METHOD FOR DEFORMING DEFORMABLE BODIES, AND DEVICES FOR THIS PURPOSE

Abstract

A method for deforming deformable bodies, preferably droplets or cells, comprising feeding a sample fluid into a microfluidic channel to create a laminar flow of the sample fluid, wherein the sample fluid transports deformable bodies, and feeding a sheath fluid into the microfluidic channel to create a laminar flow of the sheath fluid such that the sheath fluid directly borders the sample fluid in a border region of the microfluidic channel and flows in the same direction as the sample fluid at least in the border region. The viscosity of the sheath fluid is greater than the viscosity of the sample fluid, and the average flow rate of the sample fluid is greater than that of the sheath fluid.

Claims

1. A method for deforming deformable bodies, preferably droplets or cells, comprising the following steps: feeding a sample fluid into a channel to create a laminar flow of the sample fluid, wherein the sample fluid transports deformable bodies, feeding a sheath fluid into the channel to create a laminar flow of the sheath fluid such that the sheath fluid directly borders the sample fluid in a border region of the channel and flows in the same direction as the sample fluid at least in the border region, wherein at the shear rates of the sheath fluid and sample fluid occurring in the channel the viscosity of the sheath fluid is greater than the viscosity of the sample fluid, wherein the deformable bodies are deformed by the forces arising in the sample fluid in the channel due to the border region.

2. The method according to claim 1, wherein the dynamic viscosity of the sheath fluid in the border region is within a range of 1 mPa s to 1 Pa s, preferably of 50 mPa s to 250 mPa s and/or wherein the dynamic viscosity of the sample fluid in the border region is within a range of 1 mPa s to 100 mPa s, preferably of 5 mPa s to 50 mPa s.

3. The method according to claim 1, wherein the average flow rate of the sample fluid is within a range of 0.1 cm/s to 10 m/s and/or wherein the average flow rate of the sheath fluid is within a range of 0.1 cm/s to 10 m/s.

4. The method according to claim 1, wherein the sample fluid is a shear-thinning liquid containing the deformable bodies.

5. The method according to claim 1, wherein the sample fluid and the sheath fluid consist of liquids which do not mix, or only do so to a minor extent, at least within the time scale during which the deformable bodies traverse the border region.

6. The method according to claim 1, wherein the sheath fluid is a Newtonian or shear-thickening liquid.

7. A method for determining the mechanical properties of deformable bodies, preferably cells, wherein deformable bodies are deformed using the method according to claim 1 and wherein the deformation of the deformable bodies is preferably measured using an optical method.

8. A method for examining the cell biological properties of cells, comprising the deformation of cells with the method according to claim 1 and the examination of the biochemical properties of cell components, in particular the cytoskeleton or organelles, based on changes in these cell components owing to deformation, wherein the changes are preferably measured using a fluorescence-based method, in particular flow cytometry.

9. A method for sorting deformable bodies, in particular cells, comprising: performing the method according to claim 7 and sorting cells based on the particular mechanical and/or cell biological properties, preferably with a flow cytometer with a sorting function.

10. The method according to claim 1, wherein prior to the start of measuring the deformation of the deformable bodies the total flow rate of the sample fluid and sheath fluid is increased from an initial value to a higher target value.

11. A device which is designed to perform the method according to claim 1.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0045] FIG. 1 is a photo of a microfluidic channel for performing a method according to the invention. A laminar flow of sample fluid is channelled into a narrow channel through two flows, which are also laminar, of sheath fluid.

[0046] FIG. 2 is a top view of a section of the microfluidic channel. Bright field picture with transmitted light focusing on the narrow channel 12.

[0047] FIG. 3 shows the behaviour of the relative width of the flow of the sample fluid to the total width of the channel depending on the flow rate. The insets show images of the channel at different flow rates.

[0048] FIG. 4 shows the deformation behaviour of a leucocyte according to the prior art.

[0049] FIG. 5 shows the deformation behaviour of a leucocyte according to the method according to the invention.

[0050] FIG. 6(a) shows a scatter plot for leucocytes according to the prior art.

[0051] FIG. 6(b) shows a scatter plot of a leucocyte according to the method according to the invention.

[0052] FIG. 7 shows measurement results as regards the dynamic viscosities of the liquids used.

DETAILED DESCRIPTIONS OF THE DRAWINGS

[0053] FIG. 1 shows a photo of the microfluidic chip used. The microfluidic channel 12 comprises an inlet 12a in which the sheath fluids 11 are fed from two sides of a sample fluid 10. The directions of flow within this channel are each depicted with arrows. As can be seen from FIG. 1, the two sheath fluids 11 surround the sample fluid 10 symmetrically. Once these sheath fluids 11 have traversed the microfluidic channel 12, they are discharged from the outlet 13.

[0054] A detailed view of the microfluidic channel 12 can be seen in FIG. 2. As is clearly evident here, the flow of sample fluid 10 is also clearly separated from the two side flows of sheath fluid 11 within the microfluidic channel 12. This is even visibly shown in a bright field picture, as can be seen in FIG. 2. To this extent, the flow of sample fluid 10 flows in a virtual channel within the microfluidic channel 12, which is delimited by the sheath fluid 11.

[0055] With regard to basic physical principles, it should be noted that in laminar flow systems, which are characterised by low Reynolds numbers (a Reynolds number of less than 1000), a flow forms along a channel in the direction of flow which has a parabolic speed profile perpendicular to the direction of flow. In particular, the edges of the channel are subject to the condition that the speed of the molecules must be 0. At the same time, the highest speed is achieved at points where the flow of molecules is least disturbed by the edges or other flows. For channels with a laminar flow, this is in the middle. At the boundary between the two fluids, the speed of these fluids is not zero.

[0056] As shown in FIG. 1, the sheath and sample fluids 11 and 10 flow from right to left, wherein the sheath fluids 11 channel the sample fluid 10. These fluids 10, 11 flow in a laminar formation through the microfluidic channel 12, which in the example shown in FIG. 2 measures 40 m40 m in the cross-section perpendicular to the direction of flow. Within the narrow virtual microfluidic channel 12 through which the sample fluid 10 flows, the speed of the flow of sample fluid 10 increases to roughly 50 cm per second in the middle of the channel. The width of the virtual channel through which the sample fluid 10 flows depends on the flow rates transmitted by syringe pumps which provide the flow of the sheath fluid 11 and the sample fluid 10, depending on the given channel size and viscosities. Syringe pumps are advantageous, as they allow precise control of the flow volume with an accuracy of nl/s. In an RT-DC experiment, the ratio between the flow rates in the sheath fluid and sample fluid is adjusted so that the cells flow through the middle of the microfluidic channel 12. With virtual channel resizing, the sheath fluid in the present embodiment consists of a polymer solution (e.g. 100 mMol polyethylene glycol 8000 in a phosphatic buffer solution) which is significantly more viscous than the sample fluid. As shown, the flows only mix slightly, as can be clearly seen from the clear edges in FIG. 2. This was confirmed by finite element simulations. Owing to the flow time of the two fluids, there was no diffusion or only minimal diffusion.

[0057] In the present embodiment, virtual channel resizing is performed as follows: The composition of the sheath fluids 11 is selected so that they have a significantly elevated viscosity compared to the sample fluid 10, although the shear rate has little bearing on this. The first experiments were carried out with polyethylene glycol (PEG) 8000 fully dissolved with a concentration von 100 mMol in a PBS (phosphate-buffered saline solution). PBS not containing notable quantities of calcium and magnesium was used. However, another liquid can, in principle, also be used. The solution has a dynamic viscosity of roughly 235 mPa s (at a shear rate of 1 1/s-10,000 1/s). This viscosity was measured with an MC302 rheometer from Anton Paar with a cone-plate system with a diameter of 50 mm, an angle of 2 degrees and with a cylinder system. The solutions in the sample flow were measured with a rolling ball viscometer (Anton Paar, Lovis2000-DMA). The flow rates at the edge of the channel are then reduced by a factor of more than 20. An analytical description of this behaviour can be found in J. Li, P. S. Sheeran, C. Kleinstreuer, Analysis of Multi-Layer Immiscible Fluid Flow in a Microchannel, J. Fluids Eng. 133, 111202 (2011).

[0058] The parabolic profile of the flow of sample fluid 10 is formed by means of a virtually smaller channel which now exists owing to the boundary area between the sample fluid 10 and the sheath fluids 11. To this end, the changes in shear rate perpendicular to the direction of flow in the flow of the sample fluid become greater compared to the RT-DC according to WO 2015/024690 A1. This causes greater shear and normal forces in the sample fluid 10 than was the case in previous RT-DC tests. The flow rates or chips could also be changed here, but this would reduce the comparability of the results. In addition, disadvantages such as frequent chip changes and/or blockages of the channels are also avoided.

[0059] The following preferred conditions for the composition of the sheath fluids 11 result from the aforementioned observations: It is of advantage if the composition of the materials and solutions for the sample fluid 10 and the sheath fluids 11 are selected so that these fluids 10, 11 do not mixowing to diffusionin the time scale during which the deformable objects are being deformed and flow through the channel.

[0060] It is further of advantage if the material/solution of the sheath fluid 11 has a higher viscosity than the material/solution of the sample fluid 10. However, it is also conceivable that the sheath fluid and sample fluid have the same viscosity at low shear rates, but the sample fluid is shear-thinning. The viscosity of the sheath fluid 11 should depend as little as possible on the shear rate (in other words, it should be Newtonian) or be shear-thickening. For PEG 8000 (100 mMol) in PBS, for example, the dynamic viscosity (measured with a rheometer) is approximately 235 mPa s over a shear rate range of 1 per second to 10,000 per second.

[0061] It is further of advantage if the material/solution of the sample fluid is shear-thinning. For methyl cellulose (0.5%) in PBS, the dynamic viscosity follows a power law with an exponent of 1 to 0.677 (Herold, ArXiv 2017, https://arxiv.org/ftp/arxiv/papers/1704/1704.00572.pdf).

[0062] Successful realisations of virtual channel resizing can be achieved with different conditions. This was shown in experiments using the compositions of sample fluid and sheath fluid listed in the table below:

TABLE-US-00001 Successful realisation Sample fluid material Sheath fluid material 1 Methyl cellulose (0.5% w/v) Polyethylene glycol 8000 in PBS, dynamic viscosity (100 mM) in PBS, dynamic 14 mPa s, shear-thinning, viscosity 235 mPa s, 285 mOsm almost Newtonian, >2800 mOsm 2 Methyl cellulose (0.7% w/v) Polyethylene glycol 8000 in PBS, dynamic viscosity (100 mM) in PBS, dynamic 21 mPa s, shear-thinning, viscosity 235 mPa s, 289 mOsm almost Newtonian, >2800 mOsm 3 Methyl cellulose (0.5% w/v) Polyethylene glycol 8000 in PBS, dynamic viscosity (20 mM) in PBS, dynamic 14 mPa s, shear-thinning, viscosity 18 mPa s, almost 285 mOsm Newtonian, approximately 600 mOsm 4 Methyl cellulose (0.5% w/v) Polyethylene glycol 6000 in PBS, dynamic viscosity (100 mM) in PBS, dynamic 14 mPa s, shear-thinning, viscosity 32 mPa s, almost 285 mOsm Newtonian, >1400 mOsm

[0063] For all realisations listed in the table, well-defined separation boundaries form at the boundaries between the sample fluid and sheath fluid at total flow rates within a range of 3 nl/s to 400 nl/s, regardless of the flow rates used. This can clearly be seen by means of bright field microscopy, as shown in FIGS. 2, 3 and 5, and is even significantly more pronounced if phase contrast microscopy is used.

[0064] In the invention, the flow rates of the sample fluid 10 and sheath fluids 11 were adjusted independently of each other using pumps. This can also be achieved by means of pressure, electroosmosis, capillary forces and hydrostatics. Using the virtual channel resizing according to the invention, the width of the flow of sample fluid 10 can be adjusted, thus directly impacting its parabolic flow profile, as previously discussed.

[0065] There are two possible ways of achieving this. Firstly, the ratio of the flow rates of the sheath fluids 11 and sample fluid 10 can be adjusted, as in the conventional RT-DC experiment according to WO 2015/024690 A1, in order to adjust the channelling of the sample fluid 10. It is important, however, that the flow profile in the microfluidic channel 12 is also dependent on the absolute flow rate. A corresponding effect is shown in FIG. 3, for example.

[0066] Secondly, the width of the flow of the sample fluid 10 can also be adjusted in virtual channel resizing by altering the composition of sample fluid and sheath fluid and the absolute channel width. The compositions influence the width of the flow across the dynamic viscosities. This behaviour is described in Li et al., equation [20].

[0067] FIG. 3 shows the behaviour of the relative width of the flow of sample fluid 10 in relation to the total width of the channel 12, respectively perpendicular to the direction of flow in the microfluidic channel 12 depending on the total or absolute flow rate. It is noticeable here that the behaviour as the flow rate increases (arrow pointing upwards to the right) is fundamentally different from the behaviour as the flow rate falls (arrow pointing downwards to the left). In particular, the ratio of the relative width of the flow of sample fluid 10 to the width of the channel 12 primarily plateaus as the flow rate rises above a certain flow rate (approximately 200 nl/s in the present case), whereas there is no such plateau as the flow rate falls. The experiment shown in FIG. 3 was performed with composition 1 (see table) in a microfluidic channel with a cross-section of 40 m40 m. The relative width is of course dependent on the edge length of the channel cross-section, as verified or predicted by Li et al., J. Fluids Eng. 2011, 111202.

[0068] It can also be seen in FIG. 3 that it is possible using virtual channel resizing to cover a further range of virtual channel widths, wherein only the pumps specifically have to be controlled. It is neither necessary to change the solutions, nor do the samples have to be changed.

[0069] In the present example, the ratio of the flow of sample fluid 10 to the flow of sheath fluid 11 is 2 to 1 for all absolute flow rates shown in FIG. 3. The relative width of the flow of sample fluid 10 can vary between 14% (at 4 nl/s) and 27% (at 250 nl/s and on increase of the pump power). The two insets in FIG. 3 illustrate how this change can be mapped visually and show phase contrast images of the respective channels 12. The sample fluid 10 consists of 0.5 percent by weight methyl cellulose in PBS, the sheath fluid of 100 mM PEG8000 in PBS.

[0070] It is therefore possible to adjust the hydrodynamic conditions to the cells to be examined, as the channel diameter can respectively be adapted as desired to the diameter of the cells. To this extent, it is possible to use a microfluidic channel 12 which is significantly larger than the cells to be examined or the bodies to be examined and then to make the channel smaller virtually so that the channel matches the cells or bodies to be examined.

[0071] A hysteresis of the relative width of the flow of sample fluid 10 relative to the width of the microfluidic channel 12 depending on the control of the pumps can further be seen in FIG. 3. Over a large interval of absolute flow rates, the relative width of the flow of sample fluid 10 is greater when the pump power is increased than when it is reduced.

[0072] To illustrate the use of virtual channel resizing, the experiment on leucocytes in a whole blood measurement is shown in FIGS. 4 to 6 below. Once taken, the blood is stored in a citrate solution and diluted for the RT-DC assay in a methyl cellulose PBS solution at a ratio of 1 to 20 for the flow of sample fluid 10. FIG. 4 shows one granulocyte and one erythrocyte (on the left and right respectively) within a 40 m40 m channel, wherein 0.6 percent by weight methyl cellulose is used in PBS, in other words wherein both the sheath fluid and sample fluid consist of the same solution. FIG. 6(a) shows a scatter plot of the leucocytes in the same test set-up. It can be seen from this scatter plot that the granulocytes only deform slightly.

[0073] FIGS. 5 and 6(b) show a similar experiment, with the material of the sheath fluid PEG 8000 (100 mmol) being in PBS. As can clearly be seen from FIG. 5, a leukocyte is clearly deformed along the direction of flow. It can be seen from the scatter plot in FIG. 6(b) that the leukocytes have a significantly increased deformation.

[0074] Consequently, it is clear that the virtually narrowed flow of sample fluid 10 leads to a significantly more marked deformation, in other words a shift in the representation shown in FIG. 6 upwards along the Y axis, in other words the cell population is divided into sub-populations. It is also apparent from the drawings in FIGS. 4 and 5 how pronounced the differences in form are.

[0075] It is clear from the examples that cells can be deformed by means of virtual channel resizing, irrespective of the respective size of the channel 12. A relevant flow behaviour is also shown in FIG. 4 of Li et al., wherein Case II in this figure corresponds in many aspects to the presently used system.

[0076] The present invention allows the width of the flow of sample fluid 10 to only be adjusted through the choice of materials of both the sheath fluid and sample fluid, the choice of volumetric flow rate of the two fluids (e.g. using corresponding syringe pumps), and through the cross-section of the channel 12. This is described in the equations [20] and [21] in Li et al., whereby equation [21] describes the shear tensions. The deformations then depend solely on a virtual channel width and the viscosity, in other words the width of the flow of sample fluid 10. Use of the invention is therefore not confined to microfluidic chips, and it can also be produced in other geometries. It is thus possible, using the respective sheath fluids and pump settings, to achieve the same effects in glass cuvettes or tubes which may have a larger diameter by up to a few millimetres but can also be filled with sample fluid and sheath fluid(s).

[0077] FIG. 7 further shows the rheological properties of the liquids used for the sheath fluid and the sample fluid. As can be seen from the figure, methyl cellulose in PBS manifests a slight shear-thinning behaviour, while PEG in PBS presents a primarily Newtonian behaviour. In particular, it is relevant that these two liquids manifest this behaviour across the relevant shear rate range (1 1/s-10,000 1/s).

[0078] As part of the present invention, the respective dynamic viscosities in the sheath fluid are equal to or greater than the dynamic viscosity of the sample fluid. However, it would, in principle, also be conceivable for the viscosity ratio to be chosen the other way around and for cells or bodies to still be deformed, even if such a design is currently regarded as being less advantageous.

[0079] A further aspect of the invention relates to the use of the method according to the invention for feeding substances into deformable bodies, in particular cells, wherein a substance to be fed in is contained in the sheath fluid and wherein parts of the deformable body are in contact with the sheath fluid, whereby this substance merges into the body owing to this contact. It is preferred here that the surface via which the deformable body is in contact with the sheath fluid is controlled and that the period of time during which the deformable body is in contact with the sheath fluid is controlled in order to control the amount of substance fed. Such a method allows the controlled feeding of substances, in particular medications, into deformable bodies, in particular cells.