Developing Three Dimensional Culture System for Notochordal Cells

Abstract

Introduction The central compartment of the intervertebral disk (IVD) is nucleus pulposus (NP) which contains two cell types, large notochordal cells (NCCs) and small cartilage-like cells. Loss of NCCs is related to disk degeneration as these cells are required for the maintenance of NP. Recently, NCCs have drawn increasing attention of many researchers from developmental biology, stem cells research, and tissue engineering because these cells are important in development and maintenance of NP. In our group, NCCs have been isolated from Foxa2mNE-Cre/Z/EG heterozygous embryos by identifying EGFP signal using FACS. However, the yield of EGFP positive cells is low (∼1%). These cells can be cultured as monolayer but all EGFP signals are gone in 1-month time. As a result, a better culture system able to support NCCs survival, growth and differentiation in 3D configuration is warranted for study of NCC biology. Materials and Methods Notochordal cells were cultured in 3D collagen microspheres. In brief, NCCs isolated from Foxa2mNE-Cre/Z/EG heterozygous embryos with EGFP signal, either FACS sorted or unsorted, were encapsulated using rat tail type I collagen (Col1) with different concentrations (0.5 mg/mL and 1 mg/mL), to form NCCs-collagen microspheres. The NCCs-collagen microspheres were cultured for 14 days for detection of EGFP signal and immunohistochemistry for type II collagen. Moreover, NCCs synthesized and deposited type IIA collagen which became a major ECM in notochord. We intend to reconstitute a microenvironment containing type IIA collagen which may favor NCC growth by transfection of cells with type IIA collagen plasmid DNA. Overexpression of type IIA collagen in transfected cells was detected by immunocytochemistry. These transfected cells were microencapsulated in type I collagen microspheres and cultured until the entrapped cells remodeled the template matrix by depositing type IIA collagen. The newly remodeled type IIA collagen-contained microspheres were then decellularized before seeding NCCs to study the effects of this matrix niche on NCCs maintenance, proliferation, and differentiation. Results After encapsulation of NCCs, either FACS sorted or unsorted, EGFP signal of NCCs were detected by confocal microscope in 14 days culture. The EGFP signal could be detected only in FASC unsorted NCCs-collagen microspheres but not in FASC sorted NCCs-collagen microspheres. It is speculated that FASC sorted NCCs may require other cells to support their growth in 3D environment. Moreover, the EGFP signal of FASC unsorted NCCs could be maintained in collagen microspheres for at least 2 weeks in different collagen concentration conditions. However, EGFP signal of NCCs in 1 mg/mL Col1 became very weak after 14 days, suggesting that lower collagen concentration (0.5 mg/mL) may favor NCC survival. Furthermore, type II collagen could be detected in NCCs-collagen microspheres, suggesting that NCCs could retain their matrix deposition function in this culture system. Moreover, the template matrix was remodeled by NCCs in collagen microspheres as type IIA collagen was detected in microspheres entrapping transfected HEK293 cells. Nevertheless, HEK293 cells grow in aggregates without contracting the collagen meshwork, microspheres may collapse after decellularization. Therefore, microspheres with dense meshwork are required and 3T3 fibroblasts are being transfected to overexpress collagen type IIA before encapsulation for future NCCs-type IIA collagen interaction studies. Conclusion Notochordal cells were able to be cultured in type I collagen microspheres without significant reduction in EGFP signal for at least 2 weeks and NCCs entrapped in type I collagen microspheres could remodel the template type I collagen matrix by depositing type II collagen. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Chan, BP. et al. Biomaterials 2007;28:4652–4666 Cheng, HW. et. al. Tissue Engeneering Part C Methods 2009;15:697–706 Hunter, CJ. et. al. Tissue Engeneering 2003;9:667–677 Roberts, S. Biochemical Society. Transactions 2002;30:864–869 Sandell LJ et al. Dev. Dyn. 1994;199:129–140 Yuan, M. et al. Spine Journal 2011;11:947–960 Yong Z. et al. Dev. Dyn. 2001;220:350–362