Skip to main content
SpringerLink
Log in

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

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

Similar content being viewed by others

References

  • Anzalone R, Iacono ML, Corrao S, Magno F, Loria T, Cappello F, Zummo G, Farina F, La Rocca G (2010) New emerging potentials for human Wharton’s jelly mesenchymal stem cells: immunological features and hepatocyte-like differentiative capacity. Stem Cells Dev 19:423–438

    Article  CAS  PubMed  Google Scholar 

  • Bao J, Fisher JE, Lillegard JB, Wang W, Amiot B, Yu Y, Dietz AB, Nahmias Y, Nyberg SL (2013) Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. Cell Transplant 22:299–308

    Article  PubMed Central  PubMed  Google Scholar 

  • Baraniak PR, McDevitt TC (2012) Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res 347:701–711

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bieback K, Kern S, Klüter H, Eichler H (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625–634

    Article  PubMed  Google Scholar 

  • Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B (2006) Aging of mesenchymal stem cell in vitro. BMC 7:14

    Article  Google Scholar 

  • Bortolomai I, Canevari S, Facetti I, De Cecco L, Castellano G, Zacchetti A, Alison MR, Miotti S (2010) Tumor initiating cells. Cell Cycle 9:1194–1206

    Article  CAS  PubMed  Google Scholar 

  • Carpenedo RL, Sargent CY, McDevitt TC (2007) Rotary suspension culture enhances the efficiency, yield, and homogeneity of embryoid body differentiation. Stem Cells 25:2224–2234

    Article  PubMed  Google Scholar 

  • Cheng NC, Wang S, Young TH (2012) The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33:1748–1758

    Article  CAS  PubMed  Google Scholar 

  • Deans RJ, Moseley AB (2000) Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 28:875–884

    Article  CAS  PubMed  Google Scholar 

  • English K, Barry FP, Field-Corbett CP, Mahon BP (2007) IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 110:91–100

    Article  CAS  PubMed  Google Scholar 

  • Francese R, Fiorina P (2010) Immunological and regenerative properties of cord blood stem cells. Clin Immunol 136:309–322

    Article  CAS  PubMed  Google Scholar 

  • Friedenstein AJ, Gorskaja J, Kulagina N (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 4:267–274

    CAS  PubMed  Google Scholar 

  • Gregory CA, Grady Gunn W, Peister A, Prockop DJ (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 329:77–84

    Article  CAS  PubMed  Google Scholar 

  • Gurkan UA, El Assal R, Yildiz SE, Sung Y, Trachtenberg AJ, Kuo WP, Demirci U (2014) Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets. Mol Pharm 11:2151–2159

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hartmann I, Hollweck T, Haffner S, Krebs M, Meiser B, Reichart B, Eissner G (2010) Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties. J Immunol Methods 363:80–89

    Article  CAS  PubMed  Google Scholar 

  • Krontiras P, Gatenholm P, Hägg DA (2014) Adipogenic differentiation of stem cells in three‐dimensional porous bacterial nanocellulose scaffolds. J Biomed Mater Res B Appl Biomater (in press)

  • Kuo TK, Hung SP, Chuang CH, Chen CT, Shih YR, Fang SC, Yang VW, Lee OK (2008) Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 134:2111–2121

    Article  PubMed Central  PubMed  Google Scholar 

  • La Rocca G, Anzalone R, Corrao S, Magno F, Loria T, Lo Iacono ML, Di Stefano A, Giannuzzi P, Marasà L, Cappello F, Zummo G, Farina F (2009) Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers. Histochem Cell Biol 131:267–282

    Article  PubMed  Google Scholar 

  • Lam MT, Longaker MT (2012) Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering. J Tissue Eng Regen Med 6 (Suppl 3):s80–s86

    Article  PubMed Central  PubMed  Google Scholar 

  • Marcus-Sekura C, Richardson JC, Harston RK, Sane N, Sheets RL (2011) Evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products. Biologicals 39:359–369

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nishiguchi A, Matsusaki M, Asano Y, Shimoda H, Akashi M (2014) Effects of angiogenic factors and 3D-microenvironments on vascularization within sandwich cultures. Biomaterials 35:4739–4748

    Article  CAS  PubMed  Google Scholar 

  • 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 284:143–147

    Article  CAS  PubMed  Google Scholar 

  • Rim J-S, Mynatt RL, Gawronska-Kozak B (2005) Mesenchymal stem cells from the outer ear: a novel adult stem cell model system for the study of adipogenesis. FASEB J 19:1205–1207

    CAS  PubMed  Google Scholar 

  • Romanov YA, Svintsitskaya VA, Smirnov VN (2003) Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 21:105–110

    Article  PubMed  Google Scholar 

  • Su H, Wang L, Huang W, Qin D, Cai J, Yao X, Feng C, Li Z, Wang Y, So KF, Pan G, Wu W, Pei D (2013) Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells. Stem Cell Res 10:338–348

    Article  CAS  PubMed  Google Scholar 

  • Trivedi P, Hematti P (2008) Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells. Exp Hematol 36:350–359

    PubMed Central  CAS  PubMed  Google Scholar 

  • Tunma S, Inthanon K, Chaiwong C, Pumchusak J, Wongkham W, Boonyawan D (2013) Improving the attachment and proliferation of umbilical cord mesenchymal stem cells on modified polystyrene by nitrogen-containing plasma. Cytotechnology 65:119–134

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfister S, Eckstein V, Ho AD (2008) Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 3:e2213

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang W, Itaka K, Ohba S, Nishiyama N, Chung UI, Yamasaki Y, Kataoka K (2009) 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials 30:2705–2715

    Article  CAS  PubMed  Google Scholar 

  • Weiss ML, Troyer DL (2006) Stem cells in the umbilical cord. Stem Cell Rev 2:155–162

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, Troyer D, McIntosh KR (2008) Immune properties of human umbilical cord Wharton’s jelly-derived cells. Stem Cells 26:2865–2874

    Article  CAS  PubMed  Google Scholar 

  • Zagoura DS, Roubelakis MG, Bitsika V, Trohatou O, Pappa KI, Kapelouzou A, Antsaklis A, Anagnou NP (2012) Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure. Gut 61:894–906

    Article  CAS  PubMed  Google Scholar 

  • Zhang B, Liu R, Shi D, Liu X, Chen Y, Dou X, Zhu X, Lu C, Liang W, Liao L, Zenke M, Zhao RC (2009) Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2–dependent regulatory dendritic cell population. Blood 113:46–57

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, 610041, China

    Yi Li, Gang Guo, Li Li, Fei Chen, Ji Bao & Yu-jun Shi

  2. Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China

    Yi Li, Gang Guo, Ji Bao, Yu-jun Shi & Hong Bu

  3. Department of Pathology, Western China Hospital, Sichuan University, Chengdu, 610041, China

    Hong Bu

Authors
  1. Yi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gang Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Li Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ji Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yu-jun Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hong Bu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hong Bu.

Additional information

This work was supported by grants from the National Key Clinical Project, National Sciences and Technology Major Project of China (2012ZX10002-017) and the Sichuan Provincial Science and Technology Support Project (2012SZ0016).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Table 1

(DOCX 12 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Guo, G., Li, L. et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res 360, 297–307 (2015). https://doi.org/10.1007/s00441-014-2055-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-014-2055-x

Keywords

Search

Navigation