VitroGel® 3D is a pure and unmodified hydrogel that allows the maximum flexibility to manipulate the 3D cell culture environment for different needs. The unmodified hydrogel matrix structure is good for cell spheroid formation, suspension cells or cells requiring low cell-matrix interactions.
VitroGel 3D is one system of the family of ready-to-use, xeno-free tunable hydrogel system which closely mimics the natural extracellular matrix (ECM) environment.
VitroGel creates a functional and optimized environment to make cells feel like at home. The hydrogel system is room temperature stable, has a neutral pH, transparent, permeable and compatible with different imaging systems. The solution transforms into a tunable hydrogel matrix by simply mixing with the cell culture medium. Cells cultured in this system can be easily harvested out with our VitroGel Cell Recovery Solution. The hydrogel can also be tuned to be injectable for in vivo studies.
From 3D cell culture, 2D cell coating to animal injection, VitroGel makes it possible to bridge the in vitro and in vivo studies with the same platform system.
- Xeno-free tunable hydrogel – unmodified
- Good for cell spheroid formation, suspension cells or cells requiring low cell-matrix interactions
- Ready-to-use at room temperature
- Tunable hydrogel: Dilute with VitroGel Dilution Solution (TYPE 1 or TYPE 2) for different concentrations
- Neutral pH
- Compatible with VitroGel® Cell Recovery Solution for easy cell harvesting
- Injectable hydrogel (Check user handbook for preparation details)
- 10 ~ 4000 Pa of G’ of regular products at dilutions. Customized high concentration product to reach over 20K Pa
- Ships room temperature. Store at 2-8°C
- Size: 2 mL and 10 mL
Handbook and Resources
Frequently Asked Questions
To see a full list of FAQ, click here. FAQ LIST
- How to prepare the cell suspension to mix the hydrogel? Shall I add serum?
If cells cultured in a complete cell culture medium, which is supplement with 10% FBS or other critical supplements, please prepare the cell suspension using the following methods before mixing it with hydrogel solution.
- Prepare the cell suspension with 2X concentration (e.g. 100K), and mix with 100% FBS at 1:1 (v/v) ratio to get 1X cell suspension (50K) with 50% FBS.
- Mix the diluted hydrogel solution with the cell suspension from above at 4:1 (v/v) ratio to get the final cells in the hydrogel at 10K with 10% FBS supplement.
If serum plan is an important role in your traditional cell culture, it is also important for 2D coating and 3D culture. Adding serum supplements in the hydrogel and adjusting the final serum concentration to the target level would support cell growth in the hydrogel system.
- How do I adjust the hydrogel formation time?
– If VitroGel needs to be diluted more than 1:3 ratio, a longer waiting time (20-30 min) may be needed for soft gel formation. Using a higher volume of cell culture medium for mixing would help to accelerate the process of hydrogel formation.- If the hydrogel solidifies too fast after mixing with culture medium (showing as small solid gel chunk), adjust the mixing ratio by using less cell culture medium. For example, if mixing 4 mL diluted hydrogel solution with 1 mL cell culture medium lead to the solid gel chuck (particles), then mixing 4 mL diluted hydrogel solution with 0.5-0.8 mL cell culture medium would help to solve the issue.- On the other hand, if the hydrogel formation is too slow, which may happen when using low hydrogel concentration at 1:3 or 1:4 dilution or using a cell culture medium with very low ionic concentration, adjust the mixing ratio by using more cell culture medium. For example, if mixing 4 mL diluted hydrogel solution with 1 mL cell culture medium lead to a slow hydrogel formation, then mixing 4 mL diluted hydrogel solution with 1.5-4 mL cell culture medium would help to solve the issue.
- How do I adjust the stiffness of the final hydrogel?
The stiffness of the final hydrogel can be adjusted by diluting the hydrogel solution before mixing with cell culture media. Our VitroGel Dilution Solution can help to adjust the hydrogel concentration. Please read the “First-time User Note” to learn how to prepare different VitroGel dilutions. If you need a higher hydrogel stiffness than the original product, please contact us at email@example.com.
- Can I harvest cells from the hydrogel after 3D culture?
Yes, the cells can be harvested after 2D coating or 3D culture by using the VitroGel Cell Recovery Solution. VitroGel™ Cell Recovery Solution is a ready-to-use, enzyme-free solution to harvest 2D or 3D cultured cells from hydrogel fast and safely. The solution is compatible with the VitroGel hydrogel system and can recover cells from VitroGel in 15 minutes. VitroGel Cell Recovery Solution is room temperature stable, has a neutral pH and works at 37 °C operating temperature. The solution can maintain high cell viability during the recovery process. Cells can be sub-culture in both 2D and 3D culture after recovery.
Data and References
3D Cell Culture Applications
Figure 1. Beta Lox 5 (BL5) cells 3D culture in VitroGel 3D system.
A. BL5 cells culture on the surface of regular tissue culture treated well plate (control); B. Normal human islets grew in suspension culture (comparison); C. 3D culture of BL5 cells in VitroGel 3D at Day 1; D. 3D culture of BL5 cells in VitroGel 3D at Day 7. Under 3D culture of VitroGel 3D, BL5 cells form islet-like structures very similar to normal human islets. The hydrogel is prepared at 1:3 dilution. The images were taken at 10X magnification.
Figure 2. CD8+ T cells 3D culture in VitroGel 3D system
CD8+ T cells culture grew in suspension culture (contorl); B. 3D culture of CD8+ T cells in VitroGel 3D at Day 7. CD8+ T cells are vibrant in 3D culture conditions of VitroGel 3D. The cells can easily move within the unmodified hydrogel matrix. The hydrogel is prepare at 1:3 dilution. The images were taken at 10X magnification.
2D Coating Applications
Figure 3. Human colon cancer cells (HCT 116) cells cultured on top of VitroGel 3D hydrogel
A thick hydrogel coating plate has been prepared by mixing VitroGel 3D with PBS at 1:1 ratio. A 300 µL mixture has been added to a well of a 24-well plate and stabilization at room temperature for 20 min before adding cells on top of the hydrogel. Cell spheroids form on the top of the hydrogel. Cells seeded at 2.5-10×105 cells/mL.
Figure 4. Comparison of long-term neuronal culture seeded onto thick hydrogel mats.
Cells are stained with Beta-III-Tubulin (green) cytoskeleton marker and their nuclei are counter-stained with DAPI (blue). Cells spread out and form neural-like networks as early as day 3 post-differentiation, with comparable efficacy between VitroGel 3D and Matrigel, based on cell survival, culture spreading and morphological analysis reached between days 7 and 9. On Matrigel mats, cell culture health and viability drops off sharply once day 9 has passed, with most cells detaching and neurites retracting by day 14 and the vast majority of cells gone by day 21. If grown onto VitroGel 3D mats, differentiated B35 neurons have a tendency to self-organize into 3D clusters very early on (Day 7), assuming a mixed 2D/3D cell culture for the first two weeks of the time-course. By Day 21, these cells have migrated into self-assembled 3D clusters, embedded into the thick hydrogel matrix, with very few cells between the clusters, but without any significant cell death.
Figure 5. Human Lymphoblastoid Priess cells cultured on top of VitroGel 3D hydrogel
A. Priess cells grown in suspension (control); B. Priess cells grown on top of VitroGel 3D at day 7. A hydrogel substance can be prepared with different stiffness by adjusting the dilution of VitroGel 3D from 1:1 to 1:3 ratio. Cells seeded on the top of the hydrogel form cell spheroids form on the top of hydrogel. The hydrogel provides a soft substance for cell to attach and grow.
Tables of successful cell types
|Cell types||Applications||Culture medium||Dilution|
|4T1 cells||3D culture||RPMI 1640 with 10% FBS||1:2|
|BL5 human beta cells||2D and 3D culture||DMEM with 10%FBS||1:3|
|CD8+ T cells||3D culture||RMPI 1640 with 10% FBS||1:3|
|HeLa cells||3D culture||DMEM with 10%FBS||1:3|
|Human Nthy-ori 3-1 cells||3D culture||RPMI 1640 with 10% FBS||1:3|
|KHOS cells||3D culture||RPMI 1640 with 10% FBS||1:1 to 1:3|
|MDA-MB-231 cells||3D culture||RPMI 1640 with 10% FBS||1:2|
|Priess human lymphoblastoid cells||2D and 3D culture||RPMI 1640 with 10% FBS||1:3|
|Red Blood Cells||3D culture||Alsever's solution||1:1 to 1:3|
|T47D cells||3D culture||RPMI 1640 with 10% FBS||1:2|
|U2OS cells||3D culture||RPMI 1640 with 10% FBS||1:1 to 1:3|
- Shen, S., Dean, D. C., Yu, Z., Hornicek, F., Kan, Q., & Duan, Z. (2020). Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067. Journal of Cancer, 11(1), 132–141. https://doi.org/10.7150/jca.35426
- Shamloo, B., Kumar, N., Owen, R. H., Reemmer, J., Ost, J., Perkins, R. S., & Shen, H.-Y. (2019). Dysregulation of adenosine kinase isoforms in breast cancer. Oncotarget, 10(68). https://doi.org/10.18632/oncotarget.27364
- Wang, F., Nan, L., Zhou, S., Liu, Y., Wang, Z., Wang, J., Feng, X., & Zhang, L. (2019). Injectable Hydrogel Combined with Nucleus Pulposus-Derived Mesenchymal Stem Cells for the Treatment of Degenerative Intervertebral Disc in Rats. Stem Cells International, 2019, 1–17. https://doi.org/10.1155/2019/8496025
- Borzi, C., Calzolari, L., Ferretti, A. M., Caleca, L., Pastorino, U., Sozzi, G., & Fortunato, O. (2019).c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a. Cell Death & Disease, 10(10). https://doi.org/10.1038/s41419-019-2003-5
- Kim, E. J., Yang, C., Lee, J., Youm, H. W., Lee, J. R., Suh, C. S., & Kim, S. H. (2019). The new biocompatible material for mouse ovarian follicle development in three-dimensional in vitro culture systems. Theriogenology. https://doi.org/10.1016/j.theriogenology.2019.12.009
- Di Donato, M., Cernera, G., Migliaccio, A., & Castoria, G. (2019). Nerve Growth Factor Induces Proliferation and Aggressiveness in Prostate Cancer Cells. Cancers, 11(6), 784. https://doi.org/10.3390/cancers11060784
- Xiao, M., Qiu, J., Kuang, R., Zhang, B., Wang, W., & Yu, Q. (2019). Synergistic effects of stromal cell-derived factor-1α and bone morphogenetic protein-2 treatment on odontogenic differentiation of human stem cells from apical papilla cultured in the VitroGel 3D system. Cell and Tissue Research, 378(2), 207–220. https://doi.org/10.1007/s00441-019-03045-3
- Thanindratarn, P., Li, X., Dean, D. C., Nelson, S. D., Hornicek, F. J., & Duan, Z. (2019). Establishment and Characterization of a Recurrent Osteosarcoma Cell Line: OSA 1777. Journal of Orthopaedic Research. https://doi.org/10.1002/jor.24528
- Li X, Seebacher NA, Xiao T, Hornicek FJ, Duan Z. Targeting regulation of cyclin dependent kinase 9 as a novel therapeutic strategy in synovial sarcoma. Journal of Orthopaedic Research®, 37(2), 510–521. https://doi.org/10.1002/jor.24189
- Hangzhan M, Nicole AS, Francis JH, Zhenfeng D. Cyclin-dependent kinase 9 (CDK9) is a novel prognostic marker and therapeutic target in osteosarcoma. EBioMedicine, 39, 182–193. https://doi.org/10.1016/j.ebiom.2018.12.022
- Akamandisa MP, Nie K, Nahta R, Hambardzumyan D, Castellino RC. Inhibition of mutant PPM1D enhances DNA damage response and growth suppressive effects of ionizing radiation in diffuse intrinsic pontine glioma. Neuro-Oncology, 21(6), 786–799. https://doi.org/10.1093
- Huang J. 3D Cell Culture on VitroGel System. HSOA Journal of Cytology and Tissue Biology. https://doi.org/10.24966/CTB-9107/S1001
- Mahauad-Fernandez WD, Okeoma CM. B49, a BST-2-based peptide, inhibits adhesion and growth of breast cancer cells. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-22364-z
- Mahauad-Fernandez WD, Naushad W, Panzner TD, Bashir A, Lal G, Okeoma CM. BST-2 promotes survival in circulation and pulmonary metastatic seeding of breast cancer cells. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-35710-y
- Seungwoo S, Jihye K, Je-Ryung L, Eun-chae J, Tae-Jin J, Wonhee L, YongKeun P. Enhancement of optical resolution in three-dimensional refractive-index tomograms of biological samples by employing micromirror-embedded coverslips. Lab on a Chip, 18(22), 3484–3491. https://doi.org/10.1039/c8lc00880a
- Powell K. Adding depth to cell culture. Science, 356(6333), 96–98. https://doi.org/10.1126/science.356.6333.96