3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P

Home/2018, Vol. 6, No. 2/3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P

Cell and Organ Transplantology. 2018; 6(2):195-201.
DOI: 10.22494/cot.v6i2.91

3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P

Kyryk V. M.1, Kuchuk O. V.1, Mamchur A. A.1, Ustymenko A. M.1, Lutsenko T. M.1, Tsupykov O. M.1,2, Yatsenko K. V.2, Skibo G. G.1,2, Bilko D. I.3, Bilko N. M.3
1State Institute of Genetic and Regenerative Medicine National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
2Bogomoletz Institute of Physiology NAS of Ukraine, Kyiv, Ukraine
3Center of molecular and cell research of National University of Kyiv-Mohyla Academy of Ministry of Education and Science of Ukraine, Kyiv, Ukraine

Abstract
Actual issues during tissue regeneration are to ensure the survival of transplanted cells at the site of their application and further activity, especially in case of local pathological alterations such as inflammation and ischemia. For this purpose, the matrices that can not only fill the defects of tissues, but also be scaffolds for cells are developed.
The aim of this study was to evaluate the effectiveness of 3D cultivation of murine adipose-derived multipotent mesenchymal stromal cells (MSMCs) in hydrogel based on carbomer 974P.
Materials and methods. MSMCs were obtained from the adipose tissue of FVB-Cg-Tg(GFPU)5Nagy/J mice transgenic for GFP gene. The cells were phenotyped by flow cytometry and directly differentiated into osteogenic and adipogenic direction to confirm multipotent phenotype. MMSCs were cultured and directly differentiated into osteogenic direction in three-dimensional hydrogel scaffolds. For hydrogel preparation we used carbomer 974P with composition of glycerol, propylene glycol, triethylamine and agarose in original proportion.
Results. The three-dimensional hydrogel based on carbomer 974P for the further engraftment with MMSCs was obtained. Modified protocols for the preparation of hydrogels based on carbomer and agarose and their rehydration by culture media for the 3D cultivation of adipose-derived MMSCs have been developed. The optimal concentration of MSMCs and the injection method for engraftment of hydrogels of the required form and size are selected. It was shown that adipose-derived MMSCs in 3D carbomer hydrogel preserve the potential of directed osteogenic differentiation.
Conclusion. Three-dimensional hydrogel based on carbomer 974P is capable to support cells, provide the necessary cytoarchitectonics, maintain intercellular interactions, which can promote further long-term survival and specialization of the graft.

Key words: adipose-derived multipotent mesenchymal stromal cells; 3-dimentional cell culture; hydrogel; carbomer 974P

Full Text PDF (eng) Full Text PDF (ua)

1. Mashkouri S, Crowley MG, Liska MG, Corey S, Borlongan CV. Utilizing pharmacotherapy and mesenchymal stem cell therapy to reduce inflammation following traumatic brain injury. Neural Regen Res. 2016; 11:1379-84.
https://doi.org/10.4103/1673-5374.191197
2. Duval K, Grover H, Han LH, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda). 2017. 32(4):266-277.
https://doi.org/10.1152/physiol.00036.2016
PMid:28615311 PMCid:PMC5545611
3. Kim SH, Turnbull J, Guimond S. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. J Endocrinol. 2011; 209(2):139-51. DOI: 10.1530/JOE-10-0377.
https://doi.org/10.1530/JOE-10-0377
4. Knight E, Przyborski S. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat. 2015; 227(6):746-756.
https://doi.org/10.1111/joa.12257
PMid:25411113 PMCid:PMC4694114
5. Feng G, Zhang J, Li Y, et al. IGF-1 C domain-modified hydrogel enhances cell therapy for AKI. J Am Soc Nephrol. 2016. 27(8):2357-2369.
https://doi.org/10.1681/ASN.2015050578
PMid:26869006 PMCid:PMC4978042
6. Baraniak PR, Cooke MT, Saeed R, et al. Stiffening of human mesenchymal stem cell spheroid microenvironments induced by incorporation of gelatin microparticles. J Mech Behav Biomed Mater. 2012. 11:63-71.
https://doi.org/10.1016/j.jmbbm.2012.02.018
PMid:22658155 PMCid:PMC3528787
7. Labusca LS. Scaffold free 3D culture of mesenchymal stem cells; implications for regenerative medicine. J Transplant Stem Cel Biol. 2015; 2(1):8-15.
8. Grigore A, Sarker B, Fabry B, et al. Behavior of encapsulated MG-63 cells in RGD and gelatine-modified alginate hydrogels. Tissue Eng. Part A. 2014. 20(15-16):2140-2150.
https://doi.org/10.1089/ten.tea.2013.0416
PMid:24813329
9. Xing Q, Qian Z, Tahtinen M, et al. Aligned Nanofibrous Cell-Derived Extracellular Matrix for Anisotropic Vascular Graft Construction. Adv Healthc Mater. 2017; 6(10). DOI: 10.1002/adhm.201601333.
https://doi.org/10.1002/adhm.201601333
10. Capulli AK, MacQueen LA, Sheehy SP, Parker KK. Fibrous scaffolds for building hearts and heart parts. Adv Drug Deliv Rev. 2016; 15(96):83-102. DOI: 10.1016/j.addr.2015.11.020.
https://doi.org/10.1016/j.addr.2015.11.020
11. Wu S, Duan B, Qin X, Butcher JT. Living nano-micro fibrous woven fabric/hydrogel composite scaffolds for heart valve engineering. Acta Biomater. 2017; 51:89-100. DOI: 10.1016/j.actbio.2017.01.051.
https://doi.org/10.1016/j.actbio.2017.01.051
12. Ye H, Zhang K, Kai D, Li Z, Loh XJ. Polyester elastomers for soft tissue engineering. Chem Soc Rev. 2018; 47(12):4545-4580. DOI: 10.1039/c8cs00161h.
https://doi.org/10.1039/C8CS00161H
13. Alhaque S, Themis M, Rashidi H. Three-dimensional cell culture: from evolution to revolution. Philos Trans R Soc Lond B Biol Sci. 2018. 373(1750):20170216.
14. Tsou YH, Khoneisser J, Huang PC, Xu X. Hydrogel as a bioactive material to regulate stem cell fate. Bioact Mater. 2016; 1(1):39-55. DOI: 10.1016/j.bioactmat.2016.05.001.
https://doi.org/10.1016/j.bioactmat.2016.05.001
15. Mei Liu, Xin Zeng, Chao Ma. Injectable hydrogels for cartilage and bone tissue engineering. Bone Res. 2017; 5:17014. DOI:10.1038/boneres.2017.14.
https://doi.org/10.1038/boneres.2017.14
16. Sarker B, Rompf J, Silva R, et al. Alginate-based hydrogels with improved adhesive properties for cell encapsulation. Int J Biol Macromol. 2015. 78:72-78.
https://doi.org/10.1016/j.ijbiomac.2015.03.061
PMid:25847839
17. Ivanovska J, Zehnder T, Lennert P, et al. Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells. Tissue Eng Part C Methods. 2016; 22(7):708-15. DOI: 10.1089/ten.TEC.2015.0452.
https://doi.org/10.1089/ten.tec.2015.0452
18. Gupta S, Vyas SP. Carbopol/chitosan based pH triggered in situ gelling system for ocular delivery of timolol maleate. Sci Pharm. 2010; 78(4):959-976.
https://doi.org/10.3797/scipharm.1001-06
PMid:21179328 PMCid:PMC3007614
19. Hayati F, Ghamsari SM, Dehghan MM, Oryan A. Effects of carbomer 940 hydrogel on burn wounds: an in vitro and in vivo study. J Dermatolog Treat. 2018; 9(6):593-599. DOI: 10.1080/09546634.2018.1426823.
https://doi.org/10.1080/09546634.2018.1426823
20. Rossi F, Santoro M, Casalini T, et al. Characterization and Degradation Behavior of Agar–Carbomer Based Hydrogels for Drug Delivery Applications: Solute Effect. Int J Mol Sci. 2011; 12(6):3394–3408. DOI:10.3390/ijms12063394.
https://doi.org/10.3390/ijms12063394
21. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 2006; 69(9):1037-42.
https://doi.org/10.1002/cyto.a.20333
PMid:16888771
22. Li J, Mareddy S, Meifang DT, et. al. A minimal common osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft. Tissue engineering. 2009; Part A. 15(9):2481-90.
23. Mishra R, Kumar A. Osteocompatibility and osteoinductive potential of supermacroporous polyvinyl alcohol-TEOS-agarose-CaCl2 (PTAgC) biocomposite cryogels. J Mater Sci Mater Med. 2014; 25(5):1327-37. DOI: 10.1007/s10856-014-5166-8.
https://doi.org/10.1007/s10856-014-5166-8
24. Ruiz-Ojeda F, Rupérez A, Gomez-Llorente C, et al. Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review. Int J Mol Sci. 2016; 17(7):1040. DOI:10.3390/ijms17071040.
https://doi.org/10.3390/ijms17071040
25. 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):315-7.
https://doi.org/10.1080/14653240600855905
PMid:16923606
26. Suga H, Matsumoto D, Eto H, et al. Functional implications of CD34 expression in human adipose-derived stem/progenitor cells. Stem Cells Dev. 2009;18(8):1201-10. doi: 10.1089/scd.2009.0003.
https://doi.org/10.1089/scd.2009.0003
27. Rojas-Ríos P, González-Reyes A. Concise review: The plasticity of stem cell niches: a general property behind tissue homeostasis and repair. Stem Cells. 2014; 32(4):852-9. DOI: 10.1002/stem.1621.
https://doi.org/10.1002/stem.1621
28. Donnelly H, Salmeron-Sanchez M, Dalby M. Designing stem cell niches for differentiation and self-renewal. J R Soc Interface. 2018; 15(145):20180388. DOI: 10.1098/rsif.2018.0388.
https://doi.org/10.1098/rsif.2018.0388
29. Geckil H, Feng Xu, Xiaohui Zhang, et al. Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond). 2010; 5(3):469-484. DOI: 10.2217/nnm.10.12.
https://doi.org/10.2217/nnm.10.12
30. Santoro M, Marchetti P, Rossi F, et al. Smart approach to evaluate drug diffusivity in injectable agar-carbomer hydrogels for drug delivery. J Phys Chem B. 2011; 115(11):2503-10. DOI: 10.1021/jp1111394.
https://doi.org/10.1021/jp1111394
31. Bruggeman KF, Williams RJ, Nisbet DR. Dynamic and Responsive Growth Factor Delivery from Electrospun and Hydrogel Tissue Engineering Materials. Adv Healthc Mater. 2018; 7(1). DOI: 10.1002/adhm.201700836.
https://doi.org/10.1002/adhm.201700836
32. Tong Z, Solanki A, Hamilos A. Application of biomaterials to advance induced pluripotent stem cell research and therapy. EMBO J. 2015; 34(8): 987-1008. DOI: 10.15252/embj.201490756.
https://doi.org/10.15252/embj.201490756
33. Jha A, Tharp K, Ye J, et al. Enhanced Survival and Engraftment of Transplanted Stem Cells using Growth Factor Sequestering Hydrogels. Biomaterials. 2015; 47: 1-12. DOI: 10.1016/j.biomaterials.2014.12.043.
https://doi.org/10.1016/j.biomaterials.2014.12.043
34. Parker J, Mitrousis N, Shoichet MS. Hydrogel for Simultaneous Tunable Growth Factor Delivery and Enhanced Viability of Encapsulated Cells in Vitro. Biomacromolecules. 2016; 17(2):476-84. DOI: 10.1021/acs.biomac.5b01366.
https://doi.org/10.1021/acs.biomac.5b01366
35. Gothard D, Smith E, Kanczler J. In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors. PLoS One. 2015; 10(12):0145080. DOI:10.1371/journal.pone.0145080.
https://doi.org/10.1371/journal.pone.0145080
36. Kuchuk O, Kyryk V. Stepwise Differentiation of Multipotent Cells from Murine Adipose Tissue in Osteogenic Direction. Problems of Cryobiology and Cryomedicine. 2012; 22(2):161-164.
37. Fröhlich M, Grayson WL, Marolt D. Bone grafts engineered from human adipose-derived stem cells in perfusion bioreactor culture. Tissue Eng Part A. 2010; 16(1):179-89. DOI: 10.1089/ten.TEA.2009.0164.
https://doi.org/10.1089/ten.tea.2009.0164
38. Burdick J, Mauck R, Gerecht S. To Serve and Protect: Hydrogels to Improve Stem Cell-Based Therapies. Cell Stem Cell. 2016; 18(1):13-5. DOI: 10.1016/j.stem.2015.12.004.
https://doi.org/10.1016/j.stem.2015.12.004
39. Negoro T, Takagaki Y, Okura H, Matsuyama A. Trends in clinical trials for articular cartilage repair by cell therapy. NPJ Regen Med. 2018; 3:17.
https://doi.org/10.1038/s41536-018-0055-2
PMid:30345076 PMCid:PMC6181982
40. Webber M, Khan O, Sydlik S. A Perspective on the Clinical Translation of Scaffolds for Tissue Engineering Ann Biomed Eng. 2015; 43(3):641-656. DOI:10.1007/s10439-014-1104-7.
https://doi.org/10.1007/s10439-014-1104-7

Kyryk V, Kuchuk O, Mamchur A, Ustymenko A, Lutsenko T, Tsupykov O, Yatsenko K, Skibo G, Bilko D, Bilko N. 3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P. Cell and Organ Transplantology. 2018; 6(2):195-201. doi:10.22494/cot.v6i2.91

Creative Commons License
Is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.