Publications

Enteric neurospheres assemble enteric nervous system networks in 3-D Life ToGro Hydrogel. Cells used: murine enteric neural stem cells (neurospheres). Methods used: microscopy of neural networks.

3-D Life Hydrogel was used to perform a migration assay on different melanoma cell lines to compare cell motility and evaluate their cell migration capacity. A small drop of Dextran-PEG hydrogel was placed in wells of a 96 well plate and cells were seeded around the gel drop. After 24 hours the gel was dissolved by 3-D Life Dextranse leaving cells intact and attached to the bottom of the well. Cell migration into the empty space left by the dissolution of the gel was subsequently recorded and quantified by image analysis.

The 3-D Life Hydrogel system was used in a microfluidic platform specifically developed for the 3-D co-cultivation of human cervical cancerous tissue (spheroids) with donor-derived cervical fibroblasts. Donor-derived neutrophils were integrated in the microfluidic system and were recruited into the hydrogel-based co-culture. The 3-D Life hydrogel demonstrated good stability without shrinkage and provided a robust 3D matrix for the cells. The treatment of the culture with cisplatin demonstrates the applicability of the platform for drug testing and the development of new (immuno) therapeutic options. Application: Hydrogel in a 3D microfluidic platform for drug discovery. Cells used: SiHa (human squamous cervical cancer cell line), primary fibroblasts (from human cervix uteri), human PMNs (polymorphonuclear leukocytes/neutrophils from whole blood), human PBMCs (peripheral blood mononucleated cells from whole blood). Methods used: Life/Dead staining (Calcein AM/PI), immunofluorescent staining, LDH assay (cytotoxicity), TUNEL Assay, Live cell tracking in the hydrogel.

Newly developed light-induced hydrogels of Cellendes (product in development) were used in a novel integrated fluorescent light sheet bioprinting and imaging system that combines high printing speed and high resolution with light sheet-based imaging. A resolution of 9 µm was achieved in printed structures which was, up to date, only reached by two photon photopolymerization. No hydrogel contraction or expansion through the bioprinting procedure was observed. Using this bioprinting system full-thickness skin constructs were generated and remained viable for 41 days. Cells used: HaCaT (human keratinocytes), Hs27 (human fibroblasts), HUVEC. Methods: diffusion through hydrogel by Fluorescence Recovery after Photobleaching (FRAP) analysis; light sheet based bioprinting; immunofluorescence staining.

Human breast cancer cells were embedded in 3-D Life Hydrogel on a novel microfluidic system to assess its applicability to investigate CAR-T cell mediated therapeutic treatments. The publication shows how CAR-T cells can migrate into the hydrogel towards the embedded tumor cell aggregates and how subsequent cytokine release can be measured. It is also shown how the chip-based hydrogel cell culture can be used to assess treatments for antagonizing the Cytokine Release Syndrome. Cells used: MDA-MB-231 (human breast cancer cell line); primary human fibroblasts, human breast cancer organoids (patient derived), human T cells, CAR-T cells. Methods used: Injection of 3-D Life Hydrogel into microchannels; fluorescence microscopy.

Using two-photon microscopy the authors developed a method to tether proteins or peptides in the hydrogel network with micrometer precision. This allows to follow the interaction and effects of immobilised proteins or peptides with cells in subcellular dimensions.

3-D Life Hydrogel was used in a microphysiological system (MPS) to build the stromal compartment of a human choroidal in vitro model (Choroid-on-Chip). The model allowed for a controlled immune cell recruitment into the melanocyte-bearing stromal compartment through a vascular monolayer. This choroid-on-chip model was shown to effectively work in efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies. Cells used: primary human melanocytes. Methods used: microphysiological system, perfusion, live/dead staining, KI-67 staining, nuclear (DAPI) staining, antibody staining, confocal microscopy and analysis.

Using the 3-D Life Hydrogel system a reproducible tumor-stroma model including macrophages, neutrophils and fibroblasts within a malignant tumor microenvironment was developed. In contrast to collagen-based matrices, where the matrix strongly contracts during a long culture period, the 3-D Life Hydrogel maintained its size. Application: 3D tumor-stroma model for drug discovery. Cells used: primary human fibroblasts; U937 lymphoblast cell line (differentiated to macrophages), HL-60 promyelocytic cell line (differentiated to neutrophils), HaCaT-ras A-5RT3 (tumor keratinocytes), HaCaT-ras A-5IL-6 (tumor keratinocytes), MCF-7 (mamma carcinoma cells), H838 (lung carcinoma cells). Methods used: hydrogel culture in 24 well transwell insert plates. Fibroblasts and immune cells were cultured in the gel, tumor epithelial cells on top of the gel. Cultures with keratinocytes were cultivated at the air-liquid-interface.

3-D Life Hydrogel was used to create an extracellular matrix-lumen interface within a microfluidic chip. The successful co-culture of tumor spheroids in the gel and the epithelial cell layer on the gel surface shows the suitability of the setup as a cellular barrier model. Application: cell-based assays in microfluidic platforms; in vitro model of cellular barriers; transepithelial cell migration. Cells Used: HT29, MDCK. Methods used: hydrogel in a microfluidic platform; transepithelial electrical resistance (TEER) measurements.

The 3-D Life Hydrogel system was used to generate hydrogels with controlled variations in local stiffness at microscale dimensions to examine mechanotransductional effects on human mesenchymal stem cells. Application: cellular mechanotransduction. Cells Used: human mesenchymal stem cells (hMSCs). Methods used: atomic force microscopy; rheology; Live/dead staining; immunostaining; paraffin embedment and sectioning; hematoxylin-eosin staining; RT-qPCR.

Chondrons prepared from articular cartilage were cultured in 3-D Life Dextran-PEG Hydrogels modified with 3-D Life RGD Peptide in a specific architectural design. The resulting 3D tissue constructs were mechanically characterized. Application: Tissue engineering. Cells Used: chondrons derived from articular cartilage. Methods used: Live-dead staining; compression loading.

Single cells are encapsulated in 3-D Life Hydrogel spheres via an oil-phase ‘‘pinching’’ process in a microfluidic device and subsequently cultured in multiwell plates to generate clones of cells. To culture mouse embryonic stem cells (mESCs) in these spheres the hydrogel components were mixed with several additives including gelatin. mESCs maintained pluripotency over at least 8 days of culture indicating the suitability of this 3D culturing system to promote stemness of ESCs even without pluripotent media. Application: single cell analysis. Cells Used: PC9 (lung adenocarcinoma, human), ZHBTc4 ESCs (mouse embryonic stem cells). Methods used: microsphere production, microsphere cultivation, scRNA-seq, fluorescent labeling of hydrogel via biotin-streptavidin attachment.

Detailed protocol on the generation of microspheres for single cell encapsulation.

The authors studied the regulation of cell migration by tuning the spatial stiffness of hydrogels. Migration velocity and cell morphology were analyzed to charaterize the dependence of cell migration on the heterogeneous stiffness in subcellular scale. Application: mechanotransduction. Cells Used: C2C12 murine myoblasts. Methods used: live/dead cell staining; cell tracking; rheological and atomic force microscopic measurements of hydrogels; scanning electron microscopy of hydrogels; fluorescent staining (phalloidin) and fluorescent antibody staining; fluorescence imaging of cells in gels.

Cellendes hydrogel is used to culture human pancreas organoids in a chemically definded 3D matrix.

Murine ventricular cardiomyocytes were embedded in disks of PVA-PEG/RGD hydrogels and placed on a stretching device (IsoStretcher) to examine the conversion of mechanical stimuli into Ca²⁺ signaling. The cardiomyocytes in the gel were loaded with the Ca²⁺ indicator Fluo-4 AM and calcium transients were recorded upon radial stretching of the cells.

3-D Life Hydrogel is used in an artificial lymph node model using a perfused bioreactor system. PBMCs and stromal cells are cultured in this model.

The Dextran-CD Hydrogel FG is used for an in vitro 3D chemotaxis assay for leukemic cells. A detailed protocol describes the setup of the assay in µ-Slides Chemotaxis (ibidi GmbH, Munich,Germany), cell tracking and quantitative analysis of the cell movement towards the chemoattractant.

Liver cancer cells were embedded in a 3-D Life Hydrogel to analyze the effect of pressure on their proliferative, migratory and invasive ability in a 3D environment.

The work shows how different stiffnesses of 3-D Life Hydrogels can direct the differentiation of endothelial progenitor cells (EPCs) into a venous or arterial phenotype. The gels injected into nude mice support the in vivo formation of functional blood vessels. Application: in vivo vasculogenesis. Cells used: murine endothelial progenitor cells. Methods used: hydrogel injection into mice; paraffin embedment of gels; antibody staining of tissue slices; dissolution of gels with dextranase and subsequent analysis of cells with qPCR and Western blotting.

The authors took an innovative approach and used the flexibility of the 3-D Life Hydrogel system to design a clustered RGD Peptide microenvironment to study patterns of cell adhesion ligands on cell behavior. Cells Used: NIH–3T3 fibroblasts, C2C12 cells (myoblasts, murine). Methods used: Live/Dead viability assay, bright-field and confocal microscopy, phalloidin staining, DAPI staining.

The paper shows a six week cultivation of chondrons in 3-D Life Dextran-PEG Hydrogel.

A novel peptide modification of 3-D Life Hydrogel was used to establish a neurosphere outgrowth assay for developmental neurotoxicity compound testing. The work shows how cell migration, differentiation to neurons and formation of neuronal networks is supported by the hydrogel.

3-D Life hydrogel supplemented with RGD Peptide was used for long term culture (14 days) of human breast epithelial cells (MCF10A). Successful lumen formation by spheroids was observed with wildtype cells and investigated with mutated cells in this type of cultures.

3-D Life Dextran-CD hydrogel modified with RGD Peptide was used to quantitatively evaluate amoeboid versus mesenchymal cell migration of glioma cells. The work shows how 3-D Life Hydrogel, in contrast to 2D culture, supports drug evaluation aiming at the efficient inhibition of both, amoeboid and mesenchymal cell migration in 3D culture. Applications: drug evaluation, cell migration analysis. Methods used: immunofluorescent staining, fluorescence microscopy, chemotaxis in ibidi µ-slides (www.ibidi.com).

Cartilage intermediate layer protein (CILP) and aggrecan and collagen II synthesis were measured in human nucleus pulposus (NP) cells in response to mechanical stimuli, including cyclic compressive stress and cyclic tensile strain. Application: Effects of mechanical stress on protein expression. Cells used: nucleus pulposus cells. Methods used: NP cells were embedded in cylinders of 3-D Life Dextran-PEG FG hydrogels (Cat. No. G90-1). The hydrogel cylinders were introduced into Bioflex 6-well compression plates and mechanical stress was applied using a FX-5000C™ Flexercell system (Flexcell International). Cells were extracted from hydrogels using 3-D Life Dextranase (Cat. No. D10-1) and subjected to RT-PCR and Western blotting analysis.

Spheroids of HepG2 cells were grown in 3-D Life Dextran-CD Hydrogels. Application: spheroid culture. Cells Used: HepG2. Methods used: Immunofluorescence, confocal microscopy.

The effect of multiaxial cell stretch on adult ventricular cardiomyocytes (CM) embedded in 3-D Life Cellendes hydrogel of different stiffnesses (Young modulus of 1 kPa, 4-9 kPa and > than 10 kPa) was examined on a cell stretch system (IsoStretcher). Single CMs were embedded in SG-PVA-PEG hydrogels modified with RGD Peptide to allow for adhesion of cells to the surrounding hydrogel. Stretch-induced Ca²⁺ influx was measured with the Ca²⁺ indicator Fluo-4 AM which was mixed into the hydrogel before gelation.

3-D Life Hydrogel was used to co-culture pancreatic islets with adipose tissue derived mesenchymal stem cells (AT-MSCs) to determine the effect of AT-MSCs on islet insulin secretion. Hydrogel cultures were examined in vitro to determine insulin secretion as well as transplanted in diabetic mice to determine the effect on blood glucose levels. The implanted hydrogel prevented passage of immune cells to the allograft, as shown after recovery of the implant and subsequent processing for histopathological examinations. Islets and cells were recovered from explanted hydrogels by dextranase treatment and subjected to RT-PCR analyses.

This book chapter describes in detail the 3-D Life Hydrogel platform technology as well as selected applications of in vitro cultures.

A disease model of kidney fibrosis was developed using 3-D Life Dextran hydrogel by co-culturing human kidney epithelial cells and human fibroblasts in two layers of hydrogels of different biomimetic modifications. The work shows a unique and successful model system for screening of new molecules capable to interfere and modulate the dialogue between epithelial and mesenchymal cells. Application: target identification and drug evaluation. Methods used: immunofluorescent staining and fluorescence imaging, drug administration, transmission electron microscopy, proliferation assay, RNA extraction for gene expression array.

The authors established two keratocystic odontogenic tumor (KCOT) cell lines which they cultured in PVA-PEG-based 3-D Life Hydrogels modified with RGD Peptide. Stainings of the actin cytoskeleton with rhodamine phalloidin and nuclei with DAPI showed spheroid formation with different characteristics depending on the patient's disease and origin of the KCOT cell line.

3-D Life Hydrogel is used to develop an artificial lymph node model using a perfused bioreactor system. PBMCs, antigen-presenting dendritic cells, and MSC-derived stromal cells are co-cultivated.

Co-culture of HUVECs and fibroblasts in 3-D Life Hydrogels to assess titanium-hydrogel-cell compatibility for future implantation strategies.

Molecular mechanisms of de novo epithelial lumen formation is studied in long term cultures of MCF10A mammary epithelial cells in 3-D Life Hydrogels.

Cancer cells and fibroblasts are analyzed alone and in co-culture in 3-D Life Hydrogels using Raman spectroscopy.

3-D Life Dextran-PEG Hydrogel was mixed with Matrigel to adjust the stiffness of Matrigel while maintaining the ligand density for cell adhesion.

Automated drug screening of tumor spheroids in 3-D Life Hydrogels. Demonstrates the different drug sensitvities of tumor cells in 2-D versus 3-D cell culture.

Preparation of hydrogel microarrays with 3-D Life Hydrogel for research and high-throughput screening.

Review on synthetic 3D culture systems, including 3-D Life Hydrogel.

3-D Life Hydrogel is used to immobilize bacteria for Raman spectroscopy.

3-D Life Hydrogel technology is used with maleimide-modified serum albumin for the cultivation of chondrogenic cells.

3-D Life Hydrogel technology used with maleimide-modified serum albumin.