Cellular senescence plays a role in the development of aging-associated degenerative diseases. Cell therapy is recognized as a candidate treatment for degenerative diseases. To achieve the

goal of cell therapy, the quality and good characteristics of cells are concerned. Cell expansion relies on two-dimensional culture, which leads to replicative senescence of expanded cells.

This study aimed to investigate the effect of cell culture surface modification using fibronectin (FN) and vitronectin (VN) in adipose-derived stem cells (ADSCs) during long-term expansion.

Our results showed that ADSCs cultured in FN and VN coatings significantly enhanced adhesion, proliferation, and slow progression of cellular senescence as indicated by lower SA-β-gal

increased cellular senescence, and induced the expression of inflammatory molecules including HMGB1 and IL-6. The understanding of FN and VN coating surface influencing ADSCs, especially

senescence characteristics, offers a promising and practical point for the cultivation of ADSCs for future use in cell-based therapies.

Senescence, a key process that causes aging, was first discovered by Hayflick and colleagues, who reported that fibroblasts during in vitro display phenotypic changes and stop growing1.

Based on their study, the Hayflick limit was established, i.e., every cell type has its own limitation of growth2. The senescent characteristics during the aging process have been determined

and include telomere shortening, the expression of cell-cycle inhibitor protein, the upregulation of senescent-associated genes, and changes in signaling pathways involved in cell growth

and differentiation3. The accumulation of senescent cells leads to tissue and organ dysfunctions, which are associated with several degenerative diseases. Currently, treatment for

degenerative diseases is challenging, but cell therapy has shown promising results in this regard. Cell therapy is recognized as a treatment approach for tissue regeneration in which living

cells are used to treat, prevent, or cure diseases or injuries. The type of cell used in cell therapy depends on the condition being treated, e.g., diseases of the immune, blood, and stem

cells4. Mesenchymal stem/stromal cells (MSCs) have shown promising therapeutic outcomes in the field of regenerative medicine5. Adipose-derived stem cells (ADSCs) are an alternative source

of MSCs that can be isolated from fat tissue by lipoaspiration or fat biopsy. They showed high potential in rapid proliferation and immunosuppressive capabilities, allowing modulation of

immune responses and potentially minimizing immune rejection in cell therapy applications5,6.

For clinical use of ADSCs, cell processing is performed under restricted regulation according to good manufacturing process (GMP)7. Many factors within culture conditions can influence

cellular properties, including culture medium, temperature, physical support, pH, and culture vessels. These factors should be considered and validated for continuing of reproducibility and

traceability. In vitro cell expansion influences the properties of ADSCs demonstrating by replicative senescent of ADSCs during culture prolongation. The extensive growth of ADSCs to reach

the therapeutic dose slowed the progression of cell growth and prolonged the population doubling time. The morphological alteration in abnormalities of the nuclear morphology8 and cell size

is indicated by the large, flattened cytoplasm with granules9. In addition, aging ADSCs increased the expression of senescence-associated β-galactosidase (SA-β-gal) and secreted

proinflammatory factors known as senescence-associated secretory phenotype (SASP) that influence the surrounding microenvironment through cell or tissue aging9. The unsatisfactory outcome of

the clinical study was raised as a major concern, and the quality of ADSCs was shown to impact the treatment outcome, leading to unsuccessful results10. However, replicative senescence can

be prevented by optimizing culture conditions, genetic modification, antioxidant supplementation, and senescence inhibitors11,12,13,14. These methods can be used individually or in

combination, depending on the specific cell culture system and desired outcomes.

Growing cells in culture vessels coated with adhesion molecules aimed to increase cell adhesion15,16. Typically, adhesion molecules, such as collagen, fibronectin (FN), laminin, and

vitronectin (VN), are used as extracellular matrix (ECM) coatings17,18,19, but these are expensive and mostly obtained from animal sources. FN and VN are found in fresh human plasma and can

be easily purified20,21. FN can attach to cells through specific binding sites and integrin receptors on the cell surface via the cell-binding domain, particularly prominent on α5β1 integrin

through the arginine-glycine-aspartic acid (RGD) sequence in tenth fibronectin type III22. Besides, VN binds to cells through its interaction with integrin receptors on the cell surface.

Specifically, it binds to αVβ3 and αVβ5 integrin through the RGD sequence and promotes cell adhesion, spreading, and migration23. A recent study demonstrated that FN and VN play crucial

roles in regulating cell cycle progression and apoptosis. FN induces cell cycle arrest by upregulating p21 and can modulate cell survival or apoptosis depending on cellular context. FN

signaling through integrins activating prosurvival pathways like PI3K-AKT to inhibit apoptosis24,25. Similarly, VN has dual roles in cell survival and apoptosis regulation by influencing

cell cycle progression and apoptosis through the PI3K-AKT pathway activated by integrin receptors. It has also been associated with apoptosis-inducing processes like Anoikis, which is

triggered by loss of cell adhesion26,27. Additionally, FN and VN have the potential to prevent cellular senescence by modulating the PI3K/AKT signaling pathway24,25,26,27. Interestingly,

coating with these human-derived proteins can improve the cell expansion process, enhance cell survival, and boost cell growth performance. However, there is a lack of clarity regarding its

role in cellular senescence.

Currently, the process of cell processing especially ADSCs needs cell expansion steps to provide a vast number of therapeutic cells. The standard practice for this process is under

development and shows variability. To meet the good quality of ADSCs, the optimized cell culture surface modification using adhesion molecules, including FN and VN might be a suitable

process to reduce the senescent cells. In this study, we investigated the senescent characteristics of ADSCs during long-term expansion. To explore the impact of these adhesion molecules on

cellular senescence, the aging-related cellular signaling pathway was investigated. The use of adhesion molecules is expected to promote cell adhesion, improve proliferation, and reduce

replicative senescence, thereby enhancing the overall MSCs culture process. The finding of this study will support the role of adhesion molecules in therapeutic cell growth, with the

encouragement of a good protocol for cell culture in a clinical setting.

To determine the cell properties of ADSCs culture in FN and VN coating, adhesion rate and PDT was performed. The various concentrations of FN and VN were investigated to determine the

appropriate concentration for this study (Supplementary Table 1). The coated surface with the lowest concentration of FN and VN which promoted adhesion and PDT was employed in this study.

The adhesion rates of ADSCs cultured in FN and VN were investigated after cell seeding for 12 h. The number of adherence cells was counted and presented as the percentage of the seeding cell

number. Our results showed that the adhesion rates of cells in wells coated with FN (91.46% ± 3.60%) and VN (93.72% ± 3.60%) were significantly higher than the control group (CTRL) (83.22% 

± 3.62) (p