3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

solution (1:20 diluted) for 45 min and rinsed with dH2O before mounted for imaging. For Oil Red O staining (LifeLine), the samples were added to propylene glycol for 2 min, incubated in Oil Red O solution for 6 min, rinsed with propylene glycol (80% v/v in distilled dH2O) for 1 min and with dH2O twice, incubated in Hematoxylin for 2 min, and then rinsed with dH2O twice before mounted for imaging.

Glycosaminoglycan (GAG) assay
Sulfated GAG content was measured using Blyscan sulfated GAG assay kit (Biocolor Ltd., Carrickfergus) according to the manufacturer's protocol. Briefly, each segment of the hydrogel matrices was homogenized (Pro Scientific Inc.), digested in 500 µl of 0.1 mg/ml pepsin in 0.1 M HCl overnight at room temperature, and neutralized with 1M NaOH. 100 μl of the digested solution was mixed with 250 μl Blyscan dye and shaken for 30 min. After centrifuging at 15,000 × g for 10 min, the precipitate was collected and added to the 600 μl dissociation reagent for 10 min to release the bound dye. The absorbance was then read at the wavelength of 656 nm and the actual amount of GAG was determined by the standard curve (the amount of GAG as a function of absorbance). The standard curve was generated with the standard samples provided in the kit.

Calcium quantitation assay
We analyzed the mineralization in the hydrogel matrices by measuring the amount of calcium with Osteogenesis Quantitation Kit (Millipore) according to the manufacturer's protocol. Briefly, each segment of the hydrogel matrices was added to 10% acetic acid in a microtube, homogenized (Pro Scientific Inc.), and shaken for 30 min. After heated at 85 ºC for 10 min, the microtube was transferred to iced water for 5 min and centrifuged at 20,000 × g for 15 min. With a pH meter (Corning), the pH of the supernatant was ensured to fall within the range of 4.1-4.5. The absorbance was read at the wavelength of 405 nm and the actual amount of calcium was determined by the standard curve generated with the standard samples in the kit.

DNA Quantitation
The quantitation of DNA content was performed with a fluorescent dye, bisBenzimide. Each segment of the hydrogel matrices was homogenized (Pro Scientific Inc.) and added to 1 µg/ml bisBenzimide solution (Sigma-Aldrich) diluted by 10 × Fluorescent Assay buffer (Sigma-Aldrich) and water (Molecular Biology Grade, Fisher Scientific). Then the fluorescent intensity was read at the excitation wavelength of 360 nm and the emission wavelength of 460 nm. The actual DNA amount was determined by the standard curve prepared by DNA standard (1 mg/ml solution of calf thymus DNA, Sigma-Aldrich).

Viability test
Relative cell viabilities of samples were measured by the AlamarBlue ® assay (Invitrogen). Briefly, 10 % (v/v) AlamarBlue reagent solution in the cell culture medium was added to each sample for 2 hours and 100 μl of each supernatant was collected into a 96 well plate. And the fluorescence of each sample was measured at the excitation wavelength at 545 nm and the emission wavelength at 590 nm. The fluorescence differences in the two wavelength were calculated. The relative viability was calculated by comparing the following value of each sample: (fluorescence difference in the supernatant) -(fluorescence difference in a negative control, 10% AlamarBlue solution) This method of measuring the cell viability in 3D hydrogel samples did not show statistical difference from the method of manual counting using Live/Dead staining (Invitrogen) 35 .  Table S1. The sets of the diffusible signal molecules used in our experiments for specific hMSC differentiation   S1. The optimal condition for chondrogenesis and osteogenesis with respect to an immobilized cue (the cellbinding domains) was determined. Histological images of hMSC-containing hydrogel matrices are shown in A for chondrogenesis stained by Safranin O stain (GAG: red) and B for osteogenesis stained by Alizarin Red Stain (calcium: red). Scale bar: 150µm. Quantitative analyses on the differentiation outcomes confirm the results seen in the histological images that the incorporation of the cell-binding domains interferes the chondrogenesis (C) while the osteogenesis is critically dependent on the presence of the cell-binding domains (D). The statistical significance and the data below detectable levels are denoted as '*' as 'v', respectively (n=3).   S3. The negative control conditions in which the hMSC cells were incubated in the molded hydrogel matrices prepared through MC casting process but without drug-releasing microparticles. The hMSC without the drug-releasing microparticles showed no differential behavior compared to the case with the drug-releasing microparticles (see Fig.  3, Fig. 5, and Fig. S2). Both hydrogel layers in A were prepared with the hydrogel networks with cell binding domains