Abstract:
The invention relates to a filtration device ( 100 ), characterized in that it includes: a first block ( 101 ) having a cavity forming a first chamber ( 110 ) comprising a bottom wall ( 111 ) having a set of microstructures including micro-walls and micro-contacts, the set of microstructures defining micro-chambers and micro-channels on the bottom wall ( 111 ) of the culture chamber; a second block ( 102 ) having a cavity forming a second chamber ( 120 ); and a filtration membrane ( 130 ), the first block ( 101 ), the membrane ( 130 ), and the second block ( 102 ) being arranged such that the membrane ( 130 ) is located between the first chamber ( 110 ) and the second chamber ( 120 ), adjacent to each of the first and second chambers ( 110, 120 ); as well as a first opening and a second opening for enabling a first fluid to pass into the second chamber ( 120 ) which is separated from the first chamber ( 110 ) by the membrane ( 130 ).

Description:
GENERAL TECHNICAL FIELD 
       [0001]    The present invention relates to reproduction in vitro of filtration phenomena. 
         [0002]    More specifically, it relates to a filtration device for a bioreactor with a membrane separating two chambers and a filtration system implementing one or more examples of such a filtration device. 
       STATE OF THE ART 
       [0003]    Transplants presently remain the most efficient solution for treating liver disorders and kidney dysfunctions. However, insufficiency of donors forces patients awaiting an organ to undergo major and regular treatments most often sources of complications. One of the great challenges of tissue engineering therefore lies in the development of artificial organs capable of replacing failing or absent organs. The patients would then see their life conditions improve and the costs of the treatments decrease. For this purpose, many investigations have been conducted in order to reproduce in vitro phenomena internal to the organs of the human and animal body, in particular filtration phenomena. 
         [0004]    To do this, it is necessary to have available bioreactors reproducing an environment favorable to the development and organization of cells, close to that of a animal or human tissue or organ on the one hand, membranes capable of reproducing the filtration phenomena for example, glomerular filtration in kidneys. Many solutions have been proposed in the prior art. 
         [0005]    Document U.S. Pat. No. 6,197,575 presents a device for cultivating cells in order to obtain artificial tissues or organs in vitro. This device comprises an enclosure with a filtration membrane separating the enclosure into two chambers, a membrane on which are arranged channels in order to receive a cell culture. Thus, the membrane playing the role of a filter is also used as a support for cultivating cells in a culture chamber, the other portion of the enclosure forming a discharge chamber. This suggests that the membrane has some rigidity. Various fluid inflow/outflow combinations for culture and discharge chambers are contemplated (see FIGS. 1 to 2c of document U.S. Pat. No. 6,197,575), each combination corresponding to a specific use of the device. 
         [0006]    The article           A MEMS-Based Renal Replacement System           published in June 2004 describes a unit for treating blood provided for application in continuous hemodialysis. The unit consists of a stack of bilayer devices each comprising a network of channels for blood circulation and a discharge chamber, both networks facing each other and separated by an ultrafiltration membrane (see  FIG. 2  of this document). The authors of this article used an algorithm for defining the morphology of a network reproducing blood circulation conditions in human blood vessels. 
         [0007]    If the presented unit gives the possibility of effectively mimicking the blood transport conditions in blood vessels, it is not without posing problems. Indeed, the blood is confined in channels with a very narrow section (a height of 35 microns) which limits the blood flow rate treated by each device of the entity and may induce clogging problems of the channels. The authors foresee not less than 100 bilayer devices required for carrying out hemodialysis. 
         [0008]    U.S. Pat No. 7,048,856 presents a compact ultrafiltration device which may be used as a bioreactor. The device comprises a chamber in which is placed an ultrafiltration membrane, said membrane separates the chamber into a filtration portion and a discharge portion, as well as a fluid inlet, a filtration fluid outlet and a fluid discharge outlet. The membrane is adapted to the fluid at the inlet and to the contemplated filtration. For example, the membrane may have pores, the size of which is selected in order to filter urea in the blood. Moreover, according to a particular embodiment shown in this document, the device may comprise an analysis chamber in which the filtered fluid is analyzed. The membrane may also receive a cell culture. The fluid inlet and outlet may be provided with pumps or valves for controlling the flow rate, optionally connected to pressure sensors. 
         [0009]    Document WO 2004/020590 describes a bioreactor intended for cultivating living cells, in which the conditions of the human body are reproduced artificially. In order to access better understanding of certain dysfunctions of the mechanisms of the body, it is necessary to develop bioreactors capable of mimicking the micro-environment of abnormal tissues in vitro. An application proposed by the document describes a chamber divided into two sub-chambers containing cells of a first type and cells of a second type respectively. Both sub-chambers are separated by a porous barrier which may be totally impervious or else pervious to certain specific cells. The bioreactor also comprises inlet/outlet accesses allowing circulation of cells, fluids or chemicals in each of the sub-chambers. By adding various substrates positioned in a suitable way in the bioreactor, it is possible to proceed with electrochemical and optical measurements. 
         [0010]    Thus, a large variety of in vitro filtration devices have been proposed in the past. 
       PRESENTATION OF THE INVENTION 
       [0011]    The invention proposes a filtration of a novel type having many advantages as compared with the solutions proposed in the prior art. 
         [0012]    For this purpose, the invention proposes according to a first aspect, a filtration device characterized in that it comprises:
       a first block having a cavity forming a first chamber including a bottom wall having a set of microstructures comprising microwalls and microbumps, the set of microstructures defining on the bottom wall of the culture chamber, microchambers and microchannels,   a second block having a cavity forming a second chamber, and   a filtration membrane,   the first block, the membrane and the second block being laid out so that the membrane is located between the first chamber and the second chamber, adjacent to each of the first and second chambers,   a first opening and a second opening for letting through a fluid into the second chamber separated from the first chamber by the membrane.       
 
         [0018]    The filtration device according to the first aspect of the invention is advantageously completed by the following features, taken alone or in any of their technically possible combinations:
       a fluid inlet in the first chamber connected to at least one portion of the microchannels and a fluid outlet in the first chamber connected to at least one portion of the microchannels are included, the microchannels forming a network connecting the fluid inlet to each microchamber and each microchamber to the fluid outlet, so as to allow fluid circulation in the microchambers of the first chamber,   the microchambers have a length dimension and a width dimension relatively to a circulation direction of the fluid, each comprised between 500 μm and 550 μm,   the microchannels have a length dimension relative to a circulation direction of the fluid comprised between 700 μm and 750 μm and a width dimension relatively to a circulation direction of the fluid comprised between 200 μm and 250 μm,   the microchambers comprise an inlet area and an outlet area, the microwalls comprising angled areas on either side of the inlet area and of the outlet area of at least one chamber, the angled areas having a width dimension relative to a circulation direction of the fluid comprised between 100 μm and 120 μm so as to define on either side of the inlet area of said chamber, partly protected areas,   the first chamber has a culture surface area in a ratio with an overall surface area of the bottom wall, comprised between 90% and 110%,   the first chamber has a culture surface area in a ratio with an overall available volume of fluid of the first chamber, comprised between 4/mm and 6/mm,   the second chamber comprises an upper wall having a set of microstructures identical with that of the bottom wall of the first chamber,   the membrane is in a flexible material,   the membrane is in a hydrophilic material,   the membrane is in a hydrophobic material,   the membrane is a barrier membrane,   the membrane is in a suitable material for allowing cells to be grown on the membrane,       
 
         [0031]    and
       a holding means having a locked configuration in which the first block and the membrane are firmly held together on the one hand, the membrane and the second block are firmly held together on the other hand, and an unlocked configuration, in which the block and the membrane may be separated from each other and/or in which the membrane and the second block may be separated from each other, the holding means being able to be switched from the locked configuration to the unlocked configuration and vice versa, is included.       
 
         [0033]    The invention also proposes, according to a second aspect, a filtration system, characterized in that it comprises:
       a filtration device according to the first aspect of the invention, and   a fluid circuit comprising circulation piping provided with a circulation means and connected to the first and second openings in the second chamber.       
 
         [0036]    The filtration system according to the second aspect of the invention is advantageously completed by the following features, taken alone or in any of their technically possible combinations:
       a fluid inlet in the first chamber connected to at least one portion of the microchannels and a fluid outlet in the first chamber connected to at least one portion of the microchannels are included, the microchannels forming a network connecting the fluid inlet to each microchamber and each microchamber to the fluid outlet, so as to allow fluid circulation in the microchambers of the first chamber, the device further comprising a fluid circuit comprising circulation piping provided with a circulation means and connected to the fluid inlet and outlet in the first chamber,   the first circuit comprises a control means for controlling fluid pressure in the first chamber,       
 
         [0039]    and
       the second circuit comprises a control means for controlling fluid pressure in the second chamber.       
 
         [0041]    According to a third aspect, the invention proposes a filtration system comprising several filtration devices according to the first aspect of the invention connected through circulation circuits. 
     
    
     
       PRESENTATION OF THE FIGURES 
         [0042]    Other features, objects and advantages of the invention will become apparent from the following description, which is purely illustrative and non-limiting, and which should be read with reference to the appended drawings wherein: 
           [0043]      FIG. 1  schematically illustrates a filtration device in a front sectional view according to a possible embodiment of the first aspect of the invention, 
           [0044]      FIG. 2  schematically illustrates a filtration device in a side sectional view according to a possible embodiment of the first aspect of the invention, 
           [0045]      FIG. 3  illustrates a three-dimensional perspective view of microstructures according to a possible embodiment of the first aspect of the invention, 
           [0046]      FIG. 4  schematically illustrates a filtration system in a side sectional view according to a possible embodiment of the second aspect of the invention, 
           [0047]      FIG. 5  illustrates a filtration device in a side sectional view according to a possible embodiment of the first aspect of the invention in which the first chamber is provided with a fluid inlet and outlet, 
           [0048]      FIG. 6  schematically illustrates a filtration device in a top sectional view according to a possible embodiment of the first aspect of the invention in which the first chamber is provided with a fluid inlet network and outlet network, 
           [0049]      FIG. 7  schematically illustrates in a top view, microstructures of the bottom wall of the first chamber, as well as dimensions of these microstructures according to a possible embodiment of the first aspect of the invention, 
           [0050]      FIGS. 8 a  and 8 b    illustrate electron microscopy images of membranes of different porosities according to possible embodiments of the first aspect of the invention, 
           [0051]      FIGS. 9 and 10  schematically illustrate a filtration system in a side sectional view according to possible embodiments of the second aspect of the invention, 
           [0052]      FIGS. 11 and 12  graphically illustrate a time-dependent change in the concentration-over-initial-concentration ratio in the second chamber during an experiment using a system according to a possible embodiment of the second aspect of the invention, 
           [0053]      FIG. 13  schematically illustrates a filtration system in a side sectional view according to possible embodiments of the second aspect of the invention, and 
           [0054]      FIG. 14  schematically illustrates an experiment for characterizing the water slope of a membrane, which experiment applies a system according to a possible embodiment of the second aspect of the invention. 
       
    
    
       [0055]    In the different figures, similar elements bear the same numerical references. 
       DETAILED DESCRIPTION 
     General Presentation 
       [0056]    With reference to  FIGS. 1 and 2 , a filtration device  100  according to the first aspect of the invention comprises a first block  101  having a cavity which forms a first chamber  110 , a second block  102  also having a cavity which forms a second chamber  102 , as well as a filtration membrane  130  positioned between the first chamber  110  and the second chamber  120  and adjacent to each of the first and second chambers  110 ,  120 . 
         [0057]    Both of these chambers may receive a fluid to be filtered or an operating fluid. As this will be described later on, the membrane  130  allows transport of material from one fluid to the other by a concentration difference or else by a pressure difference between the first and the second chamber, or further by any other filtration cause known to one skilled in the art. 
         [0058]    As a non-limiting example, the first and second blocks  101 ,  102  may consist of glass, silica or advantageously polymers such as polymethyl methacrylate (PMMA) or polydimethylsiloxane (PDMS) or a mixture thereof. PDMS has the advantage of being porous to oxygen. Both blocks may consist of the same material or else be in different materials. 
         [0059]    The second chamber  120  is intended to receive a circulating fluid. For this purpose, the device  100  comprises a first opening  122  and a second opening  123  for letting through a fluid. It further has an upper wall  121 . 
         [0060]    By           membrane           is meant a wall separating two media. A membrane has a porosity depending on the size of its pores. In this case, according to the first aspect of the invention, the membrane separates the first chamber  110  and the second chamber  120  and defines a single closed space in complementarity with each of these chambers  110 ,  120 . In particular, in the absence of a pressure difference between both chambers  110 ,  120 , the membrane has no contact point with the bottom wall  111  of the first chamber  110 , nor with the upper wall  121  of the second chamber  120 . 
         [0061]    As this will be described in detail later on, the first opening  122  (the second opening  123  respectively) may be a fluid inlet in the second chamber  120  (a fluid outlet of the second chamber  120 , respectively) or else a fluid outlet of the second chamber  120  (a fluid inlet in the second chamber  120 , respectively) depending on the direction of the flow provided in chamber  120 . 
         [0062]    The first chamber  110  has a bottom wall  111  preferentially having a substantially rectangular shape which defines a length and a width. 
         [0063]    As a non limiting example, the length and the width of the bottom wall may measure 13.25 mm and 11.23 mm, respectively. 
         [0064]    In the following, the horizontal is defined as being parallel to the bottom wall  111  and the vertical as orthogonal to the wall  111  and directed from the wall  111  towards the membrane  130 . Thus, it will be stated that the bottom wall  111  is horizontal and located below the membrane  130 , itself located below the upper wall  121 . Moreover, the longitudinal direction of the bottom wall  111  will be noted as d 1 . 
         [0065]    These definitions have the purpose of clarifying the remainder of the text and should by no means be interpreted as a limitation of the position of use of the device according to the first aspect of the invention in any co-ordinate system. 
       Microstructures for Cultivating Cells 
       [0066]    The first chamber  110  is intended to receive a cell culture. For this purpose, the bottom wall  111  has a set of microstructures illustrated in three dimensions in  FIG. 3 . These microstructures were developed by the applicant and have already been the subject, in their form, of a thesis report: “Développement et caractérisation d&#39;une puce a cellules pour le criblage d&#39;agents toxiques” (Development and characterization of cell chip for screening toxic agents). 
         [0067]    Moreover, the applicant filed on Jun. 23, 2009 a patent application FR 0954288 claiming advantageous dimensions of these microstructures, in particular with view to application to a liver cell culture. 
         [0068]    The set of microstructures comprises:
       bumps  111 . 4  with micrometric dimensions designated as “microbumps” in the following, and   walls  111 . 3  of micrometric dimensions designated as “microwalls” in the following,       
 
         [0071]    By “overall surface area” of the bottom wall  111  is meant the surface area which the bottom wall  111  would have if the microstructures were projected onto the wall  111  in the vertical direction. This is the surface area of the rectangle formed by the wall  111 . 
         [0072]    The microwalls  111 . 3  have arrow-shaped portions and straight portions and extend over the whole length of the bottom wall  111 . 
         [0073]    A microwall  111 . 3  defines microchambers  111 . 0  at its arrow-shaped portions in complementarity with another microwall  111 . 3 , and microchannels  111 . 2  at its straight portions in complementarity with microbumps  111 . 4  as illustrated in  FIG. 2 . 
         [0074]    Each microbump  111 . 4  defines two microchannels located on either side of the microbump, each in complementarity with a microwall. 
         [0075]    The bottom wall  111  of the first chamber  110  thus comprises periodic lines in its longitudinal direction d 1 , over the whole of its width. Each line comprises an alternation of microchambers  111 . 0  and of microchannels  111 . 2 , both lines being separated by a microwall  111 . 3 . 
         [0076]    As a non-limiting example, each line may comprise nine microchambers  111 . 0  and eight bumps  111 . 4 —each corresponding to two microchannels  111 . 2 —in alternation, the bottom wall  111  comprises a total of 15 lines. 
         [0077]    Such a device has on the bottom wall  111  a geometry favorable to the development of cells, in particular as compared with planar culture devices such as Petri dishes. The microstructures allow organization of the cells in three dimensions provided that they are supplied with nutritious fluid containing elements required for development of the cells, in particular oxygen or glucose. 
       Cell Culture in a Stagnating Fluid 
       [0078]    For this purpose, the device according to the first aspect of the invention allows feeding of the cells without a circulation of nutritious fluid being necessary in the first chamber. Indeed, by placing stagnating fluid in the first chamber where the cells are brought to development, and by circulating a nutritious fluid in the second chamber, the elements required for development of the cells (in particular glucose) pass into the stagnating fluid, if the membrane  130  is selected suitably. In particular, it is possible to select a membrane for which the porosity is suitable for letting through glucose. 
         [0079]    Thus, the device  100  according to the first aspect of the invention, finds a first application in the cultivation of cells. Indeed, a circulating nutritious fluid may carry away cells and break up the structures which is an obstacle to the development of cells and limits their activity. The device  100  described above gives the possibility of going beyond this difficulty by proposing a feeding solution without any circulation of fluid in direct contact with the cells. 
         [0080]    However, the pressure of the fluid circulating in the second chamber may cause deformation of the membrane  130  which then moves nearer to the bottom wall  111 . The membrane  130  is then a mechanical threat for the developing cells; it risks breaking up the structures of the cells and tearing them off the wall  111 . 
         [0081]    Now, the microstructures protect the cells from this harmful effect. Indeed, even if the membrane  130  would come into contact with the bottom wall  111 , it would be in contact with the microwalls  111 . 3  and the microbumps  111 . 4 , the cells being always able to develop in the microchambers  111 . 0  and the microchannels  111 . 2 . Thus, the microchambers and the microchannels form a structure not only favorable for development of the cells, but also protected in the case when the membrane  130  deforms as far as the bottom wall  111 . 
         [0082]    The thereby described device  100  may be used in a filtration system according to the second aspect of the invention as this is illustrated in  FIG. 4 . The system further comprises a device  100 , a fluid circuit  300  comprising circulation piping  310  provided with a circulation means  320 . By           piping           is meant a set of one or more pipes. These pipes may be flexible or rigid, and consist of any suitable material known to one skilled in the art. The fluid circuit  300  is connected to the first and second openings  122 ,  123  in the second chamber  120 , via respective passages  151  and  152  in the second block  102 . 
         [0083]    The circulation means  320  is preferentially connected to a supply  340  of nutritious medium, and may comprise a liquid pump, a peristaltic pump, a set of valves, for example solenoid valves, or any other suitable means know to one skilled in the art. 
         [0084]    Further, the circuit  300  preferentially comprises a discharge conduit  350  for the nutritious fluid after its passing into the second chamber  120 . 
         [0085]    Such a system according to the second aspect of the invention is not limited to this illustration in which the circuit  300  is open, and in particular extends to any system in which the circuit  300  is closed and optionally comprises a means for regenerating nutritious fluid. 
       Fluid Circulation in the First Chamber 
       [0086]    Moreover, the device  100  according to the first aspect of the invention is not limited to the description made of it up to now. Alternatively, provision is made for the possibility of also circulating a fluid in the first chamber  110 . 
         [0087]    For this purpose, the device  100  according to the first aspect of the invention, further comprises a fluid inlet  112  and a fluid outlet  113  as illustrated in  FIG. 5 . 
         [0088]    The fluid inlet  112  is connected to at least one portion of the microchannels  111 . 2  via an inlet network  114 . The inlet network  114  comprises successive branches for supplying each of the lines of the bottom wall  111  of the first chamber from the fluid inlet  112 . 
         [0089]    The fluid is intended to circulate at the microchannels  111 . 2  and the microchambers  111 . 0 , and above the microstructures in the first chamber  110 , for example for feeding developing cells. 
         [0090]    The fluid outlet  113  is connected to at least one portion of the microchannels via an outlet network  115 . The outlet network  115  comprises successive confluence points for connecting each of the lines of the bottom wall  111  of the chamber  110  to the fluid outlet  113 . The inlet  114  and outlet  115  networks of the bottom wall are illustrated in  FIG. 6 , in this advantageous alternative of the invention. 
         [0091]    Thus, the microchannels  111 . 2  form a network connecting the fluid inlet  21  to each microchamber  111 . 0 —via the inlet network  114 —and each microchamber to the fluid outlet  113 —via the outlet network  115 . The microchambers  111 . 0  preferentially comprise an inlet area  115  and an outlet area  116  for allowing circulation of the fluid substantially in the direction d 1 —the longitudinal direction of the wall  111 . 
         [0092]    Many applications of the device according to this advantageous alternative of the first aspect of the invention are contemplated. 
         [0093]    For example, provision may be made for circulating in the chamber  110  a fluid containing molecules to be tested on the cells. This application is particularly of interest in the screening of toxic substances for human beings: a human cell tissue is cultivated, fed through the membrane with a nutritious fluid circulating in a chamber  120  and directly exposed to test molecules in the chamber  110 . 
         [0094]    Moreover, is possible to circulate in the chamber  110  a discharge fluid for continuously removing cell secretions. 
         [0095]    Another possible application is mechanical stimulation of the cells. Certain cells, such as endothelial cells are naturally subject to flow conditions like blood. These cells are naturally activated by friction, which may be reproduced by circulation of the fluid in the first chamber  110 . 
       Dimensions of the Microstructures 
       [0096]    Advantageous dimensions of the microstructures will now be described according to a possible embodiment of the first aspect of the invention with reference to  FIG. 7 . 
         [0097]    In this advantageous alternative, the microchambers  111 . 0  have a length dimension and a width dimension relatively to the direction d 1 , each comprised between 500 μm and 550 μm, preferentially 520 μm. Thus, the dimensions of the microchambers  111 . 0  of the filtration device  100  are advantageously 520 μm×520 μm×100 μm. 
         [0098]    These dimensions are particularly favorable for developing liver cells in the microchambers  111 . 0 . 
         [0099]    Still advantageously, the microchannels  111 . 2  have relatively to the direction d 1 , a length dimension comprised between 700 μm and 750 μm, preferentially 720 μm, and a width dimension comprised between 200 μm and 250 μm, preferentially 220 μm. Thus, the dimensions of the microchannels  111 . 2  of the device  100  are advantageously 720 μm×220 μm×100 μm. 
         [0100]    The thereby dimensioned microchannels  111 . 2  facilitate development of liver cells; in particular they allow migration of a piece of a liver organ through the network of microchannels. 
         [0101]    Moreover, in  FIG. 6 , several other characteristic dimensions of the microwalls  111 . 3 , microbumps  111 . 4  and microchannels  111 . 2  are illustrated for a possible embodiment of the filtration device  100  of the first aspect of the invention. 
         [0102]    According to an advantageous alternative, the microwalls  111 . 3  comprise angled areas  111 . 7  on either side of the inlet area  111 . 5  and of the outlet area  111 . 6  of at least one chamber  111 . 0 , as illustrated in  FIGS. 4 and 6 . 
         [0103]    These angled areas  111 . 7  have a width dimension relatively to the direction d 1  advantageously comprised between 100 μm and 120 μm, preferentially 110 μm. In particular, they have an edge transverse to the fluid circulation direction d 1 . The angled areas  111 . 7  define, relatively to the direction d 1 , partly protected areas on either side of the inlet area  111 . 5  of said chamber  110 , i.e. areas where the circulation of the fluid is suddenly slowed down. 
         [0104]    Thus, the cells developing in such partly protected areas are unlikely to be carried off by the fluid circulating in the microchamber  111 . 0 . 
         [0105]    Naturally, the extent of an area where the cells are protected from being carried off by the fluid depends on circulation conditions, in particular on the flow rate and on the shearing. 
         [0106]    Nevertheless, the partly protected areas according to this advantageous alternative of the first aspect of the invention have an edge transverse to the direction d 1  with a width of at least 100 μm. 
         [0107]    They thereby allow aggregation of the cells at corners  111 . 8  of the microchamber  111 . 0  positioned transversely on either side and downstream from the inlet area  111 . 5  relative to the direction d 1 . In particular, liver cells may aggregate as a spheroid of a large diameter of the order of 100 μm, a favorable shape for good cell activity. 
         [0108]    Generally, the network of microchannels allows the cells to develop and to aggregate in three-dimensional structures at development areas, i.e.:
       the portion of the bottom wall  111  at the microchambers  111 . 0 ,   the portion of the bottom wall  111  at the microchannels  111 . 2 ,   the side walls of the microwalls  111 . 3 , and   the side walls of the microbumps  111 . 4 .       
 
         [0113]    By culture surface area is designated the whole of these development areas. Advantageously, the ratio between the culture surface area and the overall surface area of the bottom wall  111  is comprised between 90% and 110%. It is preferentially equal to 100% to within an accuracy of 1%. 
         [0114]    As a non-limiting numerical example, the culture surface area may be broken down in the following way (for an overall surface area of the bottom wall of 149 mm 2 ):
       surface area of the bottom wall at the microchambers  111 . 0  and at the microchannels  111 . 2 : 96.5 mm 2 ,   surface area of the side walls of the microwalls  111 . 3 : 36.5 mm 2      surface area of the side walls of the microbumps  111 . 4 : 18 mm 2 .       
 
         [0118]    The culture surface area is therefore 151 mm 2 . 
         [0119]    The ratio of the culture surface area over the overall surface area of the bottom wall is therefore, in this example 101%. 
         [0120]    Thus, the microstructures on the bottom wall  111  almost do not modify the surface area available for the culture relatively to the overall surface area of the bottom wall  111 , while allowing three-dimensional development. 
         [0121]    Further, cells may also develop on the upper surfaces of the microwalls  111 . 3  and of the microbumps  111 . 4 , although such areas are not particularly favorable for three-dimensional development. 
         [0122]    The culture surface area defined earlier to which are added the upper surface areas of the microwalls  111 . 3  and of the microbumps  111 . 4  is then called a &lt;&lt;total culture surface area&lt;&lt;. 
         [0123]    In the previous numerical example, these upper surface areas are 52.5 mm 2  and the total culture surface area is 203.5 mm 2  and the ratio between the total culture surface area and the overall surface area of the bottom wall is 137%. 
         [0124]    Still advantageously, the culture chamber  10  has a volume and the ratio R 2  between the total culture surface area and the volume of the culture chamber  10  is comprised between 4 mm −1  and 6 mm −1 . 
         [0125]    If the volume is too small relatively to the total culture surface area (R 2 &gt;6 mm −1 ), the cells risk being confined and not having sufficient nutrients distributed by the fluid, which is harmful to their development. 
         [0126]    Moreover, a too large volume relatively to the total culture surface area (R 2 &lt;4 mm −1 ), is uninteresting; it is actually preferable to have miniaturized devices. 
         [0127]    By volume of the first chamber  110  is meant the available volume for the passage of the fluid; the volume occupied by the microwalls  111 . 3  and the microbumps  111 . 4  is therefore excluded. 
         [0128]    By taking up the values of the preceding numerical example, and for a chamber height of 0.2 mm, the volume of the culture chamber  110  may be determined: 149×0.2−52.5×0.1=24.55 mm 3 . 
         [0129]    The ratio between the total culture surface area and the volume of the first chamber  110  is then 203.5 mm 2 /24.55 mm 3 =8.29 mm −1 . 
         [0130]    Advantageously, the upper surface  121  of the second chamber  120  also has microstructures. These microstructures may have all the advantageous alternatives of the microstructures detailed up to now relating to the bottom wall  111  of the first chamber  110 . The microstructures may be of identical dimensions on the lower  111  and upper  121  walls, or else of different dimensions. This alternative is particularly of interest for an application with a view to cultivating cells in both chambers. 
       Contemplated Membranes Within the Scope of the Invention 
       [0131]    Different characteristics of the membrane  120  contemplated within the scope of the invention will now be described in more detail. 
         [0132]    The membrane  130  is preferably in a flexible material and may thus be slightly deformable depending on the pressure prevailing in each of the chambers  110 ,  120 . As this was seen earlier, the microstructures prevent the membrane  130  from adhering to the walls and protect the culture cells from a possible contact with the membrane. 
         [0133]    The membrane  130  may be hydrophilic or hydrophobic, and will be selected depending on the targeted application (dialysis, cell culture, . . . ). 
         [0134]    A membrane is said to be hydrophilic when there is an interaction of the terminal groups with water through a hydrogen bond. Hydrophilic membranes like cellulose membranes have good diffusion and as they have a low adsorption of the proteins, they have good convection; on the other hand the biocompatibility is poor. They are used in dialysis for letting through water and very small solutes. 
         [0135]    Hydrophobic membranes, like synthetic membranes, have a lower diffusion but a higher ultrafiltration coefficient because of their porous structure which counterbalances the negative effect of the adsorption of proteins; the latter is at the origin of better biocompatibility. They are often used in hemodiafiltration. The biocompatibility of the membranes leads to many applications of these membranes with cell culture. 
         [0136]    The membranes may be microstructured with microstructures of the micropore type (see  FIGS. 8 a  and 8 b   ) but also with micropores which may also assume the shape of microchannels or micropillars or microgeometries, for example geometries as illustrated in  FIG. 7 . 
         [0137]    Advantageously, the membrane  130  may be hydrophilic, which limits adhesion of bacteria and of proteins and reduces the resistance to the passing of a fluid in the pores of the membrane  130 . 
         [0138]    Alternatively, the membrane  130  may be hydrophobic, which limits adhesion of bacteria, facilitates adhesion of proteins and increases the resistance to the passing of fluid in the pores. 
         [0139]    Different porosities may be used for the membrane  130  depending on the size of the elements to be filtered. According to an advantageous alternative, the membrane  130  is preferably a barrier membrane, i.e. it only allows diffusion of small molecules or gases and prevents the passing of fluids from one chamber  110 ,  120  to the other chamber  120 ,  110 .  FIGS. 8 a  and 8 b    show images taken by electron microscopy of two exemplary filtration membranes  130   a,    130   b  in polyethersulfone with a respective porosity of 40,000 Da and 500,000 Da. 
         [0140]    According to a possible embodiment of the invention, the membrane  130  is selected so as to allow cultivation of cells on the membrane  130 . In particular, provision is made for advantageously cultivating model cells of biological barriers (for example the intestinal barrier or a brain barrier) on the membrane, such as MDCK or Caco-2 cells. 
         [0141]    Preferentially, but in a non limiting way, the membrane  130  has a surface area of the order of 1 cm 2 . 
       Separation of the Blocks and of the Membrane 
       [0142]    Advantageously, the filtration device  100  according to the first aspect of the invention comprises a holding means  160  for holding together the first block  101 , the membrane  130  and the second block  102  in this order. 
         [0143]    The means  160  has a locked configuration in which the first block  101  and the membrane  130  are held firmly together, on the one hand, the membrane  130  and the second block are held firmly together on the other hand, and an unlocked configuration, in which the block  101  and the membrane  130  may be separated from each other and/or in which the membrane  130  and the second block  102  may be separated from each other. 
         [0144]    Further, the holding means  160  may be switched from the locked configuration to the unlocked configuration and vice versa, for example by action of a user. 
         [0145]    The means  160  may comprise screws crossing the first block  101 , the membrane  130  and the second block  102  over the whole of their height, as illustrated in  FIG. 1 . The means  160  may also comprise stops, a vice or any other suitable means known to one skilled in the art. 
         [0146]    Such a holding means  160  has several advantages. It allows the separation of the device  100  into its constituents and the possibility of then rebuilding it. Thus, it is possible to access the chambers  110  and  120  as well as the membrane  130 , without making the device  100  unusable. 
         [0147]    This is most particularly useful for cleaning the chambers of the molecules to be filtered which are adsorbed on the surfaces and for sterilizing them, for example, with an autoclave or by any other suitable cleaning method known to one skilled in the art. 
         [0148]    Moreover, the membrane  130  may be recovered for subjecting it to analysis, such as measurements of transmembrane electric resistance, recovery of the membrane in order to produce fluorescent markings on the cells, impedance analysis, or any other useful analysis known to one skilled in the art. In the case of a membrane receiving a cell culture, the means  160  also provides the possibility of directly accessing the cell culture while avoiding the discharge of this culture from the device  100  with a fluid which would destroy the culture structure. 
         [0149]    Further, the membrane may be replaced with a new membrane for repetitive experiments without requiring cleaning. It is thus possible to repeat an experiment by changing the membrane  130  while keeping intact the cell contents of the chambers  110 ,  120 . 
         [0150]    Conversely, a same membrane  130  may be transplanted from one device  100  to another and be the subject of experiments with chambers  110 ,  120  with a geometry of different microstructures. This may be useful for characterizing the effect of the microstructures on the filtration or on a cell culture on the membrane  130 . 
       A Filtration System With Two Fluid Circuits 
       [0151]    A filtration system according to several possible embodiments of the second aspect of the invention will now be described with reference to  FIGS. 9 to 11 . 
         [0152]    In the alternatives considered below, the device  100  integrated into the filtration system comprises a fluid inlet  112  and a fluid outlet  113  in the first chamber  110 . 
         [0153]    The system comprises a fluid circuit  200  connected to the first chamber  110 , similar to the circuit  300  connected to the second chamber  120  which has already been described above. The fluid circuit  200  comprises circulation piping  210  provided with a circulation means  220  and is connected to the inlets  112  and outlet  113  in the first chamber  110 . The fluid circuit  200  is connected to the fluid inlets  112  and outlet  113  in the first chamber  110  via respective passages  141  and  142  in the first block  101 . 
         [0154]    The circulation means  320  is preferentially connected to a fluid supply  240  and may comprise a liquid pump, a peristaltic pump, a set of valves, for example solenoid valves, or any other suitable means known to one skilled in the art. 
         [0155]    Further, the circuit  200  preferentially comprises a discharge conduit  250  for the fluid after passing in the first chamber  110 . 
         [0156]    Such a system according to the second aspect of the invention is not limited to this illustration in which the circuit  200  is open, and in particular extends to any system in which the circuit  200  is closed and optionally comprises a means for regenerating the fluid. 
         [0157]    Moreover, the fluids circulating in the circuits  200  and/or  300  are advantageously temperature-controlled. For example, the supplies  240  and/or  340  may be arranged in thermostated baths (not shown). Alternatively, or cumulatively, any other means for controlling the temperature of the circuits  200  and/or  300  known to one skilled in the art may be contemplated. 
         [0158]    In the alternative embodiment illustrated in  FIG. 9  the fluids contained in the lower  110  and upper  120  chambers circulate as co-currents. According to this configuration, the first opening  122  is a fluid outlet of the second chamber  120  and the second opening  123  is a fluid inlet in the second chamber  120 . 
         [0159]    Moreover, in  FIG. 10 , an alternative embodiment is illustrated in which the fluids contained in the lower  110  and upper  120  chambers circulate as countercurrents. According to this configuration, the first opening  120  is a fluid inlet in the second chamber  120  and the second opening  123  is a fluid outlet of the second chamber  120 . 
         [0160]    The systems described by  FIGS. 9 and 10  notably find application in the field of hemodialysis. Blood to be treated may circulate in the second chamber  120  and be cleared of certain components ordinarily eliminated by a functional kidney, by filtration through the membrane  130 . A dialysis fluid contained in the first chamber  110  may then discharge the filtered elements and ensure provision of glucose for the patient. 
         [0161]    Such systems may also be used for characterizing a filtration membrane  130 . For example, it is possible to evaluate a diffusion coefficient of a molecule for a given membrane  130  from a physical model and measurements of concentration of the molecule in the first chamber  110  and/or the second chamber  120  versus time. The co-current system may be used for calibrating the model and estimating the diffiusion coefficient, and the countercurrent system may be used for checking the diffusion coefficient or vice versa. 
         [0162]    In particular, the applicant modeled the time-dependent change of the concentration in the first chamber  110 , in the case when a fluid to be filtered circulates in the second chamber  120 , with the following equations:
       in the case of a co-current flow (like in  FIG. 9 ):       
 
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             in the case of a counter-current flow (like in  FIG. 10 ): 
           
         
       
     
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   C 1 (t) represents the concentration in the first chamber  110  as a function of time t,   L represents the (common) length of the first and second chambers  110 ,  120 ,   b represents the (common) width of the first and second chambers  110 ,  120 ,   Q f  represents the filtration flow rate,   V 1  represents the volume of the first chamber  110 ,   V 2  represents the volume of the second chamber  120 ,   C 1o  represents the initial concentration in the first chamber  110 ,   C 1∞  represents the equilibrium concentration in the first chamber  110 ,   D m  represents the diffusion coefficient of the molecule to be filtered for the membrane  130 , and   δ represents the thickness of the membrane.   
 
         [0175]    By means of these models, it is possible to determine the values of the diffusion coefficient D m  of the molecules tested for a given membrane  130 . For this, for example it is possible to operate in the co-current mode and measure the disappearance of the molecule in the second chamber  120  (and therefore its appearance in the first chamber  110 ). 
         [0176]    In  FIGS. 11 and 12  the change in the concentration-over-initial-concentration-in-the-second-chamber  120  ratio is illustrated versus time for given experimental conditions, for urea ( FIG. 11 ) and vitamin B12 ( FIG. 12 ) respectively, with several types of membrane. Moreover, the applicant has also determined this ratio for albumin (graph not shown). 
         [0177]    The experimental results are illustrated, for an experiment with a membrane of high porosity (of the 8F type) by icons □, Δ, and for an experiment with a membrane of low porosity (of the 1 FPH type) by icons: *, ◯. A regression of the analytic model is then made for example with a minimization by least squares, in which it is possible to obtain experimentally the diffusion coefficients in (m 2 /s) listed in the following table: 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Urea 
                 Vitamin B12 
                 Albumin 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1 FPH 
                 2 × 10 −12  m 2 /s 
                 4 × 10 −13  m 2 /s 
                 9 × 10 −14  m 2 /s 
               
               
                   
                 8F 
                 2 × 10 −11  m 2 /s 
                 6 × 10 −12  m 2 /s 
                 2 × 10 −13  m 2 /s 
               
               
                   
                   
               
             
          
         
       
     
         [0178]    Thus, the larger the molecule, the smaller is the diffusion coefficient. Further, the more porous the membrane, the larger is the diffusion coefficient. 
         [0179]    The validity of the diffusion coefficient was then proved by means of the countercurrent model. 
         [0180]    Thus, it is possible to characterize a membrane with a microsystem requiring a membrane surface area of the order of one cm 2 . If necessary, the estimated characteristics may be extrapolated on membranes of larger size. 
       Pressure Control in the Chambers 
       [0181]    With reference to  FIG. 13 , the circulation circuit  200  advantageously comprises a fluidic pressure control means  230  in the first chamber  110 , or else the circulation circuit  300  advantageously comprises a fluidic pressure control means  330  in the second chamber  120 , or else both circuits  200 ,  300  each comprise a pressure control means  230 ,  330  in their associated chamber  110 ,  120 . 
         [0182]    Preferentially, but not as a limitation, the control means  230  ( 330  respectively) comprises pressure sensors  231 ,  232  ( 331 ,  332  respectively), for detecting pressure of the fluid in the passages  141 ,  142  in the first block  101  ( 151 ,  152  in the second block  102 , respectively) or at the fluid inlet and outlet  112 ,  113  (at the first and second openings  122 ,  123 , respectively). The means  230  ( 330  respectively) further comprises actuators  233 ,  234  ( 333 ,  334 , respectively) positioned on the piping  210  ( 310  respectively) in order to modify the pressure at the inlet and outlet of the fluid in the first chamber  110  (in the second chamber  120 , respectively). These actuators may for example be solenoid valves controlled by a unit (not shown) for processing data from pressure sensors or any other suitable means known to one skilled in the art. 
         [0183]    The pressure controls may be carried out by the processing unit of each control means  230 ,  330  by stabilizing the pressure around a fixed or variable set value for example by applying a proportional controller, a proportional-integral controller, an open loop (in which case the pressure sensors are not used) or any other suitable control loop known to one skilled in the art of system control. 
         [0184]    The control means  230 ,  330  of the circuits  200 ,  300  may be similar or different according to the needs of the considered application for the filtration system according to the second aspect of the invention. 
         [0185]    They prove to be very useful for monitoring certain parameters during the filtration such as the discard rate or the diffusion coefficient. The conditions of flow rates and transmembrane flow may thus be closely controlled. 
         [0186]    The pressure difference may be maintained at a determined value so that the membrane will not adhere onto the walls. 
         [0187]    Moreover, the means  230 ,  330  allow control of the conditions of flow rates in the chambers  110 ,  120  and the transmembrane flow during filtration. This allows control of the parameters of the filtration for a given solute, such as the discard rate, i.e. the percentage of dissolved material retained by the membrane, and the diffusion coefficient of the solute through the membrane  130 . 
         [0188]    Thus, the means  230 ,  330  give the possibility of proceeding with repetitive experiments under the same conditions of flow rate and of transmembrane flow. 
         [0189]    A particularly interesting application of this advantageous effect is to test different membranes under similar experimental conditions, which allows characterization of the properties of the membranes, for example the water slope—i.e. the hydraulic permeability of the membranes to pure water—for example via the experiment illustrated in  FIG. 14 . 
         [0190]    In this experiment, the fluid circulating in the second chamber  120  is water. There is no cell culture in the first chamber  110 , a first chamber  110  which is further isolated from the supply  240  and from the circulation means  220 , by a means  250  for short-circuiting the first circuit  200 . Alternatively, it may be considered in this example that the first circuit  200  does not comprise any supply  240  nor any circulation means  220 , in which case the short-circuiting means  250  is unnecessary. In order to illustrate the latter, the supply  240  and the means  220  are surrounded by a rectangle in dotted lines in  FIG. 14 . 
         [0191]    Further, the discharge conduit  350  of the second circuit  300  is blocked for water (for example a closed tap). Thus, the water circulating in the second chamber through the second opening  152  can only flow out of the device  100  through the passage  140  of the first block  101 , which passage  142  is connected to the discharge conduit  250  of the first circuit  200  through the piping  210 . 
         [0192]    Moreover, the system according to this alternative of the second aspect of the invention is associated with a device  400  for measuring the mass of water, comprising a container  410  connected to the discharge conduit  250 , scales  420  and a processing unit  430  interacting with the scales  420  and intended for determining the mass of water contained in the container  410  versus time. Moreover, the processing unit receives information from means  230 ,  330  for controlling pressure in both circuits  200 ,  300 . 
         [0193]    Thus, the device  400  is capable of evaluating the mass of water filtering through the membrane  130  versus time and the transmembrane pressure, which allows determination of the water slope of the membrane  130 . 
         [0194]    The experiment was conducted by the applicant on the membranes  130   a  and  130   b  illustrated in  FIGS. 8 a  and 8 b    respectively. The determined water slopes are 8 mL/(min·bar·cm 3 ) and 80 mL/(min·bar·cm 3 ), respectively. 
         [0195]    Another application is to measure the pressure drops in the first chamber  110  (and in the second chamber  120  if it receives a cell culture), which pressure drops give an indication on the variations of the number of culture cells in the chamber. 
       Devices Connected in Series 
       [0196]    Finally, it is possible to contemplate the putting of several filtration devices  100  in series thereby allowing the operating steps of a kidney to be reproduced entirely, each device reproducing a particular function. More generally, several devices  100  may be used in series for reproducing the interactions between a fluid and various cell tissues in the body. 
       Conclusion 
       [0197]    The invention has many advantages. The system according to the second aspect of the invention allows control of filtration as compared with larger systems. The circuits for the fluids do not experience turbulence and only very little edge effects, whether this be in the piping as in the chambers of the device according to the first aspect of the invention. With the means for controlling pressure and the membrane, it is possible to maintain uniform parameters (pressure, temperature, composition and concentration of the fluids) so that the observed results may easily be extrapolated to systems with similar parameters. Thus, the invention is a great step towards in vitro reproduction of phenomena of the human or animal body, as well as towards making artificial organs.