Abstract
In various embodiments a biocompatible composite membrane for in vitro cell culturing comprising a first material, which is non-water soluble and a water soluble second material is provided, wherein the composite membrane comprises a porous scaffold and a filling layer, the scaffold comprising the first material and the filling layer comprising the second material. Further, a method for fabricating the membrane, a bioreactor for use of the membrane in cell-stretch experiments and a corresponding method for investigating cells attached to the biocompatible composite membrane are also provided.
Claims
1. A biocompatible composite membrane for in vitro cell culturing comprising a non-water soluble, first material and a water soluble second material, wherein the composite membrane comprises a porous scaffold and a filling layer, the scaffold comprising the first material and the filling layer comprising the second material.
2. Composite membrane of claim 1, wherein the porous scaffold is obtained by electrospinning of a substance comprising the first material; and wherein preferably the composite membrane is obtained by spin coating of the second material onto the scaffold.
3. Method of fabricating of a biocompatible composite membrane, comprising: providing a porous scaffold by electrospinning of a substance comprising a first material; providing a filling layer comprising a second material on the substrate.
4. Method of claim 3, further comprising: providing a desorption layer, preferably by spin coating, on a surface of a target onto which the substance is electrospun, the desorption layer preferably comprising the second material; wherein preferably the desorption layer has a thickness of less than 500 nm, preferably less than 300 nm.
5. Composite membrane of claim 1, wherein the composite membrane comprises conglomerates of the second material which are embedded, as the filling layer, in the porous scaffold.
6. Composite membrane of claim 5, wherein the composite membrane is obtained by spin coating of a substance comprising the first material and the second material.
7. Composite membrane of any one of claims 1, 2, 5, 6, wherein the porous scaffold has: a thickness of less than 10 m, preferably less than 5 m, most preferably less than 1 m; preferably a volume porosity of 1 to 50%, more preferably of 5 to 45%, even more preferably of 10 to 40%; preferably an area- or volume-weighted median pore diameter between 0.5 and 20 m, preferably between 1 and 15 m, even more preferably between 2 and 10 m; preferably an elasticity of less than 5 MPa, more preferably less than 1 MPa, even more preferably less than 0.1 MPa.
8. Composite membrane of any one of claims 1, 2, 5-7, wherein the thickness of the filling layer is equal to or smaller than that of the fibrous scaffold, preferably less than 70% of the thickness of the porous scaffold , even more preferably less than 30% of the thickness of the porous scaffold and the duration for complete dissolution of the covering layer in water is more than 12 hours and less than 2 weeks, preferably more than 1 day and less than 1 week, even more preferably more than 1.5 days and less than 4 days.
9. Composite membrane of any one of claims 1, 2, 5-8, wherein the first material comprises a hydrophobic material, preferably polycaprolactone; wherein preferably the second material is wettable and preferably comprises gelatin.
10. Method of fabricating a biocompatible composite membrane for in vitro cell culturing, comprising: spin coating of a substance comprising a first material and a second material, wherein the second material forms conglomerates which are distributed in a layer comprising the first material.
11. A bioreactor for investigating cells attached to an elastic membrane, preferably a biocompatible composite membrane of any one of the preceding claims, under stretch conditions, the bioreactor comprising: 1) a housing, having a first portion and a second portion configured to be engaged with each other and defining an inner volume; 2) the membrane, being positioned such that it divides the inner volume of the housing into a first volume, the first volume being in contact with a first surface of the membrane, and a second volume, the second volume being in contact with a second surface of the membrane; 3) a first pressure sensor for determining a pressure in the first volume; 4) a second pressure sensor for determining a pressure in the second volume; and 5) means for actively, preferably cyclically, adjusting a pressure in the first volume or the second volume.
12. Bioreactor of claim 11, wherein the means for actively adjusting a pressure in the first volume or the second volume comprises a fluid pump coupled to the respective volume via an access port.
13. Method for investigating cells attached to a membrane, preferably by means of the bioreactor according to any one of the preceding claims, comprising: determining the elastic modulus of the membrane while actively adjusting the pressure in the first volume or in the second volume.
14. Method of claim 13, wherein investigating the cells further includes: measuring the pressure in the respectively other volume, preferably over time while the pressure in the first or second volume is adjusted, preferably cyclically and preferably with positive differential pressure.
15. Method of claim 13 or 14, further comprising: providing a liquid in the second volume such that the second volume is at least partly filled with the liquid and the liquid is in direct contact with the membrane.
Description
[0066] In the following, embodiments of the present invention will be described in more detail with reference to the appended figures.
[0067] FIG. 1A shows a cell culture under submerged cell culture conditions.
[0068] FIG. 1B shows a cell culture under ALI conditions during the first phase of cell experiments with the membrane according to various embodiments in its first configuration.
[0069] FIG. 1C shows a cell culture under ALI conditions during the second phase of cell experiments with the membrane according to various embodiments in its second configuration.
[0070] FIG. 2A is a Scanning Electron Microscopy (SEM) image showing the structure of an exemplary membrane according to various first embodiments during the second phase of cell experiments (second configuration).
[0071] FIG. 2B is an SEM image showing the membrane structure of the membrane according to various first embodiments from FIG. 2A during the first phase of cell experiments (first configuration).
[0072] FIG. 2C is a cross-sectional SEM image of an exemplary membrane according to various first embodiments during the first phase of cell experiments.
[0073] FIG. 3A is a SEM image showing the structure of an exemplary membrane according to various second embodiments in its configuration for the first phase of cell experiments.
[0074] FIG. 3B is a cross-sectional SEM image of an exemplary membrane according to various second embodiments in its configuration for the first phase of cell experiments as depicted in FIG. 3A.
[0075] FIG. 3C is an SEM image showing the membrane structure of the membrane according to various second embodiments as depicted in FIG. 3A in its configuration for the second phase of cell experiments.
[0076] FIG. 3D is a cross-sectional image taken by focused ion beam (FIB) SEM tomography of an exemplary membrane according to various second embodiments in its configuration for the second phase of cell experiments as depicted in FIG. 3C.
[0077] FIG. 4A shows an embodiment of a bioreactor according to various embodiments.
[0078] FIG. 4B shows an experimental setup based on an exemplary bioreactor according to various embodiments.
[0079] FIG. 4C shows a further experimental setup based on an exemplary bioreactor according to various embodiments.
[0080] FIG. 4D shows a further experimental setup based on an exemplary bioreactor according to various embodiments.
[0081] FIG. 4E shows a photo of an embodiment of a bioreactor according to various embodiments.
[0082] FIG. 1A shows a standard cell culture 3 under submerged cell culture conditions, as known from the state of the art. In experimental setups of that nature, the cell culture 3 comprising a layer of cells 5 is arranged at the bottom of a suitable container 1, e.g. a plastic well, and is completely covered with the cell culture medium 2. As already noted in the introductory section, from the perspective of the cells 5, the submerged cell culture 3 does not replicate the physiological conditions of the epithelium in the lungs and is therefore for predictive inhalation therapy or inhalation toxicology experiments than more physiologic cell culture models. In contrast to the conditions in a real lung, aerosolized drugs or toxins cannot be deposited directly onto the lung epithelium but have to diffuse through and potentially interact with the cell culture medium 2 first in order to reach the submerged cells 5.
[0083] To remedy the deficiency inherent in the submerged cell culture 3 depicted in FIG. 1A, the air liquid interface (ALI) has been developed. The corresponding experimental setup is shown in FIG. 1C. In order to simulate an ALI for the cells, the cells 5 are also located in a suitable container 1. However, in contrast to the submerged cell culture 3, the cells 5 in an ALI culture 9 are not submerged in the cell culture medium 2, but instead they are cultured on the top side of a suitable membrane 4, preferably the membrane according to various embodiments, and the cells on the membrane being in contact with the cell culture medium 2 provided on the opposite side of the membrane 4. The membrane 4 is held in place by a membrane holder 7. By arranging the cells 5 on the membrane 4, a more physiological cell culture can be obtained: The top, air-facing surface of the cells 5 corresponds to the apical surface of the epithelial cell culture which in real conditions is exposed to the external environment (air-filled cavity) of an internal organ inside the body, e.g. the alveolus inside the lung, whereas the bottom surface of the ALI cell culture 9 corresponds the basal side which is located closer to the surface of the membrane 4 and also closer to the cell culture medium 2. The ALI setup shown in FIG. 1C is more biomimetic than the submerged cell culture setup depicted in FIG. 1A since it provides more physiological drug/toxin delivery and cell conditions by allowing for direct aerosol deposition from the air onto the confluent cell layer 9 and polarization of the confluent cell layer 6 (i.e. air-facing side of cells is biologically differentprotected against drying out at the airfrom liquid/medium facing side), respectively.
[0084] The more physiologic ALI cell culture as required for the second phase of the ALI experiment (FIG. 1C) requires an initial cell seeding and growth phase, which was referred to as first phase of the ALI experiment above and is shown in FIG. 1B. During this phase a relatively small number of cells 5 is seeded on the membrane 4 and cultured under submerged culture conditions with medium on the apical (top) and basal (bottom) side. Under these conditions, the cells 5 of the initially non-confluent cell layer 6, i.e. a cell layer having gaps 8, will proliferate and the resulting new cells will fill the gaps 8 until the confluent cell layer 9 is formed, as shown in FIG. 1C. During cell proliferation cells are motile and they may migrate into and/or through the membrane 4, if the membrane 4 contains large enough pores (typically larger than ca. 3 m), which is not desirable since in the lung the epithelial layer is formed on the apical side only. Hence, during the first phase of the ALI cell experiment a non-porous membrane 4, such as the membrane according to various embodiments in its first configuration, is beneficial. Once a confluent cell layer 9 has been formed, all cells 5 have contact with neighboring cells. This inhibits motility of the cells and thus the membrane 4 can now have pores without cells migrating and/or though the membrane 4 according to various embodiments in its second configuration, which is the prerequisite for air-lifting of the cells as required for the second phase of the ALI cell experiment depicted in FIG. 1C.
[0085] As already described, the membrane according to various embodiments has been conceived in order to provide an optimal environment to the cells 5 during the two distinct phases of cell stretch experiments. FIGS. 2A-2C are SEM images showing the structure of an exemplary membrane 4 according to various first embodiments which may be preferably manufactured by electrospinning, as described above. FIG. 2A shows the structure of an exemplary membrane 4 according to various first embodiments configured for the second phase of cell experiments (FIG. 1C), while FIG. 2B shows the same membrane 4 configured for the first phase of cell experiments (FIG. 1B). The membrane 4 according to various first embodiments comprises electrospun fibers comprising at least PCL as the first material, preferably a mixture of PCL and gelatin as the second material. The average fiber diameter is 1827 nm. In the image of FIG. 2B the membrane 4 is shown in a state after it has been spin-coated with a covering layer of the second material which plays the role of the sacrificial material. That is, the layer of the second material is dissolved, in analogy to the membrane according to various second embodiments, when the membrane 4 according to various first embodiments transitions from its first configuration to its second configuration. The scale bar in both figures is 1 m.
[0086] FIG. 2C is an SEM image showing a cross-sectional view of an exemplary membrane 4 according to various first embodiments in its first configuration for use in the first phase. The scale bar in the image represents a scale of 2 m. The membrane 4 comprises the scaffold 21 or the electrospun membrane comprising electrospun fibers which themselves comprise a mixture of PCL as the first material and gelatin as the second material. The scaffold 21 is covered with a covering layer 22 (sacrificial layer) comprising gelatin as the second material. The exemplary membrane 4 shown in FIG. 2C in its configuration for the first phase has an average thickness of 1.30 m0.16 m. The same membrane 4 in its configuration for the second phase, i.e. without the covering layer 22, as shown in FIG. 2A, has an average thickness of 0.980.16 m.
[0087] FIG. 3A is an SEM image showing the structure of an exemplary membrane 4 according to various second embodiments during the first phase of cell experiments (FIG. 1B) which may be preferably manufactured by spin coating as described above. The membrane 4 according to various second embodiments comprises an inhomogeneous mixture of the first material and the second material, wherein the second material is provided in the form of conglomerates of various sizes. In the bottom right corner of the SEM image an enlarged portion of the membrane 4 is shown. In the enlarged image, an agglomeration of conglomerates 31 of the second material can be seen, which may be seen to correspond to circular islands, which extend into the membrane 4 to form a three-dimensional interconnected network of conglomerates as illustrated below (FIG. 3C and 3D).
[0088] FIG. 3B is an SEM image showing a cross sectional view of an exemplary membrane 4 according to various second embodiments in its configuration for use in the first phase (FIG. 3A). As can be deduced from the scale bar having a length of 5 m, the thickness of the membrane 4 is approximately 6 m. The lack of visible conglomerates 31 in the image as seen in FIG. 3A originates from the difference in resolution of both figures (indicated by scale bars) and the change in perspective requiring tilting the membrane 4 as compared to its orientation relative to the imaging sensor in FIG. 3B. This results in loss of some information, such as the visualization of the conglomerate structure of the membrane 4, which is evident in FIG. 3A.
[0089] FIG. 3C is an SEM image showing the membrane from FIG. 3A, but in its configuration for use in the second phase (FIG. 1C). The structure of the membrane 4 configured for the second phase is characterized by pores 32, which resemble crater-like structures. The pores correspond to void spaces in the membrane 4 which are formed after removal of the conglomerates 31 of the second material from the membrane 4. The removal of the conglomerates 31 of the second material, i.e. the transition of the membrane 4 from its configuration for the first phase (FIG. 1B) to its configuration for the second phase, takes place trough dissolution of the conglomerates 31 of the second material. For that reason, the islands or conglomerates 31 of the second material may be seen to correspond to a sacrificial material which is removed by dissolution from the membrane 4 to introduce porosity to the membrane 4. In doing so, not only porosity is introduced but also the elastomechanical properties of the membrane 4 are changed i.e. the elasticity of the membrane is increased. The scale bars shown in FIG. 3A and 3C are 100 m long. As mentioned above, the membrane 4 according to various second embodiments may be preferably manufactured by spin coating and it may comprise a basal layer comprising PCL as first material and conglomerates of gelatin as the second (sacrificial) material.
[0090] FIG. 3D is a cross-sectional image taken by focused ion beam (FIB) SEM tomography of an exemplary membrane according to various first embodiments in its configuration for the second phase of cell experiments (FIG. 3C). As can be deduced from the scale bar having a length of 1 m, the large pores 32 form a three-dimensional network of pores 32 with a diameter of approximately 1-5 m throughout the entire membrane 4. In the image, the pores 32 are shown to be open to the first (apical) surface 34 of the membrane 4. The second (basal) surface 33 of the membrane 4 is indicated by the lower dashed line. The region between the upper dashed 35 line and the lower dashed line 33 is the cross-view onto the membrane 4.
[0091] In FIG. 4A, a schematic depiction of a bioreactor 40 according to various embodiments is shown. The bioreactor 40 may be subdivided structurally into a first volume 42, which is in contact with the first surface of the membrane 4 and practically defined by the first portion 402 (e.g. a top portion) of the housing of the bioreactor 40, and a second volume 43, which is in contact with the second surface of the membrane 4 and practically defined by a second portion 403 of the housing of the bioreactor 40. The membrane 4 is held by a membrane holder 7 such that it is arranged between the first volume 42 and the second volume 43. A nebulizer N is arranged above the first volume. The nebulizer N is configured to generate a mist which may be deposited on the cells arranged on the first surface of the membrane 4 for drug/toxin experiments. The nebulizer N may be also configured to provide a continuous measurement of the pressure in the first volume 42. During the experiments, the culture medium is provided to the second volume 43, whereas the first volume 42 is filled with air. In the lower portion of FIG. 4A an additional perspective view on the membrane holder 7 and the second portion 403 of the housing is depicted. The bioreactor 40 may have a generally cylindrical cross-section.
[0092] FIGS. 4B-4D show different configurations of the bioreactor 40 according to various embodiments for investigating the membrane and consequently the cells arranged thereon. In general, as already described, the bioreactor 40 comprises a housing 41, having the first portion and the second portion configured to be engaged with each other and defining an inner volume. Inside the housing 41, the bioreactor 40 comprises the membrane 4 according to various embodiments. In a top view, the bioreactor 40 may have a cylindrical geometry. The membrane 4 is held or spanned across the inner volume of the bioreactor 40 such that it divides the inner volume of the housing into the first volume 42, the first volume 42 being in contact with a first surface of the membrane 4, and a second volume 43, the second volume 43 being in contact with the second surface of the membrane 4. A membrane holder 7 may be used to install the membrane 4 inside the housing 41, wherein the membrane holder 7 may comprise a plastic ring in which the membrane 4 is installed. The membrane holder 7 may be provided inside the housing 41 or may be an adapter which is arranged, together with the membrane 4 installed therein, inside the bioreactor 40. As can be taken from FIGS. 4B-4D, the first surface of the membrane 4 corresponds to its upper surface and may be seen to correspond to the apical side thereof, wherein its second surface corresponds to its bottom surface and may be seen to correspond to its basal side. The cell culture arranged on the membrane 4 is not shown in FIGS. 4B-4D. The bioreactor 40 further comprises a first pressure sensor for determining a first pressure p.sub.1 in the first volume 42 and a second pressure sensor for determining a second pressure p.sub.2 in the second volume 43. The first pressure sensor may be installed in the first volume 42 of the bioreactor 40, the second sensor may be installed in the second volume 43 of the bioreactor 40 or in an air-filled headspace 46 of a separate reservoir which is coupled to the second volume 43. The bioreactor 40 further comprises a means 49 for actively, preferably cyclically, adjusting the pressure in the first volume 42 or the second volume 43 (pressure unit in the following). During the cell stretch experiments, a cell culture medium 44 is provided in the second volume 43, wherein the bottom surface of the membrane 4 is in contact with the cell culture medium 44. In general, the bioreactor 40 according to various embodiments may be used for investigating cells attached to or seeded on the membrane 4 under (preferably cyclic) stretch conditions at ALI. The membrane 4 used for this purpose is the biocompatible composite membrane according to various embodiments.
[0093] In the embodiment of the bioreactor 40 shown in FIG. 4B, a reservoir for a liquid 47 (liquid or medium reservoir) is provided and coupled to the second volume 43 of the bioreactor 40 via a connecting tubing. At the same time, the air-filled headspace in the liquid reservoir 47 is couped to the first volume 42 via the liquid-filled volume comprising the second volume 43, the liquid-filled part of the volume of the fluid reservoir 47 and the connecting tubing. The pressure unit 49 corresponds to an air flow regulation means, e.g. a compressor, and is used to increase the first pressure p.sub.1 above ambient pressure in a cyclic manner. The application of a positive pressure to the top side of the membrane 4 causes deformation of the membrane 4 which in turn leads to a displacement of a certain volume of the culture medium 44. Due to its incompressibility, the culture medium 44 is transferred into the medium reservoir 47 where the second pressure p.sub.2 is monitored by the second pressure sensor. In FIG. 4B (as well as in FIGS. 4C and 4D) the membrane 4 is also indicated in its stretched state and denoted by reference sign 4*. When no pressure is applied to the membrane 4, i.e. when the first pressure p.sub.1 corresponds to the ambient pressure, the membrane 4 is in its unstretched or equilibrium state, resting on the surface of the culture medium 44. In the experimental setup shown in FIG. 4B, the culture medium 44 fills the entire second volume 44. That is, the entire bottom surface of the membrane 4 is in contact with the culture medium 44. The fluid reservoir 47 may be installed at an appropriate elevation relative to an access port at which the fluid reservoir 47 is coupled to the second volume 43 of the bioreactor 40 in order to adjust the level of the culture medium 44 in the second volume 43 such that no hydrostatic pressure is exerted on the membrane 4. In addition, the fluid reservoir comprises an air-filled headspace 46. The arrows in FIG. 4B indicate the direction in which the force exerted by the increased pressure p.sub.1 generated by the pressure unit 49 acts on the membrane 4. In FIG. 4B the dashed volume 44* represents the portion by which the culture medium 44 inside the liquid reservoir 47 fluctuates, caused by the stretch of the membrane 4 which displaces the correspondingly equal volume of culture medium 44 from the second volume 43 and then, when it relaxes to its equilibrium state, it allows that same amount of culture medium 44 back into the second volume 43. The fluctuation of culture medium 44 inside the fluid reservoir 47 translates into a fluctuation of the second pressure p.sub.2 as the air-filled headspace is being compressed and decompressed by the relocation of culture medium 44 from and back into the second volume 43. The inset 50 indicates the resulting cyclic change of the second pressure p.sub.2 in the air-filled headspace 46, with the x-axis 51 denoting time and the y-axis 52 denoting pressure.
[0094] In the alternative embodiment of the bioreactor 40 shown in FIG. 4C the second pressure sensor is arranged in the second volume 43 of the bioreactor 40 such that the second pressure p.sub.2 is measured directly in the second volume 43. In order to facilitate measurement of the second pressure p.sub.2, the culture medium 44 does not fill the entire second volume 43 such that an air-filled headspace 46 is present in the second volume 43. The inset 50, which has been introduced in FIG. 4B, indicates that the pressure monitoring is performed based on the second pressure p.sub.2. Also indicated is the dashed volume 44* by which the air-filled headspace 46 fluctuates due to the stretch of the membrane 4 into its stretched state 4* and the resulting change of the level of the culture medium 44 which compresses the air in the air-filled headspace 46. In FIG. 4C, the membrane holding means 7 includes a tapered membrane holder (similar to standard transwell inserts) which is made of an airtight material. Therefore, the air-filled headspace 46 is held airtight or sealed, so to speak, by the culture medium sealing the pores of the cell-covered membrane 44. The pressure unit 49 may be the same as in the configuration shown in FIG. 4B. The arrows in FIG. 4C indicate the direction in which the force exerted by the increased pressure p.sub.1 generated by the pressure unit 49 acts on the membrane 4.
[0095] In the further embodiment of the bioreactor 40 shown in FIG. 4D, the first pressure p.sub.1 is increased by applying pressure directly to the culture medium 44 by means of a suitably configured pressure unit 49. In this embodiment, the additional pressure is applied to the second volume 43 and the first pressure p.sub.1 in the first volume 42 is monitored. For example, the pressure unit 49 may be an actuator which is configured to move a part of the housing 41 of the bioreactor 40, e.g. a portion of its bottom or side wall, thus applying pressure to the culture medium 44. Alternatively, the pressure unit 49 may include a fluid reservoir in connection with a pump which is configured to provide additional culture medium 44 into the second volume 43. By applying pressure to the culture medium 44, the membrane 4 is brought into its stretched state 4*. The arrows in FIG. 4D indicate the direction in which the force exerted by the pressure of the culture medium 44 acts on the membrane 4. As indicated by the inset 50, the pressure monitoring is performed based on the measurement of the first pressure p.sub.1 in the first volume 42.
[0096] Monitoring of the pressure over time allows for monitoring of the pressure-based strain/elasticity relation of the membrane 4. In the embodiments of the bioreactor 40 shown in FIGS. 4B and 4C the pressure monitoring is performed by observing the second pressure p.sub.2 and in the embodiment of the bioreactor 40 shown in FIG. 4D the pressure monitoring is performed by observing the first pressure p.sub.1.
[0097] In general, stretch and the elastic modulus of the cell-covered or the plane membrane 4 can be measured using two pressure sensors (e.g. PMX5050, Freescale Semiconductor) which are provided in the apical compartment (first volume) and the basal compartment, respectively, i.e. in the air volume in the liquid reservoir 47, as shown in FIG. 4B, or in the air volume 46 provided directly in the second volume 43, as shown in FIG. 4C. In the embodiment shown in FIG. 4D, the second pressure p.sub.2 may be determined from the direct application of pressure to the culture medium 44 or the displaced medium volume 44* is known from the operating conditions of the pressure unit 49. In general, independent of the actual setup chosen for the pressure measurement, from the difference of the elastic modulus of the cell-covered and the blank membrane 4, the elastic modulus of the cell layer itself can be calculated.
[0098] FIG. 4E shows an image of an exemplary bioreactor 40. The bioreactor 40 has a cylindrical shape and the middle and lower portion of the housing comprising the first volume 42 and the second volume 43 with a diameter of approximately 3 cm, respectively. The membrane 4 which is inserted in the housing may have a diameter of that order as well. The nebulizer N is arranged a few centimeters above the top of the first volume 42 to provide for a homogenous deposition of liquid aerosol onto the membrane 4 and the cells arranged thereon. The scale bars shown at the right side and at the bottom of the image are centimeter scales, wherein additional double-arrows 60 have been added, each marking a length of 1 cm.
[0099] In the following, the monitoring method will be explained based on the exemplary setup of the bioreactor 40 according to various embodiments shown in FIG. 4B. Once a positive pressure is applied to the first volume 42, the membrane 4 expands or is stretched into its stretched form 4* and pushes the culture medium 44 from the second volume 43 into the medium reservoir 47. The air-filled part of the second volume 43, which is represented by the air-filled headspace 46 in the embodiments shown in FIG. 4B and FIG. 4C, is compressed (V) and leads to an increase of pressure p.sub.2=p.sub.2p.sub.0 in the headspace of the second volume, wherein p.sub.0 corresponds to the pressure in both the first volume 42 and the air volume 46 in the second volume when the membrane is in a relaxed state. The volume change V may be calculated from
[00001]
wherein V.sub.2,0 corresponds to the air-filled part of the second volume 43 (i.e. its initial size) when no pressure is applied by the pressure unit 49, which is represented by the air-filled headspace 46 in the embodiments shown in FIGS. 4B and 4C. For the analysis, the shape of the stretched membrane 4* is approximated by a half dome geometry.
[0100] This volume change V is caused by the membrane displacement, which may be calculated from the volume of a spherical cap, which may be calculated from the radius a of the base of the dome (here: a is the radius of the membrane 4 prior to application of a pressure by pressure unit 49) and the height h of the dome for the membrane in its stretched state 4* (both a and h are indicated in the embodiment of the bioreactor depicted in FIG. 4B) in the following manner:
[00002]
[0101] Since V is known from the first equation and a is known from the geometry of the membrane 4, the height of the dome h can be derived from this equation and thus the relative change in the membrane area transitioning from its non-stretched 4 into its stretched state 4* may be calculated (see equation 4).
[0102] The elastic modulus (Young's modulus) E, measured in kPa, of the membrane 4 can then be calculated from the following equation
[00003]
For membrane only
[00004]
For cell-covered membrane
[00005]
where the subscripts m and m+c refer to parameters during membrane only (no cell layer on membrane) and cell-covered membrane stretch experiments, respectively, while subscript c denotes parameters related to the cell layer only. p.sub.12 is the difference between p.sub.1 and p.sub.2 (p.sub.1 and p.sub.2 are indicated in the embodiments of the bioreactor depicted in FIG. 4B and 4C) and corresponds to the force per area which is applied to the membrane 4, h is the height of the dome-shaped membrane, i.e. the elastic displacement (amplitude) of the center point of the membrane 4 during stretch, a is the radius of the non-stretched membrane 4 and its thickness tin its stretched state (at any given p.sub.12). From the above equation, Young's modulus E of the membrane 4 can be calculated for each stretch cycle. The elastic modulus of the cell-layer itself (E.sub.c) can be calculated from the difference of the elastic modulus of the cell-covered membrane (E.sub.c+m) and that of the blank membrane (E.sub.m) here expressed as difference of equation 3b and 3a.
[0103] Moreover, the change in (amplitude of) membrane surface area during stretch, often referred to as amplitude of membrane stretch, can be calculated according to
[00006]
where S.sub.0 is the surface area of the membrane 4 in its relaxed state (S.sub.0=a.sup.2).
[0104] This mathematical approach may be used with the embodiments of the bioreactor 40 shown in FIG. 4B and FIG. 4C. In FIG. 4C, the medium reservoir 47 from FIG. 4B is eliminated and the second pressure p.sub.2 is measured in the air-filled headspace 46 which is enclosed by culture medium 44, a portion of the inner wall of the second volume 43 of the housing 41 and the bottom side of the tapered membrane holder 7. Thus, the air-filled headspace 46 functions as a probe provided in the second volume 43 or in the fluid reservoir 47 coupled to the second volume 44 which is used to determine the second pressure P.sub.2.
[0105] In FIG. 4D, the real-time stretch profile of the membrane 4 can be monitored in analogy to the other two embodiments by realizing that the profile of the stretched membrane 4* is inverted and will thus be observed as extending into the first volume 42 and not into the second volume 43, as is the case in FIG. 4B and FIG. 4C. Essentially, the embodiment of the bioreactor shown in FIG. 4D corresponds to an inverse of the bioreactor 40 shown in FIG. 4B, in that a positive pressure p.sub.2 is applied in the second volume 43 and the resulting change of pressure p.sub.1 in the first volume 42 is measured. In this embodiment of the bioreactor 40 (FIG. 4D) a suitably configured pressure unit 49 may be an actuator which is configured to move a part of the housing 41 of the bioreactor 40, e.g. a portion of its bottom or side wall, thus applying pressure to the culture medium 44. Alternatively, the pressure unit 49 may include a fluid reservoir in connection with a pump which is configured to provide additional culture medium 44 into the second volume 43. By applying pressure to the culture medium 44 via imposing a known volume change V to the second volume 43 or the media in the second volume 44, the vertical displacement h of the center point of the membrane when transitioning from its relaxed state 4 into its stretched state 4* may be obtained by numerically solving equation 2 for h. Once h is known, equations 3, 3a and 3b can be applied for obtaining the elastic modulus of the (cell-covered) membrane using
p.sub.12=p.sub.1=p.sub.1p.sub.0, (5)
where p.sub.0 and p.sub.1 refer to the pressure in the first volume 42 when the membrane is in its relaxed state 4 or its stretched state 4*, respectively.
[0106] Real-time monitoring of the elastic modulus (equation 3) requires monitoring of both p.sub.2 and p.sub.1 (FIG. 4B and 4C) or of both p.sub.1 and V (FIG. 4D) which can be done, but is not shown here.
[0107] Overall, it is further noted that instead of applying a positive pressure to the first volume 42 or the second volume 43, i.e. a pressure which is above ambient pressure, a negative pressure can be also applied to the first volume 42 or the second volume 43. In other words, the membrane 4 may be deformed into a stretched membrane 4* by pulling on it instead of pushing it. The terms negative and positive with respect to the pressure merely indicate the magnitude of the applied pressure relative to the ambient pressure p.sub.0. In this context it is important to note that, the pressure p.sub.0 refers to the pressure of the first and the second volume, when the membrane is in its relaxed state 4. Mostly for practical reasons, p.sub.0 is referred to as ambient pressure (approximately 1 bar at sea level), but it may be set above or below the ambient atmospheric pressure. This may be achieved by statically increasing or decreasing the background pressure in the first volume 42 and/or the second volume 43. In one exemplary scenario, the whole bioreactor 40 may be placed in a pressure chamber in which the ambient or surrounding pressure p.sub.0 may be set according to need. Increase or decrease of the ambient pressure p.sub.0 may be particularly useful to study lung tissue at surrounding pressures different from the normal atmospheric pressure, e.g. at higher pressures which occur during diving or at lower pressures which occur during flight in an airplane.
[0108] Furthermore, even though the experimental scenarios described herein only referred to the cells being seeded and forming a confluent layer on the first side of the membrane, the membrane according to various embodiments may be also used in experimental scenarios in which cells are seeded on both sides to obtain different cell types on the two sides of the membrane. Frequently used co-culture cell models consist of epithelial cells apically (on the first surface of the membrane) and endothelial cells basally (i.e. on the second surface of the membrane). During initial growth and subsequent proliferation both cell types should remain separated to ensure that both cell types form separate confluent monolayers and do not mix during the formation of the confluent monolayers. For this process, a non-porous membrane is required, such as the membrane according to various embodiments in its configuration for the first phase. Additionally, thick electrospun membranes (thickness of 70 m or more) have been shown to have the cells form a multi-layered cell structure not only on the surface of the membrane, but also deep within the membrane. This is also not desirable for a physiologic representation of an alveolar air-blood barrier, which consists of monolayers located at the ALI, and may be effectively avoided using the membrane according to various embodiments. An experimental configuration with cell layers on both sides of the membrane may be seen to represent an even more realistic alveolar air-blood barrier as compared to a setup comprising only one cell layer on the first surface of the membrane. Additional experimental configurations may include further cell types (e.g. macrophages, fibroblasts) and even more than two different cell types may be seeded either stacked on the first surface of the membrane and/or the second surface of the membrane or within the membrane.
[0109] The method for stretch/elasticity monitoring in real-time described herein is based on the realization that characteristic parameters of cyclic membrane stretch can be derived during cyclic membrane stretch from the pressure change in one or two air-filled volumes located in the first and/or second volume which are separated by the membrane and by using basic principles of physics. This well-known concept is applied to the specific conditions and requirements of a bioreactor such as the use of the perforated membrane according to various embodiments and contact between membrane and cell culture medium from one side (here the basal side). This approach allows for real-time monitoring of membrane elasticity, frequency of stretch and increase of membrane area during stretch by means of the rather technically simple measurement of two pressures during cell stretch.
[0110] In case the pressure is monitored only in one of the two volumes provided inside the bioreactor 40 and the displaced media volume during stretch 46 is not monitored otherwise (as described the embodiment depicted in FIG. 4D), one can only monitor the stretch parameters (amplitude and frequency), but not membrane elasticity.
[0111] It is also noted that the approach described above also works if the medium is in contact with the membrane from both sides. However, such a scenario corresponds to submerged cell culture conditions and not to the desired ALI conditions.
[0112] For the above approach to provide useful and sensible results, the membrane should be free of leaks. That is, that no gas/liquid should be allowed to pass across the membrane during the stretch cycles. For a perforated membrane with micron-sized pores as used here during the cell stretch experiments, this condition is not met by air, but is well satisfied for the aqueous cell culture medium due to its higher viscosity as a fluid. Thus, for the method disclosed herein, it is advantageous to bring the membrane in contact with liquid i.e. cell culture medium, which has a higher viscosity than gas/air and may therefore not pass through the pores of the membrane during stretch cycles. In that manner, the perforated membrane is effectively sealed during any given stretch cycle.
