
• the tissue that is processed by the device includes tumor tissue, although other tissue types are contemplated. Tissue obtained from different organs may also be treated. Examples include liver tissue, kidney tissue, pancreas tissue, spleen tissue, skin tissue, heart tissue, and the like.
• the fluid may be pumped into the microfluidic device using a pump.
• the cells or smaller aggregates of tissue may be collected from the outlet of the microfluidic device.
• the collected output from the microfluidic device is recirculated back into the input of the microfluidic device.
• the tissue is loaded into the sample chamber by using a sample port.
• the sample is loaded by inserting a needle into the sample port and depositing the tissue in the sample chamber.
• a plug, cap, or lid covers the sample chamber and can be removed/secured to the microfluidic device.
• a method of processing tissue in a microfluidic device that is formed in a substrate or chip having formed therein an inlet, an outlet, and a sample chamber dimensioned to hold the tissue sample, the sample chamber fluidically coupled at one side to a plurality of upstream hydro-mincing microfluidic channels disposed in the substrate or chip and further fluidically coupled to the inlet and coupled at another side of the sample chamber to a plurality of downstream sieve microfluidic channels disposed in the substrate or chip and further fluidically coupled to the outlet.
• the method includes placing the tissue within the sample chamber and flowing a fluid containing a digestive enzyme into the inlet.
• FIG. 3 illustrates a photographic image of a laser-etched acrylic sheet containing the chamber for loading tissue samples and fluidic “mincing” channels including upstream (left) for hydro-mincing and downstream (right) sieves (sieve gates).
• FIG. 4 illustrates an exploded view of a microfluidic device according to one embodiment that includes a polymer or elastic gasket layer sandwiched between two acrylic sheets or other hard plastic sheets. Hose barbs are illustrated in the top layer and nylon screws are used to hold the device together.
• FIG. 5 illustrates a photograph of the fully assembled device illustrated in FIG. 4.
• FIG. 6 schematically illustrates the experimental set-up used for digestion experiments using the microfluidic device for the processing and digestion of tissue. Flow was driven by a peristaltic pump and tissue digestion was visually monitored with a camera mounted above the device.
• FIG. 8A illustrates an exploded view of another embodiment of a microfluidic device for the processing and digestion of tissue according to one embodiment.
• FIG. 8B illustrates a top or plan view of the microfluidic device of FIG. 8 A.
• FIG. 9A illustrates an exploded view of another embodiment of a microfluidic device for the processing and digestion of tissue according to one embodiment.
• FIG. 9B illustrates a top or plan view of the microfluidic device of FIG. 9A.
• FIG. 11 illustrates an exploded view of another embodiment of a microfluidic device for the processing and digestion of tissue according to one embodiment.
• FIG. 12 illustrates an exploded view of another embodiment of a microfluidic device for the processing and digestion of tissue according to one embodiment.
• FIG. 13A is a photographic image of a tissue core obtained using a Tru-CutTM biopsy needle and placed inside the tissue chamber.
• FIG. 13B illustrates time-lapse images of tissue digestion for devices with 3, 5, and 7 hydro-mincing microfluidic channels.
• the fluid contained collagenase, and was pumped through the device at 20 mL/min.
• FIG. 13C illustrates a graph showing tissue loss as a function of time. Tissue loss was quantified from images based on mean gray value and overall tissue area. Trends were similar for each design, but variability was lowest for 3 hydro-mincing microfluidic channels.
• FIG. 13D illustrates micrograph images of device effluents after 30 min operation. Top is seven (7) hydro-mincing microfluidic channels. Middle is five (5) hydro-mincing microfluidic channels. Bottom is three (3) is hydro-mincing microfluidic channels. Scale bar is 100 pm. Error bars represent standard errors from at least three independent experiments.
• FIG. 14 illustrates an image processing algorithm used to monitor tissue digestion.
• FIG. 15A illustrates photographic images of mouse liver (top right) and kidneys (bottom right) were freshly harvested and cut into 1 cm long x 1 mm diameter pieces and placed within the device sample chamber.
• FIG. 15B illustrate time-lapsed images of tissue digestion for devices containing 3 hydro-mincing microfluidic channels. Tissue size and density both decreased over time as digestion progressed.
• FIG. 15C illustrates a graph of tissue loss quantified from images based on mean gray value and overall tissue area, with liver and kidney samples demonstrating similar trends.
• FIG. 15D illustrates a graph illustrating the results of the Cy QUANT® assay was used to directly quantify cell suspensions obtained by digestion only, scalpel mincing and digestion, or device treatment lasting for a total of 15, 30, or 60 min.
• the CyQUANT® signal increased with treatment time, and was higher overall for kidney samples. Signals from device treated samples were consistently higher than minced controls, similar to gDNA and cell counting results presented in FIG. 3 of the main text. Error bars represent standard errors from at least three independent experiments. * indicates p ⁇ 0.05 relative to minced control at the same digestion time.
• FIG. 16A illustrates a graph showing the amount of genomic DNA (gDNA) that was extracted and quantified from kidney and liver tissue cell suspensions obtained by digestion only, scalpel mincing and digestion, or device treatment lasting for a total of 15, 30, or 60 min.
• gDNA genomic DNA
• FIG. 16A illustrates a graph showing the amount of genomic DNA (gDNA) that was extracted and quantified from kidney and liver tissue cell suspensions obtained by digestion only, scalpel mincing and digestion, or device treatment lasting for a total of 15, 30, or 60 min.
• gDNA genomic DNA
• FIG. 16A illustrates a graph showing the amount of genomic DNA (gDNA) that was extracted and quantified from kidney and liver tissue cell suspensions obtained by digestion only, scalpel mincing and digestion, or device treatment lasting for a total of 15, 30, or 60 min.
• gDNA increased with treatment time, and overall was higher for kidney samples.
• Device treatment consistently provided more gDNA than minced controls at the same time point. In most cases, gDNA was also higher than the next digestion time
• FIG. 16B illustrates a graph showing cell counter results, showing that single cell numbers largely matched gDNA findings but with higher variability. Also, liver values were now similarly comparable to kidney, suggesting that kidney suspensions may have contained more aggregates. Error bars represent standard errors from at least three independent experiments. * indicates p ⁇ 0.05 relative to minced control at the same digestion time.
• FIG. 16C illustrates micrographs of minced controls and device effluents after lysing red blood cells. Note the large number of aggregates in the controls, particularly at 60 min. Scale bar is 100 pm.
• FIG. 17 illustrates FACS gating data for cell suspensions obtained from digested mouse and liver and kidney samples.
• Cell suspensions obtained from digested mouse liver and kidney samples were stained with the four-probe panel listed in Table 1 and analyzed using flow cytometry. Controls were treated only with an isotype matched (IgG2b), PE- conjugated antibody. Acquired data was assessed using a sequential gating scheme.
• an FSC-A vs. SSC-A gate (Gate 1) was used to exclude debris near the origin.
• Gate 2 was based on FSC-A vs. FSC-H, and was used to select single cells.
• Gate 3 distinguished CD45+ leukocytes based on CD45-PE signals in FL2-A vs.
• the CD45- cell subset was further divided into anucleate RBCs and nucleated tissue cell subsets based on signals from the Draq5 nuclear stain in FL4-A vs. SSC-H plots.
• the cellularity of nucleated tissue cells of interest was validated based on the signal of the cell membrane dye CellMaskTM Green in FLI-A vs. FSC-H plots.
• live and dead tissue cells were discriminated based on 7AAD signals in FL3-A vs. SSC-H plots. All gates were established using the minced control that was digested for 60 min. Heat treated cells were used as a positive control to confirm appropriate 7AAD signals for dead cells.
• FIG. 18A illustrates flow cytometry results of mouse kidney suspensions. Flow cytometry was used to identify and quantify the number of leukocytes, red blood cells, and single tissue cells in the suspensions obtained from minced controls or device treatment. Relative numbers of each cell type are shown. Red blood cells comprised the highest percentage of almost all populations, and there was no statistically significant change in population compositions across all minced control and device conditions.
• FIG. 18B illustrates flow cytometry results of mouse liver suspensions. Flow cytometry was used to identify and quantify the number of leukocytes, red blood cells, and single tissue cells in the suspensions obtained from minced controls or device treatment. Relative numbers of each cell type are shown. Red blood cells comprised the highest percentage of almost all populations, and there was no statistically significant change in population compositions across all minced control and device conditions.
• FIG. 18C illustrates a graph of total and live tissue cell numbers per mg of tissue for kidney samples.
• FIG. 18D illustrates a graph of total and live tissue cell numbers per mg of tissue for liver samples.
• tissue cell recovery increased with digestion time for minced controls, but did not change significantly with device processing beyond 10 min.
• all device conditions yielded more cells than minced controls that were digested for up to 30 min. Viability remained >80% for all but the longest time points, which reached as low as 70%.
• the x-axis for FIGS. 18A and 18B is the same as FIGS. 18C and 18D.
• Error bars represent standard errors from at least three independent experiments. * indicates p ⁇ 0.05 relative to minced control at the same digestion time. # indicates p ⁇ 0.05 compared to minced control digested for 15 min.
• FIG. 19 illustrates a graph illustrating mouse kidney and liver cell viability data. Cell viability was similar for device treated conditions relative to minced counterparts, demonstrating minimal effect of device treatment. Error bars represent standard errors from at least three independent experiments.
• FIG. 20 shows an embodiment of the microfluidic device of the present invention having an equal number of upstream hydro-mincing microfluidic channels and downstream sieve microfluidic channels, causing the device to be symmetrical.
• FIG. 21 A shows a photograph of a plurality of microfluidic devices coupled in parallel at the sample chamber in order to process larger sample sizes.
• FIG. 21B shows a schematic of a plurality of microfluidic devices coupled in parallel at the sample chamber in order to process larger sample sizes.
• FIG. 22 shows a schematic of an embodiment of the present invention wherein the outlet is fluidly connected to a junction for directing pieces of digested tissue sample through an exit tube out of the system or recirculated back into the inlet of the device in order to further process the tissue sample.
• a digestive enzyme source is additionally fluidly connected to a digestive enzyme source for providing additional digestive enzyme to be introduced to the inlet of the microfluidic device to replace digested tissue samples that exit the system through the outlet.
• FIG. 1 illustrates microfluidic device 10 for the processing and digestion of tissue according to one embodiment.
• the microfluidic device 10 is designed to process tissue obtained from a mammalian subject (e.g., human).
• the microfluidic device 10 has particular applicability for the processing and digestion of tumor tissues, although other tissue types may be processed using the microfluidic device 10.
• the microfluidic device 10 is formed in a substrate or chip structure 12 which as explained herein may be formed using multiple layers that are assembled together to form the microfluidic device 10.
• the microfluidic device 10 includes three primary features that are defined or formed in the substrate or chip structure 12.
• sample chamber 14 that holds a sample 16 in place while fluid containing proteolytic enzymes or other digestive agents is passed into the sample chamber 14 and onto the surface of the sample 16.
• the sample chamber 14 thus maintains the sample 16 in a generally fixed location in the substrate or chip structure 12. By having the sample 16 be retained in the sample chamber 14, this promotes sample mixing, enhances enzymatic activity, and as explained below applies hydrodynamic shear forces to mechanically dislodge cells and aggregates from the larger sample 16.
• the present invention features a system for the processing of a sample 16 into cellular suspensions.
• the system may include a tissue sample 16 having a size within the range of 1 mm 3 to 50 mm 3 .
• the system may further comprise a microfluidic device 10.
• the microfluidic device 10 is formed as a substrate or chip 12.
• the substrate or chip 12 may have an inlet 22, an outlet 28, and a sample chamber 14 dimensioned to hold the tissue sample 16 formed therein.
• the sample chamber 14 may be fluidically coupled at a first side to a plurality of upstream hydro-mincing microfluidic channels 18 disposed in the substrate or chip 12.
• the upstream hydro-mincing microfluidic channels 18 may be further fluidically coupled to the inlet 22.
• the length of the sample chamber 14 may be less than 50 cm, and the height of the sample chamber 14 may be less than 5 cm.
• the number of upstream hydro-mincing microfluidic channels 18 may be equal to the number of downstream sieve microfluidic channels 24 (see FIG. 20). The number of upstream hydro mincing microfluidic channels 18 and downstream sieve microfluidic channels 24 and the width of the sample chamber 14 may depend on the size of the tissue sample 16 being processed. Tissue samples 16 with a smaller width may be processed more effectively than tissue samples 16 with a larger width.
• a first instance of the microfluidic device 10 may be coupled to at most two additional instances of the microfluidic device 10.
• the coupling may occur at a third side of the sample chamber 14 (e.g., a side), a fourth side of the sample chamber 14 (e.g., another side), or a combination thereof depending on how many instances of the microfluidic device 10 are coupled to the first instance of the microfluidic device 10.
• These different instances may be contained in the same substrate or chip 12 as seen in FIGS. 21 A and 12B.
• FIGS. 21A-21B show embodiments of the aforementioned parallel coupling of microfluidic devices 10.
• tissue sample 16 is placed in the sample chamber 14 of each instance of the microfluidic device.
• the type of tissue sample 16 processed by the present invention may be selected from a group comprising kidney tissue, liver tissue, heart tissue, lung tissue, breast tumor tissue, spleen tissue, and pancreas tissue.
• the microfluidic device 10 in one embodiment, was designed to process samples obtained from core needle biopsies, directly into cell suspensions without the need for manual processing steps such as scalpel mincing. However, in other embodiments, the microfluidic device 10 processes a larger tissue sample after the sample has been subject to some mechanical processing (e.g., scalpel mincing).
• the particular size of the sample chamber 14 may vary depending on the size of the sample 16. Typically, the width of the sample chamber 14 may be within the range of about 0.5 mm and about 10 mm, the length of the sample chamber 14 is less than 2 cm, and the height of the sample chamber 14 is less than 1 cm. For example, with reference to FIG.
• the sample chamber 14 has a width 2 mm or less, the length of the sample chamber 14 is less than 2 cm, and the height of the sample chamber 14 is less than 2 mm (height dimension is perpendicular to the plane of the page). Fluid flows in the direction of arrows A along the width of the sample chamber 14.
• the width of the sample chamber 14 is around 1.5 mm or less, the length of the sample chamber 14 is around 1 cm or less (e.g., approximately the size of a Tru- Cut® core biopsy needle; about 1 cm long x 1 mm diameter tissue), and the height of the chamber is less than 2 mm (e.g., around 1 mm).
• the sample chamber 14 may be much smaller, for example, and can accommodate samples 16 having a longest dimension of around 1 mm. For example, when the sample 16 has been subject to some mechanical processing (e.g., mincing) the sample chamber 14 may have a much smaller size.
• the height of the sample chamber 14 may vary but is typically less than 1 cm and more typically less than 2 mm (e.g., a height of 1 mm may be used as described herein).
• the sample chamber 14 is dimensioned to receive a sample 16 directly obtained from a tissue biopsy device such as the Tru-Cut® core biopsy needle or similar devices.
• the second feature of the microfluidic device 10 includes a plurality of hydro mincing microfluidic channels 18 located upstream of the sample chamber 14 which focus fluid into high velocity jets that are directed into the sample 16 retained in the sample chamber 14.
• the hydro-mincing microfluidic channels 18 as seen in FIG. 1 are fluidically coupled with an inlet channel 20 that receives fluid from an inlet 22. Fluid thus moves through the microfluidic device 10 in the direction of arrows A.
• the hydro-mincing microfluidic channels 18 produce fluid jets that concentrate hydrodynamic shear forces at discrete locations on the sample 16, breaking the sample 16 down mechanically and delivering proteolytic enzymes deep inside the sample 16 (i.e., tissue).
• hydro-mincing microfluidic channels 18 This is analogous to manually mincing the tissue with a scalpel, and hence these are referred to as hydro-mincing microfluidic channels 18.
• the width of the hydro-mincing microfluidic channels 18 may vary but is generally within the range of about 50 pm to about 1 mm.
• a width of the hydro-mincing microfluidic channels 18 within the range of about 100 pm to about 200 pm may be typical, although other dimensions outside this specific range may be used.
• a plurality of downstream sieve microfluidic channels 24 are located downstream of the sample chamber 14 to act as a sieve that selectively retains larger pieces of tissue and cellular aggregates for further digestion.
• the downstream sieve microfluidic channels 24 form sieve gates that retain the larger sized tissue portions and cellular aggregates to prevent them from passing further downstream. Smaller aggregates and single cells are, however, allowed to pass out of the device 10 for collection or potentially for further microfluidic processing.
• the cells that leave the device 10 may be subject to downstream cell sorting and/or analysis to create point-of-care platforms for cell- based diagnostics and therapies.
• the downstream sieve microfluidic channels 24 are spaced evenly along the side or end of the sample chamber 14 to firmly secure the sample 16 in place in the sample chamber 14 and minimize backpressure.
• the width of the downstream sieve microfluidic channels 24 may vary but may be within the range of about 10 pm and 1 mm. More typically, the downstream sieve microfluidic channels 24 have a width within the range of about 100 pm to about 1 mm. For example, in some embodiments a width within the range of 500 pm to 1mm is useful.
• a channel width of 500 pm for the downstream sieve microfluidic channels 24 was used and the device could comfortably accommodate seven (7) such channels across the width of the sample chamber 16.
• the plurality of downstream sieve microfluidic channels 24 lead to a common outlet channel 26 that extends to an outlet 28 where fluid can leave the microfluidic device 10.
• the width of the downstream sieve microfluidic channels 24 may be larger than the width of the hydro-mincing microfluidic channels 18. In other embodiments, the width of the downstream sieve microfluidic channels 24 may be smaller than the width of the hydro-mincing microfluidic channels 18. In still other embodiments, the width of the downstream sieve microfluidic channels 24 may be substantially the same as the width of the hydro-mincing microfluidic channels 18.
• FIG. 2 illustrates one such embodiment where the output from a first device (i.e., Device# 1; microfluidic device 10) is then input into a second downstream device 100 (Device #2) for additional tissue dissociation.
• a first device i.e., Device# 1; microfluidic device 10.
• the microfluidic device 10 is coupled to another tissue dissociation device 100 like that illustrated in the ‘678 patent.
• tissue dissociation device 100 a series of stages of microfluidic channels with decreasing dimensions and having a series of expansion and constriction regions (illustrated in FIG. 2) are used to impart shearing forces on cell clusters and aggregates to dissociate tissue.
• the microfluidic device 10 can be coupled to such as device 100 as is illustrated in FIG. 2.
• the output of the first microfluidic device 10 may be coupled to the input of the second microfluidic device 100 that is used for further tissue dissociation.
• a valve 30 may be located between the two devices (10, 100) to allow selective flow through the second tissue dissociation device 100.
• a pump 32 is illustrated that is used to recirculate flow between one or both of the microfluidic devices 10, 100. It should be understood, however, that rather than recirculating flow as illustrated, flow may pass directly through the devices without any recirculation.
• the outlet 28 of the microfluidic device 10 may be fluidly connected to a junction.
• the junction may be fluidly connected to both an exit tube 31 and a recirculation tube 33.
• the exit tube 31 may be configured such that the tissue sample 16 may be directed by the pump 32 through the exit tube 31 to a collection chamber.
• the recirculation tube 33 may be fluidly connected to the inlet 22 of the microfluidic device 10 and configured such that the digestive enzyme fluid directed through the microfluidic device 10 may be directed by the pump 32 through the recirculation tube 33 to the inlet 22.
• the recirculation tube 33 may additionally be fluidly connected to a digestive enzyme source for providing additional digestive enzyme to be introduced into the microfluidic device 10 (see FIG. 22). This process is used to further process the tissue sample 16.
• FIG. 3 illustrates a photograph of the microfluidic device 10 formed in hard acrylic sheets showing three (3) hydro-mincing microfluidic channels 18 and seven (7) downstream sieve microfluidic channels 24.
• materials for the microfluidic device 10 include polyethylene terephthalate (PET).
• the microfluidic device 10 an upper substrate layer 12a formed from PET that includes the barbed ends or tubing connections 34 (e.g., hose barbs) that are fluidically coupled to the inlet 22 and outlet 28.
• the microfluidic device 10 further includes a lower substrate 12b that has the microfluidic features formed therein. This includes the inlet 22, inlet channel 20, sample chamber 14, downstream sieve microfluidic channels 24, outlet channel 26, and outlet 28 of FIG. 1.
• a polydimethylsiloxane (PDMS) gasket 12c having holes or vias 35 (for fluid access) is sandwiched between the upper substrate 12a and the lower substrate 12b which collectively together form the substrate/chip 12.
• Other elastic or polymers may be used for the gasket 12c.
• fasteners 36 e.g., screws
• FIG. 5 A block diagram illustrating an exemplary computing environment in this particular embodiment.
• a peristaltic pump 32 was used to recirculate fluid through the device to conserve proteolytic enzyme solution.
• the flow may be continuous or the cells removed prior to recirculation in the device.
• a camera 38 was mounted above the microfluidic device 10 to monitor the progress of tissue digestion.
• FIGS. 8 A, 8B, 9 A, and 9B illustrate two alternative embodiments of a microfluidic device 10 that includes a feature to aid in loading the tissue or other sample into the sample chamber 16.
• the microfluidic device 10 includes a plurality of layers 12a, 12b, 12c that may be pressure laminated together with the aid of an adhesive or glue applied to the interface between adjacent layers to form the microfluidic device 10.
• the layers 12a, 12b, 12c may be pressure laminated together with the aid of an adhesive or glue applied to the interface between adjacent layers to form the microfluidic device 10.
• FIGS. 8A and 8B an open window 40 is provided on top layer 12a of the microfluidic device 10 through which sample 16 can be loaded using forceps.
• a plug 42 that is formed from silicon rubber material or the like is then placed in the window and secured with adhesive tape or glue.
• the middle layer 12c includes a hole or via 44 formed therein so that the sample 16 can be loaded into the sample chamber 14 formed in the first substrate 12a.
• the microfluidic device 10 has a sample port 46 located in the side of the microfluidic device 10 that can be used to load a sample 16 into the sample chamber 14.
• This design enables access through the side of the microfluidic device 10 by penetrating a silicon rubber septum 48 with a needle 50 as seen in FIG. 9B.
• this needle 50 would be the same one used to extract the sample 16 (e.g., tissue), such as a core needle or punch biopsy. After penetrating the septum 48, the needle 50 would seat the sample 16 in the sample chamber 14 and remain in place (penetrating the septum 48) to seal the microfluidic device 10.
• the septum 48 may be a self-sealing septum 48 such that the needle 50 can be removed from the microfluidic device 10 without leakage of fluid or other contents outside of the microfluidic device 10.
• FIG. 10 illustrates one alternative embodiment of a microfluidic device 10 where one or more of the hydro-mincing microfluidic channels 18 can be selectively turned on or off by way of individual valves 54.
• the direction of fluid flow is indicated by arrow A in FIG. 10.
• the option is provided to mince a large area of the sample 16 by sequentially using a small number of hydro-mincing microfluidic channels 18.
• one or few of the hydro-mincing microfluidic channels 18 may be opened at any particular time so that high jetting or shearing forces are imparted on the sample 16.
• the valve(s) 54 may then be closed and another valve 54 or set of valves 54 that are aimed at a different region of sample 16 can then be turned on.
• the valves 54 may include microfluidic valves 54 that are known in the art.
• microfluidic valves 54 that use a deformable membrane to actuate flow in a channel are known and may be used as one example. This process may continue for any number of cycles to mince the entire sample 16.
• FIG. 11 illustrates another embodiment of the microfluidic device 10.
• This microfluidic device 10 is formed from a multi-layer construction using hard acrylic or PET as described herein.
• the microfluidic device 10 is formed from multiple layers 60a, 60b, 60c, 60d, 60e, and 60f.
• the layers 60a, 60b, 60c, 60d, 60e, and 60f are pressure laminated together with the aid of an adhesive or glue (e.g., silicone or acrylic-based glues or adhesives) applied to the interface between adjacent layers to form the microfluidic device 10.
• Layer 60a serves as a base or bottom layer.
• Layer 60b has formed therein the inlet channels 20, outlet channels 26, as well as the hydro-mincing microfluidic channels 18 and downstream sieve microfluidic channels 24.
• Layer 60c has formed therein vias 62 that communicate with the inlet channels 20 and outlet channels 26 in layer 60b as well as apertures or holes 64 that provide access for the inlet 22 and the outlet 28.
• Layer 60d includes the sample chamber 14 formed therein that communicates with the vias 62 in layer 60c.
• Layer 60d further includes apertures or holes 64.
• Layer 60e includes in addition to the apertures or holes 64 a via 66 that provides access to the sample chamber 14. This via 66 may have a diameter of around 1 mm which is sized to accommodate sample 16 that has been mechanically processed (e.g., minced).
• Layer 60f is the top layer and includes barbed ends 34 that provide fluid access in/out of the microfluidic device 10.
• the layer 60f includes a loading port 68 that communicates with the via 66 and into the sample chamber 14.
• a cap (not shown) may be placed over the loading port 68 after loading so that fluid and tissue/cells remain inside the microfluidic device 10.
• the loading port 68 may be configured as a Luer end that interfaces with a syringe or the like for loading.
• minced sample 16 e.g., minced tissue
• the downstream sieve microfluidic channels 24 that communicate with the outlet channels 26 may include a filtering capability that restrict the passage of larger pieces of sample 16 from flowing downstream in the device 10. Filtering may also be provided by the vias 62.
• FIG. 12 illustrates another embodiment of the microfluidic device 10.
• This microfluidic device 10 is formed from a multi-layer construction using hard acrylic or PET as described herein.
• the microfluidic device 10 is formed from multiple layers 70a, 70b, 70c, 70d and includes a cap or lid 72 that, as explained herein, is used to close the device after the sample 16 has been loaded into the microfluidic device 10.
• the layers 70a, 70b, 70c, 70d are pressure laminated together with the aid of an adhesive or glue applied to the interface between adjacent layers to form the microfluidic device 10.
• Layer 70a serves as a base or bottom layer.
• Layer 70b has formed therein the inlet channels 20 and outlet channels 26.
• Layer 70c has formed therein vias 74 that communicate with the inlet channels 20 and outlet channels 26 in layer 70b as well as apertures or holes 76 that provide access for the inlet 22 and the outlet 28.
• Layer 70d includes the sample chamber 14 formed therein that communicates with the vias 74 in layer 70c.
• Layer 70d also includes apertures or holes 78 that can accommodate barbed ends 34 (not illustrated in FIG. 12) such as those illustrated in FIGS. 4, 5, 8A, 8B, 9A, 9B, and 11.
• the sample 16 can be loaded directly into the sample chamber 14.
• the cap or lid 72 is then affixed to the layer 70d above the sample chamber 14 to seal the sample chamber 14 from the external environment of the microfluidic device 10.
• the cap or lid 72 may be secured to the layer 70d using an adhesive or the like.
• the cap or like 72 may be removable so that the microfluidic device 10 can be used multiple times.
• the cap or lid 72 is secured to the layer 70d in a permanent manner.
• the cap or lid 72 may be secured to the layer 70d using one or more fasteners (not shown) such as clamps, screws, bands, clips, or the like.
• tissue were cut into -1 cm x 1 mm x 1 mm pieces with a scalpel (see FIG. 15 A) and weighed. Digestion device experiments were then conducted as described for beef liver, with collagenase recirculated for either 15 or 30 min before sample collection. Images were again taken every 5 min and processed to monitor tissue loss, which was similar to beef liver (see FIGS. 15B and 15C). Controls were further minced with a scalpel into ⁇ 1 mm 3 pieces before digesting with collagenase for 15, 30, or 60 min in a conical tube. These samples were constantly agitated, and vortexed every 5 min. A separate control was included in which the tissue was not minced, only digested for 30 min.
• gDNA total genomic DNA extracted using a QIAamp® DNA kit.
• gDNA progressively increased with digestion time (FIG. 16A).
• Kidney samples yielded approximately 100 ng gDNA per mg of tissue after 60 min digestion, while liver was less than half this value. Slightly less gDNA was obtained from the un-minced controls, but differences were not significant.
• Device treatment yielded dramatically more gDNA than controls when compared at the same digestion time.
• This panel enabled distinction of tissue cells from non-cellular debris, anucleated red blood cells, and leukocytes, while simultaneously assessing viability.
• Stained cell suspensions were analyzed with a BD AccuriTM Flow Cytometer to obtain the number of each cell type using the gating protocol described in the methods section and shown in FIG. 17. Comparing the relative numbers for each cell type (FIG. 18A and 18B), red blood cells constituted the majority of all but the minced control that was digested for 15 min. Unexpectedly, red blood cell percentage increased slightly as the tissue was digested more thoroughly, although this effect was not significant. Leukocyte percentage remained stable, decreasing slightly with digestion time.
• Tissue cell counts which are expected to predominantly be epithelial, were quantified for kidney and liver samples and are presented in FIG. 18C and 18D; respectively.
• Tissue cell numbers were 2 to 5 times higher for kidney than liver for the minced controls, which both increased with digestion time. The increases were more than an order of magnitude between 15 to 30 min, and 5-fold between 30 to 60 min.
• With device treatment there was little change between 10 and 15 min time points, although 10 min was associated with high variability for kidney samples. Extending processing time to 30 min increased cell number by only -50% for both tissue types, although differences were not significant.
• device treatment again provided superior results at the same digestion time point. For kidney, cell number differences were 30-fold at 15 min and 4-fold at 30 min.
• Viability was approximately 80% for all kidney samples except the minced control that was digested for 60 min and 30 min device cases, which both dropped to 70% (see FIG. 19).
• viability was approximately 90% for the minced controls, 80% for 10 and 15 min device treatments, and 70% for 30 min device treatment.
• the number of live tissue cells obtained from each condition is also presented in FIGS. 18C and 18D.
• 30 min device treatment produced approximately the same number of live single tissue cells as the minced control that was digested for 60 min.
• the 10 and 15 min device treatments produced around half of this value, but in a fraction of the time.
• the number of live, single tissue cells did not increase with device treatment beyond 10 min.
• microfluidic digestion device performed better for kidney samples despite the fact that this tissue type is generally considered to be more difficult to dissociate. This is likely due to the combination of kidney cells being more robust and the denser kidney tissue requiring higher shear forces to be dissociated.
• a microfluidic device 10 is disclosed that is used to extract or isolate single cells from cm x mm-scale tissues using the combination of hydrodynamic shear forces and proteolytic digestion.
• improvements in recovery of DNA and single tissue cells were consistently observed relative to standard methods that require mincing with a scalpel.
• Device performance at short processing times was particularly exciting, as a 10 min treatment yielded results that were within 50% of scalpel mincing and digesting for 1 hour, but with improved viability. Recovery improvements were most striking for DNA, suggesting that the current device design may have left a significant number of cells within small aggregates or clusters.
• Improvements in device function and operation may be found in improving hydro mincing such as decreasing channel dimensions, increasing flow rate, and installing valves 54 such as illustrated in FIG. 10 to direct flow to different regions of the tissue. These approaches could also improve aggregate dissociation of tissue.
• the digestion device may be paired with another dissociation device 100 such as that illustrated in FIG. 2 such as the branching channel array with hydrodynamic micro-scalpels.
• the microfluidic device 10 may be used with other tissues such as solid tumors from various cancer types for diagnostic purposes and other healthy tissues such as skin, heart, and fat for use in tissue engineering and regenerative medicine. That is to say, various tissue types may be used in connection with the microfluidic device 10. This includes diseased tissue such as cancerous tissue or it may include healthy tissue. Moreover, the tissues or samples may be obtained from a number of different organs or tissue types.
• Tissue models Beef liver was purchased from a local butcher and tissue cores were extracted by using a Tru-CutTM biopsy needle (CareFusion, Vernon Hills, IL) in a manner analogous to obtaining a clinical biopsy. Briefly, the obturator was retracted to cover the specimen notch and the cannula handle was held firmly while the needle was inserted into the tissue. The obturator was quickly advanced as far as permitted to position the specimen notch in the tissue and the cannula handle was quickly advanced to cut the tissue. Tissue obtained in the specimen notch was then transferred to device using tweezers.
• Tru-CutTM biopsy needle CareFusion, Vernon Hills, IL
• Mouse liver and kidneys were harvested from sacrificed C57B/6 or BALB/c mice (Jackson Laboratory, Bar Harbor, ME) that were deemed waste from a research study approved by the University of California, Irvine, Institutional Animal Care and Use Committee (courtesy of Dr. Angela G. Fleischman). Animal organs were cut with a scalpel into 1 cm long x 1 mm diameter pieces, and the mass of each was recorded. Mouse kidneys were sliced in a symmetrical fashion to obtain histologically similar portions that included both cortex and medulla.
• the digestion device was first primed with 200 pL collagenase type I (Stemcell Technologies, Vancouver, BC) and heated to 37 °C inside an incubator to ensure optimal enzymatic conditions. Tissue was then placed inside the chamber before the device was assembled, secured with nylon screws, and filled with 1 mL collagenase. Experiments were initiated by flowing fluid through the device at 20 mL/min with the peristaltic pump, and every 5 min the flow was reversed to clear tissue from the downstream sieve gates. Device effluents were collected by pumping directly into a conical tube.
• Controls were digested in a conical tube that contained 1 mL collagenase, either with or without prior mincing with a scalpel into ⁇ 1 mm 3 pieces. Tubes were placed inside a 37 °C incubator and gently agitated on a rotating mixer. Every 5 min, the tubes were vortexed to mechanically disrupt tissue and maximize digestion. At the conclusion of digestion procedures, all cell suspensions were repeatedly vortexed and pipetted to mechanically disrupt aggregates and treated with DNase I (10 pL; Roche, Indianapolis, IN) at 37 °C for 5 min.
• DNase I (10 pL; Roche, Indianapolis, IN
• Image analysis to monitor tissue digestion During device operation, images of the tissue were captured every 5 min using a camera mounted directly above the device as illustrated in FIG. 6. Raw images were processed using ImageJ by first converting to binary to identify the borders of the tissue (see FIG. 14). Mean gray value was then determined within the tissue border, and multiplied by the area to obtain a single metric accounting for tissue size and density. Results at each time point were normalized by the initial value prior to the experiment, and presented as percent tissue remaining.
• DNA content of digested cell suspensions was assessed by extraction and purification, as well as direct assessment within cells using a fluorescent DNA stain. For both cases, samples were first filtered using a 70 pm cell strainer to remove remaining tissue and large aggregates. Purified genomic DNA (gDNA) was isolated using the QIAamp® DNA Mini Kit (Qiagen, Germantown, MD) according to manufacturer’s instructions and quantified using a Nanodrop ND-1000 (Thermo Fisher, Waltham, MA). DNA within cells was labelled using the CyQUANT® NF Cell Proliferation Assay Kit (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions.
• samples were suspended in HBSS supplemented with 35mg/L sodium bicarbonate and 20 mM HEPES and added to an opaque 96-well plate (Coming, Coming, NY) in triplicate.
• An equal volume of CyQUANT® dye was then added to each well, incubated at 37°C for 40 minutes under continuous mixing at 200 RPM, and fluorescence signal was quantified using a Synergy 2 plate reader (BioTek, Winooski, VT).
• Wells containing only HBSS and CyQUANT® dye were used for background subtraction. gDNA and fluorescence intensities were normalized by the initial tissue mass.
• Compensation was determined using the kidney and liver tissues that were minced with a scalpel and digested for 60 mi, which were aliquoted into four different preparations to obtain distinct positive and negative subsets for each probe.
• the four preparations included cell fractions with: 1) negative control CompBeads (3.0-3.4 pm diameter, BD Biosciences, San Jose, CA) and CellMaskTM Green membrane stain, 2) RBCs lysed and CD45-PE antibody, 3) live and dead (heat-killed at 55 °C for 30 min) cells with 7AAD stain, and 4) Draq5 stain. Gates encompassing the positive and negative subpopulations within each compensation sample were inputted into FlowJo to automatically calculate the compensation matrix.
• a SSC-A vs. FSC-A gate was created to select all cellular events and exclude debris from further analysis. Multicellular aggregates were removed from the analysis population to focus only on single cells using an FSC-H vs. FSC-A gate.
• Leukocytes were first distinguished from the single cell population based on CD45 expression (FL2-A or PE vs. SSC-H). Anucleate red blood cells were distinguished by their absence of Draq5 nuclear stain (FL4-A or Draq5 vs. SSC-H).

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