Microfluidic system and method for isolating and quantifying at least one sub-population of cells from a population of cells

Abstract

Disclosed are a microfluidic system (1) configured to receive cell populations and further configured to simultaneously isolate and quantify at least one sub-population of cells for each cell population, and related methods of using the system. The system comprises a substrate having networks of microchannels comprising a first sorting unit configured to isolate, by magnetic attraction, cells of interest in the population in at least one first sorting microchannel. The network comprises a second unit for simultaneous sorting and counting comprising at least one second sorting microchannel defined by a closed wall having an inner face provided with at least one functionalised electrode configured to trap a sub-population. The second unit further comprising means for counting the sub-population by impedance spectroscopy. The second sorting microchannel can have at least one pair of opposing functionalised electrodes and at least one pair of second micro-coils for trapping the cells of interest arranged in the wall facing the electrodes and controlling successive attraction/release cycles that alternate between the micro-coils.

Claims

1. A microfluidic system configured to receive populations of cells and configured to simultaneously isolate and quantify for each of said populations at least one sub-population of cells expressing a first marker having affinity for a second substance, said system including a substrate in which is etched a network of microchannels comprising a first sorting unit comprising magnetic trapping means to isolate by magnetic attraction magnetic microbeads bound to cells of interest of said population of cells in at least one first sorting microchannel, said network comprising a second simultaneous sorting and counting unit including at least one second sorting microchannel which directly or indirectly communicates with said at least one first sorting microchannel and which is defined by a closed wall having an internal face intended to be in contact with said cells of interest bound to said magnetic microbeads, said internal face being provided with at least one pair of electrodes functionalized with said second substance to specifically trap said sub-population of cells from among said cells of interest bound to said magnetic microbeads, said second sorting and counting unit including counting means by electrochemical impedance spectroscopy of said or each sub-population trapped on said pair of electrodes, wherein said at least one pair of said electrodes laid out facing each other on two opposite sides of said internal face, and with at least one associated pair of second electrically powered trapping micro-coils which are configured to bring said cells of interest bound to said magnetic microbeads in close contact with said electrodes, which are laid out in said wall and facing said respective electrodes to command successive and alternating attraction and release cycles between the micro-coils of said at least one pair of second micro-coils.

2. The microfluidic system according to claim 1, wherein said first sorting unit comprises: a first microchannel for bringing the populations of cells which opens into said at least one first sorting microchannel, and a second microchannel configured to receive said magnetic microbeads and introduce said magnetic microbeads into said at least one first sorting microchannel which specifically bind with said cells of interest.

3. The microfluidic system according to claim 2, said system comprising said magnetic micro-beads functionalized with a first substance capable of specifically recognizing a first marker of said cells of interest, and wherein said magnetic trapping means comprise at least one first electrically powered micro-coil.

4. The microfluidic system according to claim 3, wherein the first marker is CD14, and wherein the first substance is an anti-CD14 antibody.

5. The microfluidic system according to claim 1, wherein the second marker is CD16, and wherein the second substance is an anti-CD16 antibody.

6. The microfluidic system according to claim 1, wherein said internal face of the wall of said at least one second sorting microchannel is provided spaced out on its length with a succession of several electrodes including said at least one pair of functionalized electrodes and then an additional non-functionalized electrode or pair of additional non-functionalized electrodes laid out facing each other on two opposite sides of said internal face in order to achieve isolations and quantifications in series of several sub-populations of cells.

7. The microfluidic system according to claim 6, wherein said second sorting and counting unit comprises a plurality of said pairs of functionalized electrodes which are laid out in series on a same said second sorting microchannel or on several second sorting microchannels, which are either functionalized identically or not and which are associated with several said pairs of second individual trapping micro-coils.

8. The microfluidic system according to claim 1, wherein said first sorting unit comprises a plurality of said first sorting microchannels, and wherein said second sorting and counting unit comprises a plurality of said second sorting microchannels which are each provided with said at least one pair of functionalized electrodes so that said system produces in parallel a plurality of different isolations and quantifications from said sub-populations of cells from different cells of interest.

9. The microfluidic system according to claim 8, wherein said second sorting and counting unit comprises a plurality of said pairs of functionalized electrodes which are laid out in parallel on a same said second sorting microchannel or on several second sorting microchannels, which are either functionalized identically or not and which are associated with as many of said pairs of second individual trapping micro-coils.

10. The microfluidic system according to claim 1, wherein said second sorting and counting unit comprises a matrix of said pairs of functionalized electrodes which are individually addressed and which are associated with as many said second individual trapping micro-coils.

11. A method for simultaneous isolation and quantification of at least one sub-population of cells from a cell population, wherein the method comprises the step of generating a flow of a sample comprising said cell population in the network of microchannels of the microfluidic system according to claim 1, with the following successive steps: (a) trapping by magnetic attraction said cells of interest in said at least one first sorting microchannel; (b) removing undesirable constituents of said sample in order to only retain the thereby trapped cells of interest; and (c) trapping said at least one sub-population of cells from among said cells of interest in said at least one second sorting microchannel by said at least one pair of functionalized electrodes and concomitant counting by electrochemical impedance spectroscopy of said at least one trapped sub-population.

12. The isolation and quantification method according to claim 11, wherein in step (a): said cells of interest are marked with the magnetic micro-beads functionalized with a first substance specifically binding with a first marker of said cells of interest, for obtaining marked magnetic complexes; and said complexes are magnetically trapped by the magnetic trapping means comprising at least one first micro-coil.

13. The isolation and quantification method according to claim 12, wherein in step (c), said marked magnetic complexes are attracted by said at least one pair of second trapping micro-coils which is laid out facing said at least one pair of electrodes functionalized with a second substance specifically binding with a second marker of said at least one sub-population of cells to be isolated, and which brings said complexes into close contact with said at least one pair of functionalized electrodes for trapping and counting the at least one trapped sub-population of cells.

14. The isolation and quantification method according to claim 13, wherein said sample containing said population of cells is a full blood sample, said cells of interest being monocytes which are magnetically isolated in step (a) with said first marker expressed by the cells of interest which is an antigen and said first marking substance which is an antibody, said sub-population of cells isolated and quantified by immunological sorting in step (c) comprising infected monocytes expressing said second marker which is an antigen, said second substance being an antibody.

15. The isolation and quantification method according to claim 11, wherein the counting by electrochemical impedance spectroscopy of step (c) is carried out in a non-Faradic mode.

16. The isolation and quantification method according to claim 11, wherein the counting by electrochemical impedance spectroscopy of step (c) is carried out in a Faradic mode.

17. The isolation and quantification method according to claim 14, wherein the antigen of the first marker is CD14, and wherein the antibody of the first substance is an anti-CD14 antibody.

18. The isolation and quantification method according to claim 14, wherein the antigen of the second marker is CD16, and wherein the antibody of the second substance is an anti-CD16 antibody.

Description

(1) Other advantages, features and details of the invention will become apparent from the additional description which follows with reference to appended drawings, only given as examples and wherein:

(2) FIG. 1 is a diagram illustrating the main steps of a method according to the invention for isolating and quantifying infected monocytes from a full blood sample,

(3) FIG. 2 is a schematic partly longitudinal sectional view of a microfluidic system according to the invention showing an upstream portion of the first sorting unit achieving marking of the cells of interest (e.g. monocytes) by the magnetic marking means,

(4) FIG. 3 is a schematic partly longitudinal sectional view of a microfluidic system according to the invention showing a first sorting microchannel of the first sorting unit carrying out the sorting by magnetic attraction of these cells of interest,

(5) FIG. 4 is a schematic partly longitudinal sectional view of a microfluidic system according to the invention showing a second sorting microchannel achieving isolation and counting of a sub-population of cells (e.g. infected monocytes) on a pair of functionalized electrodes and then of other cells of interest on a non-functionalized electrode,

(6) FIG. 5 is a diagram schematically illustrating the counting method by electrochemical impedance spectroscopy of the sub-population trapped in this second sorting microchannel,

(7) FIG. 6 is a schematic partly cross-sectional view of a microfluidic system according to an example of the invention showing this second sorting microchannel provided with a micro-coil associated with a functionalized electrode for trapping the sub-population,

(8) FIG. 7 is a schematic partly cross-sectional view of a microfluidic system according to another example of the invention showing, as an alternative of FIG. 6, the second sorting microchannel provided with two micro-coils associated with the functionalized trapping electrode,

(9) FIG. 8 is a schematic view showing, in a second sorting microchannel, a parallel layout of functionalized electrodes associated with a single collective trapping micro-coil,

(10) FIG. 9 is a schematic view showing as an alternative of FIG. 8, in a second sorting microchannel, a layout in series of functionalized electrodes associated with a single collective trapping micro-coil,

(11) FIG. 10 is a schematic view showing as an alternative of FIGS. 8 and 9, in a second sorting microchannel, a matrix of functionalized electrodes according to the invention which are individually addressed and which are respectively associated with trapping micro-coils,

(12) FIG. 11 is a schematic view showing as an alternative of FIG. 10, in a second sorting microchannel, a matrix of functionalized electrodes which are individually addressed and which are associated with a single collective trapping micro-coil,

(13) FIG. 12 is a bar graph illustrating the influence of the surface (area in cm.sup.2 in abscissa) of a functionalized electrode according to the invention on the sensitivity of the counting measurement (expressed in ordinates by the relative impedance variation Z in %), and

(14) FIG. 13 is a graph of the Nyquist diagram type illustrating the variation of imaginary impedance (Z.sub.im, in Ohms) versus the real impedance (Zr, in Ohms) and the concentration of cells.

(15) A microfluidic system 1, 101 according to the invention (partly visible in FIGS. 2, 3, 4, 6, 7) may be advantageously used as illustrated in FIG. 1, which relates to the preferential example of the invention applying immunological sorting of the cells of interest according to several antigens. In order to better understand the principle of the invention, the example of simultaneous sorting and counting of the infected monocytes was taken, it being specified that this example is not limiting.

(16) A first sorting unit U.sub.1 (visible in FIGS. 2 and 3) is, by a first sorting operation A, intended to remove at best the constituents of a full blood sample C1 in order to only retain the monocytes C2 forming the cells of interest in this example. This sorting operation A is carried out subsequently to a step for marking M by means of magnetic micro- or nano-beads b1 functionalized with antibodies capable of recognizing the C2 monocytes (such as for example, anti-CD14 antibodies may recognize the corresponding antigen CD14 expressed by all the monocytes C2). The bead/blood cells mixture is produced on the device 1, 101 via microchannels as slots, for example. The thereby marked monocytes C2 are then retained during a first sorting operation T with at least one first electrically powered micro-coil 6 and laid out under a first sorting microchannel 5 (see FIG. 3), while the other constituents C3 of the full blood C1 (such as granulocytes, lymphocytes, red corpuscles, notably) are eluted.

(17) The marked monocytes C2 are then released and brought towards a second sorting and counting unit U.sub.2 (visible in FIGS. 4 and 6-7), where they are sorted more specifically (second sorting step B) depending on other antigens expressed at their surface (such as for example the antigen CD16 expressed by the infected monocytes C4 forming the sub-population to be isolated and to be quantified) and counted. According to the contemplated application, several antigens may be targeted in parallel.

(18) In this second unit U.sub.2, the infected monocytes C4 are trapped inside a second sorting microchannel 10 on a functionalized electrode E (see FIGS. 1, 5, 6 and 7) or else on a pair of functionalized electrodes E1 and E2 facing each other in the example of FIG. 4, the functionalization being achieved with an antibody capable of binding with an antigen of interest (for example an anti-CD16 antibody for the CD16 antigen of the infected monocytes C2). In order to maximize trapping, a single second micro-coil 10a is installed below the electrode E or else, according to the invention, a pair of second electrically powered micro-coils 11 and 12 respectively around functionalized electrodes facing each other E1 and E2, in order to attract the infected monocytes C4 on the electrodes E or E1, E2 and thus increase their probability of interaction with their functionalized surface. The cells not specifically attached onto these electrodes E or E1, E2 are then released and optionally attracted on other electrodes functionalized by other antibodies, depending on the desired application.

(19) The infected monocytes C4 or more generally said or each sub-population C4 of cells trapped on the corresponding functionalized electrode are counted by means of the electrically insulating property of this sub-population C4, by electrochemical impedance spectroscopy.

(20) FIGS. 6 and 7 show as a cross-section two microfluidic systems 1, 101 according to the invention which comprise a substrate 1a, 101a and which integrate: for the one of FIG. 6, said or each functionalized electrode E (for example in gold) coating the internal face 9 of said or each microchannel 10 and associated with a single micro-coil 10a for example in copper which is integrated to the wall 8 of the microchannel 10 below this internal face 9, and for the one of FIG. 7, said or each electrode E (for example in gold) which is associated with two micro-coils 10b and 10c for example also in copper, these micro-coils 10b and 10c being integrated to the wall 8 of said or each microchannel 10 on either side of the latter (i.e. above and below the upper and lower sides of its internal face 9 of rectangular section).

(21) The wall 8 of said or each microchannel 10 is for example made in a photosensitive polymer (e.g. of the SU8 type). Further, in FIGS. 6 and 7, the electric connections 10a, 10b, 10c (e.g. in copper) are visible respectively for powering the micro-coils 10a, 10b, 10c.

(22) More specifically in connection with the first sorting unit U.sub.1, in FIG. 2 it is seen that the step M for marking the cells of interest C2 (e.g. monocytes) is applied by mixing by means of two separate microchannels 2 and 3 which respectively convey in the lower microchannel 2, the population of cells C1 to be analysed (e.g. full blood) and, in the upper microchannel 3, a buffer solution containing the magnetic micro- or nano-beads b1 functionalized beforehand a t the surface with antibodies for example anti-CD14 antibodies. It is possible to use optionally active or passive mixtures, as well as optionally an encapsulation of the beads b1 and of the cells in drops, for example. These microchannels 2 and 3 join up in a junction area 4 opening into the first sorting microchannel 5 visible in FIG. 3 within the first sorting unit U.sub.1.

(23) The application of the sorting operation A by magnetic attraction is thus visible in FIG. 3, which is carried out by means of the micro-coil 6 which is laid out outside the microchannel 5, for example by being integrated to the closed wall 7 of this microchannel 5. The activation of the micro-coil 6 gives the possibility of trapping beads b1/monocyte C2 complexes.

(24) Said or each first sorting microchannel 5 is then rinsed in order to remove all the constituents of the blood C3 other than the thereby trapped monocytes C2. This purification step simply allows removal of all the non-desired species, in order to obtain a solution for which the physico-chemical properties are known and under control. After this rinsing, the beads b1/monocyte C2 complexes are released into the solution and are carried away by the flow towards the second sorting and counting unit U.sub.2.

(25) In the example of FIG. 4, it is seen that this unit U.sub.2 comprises the pair of functionalized electrodes E1 and E2 on the internal face 9 of the closed wall 8 of a second sorting microchannel 10, and the associated pair of micro-coils 11 and 12 in order to attract, from the whole of the marked complexes, the infected monocytes C4 onto these electrodes E1 and E2. These micro-coils 11 and 12 are for example integrated to the wall 8 (thus being positioned outside the space internal to the microchannel 10) and attract these infected monocytes C4 expressing the CD16 antigens into close contact with the surface of these functionalized electrodes, in this example by the anti-CD16 antibodies. During deactivation of the micro-coils 11 and 12, only the monocytes expressing the CD16 antigens and having established antigen-antibody interactions remain attached on the electrodes E1 and E2, the other monocytes C5 being carried away by the downstream flow of the microchannel 10. After this trapping of the infected monocytes, their number is directly counted by the aforementioned technique of impedance spectroscopy.

(26) Generally, it will be noted that the probability of trapping said or each sub-population to be quantified, such as these infected monocytes, may be maximized in different ways. In particular, the layout of both micro-coils 11 and 12 respectively below and above the microchannel 10 gives the possibility of achieving successive and alternating attraction-release cycles between both micro-coils 11 and 12 for obtaining maximum trapping efficiency. Alternatively or as a combination with this preferential layout of electrodes E1 and E2 facing each other, it is possible to provide several functionalized electrodes and associated coils which are positioned one after the other, in order to catch up with the cells which would not have been trapped by these functionalized electrodes.

(27) Finally and so as to determine the CD16+/CD14+ ratio of the monocytes, said or each second sorting microchannel 10 according to the invention may further be provided, always on the internal face 9 of its wall 8, with at least one ultimate non-functionalized electrode E3 coupled with at least one ultimate micro-coil 13 facing it positioned in this wall 8, out of the space internal to the microchannel 10. In this way, it is possible to immobilize by magnetic trapping on this electrode E3 the CD14+ monocytes which are not immunologically trapped by the electrodes E1 and E2 provided upstream, and to count these CD14+ monocytes with the same impedance spectroscopy technique.

(28) FIG. 5 illustrates this counting technique by electrochemical impedance spectroscopy (EIS), which consists of measuring the impedance of the interface between said or each functionalized electrode E1, E2 and the layer of cells trapped at its surface (e.g. layer of infected monocytes C4), by imposing a small alternating potential U difference and by measuring the current resulting from this at various frequencies, by means of a counter-electrode or reference electrode E. The impedance Z is given by the voltage U/current I ratio and is given in a known way by the formula Z=U/l=|Z| (cos +i sin ).

(29) Electrochemical impedance spectroscopy is of particular interest in the case of cell layers, the cells having excellent electrically insulating properties. A change in impedance may either result from a change in the coverage level of the electrode E, E1, E2 (this is the case when a cell adheres, grows, dies or migrates at the surface of the electrode), or a variation of the electrically insulating property of the cell layer C4. Therefore, by measuring the impedance Z at the cell C4/electrode E, E1, E2 interface for a wide range of frequencies, it is possible, by modelling of the obtained diagrams (Nyquist diagrams Zim=f (Zr)) in an equivalent electric circuit consisting of resistors and capacitors, to infer the coverage level of the cells and therefore the number of trapped cells C4.

(30) Two distinct methods exist for these measurements: The impedance measurements conducted in the presence of a redox species which plays the role of a probe (Fe(CN).sub.6.sup.3/4 for example). In this case, the capability of the probe of being reduced or oxidized at the electrode E, E1, E2 is inferred from impedance measurements by evaluating the resistance to charge transfer which varies depending on the more or less electrically insulating nature of the deposited cell layer. In this case, this is referred to as Faradic impedance; and The measurements conducted in a neutral medium (e.g. in a buffer solution or a culture medium) which give the possibility of accessing the phenomena occurring at the cell film. In this case this is referred to as non-Faradic impedance, and it is in this case, the resistance of the film which changes over time in the presence of the trapped cells.

(31) In the present invention, it is the impedance measurement in a non-Faradic mode which is preferentially applied for counting the cells C4 trapped on the electrode E1, E2, because this measurement proves to be more sensitive.

(32) FIGS. 8-11 illustrate examples of a layout of functionalized electrodes Ea, Eb, Ec, Ed, Ee, Ef, Eg, Eh, etc. in a microfluidic system 1,101 according to the invention. These electrodes Ea, . . . , Eh may be provided in separate microchannels 10 but also in a same microchannel 10.

(33) In FIG. 8, the electrodes Ea, Eb, Ec, Ed, either identically functionalized or not, are laid out in parallel while being associated with a collective trapping micro-coil 10A (according to the invention, individual trapping micro-coils may be used).

(34) In FIG. 9, the electrodes Ea, Eb, either functionalized identically or not, are laid out in series while being associated with a collective trapping micro-coil 10A (according to the invention individual trapping micro-coils may be used).

(35) In FIG. 10, the electrodes Ea, . . . , Eh, either identically functionalized or not, are laid out as a matrix, individually addressed and associated with individual trapping micro-coils 10A, . . . , 10H.

(36) In FIG. 11, the electrodes Ea, . . . , Eh, either identically functionalized or not, are laid out as a matrix, individually addressed and associated with a collective trapping micro-coil 10A.

(37) According to the size of each electrode Ea, . . . , Eh, an analogue operation (obtained signal proportional to the number of trapped cells) or digital operation may be contemplated. Indeed, as the electrode has a size of the order of magnitude of that of the cell, it then only detects one cell at a time and the signal will therefore be binary: 1 or 0.

(38) The Applicant conducted tests dealing with the sensitivity of the counting by electrochemical impedance spectroscopy at each electrode E, E1, E2, according to the area of the latter (FIG. 12), as well as with the variation of impedance measured according to the number of trapped cells on each of these electrodes E, E1, E2.

(39) The graph of FIG. 12 shows that the sensitivity of the measurement for the counting of each sub-population of cells C4 according to the invention (variation of impedance measured in % for this counting) is all the higher since the area of each functionalized trapping electrode E, E1, E2 is more reduced (area of 0.04 cm.sup.2 for the maximum value of Z close to 90%).

(40) The graph of FIG. 13 (with the frequencies used varying between 0.1 Hz and 50 kHz for the lowest impedances on the bottom left) shows that the measured impedance is all the higher since the concentration of cells on the electrode E, E1, E2 is greater, as shown by the values of concentrations of cells (framed) which appear on the side of each curve and which attain 1,000,000 for the upper curve (as compared with the lower curve relating to zero concentration of cells, and of the intermediate curves where the number of cells is successively 1,000, 10,000, 50,000 and 100,000).

(41) The Applicant further varied, for two diameters of the microbeads b1 used for the marking (2.3 m and 4.5 m), the flow pressure (from 5 to 10 mbars) and the number of active micro-coils in the second sorting microchannel 10 (from one to three micro-coils, with a 100 mA intensity of the power supply current of the micro-coils), for comparing according to these parameters the efficiency of the deviation and of the trapping or the separation of the sole magnetic microbeads according to the pressure of the flow stream in the microfluidic system 1,101.

(42) The obtained results are listed in the tables hereafter.

(43) TABLE-US-00001 TABLE 1 beads with a diameter of 2.3 m Pressure Separation 5 mbars 87% 6 mbars 86.5% 7 mbars 73%

(44) At 10 mbars, one has very high flow velocity and zero trapping.

(45) TABLE-US-00002 TABLE 2 beads with a diameter of 4.5 m, pressure of 7 mbars Number of active micro-coils Trapping Separation 1 80% 100% 2 100% 100% 3 100% 100%

(46) TABLE-US-00003 TABLE 3 beads with a diameter of 4.5 m, pressure of 8 mbars Number of active micro-coils Trapping Separation 1 0% 74% 2 32% 71% 3 42% 88%

(47) These results notably show the advantage of using at least two active micro-coils and a flow pressure comprised between about 5 mbars and 8 mbars, in order to apply the isolation and counting method according to the invention.