Cell and Tissue Research

, Volume 358, Issue 2, pp 395–405 | Cite as

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

  • Christina L. Rettinger
  • Andrea B. Fourcaudot
  • Seok J. Hong
  • Thomas A. Mustoe
  • Robert G. Hale
  • Kai P. LeungEmail author
Regular Article


Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2’-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.


Mesenchymal stem cells Adipose-derived stem cells Cell aggregates/spheroids Three-dimensional culture Wound healing Scarring Rabbit 



The authors thank Dr. Wei Xu for the isolation of rabbit dermal fibroblasts. TEM samples were processed and imaged in the Electron Microscopy Core Facility, Department of Pathology, The University of Texas Health Science Center, San Antonio, Tex., USA. The authors are also grateful to Dr. Tao You for assistance with generating the SEM images.

Supplementary material

441_2014_1939_Fig7_ESM.gif (50 kb)
Supplemental Figure 1

Confocal microscopic images of Live/Dead stain on ASC aggregates of three representative cell densities that have been maintained for 7 days in culture: (a) 250 cells/aggregate, (b) 1000 cells/aggregate, (c) 16000 cells/aggregate. Live or viable cells were stained green (calcein AM), whereas dead or compromised cells were stained red (ethidium homodimer-1). Non-viable cells were evident in ASC aggregates that were larger in size (greater than 4000 cells/aggregate). Magnification of images is 200×. (GIF 49 kb)

441_2014_1939_MOESM1_ESM.tif (1.7 mb)
High resolution (TIFF 1703 kb)
441_2014_1939_Fig8_ESM.gif (93 kb)
Supplemental Figure 2

Fluorescent images of ASC aggregates (at 500 cells/aggregate) labeled with EdU and counterstained with Hoechst after 7 days in culture (bottom panel, d-f). Images of ASC monolayers labeled with EdU and counterstained with Hoechst (top panel, a-c). Limited cell proliferation (low abundance of EdU-positive cells) was observed in ASC aggregates throughout the experiment, regardless of the size of the aggregates. Magnification of images is 100× (GIF 92 kb)

441_2014_1939_MOESM2_ESM.tif (3.9 mb)
High resolution (TIFF 4043 kb)


  1. Amos PJ, Kapur SK, Stapor PC, Shang H, Bekiranov S, Khurgel M, Rodeheaver GT, Peirce SM, Katz AJ (2010) Human adipose-derived stromal cells accelerate diabetic wound healing: impact of cell formulation and delivery. Tissue Eng A 16:1595–1606. doi: 10.1089/ten.TEA.2009.0616 CrossRefGoogle Scholar
  2. Baharvand H, Hashemi SM, Shahsavani M (2008) Differentiation of human embryonic stem cells into functional hepatocyte-like cells in a serum-free adherent culture condition. Differentiation 76:465–477. doi: 10.1111/j.1432-0436.2007.00252.x PubMedCrossRefGoogle Scholar
  3. Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R (2000) Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 28:707–715PubMedCrossRefGoogle Scholar
  4. Baraniak PR, McDevitt TC (2012) Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res 347:701–711. doi: 10.1007/s00441-011-1215-5 PubMedCrossRefPubMedCentralGoogle Scholar
  5. Bartosh TJ, Ylostalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K, Lee RH, Choi H, Prockop DJ (2010) Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107:13724–13729. doi: 10.1073/pnas.1008117107 PubMedCrossRefPubMedCentralGoogle Scholar
  6. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22:675–682. doi: 10.1634/stemcells.22-5-675 PubMedCrossRefGoogle Scholar
  7. Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME (1992) Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 102:341–351PubMedGoogle Scholar
  8. Bieback K, Kern S, Kluter H, Eichler H (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625–634. doi: 10.1634/stemcells.22-4-625 PubMedCrossRefGoogle Scholar
  9. Bittinger F, Brochhausen C, Skarke C, Kohler H, Kirkpatrick CJ (1997) Reconstruction of peritoneal-like structure in three-dimensional collagen gel matrix culture. Exp Cell Res 236:155–160. doi: 10.1006/excr.1997.3724 PubMedCrossRefGoogle Scholar
  10. Caplan AI (1991) Mesenchymal stem cells. J Orthop Res 9:641–650. doi: 10.1002/jor.1100090504 PubMedCrossRefGoogle Scholar
  11. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV (1994) Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286PubMedGoogle Scholar
  12. Colwell AS, Beanes SR, Soo C, Dang C, Ting K, Longaker MT, Atkinson JB, Lorenz HP (2005) Increased angiogenesis and expression of vascular endothelial growth factor during scarless repair. Plast Reconstr Surg 115:204–212PubMedGoogle Scholar
  13. Cook MM, Futrega K, Osiecki M, Kabiri M, Kul B, Rice A, Atkinson K, Brooke G, Doran M (2012) Micromarrows-three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells. Tissue Eng Part C Methods 18:319–328. doi: 10.1089/ten.TEC.2011.0159 PubMedCrossRefGoogle Scholar
  14. Cukierman E, Pankov R, Stevens DR, Yamada KM (2001) Taking cell-matrix adhesions to the third dimension. Science 294:1708–1712. doi: 10.1126/science.1064829 PubMedCrossRefGoogle Scholar
  15. Curcio E, Salerno S, Barbieri G, De Bartolo L, Drioli E, Bader A (2007) Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system. Biomaterials 28:5487–5497. doi: 10.1016/j.biomaterials.2007.08.033 PubMedCrossRefGoogle Scholar
  16. Davidson JM (1998) Animal models for wound repair. Arch Dermatol Res 290 (Suppl):S1–S11PubMedCrossRefGoogle Scholar
  17. Dykstra M, Reuss LE (2003) Biological electron microscopy: theory, techniques, and troubleshooting. Springer, New YorkCrossRefGoogle Scholar
  18. Fukuda J, Okamura K, Nakazawa K, Ijima H, Yamashita Y, Shimada M, Shirabe K, Tsujita E, Sugimachi K, Funatsu K (2003) Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2). Cell Transplant 12:51–58PubMedCrossRefGoogle Scholar
  19. Glicklis R, Merchuk JC, Cohen S (2004) Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions. Biotechnol Bioeng 86:672–680. doi: 10.1002/bit.20086 PubMedCrossRefGoogle Scholar
  20. Hong SJ, Jia SX, Xie P, Xu W, Leung KP, Mustoe TA, Galiano RD (2013) Topically delivered adipose derived stem cells show an activated-fibroblast phenotype and enhance granulation tissue formation in skin wounds. PLoS ONE 8:e55640. doi: 10.1371/journal.pone.0055640 PubMedCrossRefPubMedCentralGoogle Scholar
  21. Hosokawa R, Nonaka K, Morifuji M, Shum L, Ohishi M (2003) TGF-beta 3 decreases type I collagen and scarring after labioplasty. J Dent Res 82:558–564PubMedCrossRefGoogle Scholar
  22. Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM (2002) Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 30:896–904PubMedCrossRefGoogle Scholar
  23. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238:265–272. doi: 10.1006/excr.1997.3858 PubMedCrossRefGoogle Scholar
  24. Kabiri M, Kul B, Lott WB, Futrega K, Ghanavi P, Upton Z, Doran MR (2012) 3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling. Biochem Biophys Res Commun 419:142–147. doi: 10.1016/j.bbrc.2012.01.017 PubMedCrossRefGoogle Scholar
  25. Kapur SK, Wang X, Shang H, Yun S, Li X, Feng G, Khurgel M, Katz AJ (2012) Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids. Biofabrication 4:025004. doi: 10.1088/1758-5082/4/2/025004 PubMedCrossRefPubMedCentralGoogle Scholar
  26. Keese CR, Wegener J, Walker SR, Giaever I (2004) Electrical wound-healing assay for cells in vitro. Proc Natl Acad Sci U S A 101:1554–1559. doi: 10.1073/pnas.0307588100 PubMedCrossRefPubMedCentralGoogle Scholar
  27. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180. doi: 10.1002/bit.10655 PubMedCrossRefGoogle Scholar
  28. Kilroy GE, Foster SJ, Wu X, Ruiz J, Sherwood S, Heifetz A, Ludlow JW, Stricker DM, Potiny S, Green P, Halvorsen YD, Cheatham B, Storms RW, Gimble JM (2007) Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol 212:702–709. doi: 10.1002/jcp.21068 PubMedCrossRefGoogle Scholar
  29. Kloeters OTA, Mustoe TA (2007) Hypertrophic scar model in the rabbit ear: a reproducible model for studying scar tissue behavior with new observations on silicone gel sheeting for scar reduction. Wound Repair Regen 15 (Suppl 1):S40–S45PubMedCrossRefGoogle Scholar
  30. Kurosawa H (2007) Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103:389–398. doi: 10.1263/jbb.103.389 PubMedCrossRefGoogle Scholar
  31. Mansilla E, Marin GH, Sturla F, Drago HE, Gil MA, Salas E, Gardiner MC, Piccinelli G, Bossi S, Salas E, Petrelli L, Iorio G, Ramos CA, Soratti C (2005) Human mesenchymal stem cells are tolerized by mice and improve skin and spinal cord injuries. Transplant Proc 37:292–294. doi: 10.1016/j.transproceed.2005.01.070 PubMedCrossRefGoogle Scholar
  32. Markway BD, Tan GK, Brooke G, Hudson JE, Cooper-White JJ, Doran MR (2010) Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant 19:29–42. doi: 10.3727/096368909X478560 PubMedCrossRefGoogle Scholar
  33. Morris DE, Wu L, Zhao LL, Bolton L, Roth SI, Ladin DA, Mustoe TA (1997) Acute and chronic animal models for excessive dermal scarring: quantitative studies. Plast Reconstr Surg 100:674–681PubMedCrossRefGoogle Scholar
  34. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147PubMedCrossRefGoogle Scholar
  35. Reiser J, Zhang XY, Hemenway CS, Mondal D, Pradhan L, La Russa VF (2005) Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther 5:1571–1584. doi: 10.1517/14712598.5.12.1571 PubMedCrossRefPubMedCentralGoogle Scholar
  36. Sisco M, Kryger ZB, O’Shaughnessy KD, Kim PS, Schultz GS, Ding XZ, Roy NK, Dean NM, Mustoe TA (2008) Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo. Wound Repair 16:661–673. doi: 10.1111/j.1524-475X.2008.00416.x CrossRefGoogle Scholar
  37. Takahashi T, Ogasawara T, Asawa Y, Mori Y, Uchinuma E, Takato T, Hoshi K (2007) Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture. Tissue Eng 13:1583–1592. doi: 10.1089/ten.2006.0322 PubMedCrossRefGoogle Scholar
  38. Timmins NE, Dietmair S, Nielsen LK (2004) Hanging-drop multicellular spheroids as a model of tumour angiogenesis. Angiogenesis 7:97–103. doi: 10.1007/s10456-004-8911-7 PubMedCrossRefGoogle Scholar
  39. Tong JZ, Sarrazin S, Cassio D, Gauthier F, Alvarez F (1994) Application of spheroid culture to human hepatocytes and maintenance of their differentiation. Biol Cell 81:77–81PubMedCrossRefGoogle Scholar
  40. Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW (2008) Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS ONE 3:e1565. doi: 10.1371/journal.pone.0001565 PubMedCrossRefPubMedCentralGoogle Scholar
  41. Wakitani S, Saito T, Caplan AI (1995) Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18:1417–1426. doi: 10.1002/mus.880181212 PubMedCrossRefGoogle Scholar
  42. Wang Y, Kim UJ, Blasioli DJ, Kim HJ, Kaplan DL (2005) In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells. Biomaterials 26:7082–7094. doi: 10.1016/j.biomaterials.2005.05.022 PubMedCrossRefGoogle Scholar
  43. Wu L, Siddiqui A, Morris DE, Cox DA, Roth SI, Mustoe TA (1997) Transforming growth factor beta 3 (TGF beta 3) accelerates wound healing without alteration of scar prominence. Histologic and competitive reverse-transcription-polymerase chain reaction studies. Arch Surg 132:753–760PubMedCrossRefGoogle Scholar
  44. Wu Y, Chen L, Scott PG, Tredget EE (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25:2648–2659. doi: 10.1634/stemcells.2007-0226 PubMedCrossRefGoogle Scholar
  45. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins K, Thomas K, Austin T, Edwards C, Cuzzourt J, Duenzl M, Lucas PA, Black AC Jr (2001) Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 264:51–62PubMedCrossRefGoogle Scholar
  46. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228. doi: 10.1089/107632701300062859 PubMedCrossRefGoogle Scholar
  47. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279–4295. doi: 10.1091/mbc.E02-02-0105 PubMedCrossRefPubMedCentralGoogle Scholar
  48. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN (2000) Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2:477–488. doi: 10.1186/ar130 PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2014

Authors and Affiliations

  • Christina L. Rettinger
    • 1
  • Andrea B. Fourcaudot
    • 1
  • Seok J. Hong
    • 2
  • Thomas A. Mustoe
    • 2
  • Robert G. Hale
    • 1
  • Kai P. Leung
    • 1
    Email author
  1. 1.Dental and Trauma Research DetachmentUnited States Army Institute of Surgical ResearchFort Sam HoustonUSA
  2. 2.Division of Plastic Surgery, Feinberg School of MedicineNorthwestern UniversityChicagoUSA

Personalised recommendations