Publications

Human breast cancer cells were embedded in 3-D Life Hydrogel on a novel microfluidic system to assess its applicability in investigating 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.

Primary human skeletal muscle cells were embedded in 3-D Life Hydrogel and submitted to constant mechanical pressure of up to 1.2 MPa. After 3 days cells were extracted from the hydrogel by dextranase digestion and analysed for changes in cell metabolism, morphology and calcium homeostasis compared to control groups. The authors developed a experimental model for clinically common skeletal muscle injury caused by static load pressure and which may help to further the study of the mechanism of action of treatment methods for such injuries.

3-D Life Hydrogel was dispensed in 150 nl droplets on 672 spots in a microarray format (Droplet Microarray, DMA, Aquarray GmbH). In one approach 50 cells were cultured in each hydrogel droplet. In another approach spheroids were formed in culture medium with the hanging drop method on the microarray slide and subsequently immobilized in hydrogel by injection of hydrogel pregel solution into the droplets. The DMA technology combined with automated 3-D Life Hydrogel dispension and image analysis on the microarray allows for HTS applications such as drug screening, migration and invasion assays while saving reagents and compounds in 2-3 orders of magnitude compared to 96- and 384-well plates.

3-D Life Hydrogel was used in a microarray format (Droplet Microarray, DMA, Aquarray GmbH) for drug-induced Differential Gene Expression Analysis (DGEA). Chronic lymphocytic leukemia (CLL) cells were cultured in 150 nl hydrogel droplets in the presence of different concentrations of the drug doxorubicin. For subsequent DGEA a protocol was adapted for the on chip sscDNA preparation from separated hydrogel droplets. Using this methodology gene expression analysis of two genes playing an important role in the regulation of cell survival and apoptosis in CLL, SYK and GADD45b, was performed. This miniaturized format of 3D cell culture in hydrogels pushes forward high-throughput screening for personalized medicine.

Enteric neurospheres form enteric nervous system networks in 3-D Life ToGro Hydrogel.

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.

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.

Spheroids of melanoma cells expressing GFP (green fluorescent protein) were embedded in 3-D Life Hydrogel to more closely resemble the 3D in vivo tumor architecture. The effect of the drug cisplatin on cell death of individual spheroids was followed in cells with or without a p53 mutation, respectively. Results clearly show how the p53 mutation conveys resistance to cell death through this drug.

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.

Spheroids of melanoma cells expressing GFP (green fluorescent protein) were embedded in 3-D Life Hydrogel. The effect of drugs on tumor growth and cell death of individual spheroids was followed over 8 days by confocal fluorescence microscopy.

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.

Spheroids of melanoma cells were injected and grown in 3-D Life Hydrogels supplemented with fibronectin. Hydrogels contained either cell-degradable CD-Link or non-degradable PEG-Link. Different stiffnesses of hydrogels were produced and determined by rheological measurements. Spheroid outgrowth was monitored in the presence of different concentrations of the drug dabrafenib, which is an inhibitor of proliferation of metastatic melanoma.

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.

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.

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.

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.

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.

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.

Mouse pancreas organoids were cultivated in 3-D Life Hydrogel crosslinked with hyaluronic acid or PEG-Link. A 3-D Life Hydrogel composition was found that showed similar mechanical characteristics (stiffness and elasticity) compared to Matrigel®. Different microscopic analyses including Raman microscopy revealed similar growth characteristics and expression patterns of specific stem cell markers between organoids grown in 3-D Life Hydrogel and Matrigel®. This confirmed the suitability of 3-D Life Hydrogel as a potential synthetic alternative to Matrigel® in organoid culture.

An organotypic in vitro model containing microtumor spheroids, macrophages, neutrophils, fibroblasts and endothelial cells, allowing for the analysis of tumor–stroma interactions, was established with 3-D Life Hydrogel. After 20 days of culture, spheroid growth, a fibroblast network and vascular sprouting was clearly visible, the latter likely to be induced by endogenous VEGF. Cultures were miniaturized on transwell 96-well plates and treated with the drug cisplatin showing a reduction of tumor spheroid size and the fibroblast network. Results suggest that the model is highly suitable as a testing platform for novel anticancer therapeutics targeting the tumor as well as the vascular compartment.

Normal and injured primary human skeletal muscle cells (HSkMCs) were embedded in 3-D Life Hydrogel and submitted to rolling manipulation, a specific mechanical stimulation used in chinese medicine, to assess its effect on injured skeletal muscle cells. After hydrogel cultures were submitted during a 3 day cultivation to rolling manipulation, cells were recovered from the hydrogels using Dextranase and subsequently treated for protein extraction. Proteomic data and Western Blot analysis showed which signalling pathways might be important pathways by which rolling manipulation ameliorates HSkMC injury.

Dextran-based 3-D Life hydrogel modified with RGD peptide was used to grow human proximal tubule cells into spheroids. In these cultures the role of the disintegrin and metalloproteinase ADAM17 was investigated in a fibrotic diabetic milieu.

3-D Life Hydrogel is used to culture human pancreas organoids (hPOs) in a chemically definded 3D matrix. Isolated pancreatic ducts formed organoids in 3-D Life Hydrogel comparable to the ones cultured in basement membrane extracts (BME 2). Using a newly developed culture medium which specifically supports the growth of human pancreatic organoids, hPOs were expanded and passaged several times using 3-D Life Dextranase. hPOs expressed similar levels of the progenitor and beta cell marker PDX1 and, although at lower levels, the ductal markers KRT19 and SOX9 when compared with hPOs cultured in BME 2. In conclusion, hPOs well maintain a pancreatic ductal identity when cultured in 3-D Life Hydrogel.

Results show that 3-D Life Hydrogel does not contribute to light scattering when illuminated with a collimated laser beam (488 nm wavelength; 1.2 diameter; 10 s). It thus would be suitable for 3D-light-scattering biosensing, a method applied in this publication.

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.

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.

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.

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.

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.

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.

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 disease and origin of the KCOT cell line.

A thiol-modified DNA was immobilized in a 3-D Life PVA-PEG hydrogel by binding it to the thiol-reactive group of PVA.

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. The formation of capillary like structures was examined on the surface of these mixed gels of different stiffness.

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.

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

The 3-D Life Hydrogel technology was used to prepare maleimide-modified serum albumin (HSA) which was crosslinked with PEG-Link and supplemented with 4% hyaluronan (HSA–HA) for the cultivation of mesenchymal stem cells, disk cells and chondrocytes. The HSA–HA hydrogel turned out to be a suitable biomaterial for the cultivation and/or differentiation of chondrogenic cells.

The 3-D Life Hydrogel technology was used to prepare maleimide-modified human or sheep serum albumin which was crosslinked with PEG-Link and supplemented with hyaluronan. Chondrocytes cultured in these gels were vital and kept their differentiation. Moreover, the hydrogel proved to be non-permissive for endothelial cells to enter the gel in CAM assays as well as after implantation in NOD/SCID mice. Thus, the hydrogel was shown to be a good candidate to suppress pathological blood vessel formation when used for chondrocyte encapsulation for autologous chondrocyte implantation.