Obtaining Human Umbilical Cord-Derived Mesenchymal Stem Cells and Cell Line Characterization: Immunophenotype, Multipotency and Cytogenetic Stability

Obtaining Human Umbilical Cord-Derived Mesenchymal Stem Cells and Cell Line Characterization: Immunophenotype, Multipotency and Cytogenetic Stability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Obtaining Human Umbilical Cord-Derived Mesenchymal Stem Cells and Cell Line Characterization: Immunophenotype, Multipotency and Cytogenetic Stability Alina Dovgalyuk, Olesia Redko, Ilona Palii, Sophia Lechachenko, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6938731/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Mesenchymal stem cells (MSCs) derived from the human umbilical cord offer a promising source for regenerative medicine due to their accessibility, low immunogenicity, and high differentiation potential. Unlike other types of MSCs, human umbilical cord MSCs (hUC-MSCs) are obtained through non-invasive procedures and raise fewer ethical concerns. This study aimed to represent our experience in isolating and characterizing hUC-MSCs in terms of their differentiation ability, immunophenotype, and genomic stability. Methods Human umbilical cord tissue was obtained with informed consent under aseptic conditions. MSCs were isolated using enzymatic disintegration of explants with collagenase I and cultured in Advanced DMEM/F12 supplemented with 2–10% fetal bovine serum. Cell morphology, proliferation rate, and viability were monitored during the first 10 passages of cultivation. Multipotency was assessed by inducing adipogenic, osteogenic, and chondrogenic differentiation using specialized culture kits, followed by histochemical staining with Oil Red O, Alizarin Red S, and Alcian Blue. Immunophenotyping was conducted via flow cytometry using antibodies against CD73, CD90, CD105 (positive markers), and CD34, CD45 (negative markers). Cytogenetic analysis was performed on passages 2, 4, 6, 8, 10 to assess genomic stability of cell line. Results Cultured hUC-MSCs displayed typical fibroblast-like morphology and adherence to plastic. Cells successfully underwent adipo, osteo, and chondrogeneic differentiation, confirmed by lineage-specific staining. Flow cytometry revealed a typical MSC immunophenotype (CD73+, CD90+, CD105+, CD34−, CD45−). Karyotype analysis demonstrated a normal diploid chromosomal number, confirming genomic stability during in vitro expansion, as well as stochastic occurrence of polyploid cells and cells with micronuclei at later passages (8–10), not indicative of genomic transformation. Conclusions Obtained hUC-MSCs exhibit stable genetic profiles, specific surface marker expression, and trilineage differentiation potential. These characteristics support their safety and effectiveness as candidates for future cell-based therapies and regenerative medicine applications. Mesenchymal stem cells umbilical cord cultivation karyotyping immunophenotyping Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND Mesenchymal stem cells (MSCs) are currently widely used in regenerative medicine. They are considered the most promising agents for the renewal of damaged cells and tissues due to their unique properties, including chemotaxis toward sites of inflammation, ability to differentiate into various cell lineages as well as to influence the regeneration of injured organs in a paracrine manner [ 1 – 2 ]. MSCs can be easily isolated from various sources, such as bone marrow, umbilical cord, adipose tissue, and dental pulp and perinatal derivatives including placenta, amnion and umbilical cord [ 3 – 4 ]. Human umbilical cord-derived MSCs (hUC-MSCs) present a good alternative to adult MSCs, as they can be obtained through a non-invasive method and are non-immunogenic [ 5 ]. Unlike embryonic and induced pluripotent stem cells, hUC-MSCs do not exhibit tumorigenicity [ 6 ]. Researchers have shown that hUC-MSCs produce various biologically active substances, including cytokines, growth factors, heat-shock proteins, miRNA, defensin, etc [ 7 – 9 ]. The presence of these molecules in their secretome and exosomes causes angiogenic, anti-apoptotic, antioxidant, antibacterial and immunomodulating effects of hUC-MSCs [1; 9–11]. HUC-MSCs due to their multipotency are able to differentiate into various cell types including not only cells of mesodermal lineage such as chondrocytes, osteocytes, and adipocytes [5; 12–13], but also cells both ectodermal (neural) [ 14 – 15 ] and endodermal (hepatocytes) lineages [ 16 – 17 ], making them ideal candidates for tissue engineering and cell therapies. Various protocols have been developed for the isolation and cultivation of hUC-MSCs, differing in the segment of the cord used (Wharton's jelly, perivascular region, or whole cord), enzymatic digestion methods, culture media composition, and passaging techniques. These variations influence the yield, proliferation rate, and biological characteristics of the resulting MSCs [2; 7–8; 12; 18–21]. In this study, we aimed to establish a human umbilical cord-derived MSC cell line and characterize their immunophenotype, differentiation potential, and chromosomal stability during the first 10 passages of cultivation. We present our optimized protocol for isolating and culturing hUC-MSCs, highlighting the procedures that ensured high cell viability, stable phenotype, and differentiation capacity. Our approach contributes to the standardization of methods suitable for both research and therapeutic applications. Methods The umbilical cord was obtained under aseptic conditions from a female newborn with informed consent of her mother after a full-term pregnancy that proceeded without complications and ended with a cesarean section. For transporting the biological material to the laboratory, the umbilical cord was placed in a transport medium containing 200 mg of ciprofloxacin, 10,000 units of penicillin, 10,000 µg of streptomycin, and 25 µg of Gibco Amphotericin B, diluted in 100 ml of PBS and immediately delivered to the lab at 4 o C [ 22 ]. For MSCs isolation, only 5–7 cm of the umbilical cord from the placental side was used. Under sterile conditions, the umbilical cord was cut into small fragments measuring 0.5–2 mm³, transferred into centrifuge tubes, and subjected to enzymatic digestion using 0.1% collagenase I (Sigma-Aldrich, USA), diluted in 2 ml of DMEM/F12 Advanced culture medium (Gibco, USA), avoiding flotation. The enzymatic digestion was carried out for 1 hour at 37°C in a water bath, with thorough mixing of the tube contents every 15 minutes. Afterwards the resulting suspension was pipetted and centrifuged for 5 minutes at 1610×g. The pellet which included partially digested tissue pieces (explants) was resuspended in 7 ml of Advanced DMEM/F12 enriched with 10% fetal bovine serum (FBS) (Gibco, USA), seeded into culture flasks with a surface area of 25 cm² containing Advanced DMEM/F12 medium supplemented with 10% FBS, 1% L-Glutamine-Penicillin-Streptomycin solution (Sigma, USA) and 240 µg/L Heparin solution (Sigma, USA) and kept inside incubators at 37°C and 5% CO₂. The resulting primary culture was assigned passage zero (P0). The first visual assessment of cell morphology and population density was performed using an inverted microscope (Delta Optical NIB-100) 48 hours after seeding to allow cells to spread out and attach themselves to the bottom of the flasks. To remove dead or damaged cells and unattached blood elements, the culture medium was completely replaced 72 hours after the cultivation start, maintaining the FBS concentration at 10%. Further analysis of the primary hUC-MSC culture was conducted 5 days after seeding. At this stage of cultivation, cell population density (confluence) was approximately 50%. At this point, the growth medium was changed again, reducing the FBS concentration to 2% and preserving the previous concentrations of L-Glutamine-Penicillin-Streptomycin Heparin supplements. Following this, every 3 days a complete medium change or replacement of ½ or ¼ volume of medium was performed while maintaining the FBS concentration. Upon reaching 80% confluence, passaging was performed by trypsinization using TrypLE Express Enzyme (Gibco, USA). The flask bottoms were rinsed with HBSS solution, TrypLE enzyme was added, and the flasks were placed in a CO₂ incubator for 5 minutes to detach the cells from the culture plastic surface. The enzyme was neutralized using conditioned medium. The collected cell suspension was centrifuged for 8 minutes at 530×g, after which the cells were seeded into new flasks at a density of 50,000 cells per 1 ml of medium, and a new passage was assigned. Cell counting in the suspension was performed using a hemocytometer after staining with the vital dye trypan blue. Cryopreservation of the cells was performed at various passages. 1 ml of a cell suspension with a concentration of 2–4 million cells per ml was added into 2 ml cryovials, followed by the addition of 1 ml of the prepared freezing medium (30% DMEM/F12 Advanced, 40% FBS, 20% conditioned medium, and 10% dimethyl sulfoxide (Sigma, USA)). After filling, the cryovials were immediately subjected to gradual cooling at a rate of 1°C per minute using a ZPM-1 programmable mobile freezer (Kharkiv, Ukraine). Samples were then stored in liquid nitrogen at -196°C. After thawing, MSCs were cultured for 24 hours in a growth medium consisting of Advanced DMEM/F12 supplemented with 10% FBS, 1% L-glutamine–penicillin–streptomycin solution, and 240 µg/L heparin solution. Afterward, the concentration of FBS was reduced to 2%. Multipotency of cultured cells was assessed at passage 8. Cells were seeded into three 6-well plates with the addition of differentiation media: StemPro Chondrogenesis Differentiation Kit, StemPro Adipogenesis Differentiation Kit, and StemPro Osteogenesis Differentiation Kit (Gibco, USA). After incubation under protocol-specified conditions (18 days for chondrogenesis, 14 days for adipogenesis, and 21 days for osteogenesis), staining with Alcian Blue, Oil Red, and Alizarin Red S was performed respectively. To assess the genetic stability of the cell line cytogenetic analysis was performed at passages 2, 4, 6, 8, 10. Chromosome preparations were obtained using a modified standard protocol [ 23 ]. The procedure was conducted on days 3 or 4 after passaging when the cell population reached the logarithmic growth phase. After incubating cells for 3 hours in colchicine at a concentration of 1×10⁻⁷ M at 37°C, trypsinization was performed for 10 minutes in a CO₂ incubator at 37°C. After enzyme neutralization, cells were treated with warm hypotonic KCl solution (0.07 M) and incubated for another 30 minutes. Afterwards cells were treated with a standard 1:3 acetic acid:methanol chromosome fixative and left on melting ice for 10 minutes. The fixative was replaced twice, and the cell suspension was dropped onto pre-freezed damp slides. Samples were stained using Giemsa Stain (Merck, Germany) for 25 minutes and analyzed using a Nikon Eclipse Ci-E microscope (Japan). In the obtained samples, numerical chromosomal abnormalities were identified, including aneuploidy and polyploidy, and the number of cells with micronuclei and the mitotic index were calculated. The frequency was determined based on 500 cells per each slide (expressed as a percentage). For each passage 6 slides were analysed. For the analysis of MSC-specific cell surface marker expression cells cryopreserved at passage 5 and subsequently thawed were used. Immunophenotyping was performed by flow cytometry using mouse anti-human monoclonal antibodies CD90 FITC, CD73 PE, CD105 PerCP-Cy5.5, CD45 FITC, CD34 PE, and HLA-DR APC-Cy7 (all from BD Biosciences, USA). To compensate for spectral overlap of fluorochromes in multicolor analysis, control samples were used: unstained, single-stained, and fluorescence minus one (FMO) controls. A total of 1×10⁶ thawed and washed cells were resuspended in 100 µL of DPBS supplemented with 1% FBS, incubated with antibodies for 30 minutes at + 4°C in the dark, then washed with 1 mL of CellWash buffer (BD Biosciences, USA) by centrifugation at 300 ×g for 5 minutes, and finally resuspended in 350 µL of DPBS for analysis. Immediately before acquisition, the cell suspension was filtered through a 70 µm cell strainer to remove aggregates. Measurements were performed on a BD FACSAria cell sorter using BD FACSDiva software v.6.1.3. At least 30,000 events were recorded per sample for analysis. Results Microscopic analysis of the primary cell lines derived from the umbilical cord performed 48 hours after seeding the cell suspension into culture flasks revealed a small amount of cells exhibiting typical fibroblast-like morphology and spreading out of partially digested tissue explants (Fig. 1 A). Cell population density was less than 10%, and a shift in the medium's pH towards acidity was observed (phenol red, a pH indicator and an essential component of Advanced DMEM/F12, turned orange). To confirm the multipotency of the obtained cell line, adipogenic, osteogenic, and chondrogenic differentiation of MSC was performed. The differentiated cells exhibited morphology and expression of markers associated with each phenotype. Adipogenic differentiation was confirmed by the accumulation of lipid droplets typical of mature adipocytes, which were visualized as brown by Oil Red O staining (Fig. 2 A). The osteogenically differentiated cell culture demonstrated signs of matrix mineralization— the presence of calcium salt deposits confirmed by staining with Alizarin Red S (Fig. 2 B). Chondrogenic differentiation was assessed by the presence of secreted glycosaminoglycans in the extracellular matrix and formation of a chondrogenic pellet, which was stained with Alcian Blue (Fig. 2 C). Flow cytometric immunophenotyping revealed a high expression level of CD90, CD73, and CD105 markers (greater than 99.1%), with no expression of the hematopoietic markers CD45, CD34, or the major histocompatibility complex (MHC) class II marker HLA-DR. (Fig. 3 ). This immunophenotypic profile is consistent with the characteristics of mesenchymal stem cells as defined by the International Society for Cellular Therapy [ 24 ]. Cytogenetic analysis of MSCs revealed a normal diploid karyotype (46 chromosomes) (Fig. 4 ). Table 1 Karyotyping and Micronucleus Test Results Passage Number Analysed Metaphases, Number % Mitotic Index Aneuploid Cells, Number Polyploid Cells, Number % Cells with Micronuclei 2 40 1.3% 0 1 (2.5%) 0% 4 32 1.1% 0 0 0% 6 26 0.9% 0 1 (3.8%) 0% 8 21 0.7% 0 1 (4.8%) 0.07% 10 19 0.6% 0 2 (10.5%) 0.16% Cytogenetic analyses including classical karyotyping and micronucleus testing were conducted to assess genomic stability. At all passages tested (P2, P4, P6, P8, and P10), no aneuploid cells were detected among the metaphases analyzed, indicating the absence of chromosomal gain or loss. A small number of polyploid cells were observed beginning at P2 (1 polyploid cell, 2.5%) and increased slightly with passage number, reaching 10.5% at P10. The overall mitotic index gradually declined over time, from 1.3% at P2 to 0.6% at P10, consistent with reduced proliferative activity during long-term culture. Notably, micronuclei were absent at early passages (P2–P6) and only began to appear at very low frequencies at P8 (0.07%) and P10 (0.16%), suggesting a minimal increase in genomic stress or DNA damage with prolonged in vitro expansion (Table 1 ). Discussion The human umbilical cord is a promising source of stem cells. Umbilical cord tissue is usually discarded after birth, and its collection does not require any invasive procedures, unlike bone marrow extraction, and does not raise the ethical concerns associated with the use of human embryonic stem cells [ 25 ]. UC-MSCs exhibit low immunogenicity, possess immunosuppressive functions, and can inhibit T-cell proliferation [ 26 – 31 ]. The differentiation potential of UC-MSCs allows them to transform into cells derived from all three germ layers under appropriate conditions. UC-MSCs express low levels of class I MHC molecules and do not express class II MHC molecules, making them suitable for allogeneic and xenogeneic transplantation [28; 32]. Animal studies have shown that hUC-MSCs may be beneficial in treating Parkinson’s disease, spinal cord injuries, certain types of cancer, and tissue fibrosis. Their therapeutic action may be linked either to the differentiation of MSCs into cells of damaged tissues or to their paracrine effects [ 33 – 42 ]. Clinical trials in humans have demonstrated that UC-MSCs improved treatment outcomes in certain immunological and neurological disorders without signs of tumor formation or immune rejection. Thus, UC-MSCs have great potential for regenerative medicine [21; 43]. Numerous studies have shown that UC-MSCs offer advantages over MSCs from other sources. UC-MSCs exhibit higher proliferation rates and greater expansion potential compared to bone marrow MSCs [ 44 ]. Therefore, UC-MSCs can be considered an ideal source of cells for cell therapy compared to bone marrow MSCs and embryonic stem cells [ 45 ]. Different cell culture media are used for culturing hUC-MSCs. Alpha-MEM (α-MEM) is a more basic medium (with nonessential amino acids, nucleosides, vitamins, etc.) but without added growth factors. Laboratories often supplement α-MEM with 10–20% FBS to culture MSCs. In many protocols α-MEM + 10% FBS is the routine expansion medium (e.g. protocols for UCMSC isolation frequently seed explants in α-MEM/10% FBS). Because α-MEM has no built-in supplements to substitute for serum, it typically requires high FBS levels to sustain proliferation [46]. Commercial kits like StemPro™ MSC SFM and MesenCult™-ACF provide defined, serum-free formulations. These media are formulated for high-density MSC expansion without FBS (often requiring specialized coatings or growth factor mixes). In cell expansion, StemPro can rival or exceed the proliferation rate of FBS-based cultures. However, recent work shows that MSCs grown in completely serum-free media can exhibit subtle shifts in phenotype. For example, Kang et al. (2024) found that MSCs expanded in StemPro™ and MesenCult™ differed in morphology, surface-marker expression and differentiation capacity compared to standard FBS-cultured MSCs. Thus, while serum-free media are excellent for eliminating animal components (a major translational goal), researchers must verify that key MSC properties (immunophenotype, multipotency) are retained when switching from serum-rich to synthetic media [ 47 ]. The basal formulation of Advanced DMEM/F12 is enriched with extra nutrients, antioxidants and lacks components that degrade. This formulation has been optimized so that MSCs maintain rapid growth with far lower serum. FBS is a potent mitogen source: it supplies hormones, growth factors (e.g. PDGF, FGF, TGF-β), attachment proteins (fibronectin/vitronectin), and binding proteins that drive MSC proliferation [ 48 ]. High serum levels (10% or more) strongly enhance MSC expansion rates and attachment. At the same time, FBS contains differentiation signals (e.g. insulin, BMPs) that can push MSCs towards lineage commitment [ 48 ]. Thus, s erum concentration is a double-edged sword: more serum induces faster growth but also elevates the risk of spontaneous differentiation or loss of stemness. In many protocols the early culture uses 10% FBS to boost cell yields, then the serum is lowered to maintain an immature phenotype. For example, in our protocol hUC-MSCs are first grown in 10% FBS for several days to drive proliferation, then switched to 2% FBS to hold the cells in a quiescent, undifferentiated state. The key advantage of Advanced DMEM/F12 is its ability to decouple proliferation from excessive serum use. Because it contains extra nutrients and buffering, MSCs can thrive even at 2% FBS. This reduces cost and variability (fewer FBS lots) and minimizes the serum-driven differentiation signals. Other media, like α-MEM, usually lack such supplements and thus require 10–20% FBS throughout, potentially leading to higher spontaneous differentiation [ 46 ]. StemPro (serum-free) eliminates FBS entirely, but at the expense of requiring specialized conditions (e.g. coating, growth factor supplements) and with reports of altered MSC properties [ 47 ]. By contrast, Advanced DMEM/F12 strikes a balance: robust growth with minimal serum, improving consistency for translational use. For clinical applications, minimizing animal-derived components is critical to safety and reproducibility. High-quality, low-serum media reduce risks of xeno-contaminants and immune reactions. Regulatory guidelines increasingly favor xeno-free or low-serum expansion systems [ 47 ]. Our protocol reflects this: after initial expansion with 10% FBS to maximize yield, MSCs are maintained at 2% FBS in Advanced DMEM/F12. This approach takes advantage of the proliferation boost from FBS when needed, then leans on the defined nature of Advanced DMEM/F12 to stabilize the culture. The Mesenchymal Stromal Cell Committee of the International Society for Cellular Therapy proposed three minimal criteria for defining human MSCs: MSCs must be plastic-adherent when maintained under standard culture conditions; MSCs must express CD105, CD73, and CD90 and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules; MSCs must be able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro [ 24 ]. CD73, also known as 5'-nucleotidase, is a cell surface enzyme that plays a key role in purinergic signaling by converting extracellular AMP to adenosine. Adenosine exhibits anti-inflammatory and immunomodulatory effects in various tissues. High expression of CD73 on MSCs is considered an indicator of their ability to modulate immune responses and suppress inflammation. This makes MSCs expressing CD73 attractive candidates for cell therapy, especially for inflammatory diseases [ 49 ]. CD90, or Thy-1, is a glycoprotein expressed on MSCs, fibroblasts, neurons, and certain other cell types. In MSCs, CD90 is associated with cell adhesion, migration, and regulation of differentiation. It also plays a role in interacting with the extracellular matrix, which is essential for tissue regeneration. Its expression in MSCs indicates the cells retain their stemness, including self-renewal and adhesion capacity. CD90 also plays a role in modulating immune responses [ 50 ]. CD105, also known as endoglin, is a glycoprotein that functions as part of the TGF-β receptor complex. It is highly expressed on the surface of MSCs and plays a role in angiogenesis. The presence of CD105 in MSCs suggests that these cells may have pro-angiogenic properties, making them potentially useful for promoting blood flow during wound healing or under ischemic conditions. Additionally, CD105’s involvement in the TGF-β signaling pathway links it to MSCs’ immunomodulatory and anti-inflammatory effects [ 51 ]. CD34 is a transmembrane protein typically found in hematopoietic stem and progenitor cells, endothelial progenitor cells, and mature cells of hematopoietic origin. For MSC characterization, the absence of CD34 is crucial as it distinguishes MSCs from hematopoietic stem cells and other cells of hematopoietic lineage. Confirmation that our MSCs are CD34-negative provides assurance that the cells indeed belong to the mesenchymal and not hematopoietic lineage [ 52 ]. CD45 is a protein tyrosine phosphatase expressed on all nucleated hematopoietic cells, including lymphocytes, granulocytes, and monocytes. The absence of CD45 in MSCs is another key criterion in MSC immunophenotyping, helping to distinguish them from immune cells. In our study, MSCs showed no CD45 expression, further confirming their non-hematopoietic identity. Ensuring that MSCs are CD45-negative is especially important for potential therapeutic applications, as it confirms that the cells do not have immune cell characteristics and, therefore, should not trigger an immune or inflammatory response upon administration, including graft-versus-host disease [ 53 ]. The immunophenotypic profile of our cells demonstrates that they possess the required markers of mesenchymal stem cells, confirming their suitability for therapeutic use. The successful differentiation of our MSCs into adipogenic, osteogenic, and chondrogenic lineages confirms their multipotency and supports their potential use in regenerative medicine and tissue engineering. The ability to differentiate into various cell types allows for the development of cell-based therapies for conditions such as osteoarthritis [ 54 – 55 ]. The genomic integrity of MSCs is a critical determinant of their safety and utility in translational and clinical applications. In this study, we assessed genomic stability of hUC-MSCs through karyotyping and micronucleus testing over multiple passages, from P2 through P10. Our findings indicate that hUC-MSCs maintained a normal diploid karyotype without detectable aneuploidy at all passages examined. While the proportion of polyploid cells increased slightly (from 2.5% at P2 to 10.5% at P10), this change remained within ranges tolerable for MSC cultures. Polyploidy, while not synonymous with malignancy, may reflect adaptation to in vitro stress or replicative aging. Importantly, no structural abnormalities or clonal chromosomal alterations were observed, suggesting that the polyploid events were stochastic rather than indicative of genomic transformation. The micronucleus assay, a sensitive method for detecting chromosomal fragments or missegregated chromosomes, further supported these findings. No micronucleated cells were detected at P2–P6, and only a very low frequency was observed at later passages (0.07% at P8 and 0.16% at P10). This minimal increase suggests that chromosomal instability was rare and not progressive within the passage range analyzed. The gradual decline in mitotic index over passages (1.3% at P2 to 0.6% at P10) reflects reduced proliferative capacity, which is expected as MSCs approach senescence. Taken together, these findings support the cytogenetic safety of hUC-MSC s up to passage 10 when cultured under serum-reduced, defined conditions using Advanced DMEM/F12 medium. This stability, combined with their immunomodulatory and regenerative capabilities, reinforces the suitability of hUC-MSCs for clinical expansion and therapeutic use. However, given the slight rise in polyploidy and micronuclei, ongoing monitoring beyond P10 is recommended to ensure long-term safety in manufacturing pipelines. The study of MSC genomic stability is gaining importance due to the active development of cell technologies and their potential applications in various medical fields. During passaging and increased culture time, MSCs may acquire de novo chromosomal alterations that can lead to abnormal cell lines. Chromosome changes can result in genomic disorders, adversely affecting the cell and potentially leading to tumor transformation [ 56 – 58 ]. Researchers believe that cytogenetic abnormalities inevitably accumulate in cell cultures, as ex vivo cells lack the immune system’s surveillance that regularly identifies and eliminates mutated cells in the body. Spontaneous mutations that occur both in vivo and in vitro can give rise to clones of transformed cells. Cytogenetic analysis of cell lines at different passages allows for monitoring the emergence of genetically altered cells to ensure the safety of the obtained material. [ 56 – 59 ]. The results of karyotype stability studies of long-term cultured MSCs remain controversial. While studying various genomic anomalies in cultured stem cells, several researchers have found aneuploidy to be one of the most significant genome alterations [ 60 – 61 ]. The International Society for Cell and Gene Therapy (ISCT) has proposed that the presence of two identical abnormal metaphases in 20 analyzed cells (10%) constitutes a threshold for the exclusion of MSCs from clinical use. Robb KP, Fitzgerald JC, Barry F, Viswanathan S. Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency [ 62 ]. In our study, none of the evaluated metaphases exceeded this threshold, confirming the absence of clonal chromosomal abnormalities and supporting the cytogenetic safety of the cultured cell lines. Our findings are consistent with those of Stultz et al. (2016), who demonstrated that while some chromosomal instability may emerge in culture, the overall frequency of aberrations did not increase between passages P2 and P4. Interestingly, in some cases, they observed a decrease in abnormalities in later passages, possibly due to the selective elimination of genetically unstable cells through mechanisms such as apoptosis or senescence. Similarly, Algorta et al. (2024) reported that polyploidy—often considered a non-clonal and transient numerical alteration—was the most common abnormality in MSC cultures but tended to diminish over successive passages, reinforcing the hypothesis that MSCs gradually adapt to culture conditions, potentially losing abnormal karyotypes over time [ 23 ]. Recent studies consistently report that hUC-MSCs retain a normal karyotype and show no evidence of genotoxicity through at least 10 passages of culture. For example, Huang et al. (2024) cultured hUC-MSCs for 30 generations under hypoxia versus normoxia and found normal diploid karyotypes in all samples; the hypoxic MSCs maintained normal karyotype and stemness even at late passages [ 64 ]. Rare chromosomal changes in hUC-MSCs have been reported only after very long culture (e.g. trisomy 10 at P30 in one clone) – well beyond typical expansion for therapy [ 65 ]. In contrast, no signal of transformation is seen: studies in mice report no tumor formation after transplanting cultured hUC-MSCs. These findings support the conclusion that properly cultured hUC-MSCs up to P10 pose minimal genomic risk [ 65 ]. Rebelatto et al. (2023) tested hUC-MSC batches (up to P5) with cytokinesis-block micronucleus (CBMN) test and soft-agar assays and found no micronuclei or chromosomal anomalies – the cell cultures were not genotoxic and produced zero micronuclei per 1000 cells [ 66 ]. When compared to other MSC sources, hUC-MSCs appear at least as stable, if not more so, under identical culture conditions. For example, a recent Brazilian study of adipose-derived MSCs found a significant rise in DNA damage by passage 5 and a sharp increase in micronucleus frequency from P7 onward [ 67 ]. By contrast, UC-MSCs showed no such increase (micronuclei remained at zero in Rebelatto’s CBMN assay) [ 66 ]. This suggests a source-specific difference: neonatal hUC-MSCs (with longer telomeres and higher telomerase) generally tolerate expansion better than adult MSCs due to their primitive source [ 68 – 69 ]. Overall, our results confirm the mesenchymal nature, multipotency of the derived hUC-MSC line, and its safety for further preclinical studies and clinical applications. Conclusions Thus, hUC-MSCs demonstrate promising differentiation potential and immunophenotypic characteristics, making them suitable candidates for regenerative medicine. The cells express standard stem cell surface markers (CD44+, CD90+, CD105+, CD34−, CD45−) as well as a maintain diploid karyotype. Their ability to differentiate into multiple lineages and their genetic stability in early passages make them a valuable resource for cell therapies in various clinical applications. Abbreviations MSCs – mesenchymal stem cells UC-MSCs - umbilical cord MSCs hUC-MSC –human umbilical cord MSCs DMEM/F12 – Dulbecco's Modified Eagle Medium/Ham's F-12 FBS – fetal bovine serum MHC – major histocompatibility complex SSC – side scatter Declarations Ethics approval and consent to participate The study received ethical approval from the Bioethics Commission of Ternopil National Medical University, as indicated in protocol #60, dated 01.09.2020. Consent for publication Not applicable Availability of data and materials The dataset analysed during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors declare that they have no potential conflicts of interest to disclose. Funding statement The study was supported by the Ministry of Health of Ukraine as a part of the state-funded research works "Investigation of the regenerative potential of cellular therapy agents in acute respiratory distress syndrome" (2021-2023, state registration number 0121U100159) and "The use of regenerative medicine technologies for the chronic wounds treatment" (2024-2026, state registration number 0124U001099). Author Contributions Alina Dovgalyuk 1 , Olesia Redko 1 , Ilona Palii 1 , Sophia Lechachenko 1 , Halyna Lavrenchuk 2 , Vitalii Kyryk 3 , Mykhaylo Korda 1 Alina Dovgalyuk (AD) , Olesia Redko (OR), Ilona Palii (IP), Sophia Lechachenko (SL), Halyna Lavrenchuk (HL), Vitalii Kyryk (VK), Mykhaylo Korda (MK) Conceptualization: AD, MK Data curation: AD, OR, IP, SL Formal analysis: AD, OR, IP, SL Funding acquisition: AD, MK Investigation: AD, OR, IP, Methodology: AD Project administration: AD, MK Resources: HL, VK, MK Supervision: AD, MK Validation: AD, HL, VK Visualization: AD, OR, IP, Writing – original draft: AD, OR, SL Writing – review & editing: AD, OR, SL Approval of final manuscript: all authors Acknowledgements Not applicable References Redko O, Dovgalyuk A, Dovbush A, Nebesna Z, Yakubyshyna L, Krynytska I. Liver injury associated with acute respiratory distress syndrome and the prospects of mesenchymal stromal cells therapy for liver failure. 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Long-term hypoxic atmosphere enhances the stemness, immunoregulatory functions, and therapeutic application of human umbilical cord mesenchymal stem cells. Bone Joint Res. 2024;13(12):763–77. 10.1302/2046-3758.1312.BJR-2024-0136.R2 . Wang, Y., Zhang, Z., Chi, Y., Zhang, Q., Xu, F., Yang, Z., … Han, Z. C. (2013). Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death & Disease, 4(12), e950-e950. DOI: 10.1038/cddis.2013.480 Rebelatto, C. L. K., Boldrini-Leite, L. M., Daga, D. R., Marsaro, D. B., Vaz, I. M.,Jamur, V. R., … Brofman, P. R. S. (2023). Quality control optimization for minimizing security risks associated with mesenchymal stromal cell-based product development.International Journal of Molecular Sciences, 24(16), 12955. DOI: 10.3390/ijms241612955. Malagutti-Ferreira MJ, Crispim BA, Barufatti A, Cardoso SS, Guarnier LP, Rodríguez FF, Ribeiro-Paes JT. Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells. Braz J Med Biol Res. 2023;56:e12713. 10.1590/1414-431X2023e12713 . Neri S. Genetic stability of mesenchymal stromal cells for regenerative medicine applications: a fundamental biosafety aspect. Int J Mol Sci. 2019;20(10):2406. 10.3390/ijms20102406 . Rajput SN, Naeem BK, Ali A, Salim A, Khan I. Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine. World J Stem Cells. 2024;16(4):410. 10.4252/wjsc.v16.i4.410 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6938731","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":476191661,"identity":"85e554f7-e18a-4fb1-80e4-764f8c084fab","order_by":0,"name":"Alina Dovgalyuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDCCAyCigIFBgr2BQQIhQlCLAVALz2GStUgkE6mF73iP8YsfBocTZ858f/DWzTYGOb4bCWwSP/BokTxzxsyyB6hltnQys3VuG4OxJFCLZA8eLQY30tIMeIBa5kkns0kDtSRuANnCQ0CL4R+QFsnDYC31IC2Sf/BqST78GGTLbAlmsJYEA6AWaXy2SJ45fIxZxiDdeGZPsrF1zjkJw5lnHjZby+DRwne8sfnjmwpr2RnHDz68nVNmI893PPngzTd4tAABGzA6mmEcUNQwNuDXwMDA/IGBoY6QolEwCkbBKBjJAAAXHFEcstAdKQAAAABJRU5ErkJggg==","orcid":"","institution":"I.Ya.Horbachevsky Ternopil National Medical University of the Ukrainian Ministry of Health","correspondingAuthor":true,"prefix":"","firstName":"Alina","middleName":"","lastName":"Dovgalyuk","suffix":""},{"id":476191667,"identity":"ce583b79-92d6-4d13-a468-c37a11ade076","order_by":1,"name":"Olesia Redko","email":"","orcid":"","institution":"I.Ya.Horbachevsky Ternopil National Medical University of the Ukrainian Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Olesia","middleName":"","lastName":"Redko","suffix":""},{"id":476191668,"identity":"aa6a4514-83c6-4535-bb45-e17c999e7065","order_by":2,"name":"Ilona Palii","email":"","orcid":"","institution":"I.Ya.Horbachevsky Ternopil National Medical University of the Ukrainian Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Ilona","middleName":"","lastName":"Palii","suffix":""},{"id":476191669,"identity":"15b1043b-d0e5-4173-beae-d909355d2579","order_by":3,"name":"Sophia Lechachenko","email":"","orcid":"","institution":"I.Ya.Horbachevsky Ternopil National Medical University of the Ukrainian Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Lechachenko","suffix":""},{"id":476191670,"identity":"12d4b667-482a-4d79-9d4f-50e1301d208b","order_by":4,"name":"Halyna Lavrenchuk","email":"","orcid":"","institution":"State Institution \"National Research Center for Radiation Medicine of National Academy of Medical Sciences of Ukraine\". Yuriia Illienka St","correspondingAuthor":false,"prefix":"","firstName":"Halyna","middleName":"","lastName":"Lavrenchuk","suffix":""},{"id":476191671,"identity":"6c34ecbd-33bd-4f47-aa2d-44798b148bd3","order_by":5,"name":"Vitalii Kyryk","email":"","orcid":"","institution":"M. D. Strazhesko National Scientific Center of Cardiology, National Academy of Medical Sciences of Ukraine","correspondingAuthor":false,"prefix":"","firstName":"Vitalii","middleName":"","lastName":"Kyryk","suffix":""},{"id":476191672,"identity":"fb28a580-010f-4b31-a573-767e1f99d45e","order_by":6,"name":"Mykhaylo Korda","email":"","orcid":"","institution":"I.Ya.Horbachevsky Ternopil National Medical University of the Ukrainian Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Mykhaylo","middleName":"","lastName":"Korda","suffix":""}],"badges":[],"createdAt":"2025-06-20 12:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6938731/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6938731/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85567487,"identity":"3e4458fa-21aa-4e46-90c8-ed0bdfb4b256","added_by":"auto","created_at":"2025-06-27 15:08:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1704708,"visible":true,"origin":"","legend":"\u003cp\u003eA–F. Morphological characteristics of hUC-MSCs at 100× magnification.\u003c/p\u003e\n\u003cp\u003e(A) hUC-MSCs migrating out from an umbilical cord tissue explant during initial culture establishment (P0). (B–F) hUC-MSCs at successive passages (P1, 2, 5, 7 and 10 respectively), displaying typical fibroblast-like morphology consistent with mesenchymal lineage. Cells remained adherent and morphologically stable across passages, with no signs of spontaneous differentiation or abnormal growth patterns.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6938731/v1/e088d0a6ce34f2e82f12367d.png"},{"id":85568092,"identity":"37531bf7-5e1e-4c12-8b42-22f123ddab7e","added_by":"auto","created_at":"2025-06-27 15:16:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1218261,"visible":true,"origin":"","legend":"\u003cp\u003eProofsof adipogenic (A), osteogenic (B) and chondrogenic © differentiation of MSCs. (A) Accumulation of lipid droplets typical of mature adipocytes is visualized in brown. Oil Red O staining. (B) Matrix mineralization. Calcium deposits are visualized in red. Alizarin Red S staining. (C)Formation of a chondrogenic pellet. Glycosaminoglycans of the extracellular matrix are visualized in blue. Alcian Blue staining.\u003c/p\u003e\n\u003cp\u003e100× magnification.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6938731/v1/99e572bf7032cc9b65a6cc6c.png"},{"id":85567491,"identity":"bba3a31f-4eaf-41db-bf08-e730086a27d6","added_by":"auto","created_at":"2025-06-27 15:08:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180747,"visible":true,"origin":"","legend":"\u003cp\u003eDot-plot histograms showing the expression of CD90, CD73, CD105, CD45, CD34, and HLA-DR markers on human umbilical cord-derived MSCs.. Flow cytometry was performed using BD FACSAria cell sorter with BD FACSDiva software v.6.1.3\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6938731/v1/77d0432229c42d031cb12a13.png"},{"id":85567496,"identity":"0ae82b67-5c48-4c04-96ea-08cd963b8164","added_by":"auto","created_at":"2025-06-27 15:08:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230695,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotograph of a metaphase spread from a human umbilical cord cell culture. Giemsa staining. 1000× magnification\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6938731/v1/37e3879fdcbbc16851b8584c.png"},{"id":86148077,"identity":"78175501-467e-477a-8cbd-55b03469ffd1","added_by":"auto","created_at":"2025-07-07 09:32:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3889934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6938731/v1/ab70629f-d6de-4176-8e5a-d9e3b6df06d6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Obtaining Human Umbilical Cord-Derived Mesenchymal Stem Cells and Cell Line Characterization: Immunophenotype, Multipotency and Cytogenetic Stability","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eMesenchymal stem cells (MSCs) are currently widely used in regenerative medicine. They are considered the most promising agents for the renewal of damaged cells and tissues due to their unique properties, including chemotaxis toward sites of inflammation, ability to differentiate into various cell lineages as well as to influence the regeneration of injured organs in a paracrine manner [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MSCs can be easily isolated from various sources, such as bone marrow, umbilical cord, adipose tissue, and dental pulp and perinatal derivatives including placenta, amnion and umbilical cord [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman umbilical cord-derived MSCs (hUC-MSCs) present a good alternative to adult MSCs, as they can be obtained through a non-invasive method and are non-immunogenic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Unlike embryonic and induced pluripotent stem cells, hUC-MSCs do not exhibit tumorigenicity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers have shown that hUC-MSCs produce various biologically active substances, including cytokines, growth factors, heat-shock proteins, miRNA, defensin, etc [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The presence of these molecules in their secretome and exosomes causes angiogenic, anti-apoptotic, antioxidant, antibacterial and immunomodulating effects of hUC-MSCs [1; 9\u0026ndash;11].\u003c/p\u003e \u003cp\u003eHUC-MSCs due to their multipotency are able to differentiate into various cell types including not only cells of mesodermal lineage such as chondrocytes, osteocytes, and adipocytes [5; 12\u0026ndash;13], but also cells both ectodermal (neural) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and endodermal (hepatocytes) lineages [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], making them ideal candidates for tissue engineering and cell therapies.\u003c/p\u003e \u003cp\u003eVarious protocols have been developed for the isolation and cultivation of hUC-MSCs, differing in the segment of the cord used (Wharton's jelly, perivascular region, or whole cord), enzymatic digestion methods, culture media composition, and passaging techniques. These variations influence the yield, proliferation rate, and biological characteristics of the resulting MSCs [2; 7\u0026ndash;8; 12; 18\u0026ndash;21].\u003c/p\u003e \u003cp\u003eIn this study, we aimed to establish a human umbilical cord-derived MSC cell line and characterize their immunophenotype, differentiation potential, and chromosomal stability during the first 10 passages of cultivation. We present our optimized protocol for isolating and culturing hUC-MSCs, highlighting the procedures that ensured high cell viability, stable phenotype, and differentiation capacity. Our approach contributes to the standardization of methods suitable for both research and therapeutic applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe umbilical cord was obtained under aseptic conditions from a female newborn with informed consent of her mother after a full-term pregnancy that proceeded without complications and ended with a cesarean section. For transporting the biological material to the laboratory, the umbilical cord was placed in a transport medium containing 200 mg of ciprofloxacin, 10,000 units of penicillin, 10,000 \u0026micro;g of streptomycin, and 25 \u0026micro;g of Gibco Amphotericin B, diluted in 100 ml of PBS and immediately delivered to the lab at 4 \u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor MSCs isolation, only 5\u0026ndash;7 cm of the umbilical cord from the placental side was used. Under sterile conditions, the umbilical cord was cut into small fragments measuring 0.5\u0026ndash;2 mm\u0026sup3;, transferred into centrifuge tubes, and subjected to enzymatic digestion using 0.1% collagenase I (Sigma-Aldrich, USA), diluted in 2 ml of DMEM/F12 Advanced culture medium (Gibco, USA), avoiding flotation.\u003c/p\u003e \u003cp\u003eThe enzymatic digestion was carried out for 1 hour at 37\u0026deg;C in a water bath, with thorough mixing of the tube contents every 15 minutes. Afterwards the resulting suspension was pipetted and centrifuged for 5 minutes at 1610\u0026times;g. The pellet which included partially digested tissue pieces (explants) was resuspended in 7 ml of Advanced DMEM/F12 enriched with 10% fetal bovine serum (FBS) (Gibco, USA), seeded into culture flasks with a surface area of 25 cm\u0026sup2; containing Advanced DMEM/F12 medium supplemented with 10% FBS, 1% L-Glutamine-Penicillin-Streptomycin solution (Sigma, USA) and 240 \u0026micro;g/L Heparin solution (Sigma, USA) and kept inside incubators at 37\u0026deg;C and 5% CO₂.\u003c/p\u003e \u003cp\u003eThe resulting primary culture was assigned passage zero (P0). The first visual assessment of cell morphology and population density was performed using an inverted microscope (Delta Optical NIB-100) 48 hours after seeding to allow cells to spread out and attach themselves to the bottom of the flasks.\u003c/p\u003e \u003cp\u003eTo remove dead or damaged cells and unattached blood elements, the culture medium was completely replaced 72 hours after the cultivation start, maintaining the FBS concentration at 10%.\u003c/p\u003e \u003cp\u003eFurther analysis of the primary hUC-MSC culture was conducted 5 days after seeding. At this stage of cultivation, cell population density (confluence) was approximately 50%. At this point, the growth medium was changed again, reducing the FBS concentration to 2% and preserving the previous concentrations of L-Glutamine-Penicillin-Streptomycin Heparin supplements. Following this, every 3 days a complete medium change or replacement of \u0026frac12; or \u0026frac14; volume of medium was performed while maintaining the FBS concentration.\u003c/p\u003e \u003cp\u003eUpon reaching 80% confluence, passaging was performed by trypsinization using TrypLE Express Enzyme (Gibco, USA). The flask bottoms were rinsed with HBSS solution, TrypLE enzyme was added, and the flasks were placed in a CO₂ incubator for 5 minutes to detach the cells from the culture plastic surface. The enzyme was neutralized using conditioned medium. The collected cell suspension was centrifuged for 8 minutes at 530\u0026times;g, after which the cells were seeded into new flasks at a density of 50,000 cells per 1 ml of medium, and a new passage was assigned. Cell counting in the suspension was performed using a hemocytometer after staining with the vital dye trypan blue.\u003c/p\u003e \u003cp\u003eCryopreservation of the cells was performed at various passages. 1 ml of a cell suspension with a concentration of 2\u0026ndash;4\u0026nbsp;million cells per ml was added into 2 ml cryovials, followed by the addition of 1 ml of the prepared freezing medium (30% DMEM/F12 Advanced, 40% FBS, 20% conditioned medium, and 10% dimethyl sulfoxide (Sigma, USA)). After filling, the cryovials were immediately subjected to gradual cooling at a rate of 1\u0026deg;C per minute using a ZPM-1 programmable mobile freezer (Kharkiv, Ukraine). Samples were then stored in liquid nitrogen at -196\u0026deg;C.\u003c/p\u003e \u003cp\u003eAfter thawing, MSCs were cultured for 24 hours in a growth medium consisting of Advanced DMEM/F12 supplemented with 10% FBS, 1% L-glutamine\u0026ndash;penicillin\u0026ndash;streptomycin solution, and 240 \u0026micro;g/L heparin solution. Afterward, the concentration of FBS was reduced to 2%.\u003c/p\u003e \u003cp\u003eMultipotency of cultured cells was assessed at passage 8. Cells were seeded into three 6-well plates with the addition of differentiation media: StemPro Chondrogenesis Differentiation Kit, StemPro Adipogenesis Differentiation Kit, and StemPro Osteogenesis Differentiation Kit (Gibco, USA). After incubation under protocol-specified conditions (18 days for chondrogenesis, 14 days for adipogenesis, and 21 days for osteogenesis), staining with Alcian Blue, Oil Red, and Alizarin Red S was performed respectively.\u003c/p\u003e \u003cp\u003eTo assess the genetic stability of the cell line cytogenetic analysis was performed at passages 2, 4, 6, 8, 10. Chromosome preparations were obtained using a modified standard protocol [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The procedure was conducted on days 3 or 4 after passaging when the cell population reached the logarithmic growth phase. After incubating cells for 3 hours in colchicine at a concentration of 1\u0026times;10⁻⁷ M at 37\u0026deg;C, trypsinization was performed for 10 minutes in a CO₂ incubator at 37\u0026deg;C. After enzyme neutralization, cells were treated with warm hypotonic KCl solution (0.07 M) and incubated for another 30 minutes. Afterwards cells were treated with a standard 1:3 acetic acid:methanol chromosome fixative and left on melting ice for 10 minutes. The fixative was replaced twice, and the cell suspension was dropped onto pre-freezed damp slides. Samples were stained using Giemsa Stain (Merck, Germany) for 25 minutes and analyzed using a Nikon Eclipse Ci-E microscope (Japan).\u003c/p\u003e \u003cp\u003eIn the obtained samples, numerical chromosomal abnormalities were identified, including aneuploidy and polyploidy, and the number of cells with micronuclei and the mitotic index were calculated. The frequency was determined based on 500 cells per each slide (expressed as a percentage). For each passage 6 slides were analysed.\u003c/p\u003e \u003cp\u003eFor the analysis of MSC-specific cell surface marker expression cells cryopreserved at passage 5 and subsequently thawed were used. Immunophenotyping was performed by flow cytometry using mouse anti-human monoclonal antibodies CD90 FITC, CD73 PE, CD105 PerCP-Cy5.5, CD45 FITC, CD34 PE, and HLA-DR APC-Cy7 (all from BD Biosciences, USA). To compensate for spectral overlap of fluorochromes in multicolor analysis, control samples were used: unstained, single-stained, and fluorescence minus one (FMO) controls. A total of 1\u0026times;10⁶ thawed and washed cells were resuspended in 100 \u0026micro;L of DPBS supplemented with 1% FBS, incubated with antibodies for 30 minutes at +\u0026thinsp;4\u0026deg;C in the dark, then washed with 1 mL of CellWash buffer (BD Biosciences, USA) by centrifugation at 300 \u0026times;g for 5 minutes, and finally resuspended in 350 \u0026micro;L of DPBS for analysis. Immediately before acquisition, the cell suspension was filtered through a 70 \u0026micro;m cell strainer to remove aggregates. Measurements were performed on a BD FACSAria cell sorter using BD FACSDiva software v.6.1.3. At least 30,000 events were recorded per sample for analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMicroscopic analysis of the primary cell lines derived from the umbilical cord performed 48 hours after seeding the cell suspension into culture flasks revealed a small amount of cells exhibiting typical fibroblast-like morphology and spreading out of partially digested tissue explants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eCell population density was less than 10%, and a shift in the medium\u0026apos;s pH towards acidity was observed (phenol red, a pH indicator and an essential component of Advanced DMEM/F12, turned orange).\u003c/p\u003e\n\u003cp\u003eTo confirm the multipotency of the obtained cell line, adipogenic, osteogenic, and chondrogenic differentiation of MSC was performed. The differentiated cells exhibited morphology and expression of markers associated with each phenotype.\u003c/p\u003e\n\u003cp\u003eAdipogenic differentiation was confirmed by the accumulation of lipid droplets typical of mature adipocytes, which were visualized as brown by Oil Red O staining (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eThe osteogenically differentiated cell culture demonstrated signs of matrix mineralization\u0026mdash; the presence of calcium salt deposits confirmed by staining with Alizarin Red S (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eChondrogenic differentiation was assessed by the presence of secreted glycosaminoglycans in the extracellular matrix and formation of a chondrogenic pellet, which was stained with Alcian Blue (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eFlow cytometric immunophenotyping revealed a high expression level of CD90, CD73, and CD105 markers (greater than 99.1%), with no expression of the hematopoietic markers CD45, CD34, or the major histocompatibility complex (MHC) class II marker HLA-DR. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This immunophenotypic profile is consistent with the characteristics of mesenchymal stem cells as defined by the International Society for Cellular Therapy [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eCytogenetic analysis of MSCs revealed a normal diploid karyotype (46 chromosomes) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eKaryotyping and Micronucleus Test Results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePassage Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAnalysed Metaphases, Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Mitotic Index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAneuploid Cells, Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolyploid Cells, Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Cells with\u003c/p\u003e\n \u003cp\u003eMicronuclei\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (3.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (4.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (10.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCytogenetic analyses including classical karyotyping and micronucleus testing were conducted to assess genomic stability. At all passages tested (P2, P4, P6, P8, and P10), no aneuploid cells were detected among the metaphases analyzed, indicating the absence of chromosomal gain or loss. A small number of polyploid cells were observed beginning at P2 (1 polyploid cell, 2.5%) and increased slightly with passage number, reaching 10.5% at P10. The overall mitotic index gradually declined over time, from 1.3% at P2 to 0.6% at P10, consistent with reduced proliferative activity during long-term culture. Notably, micronuclei were absent at early passages (P2\u0026ndash;P6) and only began to appear at very low frequencies at P8 (0.07%) and P10 (0.16%), suggesting a minimal increase in genomic stress or DNA damage with prolonged in vitro expansion (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe human umbilical cord is a promising source of stem cells.\u003c/p\u003e \u003cp\u003eUmbilical cord tissue is usually discarded after birth, and its collection does not require any invasive procedures, unlike bone marrow extraction, and does not raise the ethical concerns associated with the use of human embryonic stem cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. UC-MSCs exhibit low immunogenicity, possess immunosuppressive functions, and can inhibit T-cell proliferation [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe differentiation potential of UC-MSCs allows them to transform into cells derived from all three germ layers under appropriate conditions. UC-MSCs express low levels of class I MHC molecules and do not express class II MHC molecules, making them suitable for allogeneic and xenogeneic transplantation [28; 32].\u003c/p\u003e \u003cp\u003eAnimal studies have shown that hUC-MSCs may be beneficial in treating Parkinson\u0026rsquo;s disease, spinal cord injuries, certain types of cancer, and tissue fibrosis. Their therapeutic action may be linked either to the differentiation of MSCs into cells of damaged tissues or to their paracrine effects [\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClinical trials in humans have demonstrated that UC-MSCs improved treatment outcomes in certain immunological and neurological disorders without signs of tumor formation or immune rejection. Thus, UC-MSCs have great potential for regenerative medicine [21; 43].\u003c/p\u003e \u003cp\u003eNumerous studies have shown that UC-MSCs offer advantages over MSCs from other sources. UC-MSCs exhibit higher proliferation rates and greater expansion potential compared to bone marrow MSCs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, UC-MSCs can be considered an ideal source of cells for cell therapy compared to bone marrow MSCs and embryonic stem cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent cell culture media are used for culturing hUC-MSCs. Alpha-MEM (α-MEM) is a more basic medium (with nonessential amino acids, nucleosides, vitamins, etc.) but without added growth factors. Laboratories often supplement α-MEM with 10\u0026ndash;20% FBS to culture MSCs. In many protocols α-MEM\u0026thinsp;+\u0026thinsp;10% FBS is the routine expansion medium (e.g. protocols for UCMSC isolation frequently seed explants in α-MEM/10% FBS). Because α-MEM has no built-in supplements to substitute for serum, it typically requires high FBS levels to sustain proliferation [46].\u003c/p\u003e \u003cp\u003eCommercial kits like StemPro\u0026trade; MSC SFM and MesenCult\u0026trade;-ACF provide defined, serum-free formulations. These media are formulated for high-density MSC expansion without FBS (often requiring specialized coatings or growth factor mixes). In cell expansion, StemPro can rival or exceed the proliferation rate of FBS-based cultures. However, recent work shows that MSCs grown in completely serum-free media can exhibit subtle shifts in phenotype. For example, Kang \u003cem\u003eet al.\u003c/em\u003e (2024) found that MSCs expanded in StemPro\u0026trade; and MesenCult\u0026trade; differed in morphology, surface-marker expression and differentiation capacity compared to standard FBS-cultured MSCs. Thus, while serum-free media are excellent for eliminating animal components (a major translational goal), researchers must verify that key MSC properties (immunophenotype, multipotency) are retained when switching from serum-rich to synthetic media [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe basal formulation of Advanced DMEM/F12 is enriched with extra nutrients, antioxidants and lacks components that degrade. This formulation has been optimized so that MSCs maintain rapid growth with far lower serum.\u003c/p\u003e \u003cp\u003eFBS is a potent mitogen source: it supplies hormones, growth factors (e.g. PDGF, FGF, TGF-β), attachment proteins (fibronectin/vitronectin), and binding proteins that drive MSC proliferation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. High serum levels (10% or more) strongly enhance MSC expansion rates and attachment. At the same time, FBS contains differentiation signals (e.g. insulin, BMPs) that can push MSCs towards lineage commitment [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, \u003cb\u003es\u003c/b\u003eerum concentration is a double-edged sword: more serum induces faster growth but also elevates the risk of spontaneous differentiation or loss of stemness. In many protocols the early culture uses 10% FBS to boost cell yields, then the serum is lowered to maintain an immature phenotype. For example, in our protocol hUC-MSCs are first grown in 10% FBS for several days to drive proliferation, then switched to 2% FBS to hold the cells in a quiescent, undifferentiated state.\u003c/p\u003e \u003cp\u003eThe key advantage of Advanced DMEM/F12 is its ability to decouple proliferation from excessive serum use. Because it contains extra nutrients and buffering, MSCs can thrive even at 2% FBS. This reduces cost and variability (fewer FBS lots) and minimizes the serum-driven differentiation signals. Other media, like α-MEM, usually lack such supplements and thus require 10\u0026ndash;20% FBS throughout, potentially leading to higher spontaneous differentiation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. StemPro (serum-free) eliminates FBS entirely, but at the expense of requiring specialized conditions (e.g. coating, growth factor supplements) and with reports of altered MSC properties [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. By contrast, Advanced DMEM/F12 strikes a balance: robust growth with minimal serum, improving consistency for translational use.\u003c/p\u003e \u003cp\u003eFor clinical applications, minimizing animal-derived components is critical to safety and reproducibility. High-quality, low-serum media reduce risks of xeno-contaminants and immune reactions. Regulatory guidelines increasingly favor xeno-free or low-serum expansion systems [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur protocol reflects this: after initial expansion with 10% FBS to maximize yield, MSCs are maintained at 2% FBS in Advanced DMEM/F12. This approach takes advantage of the proliferation boost from FBS when needed, then leans on the defined nature of Advanced DMEM/F12 to stabilize the culture.\u003c/p\u003e \u003cp\u003eThe Mesenchymal Stromal Cell Committee of the International Society for Cellular Therapy proposed three minimal criteria for defining human MSCs:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMSCs must be plastic-adherent when maintained under standard culture conditions;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMSCs must express CD105, CD73, and CD90 and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMSCs must be able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eCD73, also known as 5'-nucleotidase, is a cell surface enzyme that plays a key role in purinergic signaling by converting extracellular AMP to adenosine. Adenosine exhibits anti-inflammatory and immunomodulatory effects in various tissues. High expression of CD73 on MSCs is considered an indicator of their ability to modulate immune responses and suppress inflammation. This makes MSCs expressing CD73 attractive candidates for cell therapy, especially for inflammatory diseases [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD90, or Thy-1, is a glycoprotein expressed on MSCs, fibroblasts, neurons, and certain other cell types. In MSCs, CD90 is associated with cell adhesion, migration, and regulation of differentiation. It also plays a role in interacting with the extracellular matrix, which is essential for tissue regeneration. Its expression in MSCs indicates the cells retain their stemness, including self-renewal and adhesion capacity. CD90 also plays a role in modulating immune responses [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD105, also known as endoglin, is a glycoprotein that functions as part of the TGF-β receptor complex. It is highly expressed on the surface of MSCs and plays a role in angiogenesis. The presence of CD105 in MSCs suggests that these cells may have pro-angiogenic properties, making them potentially useful for promoting blood flow during wound healing or under ischemic conditions. Additionally, CD105\u0026rsquo;s involvement in the TGF-β signaling pathway links it to MSCs\u0026rsquo; immunomodulatory and anti-inflammatory effects [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD34 is a transmembrane protein typically found in hematopoietic stem and progenitor cells, endothelial progenitor cells, and mature cells of hematopoietic origin. For MSC characterization, the absence of CD34 is crucial as it distinguishes MSCs from hematopoietic stem cells and other cells of hematopoietic lineage. Confirmation that our MSCs are CD34-negative provides assurance that the cells indeed belong to the mesenchymal and not hematopoietic lineage [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD45 is a protein tyrosine phosphatase expressed on all nucleated hematopoietic cells, including lymphocytes, granulocytes, and monocytes. The absence of CD45 in MSCs is another key criterion in MSC immunophenotyping, helping to distinguish them from immune cells. In our study, MSCs showed no CD45 expression, further confirming their non-hematopoietic identity. Ensuring that MSCs are CD45-negative is especially important for potential therapeutic applications, as it confirms that the cells do not have immune cell characteristics and, therefore, should not trigger an immune or inflammatory response upon administration, including graft-versus-host disease [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe immunophenotypic profile of our cells demonstrates that they possess the required markers of mesenchymal stem cells, confirming their suitability for therapeutic use.\u003c/p\u003e \u003cp\u003eThe successful differentiation of our MSCs into adipogenic, osteogenic, and chondrogenic lineages confirms their multipotency and supports their potential use in regenerative medicine and tissue engineering. The ability to differentiate into various cell types allows for the development of cell-based therapies for conditions such as osteoarthritis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genomic integrity of MSCs is a critical determinant of their safety and utility in translational and clinical applications. In this study, we assessed genomic stability of hUC-MSCs through karyotyping and micronucleus testing over multiple passages, from P2 through P10. Our findings indicate that hUC-MSCs maintained a normal diploid karyotype without detectable aneuploidy at all passages examined.\u003c/p\u003e \u003cp\u003eWhile the proportion of polyploid cells increased slightly (from 2.5% at P2 to 10.5% at P10), this change remained within ranges tolerable for MSC cultures. Polyploidy, while not synonymous with malignancy, may reflect adaptation to in vitro stress or replicative aging. Importantly, no structural abnormalities or clonal chromosomal alterations were observed, suggesting that the polyploid events were stochastic rather than indicative of genomic transformation.\u003c/p\u003e \u003cp\u003eThe micronucleus assay, a sensitive method for detecting chromosomal fragments or missegregated chromosomes, further supported these findings. No micronucleated cells were detected at P2\u0026ndash;P6, and only a very low frequency was observed at later passages (0.07% at P8 and 0.16% at P10). This minimal increase suggests that chromosomal instability was rare and not progressive within the passage range analyzed.\u003c/p\u003e \u003cp\u003eThe gradual decline in mitotic index over passages (1.3% at P2 to 0.6% at P10) reflects reduced proliferative capacity, which is expected as MSCs approach senescence.\u003c/p\u003e \u003cp\u003eTaken together, these findings support the cytogenetic safety of hUC-MSC\u003cb\u003es\u003c/b\u003e up to passage 10 when cultured under serum-reduced, defined conditions using Advanced DMEM/F12 medium. This stability, combined with their immunomodulatory and regenerative capabilities, reinforces the suitability of hUC-MSCs for clinical expansion and therapeutic use. However, given the slight rise in polyploidy and micronuclei, ongoing monitoring beyond P10 is recommended to ensure long-term safety in manufacturing pipelines.\u003c/p\u003e \u003cp\u003eThe study of MSC genomic stability is gaining importance due to the active development of cell technologies and their potential applications in various medical fields. During passaging and increased culture time, MSCs may acquire de novo chromosomal alterations that can lead to abnormal cell lines. Chromosome changes can result in genomic disorders, adversely affecting the cell and potentially leading to tumor transformation [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers believe that cytogenetic abnormalities inevitably accumulate in cell cultures, as ex vivo cells lack the immune system\u0026rsquo;s surveillance that regularly identifies and eliminates mutated cells in the body. Spontaneous mutations that occur both in vivo and in vitro can give rise to clones of transformed cells. Cytogenetic analysis of cell lines at different passages allows for monitoring the emergence of genetically altered cells to ensure the safety of the obtained material. [\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of karyotype stability studies of long-term cultured MSCs remain controversial. While studying various genomic anomalies in cultured stem cells, several researchers have found aneuploidy to be one of the most significant genome alterations [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe International Society for Cell and Gene Therapy (ISCT) has proposed that the presence of two identical abnormal metaphases in 20 analyzed cells (10%) constitutes a threshold for the exclusion of MSCs from clinical use.\u003c/p\u003e \u003cp\u003eRobb KP, Fitzgerald JC, Barry F, Viswanathan S. Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, none of the evaluated metaphases exceeded this threshold, confirming the absence of clonal chromosomal abnormalities and supporting the cytogenetic safety of the cultured cell lines.\u003c/p\u003e \u003cp\u003eOur findings are consistent with those of Stultz et al. (2016), who demonstrated that while some chromosomal instability may emerge in culture, the overall frequency of aberrations did not increase between passages P2 and P4. Interestingly, in some cases, they observed a decrease in abnormalities in later passages, possibly due to the selective elimination of genetically unstable cells through mechanisms such as apoptosis or senescence.\u003c/p\u003e \u003cp\u003eSimilarly, Algorta et al. (2024) reported that polyploidy\u0026mdash;often considered a non-clonal and transient numerical alteration\u0026mdash;was the most common abnormality in MSC cultures but tended to diminish over successive passages, reinforcing the hypothesis that MSCs gradually adapt to culture conditions, potentially losing abnormal karyotypes over time [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies consistently report that hUC-MSCs retain a normal karyotype and show no evidence of genotoxicity through at least 10 passages of culture. For example, Huang et al. (2024) cultured hUC-MSCs for 30 generations under hypoxia versus normoxia and found normal diploid karyotypes in all samples; the hypoxic MSCs maintained normal karyotype and stemness even at late passages [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRare chromosomal changes in hUC-MSCs have been reported only after very long culture (e.g. trisomy 10 at P30 in one clone) \u0026ndash; well beyond typical expansion for therapy [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, no signal of transformation is seen: studies in mice report no tumor formation after transplanting cultured hUC-MSCs. These findings support the conclusion that properly cultured hUC-MSCs up to P10 pose minimal genomic risk [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRebelatto et al. (2023) tested hUC-MSC batches (up to P5) with cytokinesis-block micronucleus (CBMN) test and soft-agar assays and found no micronuclei or chromosomal anomalies \u0026ndash; the cell cultures were not genotoxic and produced zero micronuclei per 1000 cells [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen compared to other MSC sources, hUC-MSCs appear at least as stable, if not more so, under identical culture conditions. For example, a recent Brazilian study of adipose-derived MSCs found a significant rise in DNA damage by passage 5 and a sharp increase in micronucleus frequency from P7 onward [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy contrast, UC-MSCs showed no such increase (micronuclei remained at zero in Rebelatto\u0026rsquo;s CBMN assay) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis suggests a source-specific difference: neonatal hUC-MSCs (with longer telomeres and higher telomerase) generally tolerate expansion better than adult MSCs due to their primitive source [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, our results confirm the mesenchymal nature, multipotency of the derived hUC-MSC line, and its safety for further preclinical studies and clinical applications.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThus, hUC-MSCs demonstrate promising differentiation potential and immunophenotypic characteristics, making them suitable candidates for regenerative medicine. The cells express standard stem cell surface markers (CD44+, CD90+, CD105+, CD34\u0026minus;, CD45\u0026minus;) as well as a maintain diploid karyotype. Their ability to differentiate into multiple lineages and their genetic stability in early passages make them a valuable resource for cell therapies in various clinical applications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMSCs – mesenchymal stem cells\u003c/p\u003e\n\u003cp\u003eUC-MSCs - umbilical cord MSCs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehUC-MSC –human umbilical cord MSCs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDMEM/F12 – Dulbecco's Modified Eagle Medium/Ham's F-12\u003c/p\u003e\n\u003cp\u003eFBS – fetal bovine serum\u003c/p\u003e\n\u003cp\u003eMHC – major histocompatibility complex\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSSC – side scatter\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study received ethical approval from the Bioethics Commission of Ternopil National Medical University, as indicated in protocol #60, dated 01.09.2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no potential conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Ministry of Health of Ukraine as a part of the state-funded research works \"Investigation of the regenerative potential of cellular therapy agents in acute respiratory distress syndrome\" (2021-2023, state registration number 0121U100159) and \"The use of regenerative medicine technologies for the chronic wounds treatment\" (2024-2026, state registration number \u0026nbsp;0124U001099).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlina Dovgalyuk\u003csup\u003e1\u003c/sup\u003e, Olesia Redko\u003csup\u003e1\u003c/sup\u003e, Ilona Palii\u003csup\u003e1\u003c/sup\u003e, Sophia\u0026nbsp;Lechachenko\u003csup\u003e1\u003c/sup\u003e,\u0026nbsp;Halyna Lavrenchuk\u003csup\u003e2\u003c/sup\u003e, Vitalii Kyryk\u003csup\u003e3\u003c/sup\u003e, Mykhaylo Korda\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAlina Dovgalyuk (AD)\u003c/u\u003e, Olesia Redko (OR), Ilona Palii (IP), Sophia Lechachenko (SL), Halyna Lavrenchuk (HL), Vitalii Kyryk (VK), Mykhaylo Korda (MK)\u003c/p\u003e\n\u003cp\u003eConceptualization: AD, MK\u003c/p\u003e\n\u003cp\u003eData curation: AD, OR, IP, SL\u003c/p\u003e\n\u003cp\u003eFormal analysis: AD, OR, IP, SL\u003c/p\u003e\n\u003cp\u003eFunding acquisition: AD, MK\u003c/p\u003e\n\u003cp\u003eInvestigation: AD, OR, IP,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: AD\u003c/p\u003e\n\u003cp\u003eProject administration: AD, MK\u003c/p\u003e\n\u003cp\u003eResources: HL, VK, MK\u003c/p\u003e\n\u003cp\u003eSupervision: AD, MK\u003c/p\u003e\n\u003cp\u003eValidation: AD, HL, VK\u003c/p\u003e\n\u003cp\u003eVisualization: AD, OR, IP,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting – original draft: AD, OR, SL\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: AD, OR, SL\u003c/p\u003e\n\u003cp\u003eApproval of final manuscript: all authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRedko O, Dovgalyuk A, Dovbush A, Nebesna Z, Yakubyshyna L, Krynytska I. 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Genetic stability of mesenchymal stromal cells for regenerative medicine applications: a fundamental biosafety aspect. Int J Mol Sci. 2019;20(10):2406. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms20102406\u003c/span\u003e\u003cspan address=\"10.3390/ijms20102406\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajput SN, Naeem BK, Ali A, Salim A, Khan I. Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine. World J Stem Cells. 2024;16(4):410. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4252/wjsc.v16.i4.410\u003c/span\u003e\u003cspan address=\"10.4252/wjsc.v16.i4.410\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, umbilical cord, cultivation, karyotyping, immunophenotyping","lastPublishedDoi":"10.21203/rs.3.rs-6938731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6938731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMesenchymal stem cells (MSCs) derived from the human umbilical cord offer a promising source for regenerative medicine due to their accessibility, low immunogenicity, and high differentiation potential. Unlike other types of MSCs, human umbilical cord MSCs (hUC-MSCs) are obtained through non-invasive procedures and raise fewer ethical concerns. This study aimed to represent our experience in isolating and characterizing hUC-MSCs in terms of their differentiation ability, immunophenotype, and genomic stability.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman umbilical cord tissue was obtained with informed consent under aseptic conditions. MSCs were isolated using enzymatic disintegration of explants with collagenase I and cultured in Advanced DMEM/F12 supplemented with 2\u0026ndash;10% fetal bovine serum. Cell morphology, proliferation rate, and viability were monitored during the first 10 passages of cultivation. Multipotency was assessed by inducing adipogenic, osteogenic, and chondrogenic differentiation using specialized culture kits, followed by histochemical staining with Oil Red O, Alizarin Red S, and Alcian Blue. Immunophenotyping was conducted via flow cytometry using antibodies against CD73, CD90, CD105 (positive markers), and CD34, CD45 (negative markers). Cytogenetic analysis was performed on passages 2, 4, 6, 8, 10 to assess genomic stability of cell line.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCultured hUC-MSCs displayed typical fibroblast-like morphology and adherence to plastic. Cells successfully underwent adipo, osteo, and chondrogeneic differentiation, confirmed by lineage-specific staining. Flow cytometry revealed a typical MSC immunophenotype (CD73+, CD90+, CD105+, CD34\u0026minus;, CD45\u0026minus;). Karyotype analysis demonstrated a normal diploid chromosomal number, confirming genomic stability during in vitro expansion, as well as stochastic occurrence of polyploid cells and cells with micronuclei at later passages (8\u0026ndash;10), not indicative of genomic transformation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eObtained hUC-MSCs exhibit stable genetic profiles, specific surface marker expression, and trilineage differentiation potential. These characteristics support their safety and effectiveness as candidates for future cell-based therapies and regenerative medicine applications.\u003c/p\u003e","manuscriptTitle":"Obtaining Human Umbilical Cord-Derived Mesenchymal Stem Cells and Cell Line Characterization: Immunophenotype, Multipotency and Cytogenetic Stability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 15:07:58","doi":"10.21203/rs.3.rs-6938731/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a3cd509-b211-465d-aead-d25c629095f7","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T09:24:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-27 15:07:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6938731","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6938731","identity":"rs-6938731","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}