Over the past decades, cell viability tests have been an essential research tool in cell biology, tissue engineering, and regenerative medicine. Assessment of cell viability is mandatory in the production and quality control of cell products for biomedical applications. Methods of viability assessment can be broadly classified according to the underlying mechanism and how the results obtained are evaluated. This article presents variants of the most commonly used tests and protocols for assessing cell viability. Their advantages and disadvantages are presented, which should be considered when planning experiments, e.g., when developing cell preparations for regenerative medicine. The authors point out the main factors influencing the choice of viability assessment method: efficiency, speed, safety, reproducibility, sample integrity, compatibility with biomaterial, and cell line type. Finally, the authors discuss separately cell viability tests that can be applied not only to 2D cell structures but also to 3D cell structures, which have recently become widespread due to more accurate modeling of biological processes.

Regenerative medicine uses cells as therapeutic agents to heal tissues and organs. It is a rapidly evolving area of research worldwide. Cell-based therapy has emerged as a pivotal treatment approach for articular cartilage defects, recognizing the limited regenerative potential of cartilage inherent to its structural biology. Given the inherent challenges associated with the standardization of cell-based drugs compared to conventional pharmaceuticals, the evaluation of their safety and efficacy in preclinical or clinical trials incurs particular considerations. In the majority of cases, autologous chondrocytes and mesenchymal stem/stromal cells derived from various tissues become key components of cell-based therapies currently available for cartilage defects. The cell-based therapies that have been approved for clinical use vary in manufacturing methods, types of cells, and use of matrices as a cell carriers in the finished product. Furthermore, clinicians routinely use a range of surgical techniques to perform a biopsy procedure for the preparation and subsequent implantation of finished cell-based products. Each cell-based treatment option available for patients with cartilage diseases offers a particular indication, benefits, and limitations, underscoring the relevance of comparative analysis of the therapies currently used in clinical practice. This will facilitate clinicians in selecting the most suitable therapy, while researchers may potentially expand the range of diagnoses for such therapies or enhance their efficacy. This review will focus on certain cell-based therapies that have currently arrived at the stages of clinical investigation and have been approved for the treatment of cartilage defects.

Cell therapy or tissue engineering is currently one of the potentially effective strategies for repairing cartilage defects. Mesenchymal stromal cells and autologous and allogeneic chondrocytes obtained from cartilage biopsies from patients undergoing arthroscopic surgery are the most commonly used cells for tissue engineering. Matrices (scaffolds) often used during cell implantation can facilitate successful cell implantation. These can be static supporting or plastic hydrogel substances obtained by various methods from natural or synthetic materials, allowing cells to be delivered to the site damaged by injection. Significant advantages of biocompatible hydrogels in cartilage injury therapy are determined based on the properties of the natural extracellular matrix and the high plasticity required for adapting to irregular surfaces of the tissue defect and allowing injectable cell delivery. These characteristics make injectable hydrogels a promising tool for cartilage bioengineering. This study highlights the progress in injectable hydrogels as carriers of different cells in the repair of cartilage tissue defects, as well as the current requirements and unresolved issues related to the use of such therapeutic approaches.

BACKGROUND: Cryopreservation is a widely used method for long-term viability preservation of cultured cells and complex cellular structures, including organoids, which serve both scientific and clinical purposes, as well as for screening applications. However, data on cryopreservation of organoids derived from differentiated induced pluripotent stem cells (iPSCs) remain limited. AIM: The work aimed to optimize slow freeze protocols for neural organoids derived from iPSC-differentiated cells and chondrospheres. METHODS: Neural organoids differentiated from iPSCs were cryopreserved on days 9, 14, 22, 29, and 43 using four different cryopreservation media. Post-thaw assessment included organoid integrity and size measurements, quantitative polymerase chain reaction for MAP2 and NES markers, and immunohistochemical (IHC) staining for βIII-tubulin, MAP2, SOX2, and proliferating cell nuclear antigen (PCNA). Chondrospheres were derived from human chondrocytes and cryopreserved 29 days after transition to 3D culture conditions in a solution of 82% DMEM + 10% FBS + 8% DMSO + 10 µM ROCK inhibitor Y27632 (Ri, Rho-associated protein kinase inhibitor). Post-thaw IHC analysis was performed to assess the expression of chondrogenic marker proteins, including aggrecan, collagen type II, and SOX9, as well as PCNA. RESULTS: Neural organoids cryopreserved between weeks 3 and 6 of differentiation retained the highest post-thaw structural integrity. Although organoid diameter decreased by 14.5% on average 1 week after thawing, size was nearly restored to the values observed on the corresponding day of differentiation for organoids not subjected to cryopreservation by week 2. Quantitative polymerase chain reaction and IHC staining confirmed maintenance of neuronal phenotype 2 weeks post-thaw. Chondrospheres showed no change in diameter over 2 weeks post-thaw and exhibited 100% preservation of organoid integrity. IHC analysis confirmed the presence of chondrocytic proteins in chondrospheres 2 weeks after thawing. CONCLUSION: Based on the study findings, the third week of differentiation was identified as the optimal stage for cryopreservation of iPSC-derived neural organoids. The cryopreservation medium composed of 82% DMEM, 10% FBS, and 8% DMSO was optimal for both neural organoids and chondrospheres.

BACKGROUND: The use of tissue-engineered products for treating hyaline cartilage injuries is currently a promising direction for regenerative medicine. The study of storage and transportation conditions of such preparations is essential in product development because it determines the viability and functional activity of the cellular component of the product. In addition, such work should be performed following the requirements of Russian legislation before using the developed drug product in clinical trials. AIM: To characterize the storage conditions for tissue-engineered preparations such as chondrospheres. METHODS: Chondrospheres were formed from human chondrocytes obtained from the biopsy material of the knee joint cartilage of patients diagnosed according to ICD-10 codes M15–M19. After the formation of chondrospheres, cell viability was examined using the metabolic dye PrestoBlue, and the expression of chondrogenic markers (SOX9, aggrecan, collagen type 1 and type 2) was preserved using real-time polymerase chain reaction under various transportation conditions, i.e., at different storage times, medium composition, and temperature. RESULTS: The analysis of the expression of chondrocyte markers confirmed that cells in chondrospheres retain a chondrocyte phenotype. In the cell viability study of tissue-engineered products under various conditions (temperature, solution, and time), the most favorable environment for storing the product was phosphate buffer saline and 0.9% sodium chloride solution (р 0.05). The metabolic activity of the cells that make up the tissue-engineered product was maintained when stored at +4 °C for up to 3 days (р 0.05). CONCLUSION: Isotonic solution and temperature regime +4 °C for not more than 2 days showed the preferable medium and conditions for storing and transporting the prototype cartilage implant as chondrospheres.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.