The Problem with Current Cell Culture Systems
Tens of thousands of academic and industrial laboratories apply cell culture to study the biology of cells that regulate health and disease, to identify and develop new drugs, and even find ways to grow replacement tissues for implantation. But there is a major problem with the way cells are cultured in most laboratories. Cells are grown on flat, unphysiologically stiff materials such as polystyrene and glass, causing them to display aberrant behaviours: flattened shape, abnormal polarisation, altered response to pharmaceutical reagents, and general loss of phenotype. Although scientists agree that traditional cell culture methods fail to recapitulate the native cellular microenvironment, there is a lack of easy-to-use and widely applicable systems that are capable of recreating such environments in the laboratory while providing consistent properties from batch-to-batch.
Conventional 2D cell culture on plastic or glass surfaces leads to aberrant cellular phenotypes and non-predictive data1
LunaGel™ Extracellular Matrix Kits
Growing Tissues rather than just Cells
LunaGel™ is a photocrosslinkable hydrogel cell culture system that recreates the natural extracellular matrix (ECM) surrounding cells in the human body, allowing researchers to grow three-dimensional microscopic tissues, rather than just cells on plastic surfaces. The major components of LunaGel™ include native ECM proteins such as collagen type I, III, IV, and V, as well as connective tissue glycoproteins and proteoglycans. LunaGel™ retains the intrinsic cell-instructive bioactivity of natural ECMs, facilitating cell attachment, proliferation, differentiation, migration, and proteolytic degradation.
Tunable Extracellular Matrix
Vary light exposure duration to easily adjust matrix stiffness
Our manufacturing and quality control procedures ensure consistent properties
Easy And Fast
Create 3D cell culture models in <15 mins
LunaGel™ ECM contains cell-instructive motifs and attachments sites
The LunaGel™ ECM is compatible with a large variety of cells types
Cells can cleave the LunaGel™ ECM
To Each Their Own
ECM Stiffness Matters
All cells in the human body are exposed to mechanical forces which regulate cell function and tissue development and each cell type is is specifically tuned to the mechanical properties of the tissue it resides in. Neuronal cells, for example, require a very soft matrix similar to brain tissue in order to thrive, while cartilage or bone cells require much stiffer environments1. The matrix properties of human tissues can also change with disease and in turn facilitate its progression. For example, normal mammary epithelial cell growth, survival, differentiation and morphogenesis are well-supported by interaction with a soft matrix similar to normal breast tissue stiffness. Following transformation during breast cancer, however, the tissue becomes progressively stiffer2 and tumour cells become significantly more contractile and hyper-responsive to matrix mechanical cues, ultimately driving epithelial to mesenchymal transition (EMT) and metastasis3,4,5. Evidently, the importance of matrix elasticity is increasingly being studied and ECM stiffness has been shown to regulate stem cell differentiation1,6, cell migration7, epithelial to mesenchymal transition (EMT)3, the induction of malignant cancer phenotypes8, cell spreading and adhesion9, calcium signalling, and many more pathophysiological and physiological cellular events.
Cells are tuned to the material properties of their native matrix.
The LunaCrosslinker™ enables cell-friendly photocrosslinking.
LunaGel™ uses visible light polymerization to create 3D cell culture models in a matter of minutes
How it works
Creating 3D cell culture models has never been simpler! A chemical modification allows the LunaGel™ ECM to be crosslinked by exposure to blue light in the LunaCrosslinker, creating cell culture models that closely mimic natural microenvironments. The LunaGel™ hydrogel system is transparent, permeable, and compatible with standard imaging systems. ECM stiffness can be adjusted to by varying the light exposure duration in the Luna Crosslinker™ to replicate physiological conditions of different healthy and diseased tissues.
3D Cell Culture
Automated Liquid Handling
High Throughput Screening
Controlled ECM for Cell Delivery
Controlling Matrix Stiffness by Light Exposure Duration
The LunaGel™ ECM offers unprecedented control over matrix stiffness covering a substantially larger range than any of the competitor products on the market. LunaGel™ SOFT employs a cell-friendly, rapid photocrosslinking process, allowing researchers to fine-tune the elastic modulus between 0.1 and 10 kPa within just a few minutes of light exposure (Figure 1). Competitor products such as basement membrane extracts or collagen rely on lengthy thermal gelation for curing (30 – 60 min) and produce matrices with elastic modulus limited to < 1 kPa which are unphysiological for most common cell types.
Figure 1: Elastic modulus of LunaGel SOFT extracellular matrices is tuned by crosslinking duration.
LunaGel samples were crosslinked by exposure to blue light in the Luna Crosslinker. Mechanical properties were assessed using unconfined compression tests.
LunaGel in Action
Application Examples of LunaGel™ SOFT
The applications of LunaGel™ are vast and span 3D cell culture, 3D biofabrication, high throughput manufacture and screening, drug delivery and many more. Below are just a few examples of what LunaGel™ ECM can be used for.
Cancer Spheroid/Organoid Culture
A number of recent high-impact papers have demonstrated that primary tumour growth, epithelial-to-mesenchymal transition (EMT), and metastasis are regulated by ECM stiffness. LunaGel™ SOFT is the ideal product to allow users to adjust the ECM stiffness according to our predefined protocols. LunaGel™ has been successfully used to culture a large variety of commonly used cancer cell lines derived from breast cancer (MCF-7, MDA-MB-231), prostate cancer (LNCaP, PC3), ovarian cancer (OV-MZ-6), liver cancer (HUH-7, C3A) and melanoma (SK-MEL-28, WM35). Below are some examples of microtumours formed by commonly used prostate and breast cancer cell lines (Figure 2).
Figure 2: Representative images of cancer spheroids generated in LunaGel™ SOFT (Meinert, et al, Prostate Cancer, 2018).
(A) LNCaP prostate cancers cells and (B) MCF-7 breast cancer cells were encapsulated in LunaGel™ (3 kPa) and cultured under standard conditions for 14 days, followed by fixation with 4% PFA and immunofluorescence staining for E-Cadherin (green) and actin (red).
Inducing physiological gene expression patterns in human hepatoma-derived (C3A) cells
Liver cells express a variety of iron regulators including transferrin receptor (TFRs) and metallothioneins (MTs) in vivo. However, the expression of these genes is largely lost during in vitro 2D monolayer culture, limiting the value of current laboratory models. Figure 3 demonstrates that the expression of key marker genes including TFR1, TFR2, and MT2 is significantly higher in LunaGel™ ECM compared to monolayer and Matrigel culture (the current market leader in 3D cell culture products), showing that in LunaGel, the liver cells are better retaining their characteristic phenotype compared to Matrigel. Furthermore, it is demonstrated that the expression of these genes is regulated by ECM stiffness, suggesting that disrupted iron homeostasis in vivo, such as hemochromatosis which is often observed in hepatocellular carcinoma patients, may be induced by changes in the ECM properties during cancer progression.
Figure 3: C3A liver cells cultured in LunaGel™ show varying levels of iron regulator gene expression depending on matrix stiffness
(a) Representative brightfield images of C3A cells after 6 days of culture in 2D monolayer, Matrigel, and LunaGel™ at varying ECM stiffness. Spheroids formed in LunaGel™ appear more regular and spherical compared to Matrigel. The expression of (b) TFR1, (c) TFR2, and (d) MT2 at day 6 was highest in LunaGel™ cultures and further regulated by ECM stiffness.
Figure 4: IGF-1:IGFBP-3:VN (TRI) complex stimulates the growth of melanoma spheroids in LunaGel™ ECM (Murekatete, et al, Scientific Reports, 2018).
SK-MEL-28 cells were seeded onto Matrigel™ or encapsulated in LunaGel™ (5 kPa). On day 14, cells were stained with FDA for visualisation and spheroid size assessment. TRI 30 = 1 ng/mL VN + 30 ng/mL IGF-I + 90 ng/mL IGFBP-3; TRI 100 = 1 ug/mL VN + 100 ng/mL IGF-I + 300 ng/mL IGFBP-3; TRI 300 = 1 μg/mL VN + 300 ng/mL IGF-I + 900 ng/mL IGFBP-3. n = 6 (2 technical repeats, 3 experimental repeats); # p < 0.05 compared to 2% FCS, #### p < 0.0001 compared to 2% FCS; **** p < 0.0001.
Investigating the effects of IGF-I:IGFBP-3:VN trimeric complexes on melanoma spheroid growth
Insulin-like growth factor 1 (IGF-1) binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation of skin keratinocytes and fibroblasts. However, the role of this trimeric complex (TRI) in melanoma progression remains largely unknown. Figure 4 shows the formation of irregular spheroids by SK-MEL-28 cells in Matrigel with a trend towards increased spheroid sizes with higher concentration of TRI. In LunaGel™, the effects were more pronounced - cells formed regular, easy to quantify, spheroids which increased significantly in size with increasing concentrations of TRI, revealing a stimulating effect of TRI on melanoma growth.
Figure 5: Response of MCF-7 and MDA-MB-231 breast cancer cells to Abraxane treatment.
Relative metabolic rate of (A) MCF-7 and (E) MDA-MB-231 breast cancer cells encapsulated in LunaGel™ SOFT and monolayer cultures following 3 days of treatment with varying concentrations of Abraxane (metabolic response of treated groups was normalised to untreated controls). Viability and spheroid/cell morphology of (B) MCF-7 and (F) MDA-MB-231 cells in untreated cultures (control) and following treatment with 300 ppm Abraxane. Quantification of (C) relative integrated fluorescence intensities (live/dead) and (D) spheroid size revealed cytotoxic effects of Abraxane treatment in embedded MCF-7 cultures. (G) Treatment of MDA-MB-231 cells reduced cell viability by > 50% compared to controls.
Drug Screening and Development – 2D vs 3D cell culture in LunaGel™
LunaGel™ ECM facilitates drug-response studies that are highly predictive of the in vivo situation. Below is an example of MCF-7 breast cancer spheroids subjected to a chemotherapeutic agent (Abraxane/human serum albumin-conjugated paclitaxel). While Abraxane treatment led to almost complete loss of viability in monolayer cultures, the metabolic response and viability of MCF-7 cells, a cell line derived from non-metastatic breast tumours, was more similar to in vivo responses when cultured in LunaGel™ (Gurski et al., 2010),(Hongisto et al., 2013) (Figure 5). Interestingly, Abraxane treatment of metastatic MDA-MB-231 breast cancer cells led to a substantially larger decrease in metabolic activity and viability, as well as a loss of metastatic cellular morphologies. The difference in cell response between MCF-7 and MDA-MB-231 cells may be related to the variances in growth and migration patterns. MCF-7 cells form tumour-like spheroids which may hinder the penetration of the drug to the cells in the spheroid core. MDA-MB-231, on the other hand, are metastatic and highly migratory, and hence often exist as single cells rather than cell clusters, in turn leading to higher drug efficiencies. Indeed, this finding is corroborated by clinical studies which clearly demonstrate more effective treatment of metastatic cancers with Abraxane compared to primary tumours, ultimately leading to the admission of Abraxane for metastatic breast cancer treatment. Ultimately, our data demonstrates the benefits of using LunaGel™ 3D assays over traditional monolayer cultures, which incorrectly predicted a high efficiency of Abraxane treatment on both, MCF-7 and MDA-MB-231 cells.
Figure 6. Determination of the IC50 of paclitaxel using LunaGel (Eggert, et al, unpublished data, 2020)
MDA-MB-231 breast cancer cells were cultured in LunaGel (5 kPa) for 7 days, and incubated with different concentrations of paclitaxel for 120 h. Metabolic activity was assessed using automated oxygen consumption measurements.
Figure 7: Application of LunaGel™ as cell delivery vehicle in an intraperitoneal animal model (Loessner, et al, Nature Protocols, 2018).
(a) Luciferase-transduced ovarian cancer cells (OV-MZ-6) were encapsulated in LunaGel™ and bioluminescence indicative of spheroid formation was confirmed at day 14 of in vitro pre-culture. (b) Bioluminescence imaging confirmed substantial tumour formation 8 weeks following implantation and ex vivo imaging of the peritoneal organs indicated the presence of metastases. Human-derived tumour load was confirmed by positive staining for human-specific nuclear mitotic apparatus protein 1 (NuMA). (c) 4 weeks following implantation, mice were treated with intraperitoneal paclitaxel injections (10 mg/kg administered twice per week) over 4 weeks, leading to decreased tumour load and metastases.
Drug Screening and Development – Automated Determination of IC50 Values of Anticancer Drugs
The IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit a given biological process or biological component by 50%. In this study, our collaborators have used automated liquid handling to produce LunaGel™ 3D cell culture samples with MDA-MB-231 breast cancer cells. The effect of paclitaxel, a chemotherapeutic agent, was studied using high-throughput screening approaches used in pharmaceutical industry. The ability to use LunaGel™ with high-throughput techniques is a clear advantage over other 3D cell culture platforms like Matrigel.
Cell Delivery/Animal Experiments
LunaGel™ products can also be used for in vivo implantation allowing the investigation of, for example, the effects of ECM stiffness on EMT and cancer metastasis. Figure 7 demonstrates that delivery of LunaGel™-embedded luciferase-labelled OV-MZ-6 ovarian cancer cells led to primary tumour formation and metastases in mice. Treatment with the drug Paclitaxel (similar to Abraxane) led to decreased tumour burden, and, in particular, reduced incidence of metastases, similar to the in vitro studies shown above.
Figure 8: Capillary-like network formation of HUVECs and MSCs in LunaGel™ SOFT
(A) HUVECs and MSCs were cultured in LunaGel™ SOFT (0.8 kPa) in the presence of VEGF, SDF-1, and FGF-2, fixed with 4% PFA and stained for endothelial cell marker CD31 (green) and actin (red). (B) HUVEC/MSC cultured in LunaGel™ SOFT form perfusable capillary networks in microfluidic chips.
Tube formation/angiogenesis assays
LunaGel™ ECM has successfully been applied for the in vitro generation of capillary-like networks formed by primary endothelial cells (human umbilical vein endothelial cells, HUVECs) and pericytes (primary human mesenchymal stem cells, MSCs) (Figure 8A). In contrast to existing tube formation assays in Matrigel, capillary-like structures can be generated by embedded cells (true 3D environment), as opposed to seeding cells on top of the hydrogel, and in addition, are stable for much longer (up to 20 days compared to 1-2 days in Matrigel). When cultured in specialised microfluidic chips, LunaGel™-embedded HUVECs/MSCs form perfusable capillaries capable of replicating physiological blood flow conditions (Figure 8B).
Figure 9: Engineered ultra-sound patterned muscle tissue in LunaGel™ (Armstrong, et al, Advanced Materials, 2018)
(a) Confocal images of patterned myoblasts stained with Calcein over 7 days of culture. (b) Relative gene expression of skeletal muscle markers MYOG and MRF4 in unpatterned (grey) and patterned (red) tissues. (c) Immunostaining for skeletal muscle markers (red) and cell nuclei (blue) at day 7. (d) Myotube length as a function of orientation angle.
3D Bioprinting/Advanced Biomanufacturing
Figure 10: Bioprinting of LunaGel™ ECM.
LunaGel ECM was allowed to gelate and extruded through a G20 needle using a Gesim BioScaffolder printing system. To demonstrate the capability of precise deposition of multiple cell types in one print, fluorescent beads of different colours were embedded in the LunaGel™ matrix.
Engineering anisotropic muscle tissue
Myoblasts (C2C12) encapsulated in LunaGel™ form functional myotubes – microscopic muscle fibers that spontaneously start twitching as they mature. In this study, myoblasts suspended in LunaGel pre-cursor solutions were first patterned to form lines using standing ultrasound waves, followed by photocrosslinking of the LunaGel matrix. This process “locked” the cells in place, allowing them to form into highly aligned muscle fibres that express key markers of skeletal muscle tissue (Figure 9) and twitch, just like real muscles.
Bioprinting – the spatially controlled deposition of cells in so-called bioinks (hydrogels) using specialised 3D printing systems - is a hot topic in research. Bioprinting holds promise for the engineering of functional tissues and advanced 3D cell culture models. Below are some examples of LunaGel™ used as a bioink.
Bioprinting of cells embedded in LunaGel™, in this example human periodontal ligament fibroblasts, retains cell viability and facilitates the spreading, migration, and proliferation of embedded cells (Figure 11).
Figure 11: Bioprinting of human fibroblasts retains high cell viability and facilitates physiological cellular behaviour (Raveendran, et al, Dental Materials, 2019)
Human periodontal fibroblasts were resuspended in LunaGel ECM and printed using Gesim BioScaffolder. Living cells appear green, dead cells appear red.
What Users Think of LunaGel™
Dr James Armstrong, Research Fellow – Imperial College London
“Dr Christoph Meinert has provided me with several sample batches of LunaGel over the last year. We have used this material for our recent publication “Engineering Anisotropic Muscle Tissue using Acoustic Cell Patterning” (Advanced Materials, 2018) and another paper currently in press at Advanced Materials. The material that they have produced is high quality and very reproducible.”
Professor Greg Monteith, School of Pharmacy – University of Queensland
“Our research group is focused on breast cancer drug target identification. We use state of the art high throughput instruments found in pharmaceutical companies such as a Fluorescence Imaging Plate Reader (FLIPR) and automated epifluorescence microscopy based high content imaging systems (ImageXpress Micro). As you know we have tried a variety of 3D cell culture systems and we have found the LunaGel system the most useful for our high throughput drug discovery methods.”
Professor Rik Thompson, Director - Translational Research Institute
“The innovative products of Gelomics will have an increasingly important role to play in the implementation of personalised medicine, providing well defined, low cost, representative environments for the propagation and testing of cancer cells.
Invasive outgrowth of breast cancer cells in 3-dimensional media has long been recognised as a hallmark of metastatic potential. Molecularly defined, semi-synthetic hydrogels have also found utility for growth analyses, particularly in combination with stromal and endothelial cells.
To this end, my group has initiated collaborations in two separate models. We also have plans to extend this to clinical samples in the near future through the recently formed Centre for Personalised Analysis of Cancers (CPAC). CPAC is based at TRI and specialises in peri-clinical testing of 3-dimensional cultures of up to 12 different cancer types towards improved personalised outcomes. Gelomics products will be of potential interest to these various tumour streams.”
Dr Phong Tran, Advance Queensland Research Fellow – Queensland University of Technology
“My research team is focused on developing advanced drug delivery methods. We use cutting edge technology to locally deliver chemotherapeutic agents directly at the tumour site in order to reduce systemic side effects. As you know, we have been using your LunaGel hydrogel materials to study the effects of our delivery technology on the survival and metabolic activity of various cancer types. We found that these materials are a true advancement to traditional culture approaches since it now permits to generation physiologically relevant tumour tissues for drug testing. We think that the hydrogels are easy-to-use and look forward to being able to purchase your hydrogel kits from your company Gelomics Pty Ltd.”
1. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677–689 (2006).
2. Keely, P. & Nain, A. Capturing relevant extracellular matrices for investigating cell migration. F1000Research 4, 1408 (2015).
3. Wei, S. C. et al. Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway. Nat. Cell Biol. 17, 678–688 (2015).
4. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).
5. Liu, F. et al. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression. J. Cell Biol. 190, 693–706 (2010).
6. Wen, J. H. et al. Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat. Mater. 13, 979–987 (2014).
7. Zaman, M. H. et al. Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc. Natl. Acad. Sci. 103, 10889–10894 (2006).
8. Chaudhuri, O. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nat. Mater. 13, 970–978 (2014).
9. Pelham, R. J. & Wang, Y. -l. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. 94, 13661–13665 (1997).
10. Soofi, S. S., Last, J. A., Liliensiek, S. J., Nealey, P. F. & Murphy, C. J. The elastic modulus of MatrigelTM as determined by atomic force microscopy. J. Struct. Biol. 167, 216–219 (2009).
11. Gurski, L. a, Petrelli, N. J., Jia, X. & Farach-carson, M. C. 3D Matrices for Anti-Cancer Drug Testing and Development. Oncol. Issues 25, 20–25 (2010).
12. Hongisto, V. et al. High-Throughput 3D Screening Reveals Differences in Drug Sensitivities between Culture Models of JIMT1 Breast Cancer Cells. PLoS One 8, (2013).