Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Summary

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.

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 PuraMatrix^9,10 (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

1. Part 1: Culture of hNPCs in PuraMatrix

1. In advance to the generation of a scaffold with a PuraMatrix concentration of 0.25% without laminin one needs to prepare the following solutions
2. Prepare a solution containing 20% sucrose and a solution containing 10% sucrose dissolved in sterile distilled water.
3. For solution 1 mix 120 μl of the 20% sucrose solution with 120 μl of distilled water in a 1.5 ml conical tube.
4. For solution 2 mix 60 μl of the PuraMatrix solution with 60 μl of 20% sucrose solution in a 1.5 ml conical tube.

For a Preparation of a scaffold with a PuraMatrix concentration....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PuraMatrix peptide hydrogel	BD Biosciences	354250
Mouse laminin I	Cultrex	400-2009090
Sucrose	Sigma-Aldrich	S9378-1KG
Normal goat serum	Dako	X0907
Triton X 100	Carl Roth Gmbh	3051.3
PBS Dulbecco	Biochrom AG	L 1825
HBSS	GIBCO, by Life Technologies	14170-088	Hanks’ Balanced Salt Solution 1X
βIII-tubulin antibody	Santa Cruz Biotechnology, Inc.	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 & Co., Inc.	7695
Trypsin/ EDTA	GIBCO, by Life Technologies	25300-054
Benzonase 250 U/μl	Merck & Co., Inc.	1.01654.0001
Trypsin Inhibitor	Sigma-Aldrich	T6522 (500 mg)
20% HSA	Octapharma	Human-Albumin Kabi 20%