MICROFLUID DEVICE, MICROFLUID DEVICE LOADING METHOD, METHOD FOR ANTIBIOTIC SENSITIVITY TESTING, METH

专利摘要:
a microfluidic device (1) comprises a substrate (10) transparent for imaging and has a plurality of spatially defined and separated cell channels (20) that are sized to accommodate monolayer cells. a respective first end (22) of the cell channels (20) is in fluid connection with an inlet flow channel (30) having a first end (32) in fluid connection with a first fluid port (31) and a second end (34) in fluid connection with a second fluid port (33). a respective second end (24) of the cell channels (20) is in fluid connection with a first end (42) of a respective wash channel (40) having a second end (44) in fluid connection with a channel. flow outlet (50). the outflow channel (50) is in fluid connection with a third fluid port (51). the wash channels (40) are too small in size to accommodate cells.
公开号:BR112017000167B1
申请号:R112017000167-5
申请日:2015-06-12
公开日:2021-08-24
发明作者:Ozden Baltekin；Dan I. Andersson
申请人:Astrego Diagnostics Ab；
IPC主号:

专利说明:

FIELD OF TECHNIQUE
[0001] The present modalities refer, in general, to microfluidic devices and, in particular, to such microfluidic devices configured for the culture and monitoring of cells. FUNDAMENTALS
[0002] Recent developments in individual cell biology have made it clear that isogenic cells can exhibit large differences in gene expression and behavior also when grown under identical conditions. The new devices are thus needed to characterize cell-to-cell differences in phenotypes over time. Such devices need to meet certain criteria in order to be an effective tool in culturing and monitoring individual cells. For example, these devices must be easy to load with cells so that phenotypic characteristics can be monitored immediately after loading. Furthermore, many different individual cells need to be cultured in parallel to characterize cell-to-cell differences. Devices must be designed to allow cell culture over a long period of time under constant and well-controlled growth conditions to monitor, for example, lineage-dependent dynamics. It is even preferable that the devices allow changing culture conditions to monitor dynamic changes in response to new test agents or culture media. For example, it could be advantageous to test different culture media on isogenic cells in parallel or to monitor the response to media changes in different cell strains in parallel.
[0003] A desired application of microfluid devices is to quickly and in parallel monitor the phenotypic response of a sample with bacteria or bacteria mixed with blood cells to gain speed in the analysis.
[0004] Another desired application, which can be combined with antibiotic sensitivity testing (AST), would be to probe genetic variability in the cell sample by first monitoring phenotypic traits and then determining genotypic traits.
[0005] A prior art microfluidic device, termed the "Mother Machine", is disclosed in Wang et al., Current Biology 2010, 20: 10991103. The Mother Machine allows monitoring of cells in many different cell channels in parallel. However, this prior art microfluidic device has several drawbacks. For example, loading cells is complicated and it is difficult to quickly change culture conditions in the microfluid device.
[0006] There is, therefore, a need for an improved microfluidic device that overcomes some, or all, of the shortcomings of prior art microfluidic devices. SUMMARY
[0007] It is an objective of the modalities to provide an improved microfluidic device.
[0008] This and other goals are achieved by modalities, as disclosed in this document.
[0009] One aspect of the embodiments relates to a microfluidic device comprising a transparent substrate for imaging and having a spatially defined and separate plurality of cell channels that are sized to accommodate monolayer cells. A respective first end of the plurality of spatially defined and separated cellular channels is in fluid connection with a flow inlet channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port. A respective second end of the plurality of spatially defined and separated cellular channels is in fluid connection with a first end of a respective first wash channel with a second end in fluid connection with an outflow channel. The outflow channel is in fluid connection with a third fluid port. The first wash channels are too small to accommodate the cells.
[0010] Another aspect of the embodiments relates to a method of charging a microfluidic device, in accordance with the above. The method comprises introducing cells and culture medium into one of a first fluid port and a second fluid port of the microfluid device to allow cells and culture medium to flow through a flow inlet channel of the microfluid device and into a plurality of spatially defined and separated cellular channels. A respective first end of the plurality of spatially defined and separated cellular channels is in fluid connection with a flow inlet channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port. The method also comprises exiting surplus cells through the other one of the first fluid port and second fluid port and exiting culture medium through the other of the first fluid port and second fluid port and through a third port in connection. of fluid with an outflow channel. A respective second end of the plurality of spatially defined and separated cellular channels is in fluid connection with a first end of a respective first wash channel with a second end in fluid connection with an outflow channel. The first wash channels are too small to accommodate the cells.
[0011] Another aspect of the modalities concerns a method for testing antibiotic susceptibility. The method comprises loading bacterial cells into a plurality of spatially defined and separate cell channels of a microfluidic device, in accordance with the above. The method also comprises exposing bacterial cells in different spatially defined and separate cell channels from the plurality of spatially defined and separate cell channels to different antibiotics and/or different concentrations of an antibiotic. The method also comprises determining the antibiotic susceptibility of bacterial cells based on a respective phenotype characteristic, preferably at least one of a respective growth rate, a respective degree of nucleoid compaction, a respective degree of metabolic activity and a respective degree of membrane integrity of bacterial cells in the plurality of spatially defined and separated cell channels.
[0012] Yet another aspect of the modalities concerns a method for genotyping cells in situ. The method comprises loading cells into a plurality of spatially defined and separate cell channels from a microfluidic device in accordance with the above. The method also comprises fixing the cells in the plurality of spatially defined and separated cell channels and in situ genotyping the cells in the plurality of spatially defined and separated cell channels.
[0013] Another aspect of the modalities concerns a method for characterizing the phenotype of cells. The method comprises loading cells into a plurality of spatially defined and separate cell channels from a microfluidic device in accordance with the above. The method also comprises culturing the cells in the plurality of spatially defined and separated cell channels and real-time monitoring of a phenotypic characteristic in the cells in the plurality of spatially defined and separated cell channels.
[0014] The microfluidic device of the modalities is easy to load with the cells and allows cell culture under constant or variable conditions. The microfluidic device allows for rapid changes in culture conditions or application of chemicals, reagents or other agents. BRIEF DESCRIPTION OF THE FIGURES
[0015] The modalities, together with additional objectives and advantages thereof, can be better understood by referring to the following description taken in conjunction with the attached drawings, in which:
[0016] Fig. 1 is an illustration of a microfluid device, according to an embodiment;
[0017] FIG. 2 is a cross-sectional view of the microfluidic device illustrated in Fig. 1 taken along line A-A;
[0018] Fig. 3 is an illustration of a microfluid device, according to another embodiment;
[0019] FIG. 4 is a cross-sectional view of the microfluidic device illustrated in Fig. 3 taken along line A-A;
[0020] Fig. 5 is an illustration of a microfluid device, according to a further embodiment;
[0021] Fig. 6 is an illustration of a microfluid device, according to yet another embodiment;
[0022] FIG. 7 is an illustration of loading and operating the microfluid device illustrated in Fig. 6;
[0023] FIG. 8 schematically illustrates a magnification of cell and reference channels in a microfluidic device;
[0024] FIG. 9 schematically illustrates a magnification of cell channels with channel identifiers in a microfluidic device;
[0025] Fig. 10 is an illustration of a microfluid device, according to an additional embodiment;
[0026] FIG. 11 schematically illustrates an enlargement of the portion in box A of the microfluidic device illustrated in Fig. 10;
[0027] FIG. 12 schematically illustrates an enlargement of the portion in the box B of the microfluidic device illustrated in Fig. 11;
[0028] FIG. 13 is a scanning electron microscope (SEM) image of a mold for casting a microfluidic device, according to an embodiment;
[0029] FIG. 14 illustrates phase contrast images of Escherichia coli cells growing in a microfluidic device, according to a modality;
[0030] FIG. 15 illustrates phase contrast images of chloramphenicol sensitive (MIC®4 μg / ml) and chloramphenicol resistant (MIC > 12 μg / ml) strains of E. coli cultured in a microfluidic device, according to a modality;
[0031] FIG. 16 is a flowchart illustrating a method of charging a microfluidic device, according to an embodiment;
[0032] FIG. 17 shows the mean growth rate response of one erythromycin-resistant strain and two erythromycin-sensitive E. coli strains when exposed to 120 μg/ml erythromycin at time = 0 min; and
[0033] FIG. 18 shows the mean growth rate response for ciprofloxacin-resistant E. coli strain when exposed to (1) no drug (labeled "X"), (2) erythromycin (120 μg/ml, labeled with diamonds) or (3) ciprofloxacin (10 μg/ml, marked with circles) at time = 0 min. DETAILED DESCRIPTION
[0034] Throughout the figures, the same reference numbers are used for similar or corresponding elements.
[0035] The present modalities refer, in general, to microfluidic devices and, in particular, to such microfluidic devices configured for the culture and monitoring of cells.
[0036] The microfluidic device of the modalities facilitate long-term cell culture under constant or variable conditions. The microfluid device can be easily loaded with cells and allows for rapid changes in growth conditions and the addition or exchange of biological or chemical agents.
[0037] The microfluidic device of the modalities can be used for individual cell culture and experiments. In such applications, individual cells are kept separate from other cells in an input sample. For example, the microfluid device can be used to maintain and culture cells of a respective cell strain from a library of multiple cell strains, separately from cells of other cell lines. Thus, each cell strain then respectively has a separate and spatially defined cell channel in the microfluidic device in which the cells can grow and be studied.
[0038] One aspect of the modalities relates to a microfluidic device, also referred to as a culture device. The microfluidic device comprising a transparent substrate for imaging and having a plurality of spatially defined and separated cell channels that are sized to accommodate the monolayer cells. A respective first end of a plurality of spatially defined and separated cellular channels is in fluid connection with a flow inlet channel, also referred to as a flow channel. The inflow channel has a first end in fluid connection with a first fluid port, also referred to as a fluid source, and a second end in fluid connection with a second fluid port, also referred to as a fluid sink. A respective second end of the plurality of spatially defined and separated cellular channels is in fluid connection with a first end of a respective first wash channel with a second end in fluid connection with an outflow channel, also referred to as a sink channel. . The outflow channel is in fluid connection with a third fluid port, which is also referred to as a wash sink. The first wash channels are too small to accommodate the cells.
[0039] Various implementation modalities of the microfluidic device will now be further described with reference to the figures.
[0040] In one embodiment, the microfluidic device, see Fig. 1, comprises a substrate 10 transparent for imaging and having a plurality of spatially defined and separated cell channels 20. The cell channels 20 are sized to accommodate monolayer cells . A respective first end 22 of cell channels 20 is in fluid connection with a flow inlet channel 30 having a first fluid port 31 at a first end 32 and a second fluid port 33 at a second end 34. wash 40 are preferably present and extend from a respective second end 24 of the cell channels 20 to a flow from the outlet channel 50. Therefore, a first wash channel 40 has a first end 42 in connection with fluid with a second end 24 of a cellular channel 20 and an opposite second end 44 in fluid connection with the outflow channel 50. The outflow channel 50 is in fluid connection with a third fluid port 51.
Substrate 10 has multiple cell channels 20 in which cells are cultured. Cellular channels 20 may be arranged in parallel as shown in Fig. 1 and shown further in the cross-sectional view taken along line AA in Fig. 1 as shown in Fig. 2. In such a case, a respective first end 22 of cell channels 20 is in fluid connection with inflow channel 30 and extends from this inflow channel 30. In order to increase the total number of cell channels 20, cellular channels 20 can extend from each longitudinal side of the flow channel 30, thus substantially doubling the number of cell channels 20 compared to just having cell channels 20 on one side of the flow channel 30, see Fig 5.
[0042] In Fig. 5, the substrate 10 of the microfluidic device 1 has two sets 2A, 2B of cell channels 20. The cell channels 20 of each such set 2A, 2B have their respective first end in fluid connection with a fluid inlet channel 30 in common with first and second fluid inlets 31, 33 at their two ends 32, 34. Each set 2A, 2B, however, has a respective outflow channel 50A, 50B as shown in Fig. 5. Thus, the second ends of the first wash channels 40 in the first set 2A are in fluid connection with a first outflow channel 50A, while the second ends of the first wash channels 40 in the second 2B are in fluid connection with a second 50B outflow channel. These two outflow channels 50A, 50B preferably share a third common output port 51.
[0043] The microfluid device 1 of Fig. 5 basically multiplexes two sets 2A, 2B as shown in Fig. 1. This means that the two sets 2A, 2B of cell channels 20 and first wash channels 40 share a a common inflow channel 30 connected at its first end 32 with the first fluid port 31 and at its second end 34 with the second fluid port 33. Each set 2A, 2B of cell channels 20 and first wash channels 40 terminates in a respective outflow channel 50A, 50B which are interconnected and connected to a third common fluid port 51.
[0044] The cells present in the cell channels 20 in the first set 2A on the left will be exposed to the same inputs of culture medium and reagents and chemicals in the first fluid port 31 as the cells present in the cell channels 20 in the second set 2B on the right in the figure.
[0045] Also more complex arrangements of cell channels 20 and inflow channels 30 are possible in substrate 10, as will be further described in this document in connection with Figs. 6-7 and 10. The important feature is that each cell channel 20 has an end 22 in fluid connection with an inflow channel 30 and that the cell channels 20 are separated to prevent cells from escaping from a cell channel 20 and enter another cell channel 20.
[0046] The 20 cell channels are sized to accommodate monolayer cells. This means that the height or diameter of the cell channels 20 is selected to be about or slightly larger than the diameter of the cells to be monitored. For example, cell channels 20 may be substantially quadratic in cross-section, as shown in Fig. 2 with a lateral channel that substantially corresponds to the cell diameter of the cells. Alternatively, the cell channels 20 may be circular or U-shaped in cross-section, with a diameter substantially corresponding to the diameter of the cell. Other cross-sectional configurations are also possible, as long as the cells could be viablely cultured, preferably monolayer, in cell channels 20. This implies that cell channels 20 can be several cells wide, but preferably just one cell high. In this case, cells can grow into cell channels that form a 2D monolayer that may be wider than a cell, but is preferably still a monolayer.
[0047] The inflow channel 30 preferably has dimensions that are significantly larger than the diameter of the cells to be monitored. This means that any cells entering the inflow channel 30 will be spouted through the inflow inlet 30, typically, to the second fluid port 33 by a preferably continuous flow of culture medium from the first fluid port. 31 through the outflow channel 30 and to the second outflow port 33. Thus, in one embodiment, the inflow channel 30 is large enough to allow cells to flow through the inflow channel 30.
[0048] Differences in cross-sectional dimensions of the inflow channel 30 and the cell channels 20 can be further exploited to separate cells from each other in a biological sample applied to microfluidic device 1. For example, the biological sample can contain cells of interest that are small enough in size to allow cells to enter cell channels 20 when flowing through fluid inlet channel 30. The biological sample may additionally comprise larger cells that are too large to enter a channel cell 20, but still small enough to flow through the inflow channel 30. When the biological sample enters the microfluidic device 1, such as through the first inlet port 31, a size separation is obtained, in which large cells flow through the cellular channels 20 in the inflow channel 30 from the first fluid port 31 towards the second fluid port 33. However, smaller target cells will become trapped in cell channels 20.
[0049] If the biological sample, additionally or alternatively, comprises very small cells that are small enough to enter the first wash channels 40, an additional or alternative size separation is obtained. Thus, while target cells enter cell channels 20 but cannot enter first wash channels 40, due to their very large size, very small cells will enter cell channels 20 and flow into first wash channels 40 and into outflow channel 50. These small cells will then exit the microfluid device 1 through the third outflow port 51.
Thus, large cells will never enter the cell channels 20, but are released through the inflow channel 30 and will go out through the second flow port 33. Small cells will not be retained in the cell channels 20 but yes they will flow through the cell channels and first wash channels 40 and then through the outflow channel 50 and the third outlet port 51. Therefore, only target cells of the correct size and size will enter and become trapped in the cell channels 20.
[0051] The actual dimensions of the cell channels 20 and the first wash channels 40 can be selected and designed based on the particular type of cells that are to be cultured and monitored in microfluidic device 1.
Generally, cells, such as a cell strain library, are seeded by adding cells, such as cells of a respective cell strain, into each cell channel 20. The cells are thus allowed to grow in a monolayer along the length of cell channels 20. In one embodiment, each cell channel 20 thus contains cells of a single cell strain and genotype. Cells in cell channels 20 can be seen as pearls on a strand if cell channel 20 is one cell wide. If cell channel 20 is wider the cells will form a 2D layer on cell channel 20.
[0053] Growing and quenching cells beyond the first end 22 of the cell channels 20 will enter the inflow channel 30 and are thus flushed out. The second and opposite end 24 of the cell channels 20 is connected to the first wash channel 40, which is sized to prevent cells from escaping from the second end 24 of the cell channel 20 and into the first wash channel 40.
[0054] In one embodiment, the first wash channels 40 may have a small dimension along their entire length, that is, from the first end 42 connected to the second end 24 of a cellular channel 20 to the second 44 connected to the channel outflow channel 50. In an alternative embodiment, first wash channel 40 comprises a channel restriction that has a size that is too small or narrow for cells present in cell channel 20 to grow or flow through the channel restriction and into of the first wash channel 40. This channel restriction is then present at the first end 42 of the first wash channel 40. The remainder of the first wash channel 40 may, in this embodiment, have a dimension that is substantially the same as the cell channels 20. Channel restriction is therefore an efficient block or obstruction for cells, but allows the culture medium and any chemicals, reagents, or agents to flow. go through the obstruction of the channel and further into the first wash channel 40 and the outflow channel 50.
[0055] Both of these exemplary modalities achieve the desired effect of preventing target-sized cells present in cell channels 20 from growing or flowing through the second end 24 of cell channels 20 and into the first wash channels 40.
[0056] In one embodiment, see Fig. 3, each cell channel 20 of the plurality of spatially defined and separated cell channels 20 is flanked along at least one of its longitudinal sides 26, 28 with a respective second wash channel 60 having a first end 62 in fluid connection with the outflow channel 30 and a second end 64 in fluid connection with the outflow channel 50. The second wash channels 60 are too small in size to accommodate the cells.
[0057] In this modality, each cell channel 20 has at least one second wash channel 60 in fluid connection with the cell channel 20 and disposed along one of its longitudinal sides 26, 28. The modality shown in Fig. 3 has second wash channels 60 arranged along both longitudinal sides 26, 28 of each cell channel 20.
[0058] In one embodiment, the first wash channels 40 and the second wash channels 60 may be interconnected, thus forming a continuous wash layer around the cell channels 20, as shown in Fig. 3. Thus, the second portion wash channels 60 from the second end 24 of the cell channels 20 to the second end 64 of the second wash channels 60 may be interconnected to adjacent first wash channels 40.
The dimension of the second wash channels 60 (or wash layer), such as depth or height, is too small to accommodate cells. This means that cells present in cell channels 20 cannot enter adjacent second wash channels 60, but will remain in cell channels 20.
[0060] FIG. 4 is a cross-sectional view of microfluidic device 1 in Fig. 3 along line A-A. This figure clearly illustrates the comparatively smaller depth of the second wash channels 60 as compared to the cellular channels 20. The wash channels 60 can have any cross-sectional configuration such as quadratic, rectangular, circular, U-shaped, etc. .
[0061] In a specific modality, the first and wash channels 40, 60 have the same depth in the substrate 10.
The cell channel 20 preferably has a substantially equal depth traveling from its first end 22 into the inflow channel 30 as to its second end 24. This depth preferably corresponds to or is slightly greater than the diameter of cell to allow the cells in a monolayer in cell channel 20. At second end 24 of cell channel 20, the depth will be less when entering first wash channel 40 which extends from second end 24 of cell channel 20 into channel flow outlet 50. This shallower depth, preferably less than the diameter of the cells, prevents cells present in cell channel 20 from entering first wash channel 40.
[0063] In an embodiment as shown in Figs. 3 and 4, substrate 10 preferably comprises structures or portions 15 that extend through the entire thickness of substrate 10, in order to increase its stability. Figs. 3 and 4 illustrate these structures 15 in the form of columns provided between some of the second wash channels 60 along the longitudinal lengths of the cell channels 20. These pillars 15 can have any shape, as long as they support the wash layer and allow flow of d. fluid. They could, for example, be rectangular, star-shaped, round or triangular and positioned evenly or irregularly. These structures 15 may be separate structures, as shown in the figures, to promote fluid flow throughout the entire wash layer, i.e. between the second wash channels 60. In an alternative approach, each column of pillars shown in the figures forms a unique structure extending the entire length between the inflow channel 30 and the outflow channel 50. Such a solution can result in a more stable substrate 10, however, at the cost of less efficient fluid flow.
[0064] The cell channels 20 and the first and second wash channels 40, 60 are preferably open channels as shown in Figs. 2 and 4. This means that a cover plate 70 is preferably positioned on the substrate 10 to form a cover for and seal the cell channels 20 and the first and second wash channels 40, 60. The cover plate 70 is, thus, disposed on a main surface 12 of the substrate 10.
[0065] Here follows a brief description of the operation of the microfluidic device 1.
[0066] During cell loading, cells with culture medium enter the first fluid port 31 or the second fluid port 33 and flow into the inlet flow channel 30. In a preferred embodiment, all inlets fluid 31, 33, 51 is opened to allow the culture media and cells to be pushed into cell channels 20. Surplus cells or cells that are too large to fit into cell channels 20 are washed out through the second fluid port 33 or the first fluid port 31, as the depth of the cell channels 20 and the second wash channels 60 (if present) is too shallow to allow the cells to reach the outflow channel 50 and reach the third fluid port 51. The culture media exits the microfluid device 1 from both the second fluid port 33 and the first fluid port 31 and the third fluid port 51. The selection capability of the size of microfluidic device 1 can be used to seed the cell channels 20 with, for example, bacteria that are separated from, for example, blood cells, which are significantly larger.
[0067] During operation of microfluidic device 1, culture medium enters the first fluid port 31 as described above. In a first embodiment, the second fluid port 33 and the third fluid port 51 are open. This means that the culture medium not only exits through the second fluid port 33, but also through the third fluid port 51. This means that the culture medium and assay agents and reagents will effectively reach all cells within the 20 cell channels. Surplus cells will flow into the inflow channel 30 and further out from the second fluid port 33, while the media flow through all the cells and into the outflow channel 50 and out. the third fluid port 51 holds all cells supplied with fresh culture medium or reagents.
[0068] In a second embodiment, the third fluid port 51 is closed so that the culture medium and excess cells exit through the second fluid port 33. This embodiment generally achieves a less efficient flow of the cell medium over the cells in the channels of the cells 20, compared to the first embodiments.
[0069] In the growth, washing and reaction steps, the third fluid port 51 is preferably open. This means that the culture medium, the washing fluid or liquid and the solution with reaction reagents enter the first fluid port 31 and flow through the flow inlet channel 30, cell channels 20 and first wash channels 40 ( and optional second flush channels 60) to outflow channel 50 and third fluid port 51. In another embodiment, second fluid port 33 is opened during the growth and flush steps. In such a case, the culture medium, fluid or washing liquid and the reaction solution with reagents can exit through the third fluid port 51 or second fluid port 33.
[0070] FIG. 6 illustrates an embodiment of a microfluidic device 1 having multiple, i.e., at least two sets 2A, 2B, 2C, 2D, of cell channels 20 and first wash channels 40 sharing a second common fluid port 33 and third fluid port 51, but have separate, i.e. individual, first fluid ports 31A, 31B, 31C, 31D. This means that different culture medium and/or reagents or chemicals can be introduced into cells present in one of the 2A sets of cell channels 20, compared to cells present in the other 2B, 2C, 2D sets of cell channels 20.
[0071] Thus, in one embodiment, the substrate 10 of the microfluid device 1 has multiple sets 2A, 2B, 2C, 2D of the plurality of spatially defined and separated cell channels 20, multiple flow input channels 30A, 30B, 30C, 30D and multi-channel output stream 50A, 50B, 50C, 50D. The respective first end 22 of the plurality of spatially defined and separated cellular channels 20 in each set 2A, 2B, 2C, 2D of the multiple sets 2A, 2B, 2C, 2D is in fluid connection with a respective inflow channel 30A, 30B , 30C, 30D of multi-channel input stream 30A, 30B, 30C, 30D. The respective second end 44 of the first wash channels 40 in each set 2A, 2B, 2C, 2D of the various sets 2A, 2B, 2C, 2D is in fluid connection with a respective outflow channel 50A, 50B, 50C, 50D multi-channel output stream 50A, 50B, 50C, 50D.
[0072] A respective first end 32A, 32B, 32C, 32D of each flow inlet channel 30A, 30B, 30C, 30D of the multiple flow inlet channels 30A, 30B, 30C, 30D is in fluid connection with a respective first fluid port 31A, 31B, 31C, 31D. Correspondingly, a respective second end 34A, 34B, 34C, 34D of each flow inlet channel 30A, 30B, 30C, 30D of multiple flow inlet channels 30A, 30B, 30C, 30D is in fluid connection with a second flow port. fluid 33 in common. Furthermore, each outflow channel 50A, 50B, 50C, 50D of multiple outflow channels 50A, 50B, 50C, 50D is in fluid connection with a third fluid port 51 in common.
[0073] The microfluid device 1 of Fig. 6 should only be considered as an example of modality of having multiples, ie at least two, 2A, 2B, 2C, 2D sets of cell channels 20. This means that the variants of microfluidic device 1 may have a substrate with M such sets, and where M is an integer greater than or equal to two.
[0074] FIG. 7 schematically illustrates an embodiment of the use of the microfluidic device 1 of Fig. 6. In one embodiment, during loading, cells and culture medium enter the second fluid port 33 with the culture medium flowing out through the third port of fluid 51 and through the different first fluid ports 31A, 31B, 31C, 31D. Surplus cells flow out through the first fluid ports 31A, 31B, 31C, 31D. In another embodiment, cells enter through first individual fluid ports 31A, 31B, 31C, 31D with excess cells exiting through second port 33 and culture medium exits through second common fluid port 33 and third common fluid port 51 .
[0075] During operation of the microfluidic device 1, the culture medium preferably enters through the first separate fluid ports 31A, 31B, 31C, 31D, thereby allowing the different culture medium to reach different sets 2A , 2B, 2C, 2D of cell channels 20 and first wash channels 40. Surplus cells are washed out through the second common fluid port 33 and culture medium flows out from the third common fluid port 51 and also through of the second common fluid port 33.
[0076] If the same culture medium is to be used for all sets 2A, 2B, 2C, 2D of cell channels 20 and there is no need to individually add chemicals, reagents or other agents to the different sets 2A, 2B, 2C, 2D, then culture medium flow can be from the second common fluid port 33 to the third common fluid port 51 and separate first fluid ports 31A, 31B, 31C, 31D.
[0077] FIG. 10 schematically illustrates a variant of the microfluidic device 1 described above in connection with Figs. 6 and 7. In this embodiment, the respective first end 32A, 32B, 32C of each inflow channel 30A, 30B, 30C of the multiple inflow channels 30A, 30B, 30C is in fluid connection with a respective first port. of fluid 31A, 31A', 31B, 31B' 31C, 31C'. The respective second end 34A, 34B, 34C of each flow inlet channel 30A, 30B, 30C of multiple flow inlet channels 30A, 30B, 30C is in fluid connection with multiple second fluid ports 33A, 33B, 33C.
[0078] This means that, in this mode, each inlet stream 30A, 30B, 30C is in fluid connection with multiple separate first fluid ports 31A, 31A', 31B, 31B', 31C, 31C' and multiple second common fluid ports 33A, 33B, 33C. The multiple second common fluid ports 33A, 33B, 33C are thus shared by all sets 2A, 2B, 2C of cellular channels, while each such sets 2A, 2B, 2C of cellular channels has its own set of multiple firsts fluid ports 31A, 31A', 31B, 31B', 31C, 31C'.
[0079] The use of multiple first fluid ports 31A, 31A', 31B, 31B', 31C, 31C' per set 2A, 2B, 2C of cellular channels and multiple second common fluid ports 33A, 33B, 33C allow one and the same microfluid device 1 is operated in different modes.
[0080] Such first mode of operation involves the use of several specific cell strains. In such a case, the different cell strains are loaded into the microfluid device 1 at the first fluid ports 31A, 31B, 31C (or 31A', 31B', 31C') with at least one of the second common fluid ports 33A, 33B, 33C as an outlet for surplus cells, while culture medium exits the microfluid device 1 through the third common fluid port 51 and the second open common fluid port(s) 33A, 33B, 33C. During operation, culture medium and any reagents, chemicals or agents are transported from at least one of the second common fluid ports 33A, 33B, 33C that was not used for cell waste during loading and exiting the device of microfluid 1 through the third common fluid port 51 and the first fluid ports 31A', 31B', 31C' (or 31A, 31B, 31C).
[0081] In a particularly preferred embodiment, it is preferred to use a first fluid port 31A, 31B, 31C of each first fluid port pair 31A, 31A', 31B, 31B', 31C, 31C' as the inlet port. cells and culture medium during loading and the other fluid port 31A', 31B', 31C' of each first pair of fluid port 31A, 31A', 31B, 31B', 31C, 31C' as media outlet port of culture during operation. Correspondingly, it is preferable to use a second common fluid port 33A of the multiple second common fluid ports 33A, 33B, 33C as the cell and culture medium outlet port during loading and another second common fluid port 33C as the cell inlet port. culture medium, reagents, chemicals or agents during operation. This approach reduces the risk of contamination of the various fluid ports and, in particular, reduces the risk of contamination of the culture medium, reagents, chemicals or agents used during operation with culture medium and cells added during loading.
[0082] A second mode of operation involves the use of a single cell strain or library of cell strains. In such a case, the cell strain or cell strain library is loaded through at least one, preferably one, second common fluid port 33A using a respective first fluid port 31A', 31B', 31C' (or 31A, 31B, 31C) as an exit port for excess cells and culture medium and the third common fluid port 51 as an exit port for the culture medium. During operation, different culture media and/or different reagents, chemicals or agents can be introduced into the other respective first fluid port 31A, 31B, 31C (or 31A', 31B', 31C'). At least one of the second common fluid inlets 33B, 33C and the third common fluid port 51 are then used as outlet ports for different culture media and/or different reagents, chemicals or agents.
[0083] In a particular embodiment, the respective second end 34A, 34B, 34C of each inflow channel 30A, 30B, 30C of the multiple inflow channels 30A, 30B, 30C is in fluid connection with the(s) ) second common fluid port(s) 33A, 33B, 33C through a respective interconnecting channel 36A, 36B, 36C. In one embodiment, each respective interconnecting channel 36A, 36B, 36C has a length that is substantially the same.
[0084] Thus, in one embodiment, the distance of the channel from each respective second end 34A, 34B, 34C of the inflow channels, 30A, 30B, 30C to the second common fluid port 33 (see Fig. 6) or ports 33A, 33B, 33C (see Fig. 10) is preferably the same for each set of cellular channels 2A, 2B, 2C. This means that the interconnecting channels 36A, 36B, 36C are used to compensate for differences in physical distances on the substrate 10 between respective fluid inlet channels 30A, 30B, 30C and the second common fluid port(s) 33A, 33B, 33C, as shown in Fig. 10. This same distance keeps the pressure over the flow the same for each set of cell channels 2A, 2B, 2C.
[0085] FIG. 11 schematically illustrates an enlargement of the portion of the microfluid device shown in Fig. 10 present in box A. Correspondingly, Fig. 12 schematically illustrates an enlargement of the portion of the microfluid device shown in Fig. 11 present in box B. Fig. 12 illustrates two optional but preferable features of the microfluid device.
[0086] First, each cellular channel 20 preferably has a respective channel identifier 11. FIG. 12 illustrates channel identifiers 11 as respective channel numbers for the different channels of cells 20. For example, cell channels 20 can be numbered from 0000 (or 0001) to 9999 for a microfluid device 1 with 10,000 (or 9,999) cellular channels 20. Identification symbols other than numbers can be used as channel identifiers 11.
Thus, in one embodiment, see Figs. 9 and 12, the substrate 10 of the microfluidic device comprises a respective channel identifier 11 for each spatially defined and separated cellular channel 20 from the plurality of spatially defined and separated cellular channels 20. The respective channel identifiers 11 are visual by image.
[0088] It is not absolutely necessary that all channels of cell 20 have a respective channel identifier 11. Thus, in one embodiment, substrate 10 comprises a respective channel identifier 11 for at least each spatially defined and separate cellular channel 20 of the plurality of spatially defined and separated cellular channels 20. The parameter N may have any integer value equal to or greater than 1, such as 1, 2, 5, 10, 20, 25, 30, 40, 50, 60, 70 , 80, 90, or 100, as illustrative but non-limiting examples.
[0089] Thus, the channel identifiers 11 are preferably visually readable using, for example, a microscope or an imaging device that takes photographs or records a video of cells present in the channels of cells 20 of the microfluid device 1. This means that the cells present in a given cell channel 20 can be identified through the channel identifier 11.
[0090] Second, the reference microfluidic device preferably comprises channels 21, see Figs. 8 and 12. For example, each n0 cell channel 20 on substrate 10 could be replaced by a reference channel 21 for some definite integer value of n. Alternatively, the first and/or the last cellular channel 20 may be in the form of a reference channel 21. Another variant is to have one or more reference channels 21 in some other position(s) of selected cell channel(s) on substrate 10.
[0091] The reference channels 21 are designed to prevent cells from entering and growing within the reference channels 21. This can be achieved by having a dimension of the reference channels 21 that is too small for the cells to enter the reference channels 21. Alternatively, channel restrictions may be present at or near the ends of reference channels 21. Such channel restrictions thus prevent cells from entering reference channels 21, but still allow the medium to culture and any chemicals, reagents or agents enter and flow through the reference channels 21.
[0092] The reference channels 21 can be used in order to obtain context, control or reference data, such as during imaging of the microfluid device and the cells present therein. For example, chemicals that have a certain fluorescence or absorption property can be added to cells in order to determine or monitor a specific cell characteristic. In such a case, the fluorescence or absorption recordings obtained from the reference channels 21 can be used as a control context or reference to determine the fluorescence or absorption in the different cell channels 20. Thus, the reference channels 21 can be used for background subtraction purposes. Another example is when cells are monitored using phase contrast microscopy, in which case the phase contrast image characteristic of the reference channel can be used to account for non-cellular context.
[0093] FIG. 8 illustrates an embodiment of such reference channels 21. In this embodiment, substrate 10 has at least one reference channel 21, arranged in parallel between and substantially with two adjacent spatially defined cell channels 20 separate from the plurality of spatially defined cell channels and separated 20. A respective first end 23 of the at least one reference channel 21 is in fluid connection with the inflow channel 30 and comprises a cell block 27 arranged so as to prevent cells from entering the, at. at least one reference channel 21, from the respective first end 23. A respective second end 25 of the at least one reference channel 21 is in fluid connection with a first end 42 of a respective first wash channel 40 which it has a second end 44 in fluid connection with the outflow channel 50.
[0094] In this embodiment, the reference channels 21 are substantially the same as the cellular channel 20 with respect to extension and dimension, but with a big difference. The reference channels 21 comprise a block of cells 27 at their respective first end 23. This block of cells 27 may be in the form of a restriction channel or a channel portion having a smaller dimension than the remainder of the reference channels 21 The channel restriction or smallest portion is selected to be too small or too narrow for cells that would otherwise enter cell channels 20. Therefore, these cells cannot enter reference channels 21 from the first end 23. The second end 25 of the reference channels 21 is connected, like the cell channels 20, to a respective first wash channel 40. This means that the cells cannot enter the Reference channels 21 from the second end 25, due to the selected dimension or the size of the first wash channels 40.
[0095] FIG. 12 illustrates another embodiment of reference channels 21. In this embodiment, substrate 10 has at least one reference channel 21, arranged in parallel between and substantially with two adjacent spatially defined cell channels 20 separate from the plurality of spatially defined separate cell channels 20. A respective first end 23 of the at least one reference channel 21 is in fluid connection with the inflow channel 30A and comprises a channel restriction 29 arranged to prevent cells from entering the at least a reference channel 21 from the respective first end 23. A respective second end 25 of the at least one reference channel 21 is in fluid connection with the outflow channel 50A and comprises a channel restriction 29 arranged to prevent the cells from entering the at least one reference channel 21 from its second end 25.
[0096] In this mode, each reference channel 21 thus comprises two constraints of the channels 29 one at or near each end 23, 25 of the reference channel 21. The dimensions of the remaining part of the reference channel 21 can thus , be substantially the same as the dimensions of the cellular channels 20.
[0097] In the above-described embodiments, the reference channels 21 are described as being arranged in channels between the two adjacent cells 20. In other embodiments, a reference channel 21 may be present at both ends of a set 2A of cellular channels 20 so that a cellular channel 20 is missing along one of its two longitudinal sides. It is also possible to have two Reference channels 21 arranged adjacent to each other.
[0098] However, it is generally preferred that reference channels 21 are evenly distributed over set 2A of cellular channels, such as by having a reference channel 21 in each at the channel position on substrate 10.
[0099] FIG. 13 is an SEM image of a mold that can be used for casting a microfluid device of the modalities. The figure shows the channel identifiers as channel numbers and shows channel restrictions used to define the reference channels and the interface between a channel and the cellular channel corresponding to the first wash.
[0100] FIG. 14 illustrates phase contrast images of Escherichia coli cells growing in a microfluid device, according to modalities taken at two different points in time. The smaller dimension of the first wash channels, such as in the form of channel restrictions at the interface between first wash channels and cellular channels, effectively prevents E. coli cells from growing out of the left in the figure, ie, from entering in the first wash channels. The channel at the bottom of the figure is an empty reference channel. Double restrictions at each end of the reference channel prevent any E. coli cells from entering the reference channel. The channel tags are visible on the phase contrast images, thus allowing the identification of the channels and the individual E. coli cells growing in them.
[0101] The microfluid device substrate can be made of any transparent material, such as plastic material, in which the structures that constitute the cell channels, the first wash channels, the first fluid port, the second fluid port, the inflow channel, the outflow channel and the third fluid port can be defined. Non-limiting examples of suitable materials include ZEONEX® and ZEONOR®, which are cyclic olefin polymers (COP), marketed by ZEON Chemicals L.P. and TOPAS®, which are cyclic olefin copolymers (COC) marketed by Topas Advanced Polymers. These materials have excellent optical characteristics, in terms of transmission and background fluorescence. They also have good flow characteristics when heated and therefore can replicate small structures that allow the formation of microfluid device substrates.
[0102] Other examples of suitable materials for the substrate include glasses, polydimethylsiloxane (PDMS), poly (methyl methacrylate) (PMMA), polycarbonate (PC), polypropylene (PP), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET) and poly (p-phenylene sulfide) (PPS).
[0103] The cover plate illustrated in Figs. 2 and 4 can be made of a variety of materials that are preferably transparent to allow imaging. Non-limiting examples include glass and plastic materials.
[0104] In operation, the microfluid device is preferably connected to a fluid collector so as to form a culture system comprising the microfluid device and the fluid collector. The fluid collector is configured to deliver the culture medium and reagents, chemicals or agents to cell channels using at least one computer-controlled pump. The fluid collector is preferably configured to allow changing of culture media, dispensing reagents, chemicals or agents using the pre-programmed computer controlled pumps. In a specific embodiment, the cell culture reagents, chemicals or agents and media can be kept at different temperatures throughout the experiment.
[0105] FIG. 16 is a flowchart illustrating a method of charging a microfluidic device, according to an embodiment. The method comprises introducing cells and culture medium into one of a first fluid port and a second fluid port of the microfluid device at step S1 to allow cells and culture medium to flow through a flow inlet channel of the microfluidic device and into a plurality of spatially defined and separated cellular channels. A respective first end of the plurality of spatially defined and separated cellular channels is in fluid connection with a flow inlet channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port. A next step, S2, comprises exiting surplus cells through the other one of the first fluid port and second fluid port and step S3 comprises exiting culture medium through the other of the first fluid port and second fluid port and through a third port in fluid connection with an outflow channel. A respective second end of the plurality of spatially defined and separated cellular channels is in fluid connection with a first end of a respective first wash channel with a second end in fluid connection with an outflow channel. The first wash channels are too small to accommodate the cells.
[0106] Steps S2 and S3 usually occur at the same time as input cells and culture medium enter the microfluidic device and its various channels.
[0107] The microfluid device of the modalities can be used for various applications. An example of such an application is the antibiotic sensitivity test (AST). For example, the microfluidic device 1 shown in Fig. 10 can be used for this purpose. In such a case, sample strain can be loaded from, for example, the second common fluid port 33A using the first fluid ports 31A', 31B', 31C' as outlet ports for excess cells and medium. of culture and the third common fluid port 51 as an outlet port for the culture medium and as a cell aspirator for the cell channels. In one example, during loading, the culture medium flows from the second common fluid port 33C to the second common fluid port 33B to prevent contamination of the second common fluid port 33C. After cell loading, the culture medium preferably flows from the second common fluid port 33C using the first fluid ports 31A', 31B', 31C' and the third common fluid port 51 as outlet ports. . At this stage, the growth of cells in cell channels can be monitored for some time, 0 minutes to days, while taking images of cells in different cell channels. AST testing begins by flowing different antibiotics or the same antibiotic at different concentrations into different 2A, 2B, 2C sets of cell channels using the first fluid inputs 31A, 31B, 31C as ports of entry, while using the second inputs common fluid ports 33A, 33B, 33C and the third common fluid port 51 as outlet ports. Susceptibility is controlled by the phenotypic response to antibiotics in different cell channels. For example, it is possible to determine the average growth rate and cell distribution for growth rate cells only by monitoring the length extension in phase contrast using automatic image analysis routines. These measurements can be calculated on cells in many cell channels such that minute phenotypic changes can be determined within a few minutes. Phenotypic changes can also be morphological changes, such as DNA compaction or changes in membrane structure or integrity. Some phenotypic changes are best studied by adding test agents, such as 4', 6-diamidino-2-phenylindole (DAPI) and SYTOX® fluorescent spots, which can be monitored in a fluorescence channel.
[0108] A phenotypic AST can be followed by an in situ genotyping test in which the presence, absence or abundance of significant genes, DNA sequences or RNA species are determined by, for example, fluorescence in situ hybridization (FISH), in situ sequencing, or isothermal amplification using, for example, isothermal loop-mediated amplification (LAMP) or hybridization and ligation of one or more specific padlock probes, followed by rolling circle amplification (RCA).
[0109] FIG. 15 are phase contrast images taken from a microfluid device during an AST test in which chloramphenicol sensitive (minimum inhibitory concentration (MIC)^4μg/ml) and chloramphenicol resistant (MIC > 12μg/ml) strains of E. coliforam loaded in different cell channels in a microfluid device corresponding to Fig 10. After cell loading both strains were exposed to 6 μg/ml of chloramphenicol. The images that are taken with 15 minutes between them clearly show that the strain sensitive to chloramphenicol increased much less than the strain resistant to chloramphenicol.
[0110] FIG. 17 shows average growth rates for three different E. coli strains when exposed to 120 µg/ml erythromycin at time 0 min, when the three strains are loaded into, respectively, sets 2A, 2B, 2C of cellular channels 20 in one device of microfluid 1 corresponding to Fig 10. The resistant strain, indicated by "x", has a MIC of 256 μg/ml, while the non-resistant strains, indicated by circles and diamonds, have a MIC ~ 12 μg/ml. The figure clearly shows that the resistant strain can be distinguished from the sensitive strains in less than 20 min.
[0111] Figure 18 shows the mean growth rate response, as determined by phase contrast time-lapse imaging, for an E. coli strain that is resistant to ciprofloxacin (DA20859 Eco gyrA1-S83L gyrA2-D87N parC -S80I MIC~30 µg/ml) loaded into respective sets 2A, 2B, 2C of channels of cells 20 in a microfluid device 1 corresponding to Fig. 1. E. coli strain was exposed to (1) no labeled drug with "x" (2) 120 μg/ml erythromycin labeled with diamonds or (3) 10 μg/ml ciprofloxacin labeled with circles. The 0 min time point corresponds to the start of treatment. The curve corresponding to treatment with the drug (erythromycin) to which the strain is susceptible may be distinguished from the curve for no treatment at <10 min. The curve corresponding to treatment with the drug to which the strain is sensitive may be distinguished from the curve corresponding to the drug (ciprofloxacin) to which the strain is resistant in <15 min.
[0112] Thus, a modality refers to a method for testing susceptibility to antibiotics. The method comprises loading bacterial cells into a plurality of spatially defined and separate cell channels from a microfluidic device, in accordance with the modalities. The method also comprises exposing bacterial cells in different spatially defined and separate cell channels from the plurality of spatially defined and separate cell channels to different antibiotics and/or a different concentration of an antibiotic. The method further comprises determining the antibiotic susceptibility of the bacterial cells on the basis of a respective phenotypic characteristic of the bacterial cells in the plurality of spatially defined and separated cell channels.
[0113] Thus, a respective phenotypic characteristic is monitored and determined for bacterial cells and where this phenotypic characteristic is representative of the antibiotic susceptibility of bacterial cells. In a preferred embodiment, the phenotypic characteristic is preferably at least one of a respective growth rate, a respective degree of nucleoid compaction, a respective degree of metabolic activity and a respective degree of membrane integrity of the bacterial cells in the plurality spatially defined and separated cell channels.
[0114] The microfluid device of the modalities can be used for the culture and monitoring of various types of cells, including, but not limited to, bacterial cells, archaea cells, eukaryotic cells such as yeast cells, mammalian cells, human cells, etc.
[0115] Modalities microfluid device can be used for cell characterization, such as a cell strain library. Characterization may be in the form of determining at least one phenotypic characteristic of cell lines and/or in situ genotyping of the genetic material of cell strains. An example of phenotypic characterization and in situ genotyping of a cell strain library is disclosed in co-pending patent application No. PCT/SE2015/050227. In particular, the microfluid device allows for monitored phenotypic traits and in-situ determined genotypes to be linked in a highly parallel fashion. This means that a vast library of cell strains with different genotypes can be processed in parallel in the microfluid device in order to connect the monitored phenotypic characteristics for the different genotypes of the cell strains.
[0116] The cell strain library can be obtained according to various techniques within genome engineering. For example, Multiplex Automated Genomic Engineering (MAGE) can be used to create several billions of different mutant genomes per day (Wang et al. Nature, 2009, 460: 894-898). Other techniques that can be used to create a cell library include protein 9 (Cas9) associated with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (Wang et al., Science, 2014, 343: 80) -84; Koike-Yusa et al., Nature Biotechnology, 2014, 32: 309-312; Zhou et al., Nature, 2014, 509: 487-491) or large-scale RNA interference (Berns et al., Nature , 2004, 428: 431-437).
[0117] The microfluidic device then allows cells from each cell strain in the library to be maintained and cultured separately from cells of other cell lines and or other genotypes. Thus, each cell strain has, respectively, a separate and spatially defined cell channel in the microfluidic device in which the cells can grow and be studied.
[0118] Cells grown in spatially defined and separated cell channels in the microfluid device can preferably be exposed to various stimuli or physical and / or chemical agents without being washed in order to monitor the response of cells to stimuli or physical agents and/or chemicals. For example, various chemical test agents, such as nutrients, drugs, antibiotics, repressors or gene expression inducers, can be added to the culture medium and thus come into contact with the cells. The phenotypic characteristics of cell strains in terms of the cell response of different cell strains to the various test agents can then be determined, for example, using microscopy. Correspondingly, the temperature, pH, pressure, flow, gases, light exposure or mechanical stress that cells are exposed to can be changed and the response of cells from different cell strains to such changes in physical conditions can be determined, for example, using microscopy.
[0119] Cell strains have different genotypes as represented by having different variable regions in at least a part of their genetic material. The variable region is normally present in the genome of cell strains. Alternatively, the variable region is present in a mobile genetic element, such as the plasmid or vector and, therefore, should not necessarily be stably incorporated into the genome of cells. In what follows, modalities are discussed primarily in relation to the variable region being present in the genome. However, alternative embodiments are possible in which the variable region and additional DNA elements mentioned herein are instead present in a plasmid or other mobile genetic element of the cell strains. The variable region, or parts of it, can also be present in unstable genetic elements such as transposons, viruses or phages. Such coding sometimes has advantages in terms of amplifying the variable sequence before fixing the cells for in situ sequencing, for example, by specifically excising or circulating parts of the variable region of the dsDNA genome before fixing the cells.
[0120] In one embodiment, the method further comprises the random seeding of cells of cell strains in spatially defined and separated cell channels in the microfluid device. The random seeding of cells is preferably carried out so that each cell channel comprises only cells of the same genotype, i.e., the same cell lineage.
[0121] An advantage of the modalities is that the genetic identity, that is, the genotype, of the cells does not need to be determined and known prior to inoculation of the cells into the microfluid device. Thus, there is no need to keep cells in the sorted cell library prior to seeding in terms of having to know the genetic identity of each cell strain and continuously monitor the position of each genotype throughout the method. This means that the present modality in clear contrast first analyzes the phenotypic traits in parallel without any knowledge of the genotype and then determines the genotypes and connects them to the phenotypic traits.
[0122] The phenotypic trait determined for each cell strain in the library is preferably a phenotypic trait corresponding to each genotype in the library. Thus, the cells in the library are genetically different cells that have a different genotype, that is, a respective genotype per cell strain. Genotype differences imply that cells will have different phenotypic characteristics corresponding to each respective genotype.
[0123] In one embodiment, the phenotypic characteristics of cells are determined through microscopy. Microscopy to monitor and determine phenotypes has several advantages over prior art technologies. For example, fluorescence microscopy allows extended cell lines times over many generations, unique molecule detection sensitivity and the possibility to monitor temporal responses to change growth conditions in any way.
[0124] Illustrative but non-limiting examples of phenotypic characteristics that can be monitored and determined, according to modalities, using microscopy include cell morphology changes, spatial and/or temporal expression patterns of various molecules, such as the ribonucleic acid (RNA) or proteins, specific metabolic levels, shelf life or growth rate, such as in response to the addition of different agents or physical or chemical stimuli, cell-to-cell variations in gene expression levels, development of the embryo, brightness of reporter proteins or RNA aptamers, etc.
[0125] The phenotypic characterization of cell strains can thus be performed in parallel under a microscope for a longer period of time if necessary. Phenotypic characterization is, moreover, performed without knowledge of the genotype of the various cell strains in the library. In clear contrast, phenotypic characterization instead determines the respective phenotypic characteristics for each spatially defined and separate cell channel in the microfluid device. For example, assuming that the relevant phenotypic trait to be determined for cell strains is gene expression of a target gene for a fluorescence reporter protein, with a variable gene regulatory region or coding sequence, following addition of a test agent. In such a case, the microscope can be used to take an image along the microfluid device where the respective gene expression levels can be determined visually. Each individual gene expression level can then be quantified to obtain a value for each respective cell channel in the microfluidic device. Thus, the output of the phenotypic characterization determination can be a list or matrix of one or more respective values for each cell channel in the microfluidic device. If each cellular channel is assigned a respective channel identifier, as previously described in this document, the phenotypic characteristic measured for the cells of a cellular channel can then be associated with the channel identifier assigned to that cellular channel.
[0126] In one embodiment, the determination of phenotypic characteristics that determine the phenotypic characteristic of each cell strain during cell culture in the microfluidic device using microscopy. Examples of the microscopy technologies that can be used in the modalities include, for example, light field microscopy, phase contrast microscopy, fluorescence microscopy, light sheet microscopy or any type of super-resolution imaging modality such as stimulated emission depletion microscopy (STED), localized photo-activated microscopy (PALM), scanning near field optical microscopy (NSOM), 4pi microscopy, structured illumination microscopy (SIM), ground state depletion microscopy (GSD) ), spectral precision distance microscopy (SPDM), stochastic optical reconstruction microscopy (STORM). In addition, Single Intracellular Particle Tracing (SPT) or Fluorescence Correlation Spectroscopy (FCS) could also be used. Microscopy analysis can be done at fixed points of time or using time-lapse imaging.
[0127] Other measurements of phenotypes are also possible, such as measuring mechanical properties using atomic force microscopy, membrane potential using indicator dyes or microelectrodes, small molecule secretion using imaging mass spectrometry or specified biosensor arrays. Near-field optical array detectors connected directly to the culture device are also possible.
[0128] Once the phenotypic characteristics of the cell strains have been determined the cells are preferably fixed in the cell channels in the microfluidic device. Cell fixation can be performed according to techniques well known in the art. For example, formaldehyde, methanol or ethanol can be used for cell fixation. In example non-limiting cells are fixed with 4% formaldehyde for about 15 minutes or 3% (w/v) paraformaldehyde in phosphate buffered saline (PBS) for about 30 minutes.
[0129] In one embodiment, fixed cells are permeabilized prior to in situ genotyping. Several protocols traditionally used for cell permeabilization can be used according to the modalities. For example, Triton X-100 (eg 0.25% Triton X-100) or another surfactant, such as a nonionic surfactant, can be used. Alternatively, ethanol, such as 70% ethanol, can be used for cell permeabilization. Other examples include hydrochloric acid, 0.1M hydrochloric acid such as, optionally combined with a protease such as pepsin, for example 0.01% pepsin, lysozyme or to degrade the bacterial cell wall.
[0130] In situ genotyping comprises in situ genotyping of at least a part of a variable region of each cell strain in the channels of cells in the microfluidic device. Therefore, it is not absolutely necessary to genotype in-situ the complete variable region of each cell strain. Accordingly, in-situ sequencing as used herein comprises in situ sequencing of at least a part of the variable region or even the entire variable region. In situ sequencing preferentially generates information showing any nucleotide differences in the variable region between different cell lines and that these nucleotide differences give rise to different phenotypes.
[0131] In one embodiment, in situ genotyping is based on fluorescent in situ sequencing technology (FISSEQ) as described, for example, in Science, 2014, 343(6177): 1360-1363. Briefly, in-cell FISSEQ cDNA amplicons are generated in fixed cells using reverse transcriptase and incorporation of aminoallyl deoxyuridine 5'-triphosphate (dUTP) during reverse transcription (RT). The cDNA is refixed using BS(PEG) 9, an amino-reactive linker with a 4 nm spacer. The cDNA fragments are then circularized prior to rolling circle amplification (RCA). BA(PEG)9 is then used to cross-link the aminoallyl dUTP-containing RCA amplicons. SOLiD sequencing via ligation can then be used to sequence the relevant sequence in the RCA amplicons to obtain the variable region nucleotide sequence.
[0132] In one embodiment, in situ variable region genotyping preferably comprises in situ sequencing by binding the variable region or at least a portion thereof to cell channels in the microfluidic device. Ligation sequencing depends on the sensitivity of deoxyribonucleic acid (DNA) ligase to base pair mismatches. Generally, the variable region to be sequenced is preferably in the form of a single, single-stranded DNA sequence, flanked at least at one end by a known sequence that will function as a primer binding anchor sequence. An anchor primer that is complementary to the known sequence is brought in to bind to the known sequence.
[0133] A mixed set of probe oligonucleotides, typically eight to nine bases in length, is then brought up, tagged, typically with a fluorescent dye, according to the position to be sequenced. Such tagged oligonucleotides hybridize to the variable region near the anchor primer and DNA ligase preferably joins an oligonucleotide to the anchor primer when its nucleotide sequence matches the unknown variable region. Based on the fluorescence produced by the molecule, the identity of the base at that position in the variable region can be inferred.
[0134] Oligonucleotide probes can also be constructed with cleavable bonds that can be cleaved after tag identification. This wants to remove the label and regenerate a 5'-phosphate at the end of the bound probe, thus allowing a new binding cycle. This cycle of linkage and cleavage can be repeated several times to read the longer sequences. This technique sequences every Qth base in the variable region, where Q is the length of the probe left behind after cleavage. In order to sequence the omitted positions of the variable region, the anchor primer and attached oligonucleotides can be removed from the variable region and another round of sequencing per ligation is started with an anchor primer that is one or more bases shorter.
[0135] Another technique is to make repeated cycles of a single binding, where the tag corresponds to different positions on the probe, followed by the removal of the anchor primer and the bound probe.
[0136] Ligation sequencing can proceed in either direction (5'-3' or 3'-5'), depending on which end of the oligonucleotide probes is blocked by the tag.
[0137] In one embodiment, the sequence that is sequenced is preferably a cDNA sequence obtained by reverse transcription of an RNA transcript obtained from the variable region. In this embodiment, the variable region is flanked by at least one known sequence to which the anchor primer will bind.
[0138] Ligation sequencing can be performed on fixed cells to achieve in situ sequencing by ligating the variable region or at least a portion thereof into cell channels in the microfluidic device, see eg Science 2014, 343 : 1360-1363 and NatureMethods 2013, 10: 857-860, the teachings of which are incorporated herein by reference with respect to performing in situ sequencing by ligation.
[0139] Briefly, in a variant, the RNA obtained from the variable region or a barcode, is copied to cDNA through reverse transcription, followed by degradation of the mRNA strand using an RNase. In a first embodiment, a padlock probe binds to cDNA with a gap between the probe ends at bases that are oriented for sequencing by ligation. This gap is filled by DNA polymerization and DNA ligation to create a DNA circle. In a second embodiment, circulating cDNA is performed by ssDNA ligation alone. In a third embodiment, dsDNA including at least a part of the variable sequence and neighboring DNA is excised from the surrounding DNA, e.g. for restriction enzymes or transposases. The excised dsDNA can then be digested by ssDNA endonucleases in order to self-anneal and ligate to form a circular DNA.
[0140] In a variant, the variable region to be sequenced is amplified directly from dsDNA, either chromosomal or genetic in a mobile element. In this case, the dsDNA can be cut with a restriction enzyme near the variable region and the dsDNA is made single-stranded by an exonuclease. This ssDNA can be amplified by a method that normally works on cDNA, but without the need to express an RNA. This method alleviates the constraints of designing DNA region close to variable region. For example, the variable region can be filled by a gapfill padlock reaction.
[0141] As an alternative to gapfill, it is possible to use a relatively long time variable region (10-25 bp) and to hybridize a set of ssDNA oligonucleotides to the variable regions. The binding energies of the oligonucleotides are chosen such that only the specific oligonucleotide binds at the annealing and binding temperature of choice. Let's say, for example, that the library contains 4000 variants encoded in a 15 bp variable region, which would allow 415-109 variants, it is possible to choose the 4000 variants in such a way that there are no cross-hybridizing probes. After annealing the oligonucleotide and the padlock probe, the probe is ligated into a circle that can be amplified.
[0142] In both cases, the DNA circle formed is amplified from target-initiated rolling circle (RCA) generating a rolling circle product (RCP) which is subjected to sequencing by ligation. An anchor primer is annealed close to the target sequence prior to ligation of oligonucleotide probes. In one embodiment, oligonucleotide probes consist of four 9-mer libraries, with eight random positions (N) and one fixed position (A, C, G or T). Each library is labeled with one of four fluorescent dyes. The best matched oligonucleotide probe at the fixed position will be incorporated by ligation along with its fluorescent label. The sample is represented by image and each RCP displays the color that corresponds to the corresponding base. The oligonucleotide probe is washed prior to applying the oligonucleotide probes to the next base. The binding, washing, imaging and removal steps are iterated until the desired number of bases is read.
[0143] In one embodiment, in situ genotyping comprises, in situ sequencing by synthesizing the variable region or at least a portion thereof in cell channels in the microfluidic device.
[0144] For example, four types of modified dNTPs containing a terminator that blocks further polymerization are added. The terminator also contains a fluorescent tag that can be detected by the camera. Unincorporated nucleotides are washed away and images of the fluorescently labeled nucleotides are taken. The fluorescent tag along with the terminator is chemically removed from the DNA allowing for the next cycle of sequencing.
[0145] The result of in situ genotyping is preferably the nucleotide sequence of the variable region or at least a portion of it for each cell strain. Each nucleotide sequence is further linked to a respective cell channel in the microfluidic device, for example using the channel identifiers. This is possible since genotyping is performed as an in situ genotyping, such as in situ sequencing by ligation or synthesis. In situ means here that genotyping is carried out in place or in position, that is, in the cell channels in the microfluidic device.
[0146] The output of the phenotyping described above was a respective phenotypic characteristic determined for each cell channel in the microfluidic device, such as in the form of a list or matrix listing the phenotypic characteristic(s) determined for each cell channel as identified by channel identifiers. The output of in situ genotyping is the nucleotide sequence determined for the variable regions of each cell channel in the microfluidic device. This output may also be in the form of a list or matrix that lists the nucleotide sequence determined for each cell channel as identified by the channel identifiers.
[0147] Each respective phenotypic trait can then be linked or associated with each respective genotype based on the cell channels in the microfluidic device. For example, the phenotypic characteristic determined for cells of the cell strain in cell channel no. P is a result of the genotype of the cells of this cell strain and this genotype is obtained from the nucleotide sequence determined for cell channel no. P. Thus, the linkage of phenotype and genotype can be achieved simply by matching the phenotypic characteristics and genotypes determined for each cell channel in the microfluidic device.
[0148] In the application described above, the modalities microfluid device is used for combined determination of phenotype and in situ genotype of a library of cell strains. The microfluidic device may alternatively be used for mere phenotype determination or mere in situ genotype determination. Therefore, it is not necessary to perform both in situ phenotype and genotype determinations of cells loaded in the microfluidic device.
[0149] Yet another aspect of the modalities, therefore, concerns a method for genotyping cells in situ. The method comprises loading cells into a plurality of spatially defined and separate cell channels from a microfluidic device, in accordance with the embodiments. The method also comprises fixing the cells in the plurality of spatially defined and separated cell channels. The method further comprises genotyping the cells in situ in the plurality of spatially defined and separated cell channels.
[0150] Another aspect of the modalities refers to a method for characterizing the phenotype of cells. The method comprises loading cells into a plurality of spatially defined and separate cell channels from a microfluidic device, in accordance with the embodiments. The method also comprises growing or culturing the cells in the plurality of spatially defined and separated cell channels. The method further comprises monitoring in real time a characteristic cell phenotype in the plurality of spatially defined and separated cell channels.
[0151] One aspect of the embodiments relates to a method for characterizing a library of a plurality of cell strains that have different variable regions in at least a part of the genetic material of the cell strains. The method comprises loading cells from cell strains from the library into a plurality of spatially defined and separate cell channels of a microfluidic device, in accordance with the modalities. The method also comprises growing or culturing the cells of the cell strains in the plurality of spatially defined and separated cell channels. The method further comprises determining a phenotypic characteristic of each cell strain in the microfluid device, preferably by real-time monitoring of a respective cell phenotype characteristic of the cell strains in the plurality of spatially defined cell channels and separated. The method further comprises fixing the cells of the cell strains in the spatially defined and separated cell channels in the microfluidic device. The method further comprises in situ genotyping of the variable region of each cell strain into spatially defined and separate cell channels in the microfluidic device. Finally, the method comprises linking each respective phenotypic characteristic to each respective genotype based on the spatially defined and separated cell channels in the microfluidic device, preferably based on the respective channel identifiers or the respective positions of the spatially defined and separated cell channels in the substrate of the microfluidic device.
[0152] In conclusion, the modalities described above are to be understood as illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes can be made to the embodiments without departing from the scope of the present invention. In particular, the different solutions of parts in different modalities can be combined in other configurations, whenever technically possible. The scope of the present invention is, however, defined by the appended claims.

权利要求:
Claims (13)
[0001]
1. Microfluidic device (1) comprising: a substrate (10) transparent for imaging and having a plurality of spatially defined and separated cell channels (20) having a dimension to accommodate monolayer cells; a first end thereof ( 22) of said plurality of spatially defined and separated cell channels (20) is in fluid connection with a flow inlet channel (30); and a respective second end (24) of said plurality of spatially defined and spaced apart cell channels (20) is in fluid connection with a first end (42) of a respective first wash channel (40) having a second end (44 ) in fluid connection with an outflow channel (50), wherein said outflow channel (50) is in fluid connection with a third fluid port (51) characterized in that said channel inlet (30) has a first end (32) in fluid connection with a first fluid port (31) and a second end (34) in fluid connection with a second fluid port (33); and said first wash channels (40) are too small in size to accommodate said cells.
[0002]
2. Microfluidic device according to claim 1, characterized in that said flow inlet channel (30) has a sufficiently large dimension to allow said cells to flow through said flow inlet channel (30 ).
[0003]
3. Microfluidic device according to any one of claims 1 or 2, characterized in that each spatially defined and separated cell channel (20) of said plurality of spatially defined and separated cell channels (20) is flanked by the along at least one of its longitudinal sides (26, 28) with a respective second wash channel (60) having a first end (62) in fluid connection with said inlet flow channel (30) and a second end. (64) in fluid connection with said outflow channel (50), wherein said second wash channels (60) are too small in size to accommodate said cells.
[0004]
4. Microfluidic device according to any one of claims 1 to 3, characterized in that said substrate (10) has at least one reference channel (21) disposed between and substantially parallel with two cell channels adjacent spatially defined and spaced apart (20) of said plurality of spatially defined and spaced apart cell channels (20); a respective first end (23) of said at least one reference channel (21) is in fluid connection with said channel of inflow (30) and comprises a block of cells (27) arranged to prevent said cells from entering said at least one reference channel (21) from said respective first end (23); and a respective second end (25) of said at least one reference channel (21) is in fluid connection with a first end (42) of a respective first wash channel (40) having a second end (44) in fluid connection. fluid with said outflow channel (50).
[0005]
5. Microfluidic device according to any of claims 1 to 3, characterized in that said substrate (10) has at least one reference channel (21) disposed between and substantially parallel with two adjacent cell channels spatially defined and separated (20) from said plurality of spatially defined and separated cell channels (20); a respective first end (23) of said at least one reference channel (21) is in fluid connection with said inlet flow channel (30) and comprises a constriction channel (29) arranged to prevent the said cells enter said at least one reference channel (21) from said respective first end (23); and a respective second end (25) of said at least one reference channel (21) is in fluid connection with said outflow channel (50) and comprises a constriction channel (29) arranged to prevent the said cells enter said at least one reference channel (21) from said respective second end (25).
[0006]
6. Microfluidic device according to any of claims 1 to 5, characterized in that said substrate (10) comprises a respective channel identifier (11) for at least each N spatially defined and separate cell channel (20) of said plurality of spatially defined and spaced apart cell channels (20); said respective channel identifiers (11) being visual by imaging.
[0007]
7. Microfluidic device according to any one of claims 1 to 6, characterized in that said substrate (1) has several sets (2A, 2B, 2C, 2D) of said plurality of spatially defined cell channels and separated (20), a plurality of inflow channels (30A, 30B, 30C, 30D) and a plurality of outflow channels (50A, 50B, 50C, 50D); spatially defined and separated cells (20) in each set (2A, 2B, 2C, 2D) of said several sets (2A, 2B, 2C, 2D) is in fluid connection with a respective flow inlet channel (30A, 30B , 30C, 30D) of said multiple inflow channels (30A, 30B, 30C, 30D); said respective second end (44) of said first wash channels (40) in each set (2A, 2B, 2C, 2D ) of said several assemblies (2A, 2B, 2C, 2D) is in fluid connection with a respective outflow channel (50 A, 50B, 50C, 50D) of said several outflow channels (50A, 50B, 50C, 50D); a respective first end (32A, 32B, 32C, 32D) of each inflow channel (30A, 30B) , 30C, 30D) of said multiple inflow channels (30A, 30B, 30C, 30D) is in fluid connection with a respective first fluid port (31A, 31B, 31C, 31D); , 34B, 34C, 34D) of each inflow inlet channel (30A, 30B, 30C, 30D) of said multiple inflow inlet channels (30A, 30B, 30C, 30D) is in fluid connection with a second inlet port. common fluid (33); and each outflow channel (50A, 50B, 50C, 50D) of said multiple outflow channels (50A, 50B, 50C, 50D) is in fluid connection with a third common fluid port (51).
[0008]
8. Microfluidic device according to claim 7, characterized in that said respective first end (32A, 32B, 32C) of each flow input channel (30A, 30B, 30C) of said multiple flow input channels (30A, 30B, 30C) is in fluid connection with multiple respective first fluid inlets (31A, 31A', 31B, 31B', 31C, 31C'); said respective second end (34A, 34B, 34C) of each channel The inlet flow port (30A, 30B, 30C) of said multiple inlet flow channels (30A, 30B, 30C) is in fluid connection with multiple second common fluid ports (33A, 33B, 33C).
[0009]
9. Microfluidic device according to any one of claims 7 or 8, characterized in that said respective second end (34A, 34B, 34C) of each inlet flow channel (30A, 30B, 30C) of said multiple flow channels (30A, 30B, 30C) are in fluid connection with said second common fluid port (33A, 33B, 33C) through a respective interconnecting channel (36A, 36B, 36C), wherein each respective channel of interconnection (36A, 36B, 36B) has substantially the same channel length.
[0010]
10. Method of loading the microfluidic device (1) as described in any one of claims 1 to 9, characterized in that said method comprises: introduction (S1) of cells and culture medium into one of a first fluid port (31) and a second fluid port (33) of said microfluidic device (1) to allow said cells and culture medium to flow through an inflow channel (30) of said microfluidic device. microfluids (1) and in a plurality of spatially defined and spaced apart cell channels (20), wherein a respective first end (22) of said plurality of spatially defined and spaced apart cell channels (20) is in fluid connection with said inflow channel (30) having a first end (32) in fluid connection with said first fluid port (31) and a second end (34) in fluid connection with said second fluid port (33 );exit (S2) of excessive cells through the other of said first fluid port (31) and second fluid port (33); outlet (S3) of the culture medium through the other of said first fluid port (31) and second fluid port (33) and through a third port (51) in fluid connection with an outflow channel (50 ), wherein a respective second end (24) of said plurality of spatially defined and spaced apart cell channels (20) is in fluid connection with a first end (42) of a respective first wash channel (40) having a second end (44) in fluid connection with said outflow channel (50), said first wash channels (40) are too small in size to accommodate said cells.
[0011]
11. Method for antibiotic sensitivity testing, characterized in that said method comprises: loading bacterial cells into a plurality of spatially defined and separated cell channels (20) of a microfluidic device (1) as described in any one of claims 1 to 9; exposing bacterial cells in different spatially defined and separated cell channels (20) of said plurality of spatially defined and separated cell channels (20) to different antibiotics and/or different concentrations of a antibiotic; and determine the antibiotic susceptibility of said bacterial cells based on a respective characteristic of phenotype, preferably, at least one of a respective growth rate, a respective degree of nucleoid compaction, a respective degree of metabolic activity and a respective degree of membrane integrity, of said bacterial cells in said plurality of spatially defined and separated cell channels (20).
[0012]
12. Method for IN SITU cell genotyping, characterized in that said method comprises: loading cells into a plurality of spatially defined and separated cell channels (20) of a microfluidic device (1) as described in any of the claims 1 to 9; attaching said cells to said plurality of spatially defined and separated cell channels (20); IN SITU egenotyping said cells in said plurality of said spatially defined and separated cell channels (20).
[0013]
13. Method for characterizing cell phenotype, characterized in that said method comprises: loading cells into a plurality of spatially defined and separated cell channels (20) of a microfluidic device (1) as described in either of claims 1 to 9; culturing said cells in said plurality of spatially defined and separated cell channels (20); monitor in real time a characteristic phenotype of said cells in said plurality of spatially defined and separated cell channels (20).

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CN106661530B|2021-06-04|

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