Advertisement

Bone-chip system to monitor osteogenic differentiation using optical imaging

  • Dmitriy SheynEmail author
  • Doron Cohn-Yakubovich
  • Shiran Ben-David
  • Sandra De Mel
  • Virginia Chan
  • Christopher Hinojosa
  • Norman Wen
  • Geraldine A. Hamilton
  • Dan Gazit
  • Zulma GazitEmail author
Research Paper
  • 155 Downloads

Abstract

Human organoids and organ-on-chip systems to predict human responses to new therapies and for the understanding of disease mechanisms are being more commonly used in translational research. We have developed a bone-chip system to study osteogenic differentiation in vitro, coupled with optical imaging approach which provides the opportunity of monitoring cell survival, proliferation and differentiation in vitro without the need to terminate the culture. We used the mesenchymal stem cell (MSC) line over-expressing bone morphogenetic protein-2 (BMP-2), under Tet-Off system, and luciferase reporter gene under constitutive promoter. Cells were seeded on chips and supplemented with osteogenic medium. Flow of media was started 24 h later, while static cultures were performed using media reservoirs. Cells grown on the bone-chips under constant flow of media showed enhanced survival/proliferation, comparing to the cells grown in static conditions; luciferase reporter gene expression and activity, reflecting the cell survival and proliferation, was quantified using bioluminescence imaging and a significant advantage to the flow system was observed. In addition, the flow had positive effect on osteogenic differentiation, when compared with static cultures. Quantitative fluorescent imaging, performed using the osteogenic extra-cellular matrix-targeted probes, showed higher osteogenic differentiation of the cells under the flow conditions. Gene expression analysis of osteogenic markers confirmed the osteogenic differentiation of the MSC-BMP2 cells. Immunofluorescent staining performed against the Osteocalcin, Col1, and BSP markers illustrated robust osteogenic differentiation in the flow culture and lessened differentiation in the static culture. To sum, the bone-chip allows monitoring cell survival, proliferation, and osteogenic differentiation using optical imaging.

Graphic abstract

Keywords

Organ-on-a-chip Mesenchymal stem cells Osteogenesis Optical Imaging 

Notes

Compliance with ethical standards

Conflict of interest

DS, DCY, SBD, SADM, VC, DG and ZG has nothing to declare. CH, NW and GAH are employees of Emulate Inc.

Supplementary material

10404_2019_2261_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 15 kb)

References

  1. Aslan H, Zilberman Y, Arbeli V, Sheyn D, Matan Y, Liebergall M et al (2006) Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration. Tissue Eng 12(4):877–889.  https://doi.org/10.1089/ten.2006.12.877 CrossRefGoogle Scholar
  2. Ben Arav A, Pelled G, Zilberman Y, Kimelman-Bleich N, Gazit Z, Schwarz EM et al (2012) Adeno-associated virus-coated allografts: a novel approach for cranioplasty. J Tissue Eng Regen Med 6(10):e43–e50.  https://doi.org/10.1002/term.1594 CrossRefGoogle Scholar
  3. Benayahu D, Kletter Y, Zipori D, Wientroub S (1989) Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo. J Cell Physiol 140(1):1–7.  https://doi.org/10.1002/jcp.1041400102 CrossRefGoogle Scholar
  4. Bergmann S, Rohde M, Schughart K, Lengeling A (2013) The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice. Gut Pathog 5(1):19.  https://doi.org/10.1186/1757-4749-5-19 CrossRefGoogle Scholar
  5. Bhatia SN, Ingber DE (2014) Microfluidic organs-on-chips. Nat Biotechnol 32(8):760–772.  https://doi.org/10.1038/nbt.2989 CrossRefGoogle Scholar
  6. Bhise NS, Ribas J, Manoharan V, Zhang YS, Polini A, Massa S et al (2014) Organ-on-a-chip platforms for studying drug delivery systems. J Control Release 190:82–93.  https://doi.org/10.1016/j.jconrel.2014.05.004 CrossRefGoogle Scholar
  7. Bhise NS, Manoharan V, Massa S, Tamayol A, Ghaderi M, Miscuglio M et al (2016) A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication 8(1):014101.  https://doi.org/10.1088/1758-5090/8/1/014101 CrossRefGoogle Scholar
  8. Chang J, Liu F, Lee M, Wu B, Ting K, Zara JN et al (2013) NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation. Proc Natl Acad Sci USA 110(23):9469–9474.  https://doi.org/10.1073/pnas.1300532110 CrossRefGoogle Scholar
  9. Cohn Yakubovich D, Tawackoli W, Sheyn D, Kallai I, Da X, Pelled G et al (2015) Computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts. J Vis Exp.  https://doi.org/10.3791/53459 CrossRefGoogle Scholar
  10. Cohn Yakubovich D, Eliav U, Yalon E, Schary Y, Sheyn D, Cook-Wiens G et al (2017a) Teriparatide attenuates scarring around murine cranial bone allograft via modulation of angiogenesis. Bone 97:192–200.  https://doi.org/10.1016/j.bone.2017.01.020 CrossRefGoogle Scholar
  11. Cohn Yakubovich D, Sheyn D, Bez M, Schary Y, Yalon E, Sirhan A et al (2017b) Systemic administration of mesenchymal stem cells combined with parathyroid hormone therapy synergistically regenerates multiple rib fractures. Stem Cell Res Ther 8(1):51.  https://doi.org/10.1186/s13287-017-0502-9 CrossRefGoogle Scholar
  12. Esch EW, Bahinski A, Huh D (2015) Organs-on-chips at the frontiers of drug discovery. Nat Rev Drug Discov 14(4):248–260.  https://doi.org/10.1038/nrd4539 CrossRefGoogle Scholar
  13. Gomes ME, Rodrigues MT, Domingues RMA, Reis RL (2017) Tissue engineering and regenerative medicine: new trends and directions—a year in review. Tissue Eng Part B Rev 23(3):211–224.  https://doi.org/10.1089/ten.TEB.2017.0081 CrossRefGoogle Scholar
  14. Hasharoni A, Zilberman Y, Turgeman G, Helm GA, Liebergall M, Gazit D (2005) Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein-2. J Neurosurg Spine 3(1):47–52CrossRefGoogle Scholar
  15. Hoemann CD, El-Gabalawy H, McKee MD (2009) In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathol Biol (Paris) 57(4):318–323.  https://doi.org/10.1016/j.patbio.2008.06.004 CrossRefGoogle Scholar
  16. Homan KA, Kolesky DB, Skylar-Scott MA, Herrmann J, Obuobi H, Moisan A et al (2016) Bioprinting of 3D convoluted renal proximal tubules on perfusable chips. Sci Rep 6:34845.  https://doi.org/10.1038/srep34845 CrossRefGoogle Scholar
  17. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE (2010) Reconstituting organ-level lung functions on a chip. Science 328(5986):1662–1668.  https://doi.org/10.1126/science.1188302 CrossRefGoogle Scholar
  18. Huh D, Hamilton GA, Ingber DE (2011) From 3D cell culture to organs-on-chips. Trends Cell Biol 21(12):745–754.  https://doi.org/10.1016/j.tcb.2011.09.005 CrossRefGoogle Scholar
  19. Huh D, Kim HJ, Fraser JP, Shea DE, Khan M, Bahinski A et al (2013) Microfabrication of human organs-on-chips. Nat Protoc 8(11):2135–2157.  https://doi.org/10.1038/nprot.2013.137 CrossRefGoogle Scholar
  20. Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. FASEB J 20(7):811–827.  https://doi.org/10.1096/fj.05-5424rev CrossRefGoogle Scholar
  21. Jain A, van der Meer AD, Papa AL, Barrile R, Lai A, Schlechter BL et al (2016) Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium. Biomed Microdevices 18(4):73.  https://doi.org/10.1007/s10544-016-0095-6 CrossRefGoogle Scholar
  22. Kim HJ, Huh D, Hamilton G, Ingber DE (2012) Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip 12(12):2165–2174.  https://doi.org/10.1039/c2lc40074j CrossRefGoogle Scholar
  23. Kim S, Lee H, Chung M, Jeon NL (2013) Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip 13(8):1489–1500.  https://doi.org/10.1039/c3lc41320a CrossRefGoogle Scholar
  24. Kimelman-Bleich N, Pelled G, Sheyn D, Kallai I, Zilberman Y, Mizrahi O et al (2009) The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo. Biomaterials 30(27):4639–4648.  https://doi.org/10.1016/j.biomaterials.2009.05.027 CrossRefGoogle Scholar
  25. Kolesky DB, Homan KA, Skylar-Scott MA, Lewis JA (2016) Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci USA 113(12):3179–3184.  https://doi.org/10.1073/pnas.1521342113 CrossRefGoogle Scholar
  26. Korin N, Kanapathipillai M, Matthews BD, Crescente M, Brill A, Mammoto T et al (2012) Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels. Science 337(6095):738–742.  https://doi.org/10.1126/science.1217815 CrossRefGoogle Scholar
  27. Liu L, Yu B, Chen J, Tang Z, Zong C, Shen D et al (2012) Different effects of intermittent and continuous fluid shear stresses on osteogenic differentiation of human mesenchymal stem cells. Biomech Model Mechanobiol 11(3–4):391–401.  https://doi.org/10.1007/s10237-011-0319-x CrossRefGoogle Scholar
  28. Maschmeyer I, Lorenz AK, Schimek K, Hasenberg T, Ramme AP, Hübner J et al (2015) A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents. Lab Chip 15(12):2688–2699.  https://doi.org/10.1039/c5lc00392j CrossRefGoogle Scholar
  29. Moore NM, Lin NJ, Gallant ND, Becker ML (2011) Synergistic enhancement of human bone marrow stromal cell proliferation and osteogenic differentiation on BMP-2-derived and RGD peptide concentration gradients. Acta Biomater 7(5):2091–2100.  https://doi.org/10.1016/j.actbio.2011.01.019 CrossRefGoogle Scholar
  30. Moutsatsos IK, Turgeman G, Zhou S, Kurkalli BG, Pelled G, Tzur L et al (2001) Exogenously regulated stem cell-mediated gene therapy for bone regeneration. Mol Ther 3(4):449–461.  https://doi.org/10.1006/mthe.2001.0291 CrossRefGoogle Scholar
  31. Ocak M, Gillman AG, Bresee J, Zhang L, Vlad AM, Müller C et al (2015) Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model. Mol Pharm 12(2):542–553.  https://doi.org/10.1021/mp500628g CrossRefGoogle Scholar
  32. Pelled G, Tai K, Sheyn D, Zilberman Y, Kumbar S, Nair LS et al (2007) Structural and nanoindentation studies of stem cell-based tissue-engineered bone. J Biomech 40(2):399–411.  https://doi.org/10.1016/j.jbiomech.2005.12.012 CrossRefGoogle Scholar
  33. Polacheck WJ, German AE, Mammoto A, Ingber DE, Kamm RD (2014) Mechanotransduction of fluid stresses governs 3D cell migration. Proc Natl Acad Sci USA 111(7):2447–2452.  https://doi.org/10.1073/pnas.1316848111 CrossRefGoogle Scholar
  34. Riahi R, Shaegh SA, Ghaderi M, Zhang YS, Shin SR, Aleman J et al (2016) Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers. Sci Rep 6:24598.  https://doi.org/10.1038/srep24598 CrossRefGoogle Scholar
  35. Ryoo HM, Lee MH, Kim YJ (2006) Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene 366(1):51–57.  https://doi.org/10.1016/j.gene.2005.10.011 CrossRefGoogle Scholar
  36. Shanmugam VK, Tassi E, Schmidt MO, McNish S, Baker S, Attinger C et al (2015) Utility of a human-mouse xenograft model and in vivo near-infrared fluorescent imaging for studying wound healing. Int Wound J 12(6):699–705.  https://doi.org/10.1111/iwj.12205 CrossRefGoogle Scholar
  37. Sheyn D, Kallai I, Tawackoli W, Cohn Yakubovich D, Oh A, Su S et al (2011) Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Mol Pharm 8(5):1592–1601.  https://doi.org/10.1021/mp200226c CrossRefGoogle Scholar
  38. Sheyn D, Yakubovich DC, Kallai I, Su S, Da X, Pelled G et al (2013) PTH promotes allograft integration in a calvarial bone defect. Mol Pharm 10(12):4462–4471.  https://doi.org/10.1021/mp400292p CrossRefGoogle Scholar
  39. Sheyn D, Shapiro G, Tawackoli W, Jun DS, Koh Y, Kang KB et al (2016) PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Mol Ther 24(2):318–330.  https://doi.org/10.1038/mt.2015.211 CrossRefGoogle Scholar
  40. Shuler ML (2017) Organ-, body- and disease-on-a-chip systems. Lab Chip 17(14):2345–2346.  https://doi.org/10.1039/c7lc90068f CrossRefGoogle Scholar
  41. Sung JH, Esch MB, Prot JM, Long CJ, Smith A, Hickman JJ et al (2013) Microfabricated mammalian organ systems and their integration into models of whole animals and humans. Lab Chip 13(7):1201–1212.  https://doi.org/10.1039/c3lc41017j CrossRefGoogle Scholar
  42. Syftestad GT, Weitzhandler M, Caplan AI (1985) Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia. Dev Biol 110(2):275–283CrossRefGoogle Scholar
  43. Tai K, Pelled G, Sheyn D, Bershteyn A, Han L, Kallai I et al (2008) Nanobiomechanics of repair bone regenerated by genetically modified mesenchymal stem cells. Tissue Eng Part A 14(10):1709–1720.  https://doi.org/10.1089/ten.tea.2007.0241 CrossRefGoogle Scholar
  44. Tobias G, Uwe H, Tobias G (2016) Pantoprazol inhibits the stimulating effect for bone formation of diclofenac in vitro evaluated by the novel method of 99m-Tc-HDP-labeling in vitro. J Nucl Med 57:1239Google Scholar
  45. Wang B, Lee WY, Huang B, Zhang JF, Wu T, Jiang X et al (2016) Secretome of human fetal mesenchymal stem cell ameliorates replicative senescen. Stem Cells Dev 25(22):1755–1766.  https://doi.org/10.1089/scd.2016.0079 CrossRefGoogle Scholar
  46. Wobma H, Vunjak-Novakovic G (2016) Tissue engineering and regenerative medicine 2015: a year in review. Tissue Eng Part B Rev 22(2):101–113.  https://doi.org/10.1089/ten.TEB.2015.0535 CrossRefGoogle Scholar
  47. Woolf EC, Curley KL, Liu Q, Turner GH, Charlton JA, Preul MC et al (2015) The ketogenic diet alters the hypoxic response and affects expression of proteins associated with angiogenesis, invasive potential and vascular permeability in a mouse glioma model. PLoS One 10(6):e0130357.  https://doi.org/10.1371/journal.pone.0130357 CrossRefGoogle Scholar
  48. Xie C, Reynolds D, Awad H, Rubery PT, Pelled G, Gazit D et al (2007) Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng 13(3):435–445CrossRefGoogle Scholar
  49. Xu H, Othman SF, Hong L, Peptan IA, Magin RL (2006) Magnetic resonance microscopy for monitoring osteogenesis in tissue-engineered construct in vitro. Phys Med Biol 51(3):719–732.  https://doi.org/10.1088/0031-9155/51/3/016 CrossRefGoogle Scholar
  50. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ (2002) Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Investig 109(11):1405–1415CrossRefGoogle Scholar
  51. Zhang Y, Gazit Z, Pelled G, Gazit D, Vunjak-Novakovic G (2011) Patterning osteogenesis by inducible gene expression in microfluidic culture systems. Integr Biol (Camb) 3(1):39–47.  https://doi.org/10.1039/c0ib00053a CrossRefGoogle Scholar
  52. Zhang YS, Arneri A, Bersini S, Shin SR, Zhu K, Goli-Malekabadi Z et al (2016) Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomaterials 110:45–59.  https://doi.org/10.1016/j.biomaterials.2016.09.003 CrossRefGoogle Scholar
  53. Zhang YS, Aleman J, Shin SR, Kilic T, Kim D, Mousavi Shaegh SA et al (2017a) Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors. Proc Natl Acad Sci USA 114(12):E2293–E2302.  https://doi.org/10.1073/pnas.1612906114 CrossRefGoogle Scholar
  54. Zhang YS, Zhang YN, Zhang W (2017b) Cancer-on-a-chip systems at the frontier of nanomedicine. Drug Discov Today.  https://doi.org/10.1016/j.drudis.2017.03.011 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Dmitriy Sheyn
    • 1
    • 2
    • 3
    • 4
    • 5
    Email author
  • Doron Cohn-Yakubovich
    • 6
  • Shiran Ben-David
    • 2
    • 4
    • 7
  • Sandra De Mel
    • 2
    • 4
    • 7
  • Virginia Chan
    • 2
    • 4
    • 7
  • Christopher Hinojosa
    • 8
  • Norman Wen
    • 8
  • Geraldine A. Hamilton
    • 8
  • Dan Gazit
    • 2
    • 3
    • 4
    • 6
    • 7
  • Zulma Gazit
    • 2
    • 3
    • 4
    • 6
    • 7
    Email author
  1. 1.Orthopedic Stem Cell Research LabCedars-Sinai Medical Center, AHSP-A8308Los AngelesUSA
  2. 2.Board of Governors Regenerative Medicine InstituteCedars-Sinai Medical CenterLos AngelesUSA
  3. 3.Department of OrthopaedicsCedars-Sinai Medical CenterLos AngelesUSA
  4. 4.Department of SurgeryCedars-Sinai Medical CenterLos AngelesUSA
  5. 5.Department of Biomedical SciencesCedars-Sinai Medical CenterLos AngelesUSA
  6. 6.Skeletal Biotech LaboratoryHebrew University of JerusalemJerusalemIsrael
  7. 7.Skeletal Regeneration ProgramCedars-Sinai Medical Center, AHSP-8304Los AngelesUSA
  8. 8.Emulate Inc.BostonUSA

Personalised recommendations