Ready-to-use sodium alginate@paper material for three-dimensional cell culture

Abstract

Background: CiGiP is a three-dimensional culture technique that encapsulates cells in paper fibers using hydrogels, providing a good idea for the development of three-dimensional cell culture. However, the hydrogel needs to be prepared in advance and then added to the paper material, which lacks convenience and hinders the widespread application of CiGiP. Objective: To prepare a ready-to-use sodium alginate@paper material and analyze its application in three-dimensional cell culture. Methods: ① 2% sodium alginate solution was dripped onto filter paper to uniformly permeate the paper material, obtaining a freshly prepared sodium alginate@paper material. A mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide was dripped onto the freshly prepared sodium alginate@paper material, followed by different concentrations (5%, 10%, 15%, 20%) of polyethylene glycol-diamine. After freeze-drying, ready-to-use sodium alginate@paper materials were obtained. The swelling rate was used to select 15% polyethylene glycol-diamine for subsequent preparation of ready-to-use sodium alginate@paper material. ② Human embryonic kidney cells (HEK 293) were cultured in the ready-to-use sodium alginate@paper material, with two-dimensional cultured cells as control. Cytotoxicity of the ready-to-use sodium alginate@paper material was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTO™ 9 green fluorescent nucleic acid dye were cultured in ready-to-use sodium alginate@paper material and freshly prepared sodium alginate@paper material, respectively. Cell adhesion was observed under confocal laser scanning microscopy and scanning electron microscopy. ③ The ready-to-use sodium alginate@paper material was stored in a clean culture dish at room temperature for 0, 40, 80, and 120 days. The microstructure, chemical structure, and porosity of the material were detected. HEK 293 cells were cultured in ready-to-use sodium alginate@paper materials stored for 0, 40, 80, and 120 days, and cytotoxicity was assessed by lactate dehydrogenase release assay. HEK 293 cells labeled with SYTO™ 9 green fluorescent nucleic acid dye were cultured in ready-to-use sodium alginate@paper materials stored for 0, 40, 80, and 120 days, and cell adhesion was observed under confocal laser scanning microscopy. Results and Conclusion: ① Lactate dehydrogenase release assay showed that the ready-to-use sodium alginate@paper material had no cytotoxicity. Confocal laser scanning microscopy showed that HEK 293 cells adhered uniformly to both materials, with no significant difference in cell adhesion between the two groups. Scanning electron microscopy showed that HEK 293 cells maintained cell-cell interactions in both materials, with no significant difference between the two groups. ② After storage for 120 days, the sodium alginate hydrogel in the ready-to-use sodium alginate@paper material did not detach from the paper fibers, and the porosity showed no significant change. Lactate dehydrogenase release assay showed that the ready-to-use sodium alginate@paper materials stored for 40, 80, and 120 days had no cytotoxicity. Confocal laser scanning microscopy showed that HEK 293 cells adhered uniformly to the ready-to-use sodium alginate@paper materials stored for 40, 80, and 120 days, with no significant difference compared to the non-stored material. These results indicate that the ready-to-use sodium alginate@paper material has good stability.

1. Introduction

Cell culture is an indispensable fundamental technique for studying cellular physiology and biochemistry. In most traditional cell culture experiments, cells adhere to the surface of culture dishes as a monolayer, growing in a two-dimensional (2D) state. 2D cell culture has advantages such as simplicity, ease of operation, and convenient observation of cell morphology and proliferation [1-4]. However, in the 2D culture state, cells only interact with each other in the horizontal direction, which cannot fully mimic the real in vivo environment, presenting certain limitations. Three-dimensional (3D) culture preserves the spatial structure of cells in vivo, making cell differentiation, proliferation, and other behaviors closer to physiological states [5-9]. In recent years, 3D cell culture has gained increasing popularity in cell culture experiments.

Hydrogels are polymer materials with a three-dimensional network structure formed by using water as the dispersion medium. Due to their good biocompatibility and biodegradability, they are widely used in the biomedical field [10-11]. Sodium alginate hydrogel is a hydrogel with high gel strength, and its formed 3D structure provides higher support [12-15], standing out among many hydrogels and becoming one of the most widely used hydrogels in cell culture. For example, Liu Xincheng et al. [16] demonstrated that adding sodium hyaluronate to sodium alginate can further enhance its properties.