Microfluidic decanting using gaseous phase for biological cell samples, in particular in sedimented circulating tumor cells

Abstract

The invention relates to a method (500) for microfluidically decanting in a chamber (130) of a microfluidic device (100), the chamber (130) comprising a sedimentation substrate (135) having recesses (137) for receiving biological cells (15), in particular circulating tumor cells (15).

Description

[0001] Description

[0002] title

[0003] Microfluidic gas-phase decantation for biological cell samples, especially for sedimented circulating tumor cells

[0004] State of the art

[0005] Traditionally, various enrichment or depletion methods are used to analyze circulating tumor cells (CTCs) to increase the relatively small number of CTCs compared to the background, as described, for example, in D. Lin et al., “Circulating tumor cells: biology and clinical significance,” Signal Transduction and Targeted Therapy, vol. 6, no. 1 , Dec. 2021. Due to the rarity and great heterogeneity of CTCs in blood, significant losses often occur, so that often only a few cells can be isolated from a few milliliters of whole blood. There are also selection-free approaches that attempt to perform preprocessing with the lowest possible loss without loss-prone centrifugation steps.The CTCs are then detected by specifically labeling the cells, particularly using fluorescently labeled antibodies directed against specific tumor markers such as EpCAM (epithelial cell adhesion molecule), as described in K. Facher et al., “Standardized quantification of circulating peripheral tumor cells from lung and breast cancer,” Clinical Chemistry and Laboratory Medicine (CCLM), vol. 43, no. 6, Jan. 2005. However, to enable microscopic detection, interfering components (e.g., lysed erythrocytes, unbound antibodies, etc.) must be washed out or diluted.

[0006] To minimize losses as much as possible, structured substrates such as silicon with microcavities can be used in which the cells are retained during washing. For example, the published patent application DE 10 2021 203 897 A1 describes a microfluidic cartridge with a microfluidic chip with a multitude of etched depressions as a sedimentation substrate (referred to therein as a carrier substrate with microcavities), into which CTCs can be sedimented and subsequently counted based on their fluorescent labels.

[0007] Cavity height is much, for example by a factor of 22, smaller than the height of the chamber above the sedimentation substrate, which is reflected in a total volume of all microcavities of approximately 4 microliters (pL) compared to a volume above the substrate, the so-called headspace, of approximately 100 pL.

[0008] For the optical detection of sedimented CTCs, the blood lysate in the headspace must first be replaced with a more transparent medium. To ensure thorough displacement of the blood lysate, a clear wash buffer is typically used to achieve a broad horizontal flow across the substrate to ensure a uniform flow profile. The wash is then continued "wet-on-wet" until the remaining blood lysate, including that from the microcavities, is diluted to such an extent that well-defined optical detection is possible.

[0009] Disclosure of the invention

[0010] Advantages of the invention

[0011] Against this background, the invention relates to a method for microfluidic decantation in a chamber of a microfluidic device. The chamber comprises a sedimentation substrate with recesses for receiving biological cells. The biological cells can be cells of human or animal origin, in particular nucleated cells, in particular circulating tumor cells. The recesses can in particular be microcavities in the sedimentation substrate, for example, as described in the published patent application DE 10 2021 203 897 A1.

[0012] In a first step of the method, a first liquid phase located above an upper side of the sedimentation substrate is displaced by a gas phase moving in the chamber. The first liquid phase can be, in particular, a blood lysate, i.e., a liquid phase comprising lysed blood components, in particular lysed erythrocytes. Preferably, the first liquid phase comprises at least a portion of a biological sample containing biological cells, some of which have been taken up into the recesses, in particular by sedimentation. The gas phase can be, in particular, air, a single gas such as nitrogen, or a gas mixture.Alternatively, the cells accommodated in the recesses can already be located in the recesses before the first liquid phase is placed above the upper side of the sedimentation substrate, for example as a result of sedimentation of these cells from another phase previously located in the chamber. The displacement of the first liquid phase preferably occurs in such a way that a first majority of the accommodated cells remain in the recesses. For this purpose, depending in particular on the shape, in particular the width and depth of the recess, a pressure on the gas phase or a pressure in the gas phase can be set such that the first liquid phase is displaced from the upper side of the sedimentation substrate and residues of the first liquid phase, including the cells located therein, preferably largely remain in the recesses.

[0013] In a second step of the process, a second liquid phase is placed above the upper surface of the sedimentation substrate. This placement can be achieved, in particular, by displacing the gas phase with the second liquid phase. The second liquid phase can preferably be a buffer, in particular a transparent one.

[0014] In a third step of the process, the amount of residues of the first liquid phase remaining in the recesses is reduced by diffusion into the second liquid phase. Due to the placement of the second liquid phase and / or (with significantly less probability) such diffuse mixing, some of the captured cells may be transported out of the recesses again. However, after completion of the third step, a second majority of the captured cells advantageously remains in the recesses, whereby the second majority corresponds to the first majority in the loss-free limiting case.

[0015] By displacing the first liquid phase with a gas phase instead of with another liquid phase, in particular with a wash buffer, mixing of two liquid phases is advantageously avoided. Thus, a smaller volume and / or a smaller amount of gas phase and thus also less time is required to completely displace the first liquid phase from the region above the sedimentation substrate compared to using the other liquid phase. In particular, this prevents undefined states (as is possible with "wet-on-wet" rinsing) from occurring during rinsing, in particular different, undefined concentrations of the first or second liquid phase in the chamber.Furthermore, the use of the gas phase also eliminates the need for an alternative oil phase to displace the first liquid phase, thus preventing unwanted and, in particular, difficult-to-remove coating of the recesses in the sedimentation substrate with an oily film without impairing the cells located in the recesses. In particular, if the removal of the first phase from the recesses is not yet sufficient for optical analysis of the cells in the recesses, the steps of displacing, placing, and preferably also reducing the volume can be repeated. In this case, an unused liquid phase is preferably used as the new second liquid phase to avoid contamination of the recesses.According to a preferred embodiment, this new second liquid phase can comprise reagents, for example, reagents for fixation, permeabilization, and / or labeling of the captured cells. In the case of a marker, this can include, in particular, a dye, for example, a fluorescent dye, for subsequent optical analysis, in particular counting, of the captured cells. Alternatively, a liquid phase with reagents, in particular markers, can only be placed over the recesses once a predetermined reduction in the residues of the first liquid phase in the recesses has already occurred.

[0016] According to a preferred embodiment of the invention, the upper side of the sedimentation substrate has a rectangular, in particular square, basic shape. The displacement of the gas phase and preferably the placement of the second liquid phase preferably takes place from the direction of one of the corners of the upper side.

[0017] According to a further preferred embodiment of the invention, the chamber has a first inlet and a second inlet, so that liquid phases can be passed into the chamber through the first inlet and preferably expelled from the chamber again through the first inlet. Furthermore, gas phases can be passed into the chamber through the second inlet and preferably expelled from the chamber again through the second inlet. The two inlets can be arranged in different walls of the chamber, in particular in opposite walls, so that the sedimentation substrate is preferably located between the two inlets. The inlets, in particular the lower end of the inlets, can be arranged at different or the same height relative to the top side of the sedimentation substrate and / or can be of different or the same size.It is particularly preferred if the chamber is designed and / or oriented in the Earth's gravitational field such that the first inlet is located below the second inlet. This has the advantage that filling the chamber with the liquid phase against gravity supports the stabilization of a meniscus of the liquid phase and thus a well-defined spread of the liquid phase in the chamber. The same advantageous effect of meniscus stabilization is also advantageously achieved when the liquid phase is displaced back through the first inlet against gravity, in particular by introducing a gas phase through the second inlet. In particular, this utilization of gravity advantageously facilitates the introduction and removal of the liquid phases by gas phases and / or the gas phases by liquid phases when the densities of the gas phases are lower than the densities of the liquid phases.

[0018] According to an advantageous embodiment, a corner of the sedimentation substrate can be oriented toward the first inlet of the chamber, particularly if the sedimentation substrate has a rectangular, in particular square, basic shape. The sedimentation substrate can thus be arranged such that fluid entering the chamber through the first inlet flows onto the sedimentation substrate via the corner, as already mentioned above. For example, two sides of the sedimentation substrate can each be oriented at an angle of 45 degrees relative to the inlet.In contrast to a transverse arrangement of the sedimentation substrate with respect to the first inlet, i.e., one oriented at approximately 90 degrees, this design has the advantage that the inlet width can be reduced, since the fluid flow from the inlet onto the sedimentation substrate does not occur directly onto an edge of the sedimentation substrate, but initially only over the corner of the sedimentation substrate. A smaller inlet width, in turn, has the advantage that a more stable meniscus of the incoming fluid can form at the inlet, thus ensuring a more uniform filling of the chamber with the fluid.

[0019] The invention also relates to a microfluidic device configured to carry out the method according to the invention. Brief description of the drawings

[0020] Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures and having a similar effect, whereby a repeated description of the elements is omitted.

[0021] It shows

[0022] Figure 1 is a flowchart of an embodiment of the method according to the invention,

[0023] Figure 2 shows an embodiment of the device according to the invention,

[0024] Figures 3a-f show cross-sectional views through embodiments of the chamber of the device according to the invention at different times or at different orientations in the Earth’s gravitational field, and

[0025] Figures 4a-c embodiments of the chamber with the

[0026] Sedimentation substrate.

[0027] Embodiments of the invention

[0028] A preferred embodiment of the method according to the invention is described below. Flowchart 500 in Figure 1 schematically shows the corresponding method steps. The method steps described here differ from the method steps described above, particularly in their numbering. Figure 2 schematically shows an embodiment of the device 100 according to the invention, which is used in this example for the method 500. Preferably, the device 100 can be based on a microfluidic cartridge described in the published patent application DE 10 2021 203 897 A1.According to a first step 501 of the method 500, a blood sample, for example comprising 100-200 microliters (μl) of whole blood from a particularly immediately preceding blood draw from a human or animal, is provided and, in a second step, introduced into an input chamber 101 of the device 100, which can then preferably be closed. For example, a number or concentration of CTCs is to be determined for this sample. Before introduction, a substance, for example plasmin, can be added to the sample to unmask the CTCs, i.e., to expose structures on the surface of the CTCs that can be detected, for example, using staining reagents.

[0029] In the input chamber 101 or in another chamber 110 of the device, hereinafter referred to as the processing chamber 110, the sample can be mixed in a second step 502 with further liquids 80 or substances 80, which can be stored in a pre-storage chamber 120 of the device 100. The liquids or substances are preferably substances for lysing erythrocytes, for example in the form of an erythrocyte lysis buffer 151. The sample thus obtained with lysed erythrocytes is hereinafter also referred to as blood lysate. The substances 80 can also comprise staining reagents as color markers, in particular for CTCs, for example in the form of a staining solution 141.

[0030] The device 130 also comprises a chamber 130, wherein the chamber 130 comprises a sedimentation substrate 135 with recesses 137 on a surface 136 of the sedimentation substrate 135 for receiving and analyzing, in particular counting, biological cells. The recesses 137 can preferably be formed as microcavities 137. The sedimentation substrate 135 can be formed, for example, as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1.

[0031] For example, the chamber has dimensions of 12.5 x 12.5 x 0.64 mm with a volume of 100 pL, and the sedimentation substrate 135 has dimensions of 13.45 x 13.45 x 0.38 mm. The recesses 137, for example, have widths and depths in the single- to double-digit micrometer range to accommodate at least one cell. These microcavities 137 can be created in the silicon substrate using semiconductor technology or microsystem technology methods, for example, by deep plasma etching, in particular using the "Bosch-DRIE" process.

[0032] In a third step 503, the blood lysate is placed as a liquid phase above the surface of the sedimentation substrate 131 so that at least some of the cells, in this example CTCs, can sediment into the recesses of the sedimentation substrate 135.

[0033] After a predetermined period of time for the sedimentation, which can be 20 minutes, for example, the blood lysate is displaced from the surface 136 of the sedimentation substrate 135 and from the chamber 130 by a gas phase 50 in a fourth step 504. Figure 3a shows a schematic lateral sectional view through the chamber 130 and through the sedimentation substrate 135. The chamber 130 has a first inlet 131 and a second inlet 132, wherein the two inlets 131, 132 are arranged in different walls of the chamber 130, in particular in opposite walls, so that the sedimentation substrate 135 is preferably located between the two inlets 131, 132. The placement of the blood lysate 10 above the sedimentation substrate 135 in the third step can be achieved in particular by introducing the blood lysate through the first inlet

[0034] 131. If the first inlet 131 is arranged closer to the floor than to the ceiling of the chamber 130, as shown, filling of the chamber 130 can occur against gravity mainly from bottom to top, so that a meniscus 11 bounding the blood lysate is stabilized by gravity. The first inlet 131 is preferably located in the lower third, preferably in the lower fifth, most preferably in the lower tenth of the chamber 130, with "bottom" being defined by the wall of the chamber 130 on which the sedimentation substrate 135 is arranged. The second inlet 132, however, is arranged in the upper region of the chamber, as shown in Figure 3a, so that fluids entering through the second inlet 132, in particular gas phases, fill the chamber 130 during intended use mainly from "top" to "bottom". Alternatively or in addition to the arrangement of the inlets 131,

[0035] 132 At different heights, as shown in Figure 3d, the device 100 including chamber 130 can, when used as intended, also be tilted in the Earth's gravitational field, for example by 30° or 45°, in order to support filling from bottom to top and displacement from top to bottom of the liquid phases. The tilting can take place before the third step 503 and preferably remain during the subsequent steps. Figures 3e and 3f show two alternative embodiments of the chamber 130, wherein in both, as shown, the sedimentation substrate 135 preferably extends to the walls of the inlets 131, 132, so that no gap remains between the sedimentation substrate 135 and the walls.As shown in Figure 3e, the first inlet 131 is arranged higher in the wall than in the example from Figures 3a-d, namely at least or exactly as high that the first inlet 131 begins flush with the surface 136 of the sedimentation substrate 135. Thus, liquid entering through the first inlet 131 can flow directly onto the surface 136 at the same height. In the example according to Figure 3f, the second inlet 132 is arranged at the same height as the first inlet 131, and thus preferably also begins flush with the surface 136 of the sedimentation substrate 135. Furthermore, the two inlets 131, 132 can be designed to be significantly larger than in the examples in Figures 13ae. Even when using a chamber 130 as shown in Figures 3e or 3f, the device 100 including chamber 130 can, when used as intended, preferably be tilted in the gravitational field, as described above.

[0036] Preferably, the displacement of the blood lysate 10 after sedimentation in the fourth step 504 also takes place, as indicated in Figure 3a, again through the first inlet 131, for which purpose the gas phase 50 displacing the blood lysate 10, for example air at a pressure of 1.3 atmospheres, is moved, in particular pumped, through the second inlet 132 into the chamber 130 and in the process pushes the blood lysate out from diagonally above to diagonally below through the first inlet 131. Figure 3a schematically shows a snapshot of the displacement of the blood lysate 10, wherein the blood lysate 10 has already been partially displaced from the surface 136 and through the second inlet 132. As shown, residues of the blood lysate 10 remain in the recesses 137 or microcavities 137 together with the sedimented cells 15 due to the adhesion, capillary and gravity forces acting there.Figure 3b shows a snapshot after the blood lysate 10 has been completely displaced from the chamber 130 and the chamber 130 is completely filled with the gas phase 50.

[0037] In the fifth step 505 of the method 500, a second liquid phase 20, in particular a buffer 20, is placed above the upper side 136 of the sedimentation substrate 135 so that the residues of the blood lysate from the recesses 137 can diffuse into the second liquid phase and are thus removed from the recesses 137 or at least reduced in quantity. In other words, such diffuse mixing effectively replaces the remaining blood lysate in the recesses 137 by strongly diluting it with the second liquid phase, as shown in Figure 3c. When using a transparent second liquid phase, transparency now also prevails in the recesses 137, which enables undisturbed optical analysis, in particular counting, of the sedimented cells. The second liquid phase 20 is preferably, like preferably all liquid phases, moved, in particular pumped, through the first inlet 131 into the chamber 130.

[0038] The second liquid phase 20 can also comprise reagents for fixation (e.g., 4% formaldehyde and / or others) and permeabilization (e.g., 0.5% Triton X-100 and / or others) and / or one or more different markers for labeling the captured cells 15, for example, fluorescently labeled antibodies for intracellular antigens such as cytokeratin, vimentin, or others, and thus, in addition to the function of diffuse mixing, also assume the function of labeling the cells. These reagents can also be stored upstream in the device 130, for example, likewise in a pre-storage chamber, in particular for mixing as needed with the second phase 20, which is preferably also stored upstream. According to a particular embodiment, two or more of these reagents can also be pre-mixed with one another and / or pre-mixed with the second liquid phase 20.

[0039] Subsequently, in a sixth step 506, the second liquid phase can again be displaced from the chamber 130, preferably by a gas phase, and also optionally, particularly if more blood lysate needs to be removed from the recesses 137, in a seventh step 507, a further liquid phase can be placed above the upper side 136 of the sedimentation substrate 135, as in the fifth step 505, for a further reduction of the first phase in the recesses 137. This further liquid phase can also comprise reagents and / or markers as described above. These two steps 506, 507 can be repeated one or more times if necessary, preferably with unused liquid and / or gaseous phases, as indicated by the dashed arrow in Figure 1.In particular, the two steps 506, 507 can be repeated multiple times to fix, wash, permeabilize, wash, and label the captured cells, particularly in the following sequence: wherein the respective liquid phase comprises, depending on the step, fixation solution, permeabilization substance, marker, or none of these reagents. For example, depending on the need, all three steps of fixation, permeabilization, and labeling can be performed with washing steps in between, or the fixation and / or labeling steps can be omitted.

[0040] Figures 4a and 4b show two variants of an arrangement of the sedimentation substrate 135 with respect to the first inlet 131. The sedimentation substrate 135 can be arranged such that a fluid flows from the first inlet 131 via an edge 138 arranged vertically with respect to the inlet, as shown in Figure 4a, or via a corner 139 of the sedimentation substrate 135 oriented toward the inlet, as shown in Figure 4b and also in Figure 4c. The sedimentation substrate 135 can have a rectangular, in particular square, base area, as shown. An orientation of the corner 139 of the sedimentation substrate 135 with respect to the first inlet 131 as in Figure 4b or 4c has the advantage of a significant reduction in dead volume compared to the vertical orientation of the sedimentation substrate 135 as in Figure 4a, whereby it is accepted that a maximum flow velocity may belower than in the latter variant.

[0041] Figures 4b and 4c also show two alternatives for connecting the first inlet 131 to two fluid channels 161, 162. Instead of or in addition to the first inlet 131, the second inlet 132 can also be designed in this way. In Figure 4b, the two fluid channels 161, 162 merge, for example in the form of a Y-junction as shown, to form a third fluid channel 163, which then opens into the first inlet 131 of the chamber, as an intermediate channel, so to speak. In contrast, the two fluid channels 161, 162 open directly into the first inlet 131 without a third fluid channel 163 (see Figure 4c). These variants can also be realized with an inlet 131 transverse to an edge 138 of the sedimentation substrate 135, as shown in Figure 4a.For example, one of the two fluid channels 161 serves to transport a first fluid, for example, the sample as (part of) the first liquid phase, and the other fluid channel 162 serves to transport a second fluid, in particular the second liquid phase. This has the advantage that dead volume can be further reduced and / or the two channels can be flushed with different fluids and / or at different times.

Claims

Claims 1. A method (500) for microfluidic decantation in a chamber (130) of a microfluidic device (100), wherein the chamber (130) has a sedimentation substrate (135) with recesses (137) for receiving biological cells (15), in particular circulating tumor cells (15), comprising the steps: • Displacing (504) a first liquid phase (10) located above an upper side of the sedimentation substrate (135) by a gas phase (50) moving in the chamber (130), wherein a first plurality of received, in particular sedimented, cells (15) remains in the recesses (137). • Placing (505) a second liquid phase (20), in particular a buffer, above the top of the sedimentation substrate (135) and • Reducing an amount of residues of the first liquid phase (10) remaining in the recesses (137) by diffusion into the second liquid phase (20), wherein a second plurality of the absorbed, in particular sedimented, cells remains in the recesses (137).

2. The method (500) according to claim 1, wherein the first liquid phase (10) comprises at least a portion of a biological sample with biological cells, and wherein the sedimented cells (15) have passed from the first liquid phase (10) into the recesses (137) by sedimentation.

3. Method (500) according to claim 1 or 2, comprising repeating the steps of displacing (505), placing (506) and preferably reducing in this order, wherein preferably further, in particular not yet used, liquid phases are used.

4. Method (500) according to claim 3, wherein, when repeated, the liquid phase in the step of placing (506) contains reagents, in particular for a Fixation, permeabilization and / or marking of the recorded cells includes 5. Method (500) according to one of the preceding claims, wherein the upper side of the sedimentation substrate (135) has a rectangular, in particular square, basic shape and wherein the displacement (505) with the gas phase (50) and preferably the placement (506) of the second liquid phase takes place from the direction of one of the corners (139) of the upper side (136).

6. The method (500) according to any one of the preceding claims, wherein the chamber (130) has a first inlet (131) and a second inlet (132), and wherein liquid phases are passed through the first inlet (131) into the chamber (130) and preferably displaced from the chamber (130) through the first inlet (131) and / or wherein gas phases (50) are passed through the second inlet (132) into the chamber (130) and preferably displaced from the chamber (130) through the second inlet (132).

7. The method (500) according to claim 6, wherein the chamber (130) is designed and / or oriented in the Earth's gravitational field such that the first inlet (131) is located below the second inlet (132).

8. Microfluidic device (100) comprising a chamber (130), wherein the chamber (130) has a sedimentation substrate (135) with recesses (137) for receiving biological cells (15), in particular circulating tumor cells (15), wherein the device (100) is in particular configured to carry out a method (500) according to one of the preceding claims.

9. Microfluidic device (100) according to claim 8, wherein a corner (139) of the sedimentation substrate (135) is oriented towards an inlet (131, 132) of the chamber (130), so that in particular a flow onto the sedimentation substrate (135) by fluid (10, 20, 50) entering the chamber (130) through the first inlet (131) takes place via the corner (139).

10. Microfluidic device (100) according to claim 8 or 9, wherein two fluid channels (161, 162) located outside the chamber (130) open directly into the first inlet (131) of the chamber (130).

Images