Biomaterials Translational ›› 2023, Vol. 4 ›› Issue (4): 270-279.doi: 10.12336/biomatertransl.2023.04.006
• RESEARCH ARTICLE • Previous Articles Next Articles
Shunshu Deng1,2,3, Fuwei Zhu1,2,3, Kai Dai1,2,3,4,*(), Jing Wang1,3,4,*(), Changsheng Liu2,3,4,5,*()
Received:
2023-09-21
Revised:
2023-11-14
Accepted:
2023-11-30
Online:
2023-12-27
Published:
2023-12-28
Contact:
Kai Dai, daikai233@foxmail.com;Jing Wang, biomatwj@163.com;Changsheng Liu, liucs@ecust.edu.cn.
Figure 1. Illustrations of osteo-organoid cell separation procedures. (A) The mouse body soaked in 75% (v/v) ethanol was placed in biosafety cabinet. (B) After removing skin and muscle, a hindlimb was completely cut off, so that the femur and tibia could be used for extraction of bone marrow-derived mesenchymal stem cells (BM-MSCs). (C) The osteo-organoid was hidden in the swollen part of the muscle (red circle) between femur and calf. (D) Osteo-organoids covered with periosteum-like tissue were separated in a 60-mm cell culture dish. (E) The surface of the blade was convex (red curve) to cut the osteo-organoids. (F) The surface of the blade was concave (red curve) to shred the osteo-organoids.
Experiment | Culture vessel | Volume of medium | Seeding density | Other suggestions |
---|---|---|---|---|
Colony forming unit-fibroblast | 6-well plate | 2 mL per well | 5 × 105 cells per well | Replace α-MEM with DMEM to prepare the complete medium |
Purification | 100-mm dish | 15 mL per dish | 1 × 107 cells per dish | Culture under the condition of 5% O2 atmosphere |
Differentiation | 6-well plate | 2 mL per well | 2 × 105 cells per well | Use α-MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin as the medium before differentiation assays |
Proliferation | 60-mm dish | 5 mL per dish | 2 × 105 cells per well | Incubate the MSC for 2 hours in a medium containing 50 μM EdU |
Table 1. Details of cell culture in different experiments
Experiment | Culture vessel | Volume of medium | Seeding density | Other suggestions |
---|---|---|---|---|
Colony forming unit-fibroblast | 6-well plate | 2 mL per well | 5 × 105 cells per well | Replace α-MEM with DMEM to prepare the complete medium |
Purification | 100-mm dish | 15 mL per dish | 1 × 107 cells per dish | Culture under the condition of 5% O2 atmosphere |
Differentiation | 6-well plate | 2 mL per well | 2 × 105 cells per well | Use α-MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin as the medium before differentiation assays |
Proliferation | 60-mm dish | 5 mL per dish | 2 × 105 cells per well | Incubate the MSC for 2 hours in a medium containing 50 μM EdU |
Characteristics | Operations |
---|---|
Adhesion of MSCs are earlier than other cells from marrow | Wash cells using Dulbecco’s phosphate-buffered saline after seeding overnight |
MSCs are easy to be digested and suspended | Time of digestion during the cell passage is not recommended to exceed 2 minutes |
Low oxygen pressure increases the proliferation and reduces spontaneous differentiation of MSCs | Expand cells under the hypoxic condition (5% O2) |
Table 2. Purification operations are based on the characteristics of MSCs
Characteristics | Operations |
---|---|
Adhesion of MSCs are earlier than other cells from marrow | Wash cells using Dulbecco’s phosphate-buffered saline after seeding overnight |
MSCs are easy to be digested and suspended | Time of digestion during the cell passage is not recommended to exceed 2 minutes |
Low oxygen pressure increases the proliferation and reduces spontaneous differentiation of MSCs | Expand cells under the hypoxic condition (5% O2) |
Figure 2. Morphology of MSCs and CFU-F assay. (A) Primary cells before (P0-before) and after (P0-after) the first refreshing of the medium, and MSCs at 80% confluence at P0 and P1. Red and blue circles indicate spindle-shaped and round cells. (B) Left: Primary cells from osteo-organoids (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds) formed larger colonies than that from bone marrow (native BM). Scale bars: 200 μm. Right: The number of colonies in each well of a standard 6-well plate. Data are expressed as mean ± SD (n = 3). ***P < 0.001 (Student’s t-test). CFU-F: colony forming unit-fibroblast; MSCs: mesenchymal stem cells; P: passage.
Figure 3. Cell surface phenotype and multilineage differentiation of MSCs at P2. (A) Flow cytometry of odMSCs (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds; BMP) and bone marrow (native). (B) Alizarin red, oil red O or Alcian blue staining after differentiation under the osteogenic, adipogenic or chondrogenic induction. The osteogenic and adipogenic differentiation of odMSCs were more obvious than that of BM-MSCs. Black triangles indicate calcified nodules and mature adipocytes. Scale bars: 200 μm. BM-MSCs: bone marrow-derived mesenchymal stem cells; MSCs: mesenchymal stem cells; odMSCs: osteo-organoid-derived mesenchymal stem cells; P2: passage 2; Sca-1: stem cell antigen-1.
Figure 4. Upper: Ageing process for MSCs from P0 to P2. Black triangles indicate SA-β-Gal positive cells. Scale bars: 200 μm. Lower: The proportions of SA-β-Gal positive cells. The proportion of cell senescence in the native BM-MSCs group (native) increased with passage, while it remained basically unchanged in the odMSCs (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds; BMP). Data are expressed as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 (two-way analysis of variance followed by Bonferroni’s multiple comparison test). BM-MSCs: bone marrow-derived mesenchymal stem cells; odMSCs: osteo-organoid-derived mesenchymal stem cells; P: passage; SA-β-Gal: senescence-associated β-galactosidase.
Source | P0 | P1 | Total |
---|---|---|---|
Osteo-organoid | 3 days | 3 days | 6 days |
Native bone marrow | 8 days | 4 days | 12 days |
Table 3. Time of mesenchymal stem cell passage from the osteo-organoid vs. native bone marrow
Source | P0 | P1 | Total |
---|---|---|---|
Osteo-organoid | 3 days | 3 days | 6 days |
Native bone marrow | 8 days | 4 days | 12 days |
Figure 5. Cell proliferation of MSCs at P2. (A, B) Scatter diagrams (A) and content of EdU+ cells (B) by flow cytometric analysis. (C, D) Fluorescence confocal image (blue: DAPI; red: EdU; C) and statistical analysis (D). Scale bars: 200 μm. Data are expressed as mean ± SD (n = 3), and were analyzed by Student’s t-test. BM: native bone marrow-derived mesenchymal stem cells; BMP: osteo-organoid-derived mesenchymal stem cells (constructed with recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds); DAPI: 4′,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2′-deoxyuridine; MSCs: mesenchymal stem cells; ns: not significant; P2: passage 2.
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