WO2010013016A2

WO2010013016A2 - Apparatus and method for sample processing or storage

Info

Publication number: WO2010013016A2
Application number: PCT/GB2009/001893
Authority: WO
Inventors: Shu Wenmiao
Original assignee: Heriot Watt University
Current assignee: Heriot Watt University
Priority date: 2008-07-31
Filing date: 2009-07-31
Publication date: 2010-02-04
Anticipated expiration: 2011-01-31
Also published as: WO2010013016A3; GB0814035D0

Abstract

An apparatus for sample processing or storage comprises an array of sample chambers (20a-20e), a plurality of gas channels or liquid channels (10a-10e) and a gas permeable membrane structure or liquid permeable membrane structure (6) between the gas channels or liquid channels (10a-10e) and the array of sample chambers (20a- 20e), arranged so that in operation gas permeates between each gas channel or liquid channel and at least one respective sample chamber through the gas permeable membrane structure or the liquid permeable membrane structure.

Description

Apparatus and method for sample processing or storage

The present invention relates to an apparatus and method for sample processing or storage, for example for biological sample culture or testing. The apparatus or method may be used for the culture of cells, tissues or organisms and the performance of bioassays on tissue samples, cells or cell derived parts or substances.

To survive long term, cultured cells and tissues not only need liquid reagents for nutrition but gases as well, such as oxygen and carbon dioxide.

In order to control the environment that the cells are grown in, vessels with cells are conventionally placed in incubators that maintain steady humidity, gas and temperature conditions. This means that in order to culture cells under a variety of gas conditions, multiple incubators are required, which has space and cost implications.

In order to study cultured cells (for example, observe them under a microscope), change the conditions that the cells are grown in or simply to refresh the nutrient- providing medium, the cells need to be removed from the incubator and media changes are usually performed manually. Removal from the incubator exposes cells to an alteration of gas, temperature and humidity conditions as well as a risk of contamination.

Biological and biochemical assays measure the effect of a substance on an organism/cell or cell derived part or substance and are commonly used in pharmacology to investigate the effects of drugs, in academic research or in diagnostics. To ensure accuracy of results, bioassays need to be performed under controlled conditions both in terms of the liquid reagents used and environmental conditions (gas and temperature).

Drug discovery relies on testing vast numbers of compounds on cell cultures in vitro, to determine effectiveness prior to animal and patient trials. These trials are costly in terms of their use of cells and reagents, and it is desirable to initially perform these on as small a format as possible, provided that this does not compromise quality. High throughput is key in drug discovery trials. Microfluidics is an ideal technology for this purpose as it allows the precise manipulation of minute quantities of liquids. A microfluidic device is defined as comprising one or more channels, with at least one dimension less than lmm for the containment and flow of fluids. Microfluidic devices therefore allow investigators to screen large numbers of potential drugs for specific effects in a variety of cell types using a limited surface area and small amounts of costly reagents.

Most of the currently available microfluidics systems are completely sealed and therefore do not allow gas permeation. This is an advantage in terms of preventing bacterial infections of cells however does not allow for long term culture of cells (prokaryotic or eukaryotic) as cells need a supply of gases to survive.

It is an aim of the present invention to provide an improved, or at least alternative, apparatus and method for sample processing or storage, for example biological sample culture or testing.

In a first, independent aspect of the invention there is provided an apparatus for sample processing or storage, comprising an array of sample chambers, a plurality of gas channels or liquid channels and a gas permeable membrane structure or liquid permeable membrane structure between the gas channels or liquid channels and the array of sample chambers, arranged so that in operation gas or liquid permeates between each gas channel or liquid channel and at least one respective sample chamber through the gas permeable membrane structure or liquid permeable membrane structure.

The sample processing may comprise biological sample processing or storage, for example biological sample culture or testing. The processing may comprise an assay process. By providing a plurality of gas or liquid channels, different gas or liquid conditions may be provided to different sample chambers in the array. Alternatively or additionally, the gas or liquid conditions for at least one of the sample chambers may be varied over time. The use of the permeable membrane ensures that stable conditions can be maintained in the sample chambers whilst also providing for the transfer of liquid or fluid to or from the sample chambers. Depending on its material and construction, the gas permeable membrane structure may be permeable to at least some liquids. Similarly, the liquid permeable membrane structure may be permeable to at least some gases. Thus, the permeation of liquid through the liquid permeable membrane structure may comprise permeation of at least some gas through the liquid permeable membrane structure. Similarly , the permeation of gas through the gas permeable membrane structure may comprise permeation of at least some liquid through the gas permeable membrane structure. Alternatively, the gas permeable membrane structure may be impermeable to substantially all liquids.

The material and construction of the membrane may be selected to provide a desired level of permeability for a selected at least one gas or liquid, for example by selecting a membrane material having a desired porosity and thickness.

The apparatus may be sealable so that in operation samples in the sample chambers are not exposed to the external environment, for example so that the samples are not exposed to the external atmosphere. Alternatively the samples may be exposed to the external atmosphere only via the gas or liquid permeable membrane structure.

The array of sample chambers and the gas or liquid channels may be arranged so that in operation, gas or liquid permeates between a first at least one sample chamber and a first one of the gas or liquid channels and gas or liquid permeates between a second at least one sample chamber and a second one of the gas or liquid channels, and substantially no gas or liquid permeates between the first at least one sample chamber and the second one of the gas or liquid channels or between the second at least one sample chamber and the first one of the gas or liquid channels. Each gas or liquid channel may comprise at least one input for inputting gas or liquid and at least one output for outputting gas or liquid, and the at least one input and at least one output for each gas or liquid channel may be separate from the at least one input and at least one output for each other gas or liquid channel.

In operation, there may be substantially no passage of gas or liquid between each one of the plurality of gas or liquid channels and each other of the plurality of gas or liquid channels.

In operation, a sample may be provided to each sample chamber. The sample provided to the sample chambers may be the same, or of the same type, or may be different or be of different types.

The samples may be whole organisms, organs, tissue samples, cell samples, samples of cell derived parts or substances, or any other kind of biological samples. A cell derived part or substance may be a part or substance that is obtained directly from the cell, or may be a part or substance that is obtained by processing (for example lysing) the cell, for example DNA, RNA, protein, organelle or membrane material.

Examples of samples include but are not limited to:- organisms ; organs; tissues (such as tumour biopsies and blood vessels); any cells or eukaryotic or prokaryotic origin such as primary cell cultures, stem cells and cell lines, and including animal, plant, yeast and bacterial cultures. The samples may be samples for a biological or biochemical assay such as, for example, blood, urine, saliva, cell derived part or substance (such as proteins, DNA, RNA, organelles such as mitochondria or ribosome, or cell or organelle membranes).

Other examples of samples include, chemicals, pharmaceuticals, and fluids of any type that are to be subject to filtering or purification.

Each sample chamber may comprise a well, or channel or part of a channel. Each sample chamber may comprise a pool or a flat base. Each sample chamber may include a growth support structure or may be coated with a substance such as collagen, gelatine, laminin, flbronectin or any other substance to assist or alter the growth of cells. Each sample chamber may be transparent over at least part of its surface, to allow optical measurement or viewing of cells in the sample chamber.

Each gas or liquid channel may be aligned with at least one sample chamber. Each sample chamber may be aligned with at least one gas or liquid chamber.

The spacing of the gas or liquid channels may be substantially equal to a spacing of the sample chambers along at least one direction of the array.

The sample chambers may be arranged in rows. The rows of sample chambers may be parallel to one another. The spacing of sample chambers along each row may be substantially equal to the spacing of the gas or liquid channels. The sample chambers and the gas or liquid channels may be in any suitable pattern or arrangement, and are not limited to being in straight lines. For example, at least one of the gas or liquid channels may be arranged as a loop or spiral in two or three dimensions, and similarly at least some of the sample chambers may be arranged in a loop or spiral pattern.

The gas or liquid channels may be positioned substantially orthogonally to the rows of sample chambers.

Each gas or liquid channel may include at least one gas or liquid chamber. By providing at least one gas or liquid chamber as part of a gas or liquid channel, an increased surface area may be provided for permeation of gas or liquid through the gas permeable membrane or liquid permeable membrane. Each gas or liquid chamber may be aligned with a sample chamber.

The apparatus may further comprise a plurality of gas or liquid control means, each gas or liquid control means for controlling the input of gas or liquid to a respective gas channel. By providing a plurality of gas or liquid control means, the gas or liquid flow through each gas or liquid channel may be controlled individually. Each gas or liquid control means may be configured to control the flow rate and/or the constitution of the gas or liquid. The gas or liquid control means may also control one or both of the pressure and temperature of the gas or liquid.

The gas or liquid control means may be configured to control the pressure or concentration of the gas or liquid, or the partial pressure or concentration of a constituent of the gas or liquid, to have a desired value. The desired value may be a value within a desired range of values. The desired value may be a desired value relative to the pressure or partial pressure or concentration of the gas or liquid or a constituent of the gas or liquid within at least one of the sample chambers. The gas or liquid control means may be configured to control the gas or liquid, or a constituent of the gas or liquid, to have a partial pressure or concentration different to a corresponding partial pressure or concentration in one or more of the sample chambers, to enable transfer of the gas or liquid or the constituent of the gas or liquid to or from at least one of the sample chambers.

The gas or liquid, or the constituent of the gas or liquid, may comprise a substance that is used in a process by a sample in one or more of the sample chambers, or that is a by-product of a process on a sample in one or more of the sample chambers. The liquid may comprise, for example, water. The gas or the constituent of the gas may comprise, for example, one or more of air, oxygen, carbon dioxide, nitrogen or nitric oxide.

The apparatus may further comprise at least one further channel for supplying fluid to the sample chambers. The fluid may be liquid or gas, and may be a sterile fluid. The liquid may be for providing nutrition to cells, and thus may comprise water, a carbon source such as glucose, balanced salt solutions, blood serum and additional amino acids for mammalian cell culture or a source of amino acids for bacterial cell culture such as yeast or beef extract. Alternatively or additionally, the fluid may comprise a drug, hormone or other substance whose effects on cells in the sample chambers it is intended to test. Alternatively or additionally, the fluid may comprise any reagents required for biological and biochemical assays which may include antibodies or dyes or any other reagents required for enzymatic, protein or DNA assays.

The at least one further channel and the gas or liquid channels may be arranged so that in operation, for each further channel, at least one sample chamber supplied by that further channel is supplied by a first one of the gas or liquid channels and at least one other sample chamber supplied by that further channel is supplied by a second one of the gas or liquid channels.

Thus, in operation, different sample chambers that are supplied with the same fluid by the further channels may be subject to different gas or liquid conditions provided by the gas or liquid channels. That may enable the effect of different gas or liquid conditions on the growth or function of cells to be determined.

The at least one further channel may be a plurality of further channels, each further channel for supplying fluid to a respective at least one of the sample chambers.

The further channels and the gas or liquid channels may be arranged so that in operation, for each gas or liquid channel, at least one sample chamber supplied by that gas or liquid channel may be supplied by a first one of the further channels and at least one other sample chamber supplied by that gas or liquid channel may be supplied by a second one of the further channels.

Each further channel may cross at least one gas or liquid channel, and may cross a plurality of gas or liquid channels. Similarly, each gas or liquid channel may cross at least one further channel and may cross a plurality of further channels.

The apparatus may further comprise at least one fluid control means, the or each fluid control means for controlling the input of fluid to a respective further channel. Each sample chamber supplied by a particular further channel may, in operation, be populated with the same sample (for example, the same cells) or the same type of sample (for example, the same type of cells). Alternatively or additionally, each sample chamber supplied by a particular gas or liquid channel may, in operation, be populated with the same samples (for example, the same cells) or the same type of samples (for example, the same type of cells). Those arrangements can provide a particularly effective way of determining the effect of different gas or liquid conditions on the same cells or the same type of cells.

For each further channel, each sample chamber supplied by that further channel may be supplied by a respective, different gas or liquid channel than each other sample chamber supplied by that further channel.

Each sample chamber may be supplied by a single one of the gas or liquid channels and/or by a single one of the further channels. Alternatively, each sample chamber may be supplied by more than one gas or liquid channel.

The or each further channel may be substantially orthogonal to the plurality of gas or liquid channels.

The at least one further channel may connect the sample chambers.

The array of sample chambers may comprise a plurality of rows of sample chambers, the sample chambers of each row connected by a respective one of the further channels.

The gas permeable membrane structure may comprise a layer of gas permeable material. The liquid permeable membrane may comprise a layer of liquid permeable material.

The apparatus may have a layered structure comprising at least one of:- a first layer of material that comprises the gas or liquid channels; a second layer that comprises the gas permeable membrane structure or liquid permeable membrane structure; and a third layer of material that comprises the array of sample chambers, wherein the second layer is between the first and third layers.

The first, second and third layers may be arranged adjacent to one another. Alternatively there may be one or more further, intervening layers. The layers may be sealed together.

The apparatus may comprise alignment means for aligning the layers. Each of at least two of the layers may comprise respective alignment means. The alignment means may be configured to co-operate with alignment means on another layer to ensure that the layers are aligned correctly. The alignment means may comprise at least one stop.

The layers may be aligned correctly when a stop of one layer is in contact with the stop of another layer. The use of stops may be particularly useful if the layers include a screw thread, and the layers may be attached by screwing together.

Each of the gas or liquid channels and the further channels may be separate so that gas or liquid cannot pass within a layer from one channel of the layer to any other channel of the layer. Alternatively, gas or liquid channels within a layer may be interconnected and/or further channels within a layer may be interconnected.

Each gas or liquid channel may comprise at least one cavity in the material of the first layer, and/or the array of sample chambers may comprise an array of cavities in the material of the third layer, and/or the at least one further channel may comprise at least one cavity in the material of the third layer.

Each gas or liquid channel cavity may be open on one side of the layer of material. In that case, the at least one gas or liquid channel cavity may be open on the side facing the layer of gas or liquid permeable material.

Alternatively, each gas or liquid channel cavity may be open on both sides of the layer of material. In that case, each gas or liquid channel cavity may be bounded by the layer of gas or liquid permeable material on one side and by a further layer of gas or liquid permeable material on the other side.

Each gas or liquid channel may be bounded by the material of the first layer and by the layer of gas or liquid permeable material. Alternatively each gas or liquid channel may be bounded by the layer of gas or liquid permeable material on one side and by a further layer of gas or liquid permeable material on the other side.

The array of cavities and/or the further channel cavity may be open on one side of the layer of material. In that case, the array of cavities and/or the further channel cavity is preferably open on the side facing the layer of gas or liquid permeable material.

Alternatively, the array of cavities and/or the further channel cavity may be open on both sides of the layer of material. In that case, the array of cavities and/or the further channel cavity may be bounded by the layer of gas or liquid permeable material on one side and on a further layer of gas or liquid permeable material on another side.

The layers of material may be substantially planar layers of material.

The apparatus may further comprise a plurality of further layers, including at least one gas or liquid permeable membrane structure layer.

The layers may be arranged so that the plurality of gas or liquid channels is bounded on both sides by gas or liquid permeable material and/or the array of sample chambers is bounded on both sides by a layer of gas or liquid permeable material.

By bounding the gas channels on both sides by gas or liquid permeable material, gas or liquid may permeate between the gas or liquid channels and both of two different layers. For example, the apparatus may further comprise a further array of sample chambers, and the plurality of gas or liquid channels may be arranged to supply gas or liquid to both the array of sample chambers and the further array of sample chambers. Similarly, by bounding the array of sample chambers on both sides by gas or liquid permeable material, gas or liquid may permeate between the array of sample chambers and two different layers. For example, the apparatus may further comprise a further plurality of gas or liquid channels, and the plurality of gas or liquid channels and the further plurality of gas or liquid channels may be arranged to supply gas or liquid to both the array of sample chambers and the further array of sample chambers.

The apparatus may be sealable such that in operation fluid may pass to or from the sample chambers only via the at least one gas or liquid channel and/or the at least one further channel. Thus, cell conditions maybe controlled accurately.

The apparatus may be constructed on any scale, dependent on the application for which it is to be used.

Advantageously the array of sample chambers may be a microfluidic array, which may provide for testing or culture of many different samples simultaneously in a compact apparatus. Each sample chamber may have a volume less than ImI, and/or less than lOμl , and/or less than 10OnI. Each gas or liquid channel and/or each further channel may have a width less than lmm, and/or less than lOOμm.

One feature of micro-scale sample chambers is that the surface area to volume ratio of the chambers is higher than for larger scale chambers, thus providing for efficient permeation of gases within and into or out of the chambers and good mixing of gases within the chambers.

Thus, a micro-scale apparatus that uses microchambers for the culture of cells, tissues and/or conduction of bioassays and microchannels for the injection and control of liquid and gasses may be provided. However, the apparatus is not limited to being a microfluidic or microscale device, and may be constructed on any scale. The layers may be silicon, glass, plasties, polymers, metal or ceramics layers. The layers may be, for example, polyethylene. The apparatus may be constructed of low- cost material and using low-cost methods and thus may be treated as disposable.

In a further independent aspect of the invention there is provided a method of processing or storing samples, comprising:- providing samples in an array of sample chambers, and inputting gas or liquid to a plurality of gas channels or liquid channels so that gas permeates between each gas channel or liquid channel and a respective at least one sample chamber through a gas permeable membrane structure or a liquid permeable membrane structure, wherein the gas permeable membrane structure or liquid permeable structure is between the gas channels or liquid channels and the array of sample chambers.

The method may be a method of culturing or testing biological samples.

The method may further comprise controlling the gas or liquid input to each of the gas or liquid channels to provide different gas or liquid conditions for at least some of the sample chambers.

Controlling the gas or liquid input may comprise controlling the flow rate and/or the constitution of the gas or liquid. Alternatively or additionally, controlling the gas or liquid input may comprise controlling one or both of the pressure and temperature of the gas or liquid.

Controlling the gas or liquid input may comprise controlling the pressure or concentration of the gas or liquid or a partial pressure or a concentration of a constituent of the gas or liquid, to have a desired value. The desired value may be a value within a desired range of values. The desired value may be a desired value relative to the pressure or partial pressure or concentration of the gas or liquid or a constituent of the gas or liquid within the or at least one of the sample chambers. The method may comprise controlling the gas or liquid, or a constituent of the gas or liquid, to have a pressure, partial pressure or concentration different to a corresponding pressure, partial pressure or concentration in one or more of the sample chambers, to enable transfer of the gas or liquid or the constituent of the gas or liquid, to or from at least one of the sample chambers.

The gas or liquid, or the constituent of the gas or liquid, may comprise a substance that is used in a process by a sample in one or more of the sample chambers, or that is a by-product of a process on a sample in one or more of the sample chambers. The liquid may comprise, for example, water. The gas or the constituent of the gas may comprise, for example, one or more of air oxygen, carbon dioxide, nitrogen, or nitric oxide.

The method may further comprise providing at least one further channel for supplying fluid to the sample chambers, and controlling the fluid input to the or each further channel.

The fluid input to the or each further channel and/or the gas or liquid input to at least one of the gas or liquid channels may be varied over time. The apparatus and method can allow a user to vary gas and liquid conditions as desired, whilst samples are installed in the apparatus.

The at least one further channel may be a plurality of further channels and controlling the fluid input may comprise controlling the fluid input to each further channel to provide different fluid conditions for at least some of the sample chambers.

A fluid condition may comprise at least one of fluid constitution, concentration, temperature and pressure.

The method may further comprise controlling the fluid input and the gas or liquid input so that at least two sample chambers that have the same fluid conditions provided by the further channels have different gas or liquid conditions provided by the gas or liquid channels and/or so that at least two sample chambers that have the same gas or liquid conditions provided by the gas or liquid channels have different fluid conditions provided by the further channels.

The apparatus and method may provide for the long-term culture of cells that allows manipulation and permeation of gases which are crucial to cell growth, development and the recreation of conditions similar to those in whole organisms. The apparatus may includes sample chambers of any size, and in particular microfluidic sample chambers.

The apparatus and method may provide accurate control of gas or liquid compositions, steady control of humidity, and also effective prevention of bacterial infection.

The apparatus and method may provide for the culture of cells under controlled gas and medium conditions. The apparatus may provide multiple chambers for the culture of cells/ biological assays, to which multiple channels supply liquid reagents, a membrane, and multiple channels for controlled gas flow.

The apparatus may be constructed on any scale, and may be a microfluidics device consisting of microchambers and microchannels in any desired arrangement.

There is also provided apparatus substantially as described herein, with reference to the accompanying drawings, and/or a method substantially as described herein, with reference to the accompanying drawings.

Any feature in one aspect of the invention may be applied to other aspects of the invention, in any appropriate combination. In particular, apparatus features may be applied to method features and vice versa.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a schematic diagram of a cell culture or testing apparatus, shown as an exploded view for clarity;

Figure 2 is a further schematic diagram of the apparatus of Figure 1; and Figure 3 is a schematic diagram of the apparatus of Figures 1 and 2, showing gas and fluid flows.

A cell culture or testing apparatus 2 is shown in Figure 1. The apparatus comprises three layers 4, 6, 8. The three layers are shown separated, in an exploded view, in Figure 1 for illustrative purposes.

The first, gas channel, layer 4 and the third, sample array, layer 8 are substrates, that may be made of any suitable materials such as silicon, glass, plastics, polymers, metal or ceramics. In this embodiment the layers 4, 8 are made of polyethylene. The second layer 6 is a semi-permeable membrane that is permeable to gasses but not liquids and that is sandwiched between the first and third layers 4, 8. In the illustrated example the semi-permeable membrane 6 is made of teflon, but may be made of any suitable liquid-impermeable, gas-permeable material. The thickness and permeability of the membrane 6 can be selected to be suitable for a particular experiment or cell culture.

Channels and chambers are fabricated in the first and third layers 4, 8. The channels and chambers can be fabricated using any suitable method, including pressing (in the case of polymers or plastics) or etching.

The first layer 4 includes a series of channels 10a- 1Oe, each of which links a series of chambers 12a-12e. Each channel includes an input 14 and an output 16. In the embodiment shown, the channels 10a- 1Oe and chambers 12a-12e are open in the direction facing the semi-permeable membrane 6 and are closed in the opposite direction.

The third layer 8 has a similar structure to the first layer 4, and also includes a series of channels 18a-18e, each of which links a series of chambers 20a-20e. Each channel includes an input 22 and an output 24. In the embodiment shown, the channels 18a- 18e and chambers 20a-20e are also open in the direction facing the semi-permeable membrane 6 and closed in the other direction.

The apparatus shown in Figure 1 may be treated as disposable, due to the low cost of the materials and methods of construction used.

The apparatus of Figures 1 and 2 is a microscale device, and the channels 18a-18e, are spaced apart by around 200μm. The width of the channels 18a-18e is around 50μm, and the volume of each chamber 20a-20e is around 10OnL. The dimensions of the channels 10a- 1Oe and chambers 12a-12e are similar to those of the channels 18a-18e and chambers 20a-20e in this embodiment.

The first and third layers 4, 8 are attached together with the semi-permeable membrane 6 sandwiched in between, as shown in Figure 2, to provide a sealed device. The layers may be attached together and sealed using any suitable method, including clamping, heat treating, ultrasonic bonding and use of adhesive. In a variant of the illustrated embodiment, the layers 4, 6, 8 are circular and include a screw thread arrangement around their edges enabling them to be screwed together.

As the layers 4, 6, 8 are sealed together, fluid may only pass into or out of the device through the inputs 14, 22 and outputs 16, 24 of each channel 10a- 1Oe 18a-18e (although gas may also pass through the semi-permeable membrane 6). Once the device is sealed each of the channels 10a- 1Oe 18a-18e is separate and fluid cannot pass within a layer 4 between one channel 1 Oa of the layer 4 and any other channel 10b-10e ofthe layer 4.

It can be seen from Figures 1 and 2 that each row 10a- 1Oe of the first layer 4 is aligned with a series of chambers 20a-20e of the third layer 8, and that each row 18a- 18e of the third layer 8 is aligned with a series of chambers 12a-12e of the first layer

4. Furthermore, the chambers of the first layer 4 and the third layer 8 are stacked accurately on top of each other, so that each chamber of the first layer 4 is aligned with a corresponding chamber of the third layer 8.

It can also be seen from Figures 1 and 2 that the first layer 4 and the third layer 8 are of similar structure but that the channels 10a- 1Oe of the first layer 4 are arranged substantially orthogonally to the channels 18a-18e of the third layer 8.

When the layers 4, 6, 8 are attached together, gas is able to permeate between the layers through the semi-permeable membrane 6. Due to the alignment of the chambers 12a-12e, 20a-20e and the mutually orthogonal arrangement of the channels 10a- 1Oe, 18a-18e, chambers linked by a common channel of one layer are aligned with chambers that form part of distinct channels of the other layer.

As the channels of the first layer 4 and the second layer 8 are separated by the membrane 6, it is possible to inject liquid into channels of one of the first and second layers whilst providing controlled gas flows into the channels of the other of the first and second layers, without the liquid and gas flows interfering with each other.

In the embodiment illustrated in Figures 1 and 2, cells are deposited in each of the chambers of the third, sample array, layer 8, which are used as sample chambers.

The samples may be deposited in each sample chamber before the attachment and sealing of the layers 4, 6, 8. That method is particularly well suited to deposition of tissue samples. Alternatively the samples may be input as a cell solution through the channels 18a-18e after attachment and sealing of the layers 4, 6, 8. That method of inputting samples is particularly well suited to the inputting of cell or cell derived parts or substances to the sample chambers.

It is important to note that the membrane 6 is made of semi-permeable material so that gas, for example oxygen and carbon dioxide, can diffuse between the gas chambers of the first layer 4 and the liquid-filled sample chambers of the third layer 8 due to the difference of gas partial pressure between the two sides of the membrane. Thus, normal cell processes such as respiration, growth and reproduction can continue in the sample chambers without restricted or otherwise affected by the build-up of gases in the sample chambers.

The device allows each gas channel of the first layer 4 to carry different gas conditions/concentrations and each liquid channel of the third layer 8 to carry different liquids, if so desired. Therefore the apparatus allows the design of experiments in such a way that each sample chamber may be subject to a unique combination of exposure to a particular gas channel of the first layer 4 and a particular liquid channel of the third layer 8.

The apparatus of Figures 1 and 2 is shown connected to liquid and gas supplies in Figure 3. Each gas channel 10a- 1Oe is connected to a respective gas controller 30a- 30b, and each liquid channel 18a-18e is connected to a respective liquid controller 30a-30b.

In the embodiment shown, each gas controller 30a-30b is connected to two gas lines 34, 36, one 34 containing air and the other 36 containing nitric oxide. Each gas controller 30a-30e comprises a plurality of valves and operates the valves to control the level of nitric oxide enrichment of the air that is input to the gas channels. Thus, controlled gas conditions are provided in the gas channels and chambers of the first, gas channel layer. The controllers 30a-30e are able to control the partial pressures of different gases in each channel.

Each liquid controller 32a-32e is connected to two liquid lines 38, 40 one containing a nutritional fluid provides nutrition for the cells, enabling growth and/or reproduction and one containing a drug solution under investigation that may have an effect on the function of the cells. Each liquid controller 32a-32e also comprises a plurality of valves and associated actuators for operating the valves to control the drug dosage to be input to the sample cells via the fluid channels 18a- 18e.

The operation of the gas controllers 30a-30e and the liquid controllers 32a-32e is controlled by a central controller (not shown). The central controller in the embodiment of Figure 3 comprises a general purpose computer programmed with suitable control and interfacing software. In alternative embodiments, the central controller is a dedicated device, for example comprising one or more application specific integrated circuits (ASICs). The central controller comprises a user interface, allowing a user to set the various parameters for controlling operation of processes on the apparatus 2, for example gas and/or liquid flow rates, gas and/or liquid compositions and concentrations, gas and/or liquid pressures and/or temperatures, variations of those or other parameters over time, and the timings and durations of such variations.

The gas controllers 30a-30e and liquid controllers 32a-32e allow the gas and liquid conditions to be controlled and varied whilst samples are present in the sample chambers and without requiring the apparatus to be dismantled and reassembled or the sample chambers to be opened and exposed to the external environment. In some culture or testing procedures, gas and liquid conditions are varied over time whilst the samples are present in the sample chambers.

The gas controller is operable, for example, to control the gas, or a constituent of the gas, to have an under-pressure or over-pressure with respect to one or more of the sample chambers, to enable transfer of the gas or the constituent of the gas to or from the or at least one of the sample chambers.

In the embodiment of Figure 3, the same cells or other samples are provided to each of the sample chambers, the oxygen concentration is varied or set to have a different value for each gas channel 10a- 1Oe and the drug dosage concentration is varied or set to have a different value for each liquid channel 18a-18e. Thus, due to the orthogonal arrangement of the gas and liquid channels, each sample chamber may be subject to a different combination of drug dosage and oxygen concentration, and the effect of every combination of drug dosage and oxygen concentration on the function of the cells or on the other samples may be studied over time. The apparatus may be used to study any combination of liquid, gas and cell or other sample characteristics, and is not limited to the example discussed above. For instance different cells or other samples may be provided in different sample chambers, and any combination of liquid and gas compositions and characteristics may be used. In one example of an arrangement for control of gas conditions that may be used for cell culture, three gas lines carrying nitrogen, carbon dioxide and oxygen are connected to the gas controllers. That allows the concentration of all of the three gases to be varied in each gas channel, and allows low oxygen conditions to be created in the sample chambers that resemble the natural environment of the cell in an organism (typically between 1-10% oxygen concentration in humans as opposed to 21% oxygen concentration in the atmosphere).

The ability to combine liquids together may be used to test drugs by providing a drug solution in one of the liquid lines, or other substances such as growth factors, hormones, or nutritional enrichment, such as a solution of glucose. Toxicity of substances can also be tested. Additionally, the liquid lines can be used to supply the required reagents for bioassays, including protein assays, DNA or RNA assays.

In the apparatus of Figures 1 to 3, the sample chambers are transparent enabling viewing or optical measurement of the cells in situ in the sample chambers. In other embodiments, the layers 4, 6, 8 are detached at the end of an experiment and cells are extracted for study. In a further embodiment, the cells may be flushed out of the apparatus at the end of an experiment by passing suitable liquid (for example containing a substance that causes the cells to detach from the sample chamber walls) through the liquid channels 18a- 18e.

The apparatus is not limited to the particular arrangement shown in Figures 1 to 3. Variations of the apparatus that use a semi-permeable material to allow gas to pass into sample chambers are also possible. For example, the design of the channels in the first and third layers 4, 8 may be varied. The layers 4, 6, 8 may also be placed together in any combination or shape. The relative sizes of the channels and chambers of the gas channel and sample array layers may be varied and may be different from one another. For example, gas chambers may be larger than the sample chambers with which they are aligned, to ensure easier alignment and homogeneous gas transfer over the area of the sample chambers. Furthermore, the gas or liquid channels in alternative embodiments are not arranged in a parallel arrangement, and similarly the channels linking the chambers are not arranged in a parallel arrangement. The gas or liquid channels are not arranged orthogonally to the channels linking the chambers in some embodiments.

In another embodiment, more than two sample array layers 8 and/or gas channel layers 4 may be used with a semi-permeable material between each. For instance two gas channel layers 4 may sandwich a central sample array layer 8, with respective semi-permeable membranes between the sample array layer 8 and each gas channel layer 4. In that embodiment, the sample chambers are open to both sides of the sample array layer 8 and gases may be provided to each sample chamber from both gas channel layers 4 simultaneously.

Alternatively, two sample array layers 8 may be provided with a gas channel layer 4 between, and separated from them by semi-permeable membranes. In that embodiment, the gas chambers are open to both sides of the gas channel layer 4 and the gas channel layer 4 may be used to provide gas to both of the sample arrays simultaneously.

The number of layers 4, 6, 8 can be increased further, and the arrangement of the layers can be varied, as desired.

In another variant, the apparatus includes gas channels and not liquid channels. In that case, the sample chambers are isolated once samples have been installed and the layers attached together. Controlled amounts of gas are supplied to the sample chambers through the gas channels.

The embodiment of Figures 1 and 2 is a microfluidics device, and includes a relatively small number of chambers and channels for illustrative purposes. However, by using microfluidic scale channels and chambers a massive array of microchambers where both liquid and gas components can be controlled precisely for high throughput cell- based assay can be provided. For example, on a 20 x 20 mm chip, 100 microchannels on each substrate can be microfabricated with a spacing of 200 μm. By combining 100 liquid conditions (with for example different drugs and ion concentrations) and 100 gas (or liquid) conditions (with for example different O₂, CO₂ or NO_x concentrations), 10,000 experiments can be performed on the same microfluidic chip. The total reagent required, however, may be less than several μL.

The apparatus may be constructed on a smaller or larger scale if desired, and is not limited to being a microfluidics device. The scale of apparatus chosen may depend on the type of cells that are being cultured or investigated, and the nature or purpose of the culture or investigation.

Any types of samples may be cultured or investigated using the apparatus, including but not limited to:- whole organisms, organs, tissue samples, cell samples, samples of cell derived parts or substances, or any other kind of biological samples. Examples of samples include but are not limited to:- organisms; organs; tissues (such as tumour biopsies and blood vessels); any cells or eukaryotic or prokaryotic origin such as primary cell cultures, stem cells and cell lines, and including animal, plant, yeast and bacterial cultures. The samples may be samples for a biological or biochemical assay such as, for example, blood, urine, saliva, cell derived part or substance (such as proteins, DNA, RNA, organelles such as mitochondria or ribosome, or cell or organelle membranes).

Although the embodiment of Figures 1 and 2 includes gas channels for the provision of gas to or from the sample chambers, and the gas permeable membrane structure is impermeable to liquid, in other embodiments the gas permeable membrane structure is a liquid-permeable membrane structure and the channels 10a- 1Oe are operated as liquid channels used to provide liquid to and/or take liquid from the sample chambers via permeation through the liquid-permeable membrane. In such embodiments, the liquid channels may be used to supply liquids such as nutritional liquids or liquids to be tested, such as toxins or drugs. Alternatively or additionally the liquid channels and liquid permeable membrane may be used to extract liquid (and/or gas) waste products or other by-products from the sample chambers. The liquid permeable membrane may be selectively permeable so that is it permeable to some types of liquid and impermeable to other types of liquid.

Examples of membrane materials include polytetrafluoroethylene (PTFE or Teflon), polyurethane, fluorinated ethylene propylene (FEP), silicone, polysulfone (PS), polyether sulfone(PES), polyacrilonitrile (PAN), polyamide, polyimide, polyethylene (PE), polypropylene (PP), polyvinylidinefluoride (PVDF), and polyvinylchloride (PVC). All of those materials can form the semi-permeable membrane. Depending on its porosity and thickness, the membrane can be made gas permeable and/or liquid permeable.

Some embodiments that include a liquid permeable membrane structure provided between a sample array layer and a liquid channel layer are used for purification or filtration. Liquid samples are provided in the sample chamber layer or in the liquid channel layer under different conditions (for example different driving pressures or temperatures) and different filtration and/or purification effects can therefore be obtained and/or the effects of the different conditions can be studied, for example in an assay process. The purification or filtration can be, for example, a water or drug purification or filtration.

It will be understood that the present invention has been described above purely by way of example, and modifications of detail can be made within the scope of the invention.

Each feature disclosed in the description, and (where appropriate) the claims and drawings may be provided independently or in any appropriate combination.

Claims

1. An apparatus for sample processing or storage, comprising an array of sample chambers, a plurality of gas channels or liquid channels and a gas permeable membrane structure or liquid permeable membrane structure between the gas channels or liquid channels and the array of sample chambers, arranged so that in operation gas or liquid permeates between each gas channel or liquid channel and at least one respective sample chamber through the gas permeable membrane structure or liquid permeable membrane structure.

2. An apparatus according to Claim 1 , for biological sample culture or testing.

3. Apparatus according to Claim 1 or 2, wherein each gas or liquid channel is aligned with at least one sample chamber.

4. Apparatus according to any preceding claim, wherein each gas or liquid channel includes at least one gas or liquid chamber.

5. Apparatus according to any preceding claim, further comprising a plurality of gas or liquid control means, each gas or liquid control means for controlling the input of gas or liquid to a respective gas or liquid channel.

6. Apparatus according to any preceding claim, further comprising at least one further channel for supplying fluid to the sample chambers.

7. Apparatus according to Claim 6, wherein the at least one further channel and the gas or liquid channels are arranged so that in operation, for each further channel, at least one sample chamber supplied by that further channel is supplied by a first one of the gas or liquid channels and at least one other sample chamber supplied by that further channel is supplied by a second one of the gas or liquid channels.

8. Apparatus according to Claim 6 or 7, wherein the or each further channel is substantially orthogonal to the plurality of gas or liquid channels.

9. Apparatus according to any of Claims 6 to 8, wherein the at least one further channel is a plurality of further channels, each further channel for supplying fluid to a respective at least one of the sample chambers.

10. Apparatus according to Claim 9, wherein the array of sample chambers comprises a plurality of rows of sample chambers, the sample chambers of each row connected by a respective one of the further channels.

11. Apparatus according to any preceding claim, having a layered structure comprising a first layer of material that comprises the gas or liquid channels, a second layer that comprises the gas or liquid permeable membrane structure and a third layer of material that comprises the array of sample chambers, wherein the second layer is between the first and third layers.

12. Apparatus according to Claim 11, wherein each gas or liquid channel comprises at least one cavity in the material of the first layer, and/or the array of sample chambers comprises an array of cavities in the material of the third layer, and/or the at least one further channel comprises at least one cavity in the material of the third layer.

13. Apparatus according to Claim 11 or 12, further comprising a plurality of further layers, including at least one gas or liquid permeable membrane structure layer.

14. Apparatus according to Claim 13, wherein the layers are arranged so that the plurality of gas or liquid channels is bounded on both sides by gas or liquid permeable material and/or the array of sample chambers is bounded on both sides by a layer of gas or liquid permeable material.

15. Apparatus according to any preceding claim, wherein the apparatus is sealable such that in operation fluid may pass to or from the sample chambers only via the at least one gas or liquid channel and/or the at least one further channel.

16. Apparatus according to any preceding claim, wherein the array of sample chambers is a microfluidic array.

17. A method of a method of processing or storing samples, comprising:- providing samples in an array of sample chambers, and inputting gas or liquid to a plurality of gas channels or liquid channels so that gas or liquid permeates between each gas channel or liquid channel and a respective at least one sample chamber through a gas permeable membrane structure or a liquid permeable membrane structure, wherein the gas permeable membrane structure or liquid permeable structure is between the gas channels or liquid channels and the array of sample chambers.

18. A method according to Claim 17, comprising controlling the gas or liquid, or a constituent of the gas or liquid, to have a partial pressure or concentration different to a corresponding a partial pressure or concentration in one or more of the sample chambers, to enable transfer of the gas or liquid, or the constituent of the gas or liquid, to or from at least one of the sample chambers.

19. A method according to Claim 18, comprising controlling the gas or liquid input to each of the gas or liquid channels to provide different gas or liquid conditions for at least some of the sample chambers.

20. A method according to Claim 18 or 19, further comprising providing at least one further channel for supplying fluid to the sample chambers, and controlling the fluid input to the or each further channel.

21. A method according to Claim 20, wherein the at least one further channel is a plurality of further channels and controlling the fluid input comprises controlling the fluid input to each further channel to provide different fluid conditions for at least some of the sample chambers.

22. A method according to Claim 20 or 21, further comprising controlling the fluid input and the gas or liquid input so that at least two sample chambers that have the same fluid conditions provided by the further channels have different gas or liquid conditions provided by the gas or liquid channels and/or so that at least two sample chambers that have the same gas or liquid conditions provided by the gas or liquid channels have different fluid conditions provided by the further channels.

23. A method according to any of Claims 17 to 22, wherein the gas or liquid comprises a substance that is used in a process by a sample in one or more of the sample chambers, or that is a by-product of a process on a sample in one or more of the sample chambers.