Cell lines are an important tool for scientific research and clinical and pharmaceutical applications. Cells are isolated from animal tissues and can be expanded in culture to study cell biology and disease. The isolated cells can be used to produce antibodies, proteins, and vaccines. Immortalized cell lines can grow in vitro and are commonly used as models for complex biology. The most frequently used type of cell culture for scientific research and clinical and pharmaceutical applications is the two-dimensional (2D) model, but recently, the popularity of the three-dimensional (3D) cultivation method is growing.

The aim of the study was to investigate the effects of chondroitin sulfate (CS) and fucoidan (F), as well as their combination (CS + F) on the growth and viability of spheroids derived from the non-tumor cell line NIH3T3 and the tumor cell line Hepa1c1c7 using a three-dimensional (3D) cell culture model. Spheroids were formed using the liquid overlay method and treated with different concentrations of substances. Flow cytometry was used to quantitatively assess the representation of live, apoptotic, necrotic, and dead cells in the spheroids after treatment. Spheroids from the non-tumor cell line NIH3T3 after treatment with CS and fucoidan were nontoxic to the cells. By light microscopy, we observed a significant proliferative effect in the case of CS and combination of fucoidan and CS (F + CS). On the contrary, in spheroids from the Hepa1c1c7 tumor cell line, after treatment with our compounds, the compounds, especially fucoidan and a combination of CS and fucoidan, showed antiproliferative effects, which confirms their synergistic antiproliferative effect. According to flow cytometry in non-tumor NIH3T3 spheroids, CS had the most significant effect on the proportion of viable cells, which confirmed its proliferative effect (66.43 ± 4.43%). In spheroids derived from the Hepa1c1c7 tumor cell line, flow cytometry revealed the highest number of dead cells in spheroids after treatment with a combination of CS and fucoidan (19.84 ± 5.80%). Based on our results, we can conclude that a combination of CS and fucoidan showed a synergistic antiproliferative effect on Hepa1c1c7 cell cultures. The 3D cell cultures provide a more physiologically relevant platform for drug testing, highlighting the importance of understanding cell–extracellular matrix interactions in cancer research.

This review explores the pharmacological potential of chondroitin sulfate and fucoidan as immunomodulatory agents targeting N-acetylgalactosaminidase (nagalase) to normalize immune responses. Nagalase, an enzyme produced by tumor and virus-infected cells, contributes to immune suppression by deactivating macrophage-activating factor. Both chondroitin sulfate and fucoidan, as representatives of glycosaminoglycans and heteropolysaccharides, exhibit significant potential in inhibiting nagalase activity, thereby restoring immune functionality. Chondroitin sulfate, a key component of the extracellular matrix, demonstrates anti-inflammatory and tissue-regenerative properties by modulating nuclear factor (NF)-κB pathways and cytokine expression. Fucoidan, a sulfated polysaccharide derived from brown seaweed, enhances immune responses through macrophage and natural killer cell activation, while also exhibiting antiviral and anticancer activities. This dual action positions these compounds as promising agents for therapeutic interventions in chronic inflammatory conditions, cancer, and infectious diseases. The synergistic effects of chondroitin sulfate and fucoidan highlight their potential to address the root causes of immune dysregulation. This review aims to elucidate the underlying mechanisms of action and explore the clinical applications of these compounds within the framework of innovative immunotherapeutic strategies. However, current evidence is limited by the predominance of preclinical studies and variability in experimental models. Well-designed clinical trials are needed to validate their efficacy for therapeutic use.

As part of experimental research, growth factor-like substances associated with MHV-68, named MHGF-68, were discovered in our laboratory. MHGF-68 activity was manifested by the ability to alter cell morphology, that is, normal phenotype to transformed, resp. suppresses the transformed phenotype of tumour cells. The aim of the experiments was to monitor the effect of MHGF-68 on the change of the cell actin cytoskeleton in the tumour cell line Hepa1c1c7, as well as the normal cell line NIH3T3, and compare conventional stationary cultivation and dynamic cultivation conditions using a LiveFlow system (In Vitro Technologies). LiveFlow is an advanced system to test the impact of different compounds on the cell cultures, which allows simulation of in vivo conditions thanks to continuous flow of cultivation medium. MHGF-68 was prepared with the infection of BHK-21 cells with MHV-68 virus under non-permissive conditions (41°C). After dynamic cultivation with MHGF-68, we observed changes in morphology on Hepa1c1c7 cells. In cells cultured in a dynamic environment, we observed more pronounced changes in cell morphology in comparison with cells cultured statically. We observed no changes in the cytoskeletal structures in the NIH 3T3 cell line affected by MHGF-68 in both types of cultivation. The advantage of LiveFlow in comparison to in vivo testing is that the experiments performed in this system are less time and money consuming. Dynamic cultivation in the LiveFlow system is suitable for optimizing experiments before testing substances in vivo .

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.