Description

The present invention relates to a method for controlling quality of a mass spectrometry imaging (MSI) method for detecting one or more target species in a tissue sample; a method of quantifying a target species in a tissue sample and a slide for MSI analysis which comprises a 3D construct.

MSI is a well-recognized technology, and has become a powerful method for tissue-based disease classification and patient stratification. The power of MSI resides in the ability to detect proteins, lipids, metabolites and drugs while preserving the information on their spatial localization. By scanning the sample with a well-focused laser beam or ion gun, individual mass spectra are recorded from predefined coordinates providing within a short time, a detailed molecular fingerprint of the tissue that is investigated. Recent instrumental improvements have resulted in spatial resolution below 10 pm and scan times of just several minutes, making it suitable to significantly lower costs and improve decision making strategies.

Different laboratories use commercial liquid protein and lipid standards as a quality control standard (QCS) in MSI experiments for slide to slide comparisons or when evaluating the robustness of a particular method. However, these standards cannot mimic the complexity of a tissue biopsy and therefore they cannot be used to evaluate the performance of a protocol when evaluating tissue molecular extraction efficiency or spatial distribution preservation of a specific method.

Other labs use animal-derived tissue or organ (and therefore limited) sections to test inter experimental variability. However, the batch-to-batch differences and tissue/animal heterogeneity prevents the translation of MSI to the clinical diagnostic laboratories.

US10,132,796 relates to a method of detecting and quantifying a target molecule in a tissue with MSI, where a tissue homogenate is used to which the target molecule is added. The tissue homogenate is conditioned and sliced, and the slice obtained is used for the analysis. Conditioning can be freezing or embedding the homogenate in gelatine or paraffin.

EP3460470 relates to a method for monitoring the quality for MSI using a control sample to be processed and measured alongside the analytical tissue sections on the same sample support. This document emphasizes the need for a homogenous control sample. This document also describes the option of embedding cells and sectioning them.

One problem associated with the use of tissue or organ sections is that there are huge batch-to-batch differences, which is not desirable in QCS. Furthermore tissue sections are generally far from homogenous, and as such not desirable as the location of the section that will be used will influence the results. Using embedded cells does not overcome these problems, as this method will still not result in sufficiently homogenous samples and still have batch-to-batch differences. Furthermore, due to embedding, spatial resolution will be lost as generally the cells will break down. More importantly however, both methods would require sectioning of the tissue or embedded cells, which is very time consuming and may introduce artefacts in the sample. Therefore, sectioning of the QCS is preferably avoided. Lastly, these methods do not sufficiently allow to control the spatial cell distribution and the possibility to combine in a controlled way multiple cells and/or other constituents on the QCS.

Thus, an ideal QCS should mimic the structure of biological tissues: a cellular component embedded in an extracellular matrix (ECM). Therefore, a proper QCS must have: 1) a similar molecular content as a cell, e.g. metabolites, lipids or proteins and 2) a three dimensional (3D) ECM-like structure to overcome the above drawbacks in the state of the art.

The present invention provides a method for controlling quality of a mass spectrometry (MS) method for detecting one or more target species in a tissue sample, comprising the steps of:

(a) mixing a predetermined amount of the one or more target species with a carrier, creating a mixture;

(b) depositing by means of a 3D printing method the mixture obtained in step (a) on a first area of a slide, creating a 3D construct;

(c) depositing the tissue sample on the slide in a second area of the slide; and

(d) performing MS analysis of both the first and second area of the slide, wherein the carrier is a substance that mimics an ECM selected from natural or synthetic biomaterial inks. Preferably the MS method is an MSI method.

It was found that by modifying 3D printing technology to create QCS for MSI, several advantages compared to prior described QCS methods were found. First of all, using 3D printing methods allows for a more controlled distribution of cells when compared to slices of tissue samples or slices of embedded cells. Second, the method allows for fine-tuning the selection of the mixture (e.g. cells and carrier) to be printed to closely resemble the tissue sample to be analysed, which is desirable in a QCS. Third, the printing method allows for very little variation when preparing multiple slides or preparing slides at different times, thereby avoiding the problem of batch to batch variation of tissue sections when used as QCS. Fourth, since one can control the printed material and use a pre-defined pattern, one can easily evaluate artefacts during sample preparation for MSI such as analyte diffusion.

The mixture obtained in step (a) is deposited by means of a 3D printing method. Recently, 3D bioprinting techniques have become available, such as ink-jet and extrusion, which use biocompatible “inks” that have the ability to print customizable self-supporting cell-laden structures for soft tissues. When used herein, the term “biomaterial ink” refers to a material suitable for use in bioprinting. The material may have an ECM-like structure as defined herein. Preferably the biomaterial ink is suitable for including cells and to be used in bioprinting. When used herein the term “bioink” or “bio-ink” (used interchangeably) refers to a material suitable for use in bioprinting which includes cells. Therefore, when used herein a bioink is defined as a biomaterial ink which includes cells.

Bioprinting is a group of new technologies that allows an accurate deposition of biomaterials, cells and other biologicals individually or in a combined way termed bioink. These materials can be selectively dispensed with different actuation techniques (e.g. pressure control, microfluidics, droplet-on-demand, laser, etc.). Single or multiple print heads with combinations of actuation techniques are currently available allowing deposition strategies such as continuous extrusion (layer-by-layer) and droplet by droplet. Bioink, cross-linkers and/or buffer solutions are placed in reservoirs or carriers and supplied to the extrusion nozzles normally equipping the print head. The crosslinking can be performed during or post-printing. The droplet or filament diameter and biomaterial composition can be tuned and combined with a specific bioprinting principle.

When used herein, the term 3D printing method may therefore refer to bioprinting, however the term may also refer to any other method where an automated dispensing system controlled by a microprocessor (e.g. a pipetting robot) is used. Therefore in an embodiment, the 3D printing method is bioprinting or a method using an automated dispensing system controlled by a microprocessor.

In order to implement the 3D printing method in MSI, the inventors have adapted the printing methods from conventional methods to overcome several technical hurdles. For example, in bioprinting generally a crosslinking agent is used. The purpose of this agent is to retain the shape of the fluid-like printed medium. For the purpose of MSI this is not desirable, because first of all the crosslinking agent might introduce unwanted salts and second the crosslinking may affect the mass spectra of the molecules to be analysed. This is particularly the case when analysing peptides or proteins. The inventors found that the crosslinking step can be avoided by applying a drying step after the depositing of the mixture by a 3D printing method. Doing so, avoided uncontrolled diffusion of the deposited mixture on the slide. Therefore in a preferred embodiment, the depositing of the mixture comprises a drying step.

Bioprinting methods generally use a buffer for the substance to be printed. It was found by the inventors that using a buffer negatively impacted the distribution of the carrier. As demonstrated in figure 10, Tris buffered saline (TBS), which is typically used for some biomaterial solution preparation for bioprinting, negatively impacts the distribution of sodium alginate, while a homogenous distribution is observed when no buffer is used (figure 11). Therefore in a preferred embodiment, the carrier is not dissolved or included in a buffer.

It was found that using the printing method described here allows for easy generation of a gradient or standard of different concentrations of the target species. The target species may for example be an analyte. This can for example be achieved by printing multiple structures and applying different analyte concentrations, or alternatively by printing structures with different concentrations of analyte premixed. An additional advantage is that by printing distinct structures which are spaced (as opposed to a solid surface as is the case in a tissue section), diffusion of the analyte can be observed. This information can then be applied to the sample in order to correct for analyte diffusion. Therefore, in a preferred embodiment the 3D construct is a grid, one or more filaments or one or more spots.

In the method of the invention, the carrier is preferably a substance that mimics an ECM. The carrier is selected from natural or synthetic biomaterial inks, wherein the natural biomaterial inks are preferably selected from the group consisting of alginates, collagen, gelatin, fibrin, silk, dextran, agarose, hyaluronic acid, chitosan and mixtures thereof; and the synthetic biomaterial inks are selected from polyethylene glycol and polyvinyl alcohol and mixtures thereof. When used herein, a substance that mimics an ECM refers to a viscous fluid or a gel-like substance suitable for embedding cells. Therefore, it is understood that the carriers listed above are dissolved in a suitable solvent to create a viscous fluid or gel-like substance. A suitable solvent for the purposes described herein may be water or an aqueous solution.

A preferred biomaterial ink is an alginate. Although a biomaterial ink such as alginate is typically combined with a crosslinker, for the purpose of the invention preferably no crosslinker is used. One such biomaterial ink is described in PCT/EP2019/080507, which is incorporated by reference in its entirety.

For optimal performance of the MSI analysis, the 3D construct comprises one or more spots, one or more filaments or one or more grids. The thickness of the deposited mixture is from 1 to 100 pm. In particular the 3D construct is a grid with a spacing of from 5x5 to 1000x1000 pm.

The predefined shape of the bioprinted construct allows to evaluate different parameters of the MSI method such as: 1) spatial resolution, 2) molecular delocalization and 3) efficiency of the analyte extraction. Finally, the morphological characteristics of the construct can be used for instrument (spatial) calibration reducing time and costs.

In an embodiment the biomaterial ink further comprises cells. The cells may be living cells or cells that have been fixed, growth arrested or otherwise treated. Preferably the cells are living cells.

The target species to be analysed can be any chemical or biological substance that can be present in a biological tissue sample. The target species is preferably selected from the group consisting of proteins, peptides, amino acids, nucleic acids, lipids, drugs, metabolites, cells and mixtures thereof. Drugs (pharmaceutically active agents) can include so called “small molecules” but can also be “biologies”, e.g. antibodies or peptides.

The method of the invention allows the simultaneous analysis of multiple target species. This can be achieved by incorporating several target species in the mixture to be deposited as a construct. The mixture can for instance be a mixture of cells with a known drug or metabolite. When the target species comprises cells, it is preferred to deposit the cells as a single cell layer. The cells are preferably homogenously distributed cells at from 10 to 10.000 cells/mm ² .

The target species may be a metabolite or analyte, in which case the metabolite or analyte is preferably included in the carrier when printed on the slide. For example, when analysing the distribution and quantity of a compound in the tissue sample the compound is preferably present in the 3D construct. The compound may be present with the carrier when printing, however depending on the compound and preparation this may diffuse. This may be particularly useful when a standard is created by printing several spots or filaments with each a predefined different concentration of the compound, and sample preparation, storage or handling might disturb tissue and control sample quality. The diffusion of the target species in the 3D construct may be used to determine the diffusion of the target species in the sample. The QCS may be designed such that from the observed target species diffusion in the 3D construct, the diffusion in the sample can be determined and the original location and/or concentration of the target species can be approximated based on this information.

In an alternative embodiment, the target species may be applied separately, meaning it is not included in the carrier. For example, it can be applied in a second step of (separate) bioprinting or by different means such as applying a solution containing the target species to the slide and/or the 3D construct.

The tissue sample is a biological tissue sample, preferably from an animal, in particular a human. Tissue (e.g. muscle, tendon, etc.) and organs (e.g. liver, kidney, brain, pancreas, skin, heart, etc.) can be used as a sample. The tissue or organ sample can be obtained by methods known by a person skilled in the art. It is generally a tissue slice with a thickness of several micrometers.

As described above, the 3D construct of the target species is deposited in one area of the slide, not overlapping with the second area where the tissue sample is deposited. The second area will generally be larger than the first area.

The slide as described above, can be any slide that is known for MS or microscopic analysis. Such slides can be made of glass, metal, e.g. stainless steel, ceramic or plastic, or of any of such materials coated with a (conductive) metal layer, such as an indium tin oxide (ITO) coated glass slide. In general, the slide has a size of about 10-150 mm in length and 10-150 mm in width. The thickness of the slide will generally be from 0.1 to 10 mm.

The MS method can be any MS method, such as tandem mass spectrometry (MSn), multiple reaction monitoring (MRM), single reaction monitoring (SRM). Preferably, the method of the invention is used with an MSI technology using ionization from different sources such as MALDI (Matrix-Assisted Laser Desorption- Ionization), LDI (Laser Desorption-Ionization), LESA (Liquid Extraction Surface Analysis), LAESI (Laser Ablation Electrospray Ionization), DESI (Electrospray Desorption-Ionization), NanoDESI, SIMS (Secondary Ion Mass Spectrometry), combined with different types of analysers, such as TOF (Time Of Flight), Orbitrap, FTICR (Fourier Transform Ion Cyclotronic Resonance).

These imaging techniques make it possible to quantify the target molecule directly on the ion density map obtained for the tissue sample, corresponding to the spatial distribution of the target molecule in said tissue sample. It is in fact possible to transfer the signal obtained on said ion density map to the corresponding dilution range.

Preferably, the MS method is an MSI method. In particular, a MALDI (matrix assisted laser desorption/ionization) MSI method is used.

Some MS techniques, such as MALDI or Matrix enhanced (ME)-SIMS, require a slice of the tissue sample to be analysed to be first covered with a matrix.

In case of a matrix assisted MSI method, the method of the invention includes a step (after step (c)) of applying a matrix onto the slide overlying at least the first and second area of the slide; before performing the MS analysis.

As the matrix, any known MALDI matrix can be used. Matrix materials for MALDI are known in the art. The matrix materials facilitate the production of intact gas-phase ions from the material in the sample to be analysed. A laser beam serves as the desorption and ionization source. The preferred matrix material is thus capable of absorbing radiation at a specific wavelength from the laser source (typically ultraviolet or infrared laser source). Further requirements are (amongst others) that it can be soluble in appropriate solvents and that it is stable in vacuum.

Examples of matrix materials are: a-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid), 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy phenyl azo) benzoic acid (HABA), succinic acid, 2,6- dihydroxy acetophenone, ferulic acid, caffeic acid (3,4-dihydroxy-cinnamic acid), 2,4,6-trihydroxy acetophenone, 3-hydroxypicolinic acid, 2-aminobenzoic acid, nicotinic acid, trans-3-indoleacrylic acid, isovanillin, dithranol and b-carboline (Norharmane).

The matrix can be applied by known methods, such as by spraying or sublimation such as for instance described in EP3618097, which is incorporated by reference in its entirety.

The method of the invention can further include a step where the tissue sample is conditioned with a known substance such as formalin, paraffin, optimal cutting temperature compound or gelatine. In such case it may be desirable to treat the 3D construct in the same way so a better comparison with the sample can be made. In this case it is preferable that the treatment (e.g. formalin, paraffin, etc.) is performed on the 3D construct on the slide, meaning after the construct is printed, so as to avoid the need to section the 3D construct.

The method of the invention, using the bioprinted 3D construct, can further be used for quantifying a target species in a tissue sample. According to this aspect of the invention, a method is provided, comprising

(a) mixing a first predetermined amount of the target species with a carrier, creating a mixture (i) ;

(b) depositing the mixture (i) on a slide in a first area of the slide;

(c) mixing a second predetermined amount of the target species with a carrier, creating a mixture (ii);

(d) depositing the mixture (ii) on a slide in a second area of the slide; optionally depositing further predetermined amounts on further areas of the slide; thus creating a calibration construct having distinctive areas of known amounts of the target species;

(e) depositing the tissue sample on the slide in a further area of the slide;

(f) performing MS analysis of the calibration construct and the tissue sample;

(g) comparing a signal obtained by MS analysis of the calibration construct with that of the tissue sample to determine the amount of the target species in the tissue sample; wherein the steps of depositing a mixture of a target species and a carrier are carried out by means of a 3D printing method, wherein the carrier is a substance that mimics an ECM selected from natural or synthetic biomaterial inks.

Thus, by depositing a calibration construct having known quantities of target species, the MS method can be calibrated and the target species in the tissue sample can be quantified. This method can also be used to create a calibration curve for a particular target species.

As described above for the quality control method, the method of depositing the construct is a 3D printing method. The different areas with the different amounts of the target species can be distinct filaments, spots, or lines in a grid.

In an embodiment, the depositing of the mixture in steps (b) and (d) and option additional steps comprises drying the mixture.

Further preferred embodiments for the target species, tissue sample, carrier and MSI method are as described above.

According to a further aspect the invention also provides a slide for MS analysis which comprises a 3D construct, wherein the construct comprises a carrier and one or more target species, wherein the carrier mimics an ECM; wherein the target species is selected from the group consisting of proteins, peptides, amino acids, nucleic acids, lipids, drugs, metabolites, cells and mixtures thereof; wherein the construct comprises one or more grids, one or more spots or one or more filaments; wherein the construct has a thickness of from 1 to 100 pm, wherein the carrier is a substance that mimics an ECM selected from natural or synthetic biomaterial inks.

The carrier is selected from natural or synthetic biomaterial inks, wherein the natural biomaterial inks are preferably selected from the group consisting of alginates, collagen, gelatin, fibrin, silk, dextran, agarose, hyaluronic acid, chitosan and mixtures thereof; and the synthetic biomaterial inks are selected from polyethylene glycol and polyvinyl alcohol and mixtures thereof.

A preferred biomaterial ink is an alginate. A preferred biomaterial ink is described in PCT/EP2019/080507, which is incorporated by reference in its entirety. The 3D construct is present on only one area of the slide such that a tissue sample can be deposited on a second area of the slide. The total surface area of the 3D construct is for instance from 0.5 x 0.5 mm to 10 x 10 mm.

According to a further aspect the present invention also relates to a method of making a slide for MS analysis which comprises a 3D construct, wherein the construct comprises a carrier and one or more target species, comprising the steps of:

(a) mixing a predetermined amount of the one or more target species with a carrier, creating a mixture;

(b) depositing by means of a 3D printing method, the mixture obtained in step (a) on a first area of a slide, creating a 3D construct which comprises one or more grids, one or more spots or one or more filaments; and which has a thickness of from 1 to 100 pm;

(c) depositing the tissue sample on the slide in a second area of the slide, wherein the carrier is a substance that mimics an ECM selected from natural or synthetic biomaterial inks.

In an embodiment the depositing of the mixture in step (b) comprises a drying step.

The different steps of the method of the invention are shown in Figure 1. A cell culture is mixed with the carrier in a tube. Subsequently, the mixture of cells and carrier is printed onto a slide. A tissue sample is added and the slide is coated with matrix.

Figure 2 shows one configuration of the slide (1), where (2) designates a tissue sample and (3) designates a 3D grid of the mixture of carrier and target species.

Similarly, Figure 3 shows one configuration of the slide (1), where (2) designates a tissue sample and (3) designates 3D spots of the mixture of carrier and target species.

In the drawings, Figure 1 shows the different steps of the process of the invention;

Figure 2 shows an example slide obtained with the process of the invention; Figure 3 shows an example obtained with the process of the invention;

Figure 4 shows an MALDI-MSI analysis of a tissue sample and a 3D standard of the invention;

Figure 5 shows different molecular distributions (grid vs. background slide) after performing principal component analysis (first component);

Figure 6 shows the distribution of a sphingomyelin lipid standard after total ion count normalization and cluster analysis (percentage of variance is shown). The scale bar represents the intensity for sphingomyelin related m/z values;

Figure 7 shows the sphingomyelin related m/z clustered channels corresponding to the image visualized in figure 6. The x axis corresponds to the m/z range and the y axis refers to intensity;

Figure 8 shows different molecular distributions (printed grid vs. background slide) after principal component analysis on an experiment of 1 million muscle cells printed;

Figure 9 shows the distribution of different lipids associated to cells after printing 2 million cells. Each colour represents a different m/z value.

Figure 10 shows the distribution of alginate in the presence of a buffer (8% alginate in TBS). The scale bar represents the intensity for sodium alginate related m/z values;

Figure 11 shows the distribution of alginate in the absence of a buffer (8% alginate in ultrapure water). The scale bar represents the intensity for sodium alginate related m/z values;

Figure 12 shows the distribution of alginate. The slide was printed with 6% alginate and 3 mg/ml caffeine. The scale bar represents the intensity for sodium alginate related m/z values;

Figure 13 shows the alginate related m/z corresponding to the image visualized in figure 12. The x axis corresponds to the m/z range and the y axis refers to intensity;

Figure 14 shows the distribution of caffeine. The slide was printed with 6% alginate and 3 mg/ml caffeine. The scale bar represents the intensity of caffeine related m/z values;

Figure 15 shows the caffeine related m/z corresponding to the image visualized in figure 14. The x axis corresponds to the m/z range and the y axis refers to intensity; Figure 16 shows the distribution of caffeine. The slide was printed with three constructs, each with a different concentration of caffeine (as depicted in the inset);

Experimental

Materials:

Microscope ITO slide obtained from Delta technologies

Biomaterial ink: Alginate solution (e.g., 3 %w/v Alginic acid sodium salt from brown algae, Sigma)

Cell suspension (e.g. 2 million cells in 1 ml of cell culture medium)

Drug/lipid solution (e.g. sphingomyelin dissolved in methanol (Avanti), caffeine dissolved in ultrapure water)

3ml cartridge (EFD)

MALDI matrix (DHB)

Acetone in solution or any other solution needed for the deposition of the matrix MALDI sprayer or sublimation device

Bioprinter: Bioscaffolder 3.1; GeSiM mbH; pressure-based bioprinting printhead

Protocol 1 (cells)

A cell suspension with the desired amount of cells is prepared. A 1 ml of hydrogel solution is prepared in a sterile 1.5 ml Eppendorf tube. The cell suspension is spun down and the culture medium is removed. The cells are re-suspended in 50 pi of cell culture medium and the cell suspension is added to the Eppendorf tube containing the hydrogel by mixing to distribute the cells evenly without introducing air bubbles. In case that small air bubbles are visible the Eppendorf is put in a centrifuge (1500 r.p.m.) during 30 - 60 seconds.

The hydrogel solution containing cells is pipetted into a pre-sterilized bioprinter disposable cartridge. By means of a plunger, the hydrogel is compressed in the bottom of the cartridge. A construct is printed on a slide according to the settings of the bioprinter. Protocol 2 (target species)

A 1 ml of hydrogel solution is prepared in a sterile 1.5 ml Eppendorf tube. A target species is added to the Eppendorf tube containing the hydrogel by mixing without introducing air bubbles. In case that small air bubbles are visible the Eppendorf is put in a centrifuge (1500 r.p.m.) during 30 - 60 seconds.

The hydrogel solution containing the target species is pipetted into a pre sterilized bioprinter disposable cartridge. By means of a plunger, the hydrogel is compressed in the bottom of the cartridge. A construct is printed on a slide according to the settings of the bioprinter.

Protocol 3 (matrix application)

50 mg DHB in 2 ml acetone is added to a sublimator (HTX Imaging). The sublimator is operated at 180 °C for 300 seconds to coat the slide with the DHB matrix.

Protocol 4 (MSI data acquisition and analysis)

The slide coated with matrix is placed in an MS instrument (e.g., Synapt-G2 Waters, Rapiflex, etc). An analysis is carried out in positive mode, with a mass range from 200 to 2000. The laser energy is set to obtain a signal higher than 10 ^L 4. The acquisition speed is set to 2 pixels/second. Raster size is set to 15 pm. The data is processed and visualized with HDimaging and in house developed software for principal component analysis ChemomeTricks toolbox for MATLAB version 2012B (The MathWorks, Natick, MA, USA).

Protocol 5 (MSI data acquisition and analysis)

Caffeine experiments (figs. 12-16):

The slide coated with matrix is placed in a Rapiflex instrument, Bruker. An analysis is carried out in positive mode, with a mass range from 175 to 1000. The laser energy is set to obtain a signal higher than 10 ^L 4 (35% power). The frequency was set to 10000 and 200 laser shots. Raster size is set to 30x30 pm. The data is processed and visualized with SCiLS (Bruker).

Example 1

An ITO slide was prepared according to protocol 2 using muscle cells. The 3D bioprinter was set to print in a corner of the glass slide a construct of 2 layers (with a 0-90) configuration with an overall area of 5x5 mm. A distance between each strand of 500 pm was used to deposit a meander layer. A second layers with a 5 pm offset in Z was deposited with a 90 degrees rotation on top of the previous one. Matrix was applied using protocol 3. An MSI analysis was carried according to protocol 4. The result is shown in Figures 5, 6 and 7.

Example 2

An ITO slide was prepared according to protocol 1 using 1 million muscle cells. The 3D printing was the same as in Example 1. The result is shown in Figure 8.

Example 3

An ITO slide was prepared according to protocol 1 using 2 million muscle cells re suspended in PBS. The 3D Bioprinter was set to print in a corner of the glass slide a construct of 2 layers (with a 0-90) configuration with an overall area of 5x5 mm. A distance between each strand of 500 pm was used to deposit a meander layer. A second layer with a 5 pm offset in Z was deposited with a 90 degrees rotation on top of the previous one.

Matrix was applied using protocol 3. An MSI analysis was carried out according to protocol 4. The result is shown in Figure 9.

Example 4

An ITO slide was prepared according to protocol 2 using 8% alginate prepared in TBS or ultrapure water. The 3D Bioprinter was set to print in a corner of the glass slide a construct of 2 layers (with a 0-90) configuration with an overall area of 5x5 mm. A distance between each strand of 500 pm was used to deposit a meander layer. A second layer with a 5 pm offset in Z was deposited with a 90 degrees rotation on top of the previous one.

Matrix was applied using protocol 3. An MSI analysis was carried out according to protocol 5. The results are shown in Figures 10 and 11.

Example 5

An ITO slide was prepared according to protocol 2, using 6% alginate and 3 mg/ml caffeine as target species. The 3D Bioprinter was set to print in a corner of the glass slide a construct of 2 layers (with a 0-90) configuration with an overall area of 7x7 mm. A distance between each strand of 500 pm was used to deposit a meander layer. A second layer with a 5 p offset in Z was deposited with a 90 degrees rotation on top of the previous one.

Matrix was applied using protocol 3. An MSI analysis was carried out according to protocol 5. The results are shown in Figures 12 to 15.

Example 6

An ITO slide was prepared according to protocol 2, using caffeine as target species at different concentrations (1 , 3 and 5 mg/ml). The 3D Bioprinter was set to print in a corner of the glass slide a construct of 2 layers (with a 0-90) configuration with an overall area of 5x5 mm. A distance between each strand of 2500 pm was used to deposit a meander layer. A second layer with a 5 pm offset in Z was deposited with a 90 degrees rotation on top of the previous one.

Matrix was applied using protocol 3. An MSI analysis was carried out according to protocol 5. The result is shown in Figure 16.