Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.
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.
1. Part 1: Culture of hNPCs in PuraMatrix
For a Preparation of a scaffold with a PuraMatrix concentration of 0.25% supplemented with laminin (8 μg per 100 μl Matrix) one needs to prepare the following solutions.
To prepare the hNPCs for encapsulation in 3D scaffolds one has to prepare the following solutions. Dilute the Benzonase in the Trypsin/EDTA solution, using a dilution factor of 1:10.000. For a Trypsin-Inhibitor/Benzonase solution mix: DMEM/F12 + Benzonase 25U/μl + 1% HSA + Trypsin-inhibitor (0.5mg/ml).
The next steps, 1.8 to 1.11, should be done as fast as possible, because of the low pH of the PuraMatrix solution, which might be harmful for the cells.
2. Part 2: Immunocytochemical staining of entire matrices
3. Part 3: Release of hNPCs from the scaffolds for flow cytometry analysis
4. Part 4: Quantification of βIII-tubulin positive cells by flow cytometry
5. Representative Results
An example of hNPCs cultured in the self-assembling hydrogel scaffold PuraMatrix is shown in figure 1. The hNPCs grow in spheric like structures in unmodified PM. In this culture conditions, cells can hardly be recognised in a transmission light, although bundles of processes between the spheres are visible (fig 1A). Depending on the culture conditions of the used cell type, the matrix can be modified e.g. by adding laminin. For the hNPC cell line ReNcell VM (Millipore) laminin is necessary to induce a growth pattern with less dense aggregates but a more homogeneous distribution of the cells (fig 1B). Independent of modifications, the matrix can be used to study e.g. neuronal differentiation. Figure 1C presents a staining of the hNPCs for the neuronal marker βIII-tubulin. In this example the cells were grown for 7 days in the matrix and subsequently differentiated for 4 days, where differentiation was induced by withdrawal of the growth factors EGF and bFGF.13 Cell bodies and a dense network of processes built up by the cells can be recognised easily. However, it is obvious that a quantification of the cell number is time consuming, as a large number of pictures of different regions of the matrix has to be analysed, to obtain a reliable data base for a statistical analysis. In fig 1D one can see an example of cells released from the matrix and subsequently platted on cover slips. These cells might be used for functional studies.
The release of the cells from the 3D-scaffolds offers the possibility to analyse different parameters like expression of marker proteins or survival rate of the cells by AnnexinV/PI staining or a TUNEL assay. Figure 2 shows an example of a flow cytometry analysis of the percentage of βIII-tubulin+ cells. The negative control (cells not marked for βIII-tubulin) is shown in figure 2A. These controls are used to determine the gate for the later detection of βIII-tubulin+ cells (fig 2B). A comparison of a manual counting of cells and an analysis by flow cytometry is shown in figure 2C. The percentage of βIII-tubulin+ cells was determined by counting the total cell number (by means of a nuclear DAPI staining) and the number of βIII-tubulin+ cells in the fluorescence pictures.
Figure 1. hNPCs cultured in the self-assembling peptide hydrogel PuraMatrix. A) hNPCs encapsulated in PuraMatrix (PM) grow in spheric, dense packed structures, where the diameter of the spheres can be up to several hundred μm. In between the spheres one can recognise bundles of processes built up by the cells (arrows). B) Cells encapsulated in PuraMatrix supplemented with laminin (PML) grow in less dense structures, more homogenously distributed. In the laminin supplemented matrix one can recognise single cells in less dense areas (arrows), however it is hardly possible to quantify the number of cells. C) hNPCs differentiated for 4 days in PML express neuronal marker like βIII-tubulin (green). To evaluate the percentage of positive cells one has to determine the total cell number by a nuclei staining like DAPI (blue). The microphotograph presents the full projection of a z-stack of pictures taken with Biozero-8000 microscope. D) Cells released from the matrix can be seeded on e.g. cover slips for further functional studies. The microphotograph shows cells cultured for 3d, subsequently to the release procedure. The staining for βIII-tubulin (red) reveals a comparable morphology to the cells hosted in the 3D scaffold.
Figure 2. Flow cytometry analysis cells released from PuraMatrix. To overcome the time consuming quantification of microphotographs we used a protocol to release cells cultured in PuraMatrix giving access to faster analysis by flow cytometry. A) Unstained cells were used as negative control, to set the gate (black frame) for the subsequently analysis of e.g. βIII-tubulin cells. B) To quantify the percentage of βIII-tubulin+ cells the same gate, set in the negative control, was used. Positive cells appear in the right part of the x-axis, where also an intermediate population was observed (dotted frame), most likely representing cell debris. C) The comparison of manual counted cells and cells counted by flow cytometry revealed a much higher proportion of positive cells, where the time dependency of the number of βIII-tubulin+ was comparable, indicating the reliability of the flow cytometry protocol.
The use of 3D-scaffolds offers the opportunity to study the development of different cell types in a cell culture situation closer to the in vivo situation. However, regarding the analysis of e.g. neuronal differentiation or functional studies one has to overcome some obstacles to gain reliable data for e.g. quantification of cell types.
Here we described the culture of hNPCs in the peptide hydrogel based scaffold PuraMatrix and the subsequently release of the cells to be used for the studies in a 2D situation providing an easy access to tools as FACS or functional assays. Recently we demonstrated the influence of the PuraMatrix concentration on the formation of the 3D-scaffold by atomic force microscopy and the influence on the survival and differentiation of the cells.13 However, one has to keep in mind that each cell type may need different culture conditions or matrices consisting of other materials e.g. matrigel or collagen.
The protocol to release the cells is based on a protocol provided by the manufacturer18 and is comparable with protocols used to prepare e.g. primary neuronal cultures where a mechanical isolation of the cells is followed by a digestion of surrounding material by enzymes. The cells tolerate this procedure and stay vital and built up processes and express neuronal marker like βIII-tubulin, once they are seeded again on a laminin coated surface. Here we performed flow cytometry studies with released cells to quantify the amount of βIII-tubulin+ cells. The comparison to a quantification done “manually” by counting cells in micrographs revealed a much higher proportion of βIII-tubulin+ cells. This is most likely due to the higher number of cells counted by the flow cytometer (50.000 per probe) in comparison to the manual analysis (several hundred per probe). However, the kinetics of the number of βIII-tubulin+ cells follows the same pattern in both samples, where we detect a decrease of βIII-tubulin+ cells over the time. This is in accordance with studies using the ReNcell VM cells.15-17 Other hurdles to overcome during a manual analysis are very dense areas of matrices which can hardly be analysed or high background of the matrix material resulting in an underestimation of the “real” cell number. We are convinced that this protocol can be adapted to other cell types providing a fast and reliable method to quantify several aspects of proliferation, differentiation or survival of the cells.
The authors have nothing to disclose.
The authors would like to thank Norman Krüger for his excellent technical support.
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% |