Abstract: BACKGROUND: Cells-in-gels-in-paper (CiGiP) is a three-dimensional cell culture technique that uses hydrogels to encapsulate cells within paper fibers. It offers a promising approach for the development of three-dimensional cell culture. However, the hydrogels must be prepared in advance and then added to the paper material, which lacks certain convenience and hinders the widespread application of CiGiP.
OBJECTIVE: To prepare a ready-to-use sodium alginate@paper material and to analyze its application in three-dimensional cell culture.
METHODS: (1) 2% sodium alginate was incorporated into paper materials, allowing it to evenly penetrate the paper material to obtain a ready-to-use sodium alginate@paper material. A mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was then dripped onto the ready-to-use sodium alginate@paper material. Polyethylene glycol-diamine at different concentrations (5%, 10%, 15%, and 20%) was then added dropwise. The prepared sodium alginate@paper material was freeze-dried to obtain the ready-to-use sodium alginate@paper material. 15% polyethylene glycol-diamine was selected based on swelling ratio for subsequent preparation of ready-to-use sodium alginate@paper materials. (2) Human embryonic kidney (HEK) 293 cells were co-cultured with the ready-to-use sodium alginate@paper materials. Cells cultured alone were used as controls. The cytotoxicity of the ready-to-use sodium alginate@paper materials was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTOTM 9 green fluorescent nucleic acid dye were plated onto the ready-to-use sodium alginate@paper materials and freshly prepared alginate@paper materials, and cell adhesion was observed under laser confocal microscopy and scanning electron microscopy. (3) The ready-to-use sodium alginate@paper materials were stored in clean culture dishes at room temperature for 0, 40, 80, and 120 days, and the micromorphology and porosity of the materials were examined. HEK 293 cells were seeded to ready-to-use sodium alginate@paper materials after storage for 0, 40, 80, and 120 days and the cytotoxicity of the ready-to-use sodium alginate@paper materials was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTOTM 9 green fluorescent nucleic acid dye were seeded onto ready-to-use sodium alginate@paper materials after storage for 0, 40, 80, and 120 days, and cell adhesion was observed under laser confocal microscopy.
RESULTS AND CONCLUSION: (1) Lactate dehydrogenase release assays demonstrated that the ready-to-use sodium alginate@paper materials were non-cytotoxic. Laser confocal microscopy revealed that HEK 293 cells adhered uniformly to both materials, with no significant difference in adhesion between the two groups. Scanning electron microscopy revealed that HEK 293 cells maintained cell-cell interactions in both materials, with no significant difference between the two groups. (2) After 120 days of storage, the sodium alginate hydrogel in the ready-to-use sodium alginate@paper materials did not detach from the paper fibers, and the porosity did not change significantly. Lactate dehydrogenase release assays demonstrated that the ready-to-use sodium alginate@paper materials were non-cytotoxic after storage for 40, 80, and 120 days. Laser confocal microscopy revealed that HEK 293 cells adhered uniformly to the ready-to-use sodium alginate@paper material after storage for 40, 80, and 120 days, with no significant difference in adhesion compared with unstored ready-to-use sodium alginate@paper. These results demonstrate the excellent stability of the ready-to-use sodium alginate@paper material.

Key words: ready-to-use, sodium alginate hydrogel, paper, three-dimensional culture, freeze-drying, crosslinking agent, cytotoxicity, cell adhesion, porosity, biomaterial