MEASUREMENTS

The present disclosure relates to the micro fluidic perfusion culture of live tissue and is particularly applicable to the culture of tissue slices and other live systems with integrated observation of their metabolism by nuclear magnetic resonance spectroscopy (NMR) and magnetic resonance imaging (MRI).

Cell-based models are used in many fields of research. For example, cell-based models are used in the early phases of drug development processes. In the discovery and development phase of drug development, cell-based models may be used to identify and validate targets, such as a gene or a protein with therapeutic and/or disease-causing characteristics. Cell-based assays may also be used in screening processes, to assess the effects of a potential drug candidate at the cellular, molecular, and/or biochemical level. In the pre-clinical phase of drug development, cell-based in vitro and ex vivo assays may be used to evaluate the efficacy, toxicity, and/or pharmacokinetic information of a potential drug candidate. For example, a potential drug candidate can be introduced into the cellculture environment and the resulting changes in the metabolism of the cells can be investigated.

Cell-based approaches are also used in infection models, such as for determining pathogenic mechanisms of infectious agents and for developing vaccines and therapeutics. In the field of personalised medicine, cell-based models can be used in, for example, cancer cell models to evaluate and design effective cancer treatments based on the specific cell types and the specific mutations in the tumour of a particular patient.

It is known to use precision cut tissue slices (PCTS) as an improvement to traditional two dimensional (2D) or three dimensional (3D) cellular models. PCTS are superior to simpler 2D or 3D cellular models because they retain tissue-like architecture and extracellular matrix and thus function more similarly to the same tissue in vivo. However, it is challenging to maintain the viability of PCTS in an ex vivo environment. PCTS require culture under tailored and highly controlled conditions. The culture of PCTS requires specialised and complex apparatus to provide, for example, a high oxygen content environment, sufficient nutrient supply, and physiological temperatures.

Previous approaches to the culture of PCTS have used micro fluidic devices to provide continuous, high-flow perfusion of the tissue slice with culture media in a high oxygen container. Other approaches have used an NMR tube-based tissue culture, with a high oxygen content gas being continually bubbled through a high volume of culture media. Changes in the biological processes of tissue cultured using such methods can be achieved by fluorescent detection of tagged molecules and/or destructive end-point analysis techniques. However, this limits flexibility for monitoring analytes and capacity for long-term data collection.

It is an object of the present disclosure to provide alternative approaches for evaluating biological processes in live tissue that have high accuracy, reliability and/or sensitivity, that provide data in real-time, and/or that have high compatibility with existing NMR/MRI systems.

According to an aspect, there is provided a microfluidic culture apparatus comprising: an insertion module configured to be inserted into a magnet of a system for performing nuclear magnetic resonance, NMR, or magnetic resonance imaging, MRI; and a flow controller configured to drive flow of a culture medium in a first flow line and of a gas in a second flow line, wherein the insertion module comprises: a first passage forming at least part of the first flow line; a culture chamber connected in series at a position along the first passage and configured to contain a sample of living tissue; a second passage forming at least part of the second flow line; and a gas exchange arrangement configured to allow gas to pass from the second passage to the first passage in a gas exchange portion of the first passage.

The combination of the first and second passages with the gas exchange arrangement allows a sample of living tissue to be kept viable inside the magnet of an NMR/MRI system for a prolonged period despite the severe spatial constraints. At the same time, the volumes of fluids in the passages can be kept sufficiently low that meaningful changes in composition caused by metabolism in the sample can be detected with high sensitivity. Such measurements can be used to investigate various aspects of the biology of samples in the apparatus in real-time and/or in a quantitative manner. By controlling the composition of culture medium flowing through the culture chamber it is possible to evaluate the effect on samples of a range of external factors (such as a drug, an active chemical, a metabolite, a gas concentration, an infectious agent, a toxin, an environmental stressor, a genetic stressor, and/or other factors).

In some embodiments, the flow controller is configured to drive flow of the culture medium in a sequence comprising a plurality of measurement phases interleaved by refreshment phases. In each measurement phase, the culture medium is driven such that all positions along a respective longitudinally-extending measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least once. In each refreshment phase, the culture medium is driven such that all of the measurement portion from a directly preceding measurement phase is replaced by culture medium that did not encounter the culture chamber during the directly preceding measurement phase.

This configuration allows nutrients to be supplied to the sample of living tissue in a way that maintains sample viability while also allowing concentrations of metabolites to build up or diminish to an extent that makes them detectable. The provision of measurement phases interleaved by refreshment phases is advantageous over supplying a continual flow of refreshment media because in a continual flow changes in analyte concentrations may not be sufficient to be easily detectable.

The refreshment phase allows the sample to be regularly provided with fresh culture medium, which helps maintain viability. If the amount of flow during the refreshment phases is sufficiently high, the first flow line will effectively contain a sequence of portions of culture medium that will be involved in measurements (referred to as measurement portions) that are separated from each other by portions of the culture medium that will not be involved in measurements and serve merely as buffers. The provision of such buffers helps to reduce cross-contamination between different measurement portions, thereby improving the accuracy and reliability of the measurements. A series of separate experiments can be carried out without requiring a complicated or time-consuming reset of the system, whilst providing accurate and reliable results.

In some embodiments, in each measurement phase, the culture medium is driven such that all positions along a respective measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least twice. Passing the same measurement portion multiple times through the culture chamber promotes accumulation of a detectable change in concentration of analytes of interest.

In some embodiments, the gas exchange arrangement comprises a gas permeable membrane at a position adjacent to the gas exchange portion of the first passage; and the gas passes from the second passage to the first passage by diffusion through the gas permeable membrane. In an embodiment, portions of the first passage and the second passage adjacent to the gas permeable membrane are on the same side of the gas permeable membrane. This arrangement allows gas to be driven through the second passage at high pressures without the high pressures causing a deformation of the gas permeable membrane that could hinder or block flow of the culture medium in the first passage. In an embodiment, the apparatus comprises two of the gas permeable membranes and the portions of the first passage and the second passage are formed in a micro fluidic chip clamped between the two gas permeable membranes. This arrangement further facilitates management of high pressures in the second gas passage by reducing or preventing unwanted deformation of the gas permeable membranes. Providing two gas permeable membranes furthermore promotes efficient gas exchange between the second passage and the first passage by allowing gas to diffuse along parallel paths.

In some embodiments, the gas exchange arrangement is configured to allow gas to pass from the second passage to the first passage in two gas exchange portions of the first passage. The two gas exchange portions are positioned on opposite sides of the culture chamber. Providing gas exchange portions on both sides of the culture chamber helps to ensure sufficient gas exchange with the culture chamber to maintain viability of the sample. This is particularly applicable where the same measurement portion is passed multiple times through the culture chamber by repeatedly switching a direction of flow in the first flow line (thereby passing each portion of the same measurement portion backwards and forwards multiple times through the culture chamber).

In some embodiments, a liquid immiscible with the culture medium is provided in series with the culture medium in the first flow line. The immiscible liquid may be used as a hydraulic transmission liquid to reduce the volume of culture medium needed in the first flow line. This may be particularly desirable where the culture medium contains expensive components or components in limited supply, such as drug candidates. Alternatively or additionally, the immiscible liquid may be provided such that one or more of the measurement portions are separated from one or more other measurement portions by the immiscible liquid. The immiscible liquid is thus used to isolate different measurement portions or groups of measurement portions from each other. Isolating measurement portions from each other using immiscible liquid further decreases the risk of cross-contamination between different measurement portions. The separated measurement portions may have different compositions relative to each other, thus allowing different experiments to be performed easily.

In some embodiments, the culture chamber is positioned to allow NMR measurements to be performed on material in the culture chamber when the insertion module is in the magnet. In some embodiments, the apparatus further comprises an analysis chamber connected in series at a position along the first passage different from the position of the culture chamber, wherein the analysis chamber is positioned to allow NMR measurements to be performed on material in the analysis chamber when the insertion module is in the magnet. Thus, NMR measurements can be carried out directly on material in the culture chamber and/or the analysis chamber. Analyses may be performed continuously without compromising the viability of the tissue and/or requiring the material for analysis to be removed from the culture system and/or subject to conditions that are outside the design of an experiment being performed.

In some embodiments, the apparatus further comprises a temperature control arrangement configured to control a temperature in the culture chamber, wherein the temperature control arrangement comprises a reservoir configured to contain a liquid or gas in thermal contact with the culture chamber. This allows in-situ temperature control when the apparatus is inserted into the magnet with minimal risk of interference with NMR techniques.

In some embodiments, the insertion module comprises a stack of two or more submodules clamped together by a cylindrical clamping body, the clamping body being moveable along the longitudinal axis relative to the stack of sub-modules, the movement causing engagement with a ramp member that provides a radially inwards clamping force. The cylindrical shape of the device allows interfacing with commercial probes of cylindrical shape. The radially inwards clamping force mediated by the ramp member allows a controlled sealing force to be applied, allowing reliable and reproducible sealing of fluidic connections with minimal risk of damage.

According to a further aspect, there is provided a method of performing measurements involving live tissue, the method comprising: using the microfluidic culture apparatus of any the disclosed embodiments to perform measurements involving live tissue while the insertion module is in a magnet of a system for performing NMR or MRI, the live tissue being in the insertion module and the measurements comprising performing NMR or MRI.

According to a further aspect, there is provided a method of performing measurements involving live tissue in a magnet of a system for performing nuclear magnetic resonance, NMR, or magnetic resonance imaging, MRI, the method comprising: positioning an insertion module in a magnet of a system for performing NMR or MRI, wherein the insertion module comprises a first passage, a culture chamber containing a sample of living tissue, and a second passage; driving flow of a culture medium in a first flow line including the first passage; driving flow of a gas in a second flow line including the second passage; and performing measurements using the system for performing NMR or MRI, wherein: the insertion module is configured to allow gas to pass through a gas permeable member from the second passage to the first passage in a gas exchange portion of the first passage.

Arrangements of the disclosure will be further described by way of example only with reference to the accompanying drawings. Figure 1 depicts a micro fluidic culture apparatus with an insertion module inserted into a magnet of a system for performing NMR or MRI;

Figure 2 is an exploded perspective view of an example insertion module comprising a stack of two or more sub-modules clamped together by a cylindrical clamping body configured to be rotated;

Figure 3 is an exploded perspective view of a further example insertion module comprising a stack of two or more sub-modules clamped together by a cylindrical clamping body configured to be moved axially without rotation;

Figure 4 schematically depicts interconnection of functional elements of a microfluidic culture apparatus including a temperature control arrangement;

Figure 5 is an exploded view of a portion of an insertion module including a microfluidic chip and gas permeable membranes;

Figure 6 is a side view of an micro fluidic chip configuration in which the first passage defines a circuit and flow is controlled by actuation of valves;

Figure 7 depicts multiple views of an example holder unit of the insertion module;

Figure 8 is a transparent exploded view of an example insertion module comprising two holder units clamping a micro fluidic chip between two gas permeable membranes;

Figure 9 is an opaque exploded view of the insertion module of Figure 8;

Figure 10 depicts a micro fluidic culture apparatus configured to supply a contrast agent to the sample;

Figure 11 depicts NMR spectra obtained from a tissue slice sample cultured inside an NMR magnet for up to 20 hours;

Figure 12 depicts analysis of D-Glucose concentration using 1H NMR spectra with tissue slice culture for more than 20 hours;

Figure 13 depicts analysis of L- Alanine, L-Glutamine, and Acetic acid concentration using 1H NMR spectra with tissue slice culture for more than 20 hours;

Figure 14 depicts 1H NMR spectra obtained from a tissue slice cultured inside an NMR magnet for up to 42 hours;

Figure 15 depicts analysis of D-Glucose concentration using 1H NMR spectra with tissue slice culture for more than 42 hours; Figure 16 depicts analysis of L- Alanine, L-Glutamine, and Acetic acid concentration using 1H NMR spectra with tissue slice culture for more than 42 hours; and

Figure 17A-H depict further measurements obtained using the micro fluidic culture apparatus.

As exemplified in Figure 1, the present disclosure relates to a micro fluidic culture apparatus comprising a flow controller 102 and an insertion module 101.

The flow controller 102 drives flow of a culture medium in a first flow line 103. The flow controller 102 drives flow of a gas in a second flow line 104. The flow controller 102 may comprise any suitable combination of data processing hardware, firmware and/or software, electronics, power supply hardware, pumps, valves, conduits, sensing apparatus, and/or any other apparatus necessary to provide the functionality described.

The insertion module 101 comprises a culture chamber 105 for containing a sample of living tissue. The culture chamber 105 may be defined in a micro fluidic chip of the insertion module 101. The sample may be a precision cut tissue slice (PCTS), for example a precision cut liver slice or a precision cut brain slice. In some embodiments, the insertion module 101 further comprises an analysis chamber 106, which may also be defined in the micro fluidic chip.

The insertion module 101 is configured (e.g., shaped and dimensioned) to be inserted into a magnet of a system for performing NMR or MRI. NMR techniques include NMR imaging and NMR spectroscopy. At least a portion of the insertion module 101 is therefore small enough to enter the bore of the magnet. At least the culture chamber 105, portions of the first and second passages (described below), and where present the analysis chamber 106, will be located on the portion of the insertion module 101 that enters the bore. The insertion module may consist of a single unit or a plurality of sub-units. Where a plurality of sub-units are provided, the first and second passages may be formed in one or more than one of the sub-units. In some embodiments, all of the microfluidic chip of the insertion module 101 enters the bore. Figure 1 depicts an example insertion module 101 in place within the magnet of an NMR spectrometer.

In some embodiments, the insertion module 101 is configured such that the culture chamber 105 will be positioned to allow measurements to be performed on material in the culture chamber when the insertion module 101 is in the magnet of the NMR or MRI system. For example, the positioning may be such that NMR imaging and/or spectroscopy can be performed on a sample in the culture chamber 105. Alternatively or additionally, MRI scanning may be performed on the sample in the culture chamber 105. In some embodiments, the insertion module 101 is configured such that an analysis chamber 106 will be positioned to allow NMR measurements (e.g., NMR spectroscopy) to be performed on material in the analysis chamber 106 when the insertion module 101 is in the magnet.

The insertion module 101 comprises a first passage 103 a. The first passage 103 a may be defined at least partly in the same micro fluidic chip as the culture chamber 105. The first passage 103 a forms at least part of the first flow line 103. The first passage 103 a may thus be in series with a portion of the first flow line 103 that is outside the insertion module 101. In some embodiments, the first passage 103 a forms the entirety of the first flow line 103 (e.g., where the first passage 103a forms a closed circuit within the insertion module 101).

The culture chamber 105 is connected in series at a position along the first passage 103a. Liquid may thus flow in sequence through a first portion of the first passage 103a, then through the culture chamber 105, and then through a second portion of the first passage 103 a. The analysis chamber 106 is connected in series at a position along the first passage 103a that is different from the position of the culture chamber 105. Thus, material for analysis can pass from the culture chamber 105 to the analysis chamber 106 via a flow of material along the first passage 103 a.

NMR and/or MRI measurements allow the anatomy and physiology of a sample of tissue to be studied. NMR imaging provides good contrast in soft tissue and does not require exposure to ionising radiation. NMR images are maps of the nuclear spin (usually 1H) density inside a tissue, weighted by parameters of the spin Liouvillian, such as relaxation times and/or chemical shift. NMR imaging can be used to map a distribution of metabolites in tissue. NMR imaging of a sample in the culture chamber 105 can thus provide information about metabolite distribution in the sample and/or changes in biological processes in the sample. The NMR may be used to observe changes in the metabolome in response to exposure of the sample to environmental and/or genetic stresses or other external stimuli, such as may be provided by a drug, an active chemical, a metabolite, a gas concentration, an infectious agent, a toxin, an environmental stressor, a genetic stressor, and/or other factors.

As the sample of living tissue carries out biological processes, metabolites accumulate in the portion of culture medium surrounding and/or perfusing the tissue. In some embodiments, a flow in the first passage 103a can move culture media that has been in contact with the tissue from the culture chamber 105 to the analysis chamber 106. Thus perfusate (culture media) comprising metabolites produced by the sample can be transferred to the analysis chamber 106. In the analysis chamber 106, the metabolic composition of the perfusate (culture media) after it has been in contact with the sample can be assessed. NMR spectroscopy of material in the analysis chamber 106 can be used to monitor concentrations of one or more analytes in the material. For example, NMR spectroscopy can monitor changes in the concentration of metabolites in the perfusate during a culture period and/or during the period of an experiment. For example, changes in the concentration of Lactic acid (L-lactic acid), Alanine (L-Alanine), Acetic acid, Glutamine (L-Glutamine), HEPES, Glucose (D-Glucose), Glutamate, aromatic amino acids (Phenylalanine, Tyrosine, and/or Tryptophan), and/or other metabolites can be monitored. The metabolic activity of a sample of living tissue inside an NMR magnet can thus be monitored in real-time. The metabolic activity may be continuously or intermittently monitored. Two or more metabolites may be analysed simultaneously or at different times. The NMR spectroscopy can thus provide a detailed quantitative analysis of metabolic activity of the sample.

In some embodiments, an NMR spectrometer is configured to accommodate the insertion module 101 by provision of a modified sample tube holder or modified transmission line NMR microprobe. Details of such an arrangement are provided for example in Sharma & Utz, J. Magn. Reson. 303 (2019) 75-81 “Modular transmission line probes for microfluidic nuclear magnetic resonance spectroscopy and imaging”. A planar insertion module 101 may be inserted into such a modified sample tube holder or transmission line microprobe. The modified transmission line microprobe can be compatible with a commercial gradient setup for MR imaging and other MR techniques that rely on pulsed field gradients. In other embodiments, the insertion module 101 is configured to be insertable into the magnet of a standard NMR or MRI system without modification. For example, an NMR spectrometer may accommodate the insertion module 101 in an unmodified sample tube holder or in an unmodified NMR probe, for example a commercial probe of cylindrical shape. An unmodified NMR spectrometer may comprise an existing commercial liquid state NMR sample exchange setup. Direct compatibility with such existing NMR/MRI systems improves convenience and/or reduces implementation cost.

In some embodiments, as exemplified in Figures 2 and 3, the insertion module 101 comprises a stack of two or more sub-modules. The sub-modules may be clamped together using a cylindrical clamping body 117. The clamping body 117 may be configured to be moveable along the longitudinal axis relative to the stack of sub-modules. The insertion module 101 may be configured such that the movement will cause engagement with a ramp member 115 that provides a radially inwards clamping force.

In the particular example of Figures 2 and 3, the sub-modules of the insertion module 101 include a holder unit 111, a first gas permeable membrane 112 (e.g., formed from PDMS), a microfluidic chip 113, a second gas permeable membrane 114 (e.g., formed from PDMS), a ramp member 115, a ramp engagement member 116 and the clamping body 117. The holder unit 111 defines ports 130 to allow fluidic connections to be made from the exterior of the insertion module 101 to conduits within the insertion module 111, such as to the first passage 103a, second passage 104a, and/or any other passages that might be needed in the insertion module 101. The ports 130 and associated connections and passages are described in further detail below with reference to Figures 7- 9.

The cylindrical shape of the clamping body 117 ensures that an outer shape of a portion of the insertion module 101 that will be inserted into the magnet is cylindrical and thereby provides compatibility with magnets of commercial NMR/MRI systems, even if the micro fluidic chip 113 is planar. In other embodiments, the micro fluidic chip 113 may be provided at least partially in cylindrical form, for example using 3D printing techniques. In the example of Figure 2, the clamping body 117 is rotatable about a longitudinal axis of the clamping body 117 and is configured such that the rotation will cause the clamping body 117 to move along the longitudinal axis and cause the engagement with the ramp member 115. In this example, the functionality is achieved by providing angled slots 121 on opposite sides of the clamping body 117. The slots 221 engage with respective protrusions 124 and 125 on the holder unit 111 and ramp engagement member 116 on opposite sides of the stack. The protrusion 124 is fixed in place such that when the protrusion 124 rides up the respective slot 221 during rotation of the clamping body 117 the clamping body 117 is forced to move axially. The axial movement pushes the ramp engagement member 116 to move axially and engage against the ramp member 115 to provide the radially inward clamping force.

In the example of Figure 3, the clamping body 117 is configured to be moved along the axis directly without rotation. The axial movement again causes a corresponding movement of the ramp engagement member 116, which engages against the ramp member 117 to provide the radially inward clamping force.

Both of the arrangements of Figures 2 and 3 have been found to reliably and reproducibly apply suitable clamping forces to the internal elements of the insertion module 101, achieving high quality fluidic sealing and minimizing the risk of damage. A further approach to providing the clamping is described below with reference to Figures 7- 9.

Referring again to Figure 1 and to Figures 4-6, the insertion module 101 further comprises a second passage 104a. The second passage 104a may be defined at least partly in the same micro fluidic chip 113 as the culture chamber 105. The second passage 104a forms at least part of the second flow line 104. The second passage 104a may thus be in series with a portion of the second flow line 104 that is outside the insertion module 101. The insertion module 101 further comprises a gas exchange arrangement configured to allow gas to pass from the second passage 104a to the first passage 103a in a gas exchange portion 103b of the first passage 103a. Components from the gas may then be carried to the culture chamber 105 by flow of culture media in the first passage 103a. The concentration of components such as oxygen and carbon dioxide in the culture chamber 105 can thereby be controlled to maintain sample viability and/or modify the environment to perform experiments, such as to simulate disease states.

Gas is delivered to the second passage 104a by providing a flow of gas in the second flow line 104. Various compositions of the gas are possible depending on the nature of the sample and the experiment to be performed. For the purposes of maintaining sample viability, the gas will typically comprise a mixture of oxygen and carbon dioxide. The gas may for example comprise 80% oxygen and 5% carbon dioxide, or 95% oxygen and 5% carbon dioxide. Other gases may additionally or alternatively be included in the second flow line 104. For example argon may be introduced to deliberately restrict a supply of oxygen to the sample.

In some embodiments, the gas exchange arrangement comprises a gas permeable member 112/114. The gas permeable member 112/114 is provided at a position adjacent to the gas exchange portion 103b of the first passage 103a. The gas permeable member 112/114 is provided at a position adjacent to the second passage 104a. The gas permeable member 112/114 may be a gas permeable membrane. The gas may pass from the second passage 104a to the first passage 103a by diffusion through the gas permeable membrane. The gas permeable member 112/114 may comprise a PDMS (polydimethylsiloxane) membrane. A PDMS membrane is permeable to oxygen and carbon dioxide.

In some embodiments, portions of the first passage 103a and the second passage 103b adjacent to the gas permeable membrane 112/114 are on the same side of the gas permeable membrane 112/114. Thus, the gas permeable membrane 112/114 is provided to one side of the portions of the first passage 103a and the second passage 103b rather than directly between them. This approach has been found to allow gas to be provided at higher pressure in the second passage, which promote efficient gas exchange, without the higher pressure causing material of the gas permeable membrane 112/114 to deform and be caused to protrude into the first passage, which could hinder or block flow of the culture medium. In some embodiments, as exemplified in Figures 2, 3, 5, 8 and 9, the apparatus comprises two of the gas permeable membranes 112/114. Providing two of the gas permeable membranes provides parallel paths for gas diffusion, thereby promoting efficient gas exchange. In an embodiment, the portions of the first passage 103a and the second passage 103b are formed in a micro fluidic chip clamped between the two gas permeable membranes 112/114. This approach further facilitates handling of high pressures in the second passage because the clamping helps to control deformation of the gas permeable membrane material by the pressurized gas.

In some embodiments, the flow controller 102 drives flow of a culture medium in a sequence comprising a plurality of measurement phases interleaved by refreshment phases.

In each measurement phase, the culture medium is driven such that all positions along a respective longitudinally-extending measurement portion of the culture medium in the first flow line 103 pass through the culture chamber 105 and the gas exchange portion 103b at least once. In some embodiments, in each measurement phase, the culture medium is driven such that all positions along a respective measurement portion in the first flow line 103 pass through the culture chamber 105 and the gas exchange portion 103b at least twice. This may be achieved for example by switching a direction of flow in the first flow line 103 at least once in each measurement phase. Thus, the flow controller 102 will drive flow in one direction in the first flow line 103 and, after the switch, in the opposite direction. The switching in direction may be repeated any number of times during each measurement phase, to provide a back and forth, alternating flow. In some embodiments, the flow is driven in a balanced way such that a total flow in one direction is substantially equal to a total flow in the opposite direction during each measurement phase. The amplitude of the flow determines the volume of the measurement portion (and the length of the measurement portion along the first flow line) in each measurement phase. It is desirable for the volume of the measurement portion to be kept as small as possible consistent with maintaining sample viability to facilitate detection of metabolites. In some embodiments the volume of each measurement portion is of a similar order of size as the volume of the first passage 103a (e.g., as the volume of the portion of the first flow line 103 that is defined by the micro fluidic chip 113). For example, the volume of each measurement portion may be in the range of 50%-200% of the volume of the first passage 103a, preferably 50%-200% of a volume of the first passage 103a defined in a micro fluidic chip 113 of the insertion module 101. Independently of the volume of the first passage 103a, it is desirable for the volume of each measurement portion to be less than about 1 OOpl, optionally less than about 50pl, optionally less than 30p.l, optionally about 25pl.

In each measurement phase, portions of the measurement portion of culture medium that have contacted the sample in the culture chamber 105 travel along the first passage 103a to the gas exchange portion 103b. At the gas exchange portion 103b, gas flows from the second passage 104a to the culture medium in the first passage 103a. Levels of components such as oxygen that have been depleted by metabolic activity of the sample may thus be replenished in the gas exchange portion 103b of the first passage 103a. The portion of the measurement portion that has been thus replenished then travels back to the sample with the reversal of the flow in the first passage 103a and thereby replenishes the environment in the culture chamber 105 and thereby promotes maintenance of sample viability.

In each refreshment phase, the culture medium is driven such that all of the measurement portion from a directly preceding measurement phase is replaced by culture medium that did not encounter the culture chamber during the directly preceding measurement phase. A fresh portion of culture medium is thus provided for use in a subsequent measurement phase. The measurement portion from a directly preceding measurement phase (which may comprise metabolites from the sample of living tissue and/or depleted nutrients and/or an altered gas composition) is displaced along the first passage 103a far enough that it will not interfere with the subsequent measurement phase. The measurement portion from a directly preceding measurement phase may be flushed partly or completely out of the first passage 103a (i.e., out of the insertion module 101). A volume of the culture medium driven through the culture chamber 105 during each refreshment phase will typically be at least as large as the volume of each measurement portion, preferably significantly larger, such as twice as large.

Providing measurement phases interleaved by refreshment phases in this manner allows for nutrients to be supplied to the sample in a way that maintains sample viability while also allowing concentrations of metabolites to build up or diminish to an extent that makes them detectable. The approach facilitates use of measurement portions having very small volumes. In some embodiments, a liquid immiscible with the culture medium is provided in series with the culture medium in the first flow line 103. The immiscible liquid is a liquid that does not mix and/or react with the measurement portion of the culture medium. The immiscible liquid may be a biocompatible substance, such as a biocompatible oil. The immiscible liquid may be a fluorinated oil for example.

The immiscible liquid may be used as a hydraulic transmission liquid to reduce the volume of culture medium needed in the first flow line 103. The immiscible liquid may thus be provided in a portion of the first flow line 103 between the first passage 103 a and a pump used to drive flow in the first flow line 103.

Alternatively or additionally, the immiscible liquid may be provided such that one or more of the measurement portions are separated from one or more other measurement portions by the immiscible liquid. The immiscible liquid may thus be used to isolate different measurement portions or groups of measurement portions from each other. In some embodiments an alternating sequence of the immiscible liquid and the culture medium is provided in the first flow line 103 with each section of culture medium comprising one or more of the measurement portions.

In some embodiments, at least a subset of the measurement portions are different portions of a single longitudinally continuous body of culture medium in the first flow line 103. These measurement portions are not therefore separated from each other by any immiscible liquid. This approach is simple to implement and as long as the passages in the vicinity of the sample are sufficiently narrow, cross contamination from diffusion along the passages will be low enough that each measurement portion can represent an independent experiment. Typically, it will be desirable for the cross-sectional area of the first passage 103a to be less than about 100 pm x 100 .m. In one implementation, the cross section of the first passage 103a is 50 pm by 100 pm. Portions of the flow line 103 outside of the insertion module 101 should also be narrow, particularly where they are close to the insertion module 101 and where the volumes of the measurement portions are comparable with or greater than the volume of the first passage 103a. The cross-sectional area of such portions of the flow line 103 may also therefore be less than about 100pm x 100 p.m. In one class of embodiment, the portions have cross-sections with diameters in the range of 40 pm to 80 pm.

In some embodiments, as exemplified in Figure 4, gas exchange is provided in two (or more) separate gas exchange portions 103b. Gas passes from the second passage 104a to the first passage 103 a in each of the gas exchange portions 103b. In some embodiments, separate gas exchange portions 103b are positioned on opposite sides of the culture chamber 105. Thus, culture medium will always be flowing into the culture chamber 105 from a respective gas exchange portion 103b regardless of the direction of flow. This arrangement promotes consistent and effective oxygenation in embodiments where the flow direction is periodically switched in the first flow line 103.

In some embodiments, as exemplified in Figure 4, the apparatus comprises a pump 150 and a backpressure source 151 (e.g., at about 1.2 bar; not shown) acting from opposite ends of the first passage 103a. The nature of the pump 150 is not particularly limited. The pump 150 may comprise a syringe pump (having a volume of about 3ml for example) or a peristaltic pump. The pump 150 may comprise plural separate pumping arrangements, as depicted schematically in Figure 1. For example, one pumping arrangement could be configured to provide back and forth flow in the first flow line 103 and a separate pumping arrangement (e.g., a syringe pump) could be configured to input fresh culture medium into the first flow line 103. The flow controller 102 drives the flow by controlling the pump 150.

In embodiments where the flow direction is switched, at least two separate openings to the first passage 103 a will be provided, allowing liquid to enter and leave the first passage 103a at different points. The embodiment of Figure 5 exemplifies a micro fluidic chip 113 compatible with such an arrangement, with separate ports 132b and 132c leading to the portion of the first passage 103a defined in the micro fluidic chip 113. Further details are given below with reference to Figures 7-9.

In other embodiments, as exemplified in Figure 6, the first passage 103a may be arranged to form a circuit within the insertion module 101. Each measurement portion of the culture medium may then be circulated in the circuit during the respective measurement phase. The insertion module 101 may comprise a plurality of valves 108 (indicated by small circles in Figure 6) to control flow in the circuit. The valves 108 may be configured to drive circulation of the culture medium. In some embodiments, the valves 108 are actuated by changing a gas pressure adjacent to the valve 108. The change in the gas pressure causes a change in shape of a deformable member of the valve 108. The deformable member may be formed by a portion of a gas permeable membrane 112 or 114 (see below). The change in shape of the deformable member exerts a driving force on the culture medium. The flow controller may drive the circulation by actuating the valves 108 at selected times. The valves 108 may be actuated in sequence along a direction of flow, with adjacent valves 108 being actuated one after the other in sequence. At least a subset of the valves 108 are typically actuated at different times. In some embodiments, two or more of the valves 108 may be actuated at the same time. In some embodiments, the actuation of the valves 108 at selected times provides a peristaltic pumping action.

Referring again to Figure 4, in some embodiments the apparatus comprises a temperature control arrangement 160 that controls a temperature in the culture chamber 105. The temperature control arrangement 160 may comprise a reservoir containing a liquid and/or gas in thermal contact with the culture chamber 105. For example, water at a specified temperature may be held or flowed through a region in thermal contact with the culture chamber 105. Heat exchange between the culture chamber 105 and the reservoir allows the culture chamber 105 to be heated or cooled as required. The sample may thus be maintained at a physiologically appropriate temperature, for example at about 37 degrees Celsius.

In some embodiments, the temperature control arrangement 160 drives a flow of liquid or gas through the reservoir and controls the temperature of the liquid or gas. A temperature sensor 161 may be provided in thermal contact with the culture chamber 105. The temperature sensor 161 may comprise a thermocouple or thermistor for example. The temperature control arrangement 160 may use an output from the temperature sensor 161 to control the temperature of the culture chamber 105. The temperature control arrangement 160 may for example control the temperature and/or rate of flow of liquid or gas through the reservoir in response to the output from the temperature sensor 161. A readout from the temperature sensor 161 may be used to control a heater for heating a liquid or gas driven through the reservoir.

In some embodiments, the liquid reservoir forms part of the insertion module 101. The liquid reservoir may be provided in a holder unit 111 of the insertion module 101 for example. An example configuration of this type is described below with reference to Figures 7-9.

Figure 7 depicts multiple views of an example holder unit 111 of the insertion module. The holder unit 111 defines a reservoir for temperature control. Figures 8 and 9 are exploded views showing how the holder unit 111 of Figure 7 can form an insertion module 101. In the example shown, two instances of the holder unit 111 are used but this is not essential. The holder units 111 are arranged to clamp the micro fluidic chip 113 between gas permeable membranes 112 and 114. Channels 135 are provided for inserting fastening elements to provide a clamping force to seal fluidic connections and/or valves. The sealing membranes (gas permeable membranes 112 and 114) may be formed from any suitably soft and sealing material, such as PDMS. The sealing membranes (gas permeable membranes 112 and 114) may be configured to operate valves and to seal fluidic connections.

The holder unit 111 comprises ports 134a, 134b for input and output of temperature-controlled liquid to the reservoir. The reservoir may comprise a channel defined in the holder unit 111. The channel may extend from port 134a to port 134b for example. Openings to the ports 134a, 134b are provided on an obliquely inclined surface for ease of access.

Ports 130a-d may be provided to allow fluidic connections to portions of the first flow line 103, second flow line 104, and/or any other flow lines inside the insertion module 101 as mentioned above. In the example shown, ports 130b and 130c are connected to opposite ends of the first passage 103 a and ports 130a and 130d are connected to opposite ends of the second passage 104a.

Referring to Figures 8 and 9 particularly, driving flow in the first passage 103 a may cause culture medium to enter the first passage 103a through port 130b and exit the first passage 103a via port 130c. The flow of culture medium from the port 130b passes through opening 131b in the holder unit 111, through passage 132b in gas permeable membrane 112 and into the portion of the first passage 103 a in the micro fluidic chip 113 via port 133b. The flow exits the micro fluidic chip 113 via port 133c, passes through passage 132c in gas permeable membrane 112, enters the holder unit 111 via opening 131c in the holder unit 111, and exits the holder unit 111 via port 130c. The flow passes through these elements in the opposite sense when the flow is reversed. In one implementation, port 130b is connected to a supply of the culture medium (e.g., a syringe pump containing the culture medium) and port 130c is connected to a waste line for disposal of used culture medium.

Driving flow of gas in the second passage 104a may cause gas to enter the second passage 104a through port 130a and exit the second passage 104a through port 130d. The flow of gas from the port 130a passes through opening 13 la in the holder unit 111, through passage 132a in gas permeable membrane 112 and into the portion of the second passage 104a defined in the micro fluidic chip 113 via port 133a. The flow exits the micro fluidic chip 113 via port 133d, passes through passage 132d in gas permeable membrane 112, enters the holder unit 111 via opening 13 Id in the holder unit 111, and exits the holder unit 111 via port 130d. The flow passes through these elements in the opposite sense when the flow is reversed. In one implementation, port 130a is connected to a gas supply (e.g., supplying carbogen) and port 130d is connected to an outflow line.

Additional ports may be provided in the holder unit 111 to allow connections to be made to a compressed air line, for example to allow actuation of valves.

Figure 10 depicts a further example of a microfluidic culture apparatus. The apparatus has a culture chamber 105 containing a sample 109 of living tissue. The apparatus may take any of the forms discussed above. The culture chamber 105 may be closed in this example using a lid member. The lid member may be removable. The lid member may be held in place by a fastening member such as adhesive tape (e.g., optical tape).

The apparatus 100 may be operated in such a manner as to ensure that the lid member is not displaced relative to the culture chamber 105. For example, a flow rate through the culture chamber 105 and first flow line 103 may be controlled so as not to exceed a predetermined maximum rate. The flow rate may be kept equal to or less than 30pl/min for example, preferably equal to or less than 20pl/min. Steps may be taken to avoid the build-up of hydrostatic pressure at the culture chamber 105. This may be achieved for example by arranging for an outflow portion of the first flow line 103 to have a relatively short length and/or to be directed in an upwards direction when the insertion module 101 is inserted into the magnet of the NMR/MRI system.

Air bubbles generate magnetic susceptibility artefacts that can degrade the quality of NMR images. In some embodiments, as exemplified in Figure 10, additional subchambers 105a are provided on either or both sides of the culture chamber 105. The volumes of the sub-chambers 105a are configured to discourage bubble formation in the culture chamber 105. In the event that bubbles do form in a first flow line 103 during operation, the bubbles may be displaced from the culture chamber 105, for example into the sub-chambers 105a, by tapping on the insertion module 101.

It is known to use contrast agents in NMR and MRI techniques to enhance certain features in the collected data and improve the quality of the images. The embodiment of Figure 10 provides an exemplary set up which may be used to supply contrast agent to the culture chamber 105. The set up may be used with any of the configurations of the insertion module 101 described above with reference to Figures 1-9. Two extrusion devices (170a, 170b), for example syringes, are provided. The first extrusion device 170a is filled with culture medium and the second extrusion device 170b is filled with contrast agent dissolved in culture media. The extrusion devices 170a, 170b are fluidically connected to one another and to the rest of the apparatus by means of a “Y” junction 171. The “Y” junction 171 fluidically connects the extrusion devices 170a, 170b to the first flow line 103. A mixture of the culture medium and the contrast agent dissolved in culture medium can thus be provided to the culture chamber 105. The flow of the culture medium and the flow of the contrast agent dissolved in culture medium may be controlled independently. For example, the relative flow rate of each of the two solutions may be changed. The flow rate of one solution may be changed whilst the flow rate of the other solution is kept the same. The flow rate of both solutions may be changed. Thus, the concentration of the contrast agent provided to the culture chamber 105 can be adjusted. The concentration of contrast agent provided during an experiment may be maintained at the same level throughout the experiment. The concentration of contrast agent provided during an experiment may be varied during the experiment. The concentration of the contrast agent can be changed in real-time. Images of the sample of living tissue (909) can thus be acquired with different contrast agent concentrations.

A range of methods may be provided that make use of the configurations described above. The methods may comprise performing measurements involving live tissue. The measurements may be made directly on tissue in the culture chamber 105 and/or on material in a separate analysis chamber 106. The methods may comprise using the apparatus of any of the embodiments described above to perform the measurements while the insertion module 101 is in a magnet of an NMR or MRI system.

The methods may comprise positioning the insertion module 101 in the magnet, providing the flow of culture medium in the first flow line 103 and first passage 103a, providing the flow of gas in the second flow line 104 and second passage 104a. The measurements may be performed using the NMR or MRI system.

In some embodiments, a composition of either or both of the culture medium in the first flow line and the gas in the second flow line is controlled to apply a controlled stimulus to the sample. The controlled stimulus may comprise simulating one or more of the following in any combination: a disease process; administration of a therapy; administration of a drug; administration of a drug candidate; administration of an infectious agent; administration of a toxin. The methods may further comprise observing the effect of the controlled stimulus on the material in the culture chamber 105.

Demonstrations of Performance

Experimental results are described below that demonstrate the capability of the apparatus to keep tissue viable and to extract continuously quantitative information on the consumption/production rates of several (up to 20) key metabolites simultaneously and non-invasively.

Figure 11 shows proton NMR spectra obtained from a mouse liver tissue slice sample using an example implementation of the microfluidic culture apparatus. Several analytes like D-Glucose, L- Alanine, L-Lactic acid, and Acetic acid are identified within the spectra. The inventors observed the change of L-Alanine, L-Lactic acid, and Acetic acid within a culture period of 20 hours. However, D-Glucose concentration remains unchanged during this time. The obtained spectra show excellent stability and resolution over the entire experiment time.

Figure 12 shows an analysis of the D-Glucose concentration in a tissue slice sample inside an example implementation of the microfluidic culture apparatus. The tissue slice was cultured for more than 20 hours. The culture medium was refreshed in a refreshment phase every 3.5 hours. Within two refreshments of the medium, tissue was put under a back and forth pumping of a 25 pL measurement portion of culture medium. During the back and forth pumping the sample was supplied with high oxygen content by provision of a suitable flow rate (8pL/min in this case) and the gas exchange arrangement described above with gas exchange portions 103b on both sides of the culture chamber 105. Simultaneously, the low volume of the culture medium (25 pL) enabled detection of the concentration change of the D-Glucose with high sensitivity.

Figure 13 shows monitoring of concentration changes of L- Alanine, L-Glutamine, and Acetic acid. L- Alanine and L-Glutamine are consumed during the back and forth pumping, whereas Acetic acid is produced up to 12 hours into the culturing.

Figure 14 shows ¹ H NMR spectra obtained using the micro fluidic culture apparatus from a tissue slice sample cultured in the NMR magnet for up to 42 hours. Several analytes like D-Glucose, L- Alanine, L-Lactic acid, and Acetic acid are identified within the spectra. The inventors observed the change of L-Alanine, L-Lactic acid, and Acetic acid within the culture period of 42 hours. First half of the experimental time (up to 22 hours), the tissue is supplied with gas mixture of Oxygen (95%) and CO2 (5%). In the second half of the experimental time, Argon is used to restrict the supply of Oxygen.

Figure 15 shows an analysis of the D-Glucose concentration in the tissue slice cultured inside the NMR magnet for more than 42 hours. The culture medium was refreshed every 3.5 hours. Between two refreshments of the medium, tissue is put under the back and forth pumping of a 25 pL measurement portion of culture medium. In the first half of the experimental time, when the tissue is supplied with oxygen and CO2, the inventors observed a significant consumption of D-Glucose during the back and forth pumping. Once the oxygen supply is removed with Argon, Glucose consumption is reduced significantly.

Figure 16 is a graph showing analysis of L- Alanine, L-Glutamine, and Acetic acid concentration using NMR spectra with a tissue slice sample cultured for more than 42 hours. L-Alanine and L-Glutamine are consumed during the back and forth pumping, whereas Acetic acid is produced up to 16 hours into the culturing. Once the oxygen supply is removed with Argon, the inventors observed production of the Acetic acid, L-Glutamine and L- Alanine.

Figures 17A-H are graphs showing the results of further measurements performed using a microfluidic culture apparatus according to an embodiment of the disclosure.

Figure 17A shows measurements of a concentration of D-Glucose as a function of time as determined from the NMR spectra obtained continuously from the same liver tissue slice over the course of 24h. Linear fits (shown by the superimposed straight lines) give the consumption rate during each episode of 3.5h, after which the culture medium was refreshed. Figure 17B shows corresponding consumption rates in each episode with 90% confidence intervals. Corresponding data is shown respectively in Figures 17C and 17D for Acetic acid, in Figures 17E and 17F for Glutamine, and in Figures 17G and 17H for L-Tyrosine. In total, time profiles of 22 different metabolites have been extracted from the NMR data in this way. The data show that production/consumption rates on the order of 0.01 mM/h can be reliably quantified from a single tissue slice.

Further disclosed embodiments

Embodiments are defined in the following numbered clauses.

1. A micro fluidic culture apparatus comprising: an insertion module configured to be inserted into a magnet of a system for performing nuclear magnetic resonance, NMR, or magnetic resonance imaging, MRI; and a flow controller configured to drive flow of a culture medium in a first flow line and of a gas in a second flow line, wherein the insertion module comprises: a first passage forming at least part of the first flow line; a culture chamber connected in series at a position along the first passage and configured to contain a sample of living tissue; a second passage forming at least part of the second flow line; and a gas exchange arrangement configured to allow gas to pass from the second passage to the first passage in a gas exchange portion of the first passage.

2. The apparatus of clause 1, wherein: the flow controller is configured to drive flow of the culture medium in a sequence comprising a plurality of measurement phases interleaved by refreshment phases; in each measurement phase, the culture medium is driven such that all positions along a respective longitudinally-extending measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least once; and in each refreshment phase, the culture medium is driven such that all of the measurement portion from a directly preceding measurement phase is replaced by culture medium that did not encounter the culture chamber during the directly preceding measurement phase.

3. The apparatus of clause 2, wherein: in each measurement phase, the culture medium is driven such that all positions along a respective measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least twice.

4. The apparatus of clause 2 or 3, wherein the volume of each measurement portion is less than 100|fl.

5. The apparatus of any of clauses 2-4, wherein a liquid immiscible with the culture medium is provided in series with the culture medium in the first flow line.

6. The apparatus of any of clauses 2-4, wherein at least a subset of the measurement portions are different portions of a single longitudinally continuous body of culture medium in the first flow line.

7. The apparatus of any of clauses 2-6, wherein the volume of each measurement portion is in the range of 50%-200% of the volume of the first passage, preferably 50%- 200% of a volume of the first passage defined in a microfluidic chip of the insertion module.

8. The apparatus of any of clauses 2-6, wherein a volume of culture medium driven through the culture chamber in the first flow line during each refreshment phase is larger than the volume of each measurement portion, preferably at least twice as large.

9. The apparatus of any preceding clause, wherein the driving of the flow of the culture medium includes switching a direction of flow in the first flow line at least once in a measurement phase.

10. The apparatus of clause 9, wherein: the apparatus comprises a pump and a backpressure source configured to act from opposite ends of the first passage; and the flow controller is configured to perform the driving of flow by controlling the pump.

11. The apparatus of clause 10, wherein the pump comprises one or more of: a syringe pump and a peristaltic pump.

12. The apparatus of any of clauses 2-8, wherein the first passage defines a circuit in the insertion module and each measurement portion of the culture medium is circulated in the circuit during the respective measurement phase.

13. The apparatus of clause 12, wherein: the insertion module further comprises a plurality of valves; and the flow controller is configured to drive the circulation by actuating the valves at selected times.

14. The apparatus of clause 13, wherein the actuation of the valves at selected times is configured to provide a peristaltic pumping action.

15. The apparatus of clause 13 or 14, wherein each valve is configured to be actuated by changing a gas pressure adjacent to the valve.

16. The apparatus of clause 15, wherein the changing of the gas pressure causes a corresponding change in the shape of a deformable member of the valve.

17. The apparatus of any preceding clause, wherein: the gas exchange arrangement comprises a gas permeable membrane at a position adjacent to the gas exchange portion of the first passage; and the gas passes from the second passage to the first passage by diffusion through the gas permeable membrane.

18. The apparatus of clause 17, wherein portions of the first passage and the second passage adjacent to the gas permeable membrane are on the same side of the gas permeable membrane.

19. The apparatus of clause 18, wherein the apparatus comprises two of the gas permeable membranes and the portions of the first passage and the second passage are formed in a microfluidic chip clamped between the two gas permeable membranes.

20. The apparatus of any preceding clause, wherein: the gas exchange arrangement is configured to allow gas to pass from the second passage to the first passage in two gas exchange portions of the first passage; and the two gas exchange portions are positioned on opposite sides of the culture chamber.

21. The apparatus of any preceding clause, wherein the culture chamber is positioned to allow NMR measurements to be performed on material in the culture chamber when the insertion module is in the magnet.

22. The apparatus of any preceding clause, further comprising an analysis chamber connected in series at a position along the first passage different from the position of the culture chamber.

23. The apparatus of clause 22, wherein the analysis chamber is positioned to allow NMR measurements to be performed on material in the analysis chamber when the insertion module is in the magnet.

24. The apparatus of any preceding clause, further comprising a temperature control arrangement configured to control a temperature in the culture chamber.

25. The apparatus of clause 24, wherein the temperature control arrangement comprises a reservoir configured to contain a liquid or gas in thermal contact with the culture chamber. 26. The apparatus of clause 25, wherein the temperature control arrangement is configured to drive a flow of liquid or gas through the reservoir and control the temperature of the liquid or gas.

27. The apparatus of clause 25 or 26, wherein the reservoir forms part of the insertion module.

28. The apparatus of any of clauses 24-27, further comprising a temperature sensor in thermal contact with the culture chamber, wherein the temperature control arrangement uses an output from the temperature sensor to control the temperature of the culture chamber.

29. The apparatus of any preceding clause, wherein the insertion module comprises a stack of two or more sub-modules clamped together by a cylindrical clamping body, the clamping body being moveable along the longitudinal axis relative to the stack of submodules, the movement causing engagement with a ramp member that provides a radially inwards clamping force.

30. A method of performing measurements involving live tissue, the method comprising: using the apparatus of any preceding clause to perform measurements involving live tissue while the insertion module is in a magnet of a system for performing NMR or MRI, the live tissue being in the insertion module and the measurements comprising performing NMR or MRI.

31. A method of performing measurements involving live tissue in a magnet of a system for performing nuclear magnetic resonance, NMR, or magnetic resonance imaging, MRI, the method comprising: positioning an insertion module in a magnet of a system for performing NMR or MRI, wherein the insertion module comprises a first passage, a culture chamber containing a sample of living tissue, and a second passage; driving flow of a culture medium in a first flow line including the first passage; driving flow of a gas in a second flow line including the second passage; and performing measurements using the system for performing NMR or MRI, wherein: the insertion module is configured to allow gas to pass through a gas permeable member from the second passage to the first passage in a gas exchange portion of the first passage.

32. The method of clause 31, wherein driving flow of the culture medium comprises driving flow of the culture medium in a sequence comprising a plurality of measurement phases interleaved by refreshment phases; in each measurement phase, the culture medium is driven such that all positions along a respective longitudinally-extending measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least once; and in the refreshment phase, the culture medium is driven such that all of the measurement portion from a directly preceding measurement phase is replaced by culture medium that did not encounter the culture chamber during the directly preceding measurement phase.

33. The method of clause 32, wherein: in each measurement phase, the culture medium is driven such that all positions along a respective longitudinally-extending measurement portion of the culture medium in the first flow line pass through the culture chamber and the gas exchange portion at least twice.

34. The method of clause 32 or 33, wherein the volume of each measurement portion is less than 1 OOpl.

35. The method of any of clauses 32-34, wherein a liquid immiscible with the culture medium is provided in series with culture medium in the first flow line.

36. The method of clause 35, wherein the immiscible liquid is provided such that one or more of the measurement portions are separated from one or more other measurement portions by the immiscible liquid, the separated measurement portions optionally having different compositions relative to each other.

37. The apparatus of any of clauses 32-36, wherein at least a subset of the measurement portions are different portions of a single longitudinally continuous body of culture medium in the first flow line. 38. The apparatus of any of clauses 32-37, wherein the volume of each measurement portion is in the range of 50%-200% of the volume of the first passage, preferably 50%- 200% of a volume of the first passage defined in a microfluidic chip of the insertion module.

39. The apparatus of any of clauses 32-38, wherein a volume of culture medium driven through the culture chamber during each refreshment phase is larger than the volume of each measurement portion, preferably at least twice as large.

40. The method of any of clauses 30-39, wherein the driving of the flow of the culture medium includes at least one switch in the direction of flow in the first flow line in a measurement phase.

41. The method of any of clauses 30-40, wherein the first passage defines a circuit in the insertion module and each measurement portion of the culture medium is circulated in the circuit during the respective measurement phase.

42. The method of clause 41, wherein the circulation is driven by actuating valves in the insertion module at selected times.

43. The method of clause 42, wherein the actuation of the valves at selected times provides a peristaltic pumping action.

44. The method of any of clauses 30-43, wherein gas passes from the second passage to the first passage in two gas exchange portions of the first passage, the two gas exchange portions being positioned on opposite sides of the culture chamber.

45. The method of any of clauses 30-44, wherein the measurements comprise NMR performed on material in the culture chamber.

46. The method of any of clauses 30-45, wherein the measurements comprise NMR performed on material in an analysis chamber, the analysis chamber being connected in series at a position along the first passage different from the position of the culture chamber.

47. The method of any of clauses 30-46, further comprising controlling a temperature in the culture chamber by driving a flow of liquid of controlled temperature through a liquid reservoir, wherein the liquid of controlled temperature is in thermal contact with the culture chamber. 48. The method of any of clauses 30-47, wherein the measurements comprise NMR and are performed during the movement of the culture medium in the first flow line during a measurement phase.

49. The method of any of clauses 30-48, further comprising controlling a composition of either or both of the culture medium in the first flow line and the gas in the second flow line to apply a controlled stimulus to the sample.

50. The method of clause 49, wherein the controlled stimulus comprises simulating one or more of the following in any combination: a disease process; administration of a therapy; administration of a drug; administration of a drug candidate; administration of an infectious agent; administration of a toxin.

51. The method of clause 49 or 50, further comprising observing the effect of the controlled stimulus on the material in the culture chamber.