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Cited by 9Mesenchymal stem cell researchHematopoietic Stem Cell TransplantationPeriodontal Regeneration and Treatments

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Optimizing the Seeding Density of Human Mononuclear Cells to Improve the Purity of Highly Proliferative Mesenchymal Stem Cells

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Nagai, Hiroyuki, Miwa, Akihiro, Yoneda, Kenji, Fujisawa, Koichi, Takami, Taro·2023·Bioengineering
DOI 10.3390/bioengineering10010102PMC PMC9855129
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Abstract

Mesenchymal stem cells (MSCs) hold considerable promise for regenerative medicine. Optimization of the seeding density of mononuclear cells (MNCs) improves the proliferative and differentiation potential of isolated MSCs. However, the underlying mechanism is unclear. We cultured human bone marrow MNCs at various seeding densities (4.0 × 104, 1.25 × 105, 2.5 × 105, 6.0 × 105, 1.25 × 106 cells/cm2) and examined MSC colony formation. At lower seeding densities (4.0 × 104, 1.25 × 105 cells/cm2), colonies varied in diameter and density, from dense to sparse. In these colonies, the proportion of highly proliferative MSCs increased over time. In contrast, lower proliferative MSCs enlarged more rapidly. Senescent cells were removed using a short detachment treatment. We found that these mechanisms increase the purity of highly proliferative MSCs. Thereafter, we compared MSCs isolated under optimized conditions with a higher density (1.25 × 106 cells/cm2). MSCs under optimized conditions exhibited significantly higher proliferative and differentiation potential into adipocytes and chondrocytes, except for osteocytes. We propose the following conditions to improve MSC quality: (1) optimizing MNC seeding density to form single-cell colonies; (2) adjusting incubation times to increase highly proliferative MSCs; and (3) establishing a detachment processing time that excludes senescent cells.

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Figure 1 Timing of densification, distribution, and morphology of mesenchymal stem cell (MSC) colonies varied with different seeding densities of mononuclear cells (MNCs). (A) MNCs were cultured at various seeding densities (4.0 × 104, 1.25 × 105, 2.5 × 105, 6.3 × 105, and 1.25 × 106 cells/cm2). MSC colonies were stained with crystal violet over time. Arrowheads indicate aggregated cells. (B) A fixed number of MNCs was seeded into culture vessels of different culture areas (24-well plate, 6-well plate, 100 mm dish), and cultured. Colonies were stained with crystal violet. (C) The pattern of colonies formed under the low MNC seeding density condition (4.0 × 104 cells/cm2). The scale bar indicates 500 µm. (a) Higher density colony, (b) medium density colony, and (c) lower density colony are shown.
Figure 1 Timing of densification, distribution, and morphology of mesenchymal stem cell (MSC) colonies varied with different seeding densities of mononuclear cells (MNCs). (A) MNCs were cultured at various seeding densities (4.0 × 104, 1.25 × 105, 2.5 × 105, 6.3 × 105, and 1.25 × 106 cells/cm2). MSC colonies were stained with crystal violet over time. Arrowheads indicate aggregated cells. (B) A fixed number of MNCs was seeded into culture vessels of different culture areas (24-well plate, 6-well plate, 100 mm dish), and cultured. Colonies were stained with crystal violet. (C) The pattern of colonies formed under the low MNC seeding density condition (4.0 × 104 cells/cm2). The scale bar indicates 500 µm. (a) Higher density colony, (b) medium density colony, and (c) lower density colony are shown.CC BY · PubMed Central
Figure 2 At lower mononuclear cell (MNC) seeding density, the ratio of fast-growing cells become predominant, depending on the superiority of their proliferative ability. (A,B) Each colony was observed over time using a phase-contrast microscope. The scale bar indicates 500 µm. Examples are shown for (A) a lower seeding density (1.25 × 105 cells/cm2) and (B) a higher seeding density (1.25 × 106 cells/cm2). (C) The number of cells per position of colonies formed at each seeding density is shown. (D) Ratio of cells in each position relative to the total cell count at a lower seeding density (1.25 × 105 cells/cm2). Each position was color-coded based on doubling time. Numbers in the graph indicate %. (E) Enlarged cells seen at lower seeding density were stained with senescence-associated β-galactosidase (SA β-Gal) stain. Arrowheads point to enlarged senescent cells. The scale bar indicates 200 µm. (F) At lower seeding density, trypsin detachment treatment (5 min) was performed. The same position was observed microscopically before and after treatment. Arrowheads point to cells remaining after detachment treatment. The scale bar indicates 200 µm.
Figure 2 At lower mononuclear cell (MNC) seeding density, the ratio of fast-growing cells become predominant, depending on the superiority of their proliferative ability. (A,B) Each colony was observed over time using a phase-contrast microscope. The scale bar indicates 500 µm. Examples are shown for (A) a lower seeding density (1.25 × 105 cells/cm2) and (B) a higher seeding density (1.25 × 106 cells/cm2). (C) The number of cells per position of colonies formed at each seeding density is shown. (D) Ratio of cells in each position relative to the total cell count at a lower seeding density (1.25 × 105 cells/cm2). Each position was color-coded based on doubling time. Numbers in the graph indicate %. (E) Enlarged cells seen at lower seeding density were stained with senescence-associated β-galactosidase (SA β-Gal) stain. Arrowheads point to enlarged senescent cells. The scale bar indicates 200 µm. (F) At lower seeding density, trypsin detachment treatment (5 min) was performed. The same position was observed microscopically before and after treatment. Arrowheads point to cells remaining after detachment treatment. The scale bar indicates 200 µm.CC BY · PubMed Central
Figure 3 Different seeding densities of mononuclear cells (MNCs) result in different numbers of CD45-positive cells remaining. (A) When colonies formed, remaining hematopoietic cells were observed by phase-contrast microscopy when seeded at different seeding densities (1.25 × 105 cells/cm2 and 1.25 × 106 cells/cm2). The scale bar indicates 500 µm. (B) Data quantified as the percentage of CD45-positive cells using FACS analysis. The error bars indicate S.E. (n = 5). p-values were calculated using the Student’s t-test.
Figure 3 Different seeding densities of mononuclear cells (MNCs) result in different numbers of CD45-positive cells remaining. (A) When colonies formed, remaining hematopoietic cells were observed by phase-contrast microscopy when seeded at different seeding densities (1.25 × 105 cells/cm2 and 1.25 × 106 cells/cm2). The scale bar indicates 500 µm. (B) Data quantified as the percentage of CD45-positive cells using FACS analysis. The error bars indicate S.E. (n = 5). p-values were calculated using the Student’s t-test.CC BY · PubMed Central
Figure 4 Evaluation of the relationship between the timing of colony formation and the characteristics of mesenchymal stem cells (MSCs) after passaging. (A) Schematic of the single cell cloning assay. Mononuclear cells (MNCs) were seeded at 1 × 104 cells/well to form one colony per well, then passaged. MSCs cultured from each colony were characterized. (B) The number of cells harvested. (C) Phase-contrast microscope images of cells isolated from colonies formed earlier (colony#1) and later (colony#5). Scale bar indicates 300 µm. (D) Boxplot shows cell size data obtained using the microscopic images. (E) Data from real-time PCR analysis of Ki67 and PCNA in Passage 3 cells. The error bars indicate S.E. (n = 3).
Figure 4 Evaluation of the relationship between the timing of colony formation and the characteristics of mesenchymal stem cells (MSCs) after passaging. (A) Schematic of the single cell cloning assay. Mononuclear cells (MNCs) were seeded at 1 × 104 cells/well to form one colony per well, then passaged. MSCs cultured from each colony were characterized. (B) The number of cells harvested. (C) Phase-contrast microscope images of cells isolated from colonies formed earlier (colony#1) and later (colony#5). Scale bar indicates 300 µm. (D) Boxplot shows cell size data obtained using the microscopic images. (E) Data from real-time PCR analysis of Ki67 and PCNA in Passage 3 cells. The error bars indicate S.E. (n = 3).CC BY · PubMed Central
Figure 5 Characterization of mesenchymal stem cells (MSCs) isolated at higher (1.25 × 106 cells/cm2) and lower (1.25 × 105 cells/cm2) seeding densities of mononuclear cells (MNCs). (A) The number of cells harvested. The error bars indicate S.E. (n = 3). (B) Doubling time of cells in each passage (Passage2–4). The error bars indicate S.E. (n = 3). (C) Phase-contrast microscopy images of cells of each passage (Passage2–4). The scale bar indicates 500 µm. (D) Boxplot shows cell size data obtained using the microscopic images. (E) Colony-forming assay results. One hundred cells of passages 2 and 3 were seeded in 60 mm dishes. After being cultured for 14 d, colonies were stained with crystal violet. (F) Data quantifying the number of colonies formed in the colony assay. The error bars indicate S.E. (n = 3). (G) Data from real-time PCR analysis of Ki67 and PCNA in Passage 2 cells. The error bars indicate S.E. (n = 3). p-values were calculated using a Student’s t-test. NS indicates not significant. (H) SAGE analysis shows the function of genes specifically expressed in MSCs obtained under low density conditions.
Figure 5 Characterization of mesenchymal stem cells (MSCs) isolated at higher (1.25 × 106 cells/cm2) and lower (1.25 × 105 cells/cm2) seeding densities of mononuclear cells (MNCs). (A) The number of cells harvested. The error bars indicate S.E. (n = 3). (B) Doubling time of cells in each passage (Passage2–4). The error bars indicate S.E. (n = 3). (C) Phase-contrast microscopy images of cells of each passage (Passage2–4). The scale bar indicates 500 µm. (D) Boxplot shows cell size data obtained using the microscopic images. (E) Colony-forming assay results. One hundred cells of passages 2 and 3 were seeded in 60 mm dishes. After being cultured for 14 d, colonies were stained with crystal violet. (F) Data quantifying the number of colonies formed in the colony assay. The error bars indicate S.E. (n = 3). (G) Data from real-time PCR analysis of Ki67 and PCNA in Passage 2 cells. The error bars indicate S.E. (n = 3). p-values were calculated using a Student’s t-test. NS indicates not significant. (H) SAGE analysis shows the function of genes specifically expressed in MSCs obtained under low density conditions.CC BY · PubMed Central
Figure 6 Comparison of surface antigens and differentiation potential of mesenchymal stem cells (MSCs) isolated at higher (1.25 × 106 cells/cm2) and lower (1.25 × 105 cells/cm2) seeding densities of mononuclear cells (MNCs). (A) Data for CD45, CD73, CD90, and CD105 surface antigens using fluorescence-activated cell sorting (FACS) analysis of cells (passage 2). (B) Images of induced differentiation of cells into osteocytes, adipocytes, and chondrocytes. The scale bar indicates 200 µm. (C) Alizarin-red-stained area of cells induced to differentiate into osteocytes for 21 d. The error bars indicate S.E. (n = 5). (D) Percentage of fatty-acid-binding protein (FABP)-positive cells induced to differentiate into adipocytes for 21 d. The error bars indicate S.E. (n = 15). (E) Size of spheres induced to differentiate into chondrocytes for 21 d. The error bars indicate S.E. (n = 6). p-values were calculated using a Student’s t-test. NS indicates not significant.
Figure 6 Comparison of surface antigens and differentiation potential of mesenchymal stem cells (MSCs) isolated at higher (1.25 × 106 cells/cm2) and lower (1.25 × 105 cells/cm2) seeding densities of mononuclear cells (MNCs). (A) Data for CD45, CD73, CD90, and CD105 surface antigens using fluorescence-activated cell sorting (FACS) analysis of cells (passage 2). (B) Images of induced differentiation of cells into osteocytes, adipocytes, and chondrocytes. The scale bar indicates 200 µm. (C) Alizarin-red-stained area of cells induced to differentiate into osteocytes for 21 d. The error bars indicate S.E. (n = 5). (D) Percentage of fatty-acid-binding protein (FABP)-positive cells induced to differentiate into adipocytes for 21 d. The error bars indicate S.E. (n = 15). (E) Size of spheres induced to differentiate into chondrocytes for 21 d. The error bars indicate S.E. (n = 6). p-values were calculated using a Student’s t-test. NS indicates not significant.CC BY · PubMed Central
Figure 7 Overview of mesenchymal stem cell (MSC) colony formation at different mononuclear cell (MNC) seeding densities. (A) Colony-formation process under conditions of low seeding density (4.0 × 104 and 1.25 × 105 cells/cm2). (B) Colony-formation process under high seeding density conditions (6.3 × 105 and 1.25 × 106 cells/cm2).
Figure 7 Overview of mesenchymal stem cell (MSC) colony formation at different mononuclear cell (MNC) seeding densities. (A) Colony-formation process under conditions of low seeding density (4.0 × 104 and 1.25 × 105 cells/cm2). (B) Colony-formation process under high seeding density conditions (6.3 × 105 and 1.25 × 106 cells/cm2).CC BY · PubMed Central

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Metadata

Authors
Nagai, H., Miwa, A., Yoneda, K., Fujisawa, K., & Takami, T.
Affiliations
  • Yamaguchi University (JP)
  • Shibuya (Japan) (JP)
Corresponding author
Takami, Taro
ORCID
Venue
Bioengineering
Publisher
MDPI AG
Published
2023-01-11
Volume
10
Issue
1
Pages
102
ISSN
2306-5354 (ISSN-L)
DOI
10.3390/bioengineering10010102
PMC
PMC9855129
OpenAlex
W4315795778
Cited by
9
References
0
Citations by year
2026: 4 · 2025: 2 · 2024: 2 · 2023: 1
FWCI
1.56
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioengineering (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Shibuya Corporation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
  • Health Sciences › Dentistry › Periodontics › Periodontal Regeneration and Treatments
Keywords
mesenchymal stem cells, seeding rate, mononuclear cells, bone marrow, colony formation, cell proliferation, senescence, adipogenesis, chondrogenic differentiation, osteogenic differentiation, cell purity

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