3-D TISSUE CULTURE BASED METHOD TO ASSESS MITOCHONDRIAL IMPAIRMENT

Abstract

The present invention relates to a method and/or assay for the assessment of the metabolic effect of a candidate compound. The method and/or assay comprises exposing one or more 3-dimensional cell culture or tissue to one or more candidate compounds, and measuring, in at least one 3-dimensional cell culture or tissue, the effect of such exposure on the 3-dimensional cell culture or tissue-specific respiration rate (MTSRR) (FIG. 1B).

Claims

1. Method and/or assay for the assessment of the metabolic effect of a candidate compound, which method and/or assay comprises a) exposing one or more 3-dimensional cell culture or tissue to one or more candidate compounds, and b) measuring, in at least one 3-dimensional cell culture or tissue, the effect of such exposure on the 3-dimensional cell culture or tissue-specific respiration rate (MTSRR), c) measuring, in the same or at least one other 3-dimensional cell culture or tissue, the effect of such exposure on cell viability and/or the cytotoxic effect (CV-CYT) in one or more cells of at least one 3-dimensional cell culture or tissue, and d) comparing the effects of steps b) and c) to provide an estimate on the mitochondrial toxicity of the one or more candidate compounds, wherein the mitochondrial toxicity is determined on the basis of the quantitative relationship between the IC.sub.50 with respect to MTSRR (IC.sub.5O.sub._.sub.MTSRR) and the IC.sub.50 with respect to CV-CYT (IC.sub.5O.sub._.sub.CV/CYT).

2. (canceled)

3. The method and/or assay according to claim 1, wherein said method and/or assay is used to determine the mitochondrial toxicity of a candidate compound.

4. The method and/or assay according to claim 1, wherein said method and/or assay is used to determine the chemotherapeutic efficacy of a candidate compound.

5. The method and/or assay of claim 1, wherein the 3-dimensional cell culture or tissue is a spheroidal cell culture.

6. The method and/or assay of any of claim 1, wherein the 3-dimensional cell culture or tissue comprises primary cells.

7. The method and/or assay of claim 1, wherein the 3-dimensional cell culture or tissue comprises hepatocytes.

8. The method and/or assay of claim 1, wherein the 3-dimensional cell culture or tissue comprises immortalized, malignant, cancerous and/or neoplastic cells.

9. The method and/or assay according to claim 1, which method and/or assay further comprises a step of determining, in the same or at least one other 3-dimensional cell culture or tissue, the effect of the exposure on the size thereof.

10. The method and/or assay according to claim 9, in which method and/or assay the size determination refers to at least one parameter selected from the group consisting of: Diameter perimeter volume area of an optical cross section.

11. The method and/or assay according to claim 8, wherein the 3-dimensional cell culture or tissue further comprises at least one cell type selected from the group consisting of: Immune cells Stroma cells Fibroblasts Cancer stem cells Immortalized, malignant, cancerous and/or neoplastic cells that are known to be resistant or sensitive to a given anti-cancer agent.

12. The method and/or assay according to claim 1, wherein the microtissue-specific respiration rate is determined by simultaneous measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), of one or more isolated 3D microtissues

13. The method and/or assay according to claim 12, wherein in the determination of the microtissue-specific respiration rate (MTSRR), the spare respiratory capacity (SPARE) is used.

14. The method and/or assay according to claim 12, wherein in the determination of the microtissue-specific respiration rate (MTSRR), the basal respiration rate (BASAL) and/or the maximal respiration rate (MAX) are also used.

15. The method and/or assay according to claim 1, wherein the measurement of cell viability/cytotoxic effect determines the residual viability of one or more cells.

16. The method and/or assay according to claim 1, wherein the effect on microtissue-specific respiration rate and/or the effect on cell viability/cytotoxic effect is determined as inhibitory concentration, preferably as half maximal inhibitory concentration (IC50).

17. The method and/or assay according to claim 1, wherein exposure step a) is carried prior to subsequent measurement steps b) and/or c).

18. The method and/or assay according to claim 1, wherein exposure step a) is carried out in one or more first vessels, while the subsequent measurement steps b) and/or c) are carried out in one or more second and/or third vessels.

19. The method and/or assay according to claim 18, wherein after exposure step a) the microtissues are transferred, by means of a suitable dispensing device, from the one or more first vessels to the one or more second and/or third vessels, to carry out measurement steps b) and/or c)

20. (canceled)

21. The method and/or assay according to claim 1, wherein IC.sub.5O-MTSRR?IC.sub.5O-CV/CYT means that the candidate compound has no specific mitochondrial toxicity, wherein IC.sub.5O-MTSRR<IC.sub.5O-CV/CYT and IC.sub.5O-MTSRR?0.75?IC.sub.5O-CV/CYT means that the candidate compound has a low risk of mitochondrial toxicity, and wherein IC.sub.5O-MTSRR<0.75?IC.sub.5O-CV/CYT means that the candidate compound has a high risk of mitochondrial toxicity

Description

FIGURES

[0172] FIG. 1: Mitochondrial Bioenergetics 2D vs. 3D. A) OCR profile and B) Maximal Respiration. **** p<0.0001 unpaired t-test, two-tailed, Means+/?s.e.m.

[0173] FIG. 2: Outline of a general workflow. The chronologic order of the different steps is just a preferred embodiment, meaning that the treatment of the 3D microtissues with the molecules to be screened can also take place synchronously with the subsequent assays on viability (ATP content) and respiration rate. Further and not depicted in the general workflow, the steps can be accompanied by a step of size determination, e.g., by digital image analysis. It is also important to mention that in some embodiments, the assays (viability, respiration rate and size) can be made with the same 3D microtissue, while in other embodiments different 3D microtissues are being used which have however been pretreated in identical fashion.

[0174] FIG. 3: Analysis example for 3D primary human hepatocyte microtissues exposed to different candidate compounds. DILI classification: Drug-induced liver injury according to Gustafsson et al, 2014 (N=negative, P=positive). CMax=concentration of candidate compound used in Gustafsson et al, 2014; CTop=concentration of candidate compound used herein; IC50.sub.CV/Cyt determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, US) as disclosed herein, IC50.sub.MTSRR determined as inhibition on spare respiratory capacity (SPARE), as disclosed herein; MOS: Margin of safety; High Mito-toxic risk: IC.sub.5O.sub._.sub.MTSRR<0.75?IC.sub.5O.sub._.sub.CV/CYT; Low Mito-toxic risk: IC.sub.5O.sub._.sub.MTSRR<IC.sub.5O.sub._.sub.CV/CYT and IC.sub.5O.sub._.sub.MTSRR?0.75?IC.sub.5O.sub._.sub.CV/CYT; No Mito-toxic risk: IC.sub.5O.sub._.sub.MTSRR?IC.sub.5O.sub._.sub.CV/CYT

[0175] Asteriks(*) show candidate drugs with respect to which the instant assay method delivers a different and more precise result with respect to mitochondrial toxicity than the general DILI classification according to Gustafsson et al., 2014. Fialuridine, which was categorized as liver toxic by Gustafsson et al. turned out to not have specific mitochondrial toxicity, while Metformin HCl, which was categorized as not liver toxic by Gustafsson et al., turned out to have a low mitochondrial toxicity.

REFERENCES

[0176] Tsiper M V et al. (2012); PloS one vol. 7 (10) p. e45226

[0177] Gustafsson et al, (2013); Tox Sciences 137(1), 189-211