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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Abstract

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Introduction

Tumor organoids are ex vivo three-dimensional (3D) organized cultures that are derived from fresh tumor tissue and provide cancer models. Tumor organoids recapitulate the biological key features of the original primary tumor1,2,3,4 and can be expanded for up to several months and cryopreserved, similar to conventional immortalized cell lines. Tumor organoids provide a biobank of patient-derived tumor models for translational/personalized medicine5 and represent an important advance in cancer cell biology systems/models. Patient-derived tumor organoids can be used as ex vivo models to predict the efficacy of (neo)adjuvant oncological/pharmacological therapies, for which cultures are established from fresh tumor tissue and drug sensitivity assays or pharmacotyping are performed on a patient-specific basis to identify effective agents for subsequent lines of therapy1,4. Furthermore, tumor organoids overcome the limitation of the availability of primary tumor tissue and, more importantly, provide an excellent alternative or complementary system to in vivo mouse models, such as patient-derived xenografts (PDX)2. The complexity of tumor organoids is increased if the primary tumor cells are combined with stromal cells that are found in the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, which mimic the functioning and complex cellularity of the primary tumor. Tumor organoids have been established for many tumor types using standardized protocols6,7,8,9,10. Organoid propagation from different solid tumors, including colorectal and breast cancer tissue, is well-established and technically affordable11,12,13,14,15.

Surgical tumor resections or tumor biopsies provide primary tumor tissue specimens. Ideally, tumor tissue specimens should come from the center of the tumor mass or the invading edge of the tumor, as well as normal-looking tissue adjacent to the tumor. Compared to conventional 2D cultures, tumor organoids require several "add-ons", including a biological basement membrane (such as Matrigel, hydrogel, or a collagen-based scaffold), which mimics the extracellular TME, and a liquid growth medium that supplies specific nutrients and growth factors and supports cell proliferation and viability in culture16.

The most basic steps in primary cell culture are washing the tissue in saline solution to prevent contamination, mechanically cutting/digesting the tumor into small pieces of 1-3 mm3, and treatment with collagenase for the enzymatic digestion of the tissue. The digested mix is then filtered to remove large tissue fragments, resuspended in a biological basement membrane such as Matrigel, and plated as domes in low-attachment culture plates to enhance non-attachment growth. The basement membrane matrix domes are covered with liquid culture medium and supplemented with glutamine and antibiotics to avoid contamination, as well as with specific growth factors depending on the tissue type7,8,9,16,17. Other relevant cells present within the bulk tumor and the TME may also be isolated, such as cancer-associated fibroblasts (CAFs) and immune cells. This technique, which has recently been reviewed18, allows the establishment of co-cultures with different cell types to study the response to therapy in a more "realistic" tumor environment. Furthermore, cell-cell interactions and the interaction between tumor cells and components of the surrounding biological matrix can be studied.

The reported success rate of tumor organoid establishment using fresh tissue from biopsies or resected gastrointestinal tumor tissue is around 50%11, and the success rate from the latter is largely dependent on the tissue type and origin4, particularly the tumor grade and overall tumor cellularity. Three-dimensional tumor models have varying complexity, from simple unicellular aggregates to highly complex engineered models consisting of various cell types. The terminology used to describe 3D cultures in the literature is highly inconsistent19,20,21, as different terms such as spheroids, tumorspheres, and organoids are used, although the difference between them is unclear. As a clear consensus on the definition has not yet been reached, in this article, a tumor organoid is described as an organized tumor cell culture embedded into a biological basement membrane.

Herein, a validated protocol is reported for the establishment of tumor organoids from fresh tissue samples originating from fresh primary resected or PDX-derived pancreatic ductal adenocarcinoma (PDAC), and this protocol can be performed in most laboratories with basic tissue culture facilities. This protocol has been adapted from several state-of-the-art reported protocols that are currently used to establish tumor organoids or tumoroids from digestive tumor tissue from the groups of David Tuveson9, Hans Clevers8, and Aurel Perren7.

This protocol does not discuss how the fresh tissue is harvested. To obtain high-quality fresh human tumor tissue, it is important to have efficient coordination between the surgeons that harvest the tissue and the pathology department that extracts the tissue sample for organoid culture. Likewise, when using PDX as a fresh tissue source, efficient coordination with the person that harvests the tissue sample is also important. It is critical to obtain the tissue sample as quickly as possible (within 30-60 min from harvesting time) in order to maintain a high quality.

Protocol

All procedures were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Universidad Autónoma de Madrid Ethics Committee (CEI 103-1958-A337) and La Comunidad de Madrid (PROEX 294/19) and in accordance with the guidelines for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS). The protocol followed the ethical principles for biomedical research with written informed consent. Prior ethical approval was obtained for the use of fresh tissue for the establishment of the tumor organoid cultures. The samples were provided by the BioBank Hospital Ramón y Cajal-IRYCIS (National Registry of Biobanks B.0000678), integrated into the Biobanks and Biomodels Platform of the ISCIII (PT20/00045), and processed following standard operating procedures with the appropriate ethical approval. The tumors were subcutaneously implanted, as previously described22, into immunocompromised 6 week old female NU-Foxn1nu nude mice (see Table of Materials) and passaged in vivo to establish PDAC PDXs.

1. Experimental preparation

  1. Handle human samples in a class II biosafety cabinet.
  2. Wear a lab coat, protective gloves, and glasses throughout the procedure to avoid infection by tissue-borne pathogens.
    ​NOTE: A minimum of 3 h is required to process the fresh tissue sample and plate the organoids and fibroblasts.
  3. Process the fresh tissue samples22 within a maximum of 24 h from harvesting, and store at 4 °C in tissue culture medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) until processing.
  4. Keep all the samples and reagents on the ice during the entire procedure, and ensure to pre-set a refrigerated centrifuge to 4 °C and use it at a low speed to avoid damaging the cell preparation.
  5. Store P1,000 and P200 pipette tips in a −20 °C freezer to use during the protocol.
  6. Aliquot the basement membrane matrix (see Table of Materials) prior to use. For this protocol, 250 mL aliquots are recommended.

2. Processing fresh primary tumor tissue to establish tumor organoids and primary fibroblasts

NOTE: A minimum of 3 h is required to process the fresh tissue sample (either primary human resectable tumors or PDXs) and to plate the tumor organoids and fibroblasts. An outline of the organoid preparation process is shown in Figure 1, from tissue digestion to the plating of the tumor organoids. Before starting the protocol, take out an aliquot of the basement membrane matrix from the −20 °C freezer, and leave it on ice for approximately 30-60 min before use.

  1. Put a 6-well culture plate in a 37 °C cell culture incubator 3 h before plating the organoids.
  2. Measure, photograph, and wash the tissue (step 1.3) with 3 mL of Advanced DMEM-F12 (Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F-12 Ham (F12), supplemented with 5% fetal bovine serum (FBS), 15 mM Hepes, 1% L-Glutamine, 1% Penicillin-Streptomycin, 125 ng/mL Amphotericin B, 0.1 mg/mL Normocin) (see Table of Materials) by pipetting up and down and then wash with 3 mL of phosphate-buffered saline (PBS) by pipetting up and down.
  3. Remove the media by aspiration from the tissue culture plate and cut the tissue into 1 mm3 pieces in a sterile tissue culture plate using two sterile blades and 2 mL of Digestion Medium (Advanced DMEM-F12 + 10 mg Collagenase IV/mL, 0.000625% Trypsin-EDTA, and 10 µg/mL DNase). Collect the tissue in a 50 mL tube with 5 mL total of Digestion Media and incubate for 1 h at 37 °C with mechanical digestion with commercially available equipment (see Table of Materials) or with periodic mixing of the sample, by pipetting the sample up and down.
  4. Add 5 mL of Advanced DMEM-F12 to inactivate the trypsin, and filter the sample through a 70 µm pore strainer to remove large debris.
  5. Add 2 mL of DMEM with 10% FBS to the top of the filter, and collect the debris and tissue remnants to establish cultures of fibroblasts (see step 5).
  6. Centrifuge the filtrate at 200-300 x g for 5 min at room temperature, and aspirate the supernatant, leaving only the cell pellet. Add 5 mL of Advanced DMEM-F12, and centrifuge for 5 min at 300 x g.
  7. Resuspend the pellet in 4 mL of ammonium-chloride potassium (ACK, see Table of Materials) lysis buffer. Pass the mixture to a 15 mL tube, and incubate at room temperature for 2 min to lyse the red blood cells.
    NOTE: This red blood cell lysis step can be removed when there is no visible evidence of contamination.
  8. Centrifuge at 300 x g for 5 min at room temperature, and aspirate the supernatant. Add 5 mL of Advanced DMEM-F12, and centrifuge for 5 min at 300 x g.
  9. Add 1 mL of commercially available cell dissociation reagent (see Table of Materials) supplemented with DNase (1 µg/mL) to the cell pellet, and incubate for 2-3 min at room temperature.
  10. Centrifuge at 300 x g for 5 min, and aspirate the supernatant. Add 5 mL of Advanced DMEM-F12, and resuspend the cell pellet.
  11. Pipette up and down 30 times with a P1,000 pipette to dissociate the cells, centrifuge for 5 min at 300 x g, and remove the supernatant.
  12. Take P1,000 and P200 pipette tips from the −20 °C freezer, and resuspend the cell pellet in the membrane matrix using a P1,000 pipette and cold tips (50 µL per drop, taking into account the pellet volume, six to seven domes per well). Take out the previously heated plate from the incubator. Set a P200 pipette to 48 µL, and use cold tips to create domes of the basement membrane matrix in the pre-heated 6-well plate.
  13. Put the plate with the basement membrane matrix-cell domes into the 37 °C 5% CO2 incubator for 15 min to solidify the basement membrane matrix.
  14. Add 2-2.5 mL of Advanced DMEM-F12, supplemented with 20 ng/mL recombinant human epidermal growth factor (EGF), 100 ng/mL human placenta growth factor (PlGF), 10 ng/mL recombinant human basic fibroblast growth factor (bFGF), 769 ng/mL insulin-like growth factor-1 (IGF-1), and 10.5 µM ROCK Inhibitor (Advanced DMEM-F12 + growth factors) (see Table of Materials).

3. Monitoring the organoids

  1. Monitor the tumor organoid culture visually for the first 7 days and then three times per week thereafter.
  2. Change the medium twice per week with Advanced DMEM-F12 containing 20 ng/mL recombinant human epidermal growth factor (EGF), 100 ng/mL human placenta growth factor (PlGF), 10 ng/mL recombinant human basic fibroblast growth factor (bFGF), 769 ng/mL insulin-like growth factor-1 (IGF-1), and 10.5 µM ROCK inhibitor (Advanced DMEM-F12 + growth factors).
  3. Capture images of the tumor organoid culture periodically on day 1, day 3, day 7, day 10, and day 15 after processing the sample and plating the culture in the matrix to observe the growth and viability.

4. Passage and cryostorage of the organoids

  1. Remove the culture medium from the 6-well plate.
  2. Add 1 mL of an appropriate cell recovery reagent (see Table of Materials) to disassociate the basement membrane matrix and obtain a cell suspension, and recover the supernatant in a 15 mL tube. If the matrix does not dissociate immediately, incubate the sample at 4 °C for 15-20 min until completely dissolved.
  3. Add 1 mL of the same cell recovery reagent used in step 4.2 to the well of the plate to recover additional dissociated cells, and add the supernatant to the same 15 mL tube as in step 4.2.
  4. Add 5 mL of cold Advanced DMEM-F12, and centrifuge at 200 x g for 5 min.
  5. Remove the supernatant, and dry the pellet carefully by removing any remaining liquid with a 5 mL pipette; avoid moving the tube as much as possible. Resuspend the cell pellet in twice the original volume of basement membrane matrix in order to obtain a passage of 1:2.
  6. For the cryostorage of the organoid cultures, resuspend the pellet obtained in step 4.5 in 1 mL of freezing medium (10% DMSO, 20% FBS, 50% DMEM-F12, 10.5 µM ROCK inhibitor), and store at −80 °C.
    ​NOTE: The volume of basement membrane matrix can be adjusted to perform different passages, such as 1:1 (using the same volume of basement membrane matrix as the original) or 1:3 (adding three times the original volume of basement membrane matrix).

5. Establishment of fibroblasts

  1. After recovering the debris and tissue remnants in 2 mL of DMEM with 10% FBS, add this to a 6-well plate, and incubate at 37 °C with 5% CO2.
  2. Remove the tissue remnants from the fibroblast cultures from step 2.5 by the aspiration of the medium 2 days or 3 days after plating, and replace with fresh DMEM with 10% FBS.
  3. Monitor the fibroblast culture visually three times per week, and change the culture medium at least twice per week using DMEM supplemented with 10% FBS.

Results

It is important to document how the tumor organoid culture progresses over time, particularly in the first few weeks, in order to estimate how the culture will behave in downstream assays. Figure 2 shows an example of optimal tumor cell isolation and tumor organoid establishment from fresh tissue over a 15 day period. Sometimes, there is a large volume of cell debris in the sample, and it is difficult to see the developing tumor organoids, as shown in Figure 3. ...

Discussion

Major advances in pharmacological cancer therapies are challenging, as the likelihood of the approval of drugs in phase I oncology clinical trials is 5.1%, which is the lowest of all disease types23. The main reason is that cancer is very heterogenous, and therefore, patient cohorts do not uniformly respond as expected to the given treatment, which highlights that a more personalized approach is needed. Two-dimensional (2D) cultures have been used in translational cancer research for many years bu...

Disclosures

None.

Acknowledgements

This study was supported by funding from the Plataforma biobancos y biomodelos - Unidades de las Plataformas ISCIII de apoyo ala I+D+i en Biomedicina y Ciencias de la Salud (PT20/00045), The European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 857381, project VISION (Strategies to strengthen scientific excellence and innovation capacity for early diagnosis of gastrointestinal cancers), Intramural call for new research projects for clinical researchers and emerging research groups IRYCIS (2021/0446), Patient Derived Organoids 2.0 Project (CIBERONC) and the TRANSCAN II project JTC 2017 call "Establishing an algorithm for the early diagnosis and follow-up of patients with pancreatic neuroendocrine tumours (NExT)", grant number 1.1.1.5/ERANET/20/03. The biological samples used in this protocol were provided by the BioBank Hospital Ramón y Cajal-IRYCIS (B.0000678) and integrated into the Biobanks and Biomodels Platform of the ISCIII (PT20/00045). We would also like to thank Yvonne Kohl, Agapi Kataki Vita Rovita, and Thorsten Knoll for their invaluable support to develop this protocol as part of the NExT and VISION projects.

Materials

NameCompanyCatalog NumberComments
6 well Costar Ultra-low Attachment platesBiofilTCP011006
70 μm pore strainerVWR732-2758
Ammonium Chloride Potassium (ACK) Lysis BufferGibcoA10492-01
Amphotericin BGibco15290018
Cell culture incubator (21% O2, 5% CO2 and 37 ºC)NuaireNU-4750E
Cell recovery solutionCorning 354253
Collagenase IVGibco17104019
DMEM/F-12 (1:1)(1X) with L-Glutamine and HEPESGibco31330-038
DNaseRoche10104159001
Fetal Bovine Serum (FBS)Corning35-079-CV
Freezing container, NalgeneMerckC1562
gentleMACS Octo DissociatorMilteny Biotec130-096-427
HEPESGibco15630056
Human Placenta Growth Factor (PlGF)enQuireBioQP6485-EC-100UG
Immunocompromised female 6-week-old NU-Foxn1nu nude miceJanvier, France 
Insulin-like growth factor-1 (IGF-1)InvitrogenRP10931
L-GlutamineCorning354235
Matrigel Basement Membrane Matrix Corning356234
NormocinInvivoGenant-nr-2
Pasteur pipettesDeltalab200007
Penicillin Streptomycin Solution (100x)Corning30-002-CI
Phosphate-Buffered Saline (PBS)Corning21-040-CV
Recombinant Human Basic Fibroblast Growth Factor (bFGF)GibcoPHG0026
Recombinant Human Epidermal Growth Factor (EGF)GibcoPHG0311
ROCK Inhibitor Y-27632 (Dihydrochloride)STEMCELL72304
StemPro Accutase Cell Dissociation ReagentGibcoA1110501
Surgical BladesNahitaFMB018
TrypsinGibco25300054

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