Skip to main content

Advertisement

SpringerLink
Log in

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation

  • Original Articles
  • Basic Science
  • Published:
Aesthetic Plastic Surgery Aims and scope Submit manuscript

Abstract

Background

Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal.

Objectives

To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy.

Methods

BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared.

Results

The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential.

Conclusions

300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy.

No Level Assigned

This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266

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
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. P Horn S Bork A Diehlmann 2008 Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis Cytotherapy 10 7 676 685

    Article  CAS  PubMed  Google Scholar 

  2. Z Gudleviciene G Kundrotas R Liudkeviciene 2014 Quick and effective method of bone marrow mesenchymal stem cell extraction Open Med (Wars) 10 1 44 49

    PubMed  Google Scholar 

  3. A Peterbauer-Scherb M Griensven van A Meinl 2010 Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration J Tissue Eng Regen Med 4 6 485 490

    CAS  PubMed  Google Scholar 

  4. F Du Q Wang L Ouyang 2021 Comparison of concentrated fresh mononuclear cells and cultured mesenchymal stem cells from bone marrow for bone regeneration Stem Cells Transl Med 10 4 598 609

    Article  CAS  PubMed  Google Scholar 

  5. T Hisatome Y Yasunaga S Yanada 2005 Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells Biomaterials 26 22 4550 4556

    Article  CAS  PubMed  Google Scholar 

  6. P Hernigou A Poignard F Beaujean 2005 Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells J Bone Joint Surg Am 87 7 1430 1437

    PubMed  Google Scholar 

  7. F Du H Wu H Li 2017 Bone marrow mononuclear cells combined with beta-tricalcium phosphate granules for alveolar cleft repair: a 12-month clinical study Sci Rep 7 1 13773

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  8. A Minamide M Yoshida M Kawakami 2005 The use of cultured bone marrow cells in type I collagen gel and porous hydroxyapatite for posterolateral lumbar spine fusion Spine 30 10 1134 1138

    Article  PubMed  Google Scholar 

  9. J Connolly R Guse L Lippiello 1989 Development of an osteogenic bone-marrow preparation J Bone Joint Surg Am 71 5 684 691

    Article  CAS  PubMed  Google Scholar 

  10. M Scarpone D Kuebler A Chambers 2019 Isolation of clinically relevant concentrations of bone marrow mesenchymal stem cells without centrifugation J Transl Med 17 1 10

    Article  PubMed  PubMed Central  Google Scholar 

  11. FH Seeger T Tonn N Krzossok 2007 Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction Eur Heart J 28 6 766 772

    Article  PubMed  Google Scholar 

  12. RT Beem van A Hirsch IM Lommerse 2008 Recovery and functional activity of mononuclear bone marrow and peripheral blood cells after different cell isolation protocols used in clinical trials for cell therapy after acute myocardial infarction EuroIntervention 4 1 133 138

    Article  PubMed  Google Scholar 

  13. C Pösel K Möller W Fröhlich 2012 Density gradient centrifugation compromises bone marrow mononuclear cell yield PLoS ONE 7 12 e50293

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  14. N Ahmadbeigi M Soleimani F Babaeijandaghi 2012 The aggregate nature of human mesenchymal stromal cells in native bone marrow Cytotherapy 14 8 917 924

    Article  CAS  PubMed  Google Scholar 

  15. S Sampson A Botto-van Bemden D Aufiero 2013 Autologous bone marrow concentrate: review and application of a novel intra-articular orthobiologic for cartilage disease Phys Sportsmed 41 3 7 18

    Article  PubMed  Google Scholar 

  16. R Schäfer MR DeBaun E Fleck 2019 Quantitation of progenitor cell populations and growth factors after bone marrow aspirate concentration J Transl Med 17 1 115

    Article  PubMed  PubMed Central  Google Scholar 

  17. AD Ho W Wagner W Franke 2008 Heterogeneity of mesenchymal stromal cell preparations Cytotherapy 10 4 320 330

    Article  CAS  PubMed  Google Scholar 

  18. DC Colter I Sekiya DJ Prockop 2001 Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells Proc Natl Acad Sci U S A 98 14 7841 7845

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. DJ Prockop I Sekiya DC Colter 2001 Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells Cytotherapy 3 5 393 396

    Article  CAS  PubMed  Google Scholar 

  20. W Wagner AD Ho 2007 Mesenchymal stem cell preparations–comparing apples and oranges Stem Cell Rev 3 4 239 248

    Article  PubMed  Google Scholar 

  21. NY Naung S Suttapreyasri S Kamolmatyakul 2014 Comparative study of different centrifugation protocols for a density gradient separation media in isolation of osteoprogenitors from bone marrow aspirate J Oral Biol Craniofac Res 4 3 160 168

    Article  PubMed  PubMed Central  Google Scholar 

  22. A Strasser E Kalmar H Niedermüller 1998 A simple method for the simultaneous separation of peripheral blood mononuclear and polymorphonuclear cells in the dog Vet Immunol Immunopathol 62 1 29 35

    Article  CAS  PubMed  Google Scholar 

  23. R Harris EO Ukaejiofo 1969 Rapid preparation of lymphocytes for tissue-typing Lancet 2 7615 327

    Article  CAS  PubMed  Google Scholar 

  24. Z Li D Mu C Liu 2020 The cell yields and biological characteristics of stromal/stem cells from lipoaspirate with different digestion loading ratio Cytotechnology 72 2 203 215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. AJ Sun L Qiao C Huang 2018 Comparison of mouse brown and white adipose-derived stem cell differentiation into pacemaker-like cells induced by TBX18 transduction Mol Med Rep 17 5 7055 7064

    CAS  PubMed  PubMed Central  Google Scholar 

  26. WJ Lee YS Hah SA Ock 2015 Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs Exp Cell Res 333 2 273 288

    Article  CAS  PubMed  Google Scholar 

  27. J Chahla CS Dean G Moatshe 2016 Concentrated bone marrow aspirate for the treatment of chondral injuries and osteoarthritis of the knee: a systematic review of outcomes Orthop J Sports Med 4 1 2325967115625481

    Article  PubMed  PubMed Central  Google Scholar 

  28. GB Kim MS Seo WT Park 2020 Bone marrow aspirate concentrate: its uses in osteoarthritis Int J Mol Sci 21 9 3224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. S Kirana B Stratmann C Prante 2012 Autologous stem cell therapy in the treatment of limb ischaemia induced chronic tissue ulcers of diabetic foot patients Int J Clin Pract 66 4 384 393

    Article  CAS  PubMed  Google Scholar 

  30. M Yang L Sheng H Li 2010 Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model Microsurgery 30 4 275 281

    Article  PubMed  Google Scholar 

  31. W Xing D Mu Q Wang 2016 Improvement of fat graft survival with autologous bone marrow aspirate and bone marrow concentrate: a one-step method Plast Reconstr Surg 137 4 676e 686e

    Article  CAS  PubMed  Google Scholar 

  32. HH Al-Ahmady AF Abd Elazeem NE Bellah Ahmed 2018 Combining autologous bone marrow mononuclear cells seeded on collagen sponge with nano hydroxyapatite, and platelet-rich fibrin: reporting a novel strategy for alveolar cleft bone regeneration J Craniomaxillofac Surg 46 9 1593 1600

    Article  PubMed  Google Scholar 

  33. DF Hergemöller AA Pelegrine PJ Pasquali 2022 Osteocalcin and runx2 expression in anterior maxillary reconstructions using bone xenografts associated to bone marrow aspirate concentrate Contemp Clin Dent 13 3 211 216

    Article  PubMed  PubMed Central  Google Scholar 

  34. S Sauerbier B Giessenhagen W Gutwerk 2013 Bone marrow aspirate concentrate used with bovine bone mineral to reconstruct vertical and horizontal mandibular defects: report of two techniques Int J Oral Maxillofac Implants 28 5 e310 e314

    Article  PubMed  Google Scholar 

  35. OM Griffith 2010 Practical techniques for centrifugal separations Thermo Fisher Scientific USA

    Google Scholar 

  36. B Assmus T Tonn FH Seeger 2010 Red blood cell contamination of the final cell product impairs the efficacy of autologous bone marrow mononuclear cell therapy J Am Coll Cardiol 55 13 1385 1394

    Article  PubMed  Google Scholar 

  37. K Shortman 1968 The separation of different cell classes from lymphoid organs. II. The purification and analysis of lymphocyte populations by equilibrium density gradient centrifugation Aust J Exp Biol Med Sci 46 4 375 396

    Article  CAS  PubMed  Google Scholar 

  38. M Radrizzani V Lo Cicero S Soncin 2014 Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function J Transl Med 12 276

    Article  PubMed  PubMed Central  Google Scholar 

  39. DQ Tan T Suda 2018 Reactive oxygen species and mitochondrial homeostasis as regulators of stem cell fate and function Antioxid Redox Signal 29 2 149 168

    Article  CAS  PubMed  Google Scholar 

  40. F Atashi A Modarressi MS Pepper 2015 The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review Stem Cells Dev 24 10 1150 1163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. M Shekarriz DM DeWire AJ Thomas Jr 1995 A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species Eur Urol 28 1 31 35

    Article  CAS  PubMed  Google Scholar 

  42. H Yu T Zhang L Cai 2011 Chamaejasmine induces apoptosis in human lung adenocarcinoma A549 cells through a Ros-mediated mitochondrial pathway Molecules 16 10 8165 8180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. T Thum J Bauersachs PA Poole-Wilson 2005 The dying stem cell hypothesis: immune modulation as a novel mechanism for progenitor cell therapy in cardiac muscle J Am Coll Cardiol 46 10 1799 1802

    Article  CAS  PubMed  Google Scholar 

  44. F Mouquet G Lemesle C Delhaye 2011 The presence of apoptotic bone marrow cells impairs the efficacy of cardiac cell therapy Cell Transplant 20 7 1087 1097

    Article  PubMed  Google Scholar 

  45. P Bosch SL Pratt SL Stice 2006 Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells Biol Reprod 74 1 46 57

    Article  CAS  PubMed  Google Scholar 

  46. M Pierini B Dozza E Lucarelli 2012 Efficient isolation and enrichment of mesenchymal stem cells from bone marrow Cytotherapy 14 6 686 693

    Article  CAS  PubMed  Google Scholar 

  47. AM Meppelink XH Wang G Bradica 2016 Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology Cytotherapy 18 6 729 739

    Article  PubMed  Google Scholar 

Download references

Funding

This study was supported by CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-052).

Author information

Authors and Affiliations

  1. The Second Department of Craniomaxillofacial Surgery, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China

    Tianyi Gu & Li Teng

  2. Cleft Lip and Palate Center, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China

    Kongying Li & Ningbei Yin

  3. Department of Aesthetic and Reconstructive Breast Surgery, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China

    Xiaoyu Zhang

  4. Research Center, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China

    Ran Xiao & Qian Wang

  5. Key Laboratory of External Tissue and Organ Regeneration, Chinese Academy of Medical Sciences, Beijing, China

    Ran Xiao & Qian Wang

Authors
  1. Tianyi Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kongying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaoyu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ran Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ningbei Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qian Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Li Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Qian Wang or Li Teng.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

All animal experiments were approved by Peking Union Medical College, Institutional Animal Care and Use Committee of Plastic Surgery Hospital (Grant No. EAEC 2021-007).

Informed Consent

For this type of study, informed consent is not required.

Additional information

Publisher's Note

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

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gu, T., Li, K., Zhang, X. et al. The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation. Aesth Plast Surg (2024). https://doi.org/10.1007/s00266-024-03892-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00266-024-03892-8

Keywords

Search

Navigation