MICROFLUIDIC DEVICE AND METHOD FOR PROCESSING PARTICLES

Abstract

A microfluidic device for processing particles, including: an elongated chamber including elongated segment(s), input seeding channel(s) and one output seeding channel defining a seeding flow transverse to the longitudinal direction of the segment, input harvest channel(s) and one output harvest channel defining a harvest flow in the longitudinal direction, wherein, for the segment, a single input seeding tree defines input seeding channels and a single output seeding tree defines output seeding channels, junctions of the input seeding channels and output seeding channels with the processing chamber being distributed along the segment on both sides of the chamber, wherein:

[00001]$\mathrm{Rs\_input}\frac{S_{21}}{\left({.Math.}_k{V}_k*S_k\right)/V_{\mathrm{TOTs}\_\mathrm{inpu}t}}$

is higher than 50, S.sub.21 being the segment cross section perpendicular to the transverse direction, (Σ.sub.k V.sub.k*S.sub.k)/V.sub.TOTs_input being, the sum of the products of the volume and the cross section of the input seeding channel, divided by the total volume V TOTs_input of the input seeding channels.

Claims

1-20. (canceled)

21. A microfluidic device for processing particles, in particular cells, comprising: an elongated processing chamber including at least one elongated segment, at least one input seeding channel and one output seeding channel configured to define a seeding flow in a transverse direction to the longitudinal direction of the segment of the processing chamber, at least one input harvest channel and one output harvest channel configured to define a harvest flow in the longitudinal direction of the segment of the processing chamber, wherein the microfluidic device comprises, for each segment of the processing chamber, a plurality of input seeding channels and a plurality of output seeding channels whose junctions with the segment of the processing chamber are distributed on both sides along the segment, the plurality of input seeding channels being defined by a single input seeding tree and the plurality of output seeding channels being defined by a single output seeding tree wherein the ratio Rs_input such that: $\mathrm{Rs\_input}\frac{S_{21}}{\left({.Math.}_k{V}_k*S_k\right)/V_{\mathrm{TOTs}\_\mathrm{inpu}t}}$ is higher than 50, where S.sub.21 is the cross section of the segment perpendicular to the transverse direction, and (Σ.sub.k V.sub.k*S.sub.k)/V.sub.TOTs_input is the sum, for all input seeding channels between the first node closest to the tree root of the input seeding tree and the segment, of the products of the volume and the cross section of the input seeding channel, divided by the total volume V.sub.TOTs_input which is the sum of the volumes of these input seeding channels.

22. The microfluidic device according to claim 21, wherein the ratio Rh_input such that: $\mathrm{Rh\_input}=\frac{W}{\left({.Math.}_j{V}_j*S_j\right)/V_{\mathrm{TOTh}\_\mathrm{inpu}t}}$ is lower than 20, where W is the cross section of the segment perpendicular to the longitudinal direction, and (Σ.sub.jV.sub.j*S.sub.j)/V.sub.TOTh_input is the sum, for all input harvest channels between the first node closest to the tree root of the input harvest tree and the segment, of the products of the volume and the cross section of the input harvest channel, divided by the total volume V.sub.TOTh_input which is the sum of the volumes of these input harvest channels.

23. The microfluidic device according to claim 21, wherein, for each segment of the processing chamber, in more than half of the channel volume of the input seeding tree linked to the segment, the ratio of the seeding shear rate indicator of the input seeding channels to the average seeding shear rate indicator of the segment is higher than 10, where the seeding shear rate indicator (SSI) of a channel or a segment is defined by: $\mathrm{SSI}=\frac{T_Q}{S*h}$ with T.sub.Q the percentage of the seeding flow flowing through the considered channel or segment, S the cross section of the channel or segment taken perpendicular to its longitudinal fiber for the seeding flow, and h the height of the channel or segment taken in the height direction.

24. The microfluidic device according to claim 21, comprising blocking means configured to: block selectively the input and output harvest channels when a seeding flow is applied to the processing chamber through the input and output seeding channels, and block selectively the input and output seeding channels when a harvest flow is applied to the processing chamber through the input and output harvest channels.

25. The microfluidic device according to claim 21, wherein the surface of the processing chamber, perpendicular to the height direction, is greater than 4 cm.sup.2.

26. The microfluidic device according to claim 21, wherein, for each segment of the processing chamber, the pitch between adjacent junctions of the seeding channels with the segment of the processing chamber is the same along the segment.

27. The microfluidic device according to claim 21, wherein, in each seeding tree, the cross section of the seeding channels decreases with increasing channel path distance from the tree root.

28. The microfluidic device according to claim 21, wherein, for each segment of the processing chamber, the ratio of the cross section (S.sub.21) of the segment perpendicular to the transverse direction to the sum of the average cross sections (S.sub.31) of the input seeding channels perfusing the segment is higher than 5.

29. The microfluidic device according to claim 21, wherein the total volume of the seeding trees is less than the total volume of the processing chamber.

30. The microfluidic device according to claim 21, wherein the junction of each input and output seeding channel with the processing chamber is located in an upper part of the processing chamber in the height direction.

31. The microfluidic device according to claim 21, wherein, for each segment of the processing chamber, the ratio of the width of the segment, taken in the transverse direction, to the height of the segment, taken in the height direction, is higher than 5.

32. The microfluidic device according to claim 21, wherein the junction of each input and output seeding channel with the processing chamber is chamfered.

33. The microfluidic device according to claim 21, wherein the seeding and harvest channels are configured such that, upon application of a harvest flow in the processing chamber, the absolute flow rate in each seeding channel is less than 10%, of the sum of the flow rates of the harvest flow applied at the junction of each input harvest channel (with the processing chamber.

34. The microfluidic device according to claim 21, wherein the height of the processing chamber, taken in the height direction, is comprised between 50 μm and 300 μm.

35. The microfluidic device according to claim 21, comprising a set of parallel segments of the processing chamber connected in a serpentine shape by fluidic connectors.

36. The microfluidic device according to claim 21, comprising a gas permeable membrane which forms at least part of the top surface and/or the bottom surface of the segment of the processing chamber.

37. The microfluidic device according to claim 36, comprising a gas chip on the outer side of the gas permeable membrane opposite from the processing chamber, the gas chip having channels or chambers closed by the gas permeable membrane.

38. A method for processing particles, in particular cells, using a microfluidic device according to claim 21, the method comprising: a step of seeding the processing chamber with particles by applying a seeding flow to the processing chamber through the input and output seeding channels, while the input and output harvest channels are blocked, a step of collecting particles from the processing chamber by applying a harvest flow to the processing chamber through the input and output harvest channels, while the input and output seeding channels are blocked.

39. The method according to claim 38, wherein the step of seeding the processing chamber is carried out by applying successive pulses of seeding flow separated by a resting time.

40. The method according to claim 38, wherein the step of collecting particles from the processing chamber is carried out by applying successively different flow rates of the harvest flow.

Description

[0094] Features and advantages of the invention will become apparent from the following description of embodiments of a microfluidic device and a method for processing particles according to the invention, this description being given merely by way of example and with reference to the appended drawings in which:

[0095] FIG. 1 is an upper view of a microfluidic device according to a first embodiment of the invention,

[0096] FIG. 2 is a cross section along the line II-II of FIG. 1,

[0097] FIG. 3 is a view similar to FIG. 1 for a second embodiment of a microfluidic device according to the invention,

[0098] FIG. 4 is a view similar to FIG. 1 for a third embodiment of a microfluidic device according to the invention,

[0099] FIG. 5 is a view similar to FIG. 1 for a fourth embodiment of a microfluidic device according to the invention,

[0100] FIGS. 6 and 7 are cross sections of two variants of a microfluidic device according to a fifth embodiment of the invention,

[0101] FIGS. 8a, 8b, 8c are cross sections of a microfluidic device equipped with a gas chip according to three variants of a sixth embodiment of the invention, and

[0102] FIGS. 9a, 9b, 9c are upper views of the gas chip in each of the variants of FIGS. 7a, 7b, 7c, respectively.

FIRST EMBODIMENT

[0103] In the first embodiment shown in FIGS. 1 and 2, the microfluidic device 1 comprises an elongated processing chamber 2 comprising a single segment 21 extending along a longitudinal axis X. The microfluidic device 1 also comprises a single input seeding tree 3, defining a plurality of input seeding channels 33, and a single output seeding tree 4, defining a plurality of output seeding channels 43. The junctions 31, 41 of the input seeding channels 33 and the output seeding channels 43 with the segment 21 of the processing chamber are distributed on both sides along the segment 21.

[0104] More specifically, in this particular embodiment, the input seeding tree 3 and the output seeding tree 4 are binary trees which are arranged facing each other on both sides of the segment 21. The input seeding channels 33 and the output seeding channels 43 are configured to define a seeding flow in a direction Y transverse to the longitudinal direction X of the segment 21. Each of the seeding binary trees 3, 4 is symmetric in terms of hydraulic resistance, which is advantageous to ensure a homogeneous seeding flow.

[0105] The pitch p between adjacent junctions 31 of the input seeding channels 33 with the segment 21 and between adjacent junctions 41 of the output seeding channels 43 with the segment 21 is the same along the segment 21, which also contributes to a homogeneous seeding flow and homogeneous perfusion of the processing chamber 2. As can be seen in FIG. 2, the junction 31, 41 of each input and output seeding channel 33, 43 with the processing chamber 2 is located in an upper part of the processing chamber in the height direction Z. Such a configuration takes advantage of inertia, under appropriate seeding flow rate, to more homogeneously distribute particles within the processing chamber after a seeding pulse. The seeding pause can then be used to allow the particles to sediment on the bottom surface 22 of the processing chamber. Oppositely, under lower flow rates, under the effect of gravity, particles can be found to follow the lower streamline F, as shown in FIG. 2, which is less advantageous notably in terms of seeding homogeneity.

[0106] In an advantageous manner, the bottom surface 22 of the segment 21 is provided with a particle-adhesive coating, such as adhesion peptides, ECM molecules or fragments, antibodies, nanobodies, cell membrane anchoring molecules. Conversely, the input and output seeding channels 33, 43 are advantageously provided with a coating to reduce the adherence of the particles, such as a hydrophilic coating based on BSA, pHEMA, PLL, PEG, carboxybetain, agar, cellulose, chitosan, or their polymers or copolymers.

[0107] In this embodiment, the ratios Rs_input and Rs_output are higher than 50, whereas the the ratios Rh_input and Rh_output are lower than 20.

[0108] In this embodiment, the ratio of the cross section S.sub.21 of the segment 21 of the processing chamber perpendicular to the transverse direction Y to the sum of the cross sections S.sub.31 of the input seeding channels 33 at the junction 31 with the processing chamber 2 is higher than 15. In addition, the ratio of the width W of the segment 21 of the processing chamber, taken in the transverse direction Y, to the height H of the segment 21, taken in the height direction Z, is higher than 20. In this way, the segment 21 of the processing chamber is a shallow segment, where laminar flow leads to a convective replacement of most of the fluid contained in the segment. The angle to the side walls of the segment 21 of the processing chamber further facilitates the complete renewal of the fluid contained in the segment 21.

[0109] The microfluidic device 1 also comprises an input harvest channel 53 and an output harvest channel 63 configured to define a harvest flow in the longitudinal direction X of the segment 21 of the processing chamber. As can be seen in FIG. 1, the junctions 51, 61 of the input harvest channel 53 and of the output harvest channel 63 with the processing chamber 2 are chamfered, which is favorable for the homogeneity of the harvest flow. The seeding and harvest channels 33, 43, 53, 63 are configured such that, upon application of a harvest flow in the segment 21 of the processing chamber, the absolute flow rate in each seeding channel 33, 43 is less than 2.5% of the flow rate of the harvest flow applied at the junction 51 of the input harvest channel 53 with the segment 21 of the processing chamber.

[0110] In this embodiment, the input seeding tree 3 has, in more than half of its channel volume, a ratio of the seeding shear rate indicator SSI of the input seeding channels 33 to the average seeding shear rate indicator SSI of the segment 21 of the processing chamber which is higher than 20, where the seeding shear rate indicator SSI of a channel or a segment is defined by:

[00008]$\mathrm{SSI}=\frac{T_Q}{S*h}=\frac{\frac{Q}{Qtot}}{S*h}$

[0111] with T.sub.Q the percentage of the seeding flow flowing through the considered channel or segment, which is equal to the ratio of the seeding flow rate Q in the considered channel or segment to the total seeding flow rate Qtot, S the cross section of the channel or segment taken perpendicular to its longitudinal fiber for the seeding flow, and h the height of the channel or segment taken in the height direction Z.

[0112] In a non-limiting example of the first embodiment: [0113] the segment 21 of the processing chamber has a height H of 100 μm and a width W of 5 mm, [0114] the cross section S.sub.31 of the seeding channels 33, 43 at the junction 31, 41 with the processing chamber 2 is 100 μm width by 50 μm height, [0115] the pitch p between adjacent junctions 31 of the input seeding channels 33 and the pitch p between adjacent junctions 41 of the output seeding channels 43 is constant and equal to 1.25 mm, [0116] the surface of the processing chamber 2, perpendicular to the height direction Z, is greater than 0.25 cm.sup.2.

[0117] In this embodiment, the upper wall 10 and the lower wall 12 of the processing chamber 2, in the height direction Z, are transparent to the visible, near IR and near UV wavelengths, for example with the upper wall 10 or bottom wall 12 being made of cyclic olefin copolymer (COC). In this way, the bioprocess can be monitored by imaging across the walls of the microfluidic device 1. In this embodiment, the upper wall 10 and the lower wall 12 of the segment 21 of the processing chamber are planar and parallel, which facilitates focus and imaging

SECOND EMBODIMENT

[0118] In the second embodiment shown in FIG. 3, elements similar to those of the first embodiment bear identical references. The microfluidic device 1 of the second embodiment differs from the first embodiment in that the single input harvest channel 53 and the single output harvest channel 63 are replaced by input and output harvest binary trees 5 and 6 having chamfered junctions 51, 61 with the segment 21 of the processing chamber 2. Compared to the first embodiment, the harvest trees 5 and 6 provide a more homogeneous harvest flow. It is noted that, in this second embodiment, the harvest trees 5, 6 and the seeding trees 3, 4 are not interchangeable. Indeed, the harvest flow provides, with a given flow rate, a much higher shear rate within the processing chamber than if the same flow rate was used to perfuse the microfluidic device with a seeding flow.

[0119] In this embodiment, the ratios Rs_input and Rs_output are higher than 50, whereas the the ratios Rh_input and Rh_output are lower than 20.

THIRD EMBODIMENT

[0120] In the third embodiment shown in FIG. 4, elements similar to those of the first embodiment bear identical references. The microfluidic device 1 of the third embodiment differs from the second embodiment in that the processing chamber 2 includes four segments 21 that are connected in a serpentine shape by fluidic connectors 24. In this third embodiment, the microfluidic device 1 comprises a single input seeding tree 3 and a single output seeding tree 4 for all the segments 21, which is advantageous both for the compacity of the microfluidic device and for the homogeneity of the seeding flow throughout the processing chamber 2. The serpentine configuration makes it possible to optimize both the capacity of the microfluidic device 1 and its size, with a relatively simple design and a relatively simple manufacturing process since there is no need for a second channel layer.

[0121] In this embodiment, the ratios Rs_input and Rs_output are higher than 50, whereas the the ratios Rh_input and Rh_output are lower than 20.

FOURTH EMBODIMENT

[0122] In the fourth embodiment shown in FIG. 5, elements similar to those of the first embodiment bear identical references. The microfluidic device 1 of the fourth embodiment differs from the third embodiment in that, for each fluidic connector 24 which joins two segments 21 of the processing chamber, the connector 24 is connected to each of the two segments 21 by means of a binary tree having chamfered junctions with the segment. Such a configuration further improves the homogeneity of the harvest flow within the microfluidic device 1.

[0123] In a non-limiting example of the fourth embodiment, the successive cross sections of the channels of the input seeding binary tree 3 may be chosen as follows, going from the root to the extremities of the tree: [0124] the first input seeding channel from the root has a width of 200 μm and a height of 200 μm, [0125] the seeding channels of the second hierarchical level from the root have a width of 159 μm and a height of 159 μm, [0126] the seeding channels of the third hierarchical level from the root have a width of 126 μm and a height of 126 μm, [0127] the seeding channels of the fourth hierarchical level from the root have a width of 100 μm and a height of 100 μm, [0128] the seeding channels of the fifth hierarchical level from the root have a width of 100 μm and a height of 70.7 μm, [0129] the seeding channels of the sixth hierarchical level from the root, which are connected to the processing chamber 2, have a width of 100 μm and a height of 50 μm.

[0130] In this embodiment, the ratios Rs_input and Rs_output are higher than 50, whereas the the ratios Rh_input and Rh_output are lower than 20.

FIFTH EMBODIMENT

[0131] In the fifth embodiment shown in FIGS. 6 and 7, elements similar to those of the fourth embodiment bear identical references. The microfluidic device 1 of the fifth embodiment differs from the fourth embodiment in that all channels and chambers of the device share a common bottom surface formed by a gas permeable membrane 8 made of silicone and having a constant thickness between 50 μm and 2 mm As a variant, the gas permeable membrane 8 may be made of any other material compatible with biomedical use, and preferably transparent. The remaining of the microfluidic device is formed by a polymer part, for example made of polystyrene (PS) or of a cyclic olefin copolymer (COC).

[0132] FIG. 6 shows a configuration where the gas permeable membrane 8 forms the upper part of the microfluidic device 1, whereas FIG. 7 shows a configuration where the gas permeable membrane 8 is covered with an additional rigid plate 80. The additional rigid plate 80 makes it possible, during flow operations, to limit the deformation of the gas permeable membrane 8. Advantageously, the rigid plate 80 is positioned above the gas permeable membrane 8 for the duration of the flow operations and an average pressure equal to or greater than the perfusion pressure is applied on the rigid plate 80 using any suitable means, in particular mechanical means. In order to allow gas exchange across the gas permeable membrane 8 outside of flow operations, the rigid plate 80 is preferably removed after flow operations.

SIXTH EMBODIMENT

[0133] In the sixth embodiment shown in FIGS. 8a, 8b, 8c and 9a, 9b, 9c, elements similar to those of the fifth embodiment bear identical references. The microfluidic device 1 of the sixth embodiment differs from the fifth embodiment in that it further comprises a gas chip 9 on the outer side of the gas permeable membrane 8, oriented outwardly relative to the segment 21 of the processing chamber, configured to control the flow or diffusion of gas. The gas chip 9 is preferably made of a rigid and transparent polymer material such as polystyrene (PS) or a cyclic olefin copolymer (COC). The gas chip 9 is closed by the gas permeable membrane 8 and comprises, positioned facing the processing chamber, a chamber 910 provided with an array of channels 92 as shown in the examples of FIGS. 8a, 9a and 8b, 9b, or with an array of pillars 911 as shown in the example of FIGS. 8c, 9c, the array of channels 92 or pillars 911 being in contact with the gas permeable membrane 8.

[0134] The height and the spacing of the channels 92 or the pillars 911 are adjusted so as to minimize the risk of collapse of the gas permeable membrane 8 in the gas chip when the pressure in the processing chamber is greater than that in the gas exchange medium, for example by a difference of 1 bar. The channels 92 or pillars 911 supporting the gas permeable membrane 8 preferably have a height less than 100 μm, preferably less than 50 μm; an aspect ratio close to 1, preferably less than 1; and a spacing of the order of their width, for example twice their width. In the case of the use of densely arranged channels 92, the channels 92 are arranged so as to provide an efficient control of the gas concentration in the gas exchange medium present in the gas chip, through diffusion and convection. As shown in the figures, low hydraulic resistance channels may be added to the gas chip along the longitudinal axis of the segment 21 of the processing chamber to allow renewal of the gas exchange medium with a lower pressure.

[0135] When the above cross sections are chosen for the input seeding binary tree 3, the same cross sections may be chosen for the output seeding binary tree 4. In addition, when the above cross sections are chosen for the input seeding binary tree 3, the distance p between neighboring seeding channels 33 perfusing a segment 21 of the processing chamber 2 at their junction with the processing chamber may be chosen to be 1 mm, the height H of the processing chamber 2 may be chosen to be 100 μm, the width W of the processing chamber 2 may be chosen to be 5 mm

[0136] Method

[0137] A method for processing cells using any one of the above embodiments of the microfluidic devices 1 comprises steps as described below.

[0138] Seeding the Processing Chamber

[0139] The seeding step by means of the microfluidic device 1 is configured to result in a homogeneous distribution of cells within the processing chamber 2. It is noted that seeding should be performed relatively quickly, in particular in the case of adherent cells, in order to avoid an impact of pre-seeding conditions on the cells, such as anoikis, undesired adhesion of cells upstream the device, aggregation.

[0140] Using the microfluidic device 1 as describe above, the cells are seeded by seeding flow pulses, also called seeding pulses, representing a volume which is usually of the order of the volume of the processing chamber 2, usually between R=40% and R=200% of the volume of the processing chamber. Then, if the cells to be seeded are suspended in a volume representing x times the volume of the processing chamber 2, it takes in the order of x/R seeding flow pulses to seed them. Additional seeding pulses called secondary seeding pulses are also performed with “cell free” medium input in order to seed the cells remaining upstream, due notably to sedimentation and dispersion.

[0141] For each successive pair of seeding pulses, the pulses are separated by a resting time, or seeding pause, allowing for the most recently seeded cells to sediment in the processing chamber 2. To speed up sedimentation, and thus the seeding process, it is advantageous to seed the cells in a medium of low to moderate viscosity, for example a medium having a kinematic viscosity of not more than five times that of water at 37° C., and negligible elasticity, for example crosslinked hydrogel is not preferred as a seeding medium. For example, for a 100 μm height, 5 mm width, 640 mm total length processing chamber: the volume of the processing chamber is 320 μL, the seeding flow rate can be chosen between 1 and 100 μL/s depending notably on cell sedimentation speed. A typical value of 30 μL/s is recommended as a first choice before further optimization, a seeding pause of 5 s to 1200 s can be used depending on the cell sedimentation speed. A typical value of between 100 s and 300 s is recommended as a first choice before further optimization. A seeding pulse of 190 μL (around 60% of the processing chamber volume) can be chosen as a first value, assuming a widespread position distribution of the seeded cells during the seeding pulse. This value is adjusted according to the actual position distribution of the seeded cells during the seeding pulse. In such a case, one and preferably two to ten secondary seeding pulses are recommended to ensure a very low rate of cells lost upstream. The number of secondary seeding pulses should be adjusted to the total volume upstream the processing chamber 2 in the seeding flow, including potential tubing and other accessories used to perfuse the microfluidic device 1.

[0142] To avoid a long seeding time, it is preferable that the cells to be seeded be suspended in a medium volume where x/R is lower than 20. A too low value such as x/R=1 is not preferable either as the dispersion of the input cell suspension may be strongly affecting the homogeneity of the seeding along the width of the processing chamber. A value of x/R between 3 and 10 is recommended. With such parameters and a seeding pause of 300 s, the initial seeding pulses are performed within approximately 30 minutes, which is an acceptable duration. Few cells travelling upstream more slowly for various reasons including sedimentation and dispersion may experience a longer duration of the seeding process which is found to not jeopardize the advantages of the invention. It is noted that this method provides a concentration of the cell suspension. This is a further advantage of the invention, since cell suspension concentration is frequently necessary in bioproduction. By integrating the concentration function within a bioprocessing device, the need for cell concentration dedicated modules is reduced, which simplifies the implementation of bioproduction protocols.

[0143] Optional Further Seeding of the Processing Chamber

[0144] When it is desired to perform two seeding steps, for example to seed successively two different cell populations in the same processing chamber, a second seeding step as described above can be performed after the first one. When it is not necessary to have a delay between the two seedings it is possible to use the second seeding initial pulses as secondary seeding pulses of the first seeding. By doing this, the invention advantageously allows for simultaneously mixing and concentrating two different cell populations.

[0145] Three or more seeding steps of the processing chamber can be performed sequentially and similarly to the second seeding described above. Then, again, the next seeding initial pulses can be used as secondary seeding pulses of the previous seeding.

[0146] Medium Renewal

[0147] Many ordinary operations of bioproduction involve partial or extensive replacement of the medium without harvesting the cells, they are called medium renewal steps hereafter.

[0148] Medium renewal steps are for example ordinarily used for cell amplification, differentiation, viral transduction (or other genetic editing method), labelling, tagging, staining, filtration, thawing, freezing (etc.) protocols.

[0149] In bioproduction, medium renewal must generally be performed accurately in terms of volumes, to result in a homogeneous medium within the processing chamber, avoid downstream loss of cells, avoid exposure to mechanical stress or other perturbations.

[0150] Medium Renewal Through the Seeding Channels

[0151] To do so with the microfluidic device 1 according to the invention, the seeding flow mode is used to distribute the flow evenly in the processing chamber 2 with a minimal stress on cells as well as a minimal risk of losing cells. For partial replacement of the medium, the added volume is injected in the processing chamber 2, and then homogenized with back and forth flow or with loop flow or with a combination of both.

[0152] During such medium renewal operations, the flow rate is chosen so as to avoid excessive pressure (too high flow rate) or too slow operation (too low flow rate). The flow rate is also chosen depending on the cells so as to result in a low risk of undesired cell loss. The allowable flow rate for these medium renewal operations depends on the potential coating of the processing chamber 2 as well as on the type of the cells. It may additionally be preferable to choose a high flow rate within the range where cell loss is negligible to allow faster operation and more efficient disposal of certain compounds such as non-cellular vesicles, cell fragments, dying or dead cells.

[0153] In certain embodiments, the flow loop can be made in part or totality of gas permeable (or semi-permeable or selectively permeable) material to provide gas exchange, in particular exchange of CO2 and O2. This type of setup is of particular interest when the gas permeability of the processing chamber 2 is low, in order to avoid a too frequent replacement of the culture medium.

[0154] In certain embodiments, it can be found advantageous to replace a small fraction of the medium frequently rather than a large fraction less frequently to reduce the fluctuation of parameters of the medium such as nutrient concentrations. Indeed, smaller and more frequent changes result in less abrupt changes of the processing parameters which can be found to result in more robust and reproducible processes.

[0155] In certain embodiments it can be found advantageous to increase the concentration of one or several species of the medium by the replacement of the medium with a smaller amount of concentrated solution rather than a larger amount of less concentrated solution. Indeed, replacing a smaller amount of the volume can avoid unnecessary elimination of medium components the concentration of which decays more slowly than those who need to be added more frequently. Additionally, this type of operation results in less dilution of the compounds secreted by cells which can be found to have important roles to maintain or evolve cell phenotype, e.g. stem cell maintenance and differentiation.

[0156] Medium Renewal Through Harvest Channels

[0157] In certain and less frequent embodiments, especially when cells are found to have a relatively high adherence to the processing chamber, the medium renewal in harvest flow mode can be found advantageous. This is notably the case when: [0158] a higher shear rate is desired to eliminate bodies such as dying cells, dead cells, non-cellular vesicles, cell fragments, platelets, red blood cells, [0159] a high shear rate is desired to trigger mechano-transduction,

[0160] In certain embodiments, these operations are performed in linear flow only, in other in loop flow only, in yet others in a combination of linear flow and loop flow.

[0161] Medium Renewal Through Both Types of Channels

[0162] When it is desired to completely wash the device to eliminate a compound such as a chemical, an enzyme, a virus, medium renewal using seeding and/or harvest channels may be performed simultaneously or sequentially and eventually repeatedly.

[0163] These operations contain at least some linear flow but they sometimes also comprise some loop flow.

[0164] Gas Concentration Maintenance Using Diffusive Exchange Across a Gas Permeable Membrane Closing the Processing Chamber

[0165] The gas concentration in the processing chamber is advantageously obtained by diffusive exchange across a gas permeable membrane, thus allowing a reduction of culture medium consumption to compensate for oxygen consumption in the case of living cells, for example. To provide such a diffusive exchange, a gas exchange medium such a gas mix of desired composition, or a liquid with adjusted dissolved gas concentration, is disposed on the outer side of the gas permeable membrane opposite from the processing chamber. To provide stable processing conditions, or to vary processing conditions in terms of gas concentration in the processing chamber, the gas exchange medium with adjusted gas concentration is renewed on the side of the gas exchange medium opposite from the processing chamber at a rate adapted to the desired speed of variation of the gas concentration in the processing chamber or adapted to compensate for gas concentration variations in the processing chamber that may be due, for example, to metabolic activity.

[0166] In embodiments comprising a gas chip, the gas exchange medium is flown through the gas chip from its input to its output using conventional means such as pumps, gas sources or exchangers. The gas chip makes it possible to have a good control of gas concentration renewal in the processing chamber as well as a reduced required volume of gas exchange medium. This is particularly interesting when gas to be diffused into the processing chamber come from a source such as a high-pressure vessel comprising high molecular purity gas.

[0167] Harvest

[0168] Harvesting can comprise the following steps, the order indicated below only a non-limiting example. The following steps can be performed or not, any number of times, in any order and in any sort of implementation: [0169] Washing of the processing chamber 2 or of the whole microfluidic device 1 according to one of the medium renewal methods, [0170] Medium renewal by injecting a cell detachment reactant into the processing chamber 2, such as an enzyme. This step can be performed according to any medium renewal method, but preferably with a method using only seeding channels 33, 43, [0171] Additional physical treatment such as temperature, vibrations, electric field, magnetic field, illumination with visible or invisible light such as ultraviolet light or combinations of the latter to favor cell detachment, [0172] Washing of the processing chamber 2. This step can be performed according to any medium renewal method but much preferably with a method using only seeding channels 33, 43, [0173] Recollection of the cells, using only harvest channels 53, 63, while the seeding channels 33, 43 are blocked as stiffly as possible at their extremities, a flow of harvest solution such as medium or buffer being applied to the microfluidic device 1 and the cells being harvested in the output. The representative shear rate of this operation is chosen to be the highest permissible considering the constraints of mechanical integrity of the microfluidic device 1 and its accessories, with respect to the resulting pressure and the constraints of cell mechanical sensitivity. Typical shear rates allowing efficient recollection of cells with a low residual adherence to the walls of the processing chamber are in the range from 100 to 10 000 s.sup.−1; however, in specific cases, such as cells very sensitive to shear or, oppositely, cells with high residual adherence to the walls of the processing chamber, values out of this range may be used, [0174] Imaging of the microfluidic device 1, and in particular of the processing chamber 2, can optionally be performed in order to evaluate the efficacy of the harvest, in particular when a lower shear rate than usual is used.

[0175] The volume flushed through the microfluidic device 1 during the recollection of the cells is at least equal to the sum of the segments 21 of the processing chamber 2, intermediary and downstream flow path volume to the recollection volume. Preferably, a volume of twice this minimal amount or greater is used. As a first value to be used before further optimization, a volume of three times the minimal volume mentioned above is recommended.

[0176] In certain embodiments, the flow rate used during the recollection of the cells is increased continuously or discontinuously. However, it is not recommended unless specific situation suggests that it would be useful. An element that may suggest the relevance of an increasing flow rate during the recollection of the cells may for example be the very high cell density within the processing chamber 2. In this case, the cell density can increase the flow rate resistance, and thus an increasing flow rate during the recollection of the cells can avoid an excessive pressure by progressively reducing the number of cells in the device. In such cases, the upstream pressure can advantageously be monitored to adjust the flow rate and avoid excessive pressure and risk of leaks.

[0177] Cell Sorting

[0178] Because cells may have different types of adherence in the processing chamber 2, to the coated or uncoated processing chamber or to the other cells, and because they may also have different shape and size, it is possible to exploit these differences within the microfluidic device 1 to selectively harvest certain cells and/or selectively retain certain cells in order to achieve cell sorting.

[0179] In some embodiments, the processing chamber 2 is previously coated with one or several compounds (binding anchors) such as an antibody, a nanobody, an ECM molecule, fragments of ECM molecules, peptides that are specifically binding to molecules (binding targets) which are more frequent at the surface of the cells one of the two groups of cells to be separated from each other than at the surface of the cells of the second group.

[0180] In some embodiments, the cells are seeded in the processing chamber 2 according to one of the methods described above. In some embodiments, small and intense back and forth pulses are applied to the processing chamber 2 after the seeding step to reduce the frequency of superposed cells. Such pulses should represent a volume inferior to 25% of the processing chamber volume and be at least as intense as seeding flow pulses.

[0181] In some embodiments, a first recollection is performed by applying a flow through the harvest channels 53, 63 while the seeding channels 33, 43 are blocked at their extremities. In such embodiments, cells having a lower adherence to the processing chamber surface, coating or to other cells adhering quite strongly are preferentially recollected resulting in enrichment in such cells in the output of the flow and a depletion of those cells within the processing chamber 2. In some such embodiments, the flow rate is increased continuously or discontinuously up to values slightly inferior to those compromising the viability of the cells of interest or to those compromising the apparatus mechanical integrity. In some such embodiments, the volume obtained from the processing chamber 2 during this flow is separated according to ranges of applied flow rate. In some such embodiments, the number of cells recollected per unit time is monitored during this operation, for example using an imaging sensor positioned at the output of the harvest channel 63. In some such embodiments, the measure of the number of cells recollected per unit time is used to tune the flow rate. In some such embodiments, the flow rate is automatically increased until the maximum value at a specific rate while the measure of the number of cells recollected per unit time is inferior to a previously defined threshold value.

[0182] In some embodiments, a harvest (second recollection) according to one of the harvest methods described above is performed after the first sorting flow is applied to recollect some, potentially all, of the cells remaining in the processing chamber 2.

[0183] In some embodiments, one of the sorted fractions collected during the first or the second recollection is seeded again in the processing chamber 2, or in the processing chamber of a similar microfluidic device, to undergo an additional sorting. Any number of such sorting repetitions can be performed to obtain the desired composition and purity of the cell population.

[0184] As can be seen from the previous examples, a microfluidic device according to the invention provides a novel geometry where a processing chamber of high capacity is connected to two sets of channels corresponding to two distinct modes of flow, i.e. a seeding flow mode and a harvest flow mode. The seeding flow mode makes it possible to obtain a homogeneous and efficient seeding of the processing chamber, with low risk of upstream and downstream particle deposition, and makes the microfluidic device capable of concentrating and washing a particle suspension, while the harvest flow mode provides a quick, efficacious and efficient harvest. By integrating these functionalities into one microfluidic device, the invention is particularly adapted to implement automated and/or miniaturized bioproduction, which has many technical advantages such as gain in particle and reactant efficiency due to the reduction of the number of transfer steps. This unique configuration has the additional advantage of being feasible with only one channel layer, without filter membranes, thus drastically reducing manufacturing complexity and cost.

[0185] The invention is not limited to the examples described and shown.

[0186] In particular, in the illustrative embodiments described above, the microfluidic device comprises a single input seeding tree and a single output seeding tree for all the segments of the processing chamber. As a variant, the microfluidic device may comprise several input seeding trees and several output seeding trees, provided that each segment of the processing chamber has a single input seeding tree and a single output seeding tree.

[0187] In addition, in the examples described above, the successive segments of the processing chamber are arranged next to each other, being parallel to one another, and connected by fluidic connectors that are transversal to the segments of the processing chamber. As a variant or in addition to this parallel arrangement, the processing chamber may comprise several segments arranged end to end and/or segments arranged transversally to one another. In particular, in the embodiments of FIGS. 4 and 5, the fluidic connectors 24 may be replaced by segments 21 of the processing chamber.

[0188] Furthermore, in the examples shown in the figures, the microfluidic device does not comprise chamfers at the junctions of the input and output seeding channels with the processing chamber. The presence of such chamfers is however advantageous and within the scope of the invention. Alternative progressive increase of seeding channel cross-section in the vicinity and toward the processing chamber, such as a fillet, can also be found advantageous to improve the seeding flow.

[0189] Additionally, in the embodiments described above, each segment of the processing chamber has a rectangular geometry and geometrically symmetric binary trees of seeding channels. However, in other embodiments of the invention, the processing chamber and the trees of seeding channels may have different shapes and arrangements. In some embodiments of the invention, several microfluidic devices according to the invention may also be stacked, the devices of the stack then being possibly connected by hydraulic manifolds. According to another variant, the processing chamber may contain pillars joining its top and bottom surfaces to reduce the risk of collapse related to the flatness of the processing chamber. Such additional pillars may be particularly advantageous in embodiments where the width of the processing chamber is much greater than its height.