WO2016034766A1 - A method and a device for measuring permeability of molecules through a sample - Google Patents

A method and a device for measuring permeability of molecules through a sample Download PDF

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Publication number
WO2016034766A1
WO2016034766A1 PCT/FI2015/050561 FI2015050561W WO2016034766A1 WO 2016034766 A1 WO2016034766 A1 WO 2016034766A1 FI 2015050561 W FI2015050561 W FI 2015050561W WO 2016034766 A1 WO2016034766 A1 WO 2016034766A1
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WO
WIPO (PCT)
Prior art keywords
sample
carrier fluid
molecules
perfusion area
inlet channel
Prior art date
Application number
PCT/FI2015/050561
Other languages
French (fr)
Inventor
Samu HEMMILÄ
Heli Skottman
Antti JYLHÄ
Pasi Kallio
Joose KREUTZER
Kati JUUTI-UUSITALO
Anni SORKIO
Ville ELLÄ
Original Assignee
Tampereen Yliopisto
Tty-Säätiö
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tampereen Yliopisto, Tty-Säätiö filed Critical Tampereen Yliopisto
Publication of WO2016034766A1 publication Critical patent/WO2016034766A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
    • G01N2013/003—Diffusion; diffusivity between liquids

Definitions

  • the invention relates generally to measuring permeability of molecules, e.g. drug molecules, through a sample, e.g. a layer of blood-retinal barrier "BRB" cells. More particularly, the invention relates to a device for measuring permeability of molecules through a sample, to a system for measuring permeability of molecules through a sample, and to a method for measuring permeability of molecules through a sample.
  • a sample e.g. a layer of blood-retinal barrier "BRB" cells.
  • RPE retinal pigment epithelial
  • BBB blood-retinal- barrier
  • a typical in vitro measurement system comprises a donor chamber for containing liquid medium which carries molecules whose ability to penetrate a sample is being studied.
  • the measurement system comprises means for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area for receiving the molecules penetrated the sample.
  • the measurement system further comprises an inlet channel for conducting carrier fluid to the perfusion area and an outlet channel for conducting the carrier fluid and also the molecules penetrated the sample out from the perfusion area to be available for further analysis.
  • Measurement systems of the kind described above are, however, not free from challenges.
  • One of the challenges is related to the tendency of the carrier medium and/or the penetrated molecules to form a hydrodynamic diffusion layer on the surface of the sample in the perfusions area.
  • the hydrodynamic diffusion layer hinders the transport of the molecules through the sample to the perfusion area and thus it may disturb the permeability measurement.
  • Publication US8393199 describes a system where a reception chamber which receives the penetrated molecules is provided with a stirrer for equalizing the concentrations of substances in the reception chamber. It is, however, straightforward to understand that the use of the stirrer complicates the measurement system significantly.
  • a new a device for measuring permeability of molecules e.g. drug molecules
  • a sample may comprise, for example but not necessarily, cell layer such as e.g. a layer of blood- retinal barrier "BRB” cells, a layer of blood-aqueous barrier cells “BAB”, a layer of blood-brain barrier cells “BBB”, intestinal epithelium, or lung epithelium.
  • cell layer such as e.g. a layer of blood- retinal barrier "BRB” cells, a layer of blood-aqueous barrier cells “BAB”, a layer of blood-brain barrier cells “BBB”, intestinal epithelium, or lung epithelium.
  • BBBB blood-brain barrier cells
  • a device comprises: - a donor chamber for containing liquid medium carrying the molecules,
  • sample holder for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area for receiving the molecules penetrated the sample, - at least one inlet channel for conducting carrier fluid to the perfusion area, and
  • the end-portion of the inlet channel connected to the perfusion area is shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid.
  • the cross-sectional flow area of the at least one outlet channel is selected with respect to the cross-sectional flow area of the at least one inlet channel so that the flow resistance of the at least one outlet channel is substantially at most equal to the flow resistance of the at least one inlet channel so as to reduce the pressure of the carrier fluid in the perfusion area, and thereby to achieve effective rinsing.
  • the rinsing prevents, or at least inhibits, the formation of a hydrodynamic diffusion layer on the surface of the sample in the perfusions area.
  • the hydrodynamic diffusion layer would hinder the transport of the molecules through the sample to the perfusion area, and thus it would disturb the permeability measurement.
  • the volume of the perfusion area is from 1 mm 3 to 1000 mm 3 , more advantageously from 1 mm 3 to 100 mm 3 , and yet more advantageously from 1 mm 3 to 10 mm 3 .
  • a smaller volume of the perfusion area is advantageous compared to a greater volume of the perfusion area in the respect that molecules which have penetrated the sample are distributed into a smaller volume in the perfusion area and thus it is easier to detect the molecules from among the carrier fluid received from the outlet channel of the device.
  • the term "perfusion” refers to transportation mechanisms of the above-mentioned molecules through the sample.
  • a particular transportation mechanism may be active or passive.
  • retinal epithelial cells a part of the blood retina barrier, express several transport molecules such as efflux proteins which have selective specificity to transport certain molecules through the epithelium. This is an example of active transportation mechanisms.
  • Passive transportation mechanisms include for example diffusion.
  • Elements of a device according to the invention can be manufactured, for example, by mold casting.
  • mold casting there is provided also a new set of molds having forms suitable for manufacturing, by mold casting, the elements of the device according to the invention.
  • a system according to the invention comprises: - a device according to the invention,
  • a method for measuring permeability of molecules through a sample comprises:
  • the concentration of the molecules in the carrier fluid can be determined.
  • figure 1 a shows an exploded view of a device according to an exemplifying and non-limiting embodiment of the invention for measuring permeability of molecules through a sample
  • figure 1 b illustrates a view of a section taken along a line A-A shown in figure 1 a
  • figures 1 c and 1 d show section views illustrating the structure and the operation of the device depicted in figures 1 a and 1 b
  • figure 1 e shows a top-view of a functional part of the upper block of the device shown in figure 1 a
  • figure 1 f shows a top-view of a functional part of the lower block of the device shown in figure 1 a
  • figure 1 g shows a view of a section taken along a line B-B shown in figures 1 e and 1 f
  • figure 1 h shows an exploded view of a sample holder of the device depicted in figures 1 a-1 g
  • Figure 1 a shows an exploded view of a device 100 according to an exemplifying and non-limiting embodiment of the invention.
  • the device comprises six mutually similar functional parts 103a, 103b, 103c, 103d, and 103e which can be used independently of each other.
  • the device comprises a first block, i.e. an upper block, 101 and a second block, i.e. a lower block, 102.
  • the device further comprises sample holders for holding samples under study.
  • one of the sample holders is denoted with a reference number 105.
  • the sample holders are placed between the first and second blocks 101 and 102 which comprise contact surfaces shaped to match the sample holders.
  • the first and second blocks 101 and 102 can be made of for example polymer or some other suitable solid material.
  • the material of the first and second blocks 101 and 102 is advantageously inert so that the material does not bind drug molecules and release any compounds, and is nontoxic to cells.
  • the material can be for example cyclo-olefin polymer "COP".
  • the sample holders are advantageously made of sufficiently soft material so as to provide adequate sealing with the first and second blocks 101 and 102 when the sample holders are pressed between the first and second blocks. Also the material of the sample holders is advantageously inert so that the material does not bind drug molecules and release any compounds, and is nontoxic to cells.
  • the sample holders can be made of for example cyclo-olefin polymer "COP", or some other suitable polymer that is advantageously inert in the above-mentioned respect and soft enough to provide the adequate sealing.
  • COP cyclo-olefin polymer
  • the sample holders and the first and second blocks 101 and 102 can be manufactured for example by injection moulding.
  • Figure 1 b illustrates a view of a section taken along a line A-A shown in figure 1 a.
  • the section plane is parallel with the yz-plane of a coordinate system 199.
  • the sample holder 105 is presented as an exploded view which shows that the sample holder comprises first and second elements 105a and 105b each having an aperture.
  • the sample holder 105 is suitable for holding a sample 1 16 so that the apertures are aligned with respect to each other, the sample covers the apertures, and the rim of the sample is pressed between the rims of the apertures.
  • Figure 1 c shows a section view corresponding to that shown in figure 1 b but figure 1 c shows a situation in which the first and second blocks 101 and 102 of the device are fastened with respect to each other with the aid of clamping means 1 15a and 1 15b so that the sample holder 105 is pressed between the first and second blocks.
  • Figure 1 d shows a magnification of a portion 1 17 of figure 1 c.
  • the first block 101 of the device is shaped to constitute a donor chamber 104.
  • the donor chamber 104 is suitable for containing liquid medium 1 18 carrying the molecules, e.g. drug molecules, under study.
  • the second block 102 of the device is shaped to constitute an inlet channel 107 and an outlet channel 108.
  • the sample holder 105 is arranged to hold the sample 1 16 so that the sample constitutes a wall between the donor chamber 104 and a perfusion area 106 which receives the molecules that have penetrated the sample 1 16. The penetration of the molecules through the sample is depicted with dashed line arrows in figure 1 d.
  • the inlet channel 107 is suitable for conducting carrier fluid to the perfusion area 106
  • the outlet channel 108 is suitable for conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area.
  • the end-portion 107a of the inlet channel connected to the perfusion area 106 is shaped to direct the carrier fluid towards the sample 1 16 so as to rinse the sample with the carrier fluid and thereby to prevent, or at least inhibit, the formation of a hydrodynamic diffusion layer that would disturb the molecule penetration through the sample.
  • the cross-sectional flow area of the outlet channel 108 is advantageously at least equal to the cross-sectional flow area of the inlet channel 107 so that the flow resistance of the outlet channel is at most equal to the flow resistance of the inlet channel so as to reduce pressure of the carrier fluid in the perfusion area and thereby to achieve effective rinsing. Furthermore, in order to achieve effective rinsing, at least the beginning-portion 108a of the outlet channel connected to the perfusion area 106 is advantageously shaped to conduct the carrier fluid downwards from the perfusion area when the devise is in its operation position where the donor chamber 104 is above the perfusion area 106. The flow of the carrier fluid is depicted with arrows in figure 1 c.
  • the end- portion 107a of the inlet channel is substantially perpendicular to the surface of the sample 1 16 facing towards the perfusion area 106.
  • the end- portion of the inlet channel forms an acute angle, e.g.5 - 60 degrees, with respect to a geometric line perpendicular to the surface of the sample.
  • the second block 102 is shaped to constitute an output chamber 1 12 for receiving, from the outlet channel 108, the carrier fluid and the molecules penetrated the sample 1 16.
  • the carrier fluid and the molecules can be taken for further analysis from the output chamber 1 12 via a passage 121 with the aid of e.g. a pipette.
  • the output chamber 1 12 is advantageously below the perfusion area 106 when the devise is in its operating position where the donor chamber 104 is above the perfusion area 106 in order that the carrier fluid contained by the output chamber 1 12 would not cause a hydrostatic pressure in the perfusion area 106.
  • the bottom of the output chamber 1 12 can be provided with a pit, not shown, having a smaller area than the whole bottom so as to slow down the drying of the carrier fluid in the output chamber.
  • the outlet channel 108 comprises an end-portion which can be connected, e.g. with a hose pipe, to an external output chamber or to an analyzer instrument.
  • Figure 1 e shows a top-view of the functional part 103a of the first block 101 of the device
  • figure 1 f shows a top-view of the functional part 103a of the second block 102 of the device.
  • the functional parts 103a-103f of the device are illustrated in figure 1 a.
  • the opening of the end-portion 107a of the inlet chamber and the opening of the beginning-portion 108a of the outlet chamber are shown in figure 1 f.
  • Figure 1 g shows a view of a section taken along a line B-B shown in figures 1 e and 1 f.
  • the line A-A which is shown in figure 1 a is shown also in figures 1 e and 1 f.
  • the exemplifying device presented above referring to figures 1 a-1 g comprises first electrodes 1 10a and 1 10b located partly in the donor chamber 104 and second electrodes 1 1 1 a and 1 1 1 b located partly in the perfusion area 106.
  • the electrodes are suitable for measuring one or more electrical quantities indicative of one or more electrical properties of the sample 1 16.
  • the measured electrical quantity can be e.g. the dc-resistance or the ac-impedance, and thus the electrical quantity is dependent on the dc-resistance or the ac-impedance of the sample 1 16.
  • the electrical quantity can be measured at many time points during a process for measuring the permeability of the molecules through the sample so as to monitor whether the sample stays in a proper condition during the permeability measurement.
  • the electrical measurement can be four-point measurement where one of the electrodes of the donor chamber and one of the electrodes of the perfusion area are current feeding electrodes for supplying current and the other two of the electrodes are voltage measurement electrodes for measuring voltage.
  • a set of molds according to an exemplifying and non-limiting embodiment of the invention has forms suitable for manufacturing, by mold casting, the first and second blocks 101 and 102 of the device.
  • Figure 1 h shows an exploded view of the sample holder 105
  • figure 1 i shows a section view of the sample holder.
  • the first element 105a of the sample holder comprises a first aperture 109a
  • the second element 105b of the sample holder comprises a second aperture 109b.
  • the sample holder is suitable for holding the sample 1 16 so that the first and second apertures are aligned with respect to each other, the sample covers the first and second apertures, and the rim of the sample is pressed between rims of the first and second apertures as illustrated in figure 1 i.
  • the first and second elements 105a and 105b of the sample holder are shaped to fit with corresponding shapes of the first and second blocks 101 and 102 of the device as illustrated e.g.
  • the sample holder is advantageously made of material which is suitable for a cell culturing platform, e.g. COP.
  • the sample holder can be used as a cell culturing platform so that the cell layer is cultured directly on a suitable support membrane held by the sample holder.
  • the sample holder is provided with openings, such as an opening 122, for ensuring that cell culturing medium can access the above-mentioned support membrane via the aperture 109b, when the sample holder is placed on a flat bottom of a standard cell culturing well plate.
  • the first and second elements 105a and 105b of the sample holder are shaped so that the sample holder is suitable for being installed with respect to the donor chamber and to the inlet and outlet channels in a first position in which a first side of the sample faces towards the donor chamber and, alternatively, in a second position in which the second, opposite, side of the sample faces towards the donor chamber. Therefore, the permeability of molecules, e.g. drug molecules, through the sample held by the sample holder can be measured in either direction/both directions.
  • molecules e.g. drug molecules
  • the first aperture 109a flares in a direction away from the sample 1 16.
  • the second aperture 109b flares in a direction away from the sample.
  • the flaring may be established by way of conical apertures.
  • the walls of the second aperture 109b, or alternatively the walls of the first aperture 109a constitute the perfusion area and the above- mentioned flaring streamlines the flow of the carrier fluid in the perfusion area.
  • a set of molds according to an exemplifying and non-limiting embodiment of the invention has forms suitable for manufacturing, by mold casting, the first and second elements 105a and 105b of the sample holder 105.
  • Figure 2 illustrates a system according to an exemplifying and non-limiting embodiment of the invention for measuring permeability of molecules, e.g. drug molecules, through a sample.
  • the sample may comprise, for example but not necessarily, a cell layer that can be for example a layer of blood-retinal barrier cells, a layer of blood-aqueous barrier cells, a layer of blood-brain barrier cells, intestinal epithelium, or lung epithelium.
  • the system comprises a device 200 according to an exemplifying embodiment of the invention for the permeability measurement.
  • the device 200 is similar to the device 100 presented above referring to figures 1 a-1 i.
  • the donor chamber 104 of the device contains liquid medium 1 18 carrying the molecules, and the sample holder 105 of the device holds the sample.
  • the system comprises a supply-system for supplying carrier fluid to the inlet channel 107 of the device.
  • the system further comprises an analyzer instrument 124 for detecting the above-mentioned molecules from among the carrier fluid received from the outlet channel 108 of the device 200.
  • the analyzer instrument 124 can be, for example, a known instrument for determining the concentration of the above- mentioned molecules in the carrier fluid.
  • the supply-system comprises an inlet chamber 1 14 containing the carrier fluid 1 19 so that the surface of the carrier fluid contained by the inlet chamber is above-the perfusion area 106 of the device.
  • the carrier fluid is supplied to the inlet channel 107 of the device with the aid of the hydrostatic pressure.
  • the supply-system further comprises an adjustable valve 120 with the aid of which the flow rate of the carrier fluid can be adjusted.
  • the supply-system comprises a pumping device for supplying the carrier fluid to the inlet channel 107 of the device 200.
  • the pumping device can be e.g. a syringe or a peristaltic pump.
  • the carrier fluid and the molecules penetrated the sample are conducted to the output chamber 1 12 of the device 200.
  • the carrier fluid and the molecules under study can be taken for further analysis from the output chamber 1 12 with the aid of e.g. a pipette 123.
  • the analyzer instrument 124 is connected, with e.g. a hose pipe, to a channel corresponding to the output channel 108.
  • Figure 3 shows a flowchart of a method according to an exemplifying and non- limiting embodiment of the invention for measuring permeability of molecules through a sample.
  • the method comprises the following actions:
  • - action 301 holding the sample so that the sample constitutes a wall between a donor chamber containing liquid medium carrying the molecules and a perfusion area receiving the molecules penetrated the sample,
  • - action 302 conducting carrier fluid to the perfusion area via at least one inlet channel, the end-portion of the inlet channel connected to the perfusion area being shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid,
  • - action 303 conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area via at least one outlet channel, a cross- sectional flow area of the at least one outlet channel being advantageously at least equal to a cross-sectional flow area of the at least one inlet channel so that the flow resistance of the at least one outlet channel is at most equal to the flow resistance of the at least one inlet channel so as to reduce pressure of the carrier fluid in the perfusion area, and - action 304: detecting the molecules from among the carrier fluid received from the outlet channel, e.g. the concentration of the molecules in the carrier fluid can be determined.
  • the beginning-portion of the outlet channel connected to the perfusion area is shaped to conduct the carrier fluid downwards from the perfusion area.
  • the end-portion of the inlet channel is substantially perpendicular to the surface of the sample facing towards the perfusion area.
  • the sample comprises a cell layer that can be for example: a layer of blood-retinal barrier cells, a layer of blood-aqueous barrier cells, a layer of blood- brain barrier cells, intestinal epithelium, or lung epithelium.

Abstract

A device for measuring permeability of molecules through a sample comprises a donor chamber (104) for containing medium carrying the molecules, a sample holder (105) for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area (106), an inlet channel (107) for conducting carrier fluid to the perfusion area, and an outlet channel (108) for conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area. The cross-sectional flow area of the outlet channel is at least equal to the cross-sectional flow area of the inlet channel, and the inlet channel is shaped to direct the carrier fluid towards the sample so as to effectively rinse the sample. The rinsing prevents, or at least inhibits, the formation of a hydrodynamic diffusion layer that would disturb the permeability measurement.

Description

A method and a device for measuring permeability of molecules through a sample
Field of the invention The invention relates generally to measuring permeability of molecules, e.g. drug molecules, through a sample, e.g. a layer of blood-retinal barrier "BRB" cells. More particularly, the invention relates to a device for measuring permeability of molecules through a sample, to a system for measuring permeability of molecules through a sample, and to a method for measuring permeability of molecules through a sample.
Background
An important property of a medicinal compound is its ability to pass through human or animal tissue. For example, in the development of drugs for the eye, it is essential to study the delivery of particular substance from the bloodstream to the retina. The photoreceptor functions of the retina are mainly supported by retinal pigment epithelial "RPE" cells. This densely pigmented epithelial layer serves as a part of a barrier between the bloodstream and the retina, called blood-retinal- barrier "BRB". Therefore, there is a need for in vitro measurement systems for measuring permeability of molecules, e.g. drug molecules, through a sample that may comprise e.g. a layer of blood-retinal barrier "BRB" cells, blood-aqueous barrier cells "BAB", blood-brain barrier cells "BBB", intestinal epithelium, or lung epithelium.
A typical in vitro measurement system comprises a donor chamber for containing liquid medium which carries molecules whose ability to penetrate a sample is being studied. The measurement system comprises means for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area for receiving the molecules penetrated the sample. The measurement system further comprises an inlet channel for conducting carrier fluid to the perfusion area and an outlet channel for conducting the carrier fluid and also the molecules penetrated the sample out from the perfusion area to be available for further analysis.
Measurement systems of the kind described above are, however, not free from challenges. One of the challenges is related to the tendency of the carrier medium and/or the penetrated molecules to form a hydrodynamic diffusion layer on the surface of the sample in the perfusions area. The hydrodynamic diffusion layer hinders the transport of the molecules through the sample to the perfusion area and thus it may disturb the permeability measurement. Publication US8393199 describes a system where a reception chamber which receives the penetrated molecules is provided with a stirrer for equalizing the concentrations of substances in the reception chamber. It is, however, straightforward to understand that the use of the stirrer complicates the measurement system significantly.
Summary
The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying and non-limiting embodiments of the invention.
In accordance with the invention, there is provided a new a device for measuring permeability of molecules, e.g. drug molecules, through a sample that may comprise, for example but not necessarily, cell layer such as e.g. a layer of blood- retinal barrier "BRB" cells, a layer of blood-aqueous barrier cells "BAB", a layer of blood-brain barrier cells "BBB", intestinal epithelium, or lung epithelium. Furthermore, the sample may comprise a support membrane which is penetrable by the molecules under consideration and which supports the cell layer mechanically.
A device according to the invention comprises: - a donor chamber for containing liquid medium carrying the molecules,
- a sample holder for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area for receiving the molecules penetrated the sample, - at least one inlet channel for conducting carrier fluid to the perfusion area, and
- at least one outlet channel for conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area.
The end-portion of the inlet channel connected to the perfusion area is shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid. The cross-sectional flow area of the at least one outlet channel is selected with respect to the cross-sectional flow area of the at least one inlet channel so that the flow resistance of the at least one outlet channel is substantially at most equal to the flow resistance of the at least one inlet channel so as to reduce the pressure of the carrier fluid in the perfusion area, and thereby to achieve effective rinsing. The rinsing prevents, or at least inhibits, the formation of a hydrodynamic diffusion layer on the surface of the sample in the perfusions area. The hydrodynamic diffusion layer would hinder the transport of the molecules through the sample to the perfusion area, and thus it would disturb the permeability measurement.
In a device according to an exemplifying and non-limiting embodiment of the invention, the volume of the perfusion area is from 1 mm3 to 1000 mm3, more advantageously from 1 mm3 to 100 mm3, and yet more advantageously from 1 mm3 to 10 mm3. A smaller volume of the perfusion area is advantageous compared to a greater volume of the perfusion area in the respect that molecules which have penetrated the sample are distributed into a smaller volume in the perfusion area and thus it is easier to detect the molecules from among the carrier fluid received from the outlet channel of the device. In this document, the term "perfusion" refers to transportation mechanisms of the above-mentioned molecules through the sample. A particular transportation mechanism may be active or passive. For example, retinal epithelial cells, a part of the blood retina barrier, express several transport molecules such as efflux proteins which have selective specificity to transport certain molecules through the epithelium. This is an example of active transportation mechanisms. Passive transportation mechanisms include for example diffusion.
Elements of a device according to the invention can be manufactured, for example, by mold casting. In accordance with the invention, there is provided also a new set of molds having forms suitable for manufacturing, by mold casting, the elements of the device according to the invention.
In accordance with the invention, there is provided also a new system for measuring permeability of molecules through a sample. A system according to the invention comprises: - a device according to the invention,
- a supply-system for supplying carrier fluid to the inlet channel of the device, and
- an analyzer instrument for detecting the molecules from among the carrier fluid received from the outlet channel of the device. In accordance with the invention, there is provided also a new method for measuring permeability of molecules through a sample. A method according to the invention comprises:
- holding the sample so that the sample constitutes a wall between a donor chamber containing liquid medium carrying the molecules and a perfusion area receiving the molecules penetrated the sample,
- conducting carrier fluid to the perfusion area via at least one inlet channel so that the carrier fluid is directed towards the sample so as to rinse the sample with the carrier fluid, - conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area via at least one outlet channel, the flow resistance of the at least one outlet channel being substantially at most equal to the flow resistance of the at least one inlet channel so as to reduce pressure of the carrier fluid in the perfusion area, and
- detecting the molecules from among the carrier fluid received from the outlet channel, e.g. the concentration of the molecules in the carrier fluid can be determined.
A number of exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims.
Various exemplifying and non-limiting embodiments of the invention both as to constructions and to methods of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.
The verbs "to comprise" and "to include" are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in dependent claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.
Brief description of figures
Exemplifying and non-limiting embodiments of the invention and their advantages are explained in greater detail below with reference to the accompanying drawings, in which: figure 1 a shows an exploded view of a device according to an exemplifying and non-limiting embodiment of the invention for measuring permeability of molecules through a sample, figure 1 b illustrates a view of a section taken along a line A-A shown in figure 1 a, figures 1 c and 1 d show section views illustrating the structure and the operation of the device depicted in figures 1 a and 1 b, figure 1 e shows a top-view of a functional part of the upper block of the device shown in figure 1 a, figure 1 f shows a top-view of a functional part of the lower block of the device shown in figure 1 a, figure 1 g shows a view of a section taken along a line B-B shown in figures 1 e and 1 f, figure 1 h shows an exploded view of a sample holder of the device depicted in figures 1 a-1 g, figure 1 i shows a section view of the sample holder depicted in figure 1 h, figure 2 illustrates a system according to an exemplifying and non-limiting embodiment of the invention for measuring permeability of molecules through a sample, and figure 3 shows a flowchart of a method according to an exemplifying and non- limiting embodiment of the invention for measuring permeability of molecules through a sample.
Description of exemplifying embodiments Figure 1 a shows an exploded view of a device 100 according to an exemplifying and non-limiting embodiment of the invention. In this exemplifying case, the device comprises six mutually similar functional parts 103a, 103b, 103c, 103d, and 103e which can be used independently of each other. The device comprises a first block, i.e. an upper block, 101 and a second block, i.e. a lower block, 102. The device further comprises sample holders for holding samples under study. In figure 1 a, one of the sample holders is denoted with a reference number 105. The sample holders are placed between the first and second blocks 101 and 102 which comprise contact surfaces shaped to match the sample holders. The first and second blocks 101 and 102 can be made of for example polymer or some other suitable solid material. The material of the first and second blocks 101 and 102 is advantageously inert so that the material does not bind drug molecules and release any compounds, and is nontoxic to cells. The material can be for example cyclo-olefin polymer "COP". The sample holders are advantageously made of sufficiently soft material so as to provide adequate sealing with the first and second blocks 101 and 102 when the sample holders are pressed between the first and second blocks. Also the material of the sample holders is advantageously inert so that the material does not bind drug molecules and release any compounds, and is nontoxic to cells. The sample holders can be made of for example cyclo-olefin polymer "COP", or some other suitable polymer that is advantageously inert in the above-mentioned respect and soft enough to provide the adequate sealing. The sample holders and the first and second blocks 101 and 102 can be manufactured for example by injection moulding.
Figure 1 b illustrates a view of a section taken along a line A-A shown in figure 1 a. The section plane is parallel with the yz-plane of a coordinate system 199. In figure 1 b, the sample holder 105 is presented as an exploded view which shows that the sample holder comprises first and second elements 105a and 105b each having an aperture. The sample holder 105 is suitable for holding a sample 1 16 so that the apertures are aligned with respect to each other, the sample covers the apertures, and the rim of the sample is pressed between the rims of the apertures.
Figure 1 c shows a section view corresponding to that shown in figure 1 b but figure 1 c shows a situation in which the first and second blocks 101 and 102 of the device are fastened with respect to each other with the aid of clamping means 1 15a and 1 15b so that the sample holder 105 is pressed between the first and second blocks. Figure 1 d shows a magnification of a portion 1 17 of figure 1 c.
The structure and the operation of the device are illustrated below with the aid of figures 1 c and 1 d. The first block 101 of the device is shaped to constitute a donor chamber 104. The donor chamber 104 is suitable for containing liquid medium 1 18 carrying the molecules, e.g. drug molecules, under study. The second block 102 of the device is shaped to constitute an inlet channel 107 and an outlet channel 108. The sample holder 105 is arranged to hold the sample 1 16 so that the sample constitutes a wall between the donor chamber 104 and a perfusion area 106 which receives the molecules that have penetrated the sample 1 16. The penetration of the molecules through the sample is depicted with dashed line arrows in figure 1 d. The inlet channel 107 is suitable for conducting carrier fluid to the perfusion area 106, and the outlet channel 108 is suitable for conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area. As illustrated in figures 1 c and 1 d, the end-portion 107a of the inlet channel connected to the perfusion area 106 is shaped to direct the carrier fluid towards the sample 1 16 so as to rinse the sample with the carrier fluid and thereby to prevent, or at least inhibit, the formation of a hydrodynamic diffusion layer that would disturb the molecule penetration through the sample. The cross-sectional flow area of the outlet channel 108 is advantageously at least equal to the cross-sectional flow area of the inlet channel 107 so that the flow resistance of the outlet channel is at most equal to the flow resistance of the inlet channel so as to reduce pressure of the carrier fluid in the perfusion area and thereby to achieve effective rinsing. Furthermore, in order to achieve effective rinsing, at least the beginning-portion 108a of the outlet channel connected to the perfusion area 106 is advantageously shaped to conduct the carrier fluid downwards from the perfusion area when the devise is in its operation position where the donor chamber 104 is above the perfusion area 106. The flow of the carrier fluid is depicted with arrows in figure 1 c.
In the exemplifying device presented above referring to figures 1 a-1 d, the end- portion 107a of the inlet channel is substantially perpendicular to the surface of the sample 1 16 facing towards the perfusion area 106. In a device according to another exemplifying and non-limiting embodiment of the invention, the end- portion of the inlet channel forms an acute angle, e.g.5 - 60 degrees, with respect to a geometric line perpendicular to the surface of the sample.
In the exemplifying device presented above referring to figures 1 a-1 d, the second block 102 is shaped to constitute an output chamber 1 12 for receiving, from the outlet channel 108, the carrier fluid and the molecules penetrated the sample 1 16. The carrier fluid and the molecules can be taken for further analysis from the output chamber 1 12 via a passage 121 with the aid of e.g. a pipette. The output chamber 1 12 is advantageously below the perfusion area 106 when the devise is in its operating position where the donor chamber 104 is above the perfusion area 106 in order that the carrier fluid contained by the output chamber 1 12 would not cause a hydrostatic pressure in the perfusion area 106. The bottom of the output chamber 1 12 can be provided with a pit, not shown, having a smaller area than the whole bottom so as to slow down the drying of the carrier fluid in the output chamber. In a device according to another exemplifying and non-limiting embodiment of the invention, the outlet channel 108 comprises an end-portion which can be connected, e.g. with a hose pipe, to an external output chamber or to an analyzer instrument.
Figure 1 e shows a top-view of the functional part 103a of the first block 101 of the device, and figure 1 f shows a top-view of the functional part 103a of the second block 102 of the device. The functional parts 103a-103f of the device are illustrated in figure 1 a. The opening of the end-portion 107a of the inlet chamber and the opening of the beginning-portion 108a of the outlet chamber are shown in figure 1 f. Figure 1 g shows a view of a section taken along a line B-B shown in figures 1 e and 1 f. For the sake of clarity, the line A-A which is shown in figure 1 a is shown also in figures 1 e and 1 f. The exemplifying device presented above referring to figures 1 a-1 g comprises first electrodes 1 10a and 1 10b located partly in the donor chamber 104 and second electrodes 1 1 1 a and 1 1 1 b located partly in the perfusion area 106. The electrodes are suitable for measuring one or more electrical quantities indicative of one or more electrical properties of the sample 1 16. The measured electrical quantity can be e.g. the dc-resistance or the ac-impedance, and thus the electrical quantity is dependent on the dc-resistance or the ac-impedance of the sample 1 16. The electrical quantity can be measured at many time points during a process for measuring the permeability of the molecules through the sample so as to monitor whether the sample stays in a proper condition during the permeability measurement. As this exemplifying device comprises two electrodes in the donor chamber and two electrodes in the perfusion area, the electrical measurement can be four-point measurement where one of the electrodes of the donor chamber and one of the electrodes of the perfusion area are current feeding electrodes for supplying current and the other two of the electrodes are voltage measurement electrodes for measuring voltage. In a device according to another exemplifying and non-limiting embodiment of the invention, there is only one electrode in the donor chamber and one in the perfusion area. In this case, the electrical measurement is two-point measurement where voltage is measured from between the same electrodes which are used for current injection.
In the exemplifying device presented above referring to figures 1 a-1 g, there is only one inlet channel connected to the perfusion area 106 and only one outlet channel connected to the perfusion area 106. In a device according to another exemplifying and non-limiting embodiment of the invention, there are two or more inlet channels connected to a perfusion area and/or two or more outlet channels connected to the perfusion area.
A set of molds according to an exemplifying and non-limiting embodiment of the invention has forms suitable for manufacturing, by mold casting, the first and second blocks 101 and 102 of the device.
Figure 1 h shows an exploded view of the sample holder 105, and figure 1 i shows a section view of the sample holder. The first element 105a of the sample holder comprises a first aperture 109a, and the second element 105b of the sample holder comprises a second aperture 109b. The sample holder is suitable for holding the sample 1 16 so that the first and second apertures are aligned with respect to each other, the sample covers the first and second apertures, and the rim of the sample is pressed between rims of the first and second apertures as illustrated in figure 1 i. The first and second elements 105a and 105b of the sample holder are shaped to fit with corresponding shapes of the first and second blocks 101 and 102 of the device as illustrated e.g. in figure 1 d. The sample holder is advantageously made of material which is suitable for a cell culturing platform, e.g. COP. In this case, the sample holder can be used as a cell culturing platform so that the cell layer is cultured directly on a suitable support membrane held by the sample holder. For this purpose, the sample holder is provided with openings, such as an opening 122, for ensuring that cell culturing medium can access the above-mentioned support membrane via the aperture 109b, when the sample holder is placed on a flat bottom of a standard cell culturing well plate.
As illustrated in figures 1 d and 1 i, the first and second elements 105a and 105b of the sample holder are shaped so that the sample holder is suitable for being installed with respect to the donor chamber and to the inlet and outlet channels in a first position in which a first side of the sample faces towards the donor chamber and, alternatively, in a second position in which the second, opposite, side of the sample faces towards the donor chamber. Therefore, the permeability of molecules, e.g. drug molecules, through the sample held by the sample holder can be measured in either direction/both directions.
As illustrated in figure 1 i, the first aperture 109a flares in a direction away from the sample 1 16. Correspondingly, the second aperture 109b flares in a direction away from the sample. The flaring may be established by way of conical apertures. As illustrated in figure 1 d, the walls of the second aperture 109b, or alternatively the walls of the first aperture 109a, constitute the perfusion area and the above- mentioned flaring streamlines the flow of the carrier fluid in the perfusion area.
A set of molds according to an exemplifying and non-limiting embodiment of the invention has forms suitable for manufacturing, by mold casting, the first and second elements 105a and 105b of the sample holder 105. Figure 2 illustrates a system according to an exemplifying and non-limiting embodiment of the invention for measuring permeability of molecules, e.g. drug molecules, through a sample. The sample may comprise, for example but not necessarily, a cell layer that can be for example a layer of blood-retinal barrier cells, a layer of blood-aqueous barrier cells, a layer of blood-brain barrier cells, intestinal epithelium, or lung epithelium. The system comprises a device 200 according to an exemplifying embodiment of the invention for the permeability measurement. In this exemplifying case, the device 200 is similar to the device 100 presented above referring to figures 1 a-1 i. In the situation shown in figure 2, the donor chamber 104 of the device contains liquid medium 1 18 carrying the molecules, and the sample holder 105 of the device holds the sample. The system comprises a supply-system for supplying carrier fluid to the inlet channel 107 of the device. The system further comprises an analyzer instrument 124 for detecting the above-mentioned molecules from among the carrier fluid received from the outlet channel 108 of the device 200. The analyzer instrument 124 can be, for example, a known instrument for determining the concentration of the above- mentioned molecules in the carrier fluid.
In the exemplifying system illustrated in figure 2, the supply-system comprises an inlet chamber 1 14 containing the carrier fluid 1 19 so that the surface of the carrier fluid contained by the inlet chamber is above-the perfusion area 106 of the device. In this case, the carrier fluid is supplied to the inlet channel 107 of the device with the aid of the hydrostatic pressure. The supply-system further comprises an adjustable valve 120 with the aid of which the flow rate of the carrier fluid can be adjusted. In a system according to another exemplifying and non-limiting embodiment of the invention, the supply-system comprises a pumping device for supplying the carrier fluid to the inlet channel 107 of the device 200. The pumping device can be e.g. a syringe or a peristaltic pump.
In the exemplifying system illustrated in figure 2, the carrier fluid and the molecules penetrated the sample are conducted to the output chamber 1 12 of the device 200. The carrier fluid and the molecules under study can be taken for further analysis from the output chamber 1 12 with the aid of e.g. a pipette 123. In a system according to another exemplifying and non-limiting embodiment of the invention, the analyzer instrument 124 is connected, with e.g. a hose pipe, to a channel corresponding to the output channel 108.
Figure 3 shows a flowchart of a method according to an exemplifying and non- limiting embodiment of the invention for measuring permeability of molecules through a sample. The method comprises the following actions:
- action 301 : holding the sample so that the sample constitutes a wall between a donor chamber containing liquid medium carrying the molecules and a perfusion area receiving the molecules penetrated the sample,
- action 302: conducting carrier fluid to the perfusion area via at least one inlet channel, the end-portion of the inlet channel connected to the perfusion area being shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid,
- action 303: conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area via at least one outlet channel, a cross- sectional flow area of the at least one outlet channel being advantageously at least equal to a cross-sectional flow area of the at least one inlet channel so that the flow resistance of the at least one outlet channel is at most equal to the flow resistance of the at least one inlet channel so as to reduce pressure of the carrier fluid in the perfusion area, and - action 304: detecting the molecules from among the carrier fluid received from the outlet channel, e.g. the concentration of the molecules in the carrier fluid can be determined.
In a method according to an exemplifying and non-limiting embodiment of the invention, the beginning-portion of the outlet channel connected to the perfusion area is shaped to conduct the carrier fluid downwards from the perfusion area.
In a method according to an exemplifying and non-limiting embodiment of the invention, the end-portion of the inlet channel is substantially perpendicular to the surface of the sample facing towards the perfusion area.
In a method according to an exemplifying and non-limiting embodiment of the invention, the sample comprises a cell layer that can be for example: a layer of blood-retinal barrier cells, a layer of blood-aqueous barrier cells, a layer of blood- brain barrier cells, intestinal epithelium, or lung epithelium.
The non-limiting, specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.

Claims

What is claimed is:
1 . A device (100) for measuring permeability of molecules through a sample, the device comprising:
- a donor chamber (104) for containing liquid medium carrying the molecules, - a sample holder (105) for holding the sample so that the sample constitutes a wall between the donor chamber and a perfusion area (106) for receiving the molecules penetrated the sample,
- at least one inlet channel (107) for conducting carrier fluid to the perfusion area, and - at least one outlet channel (108) for conducting the carrier fluid and the molecules penetrated the sample out from the perfusion area, a cross- sectional flow area of the at least one outlet channel being selected with respect to a cross-sectional flow area of the at least one inlet channel so that flow resistance of the at least one outlet channel is substantially at most equal to flow resistance of the at least one inlet channel so as to reduce pressure of the carrier fluid in the perfusion area, characterized in that an end-portion (107a) of the inlet channel connected to the perfusion area is shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid.
2. A device according to claim 1 , wherein a beginning-portion (108a) of the outlet channel connected to the perfusion area is shaped to conduct the carrier fluid downwards from the perfusion area when the devise is in a position where the donor chamber is above the perfusion area.
3. A device according to claim 1 or 2, wherein the end-portion (107a) of the inlet channel is substantially perpendicular to a surface the sample facing towards the perfusion area.
4. A device according to any of claims 1 -3, wherein the sample holder (105) comprises a first element (105a) having a first aperture (109a) and a second element (105b) having a second aperture (109b), the sample holder being suitable for holding the sample so that the first and second apertures are aligned with respect to each other, the sample covers the first and second apertures, and a rim of the sample is pressed between rims of the first and second apertures.
5. A device according to any of claims 1 -4, wherein the sample holder is shaped to be suitable for being installed with respect to the donor chamber and to the inlet and outlet channels in a first position in which a first side of the sample faces towards the donor chamber and, alternatively, in a second position in which a second side of the sample faces towards the donor chamber.
6. A device according to any of claims 1 -5, wherein the device comprises at least one first electrode (1 10a, 1 10b) located partly in the donor chamber and at least one second electrode (1 1 1 a, 1 1 1 b) located partly in the perfusion area for measuring electrical quantities indicative of electrical properties of the sample.
7. A device according to any of claims 1 -6, wherein the device comprises a first block (101 ) shaped to constitute the donor chamber and a second block (102) shaped to constitute the inlet and outlet channels, the first and second blocks comprising contact surfaces shaped to match the sample holder (105) when the sample holder is between the first and second blocks.
8. A device according to claim 7, wherein the second block is shaped to constitute an output chamber (1 12) for receiving, from the outlet channel, the carrier fluid and the molecules penetrated the sample.
9. A set of molds having forms suitable for manufacturing, by mold casting, the first and second blocks of a device according to claim 7 or 8.
10. A system for measuring permeability of molecules through a sample, the system comprising:
- a device (200) according to any of claims 1 -8,
- a supply-system for supplying carrier fluid to the inlet channel of the device, and - an analyzer instrument (124) for detecting the molecules from among the carrier fluid received from the outlet channel of the device.
1 1 . A system according to claim 10, wherein the supply-system comprises an inlet chamber (1 14) for containing the carrier fluid so that a surface of the carrier fluid contained by the inlet chamber is above the perfusion area of the device so as to supply the carrier fluid to the inlet channel of the device with the aid of hydrostatic pressure.
12. A system according to claim 10, wherein the supply-system comprises a pumping device for supplying the carrier fluid to the inlet channel of the device.
13. A system according to any of claims 10-12, wherein the system comprises an output chamber (1 12) for receiving, from the outlet channel of the device, the carrier fluid and the molecules penetrated the sample.
14. A system according to any of claims 10-12, wherein the outlet channel of the device is connected in a fluid conductive way to the analyzer instrument.
15. A method for measuring permeability of molecules through a sample, the method comprising:
- holding (301 ) the sample so that the sample constitutes a wall between a donor chamber containing liquid medium carrying the molecules and a perfusion area receiving the molecules penetrated the sample, - conducting (302) carrier fluid to the perfusion area via at least one inlet channel,
- conducting (303) the carrier fluid and the molecules penetrated the sample out from the perfusion area via at least one outlet channel, a cross-sectional flow area of the at least one outlet channel being selected with respect to a cross-sectional flow area of the at least one inlet channel so that flow resistance of the at least one outlet channel is substantially at most equal to flow resistance of the at least one inlet channel so as to reduce pressure of the carrier fluid in the perfusion area, and - detecting (304) the molecules from among the carrier fluid received from the outlet channel, characterized in that an end-portion of the inlet channel connected to the perfusion area is shaped to direct the carrier fluid towards the sample so as to rinse the sample with the carrier fluid.
16. A method according to claim 15, wherein a beginning-portion of the outlet channel connected to the perfusion area is shaped to conduct the carrier fluid downwards from the perfusion area.
17. A method according to claim 15 or 16, wherein the end-portion of the inlet channel is substantially perpendicular to a surface of the sample facing towards the perfusion area.
18. A method according to any of claims 15-17, wherein the sample comprises a cell layer.
19. A method according to claim 18, wherein the cell layer is one of the following: a layer of blood-retinal barrier cells, a layer of blood-aqueous barrier cells, a layer of blood-brain barrier cells, intestinal epithelium, lung epithelium.
PCT/FI2015/050561 2014-09-04 2015-08-31 A method and a device for measuring permeability of molecules through a sample WO2016034766A1 (en)

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Cited By (2)

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CN108204935A (en) * 2018-01-10 2018-06-26 中南大学 The experimental provision and experimental method of diffusion pressure microcosmic mechanism during characterization slip casting
WO2019177534A1 (en) * 2018-03-12 2019-09-19 Skinetrate Pte. Ltd. Apparatus for determination of diffusion or permeation through a specimen, method of forming the same and method for controlling the same

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US6360588B1 (en) * 1998-10-27 2002-03-26 Edward Allan Ross Materials and methods for the analysis of substances passing through a membrane
US8393199B2 (en) 2007-01-31 2013-03-12 Cosmed Pharmaceutical Co., Ltd. Apparatus for measuring diffusion of transdermal absorption preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6360588B1 (en) * 1998-10-27 2002-03-26 Edward Allan Ross Materials and methods for the analysis of substances passing through a membrane
US8393199B2 (en) 2007-01-31 2013-03-12 Cosmed Pharmaceutical Co., Ltd. Apparatus for measuring diffusion of transdermal absorption preparation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108204935A (en) * 2018-01-10 2018-06-26 中南大学 The experimental provision and experimental method of diffusion pressure microcosmic mechanism during characterization slip casting
WO2019177534A1 (en) * 2018-03-12 2019-09-19 Skinetrate Pte. Ltd. Apparatus for determination of diffusion or permeation through a specimen, method of forming the same and method for controlling the same

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