Patent Application
20230415157
The technical field relates to microfluidic devices that are used to process tissue specimens or tissue samples into cellular suspensions.
Tissues are highly complex ecosystems containing a diverse array of cell subtypes. Significant variation can also arise within a given subtype due to differences in activation state, genetic mutations, epigenetic distinctions, stochastic events, and microenvironmental factors. This has led to a rapid growth in studies attempting to capture cellular heterogeneity, and thereby gain a better understanding of tissue and organ development, normal function, and disease pathogenesis. For example, in the context of cancer, intratumor heterogeneity is a key indicator of disease progression, metastasis, and the development of drug resistance. High-throughput single cell analysis methods such as flow cytometry, mass cytometry, and single cell RNA sequencing (scRNA-seq) are ideal for identifying single cells in a comprehensive manner based on molecular information, and these methods have already begun to transform our understanding of complex tissues by enabling identification of previously unknown cell types and states.
However, a critical barrier to these efforts is the need to first process tissues into a suspension of single cells. Current methods involve mincing, digestion, disaggregation, and filtering that are labor intensive, time-consuming, inefficient, and highly variable. Thus, new approaches and technologies are critically needed to ensure reliability and wide-spread adoption of single cell analysis methods for tissues. This would be particularly important for translating single cell diagnostics to human specimens in clinical settings. Moreover, improved tissue dissociation would make it faster and easier to extract primary cells for ex vivo drug screening, engineered tissue constructs, and stem/progenitor cell therapies. Patient-derived organ-on-a-chip models, which seek to recapitulate complex native tissues for personalized drug testing, are a particularly exciting future direction that could be enabled by improved tissue dissociation.
scRNA-seq has recently emerged as a powerful and widely adaptable analysis technique that provides the full transcriptome of individual cells. This has enabled comprehensive cell reference maps, or atlases, to be generated for normal and diseased tissues, as well as identification of previously unknown cell subtypes or functional states. For example, an atlas recently generated for normal murine kidney uncovered a new collecting duct cell with a transitional phenotype and unexpected level of cellular plasticity. Moreover, an atlas of primary human breast epithelium linked distinct epithelial cell populations to known breast cancer subtypes, suggesting that these subtypes may develop from different cells of origin. For melanoma, scRNA-seq was used to identify three transcriptionally distinct states, one of which was drug sensitive, and further demonstrated that drug resistance could be delayed using computationally optimized therapy schedules. While scRNA-seq is clearly a powerful diagnostic modality, the mechanical process of breaking down the tissue into single cells can introduce confounding factors that may negatively influence data quality and reliability. One factor is the lack of standardization, which can lead to substantial variation across different research groups and tissue types. Another significant concern is that incomplete break down could bias results towards cell types that are easier to liberate. A recent study utilizing single nuclei RNA sequencing (snRNA-seq) with murine kidney samples found that endothelial cells and mesangial cells were underrepresented in scRNA-seq data. Finally, lengthy enzymatic digestion times have been shown to alter transcriptomic signatures and generate stress responses that interfere with cell classification. Addressing these concerns would help propel the exciting field of scRNA-seq into the future for tissue atlasing and disease diagnostics.
Microfluidic technologies have advanced the fields of biology and medicine by miniaturizing devices to the scale of cellular samples and enabling precise sample manipulation. Most of this work has focused on manipulating and analyzing single cells. Only a small number of studies have addressed tissue processing, and even fewer have focused on breaking down tissue into smaller constituents. For example, microfluidic devices have been developed that specifically focused on breaking down cellular aggregates into single cells. This dissociation device contained a network of branching channels that progressively decreased in size down to ˜100 μm, and contained repeated expansions and constrictions to break down aggregates using shear forces. Details regarding such devices may be found in Qiu, X. et al., Microfluidic device for mechanical dissociation of cancer cell aggregates into single cells, Lab Chip 15, 339-350 (2015) and Qiu, X. et al., Microfluidic channel optimization to improve hydrodynamic dissociation of cell aggregates and tissue, Nat. Sci. Reports 8, 2774 (2018).
A device was then developed for on-chip tissue digestion using the combination of shear forces and proteolytic enzymes. Finally, a filter device was developed containing nylon mesh membranes that removed large tissue fragments, while also dissociating smaller cell aggregates and clusters. See Qiu, X. et al., Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells, Lab Chip 18, 2776-2786 (2018). The microfluidic digestion, dissociation, and filter devices each enhanced single cell recovery when operated independently. To date, however, these technologies have not been combined to maximize performance and execute a complete tissue processing workflow on-chip. Moreover, there has been no validations of microfluidically-processed cell suspensions using scRNA-seq.
In one embodiment, a microfluidic platform or system is disclosed that includes three different tissue-processing technologies (digestion, disaggregation, and filtration) that enhances break-down and produces single cell suspensions that are immediately ready for downstream single cell analysis or other use. First, the system uses a digestion device that can be loaded with minced tissue and operated with minimal user interaction. Next, in a separate device that is fluidically coupled to the digestion device integrates or combines tissue dissociation and filter technologies into a single unit. The two-device platform was optimized using murine kidney to produce single cells more quickly and in higher numbers than traditional methods. Using the optimized protocol, different tissue types were evaluated using two single cell analysis methods. For murine kidney and breast tumor tissues, microfluidic processing can produce ˜2.5-fold more epithelial cells and leukocytes, and >5-fold more endothelial cells, without affecting viability. Using scRNA-seq, it was shown that device processed samples are highly enriched for endothelial cells, fibroblasts, and basal epithelium. It was also demonstrated that stress responses are not induced in any cell type, and can even be reduced if shorter processing times are employed. For murine liver and heart, significant single cell numbers are obtained after only 15 min, and even as short as 1 minute. Interestingly, it was found that substantially more hepatocytes and cardiomyocytes are obtained if sample is recovered at discrete intervals, most likely because these cell types are sensitive to shear forces. Importantly, the microfluidic platform can significantly shorten processing time or enhance single cell recovery for all tissue types studies, and in some cases accomplish both, without affecting viability. Furthermore, the entire tissue processing workflow is performed in an automated and reliable fashion. Thus, the microfluidic platform holds exciting potential to advance diverse applications that require the liberation of single cells from tissues.
In one embodiment, a microfluidic system for processing a tissue sample is disclosed that digests, dissociates, and optionally filters tissue. The system includes a microfluidic digestion device having an inlet and an outlet and a flow path defined between the inlet and the outlet, the flow path comprising a tissue chamber configured to hold the tissue sample and a plurality of upstream fluidic channels communicating with the tissue chamber on the inlet side of the flow path and a plurality of downstream fluidic channels communicating with the tissue chamber on the outlet side of the flow path. A first pump is configured to pump a buffer-containing fluid and/or an enzyme-containing fluid into the inlet of the microfluidic digestion device (while the tissue is present in the tissue chamber). The system further includes a microfluidic dissociation/filter device comprising an inlet, a first outlet, a second outlet, and a flow path defined between the inlet and the outlet, the flow path comprising a plurality of furcating dissociation channels having a plurality of expansion and constriction regions disposed along a length thereof, wherein one or more filters are disposed in the flow path downstream of the plurality of furcating dissociation channels. Either of the first and second outlets may be selectively closed to permit flow through the dissociation region of the device only or flow through the dissociation region of the device plus the filter region(s) of the device. A second pump is configured to pump a buffer-containing fluid into the inlet of the microfluidic dissociation/filter device (along with processed tissue solution from the microfluidic digestion device). The outlet of the microfluidic digestion device is fluidically coupled to the inlet of the microfluidic dissociation/filter device. Thus, fluid containing digested tissue passes from the microfluidic digestion device to the microfluidic dissociation/filter device.
In one embodiment, a method of using the microfluidic system includes the operations of: loading the tissue sample into the tissue chamber of the microfluidic digestion device; pumping the buffer-containing fluid and/or an enzyme-containing fluid into the inlet of the microfluidic digestion device with the first pump; transferring fluid containing processed tissue sample to the microfluidic dissociation/filter device; pumping the buffer-containing fluid into the inlet of the microfluidic dissociation/filter device along with the tissue processed with the microfluidic dissociation device; and collecting effluent from the second outlet of the microfluidic dissociation/filter device.
The tissue chamber 20 may be square or rectangular shaped. An exemplary size for the tissue chamber 20 may be chamber that has a 5 mm length and 8 mm width with a height of 1.5 mm. In other embodiments, the tissue chamber 20 may be larger to accommodate larger tissue samples. For example, the tissue chamber 30 may be rectangular-shaped and dimensioned to accommodate a sliver or larger piece of tissue. The tissue chamber 30 may have a length of several centimeters (e.g., around 2-3 cm) and even up to about 10 cm. The first pump 18 may be connected to the digestion device 12 via tubing or conduit 24 as illustrated in FIG. TA. In response to fluid flow into the digestion device 12 via the first pump 18, fluidic channels 28 direct hydrodynamic shear forces and proteolytic enzymes (if contained in fluid), while also retaining minced tissue pieces in the tissue chamber 20. In one embodiment, the number of upstream fluidic channels 28 are equal to the number of downstream fluidic channels 28 (e.g., four such fluidic channels 28 are illustrated in FIG. TA). In this embodiment, the upstream fluidic channels 28 are symmetrical with the downstream fluidic channels 28. Other numbers of fluidic channels 28 may be used. The width of the upstream/downstream fluidic channels 28 may be the same in some embodiments. An exemplary width of the fluidic channels 28 is about 250 μm, although as explained herein other dimensions of the fluidic channels 28 may be used. The length of the fluidic channels 28 may be several millimeters (e.g., 4 mm). The fluidic channels 28 are separated from one another by about 1 mm to ensure reliable fabrication and integrity. The fluidic channels 28 enlarge in the region where they join the underlying via layer (i.e., flare), which was also intended to prevent clogging.
The number of fluidic channels 28 may vary depending on the size of the tissue chamber 20. For example,
The tissue sample (e.g., minced tissue) is loaded into the digestion device 12 via a port 30 (e.g., luer port) as seen in
Still referring to
In one preferred embodiment, the width at the expansion regions 54 within a particular stage is 3× the width of the constriction region 56. Thus, in one embodiment, the first stage (single dissociation channel 52) has a constriction region 56 width that is 2 mm and width of the expansion region(s) 54 is 6 mm. In the second stage, the constriction region 56 width is 1 mm while the width of the expansion regions 54 is 3 mm. In the third stage, the constriction region 56 width is 0.5 mm while the width of the expansion regions 54 is 1.5 mm. In the fourth stage, the constriction region 56 width is 0.25 mm while the width of the expansion regions 54 is 0.75 mm. In the fifth stage, the constriction region 56 width is ˜0.125 mm while the width of the expansion regions 54 is ˜0.375 mm. After the last stage of dissociation channels 52, the channels collect fluid to a common collection region 58. As discussed below, the fluid containing the processed tissue may then be directed either out of the dissociation/filtration device 40 (without filtration) or through filter media for filtration.
Still referring to
In a second flow path, the processed tissue fragments and cell aggregates are then directed through two different filters 62, 64 (e.g., nylon mesh filters). The flow along the second flow path may be accomplished by plugging or capping the flow from the first outlet 48 which then forces the fluid (and contents) along the second flow path in response to pumping by first pump 18 and/or second pump 44. The first filter 62 in the flow path may have a larger pore size (e.g., ˜50-100 μm) than the second filter 64 (e.g., ˜15-50 μm) in the flow path to allow for first filtering of larger sized tissue fragments and cell aggregates followed by a smaller filter mesh with smaller pore size. Typically, the pores range in size from about 5 μm to about 1,000 μm and more preferably within the range from about 10 μm to about 1,000 μm or from about 5 μm to about 100 μm. In one embodiment, the first filter membrane 62 has pores having diameters of d1 and the second filter membrane 64 has pores having diameters of d2, wherein d1>d2. A second pump 44 is coupled to the dissociation/filter device 44 via conduits or tubing 24. A fourth valve 66 is provided to allow for the recirculation of flow from the dissociation/filter device 40 and also for adding buffer or other fluid into the dissociation/filter device 40. The second outlet 50 carries fluid that has passed through the filters 62, 64. This fluid typically contains single cells that are output from the dissociation/filter device 40.
The dissociation/filter device 40 may also formed from multiple layers 80a-80g (e.g., seven layers). As seen in
To use the system 10, a sample of tissue is placed in the tissue chamber 20. The tissue that is processed is preferably minced prior to placement in the tissue chamber 20 (e.g., scalpel mincing tissue into pieces with sizes of ˜1 mm3). The sample of tissue may include any type of mammalian tissue including, for example, kidney, liver, heart, mammary tissue. The tissue may be healthy or diseased. The digestion device 12 is then primed with buffer and enzyme with the first pump 18. The first pump 18 is preferably a peristaltic pump. The port 30 is then sealed with a stopcock or the like and fluid is then recirculated through the digestion device 12 with the first pump 18. The flow rate through the digestion device 12 may vary but is generally within the range of about 10 to about 100 mL/min. The recirculation may take place for several minutes to up to an hour or more. In some embodiments, the recirculation flow is maintained over this entire time period (i.e., static flow operation). In other embodiments, the digestion device 12 is run using an interval operation where the tissue is processed for short time periods, eluting the cell suspension, replacing the enzyme (e.g., collagenase) in the digestion device 12 and then continuing recirculation.
While the digestion device 12 disclosed herein uses a luer port 30 other ports may be used. In addition, in still other embodiments, the top layer 70a of the digestion device 12 may include a lid or cap that can be secured to the remainder of the digestion device 12 to load tissue inside the tissue chamber 20. The lid or cap may be secured using one or more fasteners or the like. Note that the device components of the system 10 (e.g., microfluidic digestion device 12 and dissociation/filter device 40) are preferably kept incubated in an incubator or temperature-controlled environment at about 37° C. to maintain optical enzymatic activity.
Once the sample has been processed with the digestion device 12, the now processed sample then moves to the dissociation/filter device 40. Fluid exits the outlet 16 and passes through tubing or conduit 24 and enters the inlet 46 of the dissociation/filter device 40. If recirculation is intended, tubing or conduit 24 connects the first outlet 48 (i.e., cross-flow outlet) to the second pump 44, while the second outlet 50 is closed off with a stopcock. Fluid is then pumped or recirculated through the dissociation channels 52 using second pump 44. This second pump 44 may include a syringe pump or a peristaltic pump. The flow rate through the dissociation/filter device 40 may vary but is generally within the range of about 5 to about 50 mL/min. For final collection of the sample, or if only a single pass through the dissociation component (i.e., dissociation channels 52) is utilized, the cross-flow outlet 48 is closed off with a stopcock valve (or cap/plug), and sample is pumped through and collected from the second outlet 50 (i.e., effluent outlet). This fluid or effluent contains single cells. The dissociation/filter device 40 may be washed with buffer to flush out and collect any remaining cells. Thus, for the dissociation/filter device 40, a single pass may be made through the dissociation channels 52 and the filters 62, 64 and out the second outlet 50. Alternatively, the sample from the digestion device 12 may recirculate through the dissociation channels 52 for a plurality of cycles followed by a pass through the filters 62, 64 and out the second outlet 50.
The microfluidic digestion device 12 and the dissociation/filter device 40 may be fluidically connected via tubing or conduit 24. Likewise, tubing or conduit 24 connect the pumps 18, 44 to the microfluidic digestion device 12 and the dissociation/filter device 40. The valves 26, 32, 42, 66 are interposed in the conduit or tubing 24 as illustrated, for example, in
Device Design and Fabrication
Minced tissue is loaded through a port at the top of the device 12, which can then be sealed using a cap or stopcock. Scalpel mincing of tissue into ˜1 mm3 pieces is ubiquitous, and therefore this format will be compatible with a wide array of tissue types and dissociation protocols. The full design layout of the new minced tissue digestion device is shown in
The dissociation/filter device 40 processes tissue fragments and cell aggregates that are small enough to leave the tissue chamber 20 of the digestion device 12. This includes disaggregation via shear forces generated within the branching channel array (i.e., dissociation channels 52) and via physical interaction with nylon mesh filters 62, 64. Here, the dissociation and filter functionality has been integrated into a single device 40 to minimize holdup volume and simplify operation. The minced digestion and integrated dissociation/filter devices 12, 40 were fabricated using a commercial laminate process, with channel features laser micro-machined into hard plastic (PMMA or PET). All layers and other components were then aligned and bonded using pressure sensitive adhesive. Photographs of the fabricated devices are shown in
Platform Optimization Using Murine Kidney
The digestion device 12 was evaluated using adult murine kidney samples. The kidney is a complex organ composed of anatomically and functionally distinct structures, and adult kidney tissue has a dense extracellular matrix that is challenging to dissociate into single cells. Freshly dissected kidneys were minced using a scalpel to ˜1 mm3 pieces and loaded into the minced digestion device 12 through the luer port 30. The device 12 and tubing 24 were then primed with PBS containing 0.25% type I collagenase, the luer input port 30 was sealed using a stopcock, and fluid was recirculated through the device 12 using a peristaltic pump 18. Flow rates of 10 and 20 mL/min were tested. After 15 or 60 min of recirculation, sample was collected, washed, and genomic DNA (gDNA) was extracted to assess total cell recovery. A control was minced and gDNA was directly extracted to provide an upper recovery limit. At 10 mL/min, gDNA was ˜15% and 60% of the control after 15 and 60 min, respectively (
Next, single cells were analyzed using flow cytometry. Cell suspensions were labeled using a panel of antibodies and fluorescent probes specific for EpCAM (epithelial cells), TER119 (red blood cells), CD45 (leukocytes), and 7-AAD (live/dead), as listed in Table 1.
It was found that single epithelial cell numbers increased with processing time, from 15 to 60 min, producing up to ˜14,000 cells/mg tissue (
The integrated dissociation/filter device 40 was then investigated if it could further enhance single cell yield following the digestion device 12 (
Single Cell Analysis of Murine Kidney
Kidney was evaluated under different digestion times using the full microfluidic platform. Endothelial cells (via CD31, Table 1) were also added to the flow cytometry panel, since they are difficult to isolate using traditional dissociation methods. Minced tissue was loaded into the digestion device 12 and processed under static (15 or 60 min) or interval (1, 15, and 60 min) formats, and then passed through the integrated dissociation/filter device 40 one time. Controls were minced, digested (15 or 60 min), disaggregated by vortexing/pipetting, and filtered using a cell strainer. Results for epithelial cells are presented in
Table 2 shows the coefficient of variation values for kidney samples at different processing conditions.
Next scRNA-seq was performed, which has been used to catalogue the diverse cell types residing within murine kidney and create atlases. Kidney tissue was processed using the system 10 and collected at 15- and 60-min intervals along with evaluation of the 60 min control. Live single cells were isolated from debris and dead cells using fluorescence-activated cell sorting (FACS), loaded onto a droplet-enabled 10× Chromium platform, and 34,034 cells were sequenced at an average depth of ˜60,000 reads/cell. scRNA-seq quality metrics are shown in Table 3 below, and were comparable across conditions.
Table 3 shows scRNA-seq metrics for kidney and breast tumor samples.
After filtering, Seurat was used to identify (
Table 4. Weighted population values for each cluster and sub-cluster in murine kidney. Population percentages for microfluidic processing in
Total endothelial cell recovery was ˜4-fold greater than the control, while other cell types were ˜2- to 2.5-fold greater, which all match flow cytometry (
Lastly, stress response genes were evaluated, which can interfere with cell identification using transcriptomic information. Induction of stress responses have been linked to conventional tissue dissociation protocols. Since a large number of genes have been implicated, a stress response score is calculated based on previous scRNA-seq work, and results are presented in
Processing and Single Cell Analysis of Murine Breast Tumor Tissue
Solid tumors can exhibit high degrees of intratumoral heterogeneity, which has been directly implicated in cancer progression, metastasis, and the development of drug resistance. This heterogeneity has successfully been captured using scRNA-seq and linked to survival for glioblastoma, drug resistance in melanoma, and prognosis for colorectal cancer. Moreover, it is expected that expanded application of scRNA-seq in clinical settings will soon emerge to provide molecular and cellular information for guiding personalized therapies. Due to abnormal extracellular matrix composition and density, however, tumor tissues are considered to be amongst the most difficult epithelial tissues to dissociate. Microfluidic processing of mammary tumors that spontaneously arise in MMTV-PyMT transgenic mice was evaluated. First, the minced digestion device 12 and integrated dissociation/filter device 40 were optimized separately. The digestion device 12 generated ˜2-fold more EpCAM+ epithelial cells than the controls after 15 and 30 min, and the difference extended to 2.5-fold after 60 min (see
Results for the full microfluidic device system 10 are shown in
Table 5 shows the coefficient of variation values for breast tumor samples at different processing conditions.
scRNA-seq was performed again using the 15- and 60-min device intervals and the 60 min control. A total 24,527 cells were sequenced at an average of ˜45,000 reads per cell. 6 clusters were identified corresponding to epithelial cells, macrophages, endothelial cells, T lymphocytes, fibroblasts, and granulocytes (
Table 6 shows the weighted population values for each cluster and sub-cluster in murine breast tumor. Population percentages for microfluidic processing in were weighted (1× for 15 min and 1.5× for 60 min) and added to create total aggregate microfluidic platform values. These were normalized by the control and used to calculate total aggregate population distributions.
Differences for the device aggregate relative to the control were ˜2-fold for epithelial cells and 2.5- to 3-fold for T lymphocytes and macrophages, which are all similar to flow cytometry results (
Finally, stress response scores were determined as described for kidney. The importance of stress responses can be heightened for tumor since some response genes, such as members of the Jun and Fos families, have been associated with metastatic progression and drug resistance. Stress response scores were similar across all cell types and conditions for tumor (
Isolation of Hepatocytes from Murine Liver
The liver plays a major role in drug metabolism and is frequently assessed for drug-induced toxicity. In fact, liver damage is one of the leading causes of post-approval drug withdrawal. Thus, in vitro screening of drugs against primary liver tissue is a critical component of preclinical testing. Increasingly, organ-on-a-chip systems are being employed to better maintain hepatocyte functionality and activity in culture settings and to enable personalized testing on patient-derived primary cells. While liver is softer and generally easier to dissociate, hepatocytes are well known to be fragile, and thus liver presents a unique dissociation challenge. As such, it was hypothesized that shorter device processing times would be effective for liver. For these experiments, murine liver was minced into 1 mm3 pieces and hepatocytes were detected based on ASGPR1 expression. Liver was first processed using the minced digestion device 12 for either 15 or 60 min. After 15 min, hepatocyte recovery was ˜4-fold higher for the device than the comparable control (
Based on the initial optimization studies, it was concluded that the microfluidic system 10 should utilize short processing times, and use the modified dissociation/filter device 40 with only the 50 μm filter 62. After 5 min digestion device processing, ˜700 hepatocytes were recovered/mg liver tissue (
Table 7 shows coefficient of variation values for liver samples at different processing conditions.
Taken together, the performance of the microfluidic system 10 with liver was quite unique relative to kidney and tumor. It is believed that this caused by the fact that fluid shear forces are needed to break down tissue, but can also damage some cell types that have already been liberated. All tissues require proper balancing of these effects. For softer tissues like liver, the balance must be shifted away from breakdown and towards preservation, particularly for sensitive hepatocytes, which can be accomplished using interval recovery. Endothelial cells and leukocytes also exhibited some sensitivity to over-processing, although to a lesser degree. It is unclear whether this finding can be generalized to other tissues, including kidney and tumor. Liver sinusoidal endothelial cells are highly specialized, with abundant fenestrae and no underlying basement membrane. These properties could also make sinusoidal endothelial cells particularly sensitive to damage. For leukocytes, there was no distinguishing between those that originated within the liver, which would predominantly be Kupffer cells, from those that came from blood, which may be less sensitive to shear. Future studies directly assessing Kupffer cells, as well as hepatic stellate cells, would be of high interest, particularly to make progress towards complex liver models that utilize multiple cell types.
Isolation of Cardiomyocytes from Murine Heart
Heart failure is another leading cause of drug withdrawal from the market, combining with liver failure to account for ˜70% of withdrawals. Thus, there is robust interest in developing heart-on-chip technologies using primary cardiomyocytes for preclinical drug screening. Cardiomyocytes have been shown to be highly sensitive to mechanical and enzymatic dissociation techniques. In addition, they are disproportionately long in one direction, on the order of 100 μm and more. For these experiments, murine heart was minced into ˜1 mm3 pieces and cardiomyocytes were detected based on Troponin T expression. Since Troponin T is an intracellular marker, a fixable viability dye was used, Zombie Violet, in place of 7-AAD. Given potential concerns about cardiomyocyte size and shape, the effect of filter pore size in the integrated dissociation/filtration device 40 was tested. After 15 min processing with the minced digestion device 12, sample was passed through the original integrated dissociation/filter device 40 with both 50 and 15 μm pore size membranes 62, 64 or the modified version with only the 50 μm membrane 62. Cell numbers and viability were similar for all conditions (see
Next, the full microfluidic system 10 was evaluated at different digestion times. Shorter processing times were used due to the potential sensitivity of cardiomyocytes. After 5 min treatment with the digestion device 12, ˜2000 cardiomyocytes were recovered per mg heart tissue (
Table 8 shows coefficient of variation values for heart samples at different processing conditions.
Viabilities for all three cells types were similar to controls (see
A novel microfluidic system 10 is disclosed that includes a digestion device 12 that facilitates loading and processing of minced specimens, as well as a newly integrated dissociation/filter device 40 that completes the dissociation workflow so that the single cell suspension is immediately ready for downstream analysis or alternative application. The new minced digestion device 12 accelerated tissue break down and produced significantly more single cells than traditional methods, while the integrated dissociation/filter device 40 increased yield further, all without affecting viability. This was determined for a diverse array of tissue types that exhibited a wide range of properties, as well as two different single cell analysis methods, flow cytometry and scRNA-seq. A novel processing scheme was used, including interval operation, which allowed the extraction of single cells at different time points during microfluidic digestion. It was found that for tissues that were physically tougher and more robust, such as kidney and tumor, microfluidic processing produced similar cell numbers in dramatically less time (15 vs 60 min), and approximately 2.5-fold more single cells in total. scRNA-seq further confirmed that endothelial cells, fibroblasts, and basal epithelial cells were highly enriched by the microfluidic system 19, with each increasing by 4- to 10-fold. Additionally, it was found that shorter digestion times were associated with lower stress responses for some cell types, but otherwise microfluidic processing did not add to the stress response in any case. These results clearly confirm that the microfluidic tissue system 10 holds exciting potential to advance scRNA-seq studies by reducing cell subtype-biasing, processing time, and/or stress response. For tissues that were softer and may contain sensitive cell types, like liver and heart, it was found that processing times could be dramatically reduced and that interval operation was critical to avoid cell damage and maximize recovery. These results will advance goals in tissue engineering and regenerative medicine, and could be particularly exciting for patient-derived organ-on-a-chip models for pharmacological studies. By focusing on minced specimens, the microfluidic tissue processing system 10 can readily be incorporated into the dissociation workflows for most, if not all, organs and tissues. Minimizing tissue pre-processing would be advantageous, and will be pursued in future work. Another future goal will be to decrease interval recovery time points to further explore protection of fragile cells, intentional enrichment of certain cell subtypes, and lowering of stress responses. Ideally, one would integrate a cell separation strategy that would make it possible to elute single cells from the platform as soon as they are generated. The microfluidic system 10 may be used for diverse tissue properties and cell subtypes. In addition, alternative proteolytic enzymes such as cold-active proteases may be used. Finally, microfluidic cell sorting and detection capabilities may be incorporated into the system 10 to create fully integrated and point-of-care technologies for cell-based diagnostics and drug testing, with a focus on human tissues for clinical applications.
Materials & Methods
Device Fabrication. Microfluidic minced digestion devices 12 and integrated dissociation/filter devices 40 were fabricated by ALine, Inc. (Rancho Dominguez, CA). Briefly, fluidic channels, vias, and openings for membranes, luer ports, and hose barbs were etched into PMMA polyethylene terephthalate (PET) layers using a CO2 laser. Nylon mesh membranes (filters 62, 64) were purchased from Amazon Small Parts (15, and 50 μm pore sizes; Seattle, WA) as large sheets and were cut to size using the CO2 laser. Device layers and other components (hose barbs, nylon mesh membranes) were then assembled, bonded using adhesive, and pressure laminated to form monolithic devices.
Murine Tissue Models. Kidney, liver, and heart were harvested from freshly sacrificed BALB/c or C57B/6 mice (Jackson Laboratory, Bar Harbor, ME) that were determined to be waste from a research study approved by the University of California, Irvine's Institutional Animal Care and Use Committee (courtesy of Dr. Angela G. Fleischman). Mammary tumors were harvested from freshly sacrificed MMTV-PyMT mice (Jackson Laboratory, Bar Harbor, ME). For kidneys, a scalpel was used to prepare ˜1 cm longט1 mm diameter strips of tissue, each containing histologically similar portions of the medulla and cortex. Tissue strips were then further minced with a scalpel to ˜1 mm3 pieces. Liver, mammary tumor, and heart were uniformly minced with a scalpel to ˜1 mm3 pieces. Minced tissue samples were then weighed and either processed with the devices as described below. Controls were placed within microcentrifuge tubes, digested at 37° C. in a shaking incubator under gentle agitation for 15, 30, or 60 min, and mechanically disaggregated by repeated pipetting and vortexing. 0.25% collagenase type I (Stemcell Technologies, Vancouver, BC) was used for both control and device-processed conditions. Finally, cell suspensions were treated with 100 Units of DNase I (Roche, Indianapolis, IN) for 10 min at 37° C. and washed by centrifugation into PBS+.
Minced Digestion Device Operation. Minced digestion devices 12 were prepared by affixing 0.05″ ID tubing 24 (Saint-Gobain, Malvern, PA) to the device inlet 14 and outlet 16 hose barbs, which was then connected to an Ismatec peristaltic pump 18 (Cole-Parmer, Werheim, Germany) with 2.62 mm ID tubing 24 (Saint-Gobain, Malvern, PA). Prior to experiments, devices 12, 40 and tubing 24 were incubated with SuperBlock (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA) at room temperature for 15 min to reduce non-specific binding of cells to channel walls and washed with PBS+. Minced pieces of tissue were loaded into the device tissue chamber 20 through the luer inlet port 30. Devices 12 and tubing 24 were then primed with 0.25% collagenase type I solution (StemCell Technologies, Vancouver, BC), and the luer port 30 was closed off using a stopcock. The experimental setup consisting of the device 12, tubing 24, and peristaltic pump 18 were then placed inside a 37° C. incubator to maintain optimal enzymatic activity. The collagenase solution was recirculated through the device 12 and tubing 24 using the peristaltic pump 18 at a flow rate of 10 or 20 mL/min for a specified time.
Quantification of DNA Recovered from Cell Suspensions. Purified genomic DNA (gDNA) content of digested kidney tissue suspensions were assessed using a Nanodrop ND-1000 (Thermo Fisher, Waltham, MA) following isolation using a QIAamp DNA Mini Kit (Qiagen, Germantown, MD) according to manufacturer instructions. gDNA for device processed samples represents the cellular contents eluted from the device after operation, while gDNA for control samples represent the total amount of gDNA present in these samples.
Integrated Dissociation/Filter Device Operation. Following processing with the minced digestion device 12, tubing 24 was connected from the outlet 16 of the minced digestion device 12 to the inlet 46 of the integrated dissociation and filtration device 40 as seen in
Analysis of Cell Suspensions using Flow Cytometry. Cell suspensions were analyzed using tissue specific flow cytometry panels shown in Table 1. For initial studies with kidney, cell suspensions were stained concurrently with 5 μg/mL anti-mouse CD45-AF488 (clone 30-F11, BioLegend, San Diego, CA), 7 μg/mL EpCAM-PE (clone G8.8, BioLegend, San Diego, CA), and 5 μg/mL TER119-AF647 (clone TER-119, BioLegend, San Diego, CA) monoclonal antibodies for 30 minutes. Samples were then washed twice with PBS+ by centrifugation, stained with 3.33 μg/mL 7-AAD viability dye (BD Biosciences, San Jose, CA) on ice for at least 10 minutes, and analyzed on a Novocyte 3000 Flow Cytometer (ACEA Biosciences, San Diego, CA). Flow cytometry data was compensated using single stained cell samples or compensation beads (Invitrogen, Waltham, MA). Gates encompassing the positive and negative subpopulations within each compensation sample were used calculate a compensation matrix in FlowJo (FlowJo, Ashland, OR). A sequential gating scheme (see
Single Cell RNA Sequencing. These studies used 12-week old mice (male, C57BL/6 for kidney; female, MMTV-PyMT for mammary tumor, both from Jackson Laboratory, Bar Harbor, ME), which were euthanized by CO2 inhalation. Kidneys and mammary tumor were dissected, minced into ˜1 mm3 pieces, and prepared as described for the microfluidic system 10 (15- and 60-min digestion device 12 intervals, single pass-through integrated dissociation/filter device 40) or control (60 min digest) using 0.25% type I collagenase. Recovered cells were centrifuged (400×g, 5 min), treated with 100 Units of DNase I for 5 min at 37° C., and washed by centrifugation into PBS+. Samples were then incubated with RBC lysis buffer for 5 min on ice, centrifuged, and resuspended in PBS+. Cells were stained with SytoxBlue (Life Technologies, Carlsbad, CA, USA) prior to FACS (FACSAria Fusion, BD Biosciences, Franklin Lakes, NJ) to remove dead cells and ambient RNA. Sorted live single cells (SytoxBlue-neg) were centrifuged and resuspended at a concentration of 1000 cells/μL in PBS with 0.04% BSA. The 10× Chromium system (10× Genomics, Pleasanton, CA) was then used for droplet-enabled scRNA-seq. Oil, cells, reagents, and beads were loaded onto an eight-channel microfluidic chip. Lanes were loaded with ˜17,000 cells from each of the samples, determined using an automated cell counter (Countess II, Invitrogen, Carlsbad, CA). Library generation for 10× Genomics Single Cell Expression v3 chemistry was then performed according to manufacturer's instructions. An Illumina NovaSeq 6000 platform (Illumina, San Diego, CA) was used to sequence the samples at a depth of ˜60,000 reads/cell for kidney and ˜45,000 reads/cell for mammary tumor. Sequencing fastq files were aligned using 10× Genomics Cell Ranger software (version 3.1.0) to an indexed mm10 reference genome. Cell Ranger Aggr was used to normalize the mapped reads for cells across the libraries for each data set. Genes that were not detected in at least 3 cells were discarded from further analysis. Cells with low (<200) or high (>3000 for kidney; >4000 for mammary tumor) unique genes expressed were also discarded, as these potentially represent low quality or doublet cells, respectively. Cells with high mitochondrial gene percentages were also discarded (>50% for kidney and >25% for mammary tumor), as these can also represent low quality or dying cells. The Seurat pipeline was used for cluster identification, principal component analysis (PCA) was performed using genes that are highly variable, density clustering was performed to identify groups, and Uniform Manifold Approximation and Projection (UMAP) plots were used to visualize the groupings. For kidney, cell clusters were annotated using two approaches. First, top differential genes in each cluster were examined to determine the cell type of the cluster based on expression of known marker genes (e.g., Kap, Napsa, and Slc27a2 for S2-S3 proximal tubules, Gpx3 for S1 proximal tubules, Emcn for endothelial cells, Slc12a1 for loop of Henle, Slc12a3 for distal convoluted tubule, etc. Second, since a well-established atlas of murine kidney was available, a cell scoring method was used to compare marker gene signatures from each of the clusters to published datasets to confirm cluster annotations (see
Cell Aggregate Studies. MCF-7 human breast cancer cells were obtained from ATCC (Manassas, VA) and cultured as recommended. Prior to experiments, confluent monolayers were briefly digested for 5 min with trypsin-EDTA, which released cells with a substantial number of aggregates. Cell suspensions were prepared for experiments by centrifugation and resuspension in PBS containing 1% BSA (PBS+). MCF-7 cells were then recirculated through the peristaltic pump system alone, or the system with a digestion device 12 or integrated dissociation/filter device 40 attached using methods described herein. For this initial study, flow was recirculated only through the dissociation portion of the integrated device 40 but not passed through the nylon filters 62, 64 of the filtration component for final sample collection in order to avoid confounding effects. To achieve this, the effluent outlet 50 of the integrated device 40 was closed off during pump operation using a stopcock. For all three tests, 5, 10, or 20 mL/min flow rates were used, and recirculation times of 0.5, 1, 4, and 10 min. Following experiments, devices 12, 40 and tubing 24 were washed with 2 mL PBS+ to flush out and collect any remaining cells. Cell counts and viability were obtained both before and after recirculation using a Moxi Flow cytometer with type MF-S cassettes (Orfo, Hailey, ID) and propidium iodide staining.
Flow cytometry gating protocol. Cell suspensions obtained from digested murine kidney, mammary tumor, liver, and heart samples were stained with the fluorescent probes listed in Table 1 and analyzed using flow cytometry. Acquired data was compensated and assessed using a sequential gating scheme (
Evaluation of Pump and Device Recirculation Using MCF-7 Cells
The effect of repeatedly recirculating cells through the peristaltic pump 18 and minced digestion device 12 using the MCF-7 human breast cancer cell line was investigated. This is a strongly cohesive cell type that retains a significant number of aggregates after routine cell culture, and thus requires more powerful dissociation methods. Prior to experiments, confluent monolayers were briefly digested with trypsin-EDTA, centrifuged, and resuspended in PBS containing 1% BSA (PBS+). Sample was then loaded into peristaltic tubing 24 that was either looped through the pump 18 or connected to a minced digestion device 12. Following recirculation for different periods of time at different flow rates, sample was collected for measurement of single cell number and viability (propidium iodide exclusion) using a Moxi flow cytometer. Results are presented in
Next recirculation through the branching channel dissociation device 40 was tested. Previous work with this technology utilized a back-and-forth approach, which was achieved using a syringe pump. Here, the integrated dissociation/filter device 40 was used with flow recirculated only through the dissociation portion and not passed through the nylon filters 62, 64 so as to avoid confounding the results. Cell numbers obtained after recirculating at 5, 10, and 20 mL/min for 0.5, 1, 4. and 10 min are presented in
Platform Optimization Using Murine Kidney
The minced digestion device 12 and integrated dissociation/filter device 40 were separately optimized using murine kidney samples, and results for epithelial cells are presented in
Single Cell Analysis of Murine Kidney
The full microfluidic system 10 or platform was evaluated using murine kidney samples, and results for epithelial cell, endothelial cell, and leukocyte numbers are presented in
scRNA-seq was performed on kidney samples, and identified seven cell clusters that are presented and analyzed in
To facilitate correlations between scRNA-seq and flow cytometry results, gene expression of EpCAM, CD31, and CD45 was inspected. EpCAM was highly expressed predominantly in the main DCT, LOH, CD, & MC cluster (
Processing and Single Cell Analysis of Murine Breast Tumor Tissue
The minced digestion device 12 and integrated dissociation/filter device 40 were separately optimized using a murine breast tumor model (transgenic MMTV-PyMT). Samples were processed using the minced digestion device 12 for 15, 30, or 60 min, and generated ˜2- to 2.5-fold more epithelial cells than controls at the same time points (
The full microfluidic system 10 was then evaluated, and results for epithelial cell, endothelial cell, and leukocyte numbers are presented in
scRNA-seq was also performed and six cell clusters were identified that are presented and analyzed in
Lastly, scRNA-seq results were correlated to flow cytometry in a similar manner as kidney. EpCAM was now well-correlated with the main epithelial cluster (
Isolation of Hepatocytes from Murine Liver
The minced digestion device 12 and integrated dissociation/filter device 40 were tested separately using murine liver, and found that the integrated device 40 decreased hepatocyte yield (
The full microfluidic system 10 (with modified single filter 62 configuration) was then evaluated, and results for hepatocyte, endothelial cell, and leukocyte numbers are presented in
Isolation of Cardiomyocytes from Murine Heart
The minced digestion device was tested, with and without the integrated dissociation/filter device 40 using murine heart. This included both the original integrated device 40 and the modified device 40 without the 15 μm filter 64 that was created for liver. It was found that after processing heart tissue for 15 min, cardiomyocyte numbers and viability were unchanged for each case (
The full microfluidic system 10 was then evaluated, and results for cardiomyocyte, endothelial cell, and leukocyte numbers are presented in
Statistics. Data are represented as the mean±standard error. Error bars represent the standard error from at least three independent experiments. P-values were calculated from at least three independent experiments using students t-test.
While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents.
This application claims priority to U.S. Provisional Patent Application No. 63/090,497 filed on Oct. 12, 2020, which is hereby incorporated by reference. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.
This invention was made with Government support under Grant No. IIP-1362165, awarded by the National Science Foundation (NSF). The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/054440 | 10/11/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63090497 | Oct 2020 | US |
