METHOD OF PREPARING CELLS FOR 3D TISSUE CULTURE

Abstract

The present invention relates to a method of preparing cells for 3D tissue culture, which method comprises the steps of plating the cells on a suitable surface, optionally, checking for their capability to adhere to said surface, discarding the cells which have not adhered to said surface, detaching the adhered cells and transferring them into a 3D tissue culture process.

Claims

1. A method of preparing cells for 3D tissue culture, which method comprises the steps of a) plating the cells on a suitable surface b) optionally, checking for their capability to adhere to said surface, c) discarding the cells which have not adhered to said surface, d) detaching the adhered cells and transferring them into a 3D tissue culture process.

2. The method of claim 1, which does not encompass the application of a viability assay throughout steps a)-d).

3. The method of claim 1, wherein the 3D tissue culture process is at least one selected from the group consisting of scaffold-based 3D tissue culture hydrogel-based 3D tissue culture cellular self-assembly 3D tissue culture, and/or 3D bioprinting,

4. The method of claim 1, wherein the cells are cryopreserved cells which are thawed before plating.

5. The method of claim 1, wherein the cells are non-frozen cells.

6. The method of claim 1, wherein the cells are selected from primary cells stem cells, tumor cells and/or immortalized cells.

7. The method of claim 1, wherein the cells are not expanded prior to, during or after plating, and/or the cells are not passaged after plating.

8. The method of claim 1, wherein the cells are mammalian cells, preferably selected from the group consisting of human cells cynomolgus cells pig cells canine cells, rat cells,

9. The method of claim 1, wherein the cells are Hepatocytes.

10. A 3D tissue culture process selected from the group consisting of scaffold-based 3D tissue culture hydrogel-based 3D tissue culture cellular self-assembly 3D tissue culture, and/or 3D bioprinting, in which process cells are used that have been prepared according to the method of any of claim 1.

11. A 3D tissue culture that has been obtained with a process according to claim 10.

12. The 3D tissue culture according to claim 11, which culture adopts the shape of a spheroid.

13. Use of a 3D tissue culture according to claim 11 for at least one purpose selected from the group consisting of: drug efficacy and/or toxicity screenings, investigative/mechanistic toxicology, target discovery/identification, drug repositioning studies, pharmacokinetics and pharmacodynamics assays, and/or regenerative medicine.

14. A 3D tissue culture that has been obtained with a process according to claim the method of claim 1.

15. Use of a 3D tissue culture according to claim 12 for at least one purpose selected from the group consisting of: drug efficacy and/or toxicity screenings, investigative/mechanistic toxicology, target discovery/identification, drug repositioning studies, pharmacokinetics and pharmacodynamics assays, and/or regenerative medicine.

Description

FIGURES

[0139] FIG. 1 Incomplete Microtissue Formation if Seeded Directly After Thawing

[0140] Human liver microtissue formation was initiated with cryopreserved, plateable primary human hepatocytes, which were seeded directly after thawing in hanging drop plates. Two different medium compositions were tested (Medium A+B). Three representative hanging drops (Nr.1, 2, 3) were imaged directly after seeding and after 1 and 4 days in hanging drops. The hepatocytes accumulated at the meniscus of the in hanging drop and formed loose aggregates until day 4 in culture. The same hepatocyte lot displayed attachment to 2D collagen-I coated culture dishes after thawing, showing the suitability of this hepatocyte for 2D culture.

[0141] FIG. 2 Vital Cell Selection Allows for Complete Microtissue Formation

[0142] Human liver microtissue formation was initiated with 5 independent lots (donor 1-5) of cryopreserved, plateable primary human hepatocytes, which were seeded directly after thawing on collagen-I coated 2D culture dishes for 24 hours. After vital cell selection, the cells were detached from the culture dish and seeded in hanging drop plates. The hepatocytes accumulated at the meniscus of the in hanging drop and formed tight microtissues (also called hepatospheres) within 5 days of culture. The microtissues were transferred to a receiver plate (GravityTRAP) and imaged for microtissue appearance and -size. All 5 hepatocyte lots showed robust and uniform microtissue formation within 5 days of culture.

[0143] FIG. 3 Reproducible Formation of Human Liver Microtissues with Vital Cell Selection of Primary Human Hepatocytes

[0144] (A) Size variation of human liver microtissues produced after pre-plating of cryopreserved human hepatocytes. The diameter human liver microtissues of 24 production runs is shown, including the standard deviation (n=6). The diameter of human liver microtissues of 24 production runs with the same hepatocyte lot was determined. The average diameter and standard deviation of the size measurement of 6 microtissues per run is shown. The microtissues showed less than 5% size variation within each production run. The average microtissue size of all 24 productions was 280 m, with an relative size deviation of less than 10% between production runs.

[0145] (B) Resulting assay variability. The intracellular ATP-content of 40 assays with human liver microtissues was quantified with CellTiter-Glo assay after 14 days. Measurements were performed in triplicates. The average RLU's from all 40 assays was set to 100%, the relative standard deviation of each measurement to the mean is depicted. Average relative standard deviation of all 40 measurements is 14.6%.

[0146] FIG. 4 Microtissue Formation of Dog Hepatocytes is Achieved Only with Vital Cell Selection

[0147] Dog liver microtissue formation was initiated with cryopreserved, plateable primary dog hepatocytes, which were seeded either directly in the hanging drops (B) or pre-plated on collagen-I coated 2D culture dishes for 24 hours (C, D). Dog liver microtissue formation was not observed after direct seeding of cryopreserved hepatocytes until 4 days in culture. Vital-cell selected dog hepatocytes formed dense microtissues within 4 days in hanging drops (C). (D) Image of a dog liver microtissue transferred from the hanging-drop to the receiver plate.

[0148] FIG. 5 Viability and Functionality of 3D Tissues Over 5 Weeks in Culture.

[0149] (A) Intra-tissue ATP quantification. ATP content per microtissue is depicted (pmol ATP/MT) as an indicator of cell viability and vitality.

[0150] (B) Quantification of secreted albumin by ELISA over time, normalized to the initial hepatocyte cell number and time

[0151] FIG. 6 Long-Term Toxicity and Inflammation-Mediated Testing with Human Liver Microtissues Produced According to the Invention

[0152] (A) Dose-response of acetaminophen toxicity after 14 days treatment (3 re-dosing) resulted in an IC.sub.50 value of 754.2 M

[0153] (B) Dose-response curve of diclofenac supplemented for 14 days (3 re-dosing) resulted in an IC.sub.50 value of 178.6 M.

[0154] (C) Quantification of IL-6 secretion with ELISA measurement. Hepatosphere was induced for 48 h with 10 1lg/ml LPS. Induction with LPS led to a tenfold increase in IL-6 secretion.

[0155] (D) Dose-response of trovafloxacin induced toxicity in presence and absence of LPS. Presence of LPS decreased the IC50 threefold from 220 (LPS) to 71 M(+LPS)

[0156] FIG. 7 Structural Integrity of Human Liver Microtissues Produced Without Pre-Plating and with Pre-Plating

[0157] Histological preprations were made from formalin fixed and paraffin embedded human liver microtissues produced either directly after thawing of the cryopreserved hepatocytes or after pre-plating of the hepatocytes for 24h on a collagen-coated cell culture dish. 3-5 um thick sections were stained with Hematoxylin and eosin (H&E). Images were taken with a 10 objective.

[0158] The upper row of FIG. 7 shows microtissues which have been created without prior pre-plating, while the low reo shows microtissues which have been created with prior pre-plating.

[0159] For donor #3 no microtissues were formed using the cryopreserved hepatocytes without the pre-plating step. For the other preparations, a high degree of necrotic areas apparent by dense eosinophilic staining and lack of nuclei within the tissues were visible in the groups without pre-plating (especially predominant for donor 2 and 4 which appear to have hardly any viable hepatocytes integrated). The pre-plating step significantly improves structural morphology of the liver microtissues.

REFERENCES

[0160] Pichard et al. (2006) Human hepatocyte culture. Meth Mol Biol 320: 283-293.

[0161] Kelm et al., Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 2003, 83, 173-180.

[0162] Kelm et al 2010, A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. Journal of Biotechnology

[0163] Ridky T W et al., Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia. Nature Medicine 2010

[0164] Cody N A et al., Influence of monolayer, spheroid, and tumor growth conditions on chromosome 3 gene expression in tumorigenic epithelial ovarian cancer cell lines. BMC Med Genomics. 2008

[0165] Kelm & Fussenegger, Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol. 2004, 22, 195-202.

[0166] Yuhas, J et al., Simplified method for production and growth of multicellular tumor spheroids. Cancer Res. 1977, 37, 3639-3643.

[0167] Metzger et al, The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. Cytotherapy. 2011 September; 13(8):1000-12.

[0168] Geckil et al, Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond). 2010 April; 5(3): 469-484.

[0169] Carletti E et al, Scaffolds for tissue engineering and 3D cell culture. Methods Mol Biol. 2011; 695:17-39.

[0170] Fey and Wrzesinski, Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line. Toxicol Sci. 2012 June; 127(2):403-11

[0171] Jensen J, Hyllner J, Bjorquist P. Human embryonic stem cell technologies and drug discovery. J Cell Physiol 2009; 219: 513-519.

[0172] Sartipy & Bjquist. Concise Review: Human Pluripotent Stem Cell-Based Models for Cardiac and Hepatic Toxicity Assessment. Stem Cells 2011; 29 (5): 744-748