Application Notes

Generation of Tumor Cell Spheroids in Miniaturized Nanoliter Droplets Using Xeno-free VitroGel Hydrogel Matrix

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Application Note

Aquarray, Leopoldshafen, Germany
TheWell Bioscience, North Brunswick, NJ 08902

The Droplet Microarray Platform

The Droplet Microarray (DMA) is made of hydrophilic spots on a superhydrophobic background that allows formation of separated and homogeneous droplets in the sub-microliter range on a planar surface. Precise filling of the DMA spots, which are typically 350-1000 µm in size, is performed using a nanoliter liquid dispenser. A capacity of 672–-6048 droplets per DMA enables highly miniaturized high- throughput screenings on the DMA platform.

Figure 1. Droplet Microarray with 672 spots of 1 mm size dispensed in 150 nL per drop.

A multitude of chemical and biological applications have been demonstrated on DMA, including on chip high-throughput chemical synthesis followed by biological screening (1), transfection-based screenings (2), zebrafish embryo screening (3), bacterial screening (4), and cell-based compound screening. Cell screening can be performed with any cell type , e.g., cell lines, stem cells or primary cells, in, adherent or suspension culture, in 2D or 3D using spheroid-based cell culture models. Formation of single spheroids has been demonstrated in different tumor cell lines using the hanging drop technique in 100-nL droplets containing 150 cells over 24-48 h (6).

The VitroGel® Hydrogel Platform

VitroGel xeno-free (animal-free) hydrogel system is a superior alternative to an animal-based extracellular matrix (ECM) by avoiding the uncertainty of unknown components to give a well-defined and biologically functional microenvironment for consistent results. The hydrogel possesses a unique rheological shear-thinning and rapid recovery property at room temperature, allowing an extremely smooth process for high-throughput dispensing and excellent uniformity of hydrogel droplets for microarray applications.
This application note focuses on the generation of tumor cell spheroids on DMA using the ready-to-use, xeno-free functional hydrogel system VitroGel® Hydrogel Matrix (VHM01).  The hydrogel is stable at room temperature, has a neutral pH, is transparent, and is liquid/nutrition permeable and optimized to support a wide range of cell types. After mixing with a cell medium, the soft-injectable hydrogel forms, which is excellent for injection or the liquid dispensing in this study.
Dispensing on the miniaturized DMA platform is challenging for liquid dispensers because of the nanoliter volumes and the small distances between spots. A number of non-contact liquid dispensers have been identified that are capable of accurately targeting spots on the DMA. In addition, dispensing of hydrogels can be challenging because of their high viscosity. Yet, as a consequence of its unique rheological property, VitroGel can maintain a long-term (hours) injectable status at room temperature or 37°C, without clogging the source well of the dispenser. Dispensing 150 nL of the VitroGel-cell mixture to a DMA with 672 spots takes approximately one minute and results in a very homogeneous pattern. The human cervix carcinoma cell line HeLa and the human melanoma cell line SK-MEL-28 form numerous viable round spheroids in the VitroGel matrix of each individual DMA droplet after 72h of culture (Figures 2 and 3).
Figure 2. HeLa spheroids were generated by dispensing VitroGel mixed with cells on DMA. Immunofluorescence staining against Vimentin (green) and KI67 (red) was performed 72 hours later as described in Material and Methods. Nuclei were stained with Hoechst. TOP: overview of one spot. BOTTOM: magnification thereof.
Figure 3. SK-MEL 28 spheroids were generated by dispensing VitroGel mixed with cells on DMA. Immunofluorescence staining against Vimentin (green) and KI67 (red) was performed 72 hours later as described in Material and Methods. Nuclei were stained with Hoechst.

Conclusion

In conclusion, the generation of tumor cell spheroids on DMA using the ready-to-use, xeno-free functional hydrogel system VitroGel Hydrogel Matrix (VHM01) was successfully performed. Two types of tumor cell spheroids were formed. Visualization was performed with immunofluorescence staining. HeLa spheroids were generated by dispensing VitroGel mixed with cells on DMA. Immunofluorescence staining against Vimentin (green) and KI67 (red) was performed 72 hours later as described in Material and Methods. Nuclei were stained with Hoechst. SK-MEL 28 spheroids were generated by dispensing VitroGel mixed with cells on DMA. Immunofluorescence staining against Vimentin (green) and KI67 (red) was performed 72 hours later as described in Material and Methods. Nuclei were stained with Hoechst.

Materials and Methods

Cell Culture
HeLa and SK-MEL 28 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS). The cells were passaged when the cultures reached 80–90% confluence.
 
3D Culture
For the 3D culture of HeLa and SK-MEL 28 cells, VitroGel Hydrogel matrix was used. Cell suspensions were prepared in DMEM medium with 30% FBS. 400 µL of VitroGel solution was mixed with 200 µL of cell suspension at a 2:1 ratio (v/v) to make the final FBS concentration of 10% for the hydrogel-cell mixture.
 
Dispensing
Hydrogel-cell mixture was dispensed on a Droplet Microarray with 672 spots of 1 mm size with the low volume nanoliter dispenser I-DOT Mini (AQ-edition). 400 µL of the hydrogel-cell mixture was loaded in the source well, and dispensing of 150 nL per spot was performed using the liquid class “VitroGel” (4 drops 253 mbar) within 66 seconds. The DMA was immediately transferred into a Petri dish containing 4 mL humidifying buffer and a humidifying pad in the lid. After 15 min of incubation at room temperature, 50 nL DMEM 10% FCS   was dispensed onto each droplet. The DMA was immediately transferred back in the humidifying dish and incubated at 37°C and in 5% CO2.
 
Immunofluorescence staining for KI67 and Vimentin on Droplet Microarray (DMA) and microscopy
Staining was performed 72 h after seeding the cells on DMA. The DMA was washed three times by immersing the DMA in ice-cold PBS in an appropriate staining jar. Cells were fixed with 4% Formalin at room temperature in the staining jar, followed by washing with TBS (3X). The DMA was then incubated in 0.1% Triton X-100 in TBS (9.9 ml TBS + 100 µl Triton X-100) for 15 min in the staining jar followed by three washing steps. To reduce unspecific binding, the DMA was incubated with Power Block (1:10 diluted in dd H2O) directly on the DMA and carefully distributed across the entire DMA using parafilm. Any remaining liquid was removed with a vacuum.
 
Finally, 150 nL of primary antibody mix was dispensed (at a max of 75 bar) on each spot of the DMA and incubated overnight at 4°C in a humidifying chamber (KI67 polyclonal antibody: Life Technologies Catalog #PA519462, 1:50 dilution in PBS; Vimentin monoclonal antibody (J144): Invitrogen Catalog #MA3-745, dilution 1:50 in PBS). After three additional washing steps with TBS-Tween, 150 nL of the secondary antibody mix (Goat anti-rabbit IgG, DyLightTM594: ThermoScientific #UJ291725, 1:250 and Goat anti-mouse IgG (H&L), DyLightTM 488: ThermoScientific #UJ291725, 1:250) and the nuclear marker Hoechst (Hoechst 33343: ThermoScientific, #62248) was dispensed on each spot and incubated for 1 h on the DMA. After three washes in TBS, the cells were embedded with Mowiol. Bright-field and fluorescence microscopy images were taken with a Keyence BZ-9000 microscope (Keyence, Osaka, Japan), with a 2X and 10X objective.
 
References

M. Benz, A. Asperger, M. Hamester, A. Welle, S. Heissler, & P.A. Levkin. A combined high-throughput and high-content platform for unified on-chip synthesis, characterization and biological screening, Nature Communications, 2020, 11 (5391).

E. Ueda, W. Feng, and P. A. Levkin. Superhydrophilic–superhydrophobic patterned surfaces as high-density cell microarrays: Optimization of reverse transfection, Healthcare Mater, 2016, 5 (2646).

A.A. Popova, D. Marcato, R. Peravali, I.A. Wehl, U. Schepers, & P.A. Levkin. Fish-microarray: miniaturized platform for single-embryo high-throughput screenings, Advanced Functional Materials, 2018, 28 (1703486).

W. Lei, K. Demir, J. Overhage, M. Grunze, T. Schwartz, & P.A. Levkin. Droplet-Microarray: Miniaturized platform for high-throughput screening of antimicrobial compounds, Advanced Biosystems, 2020, 4 (2000073).

A.A. Popova, S. Dietrich, W. Huber, M. Reischl, R. Peravali, & P.A. Levkin. Miniaturized drug sensitivity and resistance test on patient-derived cells using Droplet-Microarray, SLAS TECHNOLOGY Translating Life Sciences Innovation, 2021, 26 (274).

Links To Products Used

VitroGel® Hydrogel Matrix

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