Its premix

For hepatocyte differentiation, cells were placed at the density of 1.5 × 10³ cells/cm² on collagen I-coated plates. At day 1, the medium was changed to stage I differentiation medium: Iscove’s modified Dulbecco’s medium with 20 ng/ml hepatocyte growth factor (Invitrogen), 10 ng/ml fibroblast growth factor 4 (Invitrogen), 1% insulin-transferrin-selenium (ITS premix, Invitrogen) and 5 mM nicotinamide (Invitrogen). At day 9, the medium was changed to stage II differentiation medium (Iscove’s modified Dulbecco’s medium with 20 ng/ml Oncostatin M, 1 μM dexamethasone, 1% ITS premix), and cells were incubated in this medium for 8 days to generate hepatocyte-like cells [12 (link)].
For function analysis, cells were stained by Periodic Acid Schiff (Sigma) following the manufacturer’s instructions. For the indocyanine green (Sigma) uptake assay, cell medium was changed to 1 mg/ml indocyanine green and incubated at 37°C for 1 hour, followed by washing with PBS three times.

A three-dimensional pellet culture was performed to evaluate sericin’s chondrogenic proliferative and differentiative properties in complex cell formation.ATDC5 cells at density 5 × 10⁵ cells/ml in a 15 ml tube were centrifuged 400 × g for 10 min at 4 °C for cell aggregation and formation as a pellet using the pellet culture technique. The pellet cells were incubated in a medium for 3 days at 37 °C with 5% CO₂. The medium was renewed every 2–3 days. The cap 15 ml tube was slightly unscrewed for gas exchange. The chondrogenic differentiation medium in this study was described from the study of Tare et al.^{81 (link)}. It comprised DMEM/F-12 (Gibco, USA), 5% FBS, 1% penicillin, and streptomycin (Gibco, USA) supplemented with 1X of ITS premix (Sigma-Aldrich, USA), 10 ng/ml TGF- $\beta$ 3, 10⁻⁸ M dexamethasone, and 100 µM ascorbate-2-phosphate.

Cells were cultured to be confluent for osteogenic and adipogenic differentiation. Osteogenic medium consisted of α-MEM and 5% FBS supplemented with 50 μg/mL L-ascorbic acid phosphate, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone (all from Sigma-Aldrich, St. Louis, MO, USA). Adipogenic medium included α-MEM and 5% FBS supplemented with 10 μg/mL insulin, 50 μM indomethacin, 0.5 mM isobutylmethaylxanthine, and 1 μM dexamethasone (all from Sigma-Aldrich). Cells were cultured for 21 days in differentiation medium, and fresh medium was replaced every other days. Calcium deposit and lipid vacuoles were stained with Alizarin red solution and Oil red O solution (all from Sigma-Aldrich), respectively. To induce chondrogenic differentiation, 1 × 10⁴ cells were centrifuged at 500g for 5 minutes at 4°C in a 15 mL conical tube. Chondrogenic medium consisted of high-glucose DMEM supplemented with 50 μg/mL ascorbate-2-phosphate, 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 1% ITS + Premix (all Sigma-Aldrich), and 10 ng/mL TGF-β3 (PeproTech, Rocky Hill, USA). Cells were cultured for 21 days, and fresh medium was replaced every other day. Spheroid-like structure was fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) and embedded in paraffin. Slides were prepared from 5 μm thick sections. Alcian blue staining (Sigma-Aldrich) was used to assess chondrogenic differentiation.

A non-tumorigenic mouse hepatocyte cell line, AML12 (America Tissue Culture Collection, CRT-2254; ATCC, Manassas, VA, USA), was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium (Gibco, Grand Island, NY, USA) containing 5 µg/mL ITS premix (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL dexamethasone (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco). According to our previous studies, primary culture rat HSC was cultivated at 37 °C in an atmosphere of 5% CO₂ in minimum essential medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco) [19 (link)]. TGF-β1 was obtained from R&D System Inc. (R&D System Inc, Minneapolis, MN, USA). Signal inhibitors were obtained from Calbiochem (Calbiochem, Cambridge, MA, USA). All chemicals were obtained from Sigma-Aldrich including melittin (MEL), unless otherwise indicated.

To induce chondrogenesis, micro mass culture system was used (Baghaban Eslaminejad et al., 2008[1 ]). Briefly, 2.5×10⁵ BM-MSCs (at P3) was pelleted using centrifuge and cultured for three weeks in chondrogenic differentiation medium (DMEM supplemented with 10 % ITS+ Premix (Sigma-Aldrich), 10−7 M dexamethasone (Sigma-Aldrich), 1 μM ascorbate-2-phosphate (Sigma-Aldrich), 1 % sodium pyruvate (Sigma-Aldrich), 10 ng/ml BMP6 (Sigma-Aldrich) and 10 ng/ml transforming growth factor-beta 1 (TGF-β1, Sigma-Aldrich). At the end of differentiation, the chondrogenic aggregates were sectioned and stained using toluidine Blue, and viewed by light microscope.

Bovine caudal IVDs were isolated and cultivated as previously described (Dudli et al., 2014 (link)). In brief, individual IVDs with endplates were harvested with a band saw after removal of the surrounding soft tissue. The Endplates were rinsed with PBS using an APEXPULSE Disposable Pulse Lavage system (Apex, Guangzhou, China). Then, the IVDs were incubated in PBS containing 10% penicillin/streptomycin (Gibco) for 15 min. Subsequently, IVDs were cultured in DMEM containing 2% fetal calf serum (FCS), 1% penicillin/streptomycin, 50 μg/ml l-ascorbic acid (Sigma-Aldrich), 1% ITS + Premix (Sigma-Aldrich) and 0.1% Primocin (InvivoGen, United States) at the humidified atmosphere (85%) with 5% CO₂ at 37°C. From day 1, the medium was changed once a day after the IVDs were cultured in our custom designed bioreactor under physiological loading (0.02–0.2 MPa; 0.2 Hz; 1 h/day) (Lang et al., 2018 (link); Zhou et al., 2021 (link)).

Porcine intestinal epithelial cell line IPEC-1, derived from the small intestine of a newborn unsuckled piglet, was cultured in Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 (1:1) medium (Life Technologies, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS, PAA Laboratories GmbH, Pasching, Austria), epidermal growth factor (5 μg/mL) (Sigma–Aldrich, St. Louis, MO, USA), insulin (10 μg/mL), transferrin (5 μg/mL), sodium selenite (5 ng/mL) (ITS Premix, Sigma) and 2 mM L-glutamine (Life Technologies). Human intestinal epithelial cells INT-407 (human embryonic intestine, ATCC CCL-6) were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) and supplemented with 10% FBS (PAA Laboratories GmbH) and 2 mM L-glutamine (Life Technologies). All cell lines were seeded in multi-well tissue culture plates (Thermo Fischer Scientific, Waltham, USA) the day before the assay, and allowed to reach confluence for the in vitro infection. The cells were maintained in an atmosphere of 5% CO₂ at 37°C.