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Russian Journal of Transplantology and Artificial Organs

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Natural silk fiber microcarriers for cell culture

https://doi.org/10.15825/1995-1191-2020-4-98-104

Abstract

The development of effective and versatile microcarriers is a pressing issue in tissue engineering and regenerative medicine.

The objective of this work is to create biocompatible fiber microparticles from the cocoons of the Bombyx  mori silkworm, and to study their structure and biological properties. 

Materials and methods. In obtaining microparticles, the Bombyx mori cocoons washed from sericin were cryo-milled in liquid nitrogen. The structure of the resulting microparticles was analyzed via scanning electron microscopy. The cytotoxicity of the obtained fibers was assessed using MTT-cell culture assay of 3T3 mouse fibroblasts. Cell adhesion analysis was performed using the Hep-G2 human hepatocarcinoma cell line. Cell visualization was performed by staining the nuclei with DAPI fluorescent dye.

Results. Natural silk microparticles were obtained in the form of cylindrical fibers with 200–400 μm average length and 15 μm diameter. It was shown that the surface of the resulting microparticles has a rough relief; no pores were found. The microparticles are non-toxic for 3T3 mouse fibroblasts, they maintain a high level of adhesion by human hepatocellular carcinoma HepG2 cells.

Conclusion. The method developed by us for fabrication of biocompatible silk fibroin microparticles in the form of fibers without using toxic reagents and significant time costs is promising for cell cultivation and delivery to the damaged area for tissue and organ regeneration.

About the Authors

M. M. Bobrova
Shumakov National Medical Research Center of Transplantology and Artificial Organs
Russian Federation
Moscow



L. A. Safonova
Shumakov National Medical Research Center of Transplantology and Artificial Organs
Russian Federation
Moscow



A. E. Efimov
Shumakov National Medical Research Center of Transplantology and Artificial Organs
Russian Federation
Moscow



O. I. Agapova
Shumakov National Medical Research Center of Transplantology and Artificial Organs
Russian Federation
Moscow



I. I. Agapov
Shumakov National Medical Research Center of Transplantology and Artificial Organs
Russian Federation

1, Shchukinskaya str., 123182, Moscow, Russian Federation

Phone: (499) 190-66-19





References

1. Asghar W, El Assal R, Shafiee H, Pitteri S, Paulmurugan R, Demirci U. Engineering cancer microenvironments for in vitro 3-D tumor models. Mater Today (Kidlington). 2015; 18 (10): 539–553. doi: 10.1016/j.mattod.2015.05.002. PMID: 28458612.

2. In JG, Foulke-Abel J, Estes MK, Zachos NC, Kovbasnjuk O, Donowitz M. Human mini-guts: new insights into intestinal physiology and host-pathogen interactions. Nat Rev Gastroenterol Hepatol. 2016; 13 (11): 633–642. doi: 10.1038/nrgastro.2016.142. PMID: 27677718.

3. Edmondson R, Broglie JJ, Adcock AF, Yang L. Threedimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014; 12 (4): 207–218. doi: 10.1089/adt.2014.573. PMID: 24831787.

4. Skardal A, Devarasetty M, Rodman C, Atala A, Soker S. Liver-Tumor Hybrid Organoids for Modeling Tumor Growth and Drug Response In Vitro. Ann Biomed Eng. 2015; 43 (10): 2361–2373. doi: 10.1007/s10439-015-1298-3. PMID: 25777294.

5. Bao J, Shi Y, Sun H, Yin X, Yang R, Li L et al. Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats. Cell Transplant. 2011; 20 (5): 753–766. doi: 10.3727/096368910X536572.

6. Soto-Gutierrez A, Zhang L, Medberry C, Fukumitsu K, Faulk D, Jiang H et al. A whole-organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods. 2011; 17 (6): 677–686. doi: 10.1089/ten.TEC.2010.0698. PMID: 21375407.

7. Zhou P, Lessa N, Estrada DC, Severson EB, Lingala S, Zern MA et al. Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice. Liver Transpl. 2011; 17 (4): 418–427. doi: 10.1002/lt.22270. PMID: 21445925.

8. Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007; 8 (10): 839–845. doi: 10.1038/nrm2236. PMID: 17684528.

9. Achilli TM, Meyer J, Morgan JR. Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol Ther. 2012; 12 (10): 1347–1360. doi: 10.1517/14712598.2012.707181. PMID: 22784238.

10. 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. doi: 10.1016/j.biotechadv.2013.03.006. PMID: 23531528.

11. Quittet MS, Touzani O, Sindji L, Cayon J, Fillesoye F, Toutain J et al. Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat. Acta Biomater. 2015; 15: 77–88. doi: 10.1016/j.actbio.2014.12.017. PMID: 25556361.

12. Georgi N, van Blitterswijk C, Karperien M. Mesenchymal stromal/stem cell-or chondrocyte-seeded microcarriers as building blocks for cartilage tissue engineering. Tissue Eng Part A. 2014; 20 (17–18): 2513–2523. doi: 10.1089/ten.TEA.2013.0681. PMID: 24621188.

13. Yang Y, Rossi FM, Putnins EE. Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture. Biomaterials. 2007; 28 (20): 3110–3120. doi: 10.1016/j.biomaterials.2007.03.015. PMID: 17433434.

14. Chen M, Wang X, Ye Z, Zhang Y, Zhou Y, Tan WS. A modular approach to the engineering of a centimetersized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers. Biomaterials. 2011; 32 (30): 7532–7542. doi: 10.1016/j.biomaterials.2011.06.054. PMID: 21774980.

15. Zhou Y, Yan Z, Zhang H, Lu W, Liu S, Huang X et al. Expansion and delivery of adipose-derived mesenchymal stem cells on three microcarriers for soft tissue regeneration. Tissue Eng Part A. 2011; 17 (23–24): 2981–2997. doi: 10.1089/ten.tea.2010.0707. PMID: 21875329.

16. Sun LY, Hsieh DK, Syu WS, Li YS, Chiu HT, Chiou TW. Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential. Cell Prolif. 2010; 43 (5): 445–456. doi: 10.1111/j.1365-2184.2010.00694.x. PMID: 20887551.

17. Agapov II, Moisenovich MM, Vasilyeva TV, Pustovalova OL, Kon’kov AS, Arkhipova AY et al. Biodegradable matrices from regenerated silk of Bombix mori. Dokl Biochem Biophys. 2010; 433: 201–204. doi: 10.1134/S1607672910040149.

18. Uebersax L, Merkle HP, Meinel L. Insulin-like growth factor I releasing silk fibroin scaffolds induce chondrogenic differentiation of human mesenchymal stem cells. J Control Release. 2008; 127 (1): 12–21. doi: 10.1016/j.jconrel.2007.11.006. PMID: 18280603.

19. Altman AM, Gupta V, Ríos CN, Alt EU, Mathur AB. Adhesion, migration and mechanics of human adiposetissue-derived stem cells on silk fibroin-chitosan matrix. Acta Biomater. 2010; 6 (4): 1388–1397. doi: 10.1016/j.actbio.2009.10.034. PMID: 19861180.

20. Kotliarova MS, Zhuikov VA, Chudinova YV Khaidapova DD, Moisenovich AM, Kon’kov AS et al. Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers. Moscow Univ Biol Sci Bull. 2016; 71: 212–217. doi: 10.3103/S0096392516040052.

21. Luetchford KA, Chaudhuri JB, De Bank PA. Silk fibroin/gelatin microcarriers as scaffolds for bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2020; 106: 110116. doi: 10.1016/j.msec.2019.110116. PMID: 31753329.

22. Moisenovich MM, Plotnikov EY, Moysenovich AM, Silachev DN, Danilina TI, Savchenko ES et al. Effect of Silk Fibroin on Neuroregeneration After Traumatic Brain Injury. Neurochem Res. 2019; 44 (10): 2261–2272. doi: 10.1007/s11064-018-2691-8. PMID: 30519983.

23. Perteghella S, Martella E, de Girolamo L, Perucca Orfei C, Pierini M, Fumagalli V et al. Fabrication of Innovative Silk/Alginate Microcarriers for Mesenchymal Stem Cell Delivery and Tissue Regeneration. Int J Mol Sci. 2017; 18 (9): 1829. doi: 10.3390/ijms18091829. PMID: 28832547.

24. Agapov II, Agapova OI, Bobrova MM, Safonova LA, Efimov AE. Mikronositel’ dlya kletok na osnove natural’nogo shelka i sposob ego polucheniya. Patent na izobretenie RU2732598 S1, 21.09.2020.

25. GOST ISO 10993-1-2011 «Izdeliya meditsinskie. Otsenka biologicheskogo deystviya meditsinskikh izdeliy. Chast’ 5. Issledovanie na tsitotoksichnost’: metody in vitro». M.: Standartinform, 2014.

26. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65 (1–2): 55–63. doi: 10.1016/0022-1759(83)90303-4. PMID: 6606682.

27. Kundu B, Rajkhowa R, Kundu SC, Wang X. Silk fibroin biomaterials for tissue regenerations. Adv Drug Deliv Rev. 2013; 65 (4): 457–470. doi: 10.1016/j.addr.2012.09.043. PMID: 23137786.

28. Zhang Q, Zhao Y, Yan S, Yang Y, Zhao H, Li M et al. Preparation of uniaxial multichannel silk fibroin scaffolds for guiding primary neurons. Acta Biomater. 2012; 8 (7): 2628–2638. doi: 10.1016/j.actbio.2012.03.033. PMID: 22465574.

29. Safonova LА, Bobrova ММ, Agapova ОI, Kotliarova МS, Arkhipova АYu, Moisenovich ММ, Agapov II. Biological Properties of regenerated Silk Fibroin Films. Sovremennye tehnologii v medicine. 2015; 7 (3): 6–13. [In English]. doi: 10.17691/stm2015.7.3.01.

30. Surguchenko VA, Ponomareva АS, Efimov АE, Nemets ЕA, Agapov II, Sevastianov VI. Characteristics of adhesion and proliferation of mouse nih/3t3 fibroblasts on the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films with different surface roughness values. Russian Journal of Transplantology and Artificial Organs. 2012; 14 (1): 72–77. [In Russ. English abstract]. doi: 10.15825/1995-1191-2012-1-72-77.

31. Servoli E, Maniglio D, Motta A, Predazzer R, Migliaresi C. Surface properties of silk fibroin films and their interaction with fibroblasts. Macromol Biosci. 2005; 5 (12): 1175–1183. doi: 10.1002/mabi.200500137. PMID: 16315185.

32. Sokolova AI, Bobrova MM, Safonova LA, Agapova OI, Moisenovich MM, Agapov II. The Relation of Biological Properties of the Silk Fibroin/Gelatin Scaffolds with the Composition and Fabrication Technology. Sovremennye tehnologii v medicine. 2016; 8 (3): 6–15. [In English]. doi: 10.17691/stm2016.8.3.01.


Review

For citations:


Bobrova M.M., Safonova L.A., Efimov A.E., Agapova O.I., Agapov I.I. Natural silk fiber microcarriers for cell culture. Russian Journal of Transplantology and Artificial Organs. 2020;22(4):98-104. https://doi.org/10.15825/1995-1191-2020-4-98-104

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ISSN 1995-1191 (Print)