Cell culturing system optimized for raman spectroscopy imaging

Abstract

The present invention relates to a biochip (100) optimized for spectroscopy imaging comprising a reservoir and a culturing chamber (110) comprising an observational interface (115) configured to allow spectroscopy measurement of the culturing chamber (110) through the observational interface (115).

Description

CELL CULTURING SYSTEM OPTIMIZED FOR RAMAN SPECTROSCOPYIMAGINGFIELD OF INVENTION

[0001] The present invention relates to a cell culturing system.

[0002] In particular, the cell culturing system is modular and is configured to allow Raman spectroscopy imaging of cells in a culturing chamber.BACKGROUND OF INVENTION

[0003] Cell culture is extensively used today as a tool to study cell development, metabolic changes and interaction with its environment. Bioreactors allow to reproduce an environment favorable to development and organization of cells, close to that of a tissue or an animal or human organ thanks to perfusion of nutritive fluid in the culturing chamber. Bioreactors are thus a powerful tool to reduce animal testing for drug development, disease control, and general monitoring of metabolic changes in cells caused by any mechanism.

[0004] In order to monitor cells growing and to study cells, a first known method is to periodically extract (collect) samples. However, systems allowing to extract samples are not satisfactory. Indeed, it is necessary to stop the perfusion to be able to take a sample of medium in the culturing chamber. Moreover, in case of multi-well plates, these systems also need to be completely opened to extract sample which implies that operation of all the parallel bioreactors must be stopped.

[0005] Moreover, for observation of a sample, specific dyes for fluorescence measurements have to be used. However, injection of dyes requires premature termination of cell culture.

[0006] To avoid use of fluorescent dyes and extraction of samples, Raman spectroscopy may be used. However, known systems for cell culture are not satisfactory for Raman spectroscopic imaging. Indeed, most of the systems configured for Raman spectroscopy are large-scale and are not adapted to laboratory environments and studies requiring high spatial resolution such as cell culture studies. Indeed, the Raman light is obstructed by materials used in the bioreactors, such as standard plastics or glass slides. One known solution to this obstruction problem is to submerge Raman probes in the bioreactors, however this is not possible in cell culture plates. Indeed, due to size and scale of cell culture plates, each culturing well can hold volumes as low as a few microliters to a few milliliters. Raman probes, on the other hand, are relatively large and bulky in comparison, making it impractical to submerge one in such a confined space without disrupting the cell environment or the sterility of the culture. Moreover, maintaining sterility is crucial in cell culture to avoid contaminating the cells with bacteria, fungi, or other microorganisms. Inserting a Raman probe directly into a well could compromise the sterile barrier, potentially leading to contamination. Finally, Raman spectroscopic imaging allows to determine a change in the cultured cells at a specific moment. Therefore, when a measure of a temporal change over a given time range is needed, multiple sessions of Raman spectroscopic imaging are needed which first increases working time of the user, second does not permit a direct comparison because of probable additional changes in the culture medium between sessions and third does not allow continuous monitoring.

[0007] Finally, known bioreactors are not environmentally friendly. Indeed, firstly, the size of the culturing chamber is determined and fixed at manufacturing. Therefore, if several culturing chamber volumes are needed, as many systems must be manufactured which increases the quantity of material needed. Moreover, cell culture plates are made of single use elements which leads to a large quantity of waste for each experiment.

[0008] There is thus a need for a modular cell culture system which allows study of continuous temporal metabolic changes in cells without interrupting cell culturing and / or which allows to reduce the quantity of waste.

[0009] To this end, a new biochip comprising an observational interface configured to allow in situ and real-time spectroscopy measurement of a cell culture chamber in the biochip has been developed.SUMMARY

[0010] This invention thus relates to a biochip comprising: a reservoir; a culturing chamber comprising a fluid inlet defining a perfusion axis, the culturing chamber being in fluid communication with the reservoir through the fluid inlet; wherein, the culturing chamber comprises an observation opening extending along the perfusion axis, the biochip further comprising an observational interface disposed on the observation opening; wherein the observational interface is configured to allow spectroscopy measurement of the culturing chamber through the observational interface.

[0011] Indeed, the position of the observation opening along the perfusion axis allows to monitor, with a single spectroscopic session, temporal metabolic changes in the cells cultured in the culturing chamber.

[0012] Indeed, the reservoir allows to perfuse inside the culturing chamber, through the fluid inlet, elements for which the cellular reaction has to be measured. Said elements thus diffuse inside the culturing chamber in the direction of the perfusion axis. Therefore, at a given time, cells closest to the fluid inlet have been submitted to the elements for a longer period than cells farthest from the fluid inlet in the direction of the perfusion axis, resulting in a gradient of cellular reactions. A single spectroscopic session performing several measurements (scanning) along the perfusion axis thus allows to obtain data representative of spatio-temporal metabolic changes in cells. This is advantageous compared to known bioreactors for which the only way to obtain data representative of temporal metabolic changes in cells is to perform several spectroscopic sessions, i.e.,several sample extractions. Also, extracting sample from the cell culture prevent access to spatial information.

[0013] Moreover, since elements diffusion is continuous, the measured temporal metabolic changes using the biochip of this disclosure is a continuous real-time monitoring of metabolic activity.

[0014] Thanks to the fact that cells of the same culturing chamber have exactly the same environment, the only varying parameter when perfusing is the injection of the perfused element. This leads to controlled environment where gradients and other conditions can be adjusted or controlled to mimic physiological states.

[0015] Moreover, the design of the biochip of this disclosure enables microscopic measurements of cell developments.

[0016] Finally, thanks to the observational interface configured to allow spectroscopy measurement, there is no need to interrupt cell culturing to perform the measurements. Indeed, the culturing chamber remains closed all along the measurement.

[0017] According to an advantageous aspect of the invention, a surface of the culturing chamber opposite the observation opening can be functionalized to promote adhesive behavior of cells.

[0018] The functionalization allows to increase the probability of cell adhesion at the bottom of the culturing chamber therefore concentrating the cell growing in the region of observation, said region of observation being defined by the position of the observation opening.

[0019] According to an advantageous aspect of the invention, the biochip further comprises: a bottom support; and an intermediate plate disposed on the bottom support and comprising a chamber hole forming walls of the culturing chamber, the bottom support and the observational interface disposed on opposite sides of the chamber hole closing the culturing chamber.

[0020] This allows to create a modular biochip wherein at least one of the parts may be reused for several experiments. This approach will not only reduce waste and operational costs but will also enhance the system’s versatility and applicability across different research settings. The intermediate plate comprising the observational interface - which is the element requiring the most accurate manufacture and thus which has the highest cost - may thus be reused after washing. The bottom support which is easily manufactured and which is the most polluted because of cell adhesion may be a single-use part.

[0021] According to an advantageous aspect of the invention, the biochip further comprises a cover configured to be disposed on the intermediate plate, the cover being configured to close the reservoir, the cover comprising an observing hole in front of the observation opening.

[0022] The cover thus allows to guaranty the sterility of the reservoir. Moreover, this prevents evaporation of the culture medium, leading to a closed system with a controlled environment.

[0023] The present invention further relates to a biochip (100) comprising: a reservoir (102); a culturing chamber (110) comprising a fluid inlet (117) defining a perfusion axis (P), the culturing chamber (110) being in fluid communication with the reservoir (102) through the fluid inlet (117); wherein, the culturing chamber (110) comprises an observation opening (112) extending along the perfusion axis (P), the biochip (100) further comprising an observational interface (115) disposed on the observation opening (112); wherein the observational interface (115) is configured to allow spectroscopy measurement of the culturing chamber (110) through the observational interface (115) absorbing less than 20%, preferably less than 10%, of light at visible, NIR and / or UVA wavelengths; wherein the biochip (100) further comprises: a bottom support (120); and an intermediate plate (130) disposed on the bottom support (120) and comprising a chamber hole (132) forming walls of the culturing chamber (110), the bottomsupport (120) and the observational interface (115) disposed on opposite sides of the chamber hole (132) closing the culturing chamber (110); wherein the biochip (100) further comprises a cover (140) configured to be disposed on the intermediate plate (130), the cover (140) being configured to close the reservoir (102), the cover (140) comprising an observing hole (145) in front of the observation opening (112).

[0024] According to an advantageous aspect of the invention, the biochip further comprises a chamber sealing element positioned between the bottom support and the intermediate plate thereby sealing a bottom of the culturing chamber.

[0025] The chamber sealing element thus prevents any leakage from the culturing chamber but also prevent the interaction of the exterior environment with the culturing chamber, reducing tremendously risks of contamination.

[0026] According to an advantageous aspect of the invention, the biochip further comprises a fastening element configured to maintain the intermediate plate abutted against the bottom support.

[0027] The fastening element thus increases the sealing between the abutted elements.

[0028] According to an advantageous aspect of the invention, the spectroscopy measurement is a Raman spectroscopy measurement, the observational interface being a plate made of a material transparent to laser light used in Raman spectroscopy, such as for example quartz, silicon (preferably a few hundred nanometers up to a few micrometers thick), calcium fluoride CaF2, barium fluoride BaF2, or fused silica.

[0029] In particular, the observational interface material should be transparent in visible range (i.e., wavelengths ranging from 380 nm to 780 nm), in the near infrared (NIR) range (i.e., wavelengths ranging from 780 nm to 1400 nm, preferably from 780 nm to 1100 nm) and / or ultraviolet A (UVA) range (i.e., wavelengths ranging from 315 nm to 380 nm). In other words, it refers to a material that absorbs less than 20%, preferably less than 10% of light at said visible, NIR and / or UVA wavelengths.

[0030] Quartz, silicon, calcium fluoride CaF2, barium fluoride BaF2, or fused silica are particularly advantageous for Raman spectroscopy due to their transparency in the visible and UVA ranges. Moreover, quartz crystals are stable and do not undergo significant changes in their properties when exposed to various environmental conditions, including temperature and humidity. This stability is essential for the accuracy and repeatability of Raman measurements. Furthermore, the observational interface material should have low intrinsic fluorescence, meaning that it does not emit significant background fluorescence when subjected to laser excitation. This is advantageous because Raman spectroscopy aims to detect the weak Raman scattering signals from the sample, and high fluorescence can interfere with these signals as it would lead to a high signal / noise ratio. The Raman peaks produced by quartz, silicon, calcium fluoride CaF2, barium fluoride BaF2, or fused silica are also usually well -separated from the Raman peaks of the sample under investigation, minimizing interference.

[0031] According to an advantageous aspect of the invention, the reservoir is disposed at an end of the culturing chamber along the perfusion axis. More particularly, the reservoir should be disposed outside of the spectroscopy light path.

[0032] This allows, contrarily to devices wherein the reservoir is disposed away from the chamber and is in fluidic communication with the chamber via tubing, to perform a homogeneous diffusion. Moreover, this allows to increase the speed of the perfusion. Finally, this allows for fine-tuning the temporal behavior of the perfusion.

[0033] This position of the reservoir allows optical spectroscopy without interference from the reservoir in the light path. Moreover, the culturing chamber can thus be observed using a microscope objective positioned close to the culturing chamber to focus on the region of observation, without any physical obstruction from the reservoir. This allows to combine two monitoring and imaging techniques: optical spectroscopy and microscopy.

[0034] According to an advantageous aspect of the invention, the biochip further comprises a chamber volume adjustment system configured to adjust a volume of the culturing chamber between at least a first volume and a second volume.

[0035] This allows to obtain a tunable volume of the culturing chamber using a single biochip instead of using several biochips with different fixed chamber volumes. Indeed, when a multi-well system comprises a plurality of biochips, the volume of each culturing chamber may thus be adjusted independently from others.

[0036] According to an advantageous aspect of the invention, the culturing chamber further comprises a piston hole, the chamber volume adjustment system comprising a piston having a piston section fitting the piston hole in a leak tight manner, the piston being movable in the culturing chamber to adjust the volume of the culturing chamber.

[0037] The use of a piston is thus a simple mechanical way to adjust the volume of the culturing chamber which allows both manual or electronical adjustment.

[0038] According to an advantageous aspect of the invention, the chamber volume adjustment system comprises an actuator configured to slide the piston along a sliding axis.

[0039] According to an advantageous aspect of the invention, the sliding axis is perpendicular to the perfusion axis.

[0040] Varying the volume of the culturing chamber perpendicularly to the perfusion axis allows to increase or reduce the effect of the perfused elements gradient on cellular reactions therefore improving the quality of the real-time monitoring, and the range of metabolic information.

[0041] Alternatively, the sliding axis could be parallel to the perfusion axis.

[0042] According to an advantageous aspect of the invention, the actuator comprises a cam.

[0043] The cam allows transforming rotary motion into linear motion. The linear motion allows to slide the piston along the sliding axis. The cam is a simple mechanism allowing to create predefined linear movements by a definition of a specific cam profile.

[0044] This invention thus relates to a multi-well system comprising at least two biochips as described in this disclosure.

[0045] This invention thus relates to a method for performing a spectroscopy measurement on a cell culture, comprising: providing a biochip of this disclosure or a multi-well system of this disclosure, wherein a cell culture is present in the culturing chamber and a perfusion fluid is present in the reservoir; using a spectrometer to acquire a spectroscopy measurement through the observational interface of the culturing chamber.DEFINITIONS

[0046] In the present invention, the following terms have the following meanings:

[0047] “Biochip” refers to any device allowing for cell culture. The biochip may be microfluidic or millifluidic. The biochip may be a bioreactor, a reactor, a flow chamber, a perfused chamber, a perfused tissue culture polystyrene (TCPS) cell culture box, or a dynamic cell culture system.

[0048] “UV” refers to light of wavelength in the range from 10 nm to 380 nm. In particular, UVA refers to the sub-range of UV from 315 nm to 380 nm.

[0049] “UVA-Visible-NIR” refers to light of wavelength in the range from 315 nm to 1400 nm.

[0050] “UVA-Visible” refers to light of wavelength in the range from 315 nm to 780 nm.

[0051] “Visible” refers to light of wavelength in the range from 380 nm to 780 nm.

[0052] “Infrared” refers to light of wavelength in the range from 780 nm to 1 mm. This range is usually split in three sub ranges. Near Infrared (NIR) corresponds to wavelength from 780 nm to 1400 nm. Short-Wavelength Infrared (SWIR) corresponds to wavelengthfrom 1400 nm to 3000 nm. Mid- wavelength Infrared (MWIR), also called Intermediate Infrared, corresponds to wavelength from 3000 nm to 5000 nm.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is a top view of a multi-well system 200 comprising six biochips 100 according to one embodiment of the invention.

[0054] Figure 2 is a side view of the multi -well system 200 of figure 1.

[0055] Figure 3 is a perspective view of the multi -well system 200 of figure 1.

[0056] Figure 4 is a section view of the multi-well system 200 of figure 3 along the plane A.

[0057] Figure 5 is an exploded top view of the multi -well system 200 of figure 1.

[0058] Figure 6 is a zoom-in view of the multi-well system 200 of figure 5 zoomed in the area B. The hidden elements are shown in transparence with dashed lines.

[0059] Figure 7 is an exploded bottom view of the multi-well system 200 of figure 1.

[0060] Figure 8 is a zoom-in view of the multi-well system 200 of figure 7 zoomed in the area C.

[0061] Figure 9 is a perspective top view of the chamber volume adjustment system comprising a cam 164a and a piston 162 according to one embodiment of the invention.

[0062] Figure 10 is a perspective bottom view of the chamber volume adjustment system of figure 9.

[0063] Figure 11 is a sequential representation of the adjustment of the volume of the culturing chamber 110 using the chamber volume adjustment system of figure 9.

[0064] Figure 12 is a representation of an observational setup with a Raman system 300 observing the cell culturing from the biochip 100 of according to one embodiment (perspective top - left - and bottom - right - views).

[0065] Figure 13 is a representation of the results of the Raman spectroscopy measurements of the cell culturing from the biochip 100 of according to one embodiment. Figure 13A shows three distinct spectra, acquired at different times but within the same region of the culturing chamber 110 during an observation period from Day 0 to Day 2 (the curve corresponding to Day 0 is represented in dark grey, the curve corresponding to Day 1 is represented in mild grey, the curve corresponding to Day 2 is represented in light grey, x-axis is Raman Shift (cm'1) and y-axis is intensity (a.u.)). Figures 13B-D are zooms on different area of the spectra shown in figure 13 A.

[0066] Figure 14 is an exploded top view of the multi-well system 200 according to one embodiment of the invention wherein the bottom of the culturing chamber comprises a calibration element 158.

[0067] Figure 15 is a top view of a multi-well system 200 according to one embodiment of the invention wherein the bottom support comprises four positioning elements 125.

[0068] Figure 16 is a zoom-in view of the multi-well system 200 of figure 5 zoomed in the area B. The hidden elements are shown in transparence with dashed lines. Each observational interface 115 comprises six sensors 118.DETAILED DESCRIPTION

[0069] This invention relates to a biochip for cell culture.

[0070] As illustrated in figures 1-3, said biochip 100 comprises: a reservoir 102; a culturing chamber 110 comprising a fluid inlet 117 defining a perfusion axis P, the culturing chamber 110 being in fluid communication with the reservoir 102 through the fluid inlet 117.

[0071] As illustrated in figures 1-3 and 6, the culturing chamber 110 comprises an observation opening 112 extending along the perfusion axis P, and the biochip 100 further comprises an observational interface 115 disposed on the observation opening 112.

[0072] The observational interface 115 is configured to allow spectroscopy measurement of the culturing chamber 110 through the observational interface 115.

[0073] The biochip 100 is advantageously configured to allow in situ and real-time spectroscopy measurement. Indeed, to perform spectroscopy measurement on the cell culture comprised in the culturing chamber 110, a primary beam of light is generated by a light source to the observation opening 112 through the observational interface 115, then a secondary beam of light (transmitted, reflected or scattered) is generated in response by the cell culture and detected by spectroscopic detectors through the observational interface 115. A detection signal representative of this secondary beam of light is determined. This detection signal is a raw spectrum, i.e., density of received light as a function of its wavelength or frequency.

[0074] The biochip 100 can also advantageously allow microscopy imaging of the cell culture in the culturing chamber 110 by placing the biochip 100 under the objective microscope so that the observational interface 115 and the objective are aligned.

[0075] Thus, the biochip 100 as described herein allows for label-free monitoring of metabolic activity of cells in a cell culture without interrupting cell culturing or submerging a spectroscopic probe in the cell culture. This gives access to monitoring metabolic changes or interaction of cells with their environment in real-time.

[0076] The spectroscopy measurement allowed by the biochip 100 may be an optical spectroscopy measurement such as, for example, UV-visible spectroscopy, infrared (IR) spectroscopy, Raman spectroscopy, or fluorescence spectroscopy.

[0077] Preferably, the biochip 100 allows in situ and real-time Raman spectroscopy measurement. Raman spectroscopy analyzes the scattering of monochromatic light by molecules and provides information about molecular vibrations. In this case, the lightsource, generating the primary beam of light, is a laser. Preferably the laser emits in a range of wavelengths from 190 nm to 1400 nm.

[0078] To allow the spectroscopy measurement, the observational interface 115 should be transparent in the range of wavelengths of the primary beam of light (i.e., beam of light generated by the light source of the spectrometer), and transparent in the range of wavelengths of the secondary beam of light (i.e., beam of light generated by the cell culture in response to the primary beam of light). Transparent in a certain range of wavelengths refers to a material that absorbs less than 20%, preferably less than 10%, of light at said range of wavelengths.

[0079] The observational interface 115 may be transparent in UV, UV A- visible, visible, NIR, and / or IR ranges of light.

[0080] The biochip 100 may have the following dimensions: length ranging from 0.5 cm to 20 cm, preferably from 1 cm to 15 cm, more preferably from 5 cm to 15 cm, width ranging from 0.5 cm to 15 cm, preferably from 1 cm to 10 cm, and / or height ranging from 170 pm to 1 cm.

[0081] The biochip 100 may have dimensions corresponding to a standard microscope stage. Advantageously, the biochip 100 may comprise at least one positioning element 125 such as pillars or holes that allow consistent placement onto a microscope stage. For example, in figure 15, the biochip 100 comprises four positioning elements 125 in the form of holes. As in figure 15, the positioning elements 125 may be disposed though or on a protrusion extending along the length of the biochip 100. The positioning elements 125 ensure reproducible positioning during Raman measurements and contribute to spatial calibration and measurement automation.

[0082] Examples of materials suitable for the culturing chamber 110 include, without being limited to, glass, polymer (plastic), calcium fluoride (CaF2), barium fluoride (BaF2), quartz, silicon or fused silica.

[0083] As the observations and / or spectroscopy measurement are performed through the observational interface 115, the observational interface 115 is preferably a plate having asection (area) larger than the observation opening 112 to totally cover the observation opening 112 (see figure 4). The observational interface 115 may be glued or abutted on the observation opening 112 to be gas tight and / or liquid tight, therefore preventing leakage or evaporation.

[0084] Examples of materials suitable for the observational interface 115 include, without being limited to, glass, polymer (plastic), calcium fluoride (CaF2), barium fluoride (BaF2), quartz, silicon or fused silica. Materials suitable for performing Raman spectroscopy on the biochip 100 preferably include, without being limited to, calcium fluoride (CaF2), barium fluoride (BaF2), quartz, silicon, fused silica as these materials are transparent to primary light beam used in Raman spectroscopy, i.e., laser light, have low autofluorescence and non-interfering Raman lines. Preferably, standard glass and polymer should be avoided in case of Raman spectroscopy.

[0085] The observational interface 115 may have a thickness ranging from 1 nm to 100 pm, preferably from 1 nm to 50 pm, more preferably from 1 nm to 100 nm. A small thickness will advantageously prevent the obstruction and disturbance of spectroscopic light. For example, for a silicone observational interface 115 with a thickness of 5 nm, 99.95% of light is transmitted leading to a loss of less than 1% for the received light after two passages through the observational interface 115. To manufacture a silicon nitride (SiN) observational interface 115 having a thickness of a few nanometers to a few tens of nanometers (for example 5 nm), SiN of desirable thickness is deposited on a silicone substrate having a thickness of several hundreds of micrometers. The observational interface 115 in such setups is created by wet-etching the SiN / Si window from the backside using Potassium Hydroxide (KOH). This allows to remove the silicone substrate and to leave only the SiN observational interface 115. The back etching may form a groove with a section in the form of a truncated triangle of 45° in the silicone substrate. The width of the observational interface 115 is thus determined by the width of the top base of the truncated triangle and the thickness of the plates. The width of the observational interface 115 formed by this technique may reach several hundreds of micrometers. For larger observational interface 115, the observational interface 115 the back etching may be performed at different parallel positions to incrementally increasethe width of the observational interface 115. This manufacturing method is useable with other ceramics as well and similar observational interfaces 115 can also be achieved with other 2D materials (such as graphene or hexa-boron nitride).

[0086] In one embodiment, the culturing chamber 110 and the observational interface 115 are manufactured as a sole piece of equipment. In this case, the whole culturing chamber 110 is transparent in the range of wavelengths of the primary beam of light.

[0087] A cell culture refers herein to cells growing by perfusing a cultivation medium, also named perfusion fluid, stored in the lateral reservoir 102 in the culturing chamber 110 where cells are cultured. The reservoir 102 facilitates fresh media storage and minimizes contamination risk.

[0088] In one embodiment, examples of cells to be cultured in the biochip 100 include, without limitation: HeLa cells, lymphocytes, escherichia coli (E. coli), bacillus subtilis, saccharomyces cerevisiae, pichia pastoris, HEK293 (Human Embryonic Kidney 293) cells, CHO (Chinese Hamster Ovary) cells, tobacco BY-2 (Bright Yellow-2) cells, arabidopsis thaliana cell culture, Sf9 cells (from Spodoptera frugiperda), Hi5 cells (from Trichoplusia ni), human induced pluripotent stem cells (hiPSCs), mesenchymal stem cells (MSCs).

[0089] The cultivation medium (or perfusion fluid) may comprise, as elements to be perfused in the culturing chamber 110, nutrients, active principles such as oxygen, nitrogen, glucose, virus, bacteria, enzymes, or drugs. The perfusion of the cell culture may be continuous or discrete with occasional releases of cultivation medium in the culturing chamber 110. The communication between the culturing chamber 110 and the reservoir 102 may therefore be continuous, or a valve may be present to open or close the communication. Then, communication may be performed if a pressure is applied on the cultivation medium stored in the reservoir 102.

[0090] A surface of the culturing chamber 110 may be functionalized to provide specific characteristics to said culturing chamber 110. To allow cell culture and cell growth, a surface of the culturing chamber 110 opposite the observation opening 112 may befunctionalized to promote adhesive behavior of cells. Functionalization may be achieved by applying a coating on at least part of at least one inner surface of the culturing chamber 110. For example, the coating may comprise a fluorinated polymer. The fluorinated polymer advantageously prevents adsorption of molecules of biological interest such as proteins and / or antibodies. For example, the coating may be a film of perfluoropolyether. For example, the coating may comprise proteins alone or in mixtures with an extracellular matrix (for example, collagen, Matrigel™, fibronectin, laminin, vimentin etc.) that cells naturally adhere to, peptide linkers such as short sequences of amino acids (for example RGD peptides) that cells can recognize and bind to, at least one specific antibody recognizing cell membrane sites (for example, CD 144, CD31 for endothelial cells, KRT7 for cholangiocytes, ALB for hepatocytes etc.), at least one cellcell adhesion protein such as Cadherin 1, self-assembled monolayer (SAM), a silane compound, a thin metallic layer (for example, gold or silver layer) that can contribute to enhance Raman signal, due to low fluorescence and light reflectance. This functionalization can also be achieved via plasma treatment, micro-texturing or nanotexturing of the surface.

[0091] Preferably, the functionalization allows to promote adhesive behavior of cells on the surface comprising said functionalization. Preferably, said surface is the surface opposite the observation opening 112. Indeed, this allows a better observation of the cells.

[0092] The observational interface 115 may comprise at least one sensor 118. Each sensor 118 may cover less than 50%, preferably less than 20%, even more preferably less than 5% of an area of the observation opening 112. The at least one sensor 118 may have a cylindrical shape or a parallel epipedal shape. The at least one sensor 118 is functionalized. For example, the sensor 118 may comprise, on its surface, a fluorinated polymer. The fluorinated polymer advantageously prevents adsorption of molecules of biological interest such as proteins and / or antibodies. For example, the sensor 118 may comprise a film of perfluoropolyether disposed on its surface. For example, the sensor 118 may comprise, on its surface, proteins alone or in mixtures with an extracellular matrix (for example, collagen, Matrigel™, fibronectin, laminin, vimentin etc.) that cells naturally adhere to, peptide linkers such as short sequences of amino acids(for example RGD peptides) that cells can recognize and bind to, at least one specific antibody recognizing cell membrane sites (for example, CD144, CD31 for endothelial cells, KRT7 for cholangiocytes, ALB for hepatocytes etc.), at least one cell-cell adhesion protein such as Cadherin 1, self-assembled monolayer (SAM), a silane compound, a thin metallic layer (for example, gold or silver layer) that can contribute to enhance Raman signal, due to low fluorescence and light reflectance. This functionalization can also be achieved via plasma treatment, micro-texturing or nano-texturing of the surface of the sensor 118. For example, when the observational interface 115 comprises a plurality of sensors 118, the sensors 118 are disposed in an array inside the observation opening 112 as represented in figure 16. This array disposition allows for both spatial and temporal information acquisition while remaining compatible with Raman measurements, due to the non-continuous nature of the SERS substrate. Each sensor 118 of the array of sensors may be functionalized differently. This advantageously enables multiplexed biochemical detection. The sensors 118 disposed in different observation interfaces 115 may be functionalized differently.

[0093] The surface of the culturing chamber 110 opposite the observation opening 112 may also be in glass or transparent plastic to allow optical spectrometry implying a primary light beam directed towards said surface.

[0094] As represented in figure 14, at least one calibration element 158 may be disposed on the surface of the culturing chamber 110 opposite the observation opening 112. The calibration element 158 advantageously allows to calibrate a focus of the Raman spectrometer. For example, the calibration element 158 allows to define a focus reference point. The calibration element 158 also advantageously allows to ensure consistent alignment and spectral reproducibility across measurements. Each calibration element 158 may cover less than 50%, preferably less than 20% event more preferably less than 5% of an area of the surface of the culturing chamber 110 opposite the observation opening 112. Each calibration element 158 is preferably planar. Each calibration element 158 is preferably made of a Raman-active material such as silicon.

[0095] The culturing chamber 110 may be defined by side walls so that the culturing chamber 110 extends perpendicularly to the perfusion axis P. The culturing chamber 110preferably has four side walls, a top and a bottom. The culturing chamber 110 may be of any size suitable for cell culture. The culturing chamber 110 has preferably a height, i.e., distance measured between top and bottom perpendicular to the perfusion axis P, inferior to length and / or width, i.e., distances measured between two side walls (along the perfusion axis P and perpendicular to the perfusion axis P respectively). The culturing chamber 110 has preferably a length, i.e., distance measured between two side walls along the perfusion axis P, inferior to width, i.e., distance measured between two side walls perpendicular to the perfusion axis P.

[0096] As illustrated in figures 1 to 3, the culturing chamber 110 has preferably a parallelepipedal shape.

[0097] The dimensions of the culturing chamber 110, including its height and width, are factors that directly impact the transportation and diffusion of nutrients and oxygen to the cells. The height of the culturing chamber 110 may typically range from 50 pm to 1 cm, though for most practical microfluidic applications, a height ranging from 0.5 mm to 5 mm, preferably from 1 mm to 2 mm considered ideal as it allows the best signal. Similarly, the width of the culturing chamber 110 may range from a few hundred microns (e.g., 200-300 pm) to 1 cm, preferably from 0.5 mm to 7 mm. The length of the culturing chamber 110 may range from 0.5 cm to 20 cm, preferably from 2 cm to 10 cm, more preferably from 2 cm to 6 cm.

[0098] The volume of the culturing chamber 110 corresponds to the capacity of the culturing chamber 110, i.e., the quantity of liquid that the culturing chamber 110 can contain which may be different from the quantity of liquid that the culturing chamber 110 actually contains. The volume of the culturing chamber 100 is thus a geometrical parameter measured from the dimensions of the culturing chamber 110. In other words, the volume of the culturing chamber 100 is not the volume of liquid inside the chamber 100. The volume of the culturing chamber 110 may range from 1.25 mm3to 4200 mm3.

[0099] As depicted in figure 6, the culturing chamber 110 comprises a fluid inlet 117 which is a channel passing through one of the culturing chamber walls, preferably a sidewall to allow fluid communication between the reservoir 102 and the cell culture in the culturing chamber 110. Cultivation medium stored in the reservoir 108 and perfused towards the culturing chamber 110 then diffuses in the cell culture along the perfusion axis P defined by the shape of the fluid inlet 117 and its position (direction, extension) inside the wall. This diffusion of perfused medium in the cell culture along the perfusion axis P creates a gradient of nutrients in the cell culture, for example an oxygen gradient, as cells near the fluid inlet 117 will be more, or faster, exposed to the nutrients than cells further away from the fluid inlet 117. The metabolic activity resulting from this diffusion gradient can then be monitored in situ and in real-time using spectroscopy measurements.

[0100] The height of the fluid inlet 117 should be inferior or equal to the height of the culturing chamber 110, preferably the height of the fluid inlet 117 is less than 1 mm. This ensures a uniform flow rate across the entire height of the culturing chamber 110, promoting even distribution and exchange of nutrients and gases within the culturing chamber 110. The width of the fluid inlet 117 should be inferior or equal to the width of the culturing chamber 110, ensuring that the culturing chamber 110 is filled adequately, facilitating the even distribution of the cultivation medium and cells throughout the culturing chamber 110.

[0101] The diffusion of perfused medium can be controlled externally if needed.

[0102] A filter may be present in the fluid inlet 117 to avoid cell migration in the reservoir 102.

[0103] The culturing chamber 110 may comprise a second fluid inlet to help mitigate the fluid perfusion and thus gradient formation. This second fluid inlet may be disposed directly opposite the first fluid inlet to the direction of the perfusion. Additionally, in this configuration, the culturing chamber 110 comprises a glass slide paired with another lateral reservoir, symmetric to the first reservoir 102.

[0104] As illustrated in figures 1 and 6, the culturing chamber 110 comprises an observation opening 112 advantageously allowing visual inspection, microscopy imaging and spectroscopy analysis of the culturing chamber 110. The observation opening 112 may be a hole in one of the culturing chamber walls, preferably in the top, thus definingthe top of the biochip 100. The observation opening 112 extends along the perfusion axis P, i.e., its largest dimension is in the direction of the perfusion axis P. Therefore, the observation opening 112 having two extremities along the perfusion axis P, a gradient is formed between the extremity closest to the fluid inlet 117 and the opposite extremity. The observation opening 112 thus allows monitoring in situ and in real-time metabolic changes happening in the cell culture. This monitoring can either be performed with a moving biochip 100 and a static spectrometer, with a static biochip 100 and a moving spectrometer, with a static biochip 100 and a static spectrometer (scanning range of the spectrometer sufficient), or with both moving biochip 100 and spectrometer.

[0105] The observation opening has a length (measured along the perfusion axis P) ranging from 1 mm to 20 mm, preferably from 5 mm to 15 mm, and a width (measured perpendicular to the perfusion axis P) ranging from 1 mm to 10 mm, preferably from4 mm to 8 mm.

[0106] The observation opening area may correspond to more than 80%, more than 90%, more than 95% of the area of the culturing chamber 110.

[0107] The culturing chamber 110 may also comprise at least one additional opening (preferably watertight but not gas tight) on the top of the culturing chamber 110. This allows to remove the bubbles and avoid bubble formation in the culturing chamber 110. In case of no additional opening, the biochip 100 may be slightly tilted to prevent air bubble formation. This step must be performed in a laminar flow hood to maintain sterility.

[0108] The reservoir 102 may be disposed at an end of the culturing chamber 110 along the perfusion axis P to avoid physical obstruction of the observations opening (see figures5 and 6). The reservoir 102 is preferably disposed outside the path of light generated by the spectrometer, this advantageously prevents unduly obstruction of light that could result in signal loss.

[0109] The length of the reservoir 102, i.e., dimension measured along the perfusion axis P, may range from 5 mm to 100 mm, preferably from 20 mm to 50 mm. The width of the reservoir 102, i.e., dimension measured perpendicular the perfusion axis P in thesame plane as length, may range from 5 mm to 100 mm, preferably from 20 mm to 50 mm. The height of the reservoir 102, i.e., dimension measured perpendicular to the perfusion axis P between the top and the bottom, may range from 0.5 mm to 50 mm, preferably from 2.5 mm to 10 mm.

[0110] The volume of the reservoir 102 may range from 500 pL to 4 mL.

[0111] The reservoir 102 may comprise a lid to reversibly close the reservoir 102. This lid advantageously offers protection against contamination and fluid evaporation: closing the reservoir 102 creates a protective barrier for the fluid in the reservoir 102, safeguarding it from potential contaminants that could compromise the integrity of the experiments. It also plays a crucial role in minimizing the evaporation of the same fluid. This is especially important in microfluidic environments where small volumes of fluid are susceptible to rapid evaporation, which can affect concentration gradients and experimental outcomes.

[0112] The biochip 100 may be manufactured in a single piece with the reservoir 102 fixed to the culturing chamber 110. The reservoir 102 and the culturing chamber 110 may be provided separately and reversibly attached together when used. This allows to reduce waste since some parts may be reused.

[0113] The biochip 100 may comprise a part forming the bottom of the reservoir 102 and / or the bottom of the culturing chamber 110.

[0114] In the example of a two-parts biochip, the biochip 100 comprises: a bottom support 120; and an intermediate plate 130 disposed on the bottom support 120.

[0115] The bottom support 120 may form the bottom of the reservoir 102 and / or the bottom of the culturing chamber 110 while the intermediate plate 130 forms the side walls of the reservoir 102 and / or of the culturing chamber 110.

[0116] In a first embodiment of the two-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the bottom support 120 and the observational interface 115 are disposed on opposite sides of thechamber hole 132 thereby forming the top and bottom of the culturing chamber 110 and closing the culturing chamber 110. Therefore, the culturing chamber 110 is formed by the intermediate plate 130 being disposed on the bottom support 120. The intermediate plate 130 may also completely comprise the reservoir 102, i.e., the bottom and the side walls of the reservoir 102 and optionally the top of the reservoir 102.

[0117] In a second embodiment, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the bottom support 120 and the observational interface 115 are disposed on opposite sides of the chamber hole 132 closing the culturing chamber 110. The intermediate plate 130 may also comprise a reservoir hole 134 forming side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102. Therefore, both the reservoir 102 and the culturing chamber 110 are formed by the intermediate plate 130 being disposed on the bottom support 120.

[0118] In a third embodiment, the intermediate plate 130 may comprise a reservoir hole 134 forming side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 being disposed on the bottom support 120. The intermediate plate 130 may also completely comprise the chamber hole 132, i.e., the bottom and the side walls of the chamber hole 132 and the observational interface 115.

[0119] In an alternative example of a two-parts biochip, the biochip 100 comprises: an intermediate plate 130; and a cover 140 disposed on the intermediate plate 130.

[0120] The cover 140 may form the top the reservoir 102 and / or the top of the culturing chamber 110 while the intermediate plate 130 may form the side walls of the reservoir 102 and / or the side walls of the culturing chamber 110.

[0121] The cover 140 may comprise an observing hole 145 in front of the observation opening 112.

[0122] In a first embodiment of this alternative two-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls and bottom of the culturing chamber 110 and the cover 140 may comprise the observational interface 115 thereby forming the top of the culturing chamber 110 and closing the culturing chamber 110. The intermediate plate 130 may also completely comprise the reservoir 102, i.e., the bottom and the side walls of the reservoir 102 and optionally the top of the reservoir 102.

[0123] In a second embodiment of this alternative two-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls and the bottom of the culturing chamber 110, and a reservoir hole 134 forming side walls and bottom of the reservoir 102. The cover 140 may comprise the observational interface 115 therefore closing the culturing chamber 110, and the cover 140 may also form the top of the reservoir 102. Thus, the reservoir 102 is formed by the cover 140 being disposed on the intermediate plate 130.

[0124] In a third embodiment of this alternative two-parts biochip, the intermediate plate 130 may comprise a reservoir hole 134 forming side walls and the bottom of the reservoir 102. Therefore, the reservoir 102 is formed by the cover 140 being disposed on the intermediate plate 130. The intermediate plate 130 may also completely comprise the chamber hole 132, i.e., the bottom and the side walls of the culturing chamber 110 and the observational interface 115.

[0125] In the example or a three parts biochip (figs 1-5, 7), the biochip 100 comprises: a bottom support 120; an intermediate plate 130 disposed on the bottom support 120; a cover 140 disposed on the intermediate plate 130, opposite to the bottom support 120.

[0126] The bottom plate may form the bottom of the reservoir 102 and / or the bottom of the culturing chamber 110 while the intermediate plate 130 forms the side walls of the reservoir 102 and / or the side walls of the culturing chamber 110, and the cover 140 forms the top of the reservoir 102 and / or the observational interface 115.

[0127] The cover 140 may comprise an observing hole 145 in front of the observation opening 112.

[0128] In a first preferred embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the bottom support 120 forming the bottom of the culturing chamber 110 and the cover 140 comprising the observational interface 115 disposed on the culturing chamber 110 opposite the bottom plate. The culturing chamber 110 is thus formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. The intermediate plate 130 may also completely comprise the reservoir 102, i.e., the bottom and the side walls of the reservoir 102 and optionally the top of the reservoir 102. In this advantageous embodiment, the bottom support 120, in particular the portion being the bottom surface of the culturing chamber 110, which is contaminated due to cell attachment and growth is single use whereas the cover 140 comprising the observational interface 115 and optionally the intermediate plate 130 may be reused.

[0129] In a second preferred embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the bottom support 120 forming the bottom of the culturing chamber 110 and the cover 140 comprising the observational interface 115 disposed on the culturing chamber 110 opposite the bottom plate. The culturing chamber 110 is thus formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. The intermediate plate 130 may also form side walls and the top of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 being disposed on the bottom support 120. In this advantageous embodiment, the bottom support 120, in particular the portion being the bottom surface of the culturing chamber 110, which is contaminated due to cell attachment and growth is single use whereas the cover 140 comprising the observational interface 115 and optionally the intermediate plate 130 may be reused.

[0130] In a third preferred embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of and the bottom of the culturing chamber 110, the cover 140 comprising the observational interface 115 beingdisposed on the culturing chamber 110 opposite the bottom plate. The culturing chamber 110 is thus formed by the cover 140 and the intermediate plate 130 being assembled. The intermediate plate 130 may also form the side walls and the top of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 being disposed on the bottom support 120. In this advantageous embodiment, the intermediate plate 130 which is contaminated due to cell attachment and growth is single use whereas the cover 140 comprising the observational interface 115 and optionally the intermediate plate 130 may be reused.

[0131] In a fourth preferred embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110 and the observational interface 115 disposed on the culturing chamber 110, the bottom support 120 forming the bottom of the culturing chamber 110. The culturing chamber 110 is thus formed by the bottom support 120 and the intermediate plate 130 being assembled. The intermediate plate 130 may also comprise a reservoir hole 134 forming the side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102, and the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 being disposed on the bottom support 120, and the cover 140 being disposed on the intermediate plate 130. In this embodiment, the cover 140 comprises an observing hole 145 in front of the observation opening 112 to avoid observational obstruction by the cover 140. In this advantageous embodiment, the bottom support 120 which is contaminated due to cell attachment and growth is single use whereas the intermediate plate 130 may be reused.

[0132] In a fifth preferred embodiment of the three-parts biochip illustrated in figure 4, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110 and the observational interface 115 disposed on the culturing chamber 110, the bottom support 120 forming the bottom of the culturing chamber 110. The culturing chamber 110 is thus formed by the bottom support 120 and the intermediate plate 130 being assembled. The intermediate plate 130 may also comprise a reservoir hole 134 forming the side walls and the bottom of the reservoir 102, the cover 140forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 and the cover 140 being assembled. In this embodiment, the cover 140 comprises an observing hole 145 in front of the observation opening 112 to avoid observational obstruction by the cover 140. In this advantageous embodiment, the bottom support 120 which is contaminated due to cell attachment and growth is single use whereas the intermediate plate 130 may be reused.

[0133] In a sixth embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming the side walls and the bottom of the culturing chamber 110 and the observational interface 115 disposed on the culturing chamber 110. The culturing chamber 110 is thus formed only in the intermediate plate 130. The intermediate plate 130 may also comprise a reservoir hole 134 forming side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102 and the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. In this embodiment, the cover 140 comprises an observing hole 145 in front of the observation opening 112 to avoid observational obstruction by the cover 140.

[0134] In a seventh embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the cover 140 comprising the observational interface 115 disposed on the culturing chamber 110 and the bottom support 120 forming the bottom of the culturing chamber 110. The culturing chamber 110 is thus formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. The intermediate plate 130 may also comprise a reservoir hole 134 forming side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102 and the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. In this advantageous embodiment, the bottom support 120 and the intermediate plate 130 which are contaminated due to cell attachment and growth are single use whereas the cover 140 may be reused.

[0135] In an eight embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls of the culturing chamber 110, the cover 140 comprising the observational interface 115 disposed on the culturing chamber 110, and the bottom support 120 forming the bottom of the culturing chamber 110. The culturing chamber 110 is thus formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. The intermediate plate 130 may also comprise a reservoir hole 134 forming side walls of the reservoir 102 and the bottom of the reservoir 102, the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 and the cover 140 being assembled. In this advantageous embodiment, the bottom support 120 and the intermediate plate 130 which are contaminated due to cell attachment and growth are single use whereas the cover 140 may be reused.

[0136] In a ninth embodiment of the three-parts biochip, the intermediate plate 130 may comprise a chamber hole 132 forming side walls and the bottom of the culturing chamber 110, the cover 140 comprising the observational interface 115 disposed on the culturing chamber 110. The culturing chamber 110 is thus formed by the intermediate plate 130 and the cover 140 being assembled. The intermediate plate 130 may also comprise a reservoir hole 134 forming side walls of the reservoir 102, the bottom support 120 forming the bottom of the reservoir 102 and the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the bottom support 120, the intermediate plate 130 and the cover 140 being assembled. In this advantageous embodiment, the intermediate plate 130 which are contaminated due to cell attachment and growth is single use whereas the cover 140 may be reused.

[0137] The features of the cover 140, the intermediate plate 130 and the bottom support 120 described hereafter may apply to all the embodiments related to the two-parts biochip and to the three-parts biochip.

[0138] Suitable materials for the cover 140 are biocompatible and also suitable for the structural integrity of the biochip 100. Examples of suitable materials for the cover 140 include, without limitation: standard polymer, for example polystyrene (PS), polypropylene (PP), or polyethylene (PE), suitable for their chemical resistance;biocompatible polymers for 3D printing, for example acrylic polyester resin, polylactic acid (PLA), polyethylene glycol diacrylate (PEGDA); ceramics, for example alumina (AI2O3) or zirconia (ZrCE); or glass.

[0139] The observing hole 145 allows access for optical measurements, i.e., allow the primary beam light to attain the cell culture. It also facilitates the approach of spectroscopy and / or microscopy objectives to the culturing chamber 110, especially to each end of the culturing chamber 110 (see figure 12). The cover 140 then does not obstruct the optical path of the spectrometer, allowing for clear and unimpeded access to the culturing chamber 110 for precise focusing and spectroscopy measurement. This also allows for the focused analysis of specific areas of interest within the biochip 100.

[0140] Suitable materials for the intermediate plate 130 are biocompatible and also suitable for the structural integrity of the biochip 100. Examples of suitable materials for the intermediate plate 130 include, without limitation: standard polymer, for example polystyrene (PS), polypropylene (PP), or polyethylene (PE), suitable for their chemical resistance; biocompatible polymers for 3D printing, for example acrylic polyester resin, polylactic acid (PLA), polyethylene glycol diacrylate (PEGDA); ceramics, for example alumina (AI2O3) or zirconia (ZrCE); or glass.

[0141] Suitable materials for the bottom support 120 are biocompatible and also suitable for the structural integrity of the biochip 100. Such materials are not necessarily transparent to primary beam of light, ideally, they are non-permeable to liquid and gas. Examples of suitable materials for the bottom support 120 include, without limitation: standard polymer, for example polystyrene (PS), polypropylene (PP), or polyethylene (PE), suitable for their chemical resistance, manufacturing ease, and application versatility; advanced optical polymers, for example cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), recognized for their excellent optical clarity and low autofluorescence, optimizing them for imaging applications; high-temperature polymers, for example polycarbonate (PC) or polyethylene terephthalate (PET), known for their high temperature and chemical resistance, ideal for sterilization processes; high-durability polymers, for example acrylonitrile butadiene styrene (ABS), silicone or polydimethylsiloxane (PDMS), valued for their toughness and flexibility, suitable forspecialized cell culture applications requiring mechanical strength or specific nonadhesive properties; biocompatible polymers for 3D printing, for example acrylic polyester resin, polylactic acid (PLA), polyethylene glycol diacrylate (PEGDA), polyethylene terephthalate glycol (PETG), or thermoplastic polyurethane (TPU), offering high-resolution fabrication capabilities, ease of sterilization, and direct cell contact compatibility; high-performance and metal-coatable polymers, for example polyetheretherketone (PEEK), polysulfone (PSU), polyethersulfone (PES), or metal- coated versions of standard and 3D printing polymers (e.g., PLA, PETG, ABS), providing structural integrity, thermal stability, and enhanced Raman signal through metal reflectance and low fluorescence; ceramics, for example alumina (AI2O3) or zirconia (ZrCE); glass or metal-coated glass, offering extreme rigidity, stability, and the option for metal coating to improve Raman spectroscopy outcomes.

[0142] When forming the bottom of the culturing chamber 110, the bottom support 120 may be functionalized for cell attachment as described hereabove.

[0143] As illustrated in figures 5 and 8, when the culturing chamber 110 is formed by the assembly of the intermediate plate 130 and the bottom support 120, a chamber sealing element 152 may be positioned between the bottom support 120 and the intermediate plate 130 thereby sealing a bottom of the culturing chamber 110. The chamber sealing element 152 is preferably liquid tight or gas tight.

[0144] When reservoir 102 is formed by the assembly of the intermediate plate 130 and the bottom support 120, a reservoir sealing element (not shown in figures) may be positioned between the bottom support 120 and the intermediate plate 130 thereby sealing a bottom of the reservoir 102. The reservoir sealing element is preferably liquid tight or gas tight.

[0145] When the reservoir 102 is formed by the assembly of the intermediate plate 130 and the cover 140, a reservoir sealing element (not shown in figures) may be positioned between the cover 140 and the intermediate plate 130 thereby sealing a top of the reservoir 102. The reservoir sealing element is preferably liquid tight or gas tight.

[0146] When reservoir 102 is formed by the assembly of the bottom support 120, the intermediate plate 130 and the cover 140, a reservoir sealing element (not shown in figures) may be positioned between the bottom support 120 and the intermediate plate 130 thereby sealing a bottom of the reservoir 102. The reservoir sealing element is preferably liquid tight or gas tight, whereas the cover 140 does not seal perfectly the reservoir 102, allowing gas diffusion therein.

[0147] The bottom support 120 and / or intermediate plate 130 may also comprise a bottom sealing element 154 disposed close to the circumference of the biochip 100 between the bottom support 120 and the intermediate plate 130 preventing leakage from the biochip 100 (figure 5). The bottom sealing element 154 is preferably liquid tight or gas tight. The bottom sealing element 154 is preferably a polymeric gasket.

[0148] The cover 140 and / or intermediate plate 130 may also comprise a top sealing element (not shown in figures) disposed close to the circumference of the biochip 100 between the cover 140 and the intermediate plate 130 preventing leakage from the biochip 100. The top sealing element is preferably liquid tight or gas tight.

[0149] The sealing elements cited hereabove may comprise a flexible and elastically deformable material such as, for example, an elastomer such as silicone and rubber. A groove may be provided in the biochip part opposite from the part comprising the sealing element so that the sealing element is nested into said groove upon sealing of the two parts. This improves the sealing of the two parts of the biochip 100 by ensuring a secure and effective seal, while not increasing the size, especially the height, of the biochip 100. This also provide enhanced durability as the sealing element is better protected from wear and tear when nesting in a groove, extending its lifespan and maintaining the integrity of the seal over time.

[0150] The biochip 100 may comprise a fastening element 150 configured to maintain the intermediate plate 130 abutted against the bottom support 120.

[0151] The biochip 100 may comprise a fastening element 150 configured to maintain the intermediate plate 130 abutted against the cover 140.

[0152] The biochip 100 may comprise a fastening element 150 configured to maintain the intermediate plate 130, the bottom support 120 and the cover 140 abutted.

[0153] The fastening element 150 may be a clamp as in figures 2 and 3. For example, the fastening element 150 may be a lever vice configured to evenly distribute a pressure along the abutted parts, may be at least one screw, may comprise magnets, may be a click system, or may include a lip or ridge that aligns with a complementary groove to create an audible snap fit when engaged, ensuring a secure seal. Additionally, the fastening element 150 could use interlocking features, such as hooks and corresponding openings, which may be further secured by magnetic forces to facilitate a strong yet detachable connection of each part of the biochip 100.

[0154] The biochip 100 may further comprise at least one chamber volume adjustment system configured to adjust a volume of the culturing chamber 110 between at least a first volume and a second volume (see figures 4 and 7-11). Volume adjustment refers to a change of the culturing chamber volume to increase or reduce said volume. The volume of the chamber is the capacity of the chamber, i.e., the quantity of liquid (such as a medium) that the culturing chamber can contain. In other words, the variation of volume corresponds to a variation of at least one of the dimensions of the culturing chamber. This is different from a variation of volume of liquid such as cultivation medium contained in the culturing chamber. In other words, the chamber volume adjustment system does not correspond to a valve or pump system configured to inject or remove liquid from the culturing chamber. The volume adjustment (or dimension adjustment) may be continuous or discrete. A discrete volume adjustment means increasing or reducing the culturing chamber volume by a predefined volume step, i.e., there are discrete adjustment volume steps. Said volume step may be ranging from 10 mm3to 100 mm3, preferably from 25 mm3to 70 mm3. The chamber volume adjustment system may be configured to perform increase or decrease of several volume steps. For example, as illustrated in figure 11, the chamber volume adjustment system comprises 3 volume steps (volume of culturing chamber hatched), meaning that three different culturing chamber volumes may be chosen, depending on the experiment or monitoring to be performed. Adjusting the culturing chamber volume advantageously allows to modulate the gradients in theculturing chamber 110 as it directly influences the diffusion rates and concentration gradients of substances within the culturing chamber 110 due to displaced liquid volume and pressured induced. Furthermore, it is possible, in this embodiment, to dynamically adjust the culturing chamber volume to a chosen volume to dynamically alter chamber conditions as the volume available will change. This can be done to simulate changes in the cells environment or to introduce different experimental stimuli.

[0155] Volume increasing may be performed by inserting, before cell culture, a height adjustment plate between the intermediate plate 130 and the bottom support 120. The height adjustment plate comprising a hole aligned with the culturing chamber 110.

[0156] In a preferred embodiment, volume reduction may be performed by inserting a given volume of a solid element in the culturing chamber 110. The volume of the culturing chamber is thus reduced by the volume of the solid element inserted in the culturing chamber. In other words, the maximal quantity of liquid that can be contained in the culturing chamber is reduced by the volume of the solid element inserted in the culturing chamber. Volume increasing may be performed by removing a given volume of a solid element from the culturing chamber 110. Preferably the insertion and removing of this solid element is performed by maintaining the sealing of the culturing chamber 110. This allows to vary the volume of the culturing chamber 110 during cell culture without interrupting the experiment. A gasket may be used to fix the solid element in place if it will not be moved during the experiment. Materials suitable for the solid element should be biocompatible and adapted for the structural integrity of the biochip 100. Examples of suitable materials for the solid element include, without limitation: standard polymer, for example polystyrene (PS), polypropylene (PP), or polyethylene (PE), suitable for their chemical resistance; biocompatible polymers for 3D printing, for example acrylic polyester resin, polylactic acid (PLA), polyethylene glycol diacrylate (PEGDA); ceramics, for example alumina (AI2O3) or zirconia (ZrCE); or glass.

[0157] Removal of the solid element from the culturing chamber 110 also allows for integration with other fluid channels. When retracted, the solid element could uncover additional openings or pathways, facilitating complex fluid manipulation tasks such as sequential reagent delivery, sample partitioning, or the creation of dynamic gradientswithin the biochip 100.

[0158] For example, the culturing chamber 110 further comprises a piston hole and the chamber volume adjustment system comprises a piston 162 having, as the solid element, a piston section fitting the piston hole, preferably in a leak tight manner. The piston hole may be disposed at different positions relative to the culturing chamber 110. For example, the piston hole may be disposed on the top of the culturing chamber 110, next to the observation opening 112. The piston hole is preferably disposed on the bottom, more preferably on at least one of side walls of the culturing chamber 110. The piston 162 being movable in the culturing chamber 110 to adjust the volume of the culturing chamber 110. For example, a piston sealing element 156 is disposed on the circumference of the piston 162. For example, a sealing element is disposed on the circumference of the piston hole. For example, a piston sealing element 156 is disposed on the circumference of the piston 162 and a second sealing element (not shown in the figures) is disposed on the circumference of the piston hole. The sealing elements may be formed with a flexible and elastically deformable material such as an elastomer, for example silicone or rubber.

[0159] The piston 162 is movable in the culturing chamber 110 to adjust the volume of the culturing chamber 110: as the piston 162 advances inside the culturing chamber 110, the dimension of the culturing chamber 110 parallel to the piston advancement is reduced and thus the volume of the culturing chamber 110 decreases; as the piston 162 is extracted from inside the culturing chamber 110, the dimension of the culturing chamber 110 parallel to the piston advancement is increased and thus the volume of the culturing chamber 110 increases.

[0160] Preferably, the piston 162 is configured to slide along a sliding axis S. The sliding axis S differs from the perfusion axis P according to a non-zero angle. The sliding axis S is preferably perpendicular to the perfusion axis P as illustrated in figure 11. A perpendicular sliding axis S will produce more pronounced gradients along the perfusion axis P. In an alternative configuration, the sliding axis S may be parallel to the perfusion axis P.

[0161] The piston 162 may be continuously moveable or moveable by steps, thus adjusting the volume of the culturing chamber 110 in a continuous or discrete manner, as explained hereabove. In this last case, the piston sealing elements 156 are only needed at the positions relative to volume steps.

[0162] The piston 162 may be moved directly by the user. Alternatively, the chamber volume adjustment system may comprise an actuator 164 configured to move the piston 162 along the sliding axis S. For example, the actuator 164 may be actuated manually or electronically.

[0163] The actuator 164 may comprise a cam 164a. In this case, the cam 164a is in contact with the piston head 162a of the piston 162 through a piston extension 162b which is a cam follower. A rotation of the cam 164a implies a translation of the piston head 162a along the sliding axis S, resulting in volume adjustment of the culturing chamber 110.

[0164] The cam 164a may be in form of an eccentrically mounted round disc or a disk with varying curvature.

[0165] The cam 164a may be in the form of a disk comprising an offset rotation axis. A fixed point on the circumference thus moves along an ellipse and the cam follower 162b has back and forth movement leading to a sliding of the piston head 162a along the sliding axis S.

[0166] As illustrated in figures 4 and 11, one chamber volume adjustment system comprises 3 volume steps, i.e., the piston 162 can be moved at 3 discrete positions to increase or decrease the culturing chamber volume. In figure 11, the rotation axis of the cam 164a at the 3 discrete positions are aligned on the dashed line for comparison.

[0167] The cam 164a may be in the form of an ellipse or all other form allowing a translation movement of the piston 162 along the sliding axis S.

[0168] The cam 164a may be rotated using a shaft 164b actuated manually or electronically.

[0169] The actuator 164 may comprise a return element such as a spring or rubber. The piston 162 and optionally the cam 164a may be disposed in a gas tight chamber set in low pressure compared to the culturing chamber 110 so that when the cam 164a does not actively push the piston 162 towards the culturing chamber 110, the piston 162 is pulled out the culturing chamber 110. This return mechanism ensures the volume adjustment system’s resilience and enables repetitive operations without manual resetting.

[0170] The actuator 164 may comprise a pressure adjustment system. In this case, the piston 162 is disposed in a gas tight chamber connected to the pressure adjustment system. As the pressure increases, overpressure is created in the gas tight chamber leading the piston 162 to be pushed inside the culturing chamber 110, resulting in its volume reduction. As the pressure decreases, lower pressure is created in the gas tight chamber leading the piston 162 to be pulled back from the culturing chamber 110, resulting in its volume increase.

[0171] The above description is for a biochip 100 comprising a single reservoir 102 and a single culturing chamber 110. However, the biochip 100 may comprise several culturing chambers 110 all in fluid communication with the same reservoir 102. The biochip 100 may also have several reservoirs 102 in fluid communication with the same culturing chamber 110 (for several media). All the above embodiments are compatible.

[0172] The invention also relates to a multi -well system 200 comprising at least two biochips 100 as described above (see figures 1 and 2).

[0173] The multi-well system 200 may comprise an array of biochips 100.

[0174] Preferably, the multi -well system 200 comprises 6 biochips 100 as illustrated in figures 1 and 2 (where a single bioship is represented by dot-dashed rectangle).

[0175] Each biochip 100 of a multi-well system 200 may comprise cooperating means configured to cooperate with cooperating means of at least another biochip 100 to fasten said biochips 100 together. Examples of said cooperating means include, without limitation, magnets, mortises, tenons, clamps, screws, or hooks and loops band.

[0176] The multi-well system 200 may have the following dimensions: length ranging from 10 mm to 200 mm, preferably from 50 mm to 150 mm, width ranging from 10 mm to 200 mm, preferably from 50 mm to 100 mm, and / or height ranging from 1 mm to 100 mm, preferably from 5 mm to 50 mm, more preferably from 10 mm to 15 mm.

[0177] For example, a multi-well system 200 comprising 6 biochips 100 has the following dimensions: length ranging from 10 mm to 200 mm, preferably from 50 mm to 150 mm, width ranging from 10 mm to 200 mm, preferably from 50 mm to 100 mm, and / or height ranging from 1 mm to 100 mm, preferably from 5 mm to 50 mm, more preferably from 10 mm to 15 mm.

[0178] In the case of a multi -well system 200 comprising biochips 100 according to two- parts or three-parts embodiments, the bottom support 120 and the cover 140 may be a single piece for several cooperating biochips 100.

[0179] The present invention also relates to a method for manufacturing a biochip 100 as described hereabove.

[0180] The biochip 100 may be 3D printed as one piece.

[0181] In the case of a biochip 100 according to two-parts or three-parts embodiments, the bottom support 120, the intermediate plate 130 and the cover 140 may be 3D printed and then assembled, using sealing elements and optionally fastening elements 150 to abut parts together.

[0182] The present invention also relates to a method for the assembly of a multi -well system 200 as described hereabove.

[0183] Said assembling method may comprise the following steps: providing at least two biochips 100; assembling the biochips 100 using cooperating means of two adjacent biochips 100.

[0184] Alternatively, said assembling method may comprise 3D printing at least two biochips 100 as a sole piece of equipment.

[0185] The present invention also relates to a method for performing a spectroscopy measurement on a cell culture, comprising: providing a biochip 100 or a multi -well system 200, wherein a cell culture is present in the culturing chamber 110 and a perfusion fluid is present in the reservoir 102; using a spectrometer to acquire a spectroscopy measurement through the observational interface 115 of the culturing chamber 110.

[0186] The acquisition of spectroscopic measurement may comprise: positioning the biochip 100 in the light path between the light source and the spectroscopic detector; generating a primary beam of light by the light source in direction of the observation opening 112 through the observational interface 115; acquiring a secondary beam of light (transmitted, reflected or scattered) generated in response by the cell culture and detected by spectroscopic detector through the observational interface 115; generating a detection signal representative of this secondary beam of light; determining a raw spectrum from the detection signal.

[0187] The present invention also relates to the use of the biochip 100 described in this disclosure.

[0188] For example, such a biochip 100 may be used for monitoring the metabolic activity of cells in a cell culture, label-free monitoring of cell culture, monitoring protein production, monitoring viral production, monitoring the manufacturing of therapeutic cells, monitoring real-time effects of virus or bacteria on cells, monitoring of the effects of drugs on cells, monitoring a disease evolution in a cell culture, biomass monitoring, monitoring of (metabolic) reactions of cells subject to any kind of stress imposed, or artificial growth monitoring.

[0189] The biochip 100 of this disclosure may also be used as a fluidic device with small gap, and hence, low diffusion / perfusion to observe effects of any liquid or particlessuspended in a liquid subject to manipulation or stimuli through the fluid inlet 117, e.g. perfusion of solvents, acids or alkaline solutions that can modify a polymeric material (particles or liquid); enzymatic reactions of polymeric micro / nanoparticles in a liquid; or a Surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS) experiment wherein colloidal noble metal nanoparticles are dispersed in a liquid in the culturing chamber 110 and molecules, biomaterial, or cells enter the culturing chamber 110 through the fluid inlet 117 producing a gradient along the perfusion axis in the culturing chamber 110 , and a concentration-resolved SERS signal can be observed by Raman spectroscopy.EXAMPLE

[0190] The present invention is further illustrated by the following example of protocol for cell culture allowing to measure the oxygen gradient by Raman spectroscopy.Materials and Methods

[0191] For this experiment a three-parts biochip 100 as illustrated in figure 4 is provided

[0192] This biochip 100 comprises: a bottom support 120; an intermediate plate 130 disposed on the bottom support 120; a cover 140 disposed on the intermediate plate 130, opposite to the bottom support 120. wherein the intermediate plate 130 comprises a chamber hole 132 forming side walls of the culturing chamber 110 and the observational interface 115 disposed on the culturing chamber 110, the bottom support 120 forming the bottom of the culturing chamber 110. The culturing chamber 110 is thus formed by the bottom support 120 and the intermediate plate 130 being assembled. The intermediate plate 130 also comprises a reservoir hole 134 forming the side walls and the bottom of the reservoir 102, the cover 140 forming the top of the reservoir 102. Therefore, the reservoir 102 is formed by the intermediate plate 130 and the cover 140 being assembled. The cover 140 comprises anobserving hole 145 in front of the observation opening 112 to avoid observational obstruction by the cover 140. The three parts are fastened together using a click system.

[0193] The culturing chamber 110 thus formed comprises a fluid inlet 117 defining a perfusion axis P, the culturing chamber 110 being in fluid communication with the reservoir 102 through the fluid inlet 117. The culturing chamber 110 further comprises an observation opening 112 extending along the perfusion axis P, and the biochip 100 further comprises a 0.2 mm thick quartz observational interface 115 disposed on the observation opening 112. The culturing chamber 110 further comprises a piston hole, the chamber volume adjustment system comprising a piston 162 having a piston section fitting the piston hole in a leak tight manner and an actuator 164 configured to slide the piston 162 along a sliding axis S, the actuator 164 being a cam 164a.

[0194] The dimensions of the reservoir 102 are: length of 32 mm, width of 15.5 mm, height of 6.25 mm. The dimensions of the culturing chamber 110 are: length of 13 mm, width of 6 mm, height of 2.45 mm.

[0195] Experiment is performed as follows:

[0196] In a first step, the intermediate plate 130 is sterilized using UV irradiation and ethanol to ensure a sterile environment and prevent contamination. At this step, the biochip 100 is manipulated in a sterilized hood.

[0197] In a second step, the volume of the culturing chamber 110 is adjusted before cell culture and a gasket is used to fix the piston 162 in place.

[0198] In a third step, the intermediate plate 130 is abutted and fastened on single use bottom support 120.

[0199] In a fourth step, cells are introduced and seeded into the culturing chamber 110, and the reservoir 102 is filled with a volume of cultivation medium needed to completely fill the culture chamber. The bubbles are removed.

[0200] After seeding, the cells are allowed time to attach to the functionalized area of the culturing chamber 110.

[0201] In a fifth step, once the cells have adhered, the remainder of the cultivation medium is added to the reservoir 102.

[0202] In a sixth step, the cover 140 is abutted and fastened to the intermediate plate 130. The biochip 100 is now ready to be removed from sterilized hood.

[0203] In a seventh step, Raman spectroscopy is performed on the biochip 100. The Raman system 300 is positioned close to observational interface 115 to focus on areas of interest within the culturing chamber 110. As represented in figure 12, the Raman system 300 irradiates the culturing chamber 110 from the top of the biochip 100 through the observational interface 115. Spatial -temporal data is collected by irradiating different points (scanning).Results

[0204] Spectra obtained from the Raman system, shown in figure 13, are analyzed using multivariate analysis techniques to monitor changes in important metabolites, biomass, pH, viability, etc., across different regions of the biochip 100.

[0205] Figure 13 A shows three distinct spectra, acquired at different times but within the same region of the culturing chamber 110 during an observation period from Day 0 to Day 2 (the curve corresponding to Day 0 is represented in dark grey, the curve corresponding to Day 1 is represented in mild grey, the curve corresponding to Day 2 is represented in light grey). These spectra reveal significant changes over time that are key to understanding the biochemical behavior of the cell culture under study. Specifically, there is an increase in the peak corresponding to lactate, which reaches its maximum intensity around 855 cm’1, suggesting an increase in lactate concentration over time (see figure 13B). Simultaneously, there is a notable decrease in the glucose peak at 1125 cm’ indicating a reduction in glucose in the sample (see figure 13D). Additionally, a significant shift of the peak from 1045 cm’1points to an acidification of the medium (see figure 13C). These spectral changes are crucial for biochemical analysis and provide valuable insights into the chemical dynamics of the cell culture being studied.REFERENCES

[0206] 100 - Biochip / / 102 - Reservoir / / 110 - Culturing chamber / / 112 - Observation opening / / 115 - Observational interface / / 117 - Fluid inlet / / 118 - Sensor / / 120 - Bottom support / / 125 - Positioning element / / 130 - Intermediate plate / / 132 - Chamber hole / / 134 - Reservoir hole / / 140 - Cover / / 145 - Observing hole / / 150 - Fastening element / / 152 - Chamber sealing element / / 154 - Bottom sealing element / / 156 - Piston sealing element / / 158 - Calibration element / / 162 - Piston / / 162a - Piston head / / 162b - Piston extension / / 164 - Actuator / / 164a - Cam / / 164b - Shaft / / 200 - Multi -well system / / 300 - Raman system / / A - sectional drawing / / B, C - Zoom-in view / / P — Perfusion axis / / S - Sliding axis

Claims

CLAIMS1. A biochip (100) comprising: a reservoir (102); a culturing chamber (110) comprising a fluid inlet (117) defining a perfusion axis (P), the culturing chamber (110) being in fluid communication with the reservoir (102) through the fluid inlet (117); wherein, the culturing chamber (110) comprises an observation opening (112) extending along the perfusion axis (P), the biochip (100) further comprising an observational interface (115) disposed on the observation opening (112); wherein the observational interface (115) is configured to allow spectroscopy measurement of the culturing chamber (110) through the observational interface (115) absorbing less than 20%, preferably less than 10%, of light at visible, NIR and / or UVA wavelengths; wherein the biochip (100) further comprises:- a bottom support (120); and- an intermediate plate (130) disposed on the bottom support (120) and comprising a chamber hole (132) forming walls of the culturing chamber (110), the bottom support (120) and the observational interface (115) disposed on opposite sides of the chamber hole (132) closing the culturing chamber (110); wherein the biochip (100) further comprises a cover (1 0) configured to be disposed on the intermediate plate (130), the cover (140) being configured to close the reservoir (102), the cover (140) comprising an observing hole (145) in front of the observation opening (112).

2. The biochip (100) according to claim 1, wherein a surface of culturing chamber (110) opposite the observation opening (112) is functionalized to promote adhesive behavior of cells.

3. The biochip (100) according to claim 1 or 2 further comprising a chamber sealing element (152) positioned between the bottom support (120) and the intermediate plate (130) thereby sealing a bottom of the culturing chamber (110).

4. The biochip (100) according to any one of claims 1 to 3, further comprising a fastening element (150) configured to maintain the intermediate plate (130) abutted against the bottom support (120).

5. The biochip (100) according to any one of claims 1 to 4, wherein the spectroscopy measurement is a Raman spectroscopy measurement, the observational interface (115) being a plate made of a material transparent to laser light used in Raman spectroscopy and emitting in a range of wavelengths from 190 nm to 1400 nm, the material absorbing less than 20%, preferably less than 10%, of light at said range of wavelengths.

6. The biochip (100) according to claim 5 wherein the observational interface (115) comprises quartz, silicon, calcium fluoride CaF2, barium fluoride BaF2, or fused silica.

7. The biochip (100) according to any one of claims 1 to 6, wherein the reservoir (102) is disposed at an end of the culturing chamber (110) along the perfusion axis (P).

8. The biochip (100) according to any one of claims 1 to 6, further comprising a chamber volume adjustment system configured to adjust a volume of the culturing chamber (110) between at least a first volume and a second volume by varying at least one dimension of the culturing chamber (110).

9. The biochip (100) according to claim 8, wherein the culturing chamber (110) further comprises a piston hole, the chamber volume adjustment system comprising a piston (162) having a piston section fitting the piston hole in a leak tight manner, the piston (162) being movable in the culturing chamber (110) to adjust the volume of the culturing chamber (110).

10. The biochip (100) according to claim 9, wherein the chamber volume adjustment system comprises an actuator (164) configured to slide the piston (162) along a sliding axis (S).

11. The biochip (100) according to claim 10, wherein the sliding axis (S) is perpendicular to the perfusion axis (P).

12. The biochip (100) according to claim 10 or 11, wherein the actuator (164) comprises a cam (164a).

13. A multi -well system (200) comprising at least two biochips (100) according to any one of claims 1 to 12.

14. A method for performing a spectroscopy measurement on a cell culture, comprising: providing a biochip (100) according to any one of claims 1 to 12 or a multi-well system (200) according to claim 13, wherein a cell culture is present in the culturing chamber (110) and a perfusion fluid is present in the reservoir ( 102); using a spectrometer to acquire a spectroscopy measurement through the observational interface (115) of the culturing chamber (110).

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