Applied Biochemistry and Biotechnology

, Volume 174, Issue 6, pp 2114–2130 | Cite as

Mass Transfer Analysis of Growth and Substance Metabolism of NSCs Cultured in Collagen-Based Scaffold In Vitro

  • Kedong SongEmail author
  • Dan Ge
  • Shui Guan
  • Chenggong Sun
  • Xuehu Ma
  • Tianqing LiuEmail author


The aim of this study is to analyze the growth and substance metabolism of neural stem cells (NSCs) cultured in biological collagen-based scaffolds. Mass transfer and metabolism model of glucose, lactic acid, and dissolved oxygen (DO) were established and solved on MATLAB platform to obtain the concentration distributions of DO, glucose, and lactic acid in culture system, respectively. Calculation results showed that the DO influenced their normal growth and metabolism of NSCs mostly in the in vitro culture within collagen-based scaffolds. This study also confirmed that 2-mm thickness of collagen scaffold was capable of in vitro cultivation and growth of NSCs with an inoculating density of 1 × 106 cells/mL.


Neural stem cells (NSCs) In vitro culture Collagen Mathematical model 



This work was supported by Fok Ying Tung Education Foundation (132027), National Science Foundation of China (31370991/31300809/31170945), Dalian Science and Technology Plan (2012E15SF174) and State Key Laboratory of Fine Chemicals (KF1111) and Fundamental Research Funds for the Central Universities (DUT14YQ106/DUT14QY20) and SRF for ROCS, SEM.


  1. 1.
    Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., et al. (1998). Neurogenesis in the adult human hippocampus. Nature Medicine, 4(11), 1313–1317.CrossRefGoogle Scholar
  2. 2.
    McKay, R. (1997). Stem cells in the central nervous system. Science, 276(5309), 66–71.CrossRefGoogle Scholar
  3. 3.
    Reynolds, B. A., & Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 255(5052), 1707–1710.CrossRefGoogle Scholar
  4. 4.
    Gage, F. H. (2000). Mammalian neural stem cells. Science, 287(5457), 1433–1438.CrossRefGoogle Scholar
  5. 5.
    Kallos, M. S., & Behie, L. A. (1999). Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors. Biotechnology and Bioengineering, 63(4), 473–483.CrossRefGoogle Scholar
  6. 6.
    Sivakumar, K. C., Dhanesh, S. B., Shobana, S., et al. (2011). A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and egf signaling pathways. OMICS, 15(10), 729–737.CrossRefGoogle Scholar
  7. 7.
    Theus MH, Ricard J, Liebl DJ. Reproducible expansion and characterization of mouse neural stem/progenitor cells in adherent cultures derived from the adult subventricular zone. Current Protocols Stem Cell Biology. Chapter 2: Unit 2D 8.Google Scholar
  8. 8.
    Liu, T., Ge, D., Cheng, F., et al. (2006). Simulation of the growth of neurosphere cultured in bioreactors. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 23(1), 147–152.Google Scholar
  9. 9.
    Buhnemann, C., Scholz, A., Bernreuther, C., et al. (2006). Neuronal differentiation of transplanted embryonic stem cell-derived precursors in stroke lesions of adult rats. Brain, 129(Pt 12), 3238–3248.CrossRefGoogle Scholar
  10. 10.
    Lee, K. Y., & Mooney, D. J. (2001). Hydrogels for tissue engineering. Chemical Reviews, 101(7), 1869–1879.CrossRefGoogle Scholar
  11. 11.
    Chen, L., Xiao, Z., Meng, Y., et al. (2012). The enhancement of cancer stem cell properties of mcf-7 cells in 3d collagen scaffolds for modeling of cancer and anti-cancer drugs. Biomaterials, 33(5), 1437–1444.CrossRefGoogle Scholar
  12. 12.
    Ma, W., Tavakoli, T., Chen, S., et al. (2008). Reconstruction of functional cortical-like tissues from neural stem and progenitor cells. Tissue Engineering Part A, 14(10), 1673–1686.CrossRefGoogle Scholar
  13. 13.
    Brito, C., Simao, D., Costa, I., et al. (2012). 3d cultures of human neural progenitor cells: dopaminergic differentiation and genetic modification. [corrected]. Methods, 56(3), 452–460.CrossRefGoogle Scholar
  14. 14.
    Doetsch, F., Caille, I., Lim, D. A., et al. (1999). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell, 97(6), 703–716.CrossRefGoogle Scholar
  15. 15.
    Cai, J., Wu, Y., Mirua, T., et al. (2002). Properties of a fetal multipotent neural stem cell (nep cell). Developmental Biology, 251(2), 221–240.CrossRefGoogle Scholar
  16. 16.
    Gritti, A., Parati, E. A., Cova, L., et al. (1996). Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. Journal of Neuroscience, 16(3), 1091–1100.Google Scholar
  17. 17.
    Svendsen, C. N., ter Borg, M. G., Armstrong, R. J., et al. (1998). A new method for the rapid and long term growth of human neural precursor cells. Journal of Neuroscience Methods, 85(2), 141–152.CrossRefGoogle Scholar
  18. 18.
    Guan, S., Ge, D., Liu, T. Q., et al. (2009). Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells. Toxicology In Vitro, 23(2), 201–208.CrossRefGoogle Scholar
  19. 19.
    Kanemura, Y., Mori, H., Kobayashi, S., et al. (2002). Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity. Journal of Neuroscience Research, 69(6), 869–879.CrossRefGoogle Scholar
  20. 20.
    Ishikawa, T., Zhu, B. L., & Maeda, H. (2006). Effect of sodium azide on the metabolic activity of cultured fetal cells. Toxicology and Industrial Health, 22(8), 337–341.Google Scholar
  21. 21.
    Akyilmaz, E., Yasa, I., & Dinckaya, E. (2006). Whole cell immobilized amperometric biosensor based on saccharomyces cerevisiae for selective determination of vitamin b1 (thiamine). Analytical Biochemistry, 354(1), 78–84.CrossRefGoogle Scholar
  22. 22.
    Selard, E., Shirazi-Adl, A., & Urban, J. P. (2003). Finite element study of nutrient diffusion in the human intervertebral disc. Spine (Phila Pa 1976), 28(17), 1945–1953. discussion 1953.CrossRefGoogle Scholar
  23. 23.
    Sengers, B. G., van Donkelaar, C. C., Oomens, C. W., et al. (2005). Computational study of culture conditions and nutrient supply in cartilage tissue engineering. Biotechnology Progress, 21(4), 1252–1261.CrossRefGoogle Scholar
  24. 24.
    Svendsen, C. N., Skepper, J., Rosser, A. E., et al. (1997). Restricted growth potential of rat neural precursors as compared to mouse. Brain Research. Developmental Brain Research, 99(2), 253–258.CrossRefGoogle Scholar
  25. 25.
    Liu, T., Dai, M. S., Ge, D., et al. (2006). Study of viability and metabolism parameters of NSPCs. Journal of Dalian University of Technology Spine, 48(6), 811–818.Google Scholar
  26. 26.
    Ozturk, S. S., Thrift, J. C., Blackie, J. D., et al. (1997). Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor. Biotechnology and Bioengineering, 53(4), 372–378.CrossRefGoogle Scholar
  27. 27.
    Patel, S. D., Papoutsakis, E. T., Winter, J. N., et al. (2000). The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures. Biotechnology Progress, 16(5), 885–892.CrossRefGoogle Scholar
  28. 28.
    Fujita, Y., Kuchimaru, T., Kadonosono, T., et al. (2012). In vivo imaging of brain ischemia using an oxygen-dependent degradative fusion protein probe. PLoS One, 7(10), e48051.CrossRefGoogle Scholar
  29. 29.
    Stippler, M., Ortiz, V., Adelson, P. D., et al. (2012). Brain tissue oxygen monitoring after severe traumatic brain injury in children: relationship to outcome and association with other clinical parameters. Journal of Neurosurgery Pediatrics, 10(5), 383–391.CrossRefGoogle Scholar
  30. 30.
    Zhu, L. L., Zhao, T., Huang, X., et al. (2011). Gene expression profiles and metabolic changes in embryonic neural progenitor cells under low oxygen. Cellular Reprogramming, 13(2), 113–120.CrossRefGoogle Scholar
  31. 31.
    Milosevic, J., Schwarz, S. C., Krohn, K., et al. (2005). Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors. Journal of Neurochemistry, 92(4), 718–729.CrossRefGoogle Scholar
  32. 32.
    Maciaczyk, J., Singec, I., Maciaczyk, D., et al. (2008). Combined use of bdnf, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells. Experimental Neurology, 213(2), 354–362.CrossRefGoogle Scholar
  33. 33.
    Clarke, L., & van der Kooy, D. (2009). Low oxygen enhances primitive and definitive neural stem cell colony formation by inhibiting distinct cell death pathways. Stem Cells, 27(8), 1879–1886.CrossRefGoogle Scholar
  34. 34.
    Cukierman, E., Pankov, R., & Yamada, K. M. (2002). Cell interactions with three-dimensional matrices. Current Opinion in Cell Biology, 14(5), 633–639.CrossRefGoogle Scholar
  35. 35.
    Irons, H. R., Cullen, D. K., Shapiro, N. P., et al. (2008). Three-dimensional neural constructs: a novel platform for neurophysiological investigation. Journal of Neural Engineering, 5(3), 333–341.CrossRefGoogle Scholar
  36. 36.
    Lee, J., Cuddihy, M. J., & Kotov, N. A. (2008). Three-dimensional cell culture matrices: state of the art. Tissue Engineering. Part B, Reviews, 14(1), 61–86.CrossRefGoogle Scholar
  37. 37.
    Mueller-Klieser, W. (1997). Three-dimensional cell cultures: from molecular mechanisms to clinical applications. American Journal of Physiology, 273(4 Pt 1), C1109–C1123.Google Scholar
  38. 38.
    Zervantonakis, I. K., Kothapalli, C. R., Chung, S., et al. (2011). Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments. Biomicrofluidics, 5(1), 13406.CrossRefGoogle Scholar
  39. 39.
    Leipzig, N. D., Wylie, R. G., Kim, H., et al. (2011). Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds. Biomaterials, 32(1), 57–64.CrossRefGoogle Scholar
  40. 40.
    Drago, F., Russo, M. S., Marazzi, R., et al. (2011). Atrial tachycardias in patients with congenital heart disease: a minimally invasive simplified approach in the use of three-dimensional electroanatomic mapping. Europace, 13(5), 689–695.CrossRefGoogle Scholar
  41. 41.
    Lai, Y., Asthana, A., Cheng, K., et al. (2011). Neural cell 3d microtissue formation is marked by cytokines’ up-regulation. PLoS One, 6(10), e26821.CrossRefGoogle Scholar
  42. 42.
    Stabenfeldt, S. E., Munglani, G., Garcia, A. J., et al. (2010). Biomimetic microenvironment modulates neural stem cell survival, migration, and differentiation. Tissue Engineering Part A, 16(12), 3747–3758.CrossRefGoogle Scholar
  43. 43.
    Itoh, T., Satou, T., Dote, K., et al. (2005). Effect of basic fibroblast growth factor on cultured rat neural stem cell in three-dimensional collagen gel. Neurological Research, 27(4), 429–432.CrossRefGoogle Scholar
  44. 44.
    O’Connor, S. M., Stenger, D. A., Shaffer, K. M., et al. (2000). Primary neural precursor cell expansion, differentiation and cytosolic ca (2+) response in three-dimensional collagen gel. Journal of Neuroscience Methods, 102(2), 187–195.CrossRefGoogle Scholar
  45. 45.
    Rodrigues, C. A., Fernandes, T. G., Diogo, M. M., et al. (2011). Stem cell cultivation in bioreactors. Biotechnology Advances, 29(6), 815–829.CrossRefGoogle Scholar
  46. 46.
    Lichtenberg, A., Dumlu, G., Walles, T., et al. (2005). A multifunctional bioreactor for three-dimensional cell (co)-culture. Biomaterials, 26(5), 555–562.CrossRefGoogle Scholar
  47. 47.
    Muller, J., Benz, K., Ahlers, M., et al. (2011). Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures. Cell Transplantation, 20(10), 1589–1602.CrossRefGoogle Scholar
  48. 48.
    Mori, Y., Kubokawa, M., Hagiwara, N., et al. (1994). Role of ph-sensitive ion channels in regulation of cell volume in opossum kidney cells. Japanese Journal of Physiology, 44(Suppl 2), S81–S86.Google Scholar
  49. 49.
    Yu, H., & Ferrier, J. (1995). Osteoclast atp receptor activation leads to a transient decrease in intracellular ph. Journal of Cell Science, 108(Pt 9), 3051–3058.Google Scholar
  50. 50.
    King, B. F., Liu, M., Townsend-Nicholson, A., et al. (2005). Antagonism of atp responses at p2x receptor subtypes by the ph indicator dye, phenol red. British Journal of Pharmacology, 145(3), 313–322.CrossRefGoogle Scholar
  51. 51.
    Motizuki, M., & Xu, Z. (2009). Importance of polarisome proteins in reorganization of actin cytoskeleton at low ph in saccharomyces cerevisiae. Journal of Biochemistry, 146(5), 705–712.CrossRefGoogle Scholar
  52. 52.
    Chen, K. C., Wu, C. H., Chang, C. Y., et al. (2008). Directed evolution of a lysosomal enzyme with enhanced activity at neutral ph by mammalian cell-surface display. Chemical Biology, 15(12), 1277–1286.CrossRefGoogle Scholar
  53. 53.
    Schultheiss, E., Weiss, S., Winterer, E., et al. (2008). Esterase autodisplay: enzyme engineering and whole-cell activity determination in microplates with ph sensors. Applied and Environmental Microbiology, 74(15), 4782–4791.CrossRefGoogle Scholar
  54. 54.
    Grineva, N. I., Akhlynina, T. V., Gerasimova, L. P., et al. (2009). Cell regulation of proliferation and differentiation ex vivo for cells containing ph chromosome in chronic myeloid leukemia. Acta Naturae, 1(3), 108–120.Google Scholar
  55. 55.
    Isfort, R. J., Cody, D. B., Stuard, S. B., et al. (1996). Calcium functions as a transcriptional and mitogenic repressor in syrian hamster embryo cells: roles of intracellular ph and calcium in controlling embryonic cell differentiation and proliferation. Experimental Cell Research, 226(2), 363–371.CrossRefGoogle Scholar
  56. 56.
    Valentin, J. E., Freytes, D. O., Grasman, J. M., et al. (2009). Oxygen diffusivity of biologic and synthetic scaffold materials for tissue engineering. Journal of Biomedical Materials Research. Part A, 91(4), 1010–1017.CrossRefGoogle Scholar
  57. 57.
    Landman, K. A., & Cai, A. Q. (2007). Cell proliferation and oxygen diffusion in a vascularising scaffold. Bulletin of Mathematical Biology, 69(7), 2405–2428.CrossRefGoogle Scholar
  58. 58.
    Chin, K., Khattak, S. F., Bhatia, S. R., et al. (2008). Hydrogel-perfluorocarbon composite scaffold promotes oxygen transport to immobilized cells. Biotechnology Progress, 24(2), 358–366.CrossRefGoogle Scholar
  59. 59.
    Kowalczyk, M., Waldron, K., Kresnowati, P., et al. (2011). Process challenges relating to hematopoietic stem cell cultivation in bioreactors. Journal of Industrial Microbiology and Biotechnology, 38(7), 761–767.CrossRefGoogle Scholar
  60. 60.
    Kehoe, D. E., Jing, D., Lock, L. T., et al. (2010). Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Engineering Part A, 16(2), 405–421.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue EngineeringDalian University of TechnologyDalianChina
  2. 2.Department of EmergencySecond Hospital Affiliated to Dalian Medical UniversityDalianChina

Personalised recommendations