Sizing of a Microfluidic Device for Confining a Sample

Abstract

The present invention relates to a method for sizing a microfluidic device for confining a sample. The sample to be confined can include cells (biological sample) or microparticles suspended in a carrier fluid medium. The present invention also relates to a method for sizing a microfluidic device for confining an explant contained in a cell culture fluid medium.

Claims

1. A method of sizing a microfluidic device intended to confine an initial sample comprising at least one population of cells or microparticles in suspension in a carrier fluid medium, the microfluidic device including: an input zone adapted to receive the carrier fluid medium containing the sample, said input zone corresponding to a cylindrical input tank of diameter D.sub.in, a confinement zone in which at least one part of the sample is confined comprising a base of surface S.sub.ch and length L.sub.ch and a side wall of height H.sub.ch, said confinement zone communicating with said input zone via a first channel of length L.sub.in, height H.sub.in and width W.sub.in, and an output zone adapted to discharge said liquid including the sample, said output zone corresponding to a cylindrical output tank of diameter D.sub.out, said output zone communicating with said confinement zone via a second channel of length L.sub.out, height H.sub.out and width W.sub.out, said method being characterized in that it consists of sizing of said device as a function of the number of cells or microparticles to be confined, comprising the following steps: A. sizing of said confinement zone as a function of the preferred amount of cells or microparticles to be confined and of the preferred coverage rate of the base of said confinement zone by said cells or microparticles, so as to define the surface D.sub.ch and the height H.sub.ch characterizing the confinement zone; B. sizing of the first channel and the second channel comprising: b1) calculation of the sedimentation speed v.sub.sedi of a particle or a cell, b2) determination of the speed v.sub.ch of the carrier fluid medium in said confinement zone as a function of the sedimentation speed v.sub.sedi of a particle or a cell as per the equation (1): $\begin{array}{cc}{V}_{\mathrm{ch}}{V}_{\mathrm{sedi}}.Math.\frac{H_{\mathrm{ch}}}{D_{\mathrm{ch}}}& \left(1\right)\end{array}$ b3) determination of the loss of charge in said device as a function of the volume of fluid medium injected Z between the input and output zones which is necessary for setting up an adequate flow in the confinement zone; b4) determination of the geometric parameters of said microfluidic device from Z and the speed v.sub.ch of the carrier fluid medium.

2. The method according to claim 1, wherein the sizing step A of said confinement zone comprises: A1) determination of the surface S.sub.ch of the base of said confinement zone as per the Stokes formula (5): $\begin{array}{cc}=\frac{N.Math..Math.r^2}{\mathrm{Sch}}& \left(5\right)\end{array}$ with being the coverage rate of the base, r being the radius of a particle or a cell, N being the number of neurons or cells to be confined, defined by the equation (6):
N=V(6) with being the concentration of cells or neurons in said sample, V being the sample volume entered in said microfluidic device at an instant t, defined as per the equation (7):
V=Qt(7) with Q being the flow rate of the sample in said microfluidic device, A2) fastening by the user of said microfluidic device of the height H.sub.ch of the wall of height H.sub.ch, as a function of the amount of preferred volume in the confinement zone and associated manufacturing restrictions.

3. The method according to claim 1, wherein the confinement zone has a cylindrical geometry with a circular base of diameter D.sub.ch, such that L.sub.ch=D.sub.ch.

4. The method according to claim 1, wherein the sedimentation speed v.sub.sedi of a particle or a cell is calculated in sub-step b1) as per the Stokes equation (8): $\begin{array}{cc}\mathrm{vsedi}=\frac{2.Math.r^2.Math.g.Math..Math.}{9.Math.}& \left(8\right)\end{array}$ with: r being the radius of a particle or a cell, being the dynamic viscosity of said carrier fluid medium, and being the difference in density between that of the particles or cells to be confined and the carrier fluid medium.

5. The method according to claim 1, wherein the determination step b4) of the geometric parameters of said microfluidic device comprises the following sub-steps: b41) choice of seven geometric parameters of said microfluidic device among the eight geometric parameters D.sub.in, H.sub.in, W.sub.in, L.sub.in, D.sub.out, H.sub.out, L.sub.out and W.sub.out; and b42) calculation of the unknown remaining geometric parameter as a function of Z and of the speed v.sub.ch of the carrier fluid medium.

6. The method according to claim 5, wherein the unknown parameter is calculated in the sub-step b42) from the equation (2) hereinbelow, for a loss in regular charge and a laminar flow of the carrier fluid medium in said microfluidic device: $\begin{array}{cc}.Math..Math.z=\frac{.Math..Math.Q}{.Math..Math.g}.Math.\left(\frac{{}_{\mathrm{in}}.Math.L_{\mathrm{in}}}{W_{\mathrm{in}}.Math.H_{\mathrm{in}}^3}+\frac{{}_{\mathrm{ch}}.Math.L_{\mathrm{ch}}}{W_{\mathrm{ch}}.Math.H_{\mathrm{ch}}^3}+\frac{{}_{\mathrm{out}}.Math.L_{\mathrm{out}}}{W_{\mathrm{out}}.Math.H_{\mathrm{out}}^3}\right)& \left(2^{}\right)\end{array}$ with: Q being the constant fluid flow rate in said device defined by the equation (9):
Q=.sub.chH.sub.chW.sub.ch(9) designates the gravitational acceleration, designates the dynamic viscosity of the carrier fluid medium, designates the density of the carrier fluid medium, L.sub.ch designates the width of the confinement zone, represents a friction coefficient, calculated for a low Reynolds number as per the equation (3): $\begin{array}{cc}=12.Math.\left(1-6.Math.{\left(\frac{2}{}\right)}^5.Math.\left(\frac{H}{W}\right)\right)& \left(3\right)\end{array}$

7. The method according to claim 6, wherein the unknown parameter is L.sub.out which is calculated according to the equation (4): $\begin{array}{cc}{L}_{\mathrm{out}}=\frac{W_{\mathrm{out}}.Math.H_{\mathrm{out}}^3}{{}_{\mathrm{out}}}.Math.\left(.Math..Math.z.Math..Math.\frac{.Math..Math.g}{.Math..Math.Q}-\frac{{}_{\mathrm{in}}.Math.L_{\mathrm{in}}}{W_{\mathrm{in}}.Math.H_{\mathrm{in}}^3}-\frac{{}_{\mathrm{ch}}.Math.L_{\mathrm{ch}}}{W_{\mathrm{ch}}.Math.H_{\mathrm{ch}}^3}\right)& \left(4\right)\end{array}$

8. The method according to claim 1, wherein the sample is a biological sample consisting of a population of cells selected from neurons and eukaryotic cells in suspension in a cellular culture medium, or in suspension in water, salt or non-salt, a solvent, a hydrogel or an organic scaffold or a polymer.

9. The method according to claim 1, wherein the sample is a non-biological sample consisting of a population of microparticles in suspension in water, salt or non-salt, a solvent, a hydrogel or an organic scaffold or a polymer, said microparticles being selected from metallic microparticles, or made of semi-conductive material, or polyethylene glycol (PEG).

10. The method according to claim 1, wherein said confinement zone is connected to at least one isolation chamber via at least one additional channel having a hydraulic resistance for passage, with no return, of the sample in said isolation chamber.

11. The method according to claim 10, wherein said isolation chamber is a confinement zone of an additional microfluidic device.

12. The method according to claim 10, wherein said isolation chamber comprises an additional sample.

13. Use of a microfluidic device such as sized according to claim 12, for studying the interaction between the initial sample in said confinement zone and the additional sample in said isolation chamber.

Description

[0094] Other advantages and particular features of the present invention will emerge from the following description given by way of non-limiting example and done in reference to the appended figures and corresponding examples:

[0095] FIG. 1 illustrates a schematic sectional view of an example of microfluidic device sized according to the first embodiment of the method of the invention, as well as a plan view of this same device;

[0096] FIG. 2 illustrates a photograph of the confinement zone (or depositing chamber) of the microfluidic device illustrated in FIG. 1;

[0097] FIG. 3 illustrates a schematic sectional view and a schematic plan view of an example of microfluidic device sized according to the second embodiment of the method of the invention.

[0098] FIGS. 4 and 5 each illustrate a photograph of the confinement zone of the microfluidic device of FIG. 3, respectively of case where the explant is a ganglion (FIG. 4) and a hippocampus (FIG. 5).

[0099] Identical elements shown in FIGS. 1 to 8 are identified by identical reference numerals. FIGS. 1 and 2 are commented on in more detail in the example 1, whereas FIGS. 3 to 5 are commented on in more detail in the example 2.

EXAMPLE 1

First Embodiment: Sizing of a Microfluidic Chip (Illustrated in FIGS. 1 and 2) in Light of its Use for the Depositing of Neurons

Device

[0100] A microfluidic device 10, sized in accordance with the method according to the invention (first embodiment) is used for the depositing of neurons.

[0101] FIG. 1 shows in particular (in profile and plan view) an example of microfluidic device 10 sized according to the first embodiment of the method of the invention. FIG. 2 shows a photograph of the depositing chamber 5 used in the case of depositing of neurons.

[0102] This microfluidic device 10 comprises an input zone 1 adapted to receive a liquid including the biological sample and an output zone 4 adapted to discharge this liquid. The input zone 1 and the output zone 4 correspond to the cylindrical tanks which have the same diameters and different heights (the height of the input zone 1 is less than the height of the output zone 4). In particular, the diameter D.sub.in of the tank, which corresponds to the input zone 1, is the same as the diameter D.sub.out of the tank which corresponds to the output zone 4. However, the input zone 1 and the output zone 4 can have different diameters. Also, the input zone 1 and/or the output zone 4 can also have forms other than the cylindrical form (for example square forms).

[0103] It should be noted that the dimensions (height and section) of the input zone 1 are selected relative to the biological sample introduced to the input zone 1: it is preferable for the dimensions of the input zone 1 to be such that it can store all the nutrients included in the cellular culture liquid and which are necessary for survival of neurons in the confinement zone (or depositing chamber) for a period ranging from 12 hours to 48 hours (but not limited to these periods), to conduct in vitro studies on these neurons.

[0104] The microfluidic device 10 of FIG. 1 further comprises a confinement zone 5 (or depositing chamber) where the biological sample is confined. More particularly, in the example of FIG. 1, the chamber 5 has a cylindrical form having a length D.sub.ch and a height H.sub.ch. It should be noted that in the example of FIG. 1, the length D.sub.ch (which corresponds to the diameter of the chamber cylindrical) is less than the diameter D.sub.in of the input zone 1 and the diameter D.sub.out of the output zone 4. However, the length D.sub.ch can be equal to or greater than the diameter D.sub.in of the input zone 1 and the diameter D.sub.out of the output zone 4. Also, it should be noted that the chamber 5 can also have other forms, for example an elongated, triangular, square or pentagonal form.

[0105] Also, it should be noted that the dimensions (height and length) of the chamber 5 are determined as a function of the volume of liquid received by the input zone 1 to be confined in this chamber 5, and they can vary from a few micrometers to a few centimeters.

[0106] Also, FIG. 1 shows that the chamber 5 is disposed between a first channel 2 and a second channel 3, the first channel 2 connecting the input zone 1 and the chamber 5 and the second channel 3 connecting the chamber 5 and the output zone 4. The first channel 2 has a height H.sub.in, a length L.sub.in and a width W.sub.in and the second channel 3 has a height H.sub.out, a length L.sub.out and a width W.sub.out. The first and the second channel have heights less than those of the input zone 1 and of the output zone 4, and they are disposed relative to each other such that the liquid received by the input zone 1 flows via the first channel 2, the chamber 5 then the second channel 3 towards the output zone 4.

[0107] It should be noted that the flow of the liquid in the microfluidic device of FIG. 1 can be stopped any time either by removing the remaining volume of liquid of the input zone 1, or by adding an equivalent volume of liquid to the output zone 4.

[0108] Also, in the example of FIG. 1, the height H.sub.in and the width W.sub.in of the first channel 2 are greater than the respective height H.sub.out and the width W.sub.out of the second channel 3 and the length L.sub.in of the first channel 2 is the same as the length L.sub.out of the second channel 3. It should be noted that in another example, the height H.sub.in and the width W.sub.in of the first channel 2 can be equal to or less than the respective height H.sub.out and the width W.sub.out of the second channel 3, and the length L.sub.in of the first channel 2 can be greater than or less than the length L.sub.out of the second channel 3.

[0109] It has been noted that the presence of the first channel 2 upstream and of the second channel 3 downstream of the confinement zone (chamber 5) in the microfluidic device 10 of FIG. 1 (the terms upstream and downstream are determined relative to the direction of the flow mentioned hereinabove of the liquid which is received by the input zone 1), and more particularly the adequate adaptation of the dimensions (i.e. the height, the length and the width) of these two channels, confines at least one part of the biological sample in the confinement zone by controlling the spatial distribution of the biological sample confined in this confinement zone. In particular, it has been noted that adaptation of the respective dimensions of the first channel 2 and of the second channel 3 as a function of the loss of charge in the microfluidic device 10 produces a microfluidic device 10 in which the speed of the flow of the liquid in the chamber 5 can be controlled, and the control of this speed allows control of the spatial distribution of the biological sample in this chamber 5 during the flow of the liquid. It should be noted that the speed of the flow of the liquid in the chamber 5 depends on the difference between the volume of liquid in the input zone 1 and the volume of liquid in the output zone 4 of the microfluidic device 10. In this way, the control of the speed of the flow of the liquid in the chamber 5 can be done by controlling this difference in volumes of the liquid between the input zone 1 and the output zone 4. The control of this difference in volumes of the liquid can be done by removing or adding a volume of liquid either to or from the input zone 1 or the output zone 4.

Sample

[0110] The sample implemented in this example is a culture medium of neurons, which comprises: [0111] 5.10.sup.7 neurons/mL, in suspension in [0112] a cellular culture liquid medium, containing Neurobasal, b27, L-cysteine and Pen/Strep.

Sizing of the Microfluidic Chip of FIGS. 1 and 2 as a Function of the Sample

[0113] An example of confinement of a population of neurons in the depositing chamber 5 of the microfluidic device 10 of FIG. 1 is illustrated by the photograph of FIG. 2: in this chip 10, the diameters of the input zone 1 and of the output zone 4 are not the same.

[0114] In particular, the photograph of FIG. 2 illustrates a scale bar 11 of a length of 200 m and cells which are confined in the chamber 5 (confinement zone). These cells are neurons of dimensions of about 10 m and a surface density of 95%.

[0115] As FIG. 2, the confinement zone is separated between four zones (see zones (A), (B), (C), (D) in FIG. 2).

[0116] As illustrated in FIG. 2, the neurons uniformly cover the surface of the confinement zone. It should be noted that to obtain this uniform confinement in the confinement zone, the dimensions of the first channel and of the second channel have been adapted to provide a microfluidic device 10 in which the flow speed V.sub.ch of the liquid in the chamber 5 can be controlled. In particular, in the case of the confinement uniform of the example of FIG. 2, the flow speed V.sub.ch of the liquid in the chamber 5 is controlled so as to be equal to or less than the sedimentation speed V.sub.sedi of the biological sample multiplied by the quotient of the height H.sub.ch and length D.sub.ch of the chamber 5 (as per the equation 1).

[0117] It should be noted that this adaptation of the dimensions of the first channel and of the second channel has been made given the losses of charge of equations (2) and (4) mentioned hereinabove, the concentration and the dimensions of the neurons and the dimensions of the input zone 1, of the output zone 4 and of the chamber 5.

[0118] In particular, the microfluidic chip 10 illustrated in FIG. 1 has the following characteristics: [0119] the input channel 2 has a height H.sub.in of 100 m, a width W.sub.in of 120 m and a length L.sub.in of 1500 m; [0120] the channel output 3 has a height H.sub.out of 20 m, a width W.sub.out of 120 m; [0121] also, the depositing chamber 5 is also cylindrical and has a height of 100 m and a diameter of 500 m; [0122] also, the input zone 1 is cylindrical; it has a height D.sub.in of 5 mm and a diameter of 4 mm, and the output zone 4 is also cylindrical and has a height of 4 mm and a diameter D.sub.out of 4.5 mm.

[0123] L.sub.out is calculated in accordance with the sizing method according to the invention, starting out from the equation (2).

[0124] In the present example, the parameters fixed at the outset were the parameters D.sub.in, H.sub.in, W.sub.in, L.sub.in, D.sub.out, H.sub.out, and W.sub.out.

[0125] But it is also possible to calculate another unknown parameter, different to L.sub.out, and fix the seven other remaining parameters.

[0126] The speed of the flow of the cellular culture liquid in the depositing chamber 5 is 41.Math.10.sup.5 m/s and this speed has been obtained by introduction of a volume of liquid of 20 L in the input zone 1.

[0127] It should be noted that in the microfluidic device of the publication by Taylor et al..sup.[1], there are no first channel and second channel connected to the Taylor confinement zone (culture chamber), as is the case in the microfluidic device 10 sized as per the present invention, and so on. In the Taylor device, it is not possible to confine the sample in the confinement zone by controlling the spatial distribution of the sample in this confinement zone.

[0128] The control of the speed of the flow of the liquid in the chamber 5 enables control of the spatial distribution of the biological sample in this chamber 5 during flow of the liquid.

Experimental Protocol

[0129] Growing cells requires a sterile medium to avoid any form of contamination. Because microfluidic chips are not sterile once they are assembled, they therefore need to be sterilized prior to use.

[0130] For this purpose, the microfluidic chip 10 used in this example for the depositing of neurons (and whereof the depositing chamber 5 is illustrated by FIG. 2), is introduced to a sterile environment (such as a hood with sterile laminar flow), and ethanol is introduced to the charging zone to replace water in the channels 2, 3 and in the depositing chamber 5. The ethanol is then rinsed with sterile water three times, before the chip 10 is exposed to UV for 30 minutes.

[0131] The surface of the depositing chamber can then be functionalized to promote the culture of cells.

[0132] Once the chip 10 is ready, a predefined volume of the sample (from 0.5 to 10 mL) is deposited in the charging zone (Input zone 1) to generate flow and begin the depositing of cells.

[0133] The flow can be stopped any time, either by removing the volume of liquid remaining in the input zone 1, or by adding an equivalent volume to the output zone (equalizing the hydrostatic pressure between the input and the output).

EXAMPLE 2

Second Embodiment: Sizing of a Microfluidic Chip (Illustrated in FIGS. 3 to 5) in Light of its Use for Depositing Explants

Device

[0134] A microfluidic device 10, sized in accordance with the method according to the invention (second embodiment), is used for depositing explants.

[0135] FIG. 3 shows in particular (in profile and plan view) an example of microfluidic device 10 sized according to the second embodiment of the method of the invention.

[0136] As illustrated in FIG. 3, the microfluidic device 10 comprises an input zone 1 adapted to receive liquid including the biological sample and an output zone 4 adapted to discharge this liquid. The input zone 1 and the output zone 4 correspond to the cylindrical tanks which have the same diameters and the same heights. In particular, the diameter D.sub.in of the tank which corresponds to the input zone 1 is the same as the diameter D.sub.out of the tank which corresponds to the output zone 4.

[0137] However, the input zone 1 and the output zone 4 can have different diameters and/or heights. Also, the input zone 1 and/or the output zone 4 can have forms other than cylindrical (for example square forms).

[0138] It should be noted that the dimensions (height and diameter) of the input zone 1 are selected relative to the dimensions of the explant (ganglion or hippocampus especially) received by this input zone 1, such that the biological sample can enter the microfluidic device 10 of FIG. 3 via this input zone 1, as is the case for the input zone 1 of the microfluidic device 10 of FIG. 1.

[0139] Also, as is the case for the input zone 1 of the microfluidic device 10 of FIG. 1, it is preferable that the dimensions of the input zone 1 of the microfluidic device 10 of FIG. 3 are adequate so they can store the number of nutrients which are included in the liquid and which are necessary for survival of the explant in the confinement zone, for a period which can be between 12 hours and 48 hours (but not limited to these periods), so as to conduct in vitro studies for these cells.

[0140] The microfluidic device 10 of FIG. 3 also comprises a confinement zone 5 where the explant is confined. This confinement zone corresponds to a space of the first channel 2 (see for example the space downstream of the first channel 2 in the example of FIGS. 4 and 5 hereinbelow) and the dimensions of this confinement zone 5 can vary from a few micrometers to a few centimeters.

[0141] Also, this microfluidic device 10 comprises a first channel 2 and a second channel 3, which have heights less than those respectively of the input zone 1 and of the output zone 4. Also, as illustrated in FIG. 3, the first channel 2 has a height H.sub.in, a length L.sub.in and a width W.sub.in and the second channel 3 has a height H.sub.out, a length L.sub.out and a width W.sub.out.

[0142] Also, the first channel 2 and the second channel 3 of the microfluidic device 10 of FIG. 3 are disposed relative to each other such that the cellular culture liquid containing the explant introduced via the input zone 1 flows via the first channel 2, the confinement zone then the second channel 3 towards the output zone 4.

[0143] It should be noted that the flow of the liquid in the microfluidic device of FIG. 3 can be stopped any time either by removing the volume of liquid remaining of the input zone 1, or by adding an equivalent volume of liquid in the output zone 4.

[0144] Also, it should be noted that the output zone 4 can be adapted for allow aspiration of the liquid to boost the speed of the liquid in the microfluidic device 10. In particular, this aspiration of the liquid can be done by using a pipette, a nozzle or a suction capillary and the dimensions of the output zone 4 are adapted so that they correspond to the dimensions of the pipette, of the nozzle or of the suction capillary.

[0145] It should be noted that as is the case for the microfluidic device 10 of FIG. 1, the respective dimensions of the first channel 2 and of the second channel 3 of the microfluidic device 10 of FIG. 3 are adapted so that, during flow of the liquid, at least one part of the biological sample is confined in the confinement zone 5 and the spatial distribution of this biological sample in the confinement zone 5 is controlled. In particular, the width W.sub.out and/or the height H.sub.out of the second channel 3 is less than the respective width W.sub.in and/or the height H.sub.in of the first channel 2 so that the second channel 3 functions as a physical barrier for the biological sample and accordingly, confinement of the biological sample is done in a space of the first channel 2, as mentioned hereinabove. According to an example, the width W.sub.out and/or the height H.sub.out of the second channel 3, apart from being less than the respective width W.sub.in and/or the height H.sub.in of the first channel 2, they are also less than the dimensions of the biological sample so that the second channel 3 can offer an improved physical barrier which is adapted to the particular dimensions of the biological sample so as to more effectively block its passage in the second channel 3.

[0146] Also, adaptation of the width and/or the height of the first channel 2 depends on the dimensions of the explant and in all cases they are preferably at least 1% larger than the dimensions of the biological sample in suspension so that the biological sample can pass through the first channel 2 without damage.

[0147] Also, with respect to the length of the first channel 2, it is preferable for this to be as sort as possible, so as to let the explant be introduced to the first channel 2, and prevent accidents along the way (unwanted adhesion of the explant to the walls of the first channel 2 before arriving at the confinement zone).

[0148] In the example of FIG. 3, the critical element for confining at least one part of the biological sample in the confinement zone 5 by controlling the spatial distribution in this confinement zone is the particular structure of the microfluidic device 10 mentioned hereinabove, and not the loss of charge as is the case for the example of FIG. 1. In this way, it has been noted that the presence of the first channel 2 and of the second channel 3 in the microfluidic device 10 of FIG. 3, and more particularly the adequate adaptation described hereinabove of the respective dimensions of these two channels 2, 3, produces a microfluidic device 10 in which the speed of the flow of the liquid in the confinement zone can be controlled to confine at least one part of the biological sample in this confinement zone by controlling the spatial distribution of this biological sample in this confinement zone. It should be noted that in this example of FIG. 3, the speed of the flow of the liquid in the confinement zone can be controlled by aspiration of the liquid mentioned hereinabove.

Samples

[0149] One of the samples implemented in this example (case of FIG. 4) is a biological sample, which comprises: [0150] a ganglion, in suspension in [0151] a cellular culture liquid medium, containing Neurobasal, b27, L-cysteine and Pen/Strep.

[0152] The other sample implemented in this example (case of FIG. 5) is also a biological sample, but which comprises: [0153] a hippocampus, in suspension in [0154] a cellular culture liquid medium, containing Neurobasal, b27, L-cysteine and Pen/Strep.

Sizing of the Microfluidic Chip of FIGS. 4 and 5 as a Function of the Size of the Explant

[0155] FIGS. 4 and 5 each illustrate a photograph representing a plan view of a confinement zone of the microfluidic device 10 schematically illustrated in FIG. 3: [0156] in FIG. 4, the confined explant 12 is a ganglion of diameter of 250 m, and [0157] in FIG. 5, the confined explant 13 is a hippocampus of a length of 3 mm and a width of 200 m.

[0158] As is evident in FIGS. 4 and 5, the confinement zone 5 corresponds to a space downstream of the first channel 2 and the height of the second channel 3 is such that passage of the explant in the second channel 3 is blocked. It should be noted that in the example of FIGS. 4 and 5, the input channel 2 has a height of 1.2 mm, a width of 2.3 mm and a length of 3 mm and the channel output 3 has a height of 100 m, a width of 100 m and a length of 4 mm. Also, for the example of FIGS. 4 and 5, the input zone (not illustrated in FIGS. 4 and 5) is cylindrical and has a height of 5 mm and a diameter of 4 mm and the output zone (not illustrated in FIGS. 4 and 5) is also cylindrical and has a height of 5 mm and a diameter and 2 mm.

[0159] In the example of FIGS. 4 and 5, the volume of liquid introduced via the input zone 1 is 40 L and the speed of the flow of the liquid in the confinement zone are caused by aspiration.

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