METHODS AND DEVICE FOR THE ANALYSIS OF TISSUE SAMPLES

Abstract

The present invention relates to methods, and devices to analyze the phenotype and/or genotype of cells obtained from tissue samples. In particular, the present invention relates to the analysis of the response of the cells as obtained to the exposure of a drug compound or combinations thereof. The methods of the present invention offer the particular advantage of being time-effective, and suitable for automatization.

Claims

1. A method for identifying a patient-specific drug or drug combination, wherein said patient suffers from, or is being diagnosed for, a neoplastic disease or tumor, said method comprising a) dissociating the cells of a patient-derived tissue sample in order to obtain dissociated cells, b) generating an array of 3D microtissues based on said dissociated cells of step a), c) contacting said array of said 3D microtissues with at least two drugs and/or combinations thereof, d) determining an effect of said drugs and/or combinations thereof on said array of said 3D microtissues, and e) identifying a patient-specific drug or drug combination based on the effect as determined, and optionally, further comprising the step of selecting said patient-specific drug or drug combination as identified.

2. The method according to claim 1, wherein dissociating said tissue sample comprises i) if required, dissecting said tissue sample into smaller pieces comprising cells, ii) treating said tissue sample with a solution comprising at least one enzyme capable of dissociating cells in said tissue sample, producing a supernatant comprising dissociated cells, and ii) removing said supernatant comprising said dissociated cells and collecting said cells, wherein steps (ii) and (iii) are repeated at least once.

3. The method according to claim 1, wherein step b) comprises adding or removing stroma cells, stromal fibroblasts, endothelial cells and immune cells to said dissociated cells, and/or wherein in step b) for each 3D microtissue a predetermined number of cells is provided, and/or wherein in step b) said 3D microtissues are generated in at least one system selected from a hanging drop system and a multiwell system.

4. The method according to claim 1, wherein the generation of said 3D microtissues does not require the use of a solubilized basement membrane preparation, and/or wherein the generation of said 3D microtissues comprises self-assembly of said cells comprised in said dissociated cells, and/or wherein the generation of said 3D microtissues comprises a maturation time of about 6 hours to 7 days, and/or wherein said 3D microtissues as generated have a size of 350 μm+/−100 μm.

5. The method according to claim 1, wherein said contacting in step c) comprises a continuous exposure to said at least two drugs and/or combinations thereof, and/or an exposure to and subsequent removal to said at least two drugs and/or combinations thereof.

6. The method according to claim 1, wherein said determining of said effect in step d) is selected from size determination of said 3D microtissue, quantification of internal reporter gene expression in said 3D microtissue, determination of the intracellular ATP content in said 3D microtissue, and determination of pre-selected biomarkers in said 3D microtissue.

7. The method according to claim 1, wherein said patient-derived tissue sample is selected from a sub-sample derived from a primary tissue sample, a primary tumor sample, and a metastasis sample, and/or wherein said tissue sample and/or the dissociated cells are frozen and re-thawed prior to the generation of said 3D microtissues.

8. The method according to claim 1, comprising providing a primary tissue sample, obtaining a subsample in addition to the patient-derived sample and subjecting said subsample to at least one of molecular profiling, histological analysis, and histochemical analysis.

9. A method for stratifying a patient with respect to a treatment with a patient-specific drug or drug combination, comprising performing the method according to claim 1, and further comprising a stratification of said patient based on said patient-specific drug or drug combination as identified.

10. A method for identifying adverse effects associated with a treatment with a patient-specific drug or drug combination in a patient, comprising performing the method according to claim 1, and further comprising the step of testing and analyzing said patient-specific drug or drug combination for adverse effects in said patient.

11. A system for identifying a patient-specific drug or drug combination, wherein said patient suffers from, or is being diagnosed for, a neoplastic disease or tumor, said system comprising a) a tissue sample dissociation unit for dissociating a patient-derived tissue sample in order to obtain dissociated cells, b) a unit for producing an array of 3D microtissues based on said dissociated cells of step a), c) a drug testing unit for contacting said array of said 3D microtissues with at least two drugs and/or combinations thereof, d) a first analysis unit for determining an effect of said drugs and/or combinations thereof on said array of said 3D microtissues, and e) a second analysis unit for identifying a patient-specific drug or drug combination based on the effect as determined, and optionally further comprising a unit for selecting said patient-specific drug or drug combination as identified.

12. The system according to claim 11, wherein said tissue sample dissociation unit comprises at least one of i) a pipetting unit, ii) an enzyme reservoir, iii) a reservoir for cell culture media, iv) a reservoir for washing solutions, v) optionally, an ultrasonic device, and vi) a centrifuge unit, and/or wherein said unit for producing an array of 3D microtissues based on said dissociated cells comprises at least one of i) a pipetting unit, ii) a cell counting unit, and, iii) a handler for microtiter plates, and/or wherein said drug testing unit comprises at least one of i) a handler for microtiter plates, ii) a pipetting unit, iii) a reservoir for cell culture media, iv) an array of reservoirs comprising at least two different drugs or combinations thereof, and iv) an incubator unit, and/or wherein said first and/or second analysis unit comprises i) a handler for microtiter plates, and/or ii) an imaging system comprising a microscope and a camera, and optionally an HR scanner.

13. The system according to claim 11, wherein said tissue sample dissociation unit and said unit for the production of an array of 3D microtissues share the same pipetting unit and/or wherein said drug testing unit and said first analysis unit share the same handler for microtiter plates.

14. The system according to claim 11, wherein said tissue sample dissociation unit and said unit for producing an array of 3D microtissues are positioned in the same housing, and/or wherein said drug testing unit and said first analysis unit are positioned in the same housing, and wherein said two housings are connected to form a discrete system and/or wherein said system is, at least in part, arranged vertically.

15. The system according to claim 11, wherein said system can be sterilized as a whole or in parts thereof, and/or wherein said system comprises means for establishing and/or maintaining sterile conditions.

16. The system according to claim 11, wherein said system comprises at least one loading port (1) comprising a loading system with a lock system (L) for a sterile loading of materials or consumables as used in the system(s) and/or unloading waste and/or products as produced in the system(s).

17. The system according to claim 16, wherein said lock system further comprises means for sterilizing the materials and/or wherein said lock system further comprises means for thawing or cooling/freezing the materials to be loaded or unloaded.

18. The system according to claim 16, wherein said lock system is adapted to specifically fit to a transport box or container, wherein said transport box or container comprises at least one port to be opened and closed inside the system.

19. The system according to claim 18, wherein said transport box or container comprises at least two different separate temperature zones.

20. A loading system with a lock system according to claim 16 or a transport box that comprises at least two different separate temperature zones.

Description

[0092] FIG. 1 illustrates the general workflow of a preferred embodiment of the method according to the invention. A tumor tissue sample (1) is obtained from a patient, for example by surgery, nuclear biopsy, small needle aspiration, core biopsy, tumor resection, liquid biopsy and/or needle aspiration. The tissue sample is further processed into a single cell solution (2) either by enzymatic degradation only or with the aid of sonification protocols. The resulting single cell suspension is used to produce microtumors in a non-adherent multi-well plate (3). After maturation, the tissues are treated for a limited time with the drugs of interest to them and the resulting effects on microtumor growth observed by appropriate non-disruptive technologies over time (4). Since the test does not destroy the microtumors, additional tests can be performed on the treated microtumors to verify the efficacy of drugs (4.1). Patient specific growth kinetics in response to different treatments are transferred to a central drug response database (5) to analyze the results and place them in a broader patient context. After the data analysis, the test results (6) are transmitted to the respective institution.

[0093] FIG. 2 shows the arrangement of a preferred microtissue production unit (system) according to the invention—embodiment without sonification. The system consist of two units the automated production unit and the drug profiling unit. In one embodiment, the microtissue/microtumor production unit consist of a fully contained (housed) and sterile environment which includes a loading port (1) to load the biopsies as well as the digestion enzymes (e.g. Collagenase, Trypsin, Elastase, Hyaluronidase, Papain, Chymotrypsin, Deoxyribonuclease I, Neutral Protease (Dispase)), culture medium and optionally supporting cells (immune cells, stroma cells, stromal Fibroblasts, endothelial cells) for the automated production process. Enzymes and medium are stored in the 4° C. zone (2). Liquids are processed with a centralized automated liquid handling system (5) such as (i) aspirating medium or enzyme solutions, (ii) addition from fresh medium, or (iii) addition of additional supporting cells. In addition to the 4° C. zone there is a 37° C. zone (6) for enzymatic digestion of the tissue samples. (9) designates the space required for consumables such as multi-well plates and centrifuge tubes. To determine the cell number for the in-process calculation of the required per well cell quantity a cell counter is integrated in the device (4) with a storage space for required consumables (8). Multi-well plates filled with the cell suspension are placed into an incubator (3) which sustains 37° C. and 5-10% CO.sub.2 environment. A robotic arm (7) is positioned centrally to service all operational units within the device. The whole device is controlled via a digital interface (10) to run the individual protocols and transfer the production data to an external data storage device for quality control assessment. The computational controlled device allows to run various production protocols depending on the tissue type processed and to maximize the number of viable cells. Exemplified in FIG. 2B are three different isolation procedures which can be processed on the device (i) a multistep sequential incubation with one single enzyme [A] with a fixed concentration. After each digestion step the cell suspension is transferred into a stop solution and fresh enzyme added to the tissue sample. (ii) a multistep sequential incubation with one single enzyme but with increasing concentrations or (iii) applying various enzymes.

[0094] FIG. 3 shows the arrangement of a preferred microtissue production unit (system) according to the invention similar to FIG. 2—embodiment with sonification. The system consist of two units the automated production unit and the drug profiling unit. In one embodiment the microtumor production unit consist of a fully contained (housed) and sterile environment which includes a loading port (1) to load the biopsies as well as the digestion enzymes (as above), culture medium and optionally supporting cells (as above) for the automated production process. Enzymes and medium are stored in the 4° C. zone (2). Liquids are processed with a centralized automated liquid handling system (5) such as (i) aspirating medium or enzyme solutions, (ii) addition from fresh medium, or (iii) addition of supplemental cells (again as above). In addition to the 4° C. zone there is a 37° C. zone (6) for enzymatic digestion of the tissue samples. The 37° C. zone is further equipped with a sonification system (11) to further facilitate the cell isolation process (increase efficiency and decrease process time). (9) designates the space required for consumables such as multi-well plates and centrifuge tubes. To determine the cell number for the in-process calculation of the required per well cell quantity a cell counter is integrated in the device (4) with a storage space for required consumables (8). Multi-well plates filled with the cell suspension are placed into an incubator (3) which sustains 37° C. and 5-10% CO.sub.2 environment. A robotic arm (7) is positioned centrally to service all operational units within the device. The computational controlled device allows to run various production protocols depending on the tissue type processed and to maximize the number of viable cells. Exemplified in FIG. 3B are three different isolation procedures which can be processed on the device with a sonification system in addition to various enzymatic profiles.

[0095] FIG. 4 shows the arrangement of another preferred microtissue production unit (system) according to the invention—embodiment with sonification. In this embodiment the microtissue drug profiling unit consist of a fully contained (housed) and sterile environment which includes a loading port (1) to load the micro-well plate containing microtumors as well as cancer-type specific maintenance medium, a pre-fabricated drug matrix and optionally additional cells required for testing. Drugs, medium and cells are stored in the 4° C. zone (2). Liquids are processed with a centralized automated liquid handling system (5) such as (i) transferring the drugs from the matrix to the microtumors, (ii) removal of the drugs, (iii) medium exchange or (iv) addition of supplemental cells. Multi-well plates filled with the cell suspension are placed into an incubator (3) which sustains 37° C. and 5-10% CO.sub.2 environment. The detection device (4) images the microplate to generate treatment-specific growth kinetics. A robotic arm (7) is positioned centrally to service all operational units within the device. Raw data are being transferred (7) to a centralized data bank for analysis.

[0096] FIG. 5 shows the arrangement of a microtissue production system according to the invention combined with a drug profiling device. The device consist of both units the automated production and the drug profiling unit. In one embodiment the device consist of a fully contained (housed) and sterile environment which includes a loading port (1) to load the biopsies as well as the digestion enzymes (as above), culture medium, drugs and optionally supporting cells (as above) for the automated production and drug testing process. Enzymes, drugs and medium are stored in the 4° C. zone (2). Liquids are processed with a centralized automated liquid handling system (5) such as (i) aspirating medium or enzyme solutions, (ii) addition from fresh medium, (iii) drug addition and removal or (iv) addition of supplemental cells. In addition to the 4° C. zone there is a 37° C. zone (6) for enzymatic digestion of the tissue samples. (9) designates the space required for consumables such as multi-well plates and centrifuge tubes. To determine the cell number for the in-process calculation of the required per well cell quantity a cell counter is integrated in the device (4) with a storage space for required consumables (8). Multi-well plates filled with the cell suspension are placed into an incubator (3) which sustains 37° C. and 5-10% CO.sub.2 environment. A robotic arm (7) is positioned centrally to service all operational units within the device. The whole device is controlled via a digital interface (10) to run the individual protocols and transfer the production data to an external data storage device for quality control assessment and data analysis.

[0097] FIG. 6 shows the arrangement of a preferred embodiment of the system according to the present invention in a vertical arrangement. In one embodiment, the individual working units and stages are organized in a vertical arrangement to minimize the required footprint of the device. Within this embodiment the liquid handling compartment can be localized at the top of the device (1) followed by the detection compartment in the middle sector (2). The incubation unit can be localized at the bottom of the device (3). All levels are operated with an automated robotic arm (4).

[0098] FIG. 7 shows a schematic exemplified workflow for testing chemotherapeutics according to the present invention. Here, the workflow to automatically test chemotherapeutics is based on tumor growth kinetics.

[0099] FIG. 8 shows a schematic exemplified workflow for testing immune-modulatory drugs. Similar to FIG. 7, the exemplified workflow to automatically test immune modulatory drugs is based on tumor growth kinetics.

[0100] FIG. 9 shows pictures of NSCLC microtumor formation four days after tissue dissociation from 4 individual NSCLC (#001 to #004) and 1 pancreatic cancer patient (PC #004). Three representative microtissues per patient are shown. Bar is 250 μm.

[0101] FIG. 10 shows the production robustness and size distribution of microtumors generated from primary tumor tissue of non-small cell lung cancer patients. 5000 cells were seed in each well of a 96-non-adhesive well plate and incubated for 4 days (example 2).

[0102] FIG. 11 shows the quantitative analysis of single compounds on pancreatic pdx microtumors 12558 (c.sub.max). Analyzed drug efficacy of single and drug combinations from PDX-derived pancreatic microtumors. On the x-axis microtumor drug impact is compared of each treated tissue prior treatment and after treatment (t11/t0), on the y-axis is the drug effect compared between treated and non-treated microtumors (t11.sub.treated/t11.sub.control). Discrimination between responder and non-responder drugs is defined by a difference of at least 20% compared to untreated control and 20% between t11 and t0 (in vitro RESIST). All drugs were concentrated according to the c.sub.max values in vivo.

[0103] FIG. 12 shows that 3D3 device efficacy results reflect in vivo efficacy. To compare whether the in vitro efficacy analysis is matching in vivo (mouse) efficacy drug response data as obtained, pancreatic patient-derived xenograft (PDX-mouse model) and pancreatic microtumors derived from PDX tumor tissue were generated. Two single drugs (Gemcitabine and Abraxane) and two drug combinations (Gemcitabine:Erlotinib and Gemcitabine:Abraxane) were tested. In vivo data points reflect the average response of 5 individual animals. In both device models, Gemcitabine has the highest efficacy as well as the other three treatments display a similar efficiency profile.

[0104] FIG. 13 shows an evaluation of synergistic combinatorial effects (½ c.sub.max) as tested. Based on IC.sub.50 values the evaluation of drug combinations is a complex procedure (Wilson et al. SLAS Techn. 2019). At least a 6×6 drug testing matrix (usually 10×10) are tested in at least triplicates to generate an IC50 matrix. Potential synergistic effects are further determined by the Chou Talalay Method (T C Chou—Cancer research, 2010—AACR). In vitro long-term efficacy testing allows direct evaluation whether two drugs in combination exhibit higher efficacy as shown for Olaparib (PARP-Inhibitor) and Trametinib (MEK-Inhibitor) (A). In accordance to the literature which has shown that in KRAS mutated cancer both drugs have synergistic effects (Sun et al. 2018 Sci Transl Med) and exhibited a synergistic effect on the KRAS mutated pancreatic microtumors from the PDX-tissues. A negative example (B) is shown for Oxaliplatin and 5FU which do not exhibit any synergistic therapeutic benefit.

[0105] FIG. 14 shows that the screening technology allows to generate harmonized data across the different drug development stages, (i) drug discovery; (ii) pre-clinical; (iii) drug development/clinical). This is exemplified for pancreatic cancer in this Figure. Microtumors from different cell sources were used and treated with cis- or oxaliplatin, respectively: (i) pancreatic cancer cell line (discovery); (ii) PDX-derived (pre-clinical) and Patient-derived (clinical). Whereas growth of the PDX and patient-derived microtumors were in a similar range, Panc-1 microtumors doubled in size.

[0106] FIG. 15 shows a sterile hatch embodiment of the lock system according to the invention.

[0107] FIG. 16 shows an example of a workflow involving the lock system according to the invention, and the contained according to the invention.

EXAMPLES

[0108] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

Example 1

[0109] Tissue Generation and Testing

[0110] A tissue biopsy sample obtained from a patient is placed into an appropriate medium, namely a transport solution comprising antibiotics. Optionally, a hypotonic solution can be added for further processing. The tissue biopsy sample material is shipped at about 4° C. For a 100 mg tissue biopsy sample, a volume of 1.5 mL of transport medium comprising the tissue sample is used.

[0111] In step 2, for a hemolysis to lyse red blood cells, the tissue sample is then subjected to sonification using a commercially sonificator at about 1 MHz, 2 Wcm.sup.−2 for 5 min. Care is taken to prevent heating of the tissue sample so as to substantially maintain the integrity of the cells, the temperature is maintained at about 4° C. The hemolysis step using sonification lasts for about five minutes and targets cells that are not embedded in the tissue environment.

[0112] After sonification, the medium is removed and replaced by a medium for tissue dissociation (step 3). For this, one or more lytic suitable enzyme(s) in an appropriate buffer system is used for several cycles, for example 4 cycles, each for about 10 to 15 minutes. The number and length of the cycles may be adjusted to the progress of the tissue digestion, for example by first using strong and then afterwards exceedingly milder conditions. The tissue dissociation takes place in a volume of about 1 ml at a temperature of about 37° C. and for about 60 minutes.

[0113] Then, a stop (STOP) solution (6 ml) containing horse serum and solubilized collagen is added to the lysis solution in order to stop the enzymatic activity. The final volume of this reaction mixture is 10 ml and the solution is kept at 4° C. The cells as obtained are washed to remove cell debris by gravity enforced sedimentation of cells for 10 minutes in 2×5 ml in production medium (DMEM+10% FCS). While the cell debris floats in the solution, the cells sediment down to the bottom of the tube. Subsequently, large tissue debris is removed by filtering 5 ml of the solution comprising the cells through a 200 μm pore size filter and further addition of 5 ml production medium. The entire step takes about 10 minutes.

[0114] Thus obtained cells are transferred to a microwell plate, for example a 384 well plate pre-loaded with production medium and subsequently incubated at a temperature of about 37° C. In each well of the multiwell plate, 25 μl of the cell suspension comprising tumor cells are editors to 50 μl production medium previously filled into the cavities of the multiwell plate. In addition, 25 μl of a solution comprising supporting cells are added to each well. In accordance with the present invention, the estimated process time of this method is about 2 to 3 hours.

[0115] According to a first option, the thus obtained plate is transferred directly into a so-called profiling device (unit) for tissue maturation (microtissue generation). As an advantage of this, the technical setup is not duplicated between a creator- and profiling-unit. This, nevertheless, delays a fast drug testing of the profiling unit, because spaces are blocked with plates which that still need to maturate for 2 to 5 days.

[0116] In a second option, a separate tissue maturation is performed within a so-called creator unit. This requires an additional incubator and imaging capacity, but does not impact the throughput of the profiler unit, which can be used for testing of other tissues.

[0117] For the characterization (profiling) of the cultured cells in the presence of a drug or combinations of drugs (workflow on the first day after plate loading: drug dosing), approximately 50 minutes are calculated, a 384-well plate is pre-loaded with maturated microtissues in Step 1 (if not maturated in the device).

[0118] In Step 2, the plate is moved to an incubator using an automated, robotic device, such as a KUKA LBR Med lightweight robot.

[0119] In Step 3, the plate is then placed in an incubator, then the plate is moved to an imaging unit (Step 4), and (Step 5), the 384-well plate is image analyzed in a reader, for example, a QC imager for 10 minutes.

[0120] Then in Step 6 the plate is moved to a liquid handling unit/station, and in Step 7 the medium is exchanged and the compound/combination is brought in contact/dosaged. The medium exchange takes about 10 minutes, and about 30 mL are necessary per 384-well plate.

[0121] In Step 8, the plate is moved again to the imaging system and in Step 9; the plate is subjected to imaging in a 384-well plate HD-imaging system for 30 minutes.

[0122] Finally, in Step 10, the plate is moved back to the incubator unit, and the microtissue cells are incubated with the drug or combination for (in this case) 8 hours.

[0123] On the first day after plate loading, the drug is removed in a process taking about 60 minutes. In Step 11, after eight hours of drug incubation of the microtissues the plate is then removed from the incubator unit. In Step 12, the plate is moved to an imager (QC on tissue formation). In Step 13, the 384-well plate is subjected to HD-imaging for 30 minutes. Subsequently, in Step 14 the plate is moved to a liquid handling station. In Step 15, the medium is exchanged two times in a period of about 20 minutes, and the compound or combination as investigated is dosed again, and filled into the 384-well plate. A total of 60 mL medium plus compound(s) are required in this step.

[0124] In Step 16, the plate is moved to the imaging system and subjected to QC-imaging for 10 minutes in Step 17. Thereafter, in Step 18, the plate is moved again to the incubator unit (Step 19) and is incubated for 24 hours before the next cycle starts.

[0125] On the second day of the determining step, the microtissue is subjected to size profiling which takes about 35 minutes. In Step 1, the plate is removed from the incubator unit, and it is moved in Step 2 to the imaging unit, where it is subjected to HD imaging for 30 minutes in Step 3. Thereafter, in Step 4, the plate is moved back to and then into the incubator unit (Step 5).

[0126] As optical readout options, the size may be taken as the primary readout and multiple parameters may be selected as secondary options, such as at least one parameter selected from diameter, perimeter, volume, and area of optical cross section.

Example 2

[0127] Method to Produce NSCLC Patient-Derived Microtumors (PMTs)

[0128] The tumor tissue sample was pre-prepared by removing of fat tissue. The target tissue size was at about 0.2-0.4 cm.sup.3. For shipping, the tumor tissue was placed into a tube containing transport medium (e.g. Dulbeco's modified Eagle Medium supplemented with suitable antibiotics). Before further use, the tumor was rinsed 3× with phosphate-buffered saline (PBS) supplemented with suitable antibiotics. After removal of the PBS, Liberase dissolved in DMEM (0.04-0.08 mg/ml) was added and incubated at 37° C. for 15 min. The enzyme supernatant was transferred into a tube pre-filled with STOP solution (DMEM+40% FCS). Then, enzymatic solution (liberase) was added, and the incubation and transfer reiterated. The rest of the tissue sample was discarded, and only the cells in the STOP solution were used. These were moved on a 200 μm cell strainer in order to remove larger undissociated tissue fragments. After sedimentation of cells in the filtrate for 5 min, the supernatant was carefully removed, and production medium (DMEM+10% FCS) was added. Then, the cells are visually counted using a microscope.

[0129] For the generation of microtumors, the cell suspension is diluted to a final concentration of 6×10.sup.4 cells per ml, and 75 μl (i.e. about 4500 cells) are added to each well in a non-adhesive 96- or 384-well plate. The plate is then incubated to form tissue, preferably for 2 to 5 days. From three individual production runs resulted in a consistent size of 260 um in average and a success rate of over 90% (FIG. 10).

Example 3

Competitive Example—Production Time

[0130] Patient-Derived Organoid Vs Patient-Derived Microtumor Production

[0131] An important parameter for the routine clinical use of technologies that provide information about a therapeutic outcome to be included in decision making is the time to availability of the information. Patients' own microtumors (PMTs) are produced without intermediate cell culture steps and are ready for drug testing within 4 days after taking tumor samples (see FIG. 9).

[0132] In contrast to the patient's own microtumors as produced according to the present invention, organoids require intermediate cell culture processes such as expansion of LGRF5+ tumor stem cells and deprivation of LGR5+ stem cells from healthy tissue before entering the drug tests. According to recent publications, the time required to produce a sufficient number of organoids could be reduced from up to 3 months to 1 month. However, this is still significantly longer than for PMTs. Another problem is that the time to test readiness of the organoids is very heterogeneous, making it difficult to integrate the process into a routine and standardized automated test process.

Example 4

Case Example Pre-Clinical Pancreatic Cancer

[0133] Anti-cancer drug testing in drug discovery, development and functional precision medicine is usually performed by calculating an IC.sub.50 (concentration when 50% of the cells have died) and/or E.sub.max (lowest concentration reaching maximum cell death) value based on a dose response curve utilizing cancer cells from cell lines or patient tumor specimens. The IC.sub.50 provides information how potent a drug is, and the E.sub.max how potent a drug is. These parameter need to be established to enable high throughput screens and select candidates which are further tested and developed. However, both parameter cannot be measured in vivo, both preclinical and clinical which makes a direct in vitro to in vivo correlation highly complex (Chantal Pauli et al. Cancer Discov. 2017). The method according to the present invention as presented here is based on the same pre-clinical and clinical outcome measure which allows a direct correlation and drug efficacy ranking. For pre-clinical correlations, efficacy of the drug is compared to of the treated microtumor as well as to the untreated control. For clinical correlations, drug efficacy is compared based on the changes in tumor size prior treatment and after treatment. Cancer cell proliferation kinetics have a significant impact on drug sensitivity (Maurice Tubiana, Acta Oncologica 1989; Benjamin Drewinko et al., Cancer Res June 1981). The extended test period (14 days) as present takes into account the effects of proliferation much more than standard drug screening formats (1-3 days).

[0134] A pre-clinical study was performed to (i) rank drugs according to their in vitro efficiency (FIG. 11) retrospectively compare drug efficiency between in vitro and in vivo assays (FIG. 12), perform drug combination testing (FIG. 13) and compare data across the whole drug discovery and development process (FIG. 14).

[0135] Microtumors were produced directly from fresh tumor patient-derived xenograft samples dissected from the mice. The tissues were dissociated into single cells and microtumors produced in a non-adhesive round bottom multiwell plate. After seven days, microtumors were treated with single drugs and several drug combinations. Over 11 days the tumor growth kinetics were continuously monitored and analyzed.

[0136] As one particularly interesting result of the analysis, Gemcitabine has shown highest efficacy whereas Oxaliplatin was least effective (FIG. 11). In between these two drugs, the other single and combination treatments rank accordingly.