EP4294568A1 - Microfluidic device for the digestion of tissues into cellular suspensions - Google Patents
Microfluidic device for the digestion of tissues into cellular suspensionsInfo
- Publication number
- EP4294568A1 EP4294568A1 EP22756955.5A EP22756955A EP4294568A1 EP 4294568 A1 EP4294568 A1 EP 4294568A1 EP 22756955 A EP22756955 A EP 22756955A EP 4294568 A1 EP4294568 A1 EP 4294568A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- tissue
- sample chamber
- microfluidic
- sample
- mincing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- 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
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- 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
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- 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
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- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- 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
- 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
- the sample chamber is further fluidically 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 downstream sieve microfluidic channels act as a sieve that firmly holds the tissue in place while also allowing smaller aggregates and cells to exit the sample chamber.
- the microfluidic device may be coupled with downstream operations such as secondary microfluidic dissociation devices to better liberate single cells from small aggregates.
- Valves may also optionally be incorporated into or associated with the upstream hydro-mincing microfluidic channels to provide a high degree of shear forces on selected or targeted areas or regions of tissue. These valves can be turned on and off to cover the entire length of tissue in the chamber.
- cell sorting and analysis components may be added to create point-of-care platforms for cell-based diagnostics and therapies.
- a microfluidic system for the processing of a tissue sample dimensioned within the range of 1 mm 3 to 50 mm 3 into cellular suspensions including a microfluidic device formed from 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 a first side to a plurality of upstream hydro-mincing microfluidic channels disposed in the substrate or chip further fluidically coupled to the inlet and coupled at a second side of the sample chamber to a plurality of downstream sieve microfluidic channels disposed in the substrate or chip further fluidically coupled to the outlet; wherein both the width of the upstream hydro mincing microfluidic and the width of the downstream sieve microfluidic channels are greater than 50 pm and are smaller than the smallest dimension of the tissue sample.
- 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. 1 illustrates a top view of one embodiment of a microfluidic device for the processing and digestion of tissue according to one embodiment.
- FIG. 2 illustrates a schematic illustration of a system that incorporates a microfluidic device for the processing and digestion of tissue with a secondary microfluidic device that is located downstream of the first device.
- 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. 7 illustrates finite-element fluid dynamics simulations showing velocity profiles in devices with different numbers of hydro-mincing microfluidic channels (3, 5, and 7). Simulation results are shown at 1 mL/min flow rate with the chamber empty and partially blocked by a model tissue. Fewer hydro-mince channels will generate stronger fluidic jets to shear the tissue, but with less overall coverage.
- 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. 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. 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.
- raw images were separately converted to binary (upper arrow) and grayscale (lower arrow) images to outline the contour and quantify mean gray value, respectively.
- the area within the tissue contour was then calculated, and multiplied by mean gray value to obtain a single metric accounting for tissue size and density.
- 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. 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. 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).
- 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 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.
- 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.
- 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).
- 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.
- 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.
- 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
- 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.
- 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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