*4.2. Cell Culture*

### 4.1.2. Synthesis of PEG Based Photopolymer with PIs 4.2.1. Chemicals

PEG-DA was used as precursor. It was mixed with PI (Irgacure, EB and EY, respectively). PI concentration varied between 1% and 6 ppm. The chemical structures of different PIs are shown in Figure 3. For the good mixing of both substances, the mixture was sonicated for around 30 min. At first, the mixture was converted in a cuvette and measured with a UV-Vis spectrometer to obtain spectra before crosslinking. Then, the mixture was dispensed on a glass slide and covered with a thin glass cover slip to achieve a flat and thin hydrogel sample. The glass-sandwich was placed under a UV-light source (366 nm) for 60 min and the glass cover slip was peeled off. A flat, thin standalone hydrogel Mouse fibroblast L929 cells were provided by Dr. Lehmann, Fraunhofer Institute for Cell Therapy and Immunology, IZI, Leipzig, Germany. RPMI 1640 medium, Trypsin, Fetal Bovine Serum (FBS) and Penicillin/Streptomycin (PS) were provided by PAA Laboratories GmbH, Austria, and cell culture plates are from SPL Live Sciences Inc., Seoul, Korea. The Incubator CB150 Series was from Binder GmbH, Germany. Phosphate Buffered Saline solution (Dulbecco's PBS) was purchased from Sigma-Aldrich Chemie, GmbH, Germany. The counter chamber was from Marienfeld Superior (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofen, Germany).

### film was received and also prepared for UV-Vis measurement. Therefore, the hydrogel was placed on a thin glass cover slip and measured with a UV-Vis spectrometer to obtain 4.2.2. Cell Culture Experiments

spectra after crosslinking. *4.2. Cell Culture*  4.2.1. Chemicals The mouse fibroblasts L929 cells were cultured in the tissue culture plate in RPMI 1640 medium with the addition of 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (PS) in a cell culture plate in an incubator at controlled temperature (37 ◦C) and CO<sup>2</sup> atmosphere (5%). The cells were grown in a cell culture plate and cell culture experiments were performed when a confluency of 75% to 95% was reached.

Mouse fibroblast L929 cells were provided by Dr. Lehmann, Fraunhofer Institute for Cell Therapy and Immunology, IZI, Leipzig, Germany. RPMI 1640 medium, Trypsin, Fetal Bovine Serum (FBS) and Penicillin/Streptomycin (PS) were provided by PAA Laboratories GmbH, Austria, and cell culture plates are from SPL Live Sciences Inc., Seoul, Korea. The Incubator CB150 Series was from Binder GmbH, Germany. Phosphate Buffered Saline solution (Dulbecco's PBS) was purchased from Sigma-Aldrich Chemie, GmbH, Germany. The counter chamber was from Marienfeld Superior (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofen, Germany). 4.2.2. Cell Culture Experiments The hydrogel samples were prior washed with water and kept in a PBS solution for around 30 min before cell culture experiments. As soon as a confluency of at least 75% was reached, the cells were washed with PBS, detached by using trypsin and, after the centrifugation process, a new medium was added on the cells and mixed properly. An amount of 10 µL of this cell medium solution was placed on a cell counter chamber in order to count the cell number by using an optical microscope and to achieve a concentration of 40,000 cells/mL. Depending on the counted cell number, the cell solution was mixed with a defined amount of new medium. The samples were placed in a TCPS plate or on the washed and precut hydrogel. The samples were then cultured within these cells for 24 h, at 37 ◦C in a 5% CO<sup>2</sup> atmosphere in a TCPS.

### 1640 medium with the addition of 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Strep-4.2.3. Live Dead Cytotoxicity Assay

tomycin (PS) in a cell culture plate in an incubator at controlled temperature (37 °C) and CO2 atmosphere (5%). The cells were grown in a cell culture plate and cell culture experiments were performed when a confluency of 75% to 95% was reached. The live dead cytotoxicity assay is a fluorescence-based method for checking the viability of cells. Hereby, the cells are stained with fluoresceindiacetete (FBS) and propidiumiodid (PI) molecules. FBS is dissociated in the cytoplasma of live cells into green fluorescence molecules and, due to the size and charge of the PI molecules, they only can

The mouse fibroblasts L929 cells were cultured in the tissue culture plate in RPMI

enter into the cell cytoplasm when the cell membranes are damaged and are bound to nucleic acids, which appear then as red fluorescence color. In this manner, the live cells appear as green-stained cells and dead cells appear as red-stained cells in a fluorescent microscope image.

For the live dead assay, a 1:1 *v*/*v* solution of PI and FBS in PBS was prepared and added into the cell culture solution in a dark environment. Immediately after the mixtures are prepared, fluorescence images are taken.

### *4.3. Analytical Instruments*

UV-Vis spectra were obtained with Cary 4000 UV-Vis Spectrometer (Agilent Technologies, Santa Clara, CA, USA). The spectral range from 300 to 900 nm was measured at room temperature. Liquid samples (before crosslinking) were converted in a cuvette for measurement. The flat, thin standalone hydrogel films (after crosslinking) were placed on a thin glass cover slip for measurement with a UV-Vis spectrometer.

The results from cell culture experiments were observed via the optical microscope from Carl Zeiss, Germany, and analyses were performed with the AxioVision V4.8.2 software (Carl Zeiss, Oberkochen, Germany).

**Author Contributions:** Conceptualization, T.S.-G. and M.C.L.; investigation, A.T. and C.B.; writing—original draft preparation, review and editing, T.S.-G.; supervision, M.C.L.; project administration and funding acquisition, T.S.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), grant number SA 2990/1-1. The APC was funded by DFG.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

