ORIGINAL RESEARCH

Cell and Organ Transplantology. 2026; 14(1):e2026141193.
DOI: 10.22494/cot.v14-1.193

Comprehensive characterization of human Wharton’s jelly mesenchymal stromal cells isolated by the combined explant-enzymatic method

Obushko R.¹, Trufanova N.¹, Revenko O.¹, Cherkashina D.¹, Lipko O.^2,3, Zubov P.¹, Petrenko O.¹

• ¹Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine
• ²Kharkiv National Medical University, Kharkiv, Ukraine
• ³Municipal non-commercial enterprise “City Clinical Hospital No. 27” of the Kharkiv City Council, Kharkiv, Ukraine

Abstract

Human mesenchymal stromal cells (MSCs) derived from Wharton’s jelly (WJ) are a premier source for regenerative medicine. However, standardized isolation protocols that ensure both high cell yield and maintained proliferative potential remain a subject of active research.
This study is aimed to evaluate the efficiency of a combined explant-enzymatic method for human WJ-MSC isolation and characterize the resulting cell populations.
Materials and methods. Cells were isolated using a combined explant-enzymatic method. Minced WJ fragments were cultured on gelatin-coated surfaces for 7 days, then explants underwent a brief (5–10 min) in situ enzymatic treatment with a trypsin-Versene solution, followed by further co-cultivation of the tissue fragments and released cells for an additional 7 days prior to the first passage. Cell characterization included colony-forming unit (CFU), immunophenotyping (CD29, CD90, CD73, CD105, CD34, CD45), directed differentiation (adipogenic and osteogenic), and secretory activity assays. Proliferative kinetics were assessed by calculating the population doubling time (PDT) across 5 passages. Secretory profile of WJ-MSCs (growth factors and cytokines regulating reparative-regenerative processes) was studied by enzyme-linked immunosorbent assay of conditioned media collected after 48 h cell incubation in minimal culture media.
Results. The combined method ensured a stable cell yield with high reproducibility. Significant inter-donor variability in primary cell yield was noted. Isolated cells fulfilled the ISCT minimal criteria, exhibiting typical fibroblast-like morphology, characteristic immunophenotype, differentiation potential, and a high proportion of CFU, indicating the preservation of primitive progenitors. Proliferative analysis showed that PDT values remained comparable and did not change significantly between passages 1 and 3. Conditioned media from WJ-MSCs contained wide range of growth factors and cytokines, with inter-donor variability: VEGF, EGF, PDGF-BB, SCF, TNF-α, and TGF-β exhibited wide fluctuations, whereas NGF-β and FGF-β were relatively stable. Overall, WJ-MSC CM were characterized by very high levels of VEGF and EGF, high PDGF-BB, elevated TNF-α and TGF-β, and the lowest levels of FGF-β.
Conclusions. The combined explant-enzymatic method is an efficient and standardized protocol for human WJ-MSC isolation, preserving high functional potency of cells for clinical and biobanking purposes.

Keywords: mesenchymal stromal cells; Wharton’s jelly of the umbilical cord; colony-forming activity; immunophenotype; directed differentiation; conditioned medium

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