Three-Dimensional (3D) Cell Culture Conditions, Present and Future Improvements


1 Orthopaedic Trauma Institute, San Francisco General Hospital, University of California, San Francisco, USA

2 School of Veterinary Medicine, University of California, Davis, USA



Context: Early development of many organs shows many morphological and molecular similarities (teeth, lung, hair, kidney and etc.). Fundamental questions in organogenesis are related to the identification of a simplified model which is able to mimic the molecular mechanisms involved in pattern organization and cell fate determination as well. Evidence Acquisition: It is widely accepted that cells behave more natively if cultured in three-dimensional conditions. Advances in 3D non-destructive, non-invasive analysis methods and improvements in the multi-scale techniques and bioreactors to obtain test and culture 3D cell aggregates have been made. On the other hand, even if 3D aggregate culture methods are able to recapitulate in vitro the cell-extracellular matrix interactions properly observed in vivo, and the synthetic/natural matrix and scaffolds have biochemical and mechanical properties, in order to mimic the native extracellular matrix, both of these systems possesses some limitations and some methodological improvements are needed. Results: The processes by which re-aggregated adult single cell types or adult and embryonic explanted tissues are able to recapitulate embryogenesis in vitro, when cultured in adhesion or embedded in 3D gels, is not surprising and is clearly under the control of a reminiscent cellular memory which recapitulates early developmental stages. Conclusions: Our underlying hypothesis is that recapitulating the three-dimensional early embryonic structure, in order to reproduce better in vitro the three-dimensional morphogenetic-like re-arrangements, would improve cells differentiation, when in vivo transplanted; moreover, it could be used as a simplified cancer disease model and reliable drug evaluation method as well.


  1. 1.Denker HW. Potentiality of embryonic stem cells: an ethical problem even with alternative stem cell sources. J Med Ethics. 2006;32(11):665–71.

    1. Shih CC, Forman SJ, Chu P, Slovak M. Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice. Stem Cells Dev. 2007;16(6):893–902.
    2. Prokhorova TA, Harkness LM, Frandsen U, Ditzel N, Schroder HD, Burns JS, et al. Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel. Stem Cells Dev. 2009;18(1):47–54.
    3. Beltrami AP, Cesselli D, Beltrami CA. Pluripotency rush! Molecular cues for pluripotency, genetic reprogramming of adult stem cells, and widely multipotent adult cells. Pharmacol Ther. 2009;124(1):23–30.
    4. Ferro F, Spelat R, D'Aurizio F, Puppato E, Pandolfi M, Beltrami AP, et al. Dental pulp stem cells differentiation reveals new insights in Oct4A dynamics. PLoS One. 2012;7(7).
    5. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.
    6. Czyz J, Wobus A. Embryonic stem cell differentiation: the role of extracellular factors. Differentiation. 2001;68(4-5):167–74.
    7. Bratt-Leal AM, Carpenedo RL, McDevitt TC. Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation. Biotechnol Prog. 2009;25(1):43–51.
    8. Holtfreter J. A study of the mechanism of gastrulation. J Exp Zool. 1944;95(2):171–212.
    9. Moscona A. Cell suspensions from organ rudiments of chick embryos. Exp Cell Res. 1952;3(3):535–9.
    10. Moscona A. The Development in Vitro of Chimeric Aggregates of Dissociated Embryonic Chick and Mouse Cells. Proc Natl Acad Sci U S A. 1957;43(1):184–94.
    11. Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng. 2007;103(5):389–98.
    12. Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent Ferro F et al. 8 Razavi Int J Med. 2014;(2):e17803 monoculture. Nat Biotechnol. 2003;21(2):183–6.
    13. Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science. 1994;265(5175):1098–101.
    14. Li X, Chen Y, Scheele S, Arman E, Haffner-Krausz R, Ekblom P, et al. Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body. J Cell Biol. 2001;153(4):811–22.
    15. Komura H, Ogita H, Ikeda W, Mizoguchi A, Miyoshi J, Takai Y. Establishment of cell polarity by afadin during the formation of embryoid bodies. Genes Cells. 2008;13(1):79–90.
    16. Daya S, Loughlin AJ, Macqueen HA. Culture and differentiation of preadipocytes in two-dimensional and three-dimensional in vitro systems. Differentiation. 2007;75(5):360–70.
    17. Pineda ET, Nerem RM, Ahsan T. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture. Cells Tissues Organs. 2013;197(5):399–410.
    18. Baraniak PR, McDevitt TC. Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res. 2012;347(3):701–11.
    19. Greiner JF, Hauser S, Widera D, Muller J, Qunneis F, Zander C, et al. Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics. Eur Cell Mater. 2011;22:403–19.
    20. Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov. 2004;3(8):711–5.
    21. Boucher RJ, Bromberg PA, Gatzy JT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physiol Respir Environ Exerc Physiol. 1980;48(1):169–76.
    22. Dunham SP. Lessons from the cat: development of vaccines against lentiviruses. Vet Immunol Immunopathol. 2006;112(1- 2):67–77.
    23. Rhim JS. Cell aggregation assay: a rapid means of evaluating and selecting in vitro transformed cells. Cancer Detect Prev. 1983;6(3):381–8.
    24. Boterberg T, Bracke ME, Bruyneel EA, Mareel MM. Cell aggregation assays. Methods Mol Med. 2001;58:33–45.
    25. Job CK. Nine-banded armadillo and leprosy research. Indian J Pathol Microbiol. 2003;46(4):541–50.
    26. Achilli TM, Meyer J, Morgan JR. Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol Ther. 2012;12(10):1347–60.
    27. Sun P, Xu Y, Du X, Ning N, Sun H, Liang W, et al. An engineered three-dimensional gastric tumor culture model for evaluating the antitumor activity of immune cells in vitro. Oncol Lett. 2013;5(2):489–94.
    28. Janorkar AV, Harris LM, Murphey BS, Sowell BL. Use of threedimensional spheroids of hepatocyte-derived reporter cells to study the effects of intracellular fat accumulation and subsequent cytokine exposure. Biotechnol Bioeng. 2011;108(5):1171–80.
    29. Peck Y, Wang DA. Three-dimensionally engineered biomimetic tissue models for in vitro drug evaluation: delivery, efficacy and toxicity. Expert Opin Drug Deliv. 2013;10(3):369–83.
    30. Ferro F, Spelat R, D'Aurizio F, Falini G, De Pol I, Pandolfi M, et al. Acellular bone colonization and aggregate culture conditions diversely influence murine periosteum mesenchymal stem cell differentiation potential in long-term in vitro osteoinductive conditions. Tissue Eng Part A. 2012;18(13-14):1509–19.
    31. Ferro F, Falini G, Spelat R, D'Aurizio F, Puppato E, Pandolfi M, et al. Biochemical and biophysical analyses of tissue-engineered bone obtained from three-dimensional culture of a subset of bone marrow mesenchymal stem cells. Tissue Eng Part A. 2010;16(12):3657–67.
    32. Ferro F, Spelat R, Falini G, Gallelli A, D'Aurizio F, Puppato E, et al. Adipose tissue-derived stem cell in vitro differentiation in a three-dimensional dental bud structure. Am J Pathol. 2011;178(5):2299–310.
    33. Ward A, Quinn KP, Bellas E, Georgakoudi I, Kaplan DL. Noninvasive metabolic imaging of engineered 3D human adipose tissue in a perfusion bioreactor. PLoS One. 2013;8(2).
    34. Rice WL, Kaplan DL, Georgakoudi I. Quantitative biomarkers of stem cell differentiation based on intrinsic two-photon excited fluorescence. J Biomed Opt. 2007;12(6):60504.
    35. Georgakoudi I, Rice WL, Hronik-Tupaj M, Kaplan DL. Optical spectroscopy and imaging for the noninvasive evaluation of engineered tissues. Tissue Eng Part B Rev. 2008;14(4):321–40.
    36. Quinn KP, Bellas E, Fourligas N, Lee K, Kaplan DL, Georgakoudi I. Characterization of metabolic changes associated with the functional development of 3D engineered tissues by non-invasive, dynamic measurement of individual cell redox ratios. Biomaterials. 2012;33(21):5341–8.
    37. Rice WL, Kaplan DL, Georgakoudi I. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation. PLoS One. 2010;5(4).
    38. Dittmar R, Potier E, van Zandvoort M, Ito K. Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence. Tissue Eng Part C Methods. 2012;18(3):198–204.
    39. Konig K, Riemann I. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt. 2003;8(3):432–9.
    40. Provenzano PP, Rueden CT, Trier SM, Yan L, Ponik SM, Inman DR, et al. Nonlinear optical imaging and spectral-lifetime computational analysis of endogenous and exogenous fluorophores in breast cancer. J Biomed Opt. 2008;13(3):31220.
    41. Canton I, Sarwar U, Kemp EH, Ryan AJ, MacNeil S, Haycock JW. Real-time detection of stress in 3D tissue-engineered constructs using NF-kappaB activation in transiently transfected human dermal fibroblast cells. Tissue Eng. 2007;13(5):1013–24.
    42. Dunkers JP, Lee YJ, Chatterjee K. Single cell viability measurements in 3D scaffolds using in situ label free imaging by optical coherence microscopy. Biomaterials. 2012;33(7):2119–26.
    43. Poirier-Quinot M, Frasca G, Wilhelm C, Luciani N, Ginefri JC, Darrasse L, et al. High-resolution 1.5-Tesla magnetic resonance imaging for tissue-engineered constructs: a noninvasive tool to assess three-dimensional scaffold architecture and cell seeding. Tissue Eng Part C Methods. 2010;16(2):185–200.
    44. Jeong GS, Jun Y, Song JH, Shin SH, Lee SH. Meniscus induced self organization of multiple deep concave wells in a microchannel for embryoid bodies generation. Lab Chip. 2012;12(1):159–66.
    45. Kang E, Choi YY, Jun Y, Chung BG, Lee SH. Development of a multilayer microfluidic array chip to culture and replate uniformsized embryoid bodies without manual cell retrieval. Lab Chip. 2010;10(20):2651–4.
    46. Bazou D, Castro A, Hoyos M. Controlled cell aggregation in a pulsed acoustic field. Ultrasonics. 2012;52(7):842–50.
    47. Vanherberghen B, Manneberg O, Christakou A, Frisk T, Ohlin M, Hertz HM, et al. Ultrasound-controlled cell aggregation in a multi-well chip. Lab Chip. 2010;10(20):2727–32.
    48. Ramasamy TS, Yu JS, Selden C, Hodgson H, Cui W. Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality. Tissue Eng Part A. 2013;19(3- 4):360–7.
    49. Nagamoto Y, Tashiro K, Takayama K, Ohashi K, Kawabata K, Sakurai F, et al. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets. Biomaterials. 2012;33(18):4526–34.
    50. Zwolinski CM, Ellison KS, Depaola N, Thompson DM. Generation of cell-derived three dimensional extracellular matrix substrates from two dimensional endothelial cell cultures. Tissue Eng Part C Methods. 2011;17(5):589–95.
    51. Fujiwara H, Hayashi Y, Sanzen N, Kobayashi R, Weber CN, Emoto T, et al. Regulation of mesodermal differentiation of mouse embryonic stem cells by basement membranes. J Biol Chem. 2007;282(40):29701–11.
    52. Shi Y, Kirwan P, Smith J, Robinson HP, Livesey FJ. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci. 2012;15(3):477–86.
    53. Rodriguez-Fraticelli AE, Auzan M, Alonso MA, Bornens M, MartinBelmonte F. Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis. J Cell Biol. 2012;198(6):1011–23.
    54. Ochsner M, Textor M, Vogel V, Smith ML. Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells Ferro F et al. Razavi Int J Med. 2014;(2):e17803 9 in response to rigidity and shape. PLoS One. 2010;5(3).
    55. Tekin H, Anaya M, Brigham MD, Nauman C, Langer R, Khademhosseini A. Stimuli-responsive microwells for formation and retrieval of cell aggregates. Lab Chip. 2010;10(18):2411–8.
    56. Lee SJ, Atala A. Scaffold technologies for controlling cell behavior in tissue engineering. Biomed Mater. 2013;8(1):10201.
    57. Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng. 2009;103(4):655–63.
    58. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol. 2005;23(1):47–55.
    59. Bernard AB, Lin CC, Anseth KS. A microwell cell culture platform for the aggregation of pancreatic beta-cells. Tissue Eng Part C Methods. 2012;18(8):583–92.
    60. Abukawa H, Zhang W, Young CS, Asrican R, Vacanti JP, Kaban LB, et al. Reconstructing mandibular defects using autologous tissueengineered tooth and bone constructs. J Oral Maxillofac Surg. 2009;67(2):335–47.
    61. Funamoto K, Zervantonakis IK, Liu Y, Ochs CJ, Kim C, Kamm RD. A novel microfluidic platform for high-resolution imaging of a three-dimensional cell culture under a controlled hypoxic environment. Lab Chip. 2012;12(22):4855–63.
    62. Keller R. Shaping the vertebrate body plan by polarized embryonic cell movements. Science. 2002;298(5600):1950–4.
    63. Davies J. Mechanisms of Morphogenesis.Burlington: Elsevier Academic Press; 2005.
    64. Rozario T, DeSimone DW. The extracellular matrix in development and morphogenesis: a dynamic view. Dev Biol. 2010;341(1):126–40.
    65. Foty RA, Pfleger CM, Forgacs G, Steinberg MS. Surface tensions of embryonic tissues predict their mutual envelopment behavior. Development. 1996;122(5):1611–20.
    66. Steinberg MS. Mechanism of tissue reconstruction by dissociated cells. II. Time-course of events. Science. 1962;137(3532):762–3.
    67. Dean DM, Morgan JR. Cytoskeletal-mediated tension modulates the directed self-assembly of microtissues. Tissue Eng Part A. 2008;14(12):1989–97.
    68. Dean DM, Rago AP, Morgan JR. Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissues. Cell Motil Cytoskeleton. 2009;66(3):129–41.
    69. Krieg M, Arboleda-Estudillo Y, Puech PH, Kafer J, Graner F, Muller DJ, et al. Tensile forces govern germ-layer organization in zebrafish. Nat Cell Biol. 2008;10(4):429–36.
    70. Pearson GW, Hunter T. Real-time imaging reveals that noninvasive mammary epithelial acini can contain motile cells. J Cell Biol. 2007;179(7):1555–67.
    71. Wang H, Lacoche S, Huang L, Xue B, Muthuswamy SK. Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly. Proc Natl Acad Sci U S A. 2013;110(1):163–8.
    72. Assal Y, Mie M, Kobatake E. The promotion of angiogenesis by growth factors integrated with ECM proteins through coiled-coil structures. Biomaterials. 2013;34(13):3315–23.
    73. Rosines E, Sampogna RV, Johkura K, Vaughn DA, Choi Y, Sakurai H, et al. Staged in vitro reconstitution and implantation of engineered rat kidney tissue. Proc Natl Acad Sci U S A. 2007;104(52):20938–43.
    74. Wolff E, Haffen K, Kieny M, Wolff E. [Tests in vitro cultures of embryonic organs in synthetic media]. J Embryol Exp Morph. 1953;1(1):55–84.
    75. Willerth SM, Arendas KJ, Gottlieb DI, Sakiyama-Elbert SE. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells. Biomaterials. 2006;27(36):5990–6003.
    76. Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010;28(8):848–55.
    77. Lee CH, Cook JL, Mendelson A, Moioli EK, Yao H, Mao JJ. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet. 2010;376(9739):440–8.
    78. Stosich MS, Moioli EK, Wu JK, Lee CH, Rohde C, Yoursef AM, et al. Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape. Methods. 2009;47(2):116–21.
    79. Belema-Bedada F, Uchida S, Martire A, Kostin S, Braun T. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell. 2008;2(6):566–75.