Method and microfluidic device for preparing a sample for analysis of biological cells, in particular circulating tumor cells

Abstract

The invention relates to a method (500) for preparing a sample for analysis of biological cells, in particular circulating tumor cells, and a microfluidic device (100), wherein a first part of the sample is directed into an analysis chamber (130) of the device (100) for analysis and a second part of the sample is directed into a parking chamber (120) of the device (100) for later removal from the device (100).

Description

State of the art

[0001] Circulating tumor cells (CTCs) are cells that can detach from the primary tumor, penetrate the peripheral bloodstream, and then reach surrounding tissue. These cells, which are present in the blood even despite surgery and therapy, are referred to as "minimum residual disease (MRD)" and are regarded as the starting point for metastases and recurrences, as described, for example, in Meng et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 10, 2004, 24, pp. 8152-8162. Their detection and quantification (CTC count) is therefore relevant for both prognostic statements and clinical management (Cristofanilli et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin. Oncol. 33, 2006, 3, p.9-14), particularly for the detection of metastasis at an early stage and for efficiency control and adjustment during a therapy procedure.

[0002] The quantification of CTCs from a so-called "liquid biopsy," i.e., a blood sample from a tumor patient, has the advantage that it can be performed as often as desired, as it is a minimally invasive procedure involving a blood draw. The greatest challenge is the very small number of CTCs relative to blood cells—especially in the MRD stage or in the very early stages of metastasis. CTC detection therefore requires highly sensitive detection methods to detect the very small number of CTCs among millions of red and white blood cells early and efficiently. A sedimentation array offers a method for quantifying CTCs from a blood sample after fluorescent staining with tumor cell-specific antibodies of one type (e.g., EpCAM-FITC) or of multiple types (e.g., EpCAM, cytokeratin, HER2-neu, etc.) using optical detection with as little loss as possible. This allows nucleated cells from a blood sample to settle in wells of a single plane after erythrocyte lysis, where they can be detected and counted with spatial resolution using optical systems. Furthermore, the capture of the cells in these wells allows for flushing with various media and reagents to remove excess lysis buffer, erythrocyte lysate, free dyes, or similar substances, and / or to add additional dye labels without the target cells changing their position or being flushed out again.The published patent application DE 10 2021 203 897 A1 describes a suitable microfluidic cartridge comprising a microfluidic chip with a plurality of etched wells as a sedimentation array, into which CTCs can be sedimented and subsequently analyzed and counted by means of their various fluorescent labels. Disclosure of the inventionAdvantages of the invention

[0003] Against this background, the invention relates to a method for preparing a sample for the analysis of biological cells. The biological cells can be cells of human or animal origin, in particular nucleated cells, especially circulating tumor cells.

[0004] In a first step of the procedure, a blood sample is provided. This blood sample may be whole blood. The sample may have been obtained from a human or animal, particularly from a blood sample taken immediately prior to the blood sample.

[0005] According to a second step, the sample is introduced into an input chamber of a microfluidic device, in particular a microfluidic cartridge, wherein the input chamber is fluidically connected or connectable to an analysis chamber and a parking chamber. "Connectable" is to be understood in particular as meaning that the fluidic connection can be enabled and interrupted, for example, via a valve. "Fluidically connected" is also to be understood in particular as such fluidic connectability. The microfluidic device can in particular be based on the microfluidic cartridge from the above-mentioned published patent application DE 10 2021 203 897 A1 or represent a further development of this cartridge. The analysis chamber comprises an analysis substrate for receiving and analyzing, in particular counting, cells from the sample.In this context, the uptake of cells can be understood as contacting the cells with the analysis substrate, in particular deposition of the cells on a surface of the analysis substrate due to sedimentation. The analysis substrate preferably comprises an array with cavities, so that the cells can enter the cavities, in particular sediment therein (and the analysis substrate and the array can also be referred to as a sedimentation substrate or sedimentation array). The invention thus also relates to such a microfluidic device, in particular a microfluidic cartridge, with an input chamber and a fluidically connected storage chamber or a fluidically connected analysis chamber with an analysis substrate, in particular a sedimentation array with cavities.

[0006] In a third step, a first part of the sample is fed into the analysis chamber and in a fourth step, a second part of the sample is fed into the parking chamber, whereby the fourth step can also take place simultaneously with or before the third step. The sample fed into the device is thus divided into at least two parts, whereby the first part is fed into the analysis chamber for analysis, while the second part is stored in the parking chamber. In a particular embodiment of the invention, the parking chamber or the analysis chamber is part of the input chamber or corresponds to the input chamber. In other words, the sample can be fed directly into the parking chamber or, alternatively, directly into the analysis chamber and, in this embodiment, after the sample has been divided, the first part is fed from the parking chamber into the analysis chamber or the second part is fed from the analysis chamber into the parking chamber.

[0007] According to a fifth step, cells of the first part of the sample that have been picked up from the analysis substrate, in particular those that have entered the wells of the array, are analyzed. The picking up of these cells or the entry of these cells into the wells can, as explained above, preferably take place by sedimentation of the cells by placing at least a part of the first part of the sample above the analysis substrate or above the wells. The analysis can, in particular, comprise a count of the picked up cells. Preferably, one or more types of cells are counted, in particular nucleated cells, in particular CTCs. The affiliation of a cell to one or more such types of cell can, in particular, be determined based on a marking, in particular one or more color markings, of the cells.For this purpose, one or more markers, particularly preferably dyes coupled to antibodies, can be added to the sample, for example fluorescent dyes coupled to antibodies, which specifically bind to selected surface structures such as surface proteins or other surface molecules, for example to the epithelial cell adhesion molecule (EpCAM) or to cytokeratin (CK) in the case of CTCs. The addition of the markers or mixing with the markers can take place before or after the division of the sample into at least two parts. For this purpose, the markers can be stored upstream in the device, for example in a chamber fluidically connected to the input chamber or the analysis chamber. If the addition or mixing takes place after the division of the sample, the addition or mixing can take place in an area, for example another chamber, upstream of the analysis chamber or in the analysis chamber, i.e. generally before or after the third step.

[0008] According to an advantageous embodiment of the invention, lysis of cells, in particular certain types of blood cells, in particular erythrocytes, is carried out in the sample, in particular in the first part of the sample. The lysis can be carried out by adding lysis substances or a lysis agent, in particular in the form of a lysis buffer. This has the advantage that the cells to be lysed, in particular the unlysed erythrocytes, do not sink to the surface of the analysis substrate, in particular not into the cavities of the array, thereby blocking the surface or the cavities for the cells to be examined, in particular to be counted. Due to the forced opening of their cell membranes, the composition of the lysed cells largely corresponds to the fluidic environment, so that the lysed cell relics no longer sink, in particular sediment.

[0009] In a sixth step of the method, at least a portion of the second sample is removed from the parking chamber, in particular for further analysis inside or outside the device. For this purpose, the device can have a preferably reversibly closable opening that is fluidically connected to the parking chamber.

[0010] The invention has the advantage that for an initial analysis of the sample, in particular the counting of relevant cells in the sample, only the first part of the sample is used and possibly consumed. The first part of the sample can be regarded as a representative part of the entire sample, so that findings from the analysis of the first part can be used for the use of the remainder, in particular the parked second part. Another advantage is that, due to the division, the first part of the sample can be omitted for further analysis. In particular, the first part of the sample and in particular the cells sedimented in the wells do not have to be removed from the analysis chamber or from the array again, which could be laborious. Furthermore, the parked second part of the sample can be prepared for further analysis in the meantime, for example by processing the second part.The processing may, in particular, comprise pretreatment of the second part for a polymerase chain reaction and / or for cell sorting / dielectrophoresis and / or for gene sequencing, for example, adding substances for these processes or removing or inactivating certain components of the second part. According to an advantageous embodiment, the second part can be temperature-controlled in the device, in particular in the parking chamber, in particular kept at a temperature within a predetermined temperature range, for example, for cooling the second part.

[0011] According to a particularly advantageous development of the invention, the second portion of the sample is mixed with a buffer. This mixing can take place in the parking chamber or in an area, for example, a chamber, outside the parking chamber, i.e., in particular, before the fourth step of conveying the second portion into the parking chamber. In particular, the buffer can be used to dilute the second portion, for example, to dilute lysis agents and / or markers, in particular dyes or dyes coupled to specific antibodies, present in the second portion.

[0012] In a particularly preferred embodiment of the invention, leukocytes in the second part of the sample are depleted. Depletion is understood in particular to mean a reduction or even neutralization of the effect of the leukocytes. This can occur in particular through damage, for example through chemical substances, in particular through lysis, or through impairment of the mobility or even immobilization of the leukocytes, in particular through binding to leukocyte-specific antibodies. This is an advantageous preparatory step for a subsequent analysis, since the large number of leukocytes can make carrying out the analysis more difficult and / or reduce the quality of an analysis result. The damaging substances and / or the antibodies can preferably be arranged in the parking chamber and / or in a channel leading to the parking chamber, in particular upstream.At least some of the antibodies can be immobilized on one or more walls and / or structures, particularly protrusions, columns, elevations, and / or depressions. This has the advantage that the leukocytes are then also indirectly immobilized on these walls or structures via binding to the antibodies and are thus effectively removed from the second part of the sample.

[0013] According to another particularly advantageous embodiment of the invention, a number of cells captured by the analysis substrate, in particular a number of cells entering cavities of the array, is determined from the first portion of the sample, preferably after a predetermined sedimentation period, and, depending on this number, a quantity of the second portion of the sample is determined for further analysis. This determined quantity of the second portion can then be sent for further analysis, either inside or, after removal from the storage chamber, outside the device. In particular, the size of the quantity can correlate positively, in particular linearly, with the number of cells determined.The more cells sought are detected in the wells, especially with a fixed sedimentation time, the higher the concentration of these cells can be assumed in the entire sample and thus also in the second part, and the lower the amount of the second part used for further analysis can be in order to nevertheless reasonably assume a minimum number of this type of cells in the amount used.

[0014] The invention, in particular the microfluidic device and the method, can preferably also be applied to other discrete components, in particular particles, in blood or other biological fluids, in particular body fluids, which can sediment onto the analysis substrate. Thus, according to an advantageous development, the preparation and analysis of biological cells according to the invention can be understood as the preparation or analysis of such discrete components or particles in the blood. Short description of the drawings

[0015] Embodiments of the invention are illustrated schematically in the drawings and explained in more detail in the following description.

[0016] It shows Fig. 1 a flowchart of an embodiment of the method according to the invention and Fig. 2 an embodiment of the device according to the invention Embodiments of the invention

[0017] In the following, a preferred embodiment of the method according to the invention is described, for which purpose the flow chart 500 in Fig. 1 schematically shows the corresponding process steps. The process steps described here differ in some respects from the process steps described above, particularly with regard to their numbering. Fig. Figure 2 schematically shows an embodiment of the device 100 according to the invention, which in this example is used 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.

[0018] As in Fig. 2, the device 100 has an input chamber 110 for receiving a sample, an analysis chamber 130 for receiving a first portion of the sample, and a parking chamber 120 for receiving a second portion of the sample. The analysis chamber 130 and the parking chamber 120 are each fluidically connected to the input chamber 110, whereby both chambers 120, 130 can also be connected directly to one another, preferably via a valve. The analysis chamber 130 comprises an analysis substrate 131 for receiving cells from the sample. For this purpose, the analysis substrate 131 can preferably be designed as a sedimentation array 131 and preferably have microcavities for receiving individual cells, for example, shaped as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1.Microcavities are understood to be recesses on a surface of the array 131, in particular the silicon substrate, wherein the recesses have widths and depths in the single- to double-digit micrometer range for accommodating at least one cell. The device 130 further comprises additional storage chambers 140, 150, 160, which are described below. All chambers can preferably be closed by valves (not shown) and filled and emptied using conventional microfluidic operations, in particular with the aid of microfluidic membrane pump chambers (not shown).

[0019] According to a first step 501 of the method 500, a blood sample, for example, comprising 100-200 microliters (µl) of whole blood from an immediately preceding blood draw from a patient, is provided and, preferably after unmasking, for example, with plasmin in the case of CTCs to remove receptor-masking entities on the surface of the cells, is introduced into the input chamber 110 of the device 100 in a second step, which can then be sealed. For example, for this sample, a number or concentration of CTCs is first determined, and then a portion of the sample is analyzed in more detail.

[0020] In the input chamber 110 or in another chamber of the device, the sample can be mixed with further liquids or substances, in particular with unmasking or staining reagents, for example in the form of different dyes bound to different specific antibodies as color markers, in particular for CTCs, for example in the form of a staining solution 141, and / or with substances for erythrocyte lysis, for example in the form of an erythrocyte lysis buffer 151, and which can be stored in a first pre-storage chamber 140 or in a second pre-storage chamber 150.

[0021] First, in a third step 503, for example, the staining solution 141 from the first pre-storage chamber 140 can be added to the blood sample. After incubating the blood sample with the staining solution 141, in a fourth step 504, erythrocyte lysis buffer 151 is pumped from the second pre-storage chamber 150 to the sample, for example, in a ratio of 1:5 (alternatively, for example, 1:10 or 1:20). This increases the sample volume to 500-1000 µL. The staining reagents and the substances for erythrocyte lysis can alternatively also be stored as components of a common buffer, also referred to as an "all-in-one" buffer, for example in the second pre-storage chamber.

[0022] The first half of this diluted sample is then pumped to the analysis chamber 130 in a fifth step 505, while the second half is directed into the parking chamber 120 in a sixth step 506. Both steps 505, 506 can also be performed in reverse order or, in particular, simultaneously. Alternatively, a different division of the sample into at least two parts is also possible, for example, directing a quarter of the sample for CTC counting to the analysis chamber 130 and directing the remaining three-quarters into the parking chamber 120. In the parking chamber 120, the second part of the sample directed there can be mixed, for example, in a 1:1 ratio with a physiological buffer 161 or a nutrient medium to mitigate further influence by the erythrocyte lysis buffer 151 and prevent negative changes to the sample. The physiological buffer 161 is preferably stored in a third pre-storage chamber 160.Alternatively or additionally, the second portion of the sample can be mixed with another, preferably also pre-stored lysis buffer, in order to lyse further cells, for example leukocytes or platelets as required. Alternatively or additionally, the parking chamber 120 can also be temperature-controlled, in particular cooled relative to room temperature, for example by contacting the parking chamber 120, in particular one or more walls of the parking chamber 120, with a cooling and / or heating element 132, for example comprising a Peltier element or based on an endothermic or exothermic chemical reaction, wherein the cooling and / or heating element can also be brought into contact with an outer wall of the device 100 outside the device 100.

[0023] In addition, the sample in this chamber 120 has sufficient time to interact with antibodies 121 preferably immobilized there, which bind leukocytes in the sample and thus also immobilize them. For example, depending on the specific antibody, 1-50 nanograms (ng), preferably 1-10 ng of antibodies can be present in the second part of the sample per expected milliliter of whole blood. These leukocyte antibodies 121 can, for example, be as described in Fig. 2, immobilized on one or more walls 122 of chambers, in particular the parking chamber 120, or channels, for example in a channel 125 leading to the parking chamber 120, for example by being spotted there and bound by means of a suitable binding chemistry known in microfluidics, in particular covalently or via hydrophobic interaction, for example as described in Lee et al., Lab Chip, 2020, 20, 912-922, https: / / doi.org / 10.1039 / C9LC01248F. These channels are preferably not used for transporting the first part of the sample to the array 131 in order to avoid losing a single cell in the first part for maximum quantification efficiency. Alternatively or additionally, at least some of the antibodies are not immobilized and are preferably stored upstream in another chamber to be introduced into the parking chamber as needed.

[0024] Preferably, in particular in the parking chamber 120 structures, especially small columns (as in Fig. 2) to which the antibodies 121 are immobilized in order to increase the reactive surface. For example, these columns have widths and heights in the two- to three-digit micrometer range, are arranged at distances in the two- to three-digit micrometer range from each other, and can be made of plastic or silicon, for example. Only the leukocytes preferentially bind to these immobilized antibodies 122, while the CTCs remain free in the second part of the sample and are subsequently analyzed with higher purity and reduced background (for example, 1-1000 CTCs per milliliter (mL) below a maximum of 10 5 preferably 10 3 up to 10 4Leukocytes / mL) can be removed for further analyses, in particular via a removal chamber 170 connected to the parking chamber 120, which preferably has a reversibly closable opening to an area outside the device 100.

[0025] Potential leukocyte antibodies are preferably directed against leukocyte surface antigens, such as CD45 (a surface antigen on all hematopoietic cells). Alternatively or additionally, other antibodies binding to CD3, CD4, CD8, CD14, CD19, or CD56 may be pre-positioned or immobilized.

[0026] Alternatively or additionally, the leukocyte antibodies can be immobilized on preferably magnetic particles of a well-defined size, in particular on so-called “beads” used in microfluidics, in order to subsequently remove the leukocytes from the second part of the sample by filtration and / or with the aid of a magnet.

[0027] In a seventh step 507, a count of the CTCs that have entered the microcavities of the sedimentation array is performed, preferably via fluorescence detection of the color-labeled CTCs as described above. The number of CTCs thus determined is extrapolated to the volume of the second part in the parking chamber 120. Thus, in an eighth step 508, a pre-characterized and leukocyte-depleted sample can be taken from the parking chamber 120. This CTC sample, with the highest possible purity, can then be used for mutation-specific PCR, either directly or after nucleic acid purification (DNA and / or RNA). Furthermore, the cells of the taken sample can be further processed in a cell sorting device, such as a dielectrophoresis chip.This processing can be used in particular to isolate cells and thus to examine them at the single-cell level, for example to confirm their tumor origin and / or to detect certain expression patterns or mutations. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] DE 10 2021 203 897 A1 [0002, 0005, 0017, 0018] Cited non-patent literature

[0000] Meng et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 10, 2004, 24, pp. 8152-8162

[0001] Cristofanilli et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin. Oncol. 33, 2006, 3, S. 9-14

[0001] Lee et al., Lab Chip, 2020,20, 912-922, https: / / doi.org / 10.1039 / C9LC01248F

[0023]

Claims

[1] Method (500) for preparing a sample for an analysis of biological cells, in particular circulating tumor cells, comprising the steps • Input (502) of a sample comprising blood into an input chamber (110) of a microfluidic device (100), wherein the input chamber (110) is or can be fluidically connected to an analysis chamber (130) and a parking chamber (120). • guiding (505) a first part of the sample into the analysis chamber (130), wherein the analysis chamber (130) has an analysis substrate (131), in particular, a sedimentation array (131). • Directing (506) a second part of the sample into the parking chamber (120). • Analyzing (507), in particular counting, cells taken up from the analysis substrate (131), in particular cells that have entered cavities of the sedimentation array (131), of the first part of the sample. • Taking (508) at least a part of the second sample from the parking chamber (120), in particular for further analysis. [2] Method (500) according to claim 1, wherein, in particular before conducting the first part and / or before conducting the second part, a lysis (504) of cells, in particular of erythrocytes, and / or one or more markings (503), in particular stainings, of cells, in particular of circulating tumor cells, in the sample, in particular in the first part and / or in the second part, takes place. [3] Method (500) according to one of the preceding claims, wherein the second part is mixed with a buffer, in particular in the parking chamber (120), in order to preferably dilute lysis substances, dyes and / or dyes preferably bound to specific antibodies in the second part. [4] Method (500) according to one of the preceding claims, wherein leukocytes in the second part of the sample are depleted, in particular by damage or impaired mobility, preferably by binding to leukocyte-specific antibodies (121). [5] Method (500) according to one of the preceding claims, wherein a number of cells taken up by the analysis substrate (131), in particular of cells entering cavities of the sedimentation array (131), is determined from the first part of the sample, preferably after a predetermined sedimentation time, and depending on this number, an amount of the second part of the sample is determined for further analysis and is preferably taken from the parking chamber (120) or the device (100). [6] Method (500) according to one of the preceding claims, wherein at least a part of the second part of the sample is further prepared for further analysis, in particular for a polymerase chain reaction and / or for cell sorting, in particular via dielectrophoresis, and / or for gene sequencing, in particular is mixed with substances. [7] Microfluidic device (100) for preparing a sample for an analysis of biological cells, in particular circulating tumor cells, in particular according to one of the preceding methods, comprising an input chamber (110) for receiving a sample comprising blood, an analysis chamber (130) for receiving a first part of the sample and a parking chamber (120) for receiving a second part of the sample, wherein the analysis chamber (130) and the parking chamber (120) are or can be fluidically connected to the input chamber (110) and wherein the analysis chamber (130) has an analysis substrate (131), in particular a sedimentation array (131) with cavities, for receiving and analyzing, in particular counting, cells from the first part. [8] Microfluidic device (100) according to claim 7, wherein the device (100) has a reversibly closable opening (170) for removing fluid from the parking chamber (120) and in particular from the device (100). [9] Microfluidic device (100) according to claim 7 or 8, wherein the device (100), preferably the parking chamber (120) and / or a channel (125) leading to the parking chamber (120), comprises antibodies (121) for binding leukocytes. [10] Microfluidic device (100) according to claim 9, wherein at least some of the antibodies (121) are immobilized on one or more walls (122) and / or structures (123), in particular elevations, in particular in the parking chamber (120) and / or in a channel (125) leading to the parking chamber (120).

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