Cell Patterning to Control the Isolation and Clustering of hNT Astrocytes in vitro

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Chapter 3 Cell Patterning to Control the Isolation and Clustering of hNT Astrocytes in vitro

Introduction

In this chapter, we examine how the parylene-C/SiO2 platform can be used to introduce structure into in vitro astrocyte cultures. Subsequently, in Chapter 6 we examine the Ca²⁺ activity of such patterned astrocyte cultures and compare and contrast this with traditional non-patterned astrocyte cultures.
Cells grown in vitro exist in an environment that is different from that which they would experience in vivo. In order for experimental results from in vitro culture to be physiologically relevant it is important to replicate, or substitute, salient features of the in vivo environment. Typically, this involves ensuring that: the cell culture substrate has the appropriate surface properties to enable cell attachment; the cells are cultured in a medium that contains the required nutrients and growth factors; the culture media is it at the correct temperature and pH; and the cells have sufficient access to oxygen. While those factors are sufficient for many cell types, the structure of cell interactions is an additional factor that is critical to the function of other cells. For example, the nature of cell-to-cell communication in neurons, through their axons and dendrites, gives rise to information processing and signal transduction throughout the body. Similarly, the structure of astrocyte interactions in the brain is thought to be important for proper the function of astrocytes. Recent literature has demonstrated that an astrocytes morphological structure is an important determinant in the intracellular propagation of Ca²⁺ signals [121, 122]. Contemporary literature suggests that astrocytes exist in networks and communicate through transient elevations in intracellular calcium [12]. On a larger scale, the structure of the interactions between astrocytes, has been demonstrated to influence the propagation of Ca²⁺ signals intercellularly. It is, therefore, important to develop cell culture techniques that are able to influence astrocyte-to-astrocyte interactions. By exerting control over the interaction between astrocytes, we expect that patterned astrocyte cultures will be able to replicate features of in vivo astrocyte behaviour. Furthermore, the level of control afforded by patterned cell cultures will help to unravel the structure-function relationship that governs the role of Ca²⁺ in astrocyte physiology.
A recently developed cell patterning technique uses the organic polymer parylene-C, deposited on SiO2, to alter the absorption of serum proteins to the cell-culture substrate. Differential protein absorption at the parylene-C and SiO2 surfaces promoted contrasting cell-adhesive and cell-repulsive areas that were able to pattern cells [66, 68]. The parylene-C/SiO2 platform has been used to effectively pattern a variety of neural cell types, including primary murine hippocampal neurons and glia [66, 70], HEK 293 cells [67] and human astrocytes and neurons derived from the NT2 cell line [85, 96].
Early patterned cultures that used the parylene-C/SiO2 platform, produced by Delivopoulos et al., were grown on long and wide parylene strips [66]. Such cultures were able to define areas where astrocytes and neurons would and would not grow. However, because the parylene-C areas were larger than the size of the glial cells used, within a large parylene-C area there was little control over how the cells grew. Later work by Hughes et al. used thin strips of parylene to isolate HEK293 cells in linear patterns. Such thin strips were able to limit cell-to-cell interactions by limiting the formation of cell processes to one dimension [67]. Similarly, Unsworth et al. demonstrated how thin strips of parylene-C could be used to isolate individual astrocytes [96].
The motivation of this chapter is, therefore, to systematically explore how varying the geometry of the parylene-C pattern can generate astrocyte cultures with defined qualities, such as isolation and clustering.
The work presented in this chapter is, in part, drawn from the paper “Patterning of functional human astrocytes onto parylene-C/SiO2 substrates for the study of Ca²⁺ dynamics in astrocytic networks.” published in the Journal of Neural Engineering in February 2018 by Brad J. Raos, Dr M. Cather Simpson, Dr Colin S. Doyle, Dr Alan F. Murray, Dr E. Scott Graham and Dr Charles P. Unsworth. The previously published results included in this chapter are largely unchanged, as allowed by the University of Auckland under the 2016 Statute and Guidelines for the Degree of Philosophy. The co-authors of this work have advised and commented on the manuscript, however, the bulk of the research and preparation for the published work was undertaken by the thesis author (see accompanying declaration).

Methods

In this section, we describe the design of parylene-C/SiO2 substrates that were used to assess the influence of the parylene-C pattern geometry. Next, we briefly describe cell culture protocols that were used in this chapter, including astrocyte differentiation and plating on parylene-C/SiO2 substrates, fluorescence labelling of astrocytes, and image acquisition and processing. Finally, we describe the statistical methods and notation that were used in this chapter.

Design of Parylene-C/SiO2 substrates

The cell-patterning substrate consisted of a grid of equally spaced square nodes of parylene-C surrounded by SiO2 (Figure 3.1). The design parameters of this pattern, the node size (dN) and the inter-node distance (dS) are indicated in Figure 3.1(a). We hypothesized that smaller nodes, due to their limited surface area, would be able to isolate single astrocytes, whereas larger nodes would be able to isolate clusters of multiple cells. The hypothesis is demonstrated visually in Figure 3.1(b).
We analysed a range of node sizes that were based on the typical sizes of hNT astrocytes in culture. Preliminary experiments were performed to determine the range of astrocyte sizes in culture. Astrocytes were plated at a density of 50 cells per mm² on Petri dishes. Images of the astrocytes were captured both immediately after plating and after 24 hours in culture. The size of an astrocyte was approximated by the maximum width of the cell. Figure 3.2(a) indicates that the mean size of astrocytes suspended in media prior to attachment was 37 ± 7 µm (s.d., n = 50). After 24 hours in culture the hNT astrocytes adopted a polygonal morphology with a large spreading cytoplasm and a mean size of 146 ± 34 µm (s.d., n = 87). Consequently, we evaluated a range of node widths between 25 and 200 µm. Similarly, we investigated inter-node distances of 50, 95, 140 and 230 µm in order to determine the minimum distance between adjacent nodes that would be required to prevent cells from growing between nodes.

Astrocyte Differentiation and Plating on Patterned Substrates

hNT astrocytes were differentiated from NT2 precursor cells according to the protocol outlined in section 2.2. Cells with a neuronal morphology were removed by selective trypsinization and discarded. Immediately prior to plating the astrocytes, the patterned parylene-C/SiO2 substrates were cleaned and activated by immersion in FBS according to the protocol outlined in section 2.4.2.
hNT astrocytes were plated in 200 µL aliquots on parylene-C/SiO2 samples at 50 cells per mm². We chose a plating density of 50 cells per mm² because that was within the range of cell densities that we observed at the end of the astrocyte differentiation – typically between 33 and 100 cells per mm². 50 cells per mm² was also approximately the cell density where hNT astrocytes would form a confluent monolayer. At higher cell densities hNT, astrocytes would still form a confluent monolayer, however, the cells adopted a more compact morphology.
Astrocytes were plated simultaneously on both non-patterned and patterned substrates. The non-patterned substrates served as a control to assess the variability in the actual plating density compared to target of 50 cells per mm². The mean cell density of astrocytes on non-patterned substrates was 48 ± 5 cells per mm² (n = 16). Consequently, we do not expect that variability in the actual cell plating density had a significant influence on the results presented in this chapter.

Fluorescence Labelling and Imaging

We quantified cell-patterning on parylene-C/SiO2 substrates after 48 hours in culture. Astrocytes were labelled with CMFDA, fixed with 4% PFA, and then counterstained with Hoechst 33258, according to the protocols that have been outlined in section 2.5.1 and section 2.5.2, respectively.
Samples were imaged on an Olympus BX53 upright microscope that was equipped with a motorized stage. Samples were illuminated by a mercury-bulb and fluorescence images were obtained using GFP and DAPI (470-495/550 nm and 360-370/410 nm, Em/Ex) fluorescence filters. Greyscale 8-bit images were captured at 10x magnification in a grid mosaic pattern that covered the entire sample.

Image Processing for Cell-Patterning Analysis

Fluorescence and brightfield images of labelled astrocytes on parylene-C/SiO2 substrates were processed to generate binary masks that represented cytoplasm areas, nuclear areas and parylene-C areas. Image processing was performed according to the protocol outlined in section 2.7.
Processed images were then used to determine the quality of cell patterning by calculating the parylene adhesion index (PAI), the silicon repulsion index (SRI), the mean number of cells per cluster and the node isolation index (NIX), according to the protocols outlined in section 2.8.

Statistical Analysis

Statistical analysis of the patterned cultures was performed in MATLAB. Null hypotheses and p-value have been reported in the text where they are relevant. The Bonferroni correction was applied where multiple comparisons were performed. The notation, _/ , has been adopted to refer to the mean value of either the PAI, the SRI, the mean cluster size or the NIX on a given patterned substrate with a node size of and an inter-node distance of .

Results

In this section, we present results that demonstrate how the parylene-C geometry influenced the quality and characteristics of astrocyte patterning. First, we consider the effect of node size, followed by the effect of inter-node spacing. Cell patterning is evaluated in terms of the PAI, the SRI, the number of astrocytes per cluster, and the NIX.

Influence of Node Size on Astrocyte Patterning

We hypothesized that the size of astrocyte clusters that we would be able would depend on the parylene node size. The influence of parylene node size on astrocyte patterning is demonstrated first in Figure 3.3 using characteristic images of astrocytes cultured on parylene-C/SiO2 substrates. That influence is then quantified in Figure 3.4 by the PAI and the SRI, in Figure 3.5 by the mean cluster size and the NIX and in Figure 3.6 by the distribution of cluster sizes. Finally, in Figure 3.7, we consider whether multiple astrocytes that had been isolated on a single node could be considered part of the same cluster.
Figure 3.4(a), below, demonstrates that the PAI increased as the node size increased. Between 25 and 125 µm the PAI increased from approximately 0.25 to 0.7 and was significantly positively correlated with node size (Correlation coefficient = 0.75, p = 0.0049). Figure 3.4(a) also demonstrates that, on larger nodes between 125 and 200 µm, the PAI was consistently approximately 0.7 and that there was no correlation between the PAI node size (Correlation coefficient = 0.13, p = 0.68). Conversely, Figure 3.4(b) demonstrates that there was no trend in the SRI as a function of node size (Correlation coefficient = 0.29, p = 0.18) with all substrates having a SRI of approximately 0.9.
Figure 3.5(a), below, demonstrates that 75 µm nodes resulted in the smallest mean cluster size of approximately 1.5. Contrary to our initial hypothesis, as the node size decreased below 75 µm, the mean cluster size increased. That result can be rationalized by considering the images of 25 and 50 µm nodes in Figure 3.3(a) and Figure 3.3(b), above. Those images demonstrate that the astrocytes showed poor conformity to the 25 and 50 µm parylene nodes and appeared to tolerate growing on the SiO2 areas. Such cell growth on SiO2 led to astrocytes bridging between nodes to connect neighbouring clusters of cells. However, consistent with our hypothesis, Figure 3.5(a) demonstrates that as the parylene node size increased above 75 µm, the mean cluster size was significantly positively correlated with node size (Correlation coefficient = 0.83, p = 1.8e-5). The images of 75 to 200 µm nodes, in Figure 3.3(c) to Figure 3.3(h), demonstrate that the increase in the mean cluster size was due to astrocytes clustering onto single nodes, rather than cells bridging between nodes. The maximum mean cluster size of 4.5 was obtained in cultures on 200 µm nodes.
Similarly, Figure 3.5(b) demonstrates how the mean number of astrocytes per node varies with node size. The mean number of astrocytes per node is different from the mean cluster size because not all clusters were located on a node. While the intent of the pattern was to isolate cells on parylene-C nodes only, the SiO2 areas were not completely cell repulsive. That lack of repulsion resulted in limited cellular growth on SiO2 areas. Figure 3.5(b) demonstrates that 25 and 50 µm nodes contained an average of 0.11 and 0.5 cells, respectively. In contrast, the mean cluster size for 25 and 50 µm nodes was 2.8 and 1.7, respectively. The difference between the mean cluster size and the number of nuclei per node on 25 and 50 µm nodes indicated that those nodes were not sufficiently large to support astrocyte growth.
The poor conformity of astrocytes to 25 and 50 µm nodes is further demonstrated in Figure 3.5(c), which indicates how the NIX varied with node size. There was a positive correlation between the NIX and node size between 25 and 75 µm (Correlation coefficient = 0.60, p = 0.057). The decrease in node isolation on small nodes is, again, attributed to astrocytes growing on the SiO2 areas and making contact with neighbouring astrocytes. No correlation between node size was found between 75 µm and 200 µm (Correlation coefficient = 0.03, p = 0.90), indicating that above 75 µm the node size is not a significant factor in determining how well astrocytes on a node were isolated from neighbouring astrocytes.
Because the mean cluster size was only an average for the entire sample, we also investigated the distribution of cluster sizes on the patterned substrates. Figure 3.6, below, demonstrates how the distribution of the size of astrocyte clusters varied with node size. Clusters of greater than 7 astrocytes accounted for less than 5% of the total number of clusters and were excluded from the graph. Figure 3.6 demonstrates that the smallest node sizes, 25-75 µm, were highly selective for single astrocytes, whereas largest node size, 200 µm, resulted in an approximately uniform distribution of cluster sizes. Intermediate node sizes, 100-175 µm, exhibited a distribution that transitioned between the two extremes. In cultures on 75 µm nodes 74% of astrocytes were present as single isolated cells. Similarly, the proportion of astrocytes in larger clusters on 75 µm nodes decreased sharply, with clusters of 3 or more astrocytes accounting for an average of less than 10%. Conversely, on 200 µm nodes, single astrocytes accounted for only 20% of clusters, while a greater proportion of larger astrocyte clusters were present.
Finally, we observed that, in a minority of cases, two astrocytes could be present on the same node without being connected. Figure 3.7(a), below, demonstrates the case where the astrocytes on a node were considered to be part of the same cluster. In contrast, Figure 3.7(b) demonstrates the case where multiple astrocytes were observed on the same parylene node but were not part of the same cluster. While multi-cluster nodes do not affect the cell clustering and isolation metrics, in Chapter 6, we examine differences in Ca²⁺ signalling between astrocytes in different patterned cultures. In Chapter 6, we assume that astrocytes on the same node are part of the same cluster and, therefore, we verify this assumption below.
We defined astrocytes to be in the same cluster if they were part of the same object in the cytoplasm mask. Figure 3.7(c) shows the percentage of nodes that contained more than one cell cluster as a function of node size. We observed that practically all nodes that were 75 µm and below contained only one cluster of cells. As the node size increased there was a greater chance that multiple disconnected clusters would be present on the same node. In Chapter 6 we analyse Ca²⁺ signalling on astrocytes that had patterned on both 75 and 150 µm nodes at an inter-node of 140 µm. Because non-connected clusters on these nodes account for 0.3% and 4% of clusters, respectively, we do not believe that multi-cluster nodes would have a significant effect on any analysis of Ca²⁺ signalling in patterned astrocytes.
Substrates with node sizes of 75 and 150 µm were chosen for further investigation of the influence of inter-node distance on astrocyte clustering and isolation.

Influence of Inter-Node Distance on Astrocyte Patterning

The influence of parylene node size on astrocyte patterning is demonstrated first in Figure 3.8 using characteristic images of astrocytes cultured on parylene-C/SiO2 substrates. That influence is then quantified in Figure 3.9 by the PAI and the SRI, in Figure 3.10 by the mean cluster size and the NIX and in Figure 3.11 by the distribution of cluster sizes.
As the inter-node distance was increased the total area of parylene-C on the sample also decreased. That would result in a greater number of cells relative to the parylene-C area and, consequently, the PAI should be expected to increase. Figure 3.9 demonstrates that the PAI was positively correlated with inter-node distance for both 75 and 150 µm nodes (75 µm – Correlation Coefficient = 0.92, p = 0.0015; 150 µm – Correlation Coefficient = 0.82, p = 9.3e-4).
No significant trend in the SRI was observed for either 75 or 150 µm nodes (75 µm – Correlation Coefficient = 0.31, p = 0.31; 150 µm – Correlation Coefficient = 0.37, p = 0.23).
Figure 3.10(a) demonstrates that the inter-node distance had no significant effect on the mean cluster size for astrocytes cultured on 75 µm parylene nodes with all substrates resulting in a mean cluster size of approximately 1.5 (Correlation Coefficient = -0.14, p = 0.66). In contrast, astrocytes cultured on 150 µm substrates showed a trend for increasing cluster size as the inter-node distance increased (Correlation Coefficient = 0.73, p = 0.007). The mean cluster size for astrocytes on 150 µm nodes at an inter-node distance of 50 µm was 2.8 and increased to 3.6 at 230 µm.

Table of Contents
Abstract 
Acknowledgements 
Table of Contents
List of Figures
List of Publications 
Glossary
Chapter 1 Introduction and Background
1.1 Thesis Outline
1.2 Astrocytes
1.3 Cell Patterning
1.4 Parylene-C/SiO2 Platform
1.5 Further Applications of Cell Patterning to Astrocyte Cultures
1.6 PEGylation
1.7 Chapter Summary
Chapter 2 Common Protocols and Methods
2.1 NT2 Cell Culture
2.2 hNT Astrocyte Differentiation from NT2 Cells
2.3 Cryopreservation of NT2 cells and hNT astrocytes
2.4 Fabrication of Parylene-C/SiO2 Substrates
2.5 Fluorescence Labelling
2.6 Calcium Image Processing
2.7 Cell Patterning Image Processing
2.8 Cell Patterning Metrics
2.9 Image Processing Sensitivity Analysis
2.10 Chapter Summary
Chapter 3 Cell Patterning to Control the Isolation and Clustering of hNT Astrocytes in vitro
3.1 Introduction
3.2 Methods
3.3 Results
3.4 Discussion
3.5 Chapter Summary
Chapter 4 Evaluation of Parylene Derivatives for use as Parylene/SiO2 Cell Patterning Substrates
4.1 Introduction
4.2 Methods
4.3 Results and Discussion
4.4 Chapter Summary
Chapter 5 PEGylation of Parylene-C/SiO2 Substrates for Improved Cell Patterning 
5.1 Introduction
5.2 Methods and Materials
5.3 Results
5.4 Discussion
5.5 Chapter Summary
Chapter 6 Analysis of Calcium Signalling in Patterned hNT Astrocytes 
6.1 Introduction
6.2 Methods and Materials
6.3 Results
6.4 Discussion
6.5 Chapter Summary
Chapter 7 Nanosecond Laser Stimulation of Ca2+ Transients in hNT Astrocytes in vitro
7.1 Introduction
7.2 Methods
7.3 Results
7.4 Discussion
7.5 Chapter Summary
Chapter 8 Conclusions 
8.1 Future Work
References
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Parylene/SiO2 Cell Patterning and Nanosecond Laser Stimulation for the Study of Calcium Signalling in Human hNT Astrocytes