Experimental study on adipose-derived stem cells amplified by silk fibroin/poly-<i>L</i>-lactic acid microcarriers <i>in vitro</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Yuanliang , MU Lin , LIN Yanxian , JIANG Haiyue ,  TENG Li

Department of Craniomaxillofacial Surgery, Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100144, P.R.China;

Corresponding author：

TENG Li, Email: tenglidr@sina.com

Keywords：

Silk fibroin-poly-L-lactic acid; microcarrier; dynamic culture; adipose-derived stem cells

DOI：

10.7507/1002-1892.202101048

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effect of silk fibroin-poly-L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs).Methods ADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells.Results ADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar (P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences (P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs.Conclusion The ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.

Citation： HUANG Yuanliang, MU Lin, LIN Yanxian, JIANG Haiyue, TENG Li. Experimental study on adipose-derived stem cells amplified by silk fibroin/poly-L-lactic acid microcarriers in vitro. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(5): 611-619. doi: 10.7507/1002-1892.202101048 Copy

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11.	孙恒, 蒋海越, 马世泽, 等. 左旋聚乳酸多孔微球体外动态培养耳郭软骨细胞的研究. 组织工程与重建外科杂志, 2019, 15(2): 81-84.
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13.	Song J, Chen Z, Murillo LL, et al. Hierarchical porous silk fibroin/poly (L-lactic acid) fibrous membranes towards vascular scaffolds. Int J Biol Macromol, 2021, 166: 1111-1120.
14.	Voga M, Drnovsek N, Novak S, et al. Silk fibroin induces chondrogenic differentiation of canine adipose-derived multipotent mesenchymal stromal cells/mesenchymal stem cells. J Tissue Eng, 2019, 10: 2041731419835056. doi: 10.1177/2041731419835056.
15.	Barlian A, Judawisastra H, Alfarafisa NM, et al. Chondrogenic differentiation of adipose-derived mesenchymal stem cells induced by L-ascorbic acid and platelet rich plasma on silk fibroin scaffold. PeerJ, 2018, 6: e5809. doi: 10.7717/peerj.5809.
16.	Shekaran A, Sim E, Tan KY, et al. Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers. BMC Biotechnol, 2015, 15: 102. doi: 10.1186/s12896-015-0219-8.
17.	Goh TK, Zhang ZY, Chen AK, et al. Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells. Biores Open Access, 2013, 2(2): 84-97.
18.	Tseng PC, Young TH, Wang TM, et al. Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension. Biomaterials, 2012, 33(2): 556-564.
19.	Lin YM, Lim JF, Lee J, et al. Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells. Cytotherapy, 2016, 18(6): 740-753.
20.	Ng EX, Wang M, Neo SH, et al. Dissolvable gelatin-based microcarriers generated through droplet microfluidics for expansion and culture of mesenchymal stromal cells. Biotechnol J, 2021, 16(3): e2000048. doi: 10.1002/biot.202000048.
21.	Ingber DE. Tensegrity II. How structural networks influence cellular information processing networks. J Cell Sci, 2003, 116(Pt 8): 1397-1408.
22.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
23.	Liu L, Yuan W, Wang J. Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress. Biomech Model Mechanobiol, 2010, 9(6): 659-670.

1. Arthur A, Gronthos S. Clinical application of bone marrow mesenchymal stem/stromal cells to repair skeletal tissue. Int J Mol Sci, 2020, 21(24): 9759. doi: 10.3390/ijms21249759.
2. Le H, Xu W, Zhuang X, et al. Mesenchymal stem cells for cartilage regeneration. J Tissue Eng, 2020, 11: 1-22.
3. Barckhausen C, Rice B, Baila S, et al. GMP-compliant expansion of clinical-grade human mesenchymal stromal/stem cells using a closed hollow fiber bioreactor. Methods Mol Biol, 2016, 1416: 389-412.
4. McGrath M, Tam E, Sladkova M, et al. GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells. Stem Cell Res Ther, 2019, 10(1): 11. doi: 10.1186/s13287-018-1119-3.
5. Chen AK, Reuveny S, Oh SK. Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction. Biotechnol Adv, 2013, 31(7): 1032-1046.
6. Wall ME, Bernacki SH, Loboa EG. Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells. Tissue Eng, 2007, 13(6): 1291-1298.
7. Gruber HE, Somayaji S, Riley F, et al. Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence. Biotech Histochem, 2012, 87(4): 303-311.
8. Elkhenany H, Amelse L, Caldwell M, et al. Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential: implications for bone tissue engineering. J Anim Sci Biotechnol, 2016, 7: 16. doi: 10.1186/s40104-016-0074-z.
9. Bonnier F, Keating ME, Wróbel TP, et al. Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models. Toxicol In Vitro, 2015, 29(1): 124-131.
10. Tsai AC, Jeske R, Chen X, et al. Influence of microenvironment on mesenchymal stem cell therapeutic potency: From planar culture to microcarriers. Front Bioeng Biotechnol, 2020, 8: 640. doi: 10.3389/fbioe.2020.00640.
11. 孙恒, 蒋海越, 马世泽, 等. 左旋聚乳酸多孔微球体外动态培养耳郭软骨细胞的研究. 组织工程与重建外科杂志, 2019, 15(2): 81-84.
12. Eggerschwiler B, Canepa DD, Pape HC, et al. Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells. Stem Cell Res Ther, 2019, 10(1): 69. doi: 10.1186/s13287-019-1170-8.
13. Song J, Chen Z, Murillo LL, et al. Hierarchical porous silk fibroin/poly (L-lactic acid) fibrous membranes towards vascular scaffolds. Int J Biol Macromol, 2021, 166: 1111-1120.
14. Voga M, Drnovsek N, Novak S, et al. Silk fibroin induces chondrogenic differentiation of canine adipose-derived multipotent mesenchymal stromal cells/mesenchymal stem cells. J Tissue Eng, 2019, 10: 2041731419835056. doi: 10.1177/2041731419835056.
15. Barlian A, Judawisastra H, Alfarafisa NM, et al. Chondrogenic differentiation of adipose-derived mesenchymal stem cells induced by L-ascorbic acid and platelet rich plasma on silk fibroin scaffold. PeerJ, 2018, 6: e5809. doi: 10.7717/peerj.5809.
16. Shekaran A, Sim E, Tan KY, et al. Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers. BMC Biotechnol, 2015, 15: 102. doi: 10.1186/s12896-015-0219-8.
17. Goh TK, Zhang ZY, Chen AK, et al. Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells. Biores Open Access, 2013, 2(2): 84-97.
18. Tseng PC, Young TH, Wang TM, et al. Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension. Biomaterials, 2012, 33(2): 556-564.
19. Lin YM, Lim JF, Lee J, et al. Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells. Cytotherapy, 2016, 18(6): 740-753.
20. Ng EX, Wang M, Neo SH, et al. Dissolvable gelatin-based microcarriers generated through droplet microfluidics for expansion and culture of mesenchymal stromal cells. Biotechnol J, 2021, 16(3): e2000048. doi: 10.1002/biot.202000048.
21. Ingber DE. Tensegrity II. How structural networks influence cellular information processing networks. J Cell Sci, 2003, 116(Pt 8): 1397-1408.
22. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
23. Liu L, Yuan W, Wang J. Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress. Biomech Model Mechanobiol, 2010, 9(6): 659-670.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study on adipose-derived stem cells amplified by silk fibroin/poly-L-lactic acid microcarriers in vitro

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