Культивирование прав нервные клетки-предшественники в 3-мерном самоорганизации пептидов Гидрогель
Summary
Здесь мы опишем использование самоорганизации 3-мерные эшафот к культуре человеческих нервных клеток-предшественников. Мы представляем протокол выпустить из клетки строительные леса, которые будут проанализированы в дальнейшем, например, с помощью проточной цитометрии. Этот протокол может быть адаптирован и к другим типам клеток проводить детальный механистически исследований.
Abstract
The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by βIII-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming.
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.
Protocol
Discussion
Использование 3D-строительные леса предлагает возможность изучать развитие различных типов клеток в культуре клеток ситуация ближе к ситуации в естественных условиях. Однако, что касается анализа, например, нейронных дифференциации или функциональные исследования нужно преодол…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить Нормана Крюгер за его отличную техническую поддержку.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| PuraMatrix peptide hydrogel | BD Bioscience | 354250 | |
| Mouse laminin I | Cultrex | 400-2009090 | |
| Sucrose | Sigma | S9378-1KG | |
| Normal goat serum | Dako | X0907 | |
| Triton X 100 | Roth | 3051.3 | |
| PBS Dulbecco | Biochrom AG | L 1825 | |
| HBSS | Gibco | 14170-088 | Hanks’ Balanced Salt Solution 1X |
| βIII-tubulin antibody | Santa Cruz | Sc-51670 | Mouse, monoclonal, 1:500 |
| Alexa Fluor 488 | Invitrogen | A 11029 | Goat α mouse, 1:1000 |
| Alexa Fluor 568 | Invitrogen | A 11031 | Goat α mouse, 1:1000 |
| Alexa Fluor 647 | Invitrogen | A 21235 | Goat α mouse, 1:1000 |
| Mowiol 4-88 Reagent | Calbiochem | 475904 | |
| Dabco | Aldrich | D2,780-2 | 1,4-Diazabicyclo[2.2.2]octane 98% |
| Cell strainer | BD Biosciences | 352350 | 70 μm pore size |
| Saponin | Merck | 7695 | |
| Trypsin/ EDTA | GIBCO | 25300-054 | |
| Benzonase 250 U/μl | Merck | 1.01654.0001 | |
| Trypsin Inhibitor | Sigma | T6522 (500 mg) | |
| 20% HSA | Octapharma | Human-Albumin Kabi 20% |
References
- Zhang, S., Gelain, F., Zhao, X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin. Cancer. Biol. 15, 413-420 (2005).
- Gelain, F., Horii, A., Zhang, S. Designer self-assembling peptide scaffolds for 3-d tissue cell cultures and regenerative medicine. Macromol. Biosci. 7, 544-551 (2007).
- Blow, N. Cell Culture: building a better matrix. Nature. Methods. 6 (8), 619-622 (2009).
- Hauser, C. A., Zhang, S. Designer self-assembling peptide nanofiber biological materials. Chem. Soc. Rev. 39, 2780-2790 (2010).
- Teng, Y. D. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc. Natl. Acad. Sci. U. S. A. 99, 3024-3029 (2002).
- Silva, G. A. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science. 303, 1352-1355 (2004).
- Gelain, F., Bottai, D., Vescovi, A., Zhang, S. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS. ONE. 1, e119-e119 (2006).
- Taraballi, F. Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures. Front Neuroengineering. 3, 1-1 (2010).
- Zhang, S., Holmes, T., Lockshin, C., Rich, A. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc. Natl. Acad. Sci. U. S. A. 90, 3334-3338 (1993).
- Zhang, S. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials. 16, 1385-1393 (1995).
- Horii, A., Wang, X., Gelain, F., Zhang, S. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration. PLoS. ONE. 2, e190-e190 (2007).
- Thonhoff, J. R., Lou, D. I., Jordan, P. M., Zhao, X., Wu, P. Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain. Res. 1187, 42-51 (2008).
- Ortinau, S. Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells. Biomed. Eng. Online. 9, 70-70 (2010).
- Abu-Yousif, A. O., Rizvi, I., Evans, C. L., Celli, J. P., Hasan, T. PuraMatrix Encapsulation of Cancer Cells. J. Vis. Exp. (34), e1692-e1692 (2009).
- Morgan, P. J. Protection of neurons derived from human neural progenitor cells by veratridine. Neuroreport. 20, 1225-1229 (2009).
- Giese, A. K. Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells. BMC. Cell Biol. 11, 94-94 (2010).
- Schmöle, A. C. Novel indolylmaleimide acts as GSK-3beta inhibitor in human neural progenitor cells. Bioorg. Med. Chem. 18, 6785-6795 (2010).
- PuraMatrix, B. D. Peptide Hydrogel. Guidelines for Use. Catalog No 354250, (2006).
