Published: June 22nd, 2021
This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.
Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.
Existing preclinical models of pancreatic ductal adenocarcinoma (PDAC) and respective metastases are poor predictors of response to treatment in patients which is a major drawback in drug development and the identification of predictive biomarkers1. Although models such as patient-derived organoids and patient-derived xenografts are promising, their use remains limited2. Major limitations of these in vitro models are the lack of the tumor microenvironment and xenografting in non-human immunocompromised species. Especially in PDAC and its metastases, the tumor microenvironment has considerably gained interest ove....
Tissue specimens were collected and processed after approval by the local ethics committee of the University of Lübeck (# 16-281).
1. Fresh tissue collection and handling
NOTE: Every unfixed human tissue specimen should be handled with caution to prevent the risk of infection from blood-borne pathogens. All patients should be tested to be negative for HIV, HBV, and HCV prior to tissue processing. Wear a protective coat and handle human tissue specimens with glove.......
Figure 1 provides an overview of the workflow to culture OTSCs from fresh, unfrozen tumor tissue. Specimens of primary PDACs and metastases were collected directly after surgical resection and stored overnight on wet ice at 4 °C in the tissue storage solution. The specimens were processed, and slices were cultured as described in the protocol. The macroscopic morphology of each OTSC did not change grossly during cultivation. However, the size of the surface area of the OTSCs decrea.......
OTSCs of fresh tumor samples are a close approximation of the tumor in situ. They maintain their baseline morphology, proliferative activity, and microenvironment during the cultivation for a defined, tissue-dependent period11,12,13. This technique enables the immediate application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as profiling of the transcriptome and pr.......
|Advanced DMEM/F-12 Medium
|Agarose Low Melt
|8% in Ringer solution
|Antibody Diluent, Background Reducing
|Bioethanol (99%, denatured)
|Citric Acid monohydrate
|Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb
|Cell Signalling Technology
|Derby Extra Double Edge Safety Razor Blades
|Eosin Y-solution 0,5% aqueous
|Eukitt Quick hardening mounting medium
|Fetal bovine serum
|Formaldehyde solution 4,5%, buffered
|Hem alum solution acid acc. to Mayer
|Sigma Aldrich (Merck)
|Hydrogen peroxide 30%
|Sigma Aldrich (Merck)
|Liquid DAB+ Substrate Chromogen System
|MACS Tissue Storage Solution
|Microscope Slides Superfrost Plus
|Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 µm
|Monoclonal mouse anti-human Cytokeratin 7 (Clone OV-TL 12/30)
|Monoclonal mouse anti-human Ki67 Clone MIB-1
|Monoclonal mouse Anti-vimentin (Clone V9)
|Negative control Mouse IgG2a
|Negative control Mouse IgG1
|Paraffin (melting temperature 56°- 58°)
|Penicillin-Streptomycin (10.000 U/ml)
|PBS pH 7,4 (1x) Flow Cytometry Grade
|Resazurin sodium salt; 10 mg/ml in PBS
|Sigma Aldrich (Merck)
|Tissue culture testplate 6
|VECTASTAIN Elite ABC-Peroxidase Kit
|Xylene (extra pure)
|ClarioStar Microplate Reader
|Paraffin Embedding Center E61110
|Rotary Microtome Microm HM355S
|Section Transfer System Microm STS
|VT 1200S Vibratom
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