Method Article

Bioprinting of Hydrogel Tumor Slices as a 3D Model for Mantle Cell Lymphoma

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DOI:

10.3791/68417

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September 12th, 2025

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1Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, 2Department of Physiology and Pharmacology, Karolinska Institute, 3Department of Clinical Pathology, Robert Bosch Hospital, 4Robert Bosch Hospital, 5Departments of Clinical Pharmacology, Pharmacy and Biochemistry, University of Tuebingen, 6iFIT Cluster of Excellence (EXC 2180), Image-guided and Functionally Instructed Tumor Therapies, University of Tuebingen, 7German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Tuebingen, 8National Center for Tumor Diseases (NCT), NCT South-West, a partnership between DKFZ, University Hospital Tuebingen, University of Tuebingen, Tuebingen, Bosch Health Campus, Stuttgart, Robert Bosch Hospital, Stuttgart, University Hospital Ulm, and Ulm University

In This Article

Summary

This protocol provides a detailed description of the generation, culture, and analysis of mantle cell lymphoma in 3D printed hydrogel tumor slices.

Abstract

Mantle cell lymphoma (MCL) is a rare, aggressive B-cell neoplasm that frequently relapses and only shows a limited response to conventional chemotherapy. A major challenge in MCL research is culturing primary MCL cells ex vivo, as cells tend to undergo spontaneous apoptosis when cultured in 2D suspension culture. Although 3D models are known to better recapitulate the in vivo situation of solid tumors, their application is still poorly explored in lymphomas. Developing 3D models that replicate the in vivo conditions of MCL within the lymph node could enhance their survival, facilitate the study of the MCL-tumor microenvironment crosstalk, and mimic the in vivo drug response. Here, a 3D printed model of MCL in the form of hydrogel tumor slices was established, along with an optimized culture method. A standardized process was developed using MCL cell lines or primary MCL cells, in which the cells are immersed in a hydrogel containing alginate, type I collagen, and basement membrane matrix by bioprinting into a gelatin support bath. The resulting MCL hydrogel tumor slices are cultured on a filter support to maintain their stability throughout the culture period. Drug treatments can be applied to the system. The response of single cells inside the hydrogel tumor slice can be tracked by four-color live 3D fluorescence imaging. Primary MCL cells demonstrated a stable viability when cultured in the hydrogel tumor slices. This protocol provides a detailed description of the generation, culture, and analysis of MCL cells in hydrogel tumor slices. By closely mimicking the tumor microenvironment and utilizing an air-liquid interface culture, the presented model enhances physiological relevance compared to the traditional 2D culture. It offers significant potential for advancing both biological and therapeutic studies of MCL.

Introduction

Mantle cell lymphoma (MCL) is a rare and aggressive B-cell non-Hodgkin lymphoma originating from the mantle zone of lymph nodes1. The mantle zone is composed of a ring of small lymphocytes surrounding the germinal center of the B cell follicle. Typically, MCL shows an aggressive clinical course, with frequent relapses after the initial chemotherapy. Due to an improved understanding of disease formation1 and the development of novel treatments, including immunochemotherapy2, Bruton's tyrosine kinase inhibitors3 or antibody-drug conjugates4, treatment options for MCL patients have led to improved response rates and a prolonged progression-free survival. However, drug resistance and relapses still render the disease incurable5. Partially, this is a consequence of its heterogeneity and the influence of the tumor microenvironment on tumor development and drug resistance6. A challenge in studying MCL is the difficulty of cultivating primary MCL cells in suspension culture7, as cells undergo spontaneous apoptosis ex vivo4,5. This highlights the need for alternative approaches to better model MCL in preclinical models.

In vivo, tumors are three-dimensional (3D) structures composed of various cellular components, including tumor cells, immune cells, and stromal cells, as well as non-cellular components such as the extracellular matrix (ECM). The ECM, which consists of macromolecules like collagen and laminin, forms a complex fibrous meshwork of elastic fibers that is highly hydrophilic, creating a gel-like environment2,8,9,10. To better replicate these in vivo conditions of tumors ex vivo, a variety of 3D models have been developed. In recent years, these models have become crucial for studying cancer pharmacology and biology, mainly focusing on solid tumors11.

To date, this field remains poorly explored in lymphomas, since hematological cancer cells have the capacity to grow both in suspension and in stromal surroundings like lymph nodes, the bone marrow, or secondary lymphoid tissues12. In vivo, MCL cells disseminate from the mantle zone of lymph nodes into the blood or the bone marrow, though they mainly proliferate within the lymph nodes from which they originate13,14. Within the lymph nodes, MCL cells are surrounded by T cells, macrophages, and mesenchymal stromal cells, all of which have been shown to control the survival or drug response of MCL cells14,15,16,17,18. Moreover, the MCL-tumor microenvironment crosstalk, as well as the porosity and mechanical properties, are important factors that can influence drug responses and resistance6,14. Thus, standardized and reproducible 3D tumor models of MCL that simulate the native microenvironment are required to study primary MCL. So far, mainly cell-based models such as spheroids or organoids of primary MCL cells have been established6,19. They successfully enhance the viability and long-term culture of primary MCL cells. However, the extracellular matrix is underrepresented in these models. In this context, advanced 3D bioprinting offers a promising approach. By directly assembling living cells and hydrogels, this technology enables the generation of physiologically relevant hydrogel-based 3D lymph node models.

Here, a 3D bioprinted model of MCL in the form of hydrogel tumor slices was established, along with an optimized culture method. Cells are embedded in collagen type I-, alginate-, and Matrigel-based hydrogel slices by freeform reversible embedding of suspended hydrogels (FRESH) bioprinting20. The hydrogel tumor slices are cultured on a filter support, which allows the MCL cells to grow in a setting that closely recapitulates the in vivo conditions of the lymph node.

This protocol provides a detailed description of the generation, culturing, and analysis of MCL cells in hydrogel tumor slices. By utilizing four-color live 3D fluorescence imaging, the viability or drug response of single cells inside the hydrogel tumor slice can be tracked. Primary MCL cells demonstrated improved survival when cultured in the hydrogel tumor slices. This model provides a tool for studying MCL biology and treatment response, paving the way towards understanding microenvironmental influences and MCL patient variability.

Protocol

The work with primary MCL cells has to be approved by the local ethics committee. In this study, the ethics committee of the Medical Faculty, Eberhard-Karls-University and University Hospital Tuebingen approved the collection and work-up of patient and healthy donor samples (project number 159/2011BO2), and informed written consent was obtained from the patients in accordance with the Declaration of Helsinki. The primary MCL cells used here were isolated from whole MCL patient lymph nodes and thus included not only MCL cells, but also other cells such as immune cells and fibroblasts.

1. Preparation of gelatin slurry for the support bath

NOTE: The gelatin slurry will serve as the support bath during the 3D-bioprinting process. It induces crosslinking of the bioink, allowing the formation of a 3D structure. The preparation of the gelatin slurry has to be conducted in sterile conditions within a cell culture hood.

  1. Prepare a 4.5% (w/v) gelatin solution in 10 mM HEPES and 11 mM CaCl2 by dissolving 6.75 g gelatin powder in 1.5 mL of 1 M HEPES, 15 mL of 110 mM CaCl2, and 133.5 mL of sterile H2O in a sterile Mason jar with a lid. Ensure the Mason jar is suitable for mixing in a high-performance mixer.
  2. Incubate the solution at 37°C for 4 h or until no smears are observed upon stirring the solution, indicating that the gelatin is dissolved completely. Cool the gelatin at 4°C overnight or for at least 12 h.
  3. Prepare a sterile wash buffer consisting of 10 mM HEPES and 14.4 mM CaCl2. Pre-cool the wash buffer to 4°C.
  4. Break the solidified 4.5% gelatin into several pieces using a sterile spoon.
  5. Fill the Mason jar containing the 4.5% gelatin completely with the pre-cooled wash buffer, ensuring the buffer remains cold throughout the process of preparing the gelatin slurry.
  6. Freeze the gelatin-containing Mason jar at -20°C for 45-60 min, until the gelatin suspension reaches 0°C, but has not yet started to form ice crystals. Use a thermometer to monitor the temperature.
  7. Attach the mixer lid and blade to the Mason jar, then mount the Mason jar onto the mixer. Blend the gelatin suspension for 120 s using the pulse setting.
    NOTE: The blender settings may have to be adjusted when using a different type of blender.
  8. Transfer the blended suspension to pre-cooled 50 mL tubes. Centrifuge at 3,800 x g for 2 min at 4°C. Ensure that the suspension is separated into a solid gelatin slurry at the bottom and the liquid wash buffer at the top.
  9. Perform the following step on ice: Wash the gelatin slurry. Aspirate the top liquid layer, trying to remove the bubbles visible on top of the wash buffer. Add 20 mL of fresh, 4°C wash buffer to the tube. Vortex the tube to mix the gelatin slurry with the wash buffer.
  10. Repeat the centrifugation and washing process at least 3x, or until no bubbles are visible at the top of the wash buffer. After the final wash, remove any residual wash buffer by aspiration. The gelatin slurry can be stored at 4°C for several weeks.

2. Preparation of cell-laden bioink

  1. Prepare a sterile 5% alginate solution 1-2 days prior to printing. Add 1 g Alginate to 20 mL of sterile PBS in a 50 mL tube. To prevent dry alginate powder residues, add the PBS in small increments, vortexing between additions. Incubate the solution either in a 37 °C water bath for several hours or overnight on a tube roller at room temperature. Once the alginate is completely dissolved, store the solution at 4 °C until needed.
  2. If working with an MCL cell line, culture the cells in suspension in a T75 flask. On the day of printing, ensure that 1.4 x 107 cells are available for the preparation of 1 mL of bioink, which will yield 9-10 hydrogel tumor slices.
  3. If working with primary MCL cells, cells do not need to be cultured in suspension culture before bioprinting. Thaw the cryopreserved primary MCL cells directly before bioprinting. Transfer the primary MCL cells into a 50 mL tube with culture medium. To remove the remaining freezing medium, immediately centrifuge the tube at 340 x g for 5 min, then remove the supernatant. Resuspend the cell pellet in 20 mL of fresh medium for cell counting.
  4. On the day of printing, prepare the bioink adapted for the cells of interest. For Jeko-1 cells, prepare a bioink composed of 0.5% (w/v) alginate, 20% (v/v) type-I collagen, 1x RPMI, and 2 mM HEPES. Mix 100 µL of 5% alginate, 200 µL of type-I collagen, 100 µL of 10x RPMI, 200 µL of 10 mM HEPES, and 350 µL of PBS for 1 mL of bioink. Cells will be added in step 2.6.
  5. For primary MCL cells, prepare a bioink composed of 0.5% (w/v) alginate, 20% (v/v) type-I collagen, 1x RPMI, and 0.5 mM HEPES. Mix 100 µL of 5% alginate, 200 µL of type-I collagen, 100 µL of 10x RPMI, 50 µL of 10 mM HEPES, and 500 µL of basement membrane matrix for 1 mL of bioink. Cells will be added in step 2.6. If working with an MCL cell line, transfer the cell suspension from the culture flask to a 50 mL tube.
  6. Mix the cells with 0.4% trypan blue in a 1:1 ratio. Count the viable cells (unstained) using a cell counter or a Neubauer chamber. Calculate the volume of cell suspension for 1 mL of bioink, which equals 1.4x107 cells. Transfer the calculated volume into a new 50 mL tube. Centrifuge at 340 x g for 5 min, then remove the supernatant. Resuspend the cell pellet in the prepared bioink to generate the final cell-laden bioink.
    NOTE: It was estimated that the cell pellet has a volume of 50 µL medium. Together with the bioink prepared in step 2.4, this will yield 1 mL of bioink.

3. In-bath bioprinting

NOTE: If possible, conduct the 3D printing process in a sterile environment, such as under a cell culture hood. The 3D printer used was modified for FRESH printing by adding a syringe pump extender, as described in Hinton et al.20.

  1. Switch on the bioprinter and connect it to the 3D-printer software. Open the .stl file containing the blueprint for the hydrogel tumor slices. Up to 5 slices can be printed simultaneously. Ensure that each hydrogel tumor slice has a diameter of 8 mm and a height of 1.5 mm. Set the fill density to 85%. Home the printer head.
    NOTE: The STL file was designed with Fusion 360 and can be found in the Supplementary Coding File 1. For 3D printing, the software Cura Version 15.04.5 was used.
  2. Transfer the previously prepared gelatin slurry into a 35 mm diameter Petri dish until it is half full. Ensure no air gaps remain in the gelatin slurry. Pull out excess water from the slurry using disposable precision wipes to generate a gelatin support bath, which is pasty and holds the shape until sufficient force is applied.
  3. Transfer the cell-laden bioink into a 2.5 mL glass syringe and connect it to a 0.8 mm blunt nozzle. Invert the syringe and carefully eject excess air and air bubbles. Once the syringe is prepared, insert it into the bioprinter. Using the 3D-printer software, extrude bioink until it starts to get ejected from the nozzle. Wipe away the ejected bioink drop using a disposable science precision wipe to prevent clogging of the nozzle.
  4. Place the gelatin support bath right below the syringe nozzle. Lower the printer head until the nozzle is positioned inside the gelatin slurry, 2 mm above the bottom of the support bath.
  5. Start the printing process via the 3D-printer software.
  6. Once the printing process is complete, remove any excess gelatin slurry from the nozzle to prevent clogging. Take out the support bath containing the hydrogel tumor slices and cover it with a lid to maintain sterility.
  7. Incubate the support bath containing the printed hydrogel tumor slices at 37 °C to allow the gelatin to melt. This step will release the printed hydrogel tumor slices from the support bath.
  8. Pre-warm the wash buffer and the cell culture medium to 37 °C. While the gelatin is melting, prepare a 6-well plate for washing the hydrogel tumor slices. Fill two wells with 10 mM HEPES,14.4 mM CaCl2 wash buffer (same buffer used for washing of the gelatin slurry), and two wells with cell culture medium.
  9. Once the gelatin has melted, use a sterile spatula to transfer the hydrogel tumor slices into the wash buffer. Wash the slices for 1 min each, first in the two wash buffer-containing wells, then in the two medium-containing wells. The cell-laden hydrogel tumor slices are now ready for culture.
  10. Keep the remaining bioink to repeat the printing process.

4. Hydrogel tumor slice culture and treatment

  1. To culture the hydrogel slices, transfer them onto a filter support (0.4 µm pore size) placed inside a 6-well plate. Culture up to 5 hydrogel slices on a single filter support. Add 1.5 mL of cell culture medium below the filter support. Add a drop of medium on top of the hydrogel tumor slices to prevent them from drying out.
  2. For the drug treatment of hydrogel tumor slices, add the desired amount of the drug to the cell culture medium. Here, hydrogel tumor slices were treated with 0.33 µM-27 µM Doxorubicin for 48 h or 72 h. Always culture a set of slices without treatment as a control.
  3. Place the 6-well plate with the hydrogel tumor slices in a cell culture incubator at 37 °C and 5% CO2 for the desired cultivation period.

5. Viability analysis of cells by trypan blue exclusion assay

  1. To assess cell viability after culture or treatment, dissolve the hydrogel tumor slices. Transfer a hydrogel tumor slice into a 1.5 mL tube. Add 500 µL per slice of a dissolving buffer containing 30 mM EDTA, 55 mM sodium citrate, and 150 mM sodium chloride (pH 6.8). Pipette up and down until the hydrogel has completely dissolved.
  2. Centrifuge the solution at 340 x g for 5 min. Aspirate the supernatant. Re-suspend cells in 500 µL of medium per dissolved hydrogel slice.
  3. Mix the cell suspension in a 1:1 ratio with 0.4% trypan blue. Count the viable (unstained) and dead cells (stained) with a cell counter or a Neubauer chamber. Record the number of live and dead cells for further analysis.

6. Four-color live 3D fluorescence imaging

NOTE: The four-color staining used here allows differentiation between living cells (TMRM-positive), apoptotic cells (Fluorogenic Caspase-3 substrate-positive), and dead cells (PicoGreen-positive)21,22,23. Nuclei are detected with DRAQ5. However, this protocol may be adjusted to any other photostable, specific fluorescence dye to detect the biomarkers, cellular structures or cell types of choice. Before staining, ensure that the microscope is able to detect the wavelengths of the fluorophores used for staining.

  1. For a four-color staining of a hydrogel tumor slice, prepare a staining solution by adding TMRM, DRAQ5, and fluorogenic Caspase-3 substrate to 1 mL of cell culture medium at a final concentration of 0.5 µM, 5 µM, and 2 µM, respectively.
  2. Using a sterile spatula, transfer a hydrogel tumor slice to a chambered glass coverslip. Add the prepared staining solution on top of the hydrogel slice until it is fully covered. Incubate the sample for 20 min at 37 °C in the dark.
  3. After the initial incubation, collect the staining solution covering the hydrogel tumor slice and add 0.7 µL/mL PicoGreen. Pipette the staining solution back to the hydrogel slice and incubate for an additional 10 min at 37 °C in the dark.
  4. After incubation, carefully remove the excess medium while leaving enough staining solution to keep the hydrogel slice moist.
  5. Analyze the slice immediately using a confocal microscope. To prevent phototoxicity and photobleaching, minimize the photon dose applied by reducing laser intensity and excitation time. The excitation/emission spectra of the stains described above can be found in Table 1.
  6. To obtain an overview of the cellular distribution of the cell in the hydrogel, capture z-sections. If z-sections have been taken during imaging, generate 3D reconstructions of the hydrogel tumor slices using the appropriate software. These reconstructions will allow for a thorough analysis of the spatial distribution and cellular status of cells in the hydrogel tumor slices.
  7. Follow the instructions and script provided in Supplementary File 1 and Supplementary File 2 to perform cell segmentation and reconstruction.

Results

The hydrogel tumor slices containing MCL cells were generated with the protocol described above. The steps of the workflow for 3D-printing and culturing the hydrogel tumor slices are shown in Figure 1A. The 3D printer used was modified for FRESH printing by the addition of a syringe pump extender as described in Hinton et al.20 (Figure 1B). Up to five slices were printed at once (Figure 1C) and cultured on one filter support (Figure 1D). Medium was added below the filter support to provide a nutrient supply to the hydrogel tumor slices. A drop of medium was added on top of each hydrogel tumor slice to prevent them from drying out. An overview of five hydrogel tumor slices cultured on one filter support is shown in Figure 1E, along with close-up images of a hydrogel slice. During a culture period of 3 days, the hydrogel tumor slices were stable and maintained their structure (Figure 2A). Directly after printing, air bubbles were present in the hydrogel tumor slices, but they disappeared during cultivation (Figure 2A).

The viability of the MCL cell line Jeko-1 cultured in hydrogel tumor slices (3D) compared to Jeko-1 cells cultured in suspension culture (2D) was assessed by trypan blue exclusion assay (Figure 2B). After 3 days of culture, 80% of cells were viable, compared to an initial viability of 88% of Jeko-1 cells before printing. Jeko-1 cells cultured in 2D maintained the initial viability of 88%. There was no significant difference between Jeko-1 cells cultured in 2D and 3D. To assess whether Jeko-1 cells proliferate in the hydrogel slices, the mean cell number of Jeko-1 cells per hydrogel slice was determined (Figure 2C). While 5x105 Jeko-1 cells were contained in each slice directly after printing (d0), this number nearly doubled to 9.3x105 cells after 3 days of culture.

The distribution and status of Jeko-1 cells cultured in hydrogel slices were assessed by four-color live fluorescence imaging (Figure 2D) directly after printing or after 3 days of culture. After printing, 3D reconstructions of the Jeko-1 hydrogel slices showed a homogeneous distribution of cells. The majority of cells were positive for TMRM, a marker for mitochondrial membrane potential22. A minority of cells were positive for Caspase-3 activity, a marker for apoptosis induction23, as well as for the dead cell marker PicoGreen21. After 3 days of culture, an increase in cell number was observed alongside the formation of cell clusters. Most cells remained TMRM-positive, while only a few cells stained positive for Caspase-3 activity or PicoGreen (Figure 2C).

To analyze whether MCL cells in hydrogel slices are responsive to chemotherapeutics, Jeko-1 cells cultured either in suspension culture (2D) or in hydrogel slices (3D) were treated with the chemotherapeutic drug Doxorubicin, which is part of the standard-of-care treatment for MCL patients24. The overall structure of the hydrogel slices was maintained during Doxorubicin treatment (Figure 3A). The viability of Jeko-1 cells cultured in hydrogel slices decreased dose-dependently in response to Doxorubicin treatment. The IC50 of Doxorubicin in Jeko-1 cells was increased when cells were cultured in hydrogel slices (IC50=5.8 µM) compared to Jeko-1 cells cultured in 2D suspension culture (IC50=2.0µM; Figure 3B). Four-color live fluorescence imaging showed a reduced TMRM- and an increased PicoGreen-signal in Jeko-1 cells cultured in hydrogel slices in response to Doxorubicin treatment, confirming a treatment-induced reduction of viability. Images obtained from four-color live fluorescence imaging were 3D-reconstructed, segmented based on the DRAQ5 nuclei signal, and analyzed using the Python Script described in Supplementary File 1. An exemplary segmentation mask is shown in Supplementary Figure 1. The cell number identified from the DRAQ5 signal was used to determine the total cell number. The cell number positive for TMRM, PicoGreen, and Caspase-3 activity was used to identify the percentage of viable, dead, or apoptotic cells, respectively. Quantifications of the four-color live fluorescence staining of Jeko-1 cells cultured in hydrogel slices for 3 days with or without 9 µM Doxorubicin showed comparable viability to the trypan blue exclusion assay (Table 2).

In addition to established MCL cell lines, primary MCL cells of three different MCL patients were cultured either in 2D suspension culture or in 3D hydrogel slices (Figure 4, Supplementary Figure 2). As verified by a pathologist, all three MCLs (Patient 1-3) were of classic/common morphology with Ki67 indices of 20%-50%. The primary MCL cells included all cell types isolated from MCL patient lymph nodes and thus contained not only MCL cells but also other types of immune cells or stromal cells. The viability of the primary MCL hydrogel slices was assessed by the trypan blue exclusion assay. After 4 days of culture, primary MCL cells in hydrogel slices maintained a viability of 43.6% (45.7% on day 3) compared to a baseline viability of 71.0% before printing (Figure 4A). This represents an increased viability compared to cells cultured in suspension (2D), which had a survival of 19.6% after four days of culture (28.4% on day 3). This viability was visualized by four-color live fluorescence imaging, in which most cells were TMRM-positive and viable, while a subpopulation was positive for Caspase-3 activity and PicoGreen, indicating apoptosis and cell death, respectively (Figure 4B). The quantification data of Figure 4B are shown in Table 2. Primary cells from two further MCL patients showed an increased survival of cells when cultured in the hydrogel slices (3D) compared to the respective 2D suspension culture (Supplementary Figure 1).

3D bioprinting process diagram using hydrogel and bioink for cell culture in research setup.
Figure 1: Workflow of 3D-printing of hydrogel tumor slices. (A) Workflow of 3D-printing and culture of cells in hydrogel tumor slices. (B) Printer set-up used for 3D-bioprinting of hydrogel slices in a gelatin support bath. (C) Printing a blueprint in the 3D-printer software. Up to five hydrogel slices were printed at once. (D) Schematic representation of the hydrogel slice culture. Hydrogel slices were cultured on a filter support in a 6-well plate. A drop of medium was added on top of each hydrogel slice to maintain their moisture. Up to five hydrogel slices can be cultured on one filter support. (E) 3D-printed hydrogel slices cultured on a filter support, as shown by an overview image or by close-up images. The scale bars represent 5 mm, 1 mm, or 200 µm as indicated. Please click here to view a larger version of this figure.

3D cell culture analysis; includes viability assay, cell count graph, and fluorescence microscopy.
Figure 2: Culture of a mantle cell lymphoma (MCL) cell line in 3D-printed hydrogel slices. (A) Close-up of a representative hydrogel slice containing the MCL cell line Jeko-1 after printing (d0) or after 3 days of culture (d3). The scale bar represents 1 mm. (B) Viability of Jeko-1 cells cultured in suspension (2D) or in hydrogel slices (3D) for 2 days as assessed by trypan blue exclusion assay. Mean ± SD of three independent bioprinting experiments is shown. (C) Jeko-1 cell number contained in each hydrogel slice directly after printing (d0) or after up to 3 days of culture (d1-d3). Mean ± SD of three independent bioprinting experiments is shown. No statistically significant difference was observed (n.s., p > 0.05, Mann-Whitney test). (D) Three-dimensional reconstruction of Jeko-1 hydrogel slices on the day of printing (d0) or after 3 days of culture (d3). The model was generated from z-stacks of a four-color live 3D fluorescence imaging. Living cells are shown in red (TMRM), apoptotic cells in yellow (Caspase-3 enzymatic activity), dead cells in green (PicoGreen), and nuclei in blue (DRAQ5). The scale bar represents 50 µm. Please click here to view a larger version of this figure.

Jeko-1 doxorubicin study: 3D cell culture (A), viability curve (B), confocal microscopy (C).
Figure 3: Drug treatment of a mantle cell lymphoma (MCL) cell line in hydrogel slices. (A) Close-up of a representative hydrogel slice containing the MCL cell line Jeko-1 treated with 3 µM Doxorubicin for 3 days (d3). The scale bar represents 1 mm. (B) Jeko-1 cells cultured in suspension (2D) or in hydrogel slices (3D) were treated with increasing concentrations of the chemotherapeutic agent Doxorubicin for 2 days. Viability of cells was assessed by the trypan blue exclusion assay. Mean ± SD of four independent bioprinting experiments is shown. (C) Three-dimensional reconstruction of Jeko-1 hydrogel slices treated with 9 µM Doxorubicin (Doxo) for 3 days (d3). The model was generated from z-stacks of a four-color live 3D fluorescence imaging. Living cells are shown in red (TMRM), apoptotic cells in yellow (Caspase-3 enzymatic activity), dead cells in green (PicoGreen), and nuclei in blue (DRAQ5). The scale bar represents 50 µm. Please click here to view a larger version of this figure.

Primary MCL cell viability bar chart and 3D fluorescence microscopy for cell activity analysis.
Figure 4: Culture of primary MCL cells in 3D-printed hydrogel slices. (A) Viability of primary MCL cells cultured in suspension (2D) or in hydrogel slices (3D) for up to 4 days, as assessed by trypan blue exclusion assay. Mean viability of one experiment is shown. (B) Three-dimensional reconstruction of primary MCL cells cultured in hydrogel slices (3D) for 3 days. Z-stacks were generated from hydrogel slices stained with a four-color live staining and analyzed using a confocal laser scanning microscope (CLSM). Living cells are shown in red (TMRM), apoptotic cells in yellow (Caspase-3 enzymatic activity), dead cells in green (PicoGreen), and nuclei in blue (DRAQ5). The scale bar represents 50 µm. Please click here to view a larger version of this figure.

StainTMRMDRAQ5NucView405 Caspase-3 substratePicoGreen
Excitation/Emission548 nm/574 nm647 nm/681 nm429 nm/ 469 nm488 nm/ 520 nm

Table 1: Excitation/Emission maxima of the stains used for four-color live 3D fluorescence imaging.

Sample% of cells
TMRM (Viable cells)PicoGreen (Dead cells)Caspase-3 (Apoptotic cells)
Jeko-1 Ctrl8323.618.3
Jeko-1 Doxo 9 µM27.681.386.6
Primary MCL Patient 175.231.133.1

Table 2: Quantification of 3D-reconstructions of four-color live staining of hydrogel tumor slices cultured. Hydrogel tumor slices were cultured for 3 days and contained either Jeko-1 cells cultured with or without treatment with 9 µM Doxorubicin or primary MCL cells (patient 1).

Supplementary Figure 1: Cell identification from 3D-reconstructions of four-color live staining. 3D-reconstructions of confocal image labeled with DRAQ5 obtained from (A) four-color live staining and (B) a segmented image on this channel generated using the Python imaging analysis pipeline. The scale bar represents 200 µm. Please click here to download this figure.

Supplementary Figure 2: Culture of primary MCL cells of patients 2 and 3 in hydrogel tumor slices. Viability of primary MCL cells of (A) patient 2 or (B) patient 3 cultured in suspension (2D) or in hydrogel slices (3D) for up to 4 days as assessed by trypan blue exclusion assay. Mean viability of one experiment is shown. Please click here to download this figure.

Supplementary File 1: Instructions for analysis of 3D reconstructions obtained from four-color live staining. Please click here to download this file.

Supplementary File 2: Python script for analysis of 3D reconstructions obtained from four-color live staining. Please click here to download this file.

Supplementary Coding File 1: Stl. file containing the blueprint for the hydrogel tumor slices.  Please click here to download this file.

Discussion

The hydrogel tumor slices described in this protocol offer an MCL model that mimics the lymph node structure, allowing the study of MCL cell survival and drug response in a 3D tumor microenvironment generated by bioprinting.

The most widely used bioprinting methods are based on extrusion, where bioink is ejected through a dispensing system. In inkjet printing, for example, bioinks are rapidly crosslinked after deposition to form 3D structures25. However, this approach comes with limitations, particularly in terms of the viscosity of the materials used and the ability to stack layers. In contrast, embedded syringe-based extrusion 3D bioprinting, such as the FRESH bioprinting used in this protocol, applies a gel-in-gel approach. This allows the printing of low-viscosity bioinks, like collagen, into stable constructs, making it possible to mimic the complex tumor microenvironment20,26. While 3D printers specifically developed for bioprinting can be expensive, the method presented here offers a more accessible entry point. It can be adapted to standard extrusion printers with relatively simple modifications. These modifications can be 3D-printed with affordable filaments, and the designs are available as open-source resources, for example, by Hinton et al.20. However, it is important to note that the preparation of the hydrogel support bath for FRESH bioprinting is highly temperature-sensitive and requires careful attention during slurry preparation.

Frequently, 3D-printed tumor models are cultured in floating systems, lacking the in vivo oxygen gradient25, or in microfluidic chip cultures27, which capture a high degree of complexity but have a low throughput and require high maintenance. These hurdles can be overcome with the hydrogel tumor slices described here. The setup of culturing up to five hydrogel tumor slices on an air-liquid interface is derived from the culture of precision-cut tumor slices of solid tumors28. The membrane of the filter support used in this protocol is composed of a polytetrafluoroethylene (PFTE) membrane with 0.4 µm pore size, which allows nutrient exchange but prevents cellular attachment or cell migration29. The culture on the PTFE membrane provides physical support for the slices to maintain their stability and prevent them from rupturing or disintegrating. Additionally, they provide an air-liquid interface, providing oxygen mainly from the air above and nutrients from the medium below the filter30. The system thus incorporates spatiotemporal variations in nutrient supply, cell proliferation, and oxygenation, as they may occur in vivo25. Cultured in the hydrogel tumor slices on a filter support, primary MCL cells maintained a viability of over 40% for 4 days, which was superior to the viability observed in 2D suspension culture (Figure 3A, Supplementary Figure 2). Further investigations should address whether a prolonged viability can be achieved by the addition of cytokines or co-cultivation with CD40L-expressing fibroblasts, as it has been described in spheroids and organoids of primary B-cell lymphoma tumor6,19,30 or co-cultures of primary MCL cells with CD40L-expressing fibroblast L cells14. Incorporating such approaches into the hydrogel tumor slices should be considered to enhance the culture of primary MCL cells as well as the similarity to the native microenvironment.

Lymph nodes contain a well-developed network of extracellular matrix fibers. Due to their pattern, they are known as reticular fibers. Mainly, these fibers are composed of collagen and basement-membrane-specific components31,32,33. To generate a matrix resembling the in vivo situation in lymph nodes, the bioink used to generate the hydrogel tumor slices is composed of collagen type I and Matrigel, which consists of the four major basement membrane ECM proteins laminin, collagen IV, entactin, and perlecan34. Further, alginate is added to the bioink to stabilize the hydrogel tumor slices, allowing for a prolonged culture and the transport from one vessel to another using a spatula. Testing different hydrogels containing collagen and alginate showed the highest cellular viability in combination with a stable and durable hydrogel slice when using a bioink with 0.5% alginate and 20% collagen (data not shown). Besides the components, the hydrogel tumor slice diameter of 0.8 cm further resembles the mean lymph node diameter of 1 cm35. Live 3D imaging of the hydrogel tumor slices further showed an even distribution of cells (Figure 2C).

Here, the ability to treat the hydrogel slices with drugs was shown by treatment with doxorubicin, which is part of the R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), a standard-of-care therapy for older MCL patient subgroups24. By comparison to 2D cultures, the influences of the tumor microenvironment on treatment effects can be studied in the hydrogel tumor slices. As shown in Figure 3, Jeko-1 cells cultured in hydrogel slices were more resistant to Doxorubicin treatment than 2D-cultured cells. This is in line with observations that the extracellular matrix can influence drug responses and frequently enhance drug resistance through modulating the tumor stiffness36, cell-cell communication37, and drug delivery38. Additionally, flow cytometric analysis, as well as immunohistochemical or multiplex immunofluorescence staining, can give deeper insights into the treatment response of single cells in the hydrogel tumor slices.

In conclusion, this protocol describesthe generation and culture of hydrogel tumor slices, which represent a 3D lymph node model mimicking the native three-dimensional lymph node structure. It holds the potential to expand the application from MCL cells to other primary lymphoma indications.

Disclosures

This study was supported by the Robert-Bosch Stiftung and the Ministry of Science, Research and Arts Baden-Württemberg (3R-Network BW).

Acknowledgements

We thank Kerstin Willecke for her excellent technical assistance and Yang Zhang for his valuable assistance with the 3D image analysis.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
µ-slide 8 Well Glass Bottomibidi GmbH80827Glass bottom slides used for live imaging of hydrogel slices
10x PBSCarl Roth GmbH + Co.KG9150.1Bioink component
10X RPMI-1640 MediumSigma-AldrichR1145Bioink component
2.5 ml Gastight Syringe Model 1002 TLLHamilton81420Syringe for bioprinting
3D Extrusion printer, PrintrBot SimplePrintrbotSelf-modified to allow for printing of bioinks
6-well plateTh. Geyer GmbH & Co.KG7696790Culture of the hydrogel slices
Bottle top vacuum filtration systems, PES membrane, 500 ml, 0.22 µm pore size, 70 mm diameter membraneCorning Inc.513-3355Sterile filtration of buffers
Calcium chloride dehydrateCarl Roth GmbH + Co.KGT885.2Component of wash buffer and gelatin slurry
Collagen I from ratCorning Inc.CLS354249Bioink component
Cura Version 15.04.5 software for 3D printingUltimakerSoftware used for 3D printing
DRAQ5Biostatus Ltd.DR50050Stain for live imaging of hydrogel slices
Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrateSigma-AldrichE5134Component of alginate dissolving buffer
EVE Automatic Cell CounterNano EnTek Inc.10027-452Cell Counter
Falcon Bacteriological Petri Dishes with Lid, 35 mm diameterFalcon351008Filled with gelatin slurry to form the support bath for bioprinting
Falcon Tubes, 50 mLSarstedt AG & Co.KG62.547.254
Fetal calf serumSigma-AldrichS0615Cell culture medium supplement
Gelatin from porcine skinSigma-AldrichG1890Main component of the gelatin slurry
HEPES, Free Acid, Molecular Biology GradeMerck KGaACAS 7365-45-9Component of wash buffer and gelatin slurry
Jeko-1 MCL cell lineATCCCRL-3006MCL cell line
KIMTECH Science Precision wipesKimberly Clark Corporation7551Used to draw excess water from the gelatin slurry and to remove droplets from the 3D-printer nozzle
LAS X software Version 3.5.7.23225LeicaSoftware used for 3D reconstructions
Leica TCS SP8 Confocal laser scanning microscopeLeicaTCS SP8Confocal microscope for live imaging
Mason Jar Regular Pint 16 ozBall CorporationG001Jar in which the gelatin slurry is prepared
Matrigel Growth Factor Reduced (GFR) Basement Membrane MatrixCorning Inc.356231Bioink component
Millicell cell culture insert, 0.4 µm pore sizeMerck KGaAPICMORG 50Filter support for hydrogel slices
NucView 405 Caspase-3 Substrate, 1 mM in 1x PBSBiotium10405Stain for live imaging of hydrogel slices
Oster Professional Series BlenderOsterBPST02-B00Blender to generate gelatin slurry
Penicillin/StreptamycinGibco15140163Supplement for MCL cell culture medium
Quant-iT PicoGreen dsDNA Assay-Kits und dsDNA-ReagenzienInvitrogenP7589Stain for live imaging of hydrogel slices
Rotina 35 RHettich24573Centrifugation of cells
ROTINA 38/38R centrifugeHettichZ720109Centrifugtion of gelatin slurry
RPMI-1640+GlutaMAXGibco61870036Base of culture medium for MCL cells
Sodium alginateFMC CooperationGQ9109901Bioink component
Sodium chlorideMerck KGaA1.06404Component of alginate dissolving buffer
Sodium citrateCarl Roth GmbH + Co.KG3580.1Component of alginate dissolving buffer
Sterican blunt application cannulas 21G 0.8x22 mmB.Braun9180109Nozzle for 3D-printing
Tetramethylrhodamin-methylester-perchlorat (TMRM)Sigma-AldrichT5428Stain for live imaging of hydrogel slices
Trypan blue, 0.4%Sigma-AldrichT8154Stain for viability analysis
Waterproof thermometerVWR620-2099Thermometer to monitor the temperature of the gelatin slurry

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3D BioprintingTumor MicroenvironmentPrimary MCL CellsCollagen BioinkGelatin Support BathDrug Response AnalysisLive Fluorescence ImagingAir Liquid Interface Culture
Bioprinting of Hydrogel Tumor Slices as a 3D Model for Mantle Cell Lymphoma
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