Exploring a novel spheroid 3D cell culture system for Tie2+ nucleus pulposus cells of the intervertebral disc
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Description
Introduction
Low back pain (LBP) is a big problem in our aging society. LBP impacts the patients quality of life and places an immense burden on the healthcare system worldwide [1]. Degeneration of the intervertebral disc (IVD) is one of the common causes of LBP. Recently, nucleus pulposus (NP) progenitor cells (NPPC) were discovered, which are positive for Angiopoietin-1 receptor (aka. Tie2+/CD202b) [2-5]. These NPPCs are a promising cell source for IVD regeneration and may spark the development of novel cell therapies. It is known, that these NPPCs are pretty rare (2-10% of all IVD cells) in human IVDs and diminish in number with increasing age [6]. The low numbers of sorted primary NPPCs necessitates an expansion of the NPPCs. It has been demonstrated, that 3D culture is superior to classic 2D culture to maintain the pluripotent phenotype of the NPPCs [3]. The goal of this research is to test the expansion and culture of bovine NPPCs cells in a novel spheroid plate.
Material and Methods
NP tissue was isolated from 5-6 discs form 4 bovine bovine tails (aged 10-13 months). NP cells were isolated by using a mild two step digestion protocol. Then, the primary NP cells were stained with Tie2 Antibody conjugated with Alexa 488 and DAPI for FACS. Sorted NP cells were expanded for 2 weeks under hypoxia (5% O2 ) and were supplemented with 2.5ng/ml FGF2 until 80% confluency. Tie2+ and Tie2- cells were then seeded in the functionalized and in the 2D control wells of the spheroid plate SP5D of Kugelmeier Ltd (see fig. 1). The cells were cultured for 2 weeks under hypoxia and FGF2. Colony forming unit-assay was performed at day 0. Cell activity, DNA/GAG content and gene expression of selected genes was measured on day 1, 8, 15.
Results
The DNA content of the Tie2+ and – spheroids (see fig. 2) increased by a factor of 1.7 and 3. The cells in the 2D plastic control wells grew faster and increased by factor of 11 and respectively 18.
Figure 1: Spheroids of bovine NP Tie2+ cells stained with Calcein AM and Ethidium homodimer in the SP5D spheroid plate by Kugelmeier, 400 cells/microwell, 1d 6h after seeding
Figure 2: Absolut DNA content/well, either 750 spheroids or 2D control in 24well plate, Tie2 + and Tie2-, cultivated under hypoxia (2% O2 ) and supplemented with FGF2
(N=1,
n=3),
means
±
SD
Conclusion
The obtained results of the pilot show that sorted bovine NP cells proliferated in all culture conditions. There seems to be a big difference in the cell proliferation rate depending on the chosen culture system (3D vs 2D). However, more biological replicates are needed for further evaluation of the results.
References
1. Spencer et al., Lancet 392:1789-858, 2018
2. Bischof et al., Applied Sciences 11: 7144, 2021
3. Guerrero et al., JOR SPINE 4:e1131, 2020.
4. Sako et al., Applied Sciences 11, 2021
5. Zhang, et al., Int. J. Mol. Sci 21: 9423, 2020
6. Sakai, et al., Nat Commun 3:1264, 2012
Acknowledgements
This study is supported by an SNF Bridge – Discovery Grant (https://data.snf.ch/grants/grant/211510). FACSLab and Life cell imaging (LCI) of the DBMR, University of Bern are gratefully acknowledged.
Low back pain (LBP) is a big problem in our aging society. LBP impacts the patients quality of life and places an immense burden on the healthcare system worldwide [1]. Degeneration of the intervertebral disc (IVD) is one of the common causes of LBP. Recently, nucleus pulposus (NP) progenitor cells (NPPC) were discovered, which are positive for Angiopoietin-1 receptor (aka. Tie2+/CD202b) [2-5]. These NPPCs are a promising cell source for IVD regeneration and may spark the development of novel cell therapies. It is known, that these NPPCs are pretty rare (2-10% of all IVD cells) in human IVDs and diminish in number with increasing age [6]. The low numbers of sorted primary NPPCs necessitates an expansion of the NPPCs. It has been demonstrated, that 3D culture is superior to classic 2D culture to maintain the pluripotent phenotype of the NPPCs [3]. The goal of this research is to test the expansion and culture of bovine NPPCs cells in a novel spheroid plate.
Material and Methods
NP tissue was isolated from 5-6 discs form 4 bovine bovine tails (aged 10-13 months). NP cells were isolated by using a mild two step digestion protocol. Then, the primary NP cells were stained with Tie2 Antibody conjugated with Alexa 488 and DAPI for FACS. Sorted NP cells were expanded for 2 weeks under hypoxia (5% O2 ) and were supplemented with 2.5ng/ml FGF2 until 80% confluency. Tie2+ and Tie2- cells were then seeded in the functionalized and in the 2D control wells of the spheroid plate SP5D of Kugelmeier Ltd (see fig. 1). The cells were cultured for 2 weeks under hypoxia and FGF2. Colony forming unit-assay was performed at day 0. Cell activity, DNA/GAG content and gene expression of selected genes was measured on day 1, 8, 15.
Results
The DNA content of the Tie2+ and – spheroids (see fig. 2) increased by a factor of 1.7 and 3. The cells in the 2D plastic control wells grew faster and increased by factor of 11 and respectively 18.
Figure 1: Spheroids of bovine NP Tie2+ cells stained with Calcein AM and Ethidium homodimer in the SP5D spheroid plate by Kugelmeier, 400 cells/microwell, 1d 6h after seeding
Figure 2: Absolut DNA content/well, either 750 spheroids or 2D control in 24well plate, Tie2 + and Tie2-, cultivated under hypoxia (2% O2 ) and supplemented with FGF2
(N=1,
n=3),
means
±
SD
Conclusion
The obtained results of the pilot show that sorted bovine NP cells proliferated in all culture conditions. There seems to be a big difference in the cell proliferation rate depending on the chosen culture system (3D vs 2D). However, more biological replicates are needed for further evaluation of the results.
References
1. Spencer et al., Lancet 392:1789-858, 2018
2. Bischof et al., Applied Sciences 11: 7144, 2021
3. Guerrero et al., JOR SPINE 4:e1131, 2020.
4. Sako et al., Applied Sciences 11, 2021
5. Zhang, et al., Int. J. Mol. Sci 21: 9423, 2020
6. Sakai, et al., Nat Commun 3:1264, 2012
Acknowledgements
This study is supported by an SNF Bridge – Discovery Grant (https://data.snf.ch/grants/grant/211510). FACSLab and Life cell imaging (LCI) of the DBMR, University of Bern are gratefully acknowledged.
Date of Publication
2023
Publication Type
Conference Item
Subject(s)
Language(s)
en
Contributor(s)
Oberli, Andrea |
Access(Rights)
restricted