SpringerLink
Log in

Encapsulated explant in novel low shear perfusion bioreactor improve cell isolation, expansion and colony forming unit

  • Published:
Cell and Tissue Banking Aims and scope Submit manuscript

Abstract

One of most important issue in the field of regenerative medicine is selection of appropriate cells, scaffolds and bioreactors. The present study aimed to investigate the appropriate method for the isolation of human UC-MSCs cells from explant cultured in alginate scaffold within novel perfusion bioreactor. MSCs were isolated with explant method and CD markers such CD73, CD31, CD90 and CD105 as were analyzed by flow cytometry. The culture chamber of the novel perfusion bioreactor was made from Plexiglas and housed the cell/scaffold constructs in the central part and the medium for the whole culture period. The flow behavior within the bioreactor chamber were performed for closed and open bypass systems. The shear stress profiles simulated using CFD modeling. The fluid flow distribution within the bioreactor chamber was performed in PBS solution containing a blue colorant. UC explants were resuspended in sodium alginate and were allowed to polymerize and placed in the perfusion bioreactor and cultured. MSCs were positive for mesenchymal markers such as CD73 and CD31. All 3D Perfusion bioreactor parts, except peristaltic pump was sterilizable by autoclaving. Results of CFD indicated very low wall shear stress on surface of culture chamber at flow rate 2 ml/min. The maximum wall shear stress was 1.10 × 10−3 m/s = 0.0110 dyne/cm2 (1 Pa = 10 dyne/cm2). The fluid flow distribution within the alginate gel initially exhibited oscillation. In comparison, when encapsulated explants were placed in the perfusion bioreactor, cell proliferation appeared faster (4.6 × 1011 ± 9.2 × 1011) than explants cultures in 2D conventional culture method (3.2 × 1011 ± 1 × 1011). Proliferated cell formed several colonies. Migration of chondrocytes to the periphery of the alginate bead was visible after 1 week of culture. Perfusion bioreactor with low shear stress and alginate hydrogel improve cell isolation and expansion and eliminate cell passaging and enhance colony forming unit of UC-MSCs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Thailand)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Alimperti S, Lei P, Wen Y, Tian J, Campbell AM, Andreadis ST (2014) Serum-free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential. Biotechnol Prog 30(4):974–983

    Article  CAS  Google Scholar 

  • Azandeh S, Mohammad Gharravi A, Orazizadeh M, Khodadi A, Hashemi Tabar M (2016) Improvement of mesenchymal stem cell differentiation into the endoderm lineage by four step sequential method in biocompatible biomaterial. BioImpacts: BI 6(1):9–13

    Article  CAS  Google Scholar 

  • Chen AKL, Chew YK, Tan HY, Reuveny S, Oh SKW (2015) Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed. Cytotherapy 17(2):163–173

    Article  Google Scholar 

  • Delaine-Smith RM, Reilly GC (2012) Mesenchymal stem cell responses to mechanical stimuli. Muscles Ligaments Tendons J 2(3):169–180

    PubMed  PubMed Central  Google Scholar 

  • Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J (2007) A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog 23(1):187–193

    Article  CAS  Google Scholar 

  • Gharravi AM, Orazizadeh M, Hashemitabar M, Ansari-Asl K, Banoni S, Alifard A et al (2013) Design and validation of perfusion bioreactor with low shear stress for tissue engineering. J Med Biol Eng 33(2):185–191

    Article  Google Scholar 

  • Gharravi AM, Orazizadeh M, Hashemitabar M (2014) Direct expansion of chondrocytes in a dynamic three-dimensional culture system: overcoming dedifferentiation effects in monolayer culture. Artif Organs 38(12):1053–1058

    Article  CAS  Google Scholar 

  • Han Y-F, Tao R, Sun T-J, Chai J-K, Xu G, Liu J (2013) Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods. Cytotechnology 65(5):819–827

    Article  CAS  Google Scholar 

  • Howard D, Buttery LD, Shakesheff KM, Roberts SJ (2008) Tissue engineering: strategies, stem cells and scaffolds. J Anat 213(1):66–72

    Article  CAS  Google Scholar 

  • Iftimia-Mander A, Hourd P, Dainty R, Thomas RJ (2013) Mesenchymal stem cell isolation from human umbilical cord tissue: understanding and minimizing variability in cell yield for process optimization. Biopreserv Biobank 11(5):291–298

    Article  CAS  Google Scholar 

  • ** HJ, Bae YK, Kim M, Kwon S-J, Jeon HB, Choi SJ et al (2013) Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci 14(9):17986–18001

    Article  Google Scholar 

  • King JA, Miller WM (2007) Bioreactor development for stem cell expansion and controlled differentiation. Curr Opin Chem Biol 11(4):394–398

    Article  CAS  Google Scholar 

  • Kretlow JD, ** YQ, Liu W, Zhang WJ, Hong TH, Zhou G et al (2008) Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 9:60

    Article  Google Scholar 

  • Kumar A, Lau W, Starly B (2017) Human mesenchymal stem cells expansion on three-dimensional (3D) printed poly-styrene (PS) scaffolds in a perfusion bioreactor. Procedia CIRP 65:115–120

    Article  Google Scholar 

  • Lam ATL, Li J, Toh JPW, Sim EJH, Chen AKL, Chan JKY et al (2017) Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors. Cytotherapy 19(3):419–432

    Article  CAS  Google Scholar 

  • Li D, Zhou J, Chowdhury F, Cheng J, Wang N, Wang F (2011) Role of mechanical factors in fate decisions of stem cells. Regener Med 6(2):229–240

    Article  Google Scholar 

  • Martin Y, Vermette P (2005) Bioreactors for tissue mass culture: design, characterization, and recent advances. Biomaterials 26(35):7481–7503

    Article  CAS  Google Scholar 

  • Nagamura-Inoue T, He H (2014) Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility. World J Stem Cells 6(2):195–202

    Article  Google Scholar 

  • O’Brien FJ (2011) Biomaterials & scaffolds for tissue engineering. Mater Today 14(3):88–95

    Article  Google Scholar 

  • Pochampally R (2008) Colony forming unit assays for MSCs. Methods Mol Biol 449:83–91

    PubMed  Google Scholar 

  • Rafiq QA, Coopman K, Nienow AW, Hewitt CJ (2016) Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors. Biotechnol J 11(4):473–486

    Article  CAS  Google Scholar 

  • Salehi-Nik N, Amoabediny G, Pouran B, Tabesh H, Shokrgozar MA, Haghighipour N et al (2013) Engineering parameters in bioreactor’s design: a critical aspect in tissue engineering. Biomed Res Int 2013:762132

    Article  Google Scholar 

  • Santos FD, Andrade PZ, Abecasis MM, Gimble JM, Chase LG, Campbell AM et al (2011) Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods 17(12):1201–1210

    Article  Google Scholar 

  • Schirmaier C, Jossen V, Kaiser SC, Jüngerkes F, Brill S, Safavi-Nab A et al (2014) Scale-up of adipose tissue-derived mesenchymal stem cell production in stirred single-use bioreactors under low-serum conditions. Eng Life Sci 14(3):292–303

    Article  CAS  Google Scholar 

  • Schop D, Janssen FW, Borgart E, de Bruijn JD, van Dijkhuizen-Radersma R (2008) Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism. J Tissue Eng Regener Med 2(2–3):126–135

    Article  CAS  Google Scholar 

  • Venkatesan J, Bhatnagar I, Manivasagan P, Kang KH, Kim SK (2015) Alginate composites for bone tissue engineering: a review. Int J Biol Macromol 72:269–281

    Article  CAS  Google Scholar 

  • Vetsch JR, Betts DC, Müller R, Hofmann S (2017) Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: a combined experimental and computational approach. PLoS ONE 12(7):e0180781

    Article  Google Scholar 

  • Wong M (2004) Alginates in tissue engineering. In: Hollander AP, Hatton PV (eds) Biopolymer methods in tissue engineering. Humana Press, Totowa, pp 77–86

    Google Scholar 

  • Yeatts AB, Choquette DT, Fisher JP (2013) Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems. Biochem Biophys Acta 1830(2):2470–2480

    Article  CAS  Google Scholar 

  • Yu Y, Li K, Bao C, Liu T, ** Y, Ren H et al (2009) Ex vitro expansion of human placenta-derived mesenchymal stem cells in stirred bioreactor. Appl Biochem Biotechnol 159(1):110–118

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study approved in the Shahroud University of Medical Sciences. Special thanks to Shahroud University of Medical Sciences for the support.

Author information

Authors and Affiliations

  1. Tissue Engineering and Stem Cells Research Center, Shahroud University of Medical Sciences, Shahroud, Iran

    Anneh mohammad Gharravi

Authors
  1. Anneh mohammad Gharravi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anneh mohammad Gharravi.

Ethics declarations

Conflict of interest

The author declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gharravi, A. Encapsulated explant in novel low shear perfusion bioreactor improve cell isolation, expansion and colony forming unit. Cell Tissue Bank 20, 25–34 (2019). https://doi.org/10.1007/s10561-019-09749-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10561-019-09749-8

Keywords

Search

Navigation