Skip to main content
SpringerLink
Log in

The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells

  • Technical Note
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Cell line passage number is an important consideration when designing an experiment. At higher passages, it is generally understood that cell health begins to decline and, when this occurs, the result can be variable data. However, there are no specific guidelines regarding optimal passage range, and this information is dependent on cell type. To explore these variabilities, low passage D1 cells were thawed (passage 3) and passaged serially until a much higher number (passage 34). Samples were taken every five passages and analyzed for alkaline phosphatase and triglyceride; also, the gene expression of both adipogenic and osteogenic markers was tested. The results indicate that the growth rate of these cells did slow down after passage 30. However, expression of the osteogenic characteristics seemed to cycle, with the highest levels seen at passage 4 and 24. The adipocyte expression levels remained the same throughout the study.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

References

  • Chung C, Iida-Klein A, Wyatt L, Rudkin G, Ishida K, Yamaguchi D, Miller T (1999) Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta 1 and bone morphogenetic protein-2. Biochem Biophys Res Commun 265:246–251

    Article  CAS  Google Scholar 

  • Clynes M (ed) (1998) Animal cell culture techniques. Springer, Berlin

  • Cui Q, Wang G-J, Balian G (1997) Steroid-induced adipogenesis in a pluripotential cell line from bone marrow. J Bone Joint Surg 79:1054–1063

    CAS  Google Scholar 

  • Devine MJ, Mierisch CM, Jang E, Anderson PC, Balian G (2002) Transplanted bone marrow cells localize to fracture callus in a mouse model. J Orthop Res 20:1232–1239

    Article  Google Scholar 

  • Diduch D, Coe M, Joyner C, Owen M, Balian G (1993) Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization. J Bone Joint Surg 75-A:92–105

    Google Scholar 

  • Freshney RI (2005) Culture of animal cells, 5th edn. Wiley, Hoboken

    Book  Google Scholar 

  • Haghi M, Young PM, Traini D, Jaiswal R, Gong J, Bebawy M (2010) Time- and passage-dependent characteristics of a calu-3 respiratory epithelial cell model. Drug Dev Ind Pharm 36:1207–1214

    Article  CAS  Google Scholar 

  • Hughes P, Marshall D, Reid Y, Parkes H, Gelber C (2007) The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques 43:575–586

    Article  CAS  Google Scholar 

  • Jensen HL, Norrild B (2000) The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection. J Virol Methods 84:139–152

    Article  CAS  Google Scholar 

  • Kim MJ, Kim CW, Choi YS, Kim MH, Park CJ, Suh Y (2012) Age-related alterations in mesenchymal stem cells related to shift in differentiation from osteogenic to adipogenic potential: implication to age-associated bone diseases and defects. Mech Ageing Dev 133:215–225

    Article  Google Scholar 

  • Li X, Jin L, Cui Q, Wang G, Balian G (2005) Steroid effects on osteogenesis through mesenchymal cell gene expression. Osteoporos Int 16:101–108

    Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Maxson S, Burg KJL (2008) Conditioned media causes increases in select osteogenic and adipogenic markers in mesenchymal stem cell cultures. J Tissue Eng Regener Med 2:147–154

    Article  CAS  Google Scholar 

  • Peterson WJ, Tachiki KH, Yamaguchi DT (2004) Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro. Cell Prolif 37:325–336

    Article  CAS  Google Scholar 

  • Prasad Chennazhy K, Krishnan LK (2005) Effect of passage number and matrix characteristics on differentiation of endothelial cells cultured for tissue engineering. Biomaterials 26:5658–5667

    Article  Google Scholar 

  • von Zglinicki T, Saretzki G, Döcke W, Lotze C (1995) Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 220:186–193

    Article  Google Scholar 

  • Wall ME, Bernacki SH, Loboa EG (2007) Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells. Tissue Eng 13:1291–1298

    Article  CAS  Google Scholar 

  • Yang CC, Ellis SE, Xu F, Burg KJL (2007) In vitro regulation of adipogenesis: tunable engineered tissues. J Tissue Eng Regener Med 1:146–153

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Funding was provided by the Clemson University Hunter Endowment.

Author information

Authors and Affiliations

  1. Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC, USA

    K. Kwist

  2. Institute for Biological Interfaces of Engineering and Department of Bioengineering, Clemson University, Clemson, SC, USA

    K. J. L. Burg

  3. Department of Mathematical Sciences, Clemson University, Clemson, SC, USA

    W. C. Bridges

  4. Department of Chemical Engineering, Kansas State University, Manhattan, KS, USA

    K. J. L. Burg

Authors
  1. K. Kwist
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. C. Bridges
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. J. L. Burg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. J. L. Burg.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kwist, K., Bridges, W.C. & Burg, K.J.L. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells. Cytotechnology 68, 1661–1667 (2016). https://doi.org/10.1007/s10616-015-9883-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-015-9883-8

Keywords

Search

Navigation