Papain

After harvesting, the constructs were weighed and then digested for 24 h in 1 mL of 0.1 M PBS containing 2.5 U of papain (Beyotime, China), 5 mM cysteine-HCl and 5 mM Na2EDTA at 60 °C. The cell number was assessed based on the DNA content using Hoechst 33258 dye (KeyGen Biotechnology Nanjing, China), as previously described [21 (link)]. Hoechst 33258 is a fluorescent nucleic acid stain used to quantitate double-stranded DNA in solution. The fluorescence of the samples was converted to cell numbers using a cell-number standard curve created previously. The glycosaminoglycan (GAG) content was determined using the 1, 9-dimethylmethylene blue (DMMB; Jianglaibio, China) dye-binding assay and a shark cartilage chondroitin sulfate reference sample (C4348; Sigma, USA). The GAG content was normalized to the cell number to assess the single-cell biosynthetic activity.

β-Galactosidase (β-Gal), horseradish peroxidase, alkaline phosphatase, papain, pepsin, and α-mannosidase were purchased from Beyotime (Shanghai, China). Lysozyme, glucose, bovine serum albumin, γ-globulin, transferrin, mannitol tryptophan and proline were purchased from Solarbio (Beijing, China). Ciprofloxacin, ampicillin, kanamycin, dimethyl sulfoxide (DMSO), Polypropylene pyrrolidone K30 (PVP-K30, MW = 40,000) and all other chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, United States). Luria-Bertani broth (LB) medium was purchased from BD Biosciences (San Jose, CA). All aqueous solutions were prepared with Milli-Q water (≥18 MΩ, Milli-Q, Millipore).