Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Isolation of Placenta-Derived MSCs. (A) Illustration of the MSC isolation process. (B) Representative morphology of MSCs during isolation. (C) Differentiation of MSCs into adipocytes (FABP-4 + ) and osteocytes (osteocalcin + ). (D) Flow cytometry analysis of MSC surface markers. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (E) Size distribution of MSCs cultured in T-25 flasks at passages 4 (P4), 6 (P6), and 8 (P8).

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Derivative Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Identifying the Optimal Spheroid Diameter. (A) Representative images of MSC (P3) spheroids cultured for 3 days with varying initial cell numbers per spheroid. (B) Quantification of spheroid diameter over the 3-day culture period. (C) Representative images of individual MSCs following 2D and 3D spheroid culture. (D,E) Cell size distribution of MSCs after 2D and 3D spheroid culture. (F) Proportions of small (≤15 µm) and large (>15 µm) MSCs after 2D and 3D spheroid culture. Spheroid culture duration in (C–F) was 72 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Determining the Optimal Culture Duration. (A) Representative images showing morphological changes in MSC (P4) spheroids over a 7-day culture period. (B) Images of individual MSCs cultured in 2D or in 25 K-cell spheroids for varying durations. (C) Changes in the diameter of 25 K-cell MSC 3D spheroids over 7 days. (D) Comparison of mean MSC diameter in 2D versus 25 K spheroid cultures over 7 days (E,F) Size distribution of MSCs within 25 K spheroids across the 7-day culture period.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effect of Chemically Defined Medium on MSC Viability and Size in Spheroid Culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P6) spheroids cultured in various media. Three representative spheroids are shown in each condition. (B) MSC size distribution in 3D cultures across different media conditions. (C,D) Anti-inflammatory capability of MSCs cultured under varying conditions. RAW-Dual™ reporter cells were stimulated with 100 ng/mL LPS and 10 ng/mL IFNγ, then treated with MSCs. Dexamethasone (Dex, 1 µg/mL) was used as a positive control. Luciferase activity (C) and mouse IL-6 (D) were measured. Spheroid culture time was 48 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Staining, Cell Culture, Positive Control, Luciferase, Activity Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effects of Alternating 2D/3D Culture on MSC Size and Immunomodulatory Function. (A) MSCs were cultured in flasks for four passages, with an additional 2-day spheroid culture following each passage. Shown are the mean cell diameters immediately after 2D culture and after subsequent spheroid culture. (B) MSC diameters from P5 to P8 using conventional 2D culture versus the alternating 2D/3D method. P4 cells served as the starting population for both conditions. Diameters were measured after harvesting from 2D flasks. (C) Comparison of MSC sizes at P5–P8 between the two culture methods. (D) Comparison of doubling times at P5–P8 between the two methods. (E,F) Macrophages were activated with LPS and IFNγ, then treated with either early-passage (P4) or late-passage (P15) MSCs derived from conventional 2D culture or the alternating 2D/3D protocol. Levels of mouse IL-6 and IL-10 were measured to assess immunomodulatory effects.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: RGD-Modified Alginate Hydrogel Tubes (AlgTubes) for MSC Culture. (A) Schematic of alginate modification with RGD peptides. (B,C) Chemistry underlying hydrogel tube formation. (D,E) Process AlgTubes using a micro-extruder: a cell suspension and alginate solution are pumped into the central and side channels, respectively, creating coaxial core-shell flows. These are extruded through a nozzle into a CaCl 2 buffer, where Ca 2+ ions crosslink the outer alginate shell, forming hydrogel tubes instantly. (F) Illustration of growing MSCs within an AlgTube. (G,H) SEM images showing the porous structure of the AlgTubes.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Suspension

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Dynamic Cell Adhesion in RGD-Modified AlgTubes. (A) MSCs were processed into RGD-modified AlgTubes. (B–D) Cells adhered to the inner surface and proliferated from day 0 to day 6. (E) On day 6, free RGD peptides were added to the culture medium, leading to cell contraction within 24 h. (F) By 48 h, cells had fully detached and formed spheroids (red arrows). (G,H) Removal of free RGD peptides from the medium resulted in MSC reattachment to the AlgTube surface and resumed cell growth (blue arrows). (I–L) As a comparison, in the absence of free RGD peptides, day 6 MSCs continued to grow and eventually covered the inner surface of the hydrogel tube.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Comparison