Embryonic stem cells (ESCs) derived from the inner cell mass of a blastocyst are capable of self-renewal (the ability to divide into daughter cells with the same capacity as the original cell) and pluripotency (the ability to differentiate into all cell lineages of an organism). To maintain these characteristics, co-cultures with feeder cells, such as a mitotically inactivated embryonic fibroblast, are commonly used (Heuer et al., 1993). However, the use of feeder cells is associated with certain well-known problems, including the risk of unknown animal-derived pathogens, difficulties in analyzing ESCs. In response, a feeder-free culture system was developed that coats the surface of a culture dish with extracellular proteins or isolated biomaterials to optimize the conditions of the medium and culture ESCs alone (Williams et al., 1988; Tielens et al., 2007). Various feeder-free ESC culture systems have been developed that employ specific coating materials (Schmidmaier et al., 2003; Foppiano et al., 2006; Egger et al., 2016), with gelatin widely as an economical option (Egger et al., 2016). However, an optimal concentration of gelatin coating for feeder-free ESC cultures has not been reported, with the result that the gelatin concentration used by researchers makes reproducibility difficult.

The atomic force microscope (AFM) is among the most potent tools for topological analysis because it simulates the surface of various matrices, including the cell surface and the ECM at the nano level (Alcaraz et al., 2018). An AFM has several is easy to use, produces high-resolution images, and is compatible with a diverse array of samples. AFM can also measure mechanical properties such as adhesion, deformation, stiffness, modulus, and material energy dissipation at the nanoscale, as well as the surface (Sugimoto et al., 2007) AFM also allows for specific structural investigations by attaching appropriate organic molecules to the tip (Rief et al., 1997). Over several decades, AFM has been used to discover various biological systems, including proteins (Shan and Wang, 2015), virus particles (Kuznetsov and McPherson, 2011), the organelle (Wang et al., 2018), the ECM (Ye et al., 2021), various tissues, and provide insight into numerous pathological/physiological processes.

In this study, we investigated the optimal concentration of gelatin coating for the culture and differentiation of ESCs and compared the surface microstructure of a culture dish according to the concentration of gelatin through AFM.

Materials

Materials such as Gelatin (cat. No. 900-70-8, Duksan), DMEM (cat. No. LM001-05, Welgene), FBS (cat. No. 30044-333, Gibco), MEM NEAA (cat. No. 11140-050, Gibco), Glutamax (cat. No. 35050-061, Gibco), 2-Mercaptoethanol (cat. No. 02194705, MP Biomedicals), Penicillin-Streptomycin (cat. No. 15140-122, Gibco), Laduviglusib (a glycogen synthase kinase 3 (GSK3) inhibitor (CHIR99021; cat. No. HY-10182, MCE)), Mirdametinib (an inhibitor of mitogen activated protein kinase (MAPK) / extracellular signal- regulated kinase (ERK) kinase (PD0325901; cat. No. HY- 10254, MCE)), ESGRO^® Recombinant Mouse LIF Protein (LIF; cat. No. ESG1107, Sigma-Aldrich), DPBS (cat. No. LB001-02, Welgene, Korea), TRI-Reagent (cat. No. FATRR 001, FAVORGEN), AMPIGENE^® cDNA Synthesis Kit (cat. No. ENZ-KIT106-0200, ENZO), TOPreal^TM qPCR 2X PreMIX (SYBR Green with low ROX; cat. No. RT500M, Enzynomics, Korea), Chloroform (cat. No. un1888, Duksan, Korea), Iso-propyl alcohol (cat. No. un1219, Duksan, Korea), a 6-well culture plate (cat. NO. 3006, SPL, Korea), Reagent Reservoir (cat. No. 95128095, Thermo Scientific^TM) and a D-Plus^TM CCK cell viability assay kit (cat. No. CCK-3000, DonginLS, Korea) were purchased. A CO₂ incubator (cat. No. NB-203XL, N-BIOTECH), Inverted Laboratory Microscope Leica DM IL LED (Leica), ASTEC Thermal Cyclers Gene Atlas (cat. No. HU-, Astec), and Rotor-Gene Q 2plex (cat. No. 9001680, QIAGEN) and Epoch 2 Microplate Spectrophotometer (cat. No. EPOCH2NSC, Agilent) were used to perform the experiments.

Young’s modulus analysis using AFM

Plastic plates (petri-dishes) were coated with different concentrations of autoclaved gelatin solutions (0%, 0.1%, 0.5%, 1%, and 2%). In brief, the gelatin solution was placed in a petri-dish to submerge the surface, where it was left for 30 minutes, and then the gelatin solution was removed and dried. AFM (NX-10, Park Systems Co.) was used to analyze the surface of each gelatin-coated petri-dish. To this end, the Young’s modulus of each gelatin-coated sample with a mixing ratio of 0%, 0.1%, 0.5%, 1%, and 2% was examined by using the Pinpoint^TM nanomechanical mode. Considering the characteristics of the sample, an AC160TS cantilever for an AFM with a spring constant (k) of 20 ± 3 N/m was used. We measured each Young’s modulus of the samples after calibrating photodetector sensitivity, force gradient correction, and spring constant (k) for accurate and quantitative measurement. In this experiment, measurement was fixed by setting the parameters of the scan speed: 25.0 μm/s, set point: 105 nN, and minimum length: 45 nm.

ESC maintenance and seeding

Mouse ESCs (E14, OG2 cell line) were cultured on a 60 mm culture dish and maintained using DMEM media supplemented with 15% FBS, 1X Non-Essential Amino Acid (NEAA), 1X Glutamax, 55 μM 2-Mercaptoethanol, 1X Penicillin-Streptomycin, 2i (3 μM CHIR99021 + 1 μM PD0325901), and 5 × 10⁴ U/mL LIF (maintenance media) in humified culture incubator (37℃ with 5% CO₂). The E14 or OG2 (5 × 10⁴ cells) were seeded into each concentration of gelatin-coated 6-well plate (0.01, 0.05, 0.1, 0.5, 1%) with 2 mL of maintenance media.

CCK assay

Cell viability was measured using a D-Plus^TM CCK cell viability assay kit. To test cell viability, E14 (1 × 10⁴ cell) was seeded into each concentration of gelatin-coated 96-well plate with 100 mL of maintenance media. After 2 days, 10 mL of D-Plus^TM CCK was added to each well, after which the plate was incubated briefly to react. Absorbance at 450 nm was measured for 4 hours at 10 minutes-intervals using an Epoch 2 Microplate Spectrophotometer maintaining 37℃. Blank wells contained only maintenance media. Cell proliferation was independently repeated three times with the same number of cells (1 × 10⁴ cell/well).

ESC differentiation

OG2 (5 × 10⁵ cell) was seeded into gelatin-coated 6-well plates of each concentration with 2 mL of maintenance media. After 8 hours, the media was removed and carefully washed with 500 mL DPBS, and then differentiation media was added for 8 days. The differentiation media was DMEM media supplemented with 15% FBS, 1X Non-Essential Amino Acid (NEAA), 1X Glutamax, 55 μM 2-Mercaptoethanol, 1X Penicillin-Streptomycin, and 1 μM retinoic acid. After 2 days, cell morphology was analyzed using an optical microscope, and the total RNA was isolated. Total RNA was harvested using TRI-Reagent consistent with the manufacturer’s manual, after which reverse transcription was performed using an AMPIGENE® cDNA Synthesis Kit according to the manufacturer’s manual.

qPCR analysis

Quantitative PCR was performed using a TOP real^TM qPCR 2X PreMIX and Rotor-Gene Q 2plex PCR machine. Each cDNA (100 ng/mL) was subjected to qPCR as template and primers were as follows; mGAPDH (forward: 5’-AATGGTGAAGGTCGGTGTGAACGG-3’, reverse: 5’-GTCTCGCTCCTGGAAGATGGTGATG-3’), mOct4 (forward: 5’-CACCATCTGTCGCTTCGAGGC-3’, reverse: 5’-CTGCACCAGGGTCTCCGATTTG-3’), mSox2 (forward: 5’-CATGAGAGCAAGTACTGGCAAG-3’, reverse: 5’-CCAACGATATCAACCTGCATGG-3’), mNanog (forward: 5’-CTTTCACCTATTAAGGTGCTTGC-3’, reverse: 5’-TGGCATCGGTTCATCATGGTA-3’), mSox17 (forward: 5’-TTCTGTACACTTTAATGAGGCTGTTC-3’, reverse: TTGTGGGAAGTGGGATCAAG-3’), mGata4 (forward: 5’-CAGCAGCAGCAGTCAAGAGATG-3’, reverse: 5’-ACCAGGCTGTTCCAAGAGTCC-3’), mBrachyuyry (forward: 5’-ATCAGAGTCCTTTGCTAGGTAG-3’, reverse: 5’-GTTACAATCTTCTGGCTATGC-3’), mNestin (forward: 5’-AACTGGCACACCTCAAGATGT-3’, reverse: 5’-TCAAGGGTATTAGGCAAGGGG-3’), mPax6 (forward: 5’-ACCAGTGTCTACCAGCCAATCC-3’, reverse: 5’-GCACGAGTATGAGGAGGTCTGA-3’). The qPCR was performed in Three-step with melt and the conditions were as follows; hold at 95℃ for 10 min, in cycle, denaturation at 95℃ for 30 s, annealing at annealing temperature for 60 s, extension at 72℃ for 60 s. Due to differences in the annealing temperature for each primer, the experiments were conducted separately as 58℃-mSox2, mNanog, mSox17, mGata4, mBrachyury, mNestin, mPax6, 62℃-mOct4. mGAPDH acted as an internal control to normalize the expression levels. We quantification the data using the 2^-ΔΔCt method.

Statistical analysis

The experimental data were expressed on the graphs as means ± the standard error of the mean (SEM). The significance of the data was determined through a one-way analysis of variance test (ANOVA) and a t-test performed with Microsoft Excel. p < 0.05 was considered significant.

Young’s modulus by AFM

To examine the effect of gelatin-coating the surface of a cell culture dish, surface roughness and the degree of hardness were measured by Young’s modulus at the nanoscale using an AFM. Our analysis showed that the mechanical property (gigapascals, GPa) decreased when 0.1% or more gelatin coated on surface of the plate (Fig. 1A). However, we confirmed that when the concentration was 0.05% or less, the deviation in the GPa value was large and was close to the GPa value of the 0% sample on average (data not shown). In addition, as a result of measuring the surface strength by gelatin coating, it was confirmed that the strength increased significantly at a gelatin coating concentration of 0.5% or more (Fig. 1B). Taken together, these results demonstrate that the gelatin coating has a definite effect on the plate surface and is significant at concentrations above 0.5%.

Figure 1.Young’s modulus analysis of gelatin-coated surfaces using an atomic force microscope. (A) The surface strength of each gelatin coating concentrations was indicated through the Pinpoint^TM nanomechanical mode. The higher the surface strength, the brighter the brown color displayed. (B) The surface roughness of each gelatin coating concentrations was indicated through the Pinpoint^TM nanomechanical mode. A total of three points of surfaces of each sample were measured, and the strength and roughness of the surface were analyzed and graphed using AFM (NX-10, Park Systems)’s Young’s modulus software. Data were presented as mean ± SEM of N = 3 experiments. (*p < 0.05, **p < 0.01, compared with uncoating group).

Effect on cell attachment of the concentration of gelatin coating

To identify the optimal concentration of gelatin-coating for the attachment of ES cells, E14 cells were seeded on each well of a petri-dish respectively coated with 0%, 0.01%, 0.05%, 0.1%, 0.5%, and 1% gelatin. After 16 hours, cell adhesion was observed morphologically; ES cells were floating and clumping together on the uncoated plate (Fig. 2A, black arrow), but on the petri-dish coated with 0.1% or more gelatin, most of the ES colonies adhered to the dish surface (Fig. 2A). To assess the effect of different concentrations of gelatin coating on cell growth, CCK analysis was performed using E14 cells. This confirmed that cell proliferation significantly increased at a 0.1% gelatin concentration or higher (Fig. 2B). In sum, gelatin coating is essential for ESC cultures, and the optimal concentration of gelatin coating is 0.1%.

Figure 2.Comparison of adhesion and proliferation ability of ES cells according to gelatin coating concentration. (A) Mouse ES cells (E14, 5 × 10⁴ cells) were seeded and incubated on petri-dishes coated with various concentrations of gelatin for 48 h, then the morphology of each ES colony was captured. Black arrow indicates clumped cell mass and white arrow indicates attached colony. (B) Mouse ES cells (E14, 5 × 10³ cells) were seeded on each concentration of gelatin-coated 96-well culture plate. Purchased cell culture dishes are coated with basic poly-L lysine. After 48 h, cell proliferation analysis was performed using CCK reagent. Data were normalized to control and presented as mean ± SEM of N = 5 experiments. The CCK analysis was independently repeated three times. **p < 0.01 vs. control (no coating).

The effect of gelatin coating on ESC differentiation

Differentiation of ESCs was induced at each concentration of gelatin to examine the additional effect of gelatin coating on ESCs. After 4 days, cell morphology had clearly changed on the surface of the culture dish coated with 0.1% or more gelatin, confirming that differentiation was successfully induced (Fig. 3A, white arrow). However, in the 0.05% or less gelatin-coated plates, ES colonies and cell morphology showed abnormal morphology (Fig. 3A, upper; black arrow). Examining the morphology of the ES colonies in more detail, some differences were clear. Distinct from the spherical colony form of the under 0.05% gelatin-coated group, at a gelatin-coating concentration of 0.1% or more, differentiated ES cell fibers were present and flat colonies were formed (Fig. 3A, lower). Additionally, we performed differentiation induction experiments using Oct4 distal enhancer-mediated GFP-labeled ESCs (OG2; Supplementary Fig. 1). As a result of checking GFP after 4 days of induction of differentiation, it was confirmed that the GFP of the OG2 cell line decreased slightly due to the gelatin coating (Fig. 3B). Taken together, our results demonstrate that gelatin coating at an optimal concentration of 0.1% or higher is required even for the differentiation of ES cells.

Figure 3.Comparison of ES cell differentiation at each gelatin coating concentration. (A) Mouse ESCs (E14, 5 × 10⁴ cells) were seeded and incubated gelatin-coated (various concentrations) 12-well culture plate. Purchased cell culture dishes were coated with basic poly-L lysine. For differentiation induction, ES cells were treated with a differentiation induction medium supplemented with 1 μM retinoic acid for 4 days. The black arrow indicates abnormal morphology in the ES colony and the white arrow indicates a normal morphology of differentiated cells. (B) OG2 ESCs (5 × 10⁴ cells) were seeded and incubated gelatin-coated (various concentrations) 12-well culture plate. For differentiation, OG2 were treated with a differentiation induction medium supplemented with 1 μM retinoic acid for 4 days. 4 days after differentiation induction, OG2 was observed using a fluorescence microscope.

qPCR was performed to confirm the effect of gelatin coating on ES cell differentiation at the level of gene expression. In all differentiated cells, expressions of the pluripotency markers Oct4, Nanog, and Sox2 had significantly decreased compared to undifferentiated cells, and all pluripotency markers had decreased in the gelatin-coating group (Fig. 4A to 4C). The expression of differentiation markers by gelatin coating showed relatively diverse patterns. The expression of Gata4 and Pax6 was significantly suppressed in the all of gelatin-coated groups (Fig. 4D and 4G), but the expression of brachyury had been significantly increased by the gelatin coating (Fig. 4F). Sox17 expression was not affected by gelatin (Fig. 4E), and Nestin expression was significantly increased only in the 1% gelatin-coated group (Fig. 4H). This experiment confirmed that each concentration of gelatin coating can exert a very specific effect on the differentiation of embryonic stem cells.

Figure 4.Identification of differentiation markers at each stage after differentiation induction of ES cells at each gelatin concentration. Mouse ES cells (OG2, 1 × 10⁵ cells) were seeded and incubated on a 6-well culture plate coated with various concentrations of gelatin. For differentiation induction, ES cells were treated with a differentiation induction medium supplemented with 1 μM retinoic acid for 4 days. (A-H) Expression levels of pluripotency (A-C) and differentiation markers (D-H) were analyzed using prepared cDNAs by q-RT PCR. The graphs of each fold change were drawn by Prism 6 (GraphPad Software). Data were normalized to controls and presented as mean ± SEM of N = 3 experiments (*p < 0.05, **p < 0.01, compared with uncoating group; ^#p < 0.01, compared with undifferentiated mouse ES cell).

The attachment of cells is a crucial problem directly related to cell growth and survival. Numerous previous studies have used coated culture dishes as a pretreatment to create the appropriate cell microenvironment in an in vitro culture, employing coating materials as diverse as gelatin (Tielens et al., 2007; Giol et al., 2019), collagen (Chen et al., 2005; Niwa et al., 2012; Liu et al., 2015), fibronectin (Cornelissen et al., 2013; De Visscher et al., 2012), and laminin (Tamura et al., 1997; El-Ghannam et al., 1998; Oyane et al., 2005; Min et al., 2013; Yamashita et al., 2015; Leino et al., 2018). Gelatin in particular is widely used because of its ease of culturing and analyzing of ESCs, and its economic advantages. However, no studies have yet reported suitable gelatin concentrations. In this study, we identified the optimal concentration of gelatin coating for the culture and differentiation of ESCs.

We specifically sought to determine whether the gelatin coating has any physical influence on cell adhesion. Using Young’s modulus analysis using AFM, which can effectively analyze nanometer scale surfaces, we were able to confirm that the gelatin coating has a definite effect on the roughness and strength of the cell attachment site. The Young’s modulus analysis revealed no significant differences in surface strength and roughness at a gelatin concentration of 0.5% or more, so the maximum value of gelatin concentration was set to 0.5% in subsequent experiments.

The petri-dish is plastic without any coating required for animal cell cultures, so cells are not able to grow attached to it. As confirmed in Fig. 2, cells could not attach and died if there was no ECM at the cell attachment site during cell culture. However, if only 0.5% or more gelatin was coated, cell culture was successfully maintained. Therefore, we propose that the gelatin coating plays a critical role in the in vitro culture of cells through its role as an ECM and that coating over a 1% concentration is superfluous.

Although the optimal gelatin coating concentration for ES cells is, as we suggest, 0.5% (as shown in Fig. 2A), some cells adhered even at a very low gelatin coating concentration of 0.01%. This fact is consistent with previous studies showing that 0.01% gelatin coating is sometimes used in the maintenance of various fibroblasts and progenitor cells, with the exception of ES cells (Lee et al., 2015). We suggest that researchers check the adhesion and characteristics of the cells they culture in advance.

ES cell research is most commonly directly related to differentiation research, and as such it is important to confirm the association between gelatin coating and the differentiation of ES cells. Inducing the differentiation of ES cells usually takes more than a week. To confirm the effect of gelatin coating on the differentiation of ESCs, we used a cell culture dish with a conventional coating. In our experiment, the effect of gelatin coating grew clearer as the concentration of gelatin increased. Our results also show that the differentiation of ES cells was normally induced at a gelatin concentration of 0.5% or more and that the shape of the ES colonies was abnormal when cultured with a lower concentration of gelatin (Fig. 3A). During the induction of ESC differentiation, the cell morphology was normally changed by gelatin coating, so we additionally confirmed the differentiation-regulating effect of gelatin using the OG2 cell line. The OG2 is a transgenic ESC in which GFP is reported by the distal enhancer of Oct4, and GFP decreases as differentiation proceeds. Since we confirmed that GFP changes according to the concentration of gelatin during induction of OG2 differentiation, we checked the expression of pluripotency markers and differentiation markers through Q-PCR to confirm more accurate changes.

An even clearer difference was observed when comparing the expression of differentiation markers as well as cell morphology analysis. Oct4, Sox2 and Nanog are representative pluripotency markers. Even after only 2 days of differentiation, the expression of these pluripotency markers rapidly decreased. The gelatin coating effect seems to act more specifically on the differentiation of these ES cells. As can be seen from our results, the expression of Oct4 and Sox2 significantly decreased even at the low gelatin coating concentration of 0.01%, but the expression of Nanog tended to decrease slightly at a gelatin coating concentration of 0.5% or more. GATA4 is a marker expressed in the early endoderm and mesoderm. In our Fig. 4D, gelatin can be seen to have significantly inhibited the expression of GATA4, but Sox17, an endoderm marker, had no change in expression by gelatin (Fig. 4E). Therefore, while we expected that gelatin would inhibit the expression of mesoderm markers, in fact we confirmed that it significantly increased the expression of brachyury, a representative mesoderm marker (Fig. 4F). It also shows inconsistent effects on the expression of the neuroectodermal markers Pax6 and Nestin (Fig. 4G and 4H). Based on these results, we suggest that additional studies on the regulation of gene expression during ES cell differentiation by gelatin are necessary. Interestingly, the brachyury expression was not increased in the gelatin no-coat group (Fig. 4F). This result supports previous studies showing that collagen IV can specifically differentiate into the mesoderm lineage (Schenke-Layland et al., 2007). Collagen is known to induce differentiation of ES cells, not only to the ICM-based three germ layers, but also to the trophoblast layer, and because gelatin is so closely related to collagen, it is assumed that they do not differentiate into mesoderm (Rozario and DeSimone, 2010).

Our findings show that 0.5% gelatin is the optimal level for ES cell adhesion, proliferation, and differentiation. Further, each concentration of gelatin-coating is expected to provide an indicator of cell culture that reduces experimental errors, including those focused-on cell differentiations through control of the cell culture environment. We also suggest that various analysis tools such as Young’s modulus using AFM are needed for studies related to cell signaling responses, and further studies of the connections between the ECM and cell membrane are needed.

None.

Conceptualization, H.J.,; methodology, H.S.; supervision, H.J.

This research was supported by a grant from the National Research Foundation of Korea (NRF), with money provided by the Korean government (MSIT) (No. 2021R1C1C1011346), and the National University Promotion Program at Jeonbuk National University in 2021.

Institutional Animal Care and Use Committee of Yonsei University (No. YWC-P120).

Not applicable.

Not applicable.

Not applicable.

No potential conflict of interest relevant to this article was reported.