Following a set period of time in the model, cell retrieval from the 3D model for separate downstream analysis of different cell types was necessary. A series of experiments was performed to optimise cell extraction from the model and subsequent cell separation to isolate individual cell populations.
3.3.3.1 Collagenase optimisation
The initial stage of cell retrieval from a 3D type I collagen gel is gel digestion. On Vivek Mudera’s recommendation (UCL), lyophilised collagenase D was used for gel digestion, allowing total cell retrieval. Cell populations (ie. MSCs and HSPCs) were subsequently separated via flow cytometry. It is essential for the cells to be isolated from the gels without compromising viability. For flow cytometry, obtaining a suspension of single cells is of particular importance to obtain discrete data for each cell, and to prevent damaging the machine during analysis.
Lyophilised collagenase type D (Roche) was reconstituted in PBS to a stock concentration of between 25 and 50 mg ml-1, stored at -20 °C. This was thawed
and diluted at point-of use to create working solutions. The volume of collagenase solution used was equal volume to the gel being digested. As collagenase is not inhibited by serum, an equal volume of 10 mM EDTA in PBS was used to quench the reaction after the given incubation time.
3.3.3.1.1 Testing collagenase on cell free gels
To ascertain the range of concentrations and incubation times that could be used for this protocol, digestion of cell-free collagen gels was tested. The manufacturer recommended a working concentration between 0.5-2.5 mg ml-1. Initial trial
concentrations of 0.5 mg ml-1, 1 mg ml-1, 2 mg ml-1,and 2.5 mg ml-1 were therefore
used. To each 500 µl volume of gel, an equal volume of collagenase was added. The incubation times chosen were 15 min, 30 min, and 60 min in accordance with manufacturer recommendations. The same volume of EDTA was added to quench the reaction after the allotted incubation time. The liquid in the digested wells was then removed to a new well plate to observe the extent of fragmentation. From Table 3.3 it is evident that even the highest concentration and incubation time was insufficient to digest the gel. Hence, the lowest concentration was
Chapter 3 99 discarded and the incubation time was increased for subsequent viability tests in 3.3.3.1.2.
Time min-1
Concentration
mg ml-1 15 30 60
0.5 Gel intact Gel intact but mostly intact Gel fragmented
1 removed media Gel visible in removed media Gel visible in removed media Gel visible in
2 removed media Gel visible in removed media Gel visible in Gel partially intact 2.5 removed media Gel visible in removed media Gel visible in Gel intact
Table 3.3 Table showing state of collagen gels after digestion with collagenase of varying concentrations and durations.
Gels that could not be easily removed from the wells are indicated by shaded cells.
3.3.3.1.2 Testing cell viability after collagenase digestion
MG63 cells were seeded as a cell suspension (i.e. not a pre-formed spheroid) into collagen gels at approximately 8 × 104 cells per gel, resulting in a dispersed cell
population throughout the gel. Collagenase at concentrations of 1 mg ml-1, 2 mg
ml-1, or 2.5 mg ml-1 was added to each gel and incubated for 30, 60, or 90 min. 10
mM EDTA was added at the end of the allotted incubation time to quench the reaction. Subsequently, the cells were stained with a calcein AM/ethidium homodimer solution to assess the viability of the cell population after the digestion.
Cells were observed floating in the media after 30 minutes of digestion; the number of floating cells increased with digestion time. At longer incubation times and higher concentrations, cells were observed beginning to adhere to the surface of the well plate, having been freed from the collagen matrix. Viability stained cells were imaged after a 1-hour incubation with the stain. Cells were counted and the percentage viability is indicated in Figure 3.7.
Figure 3.7 MG63 cell viability after digestion of collagen gels with collagenase at different concentrations.
A calcein/ethidium homodimer viability test was performed following digestion and samples were imaged on a fluorescence microscope. The numbers of cells exhibiting green (live) and red (dead) fluorescence were counted, and percentage viability determined for each image. Values are mean percentage viability 95 % confidence interval (CI). Mean percentage viability was tested between the concentrations of collagenase at each time point using two- way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.
Two-way ANOVA was used to compare the percentage viabilities to the 0 mg ml-1
control at each time point. Statistically significant differences from the control were observed for 2 and 2.5 mg ml-1 at 30 min and 1, 2, and 2.5 mg ml-1 at 60 min.
However, at 90 min viability was not significantly different from the control at any concentration. As the viability of cells at all time points and concentrations remained above 80%, this was considered sufficient to obtain a large enough number of cells for RNA extraction and subsequent Fluidigm analysis. A concentration of 2.5 mg ml-1 collagenase D and an incubation time of 90 min were
therefore used for all subsequent digestions.
30
60
90
0
20
40
60
80
100
120
Incubation time/min
P
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%
0 mg/ml
1 mg/ml
2 mg/ml
2.5 mg/ml
**
*
*
***
*
Chapter 3 101
3.3.3.1.3 Spheroid dissociation after collagen digestion
As the MSCs were to be cultured as spheroids within the collagen gel, an effective method of dissociating the spheroids post-collagenase digestion was investigated. As cell-cell contacts within the spheroid are not easily dissociated, both enzymatic and mechanical methods were tested. Spheroids were removed from the supernatant following digestion by centrifugation, and then subjected to dissociation. Two methods of mechanical dissociation and incubation with trypsin were tested (Figure 3.8):
1. Needle: spheroids were taken up into a 21-gauge 40 mm needle and vigorously agitated up and down.
2. Pipette tip: spheroids were taken up into a 200 µl pipette tip and vigorously agitated up and down.
3. Trypsin: spheroids were enzymatically digested for either 2 or 5 min.
Following this treatment, the spheroids were seeded into a fresh tissue culture vessel. After 24 hours, they were stained with Coomassie Blue and imaged on a light microscope (Figure 3.8).
Figure 3.8 Methods for dissociation of spheroids after digestion of collagen gels.
Spheroids were subjected to mechanical or enzymatic dissociation, and the resulting cell suspension was seeded onto a fresh surface. After 24 hours, these were stained with Coomassie Blue. Scale bar = 200 µm.
Successful dissociation was indicted by small size of nanoparticle/cell clumps (dark areas in the images) and confluence of the cell monolayer. The pipette tip method was the least effective, with a disperse cell layer and multiple large cell aggregates. The 2-min trypsin digestion was slightly more effective but also produced large aggregates. No differences were observed between the 5-min trypsin incubation and the needle method: both produced a confluent cell layer, with multiple small aggregates. For the remainder of the project, a needle was used for resuspension, as it does not involve chemical treatment of the cells.
3.3.3.1.4 Investigating the effects of collagenase on a monolayer
For dissociation of the eventual triple co-culture model (Figure 5.2), ensuring that human umbilical vein endothelial cells (HUVECs) or osteoblasts (OBs) can be isolated from the model in addition to MSCs and HSPCs is important, so that they can be analysed separately. If collagenase has no effect on a monolayer beneath a gel, then once the digested gel containing spheroids and HSPCs has been removed, the co-cultured HUVEC/OB monolayer beneath can be extracted using
needle
pipette tip
Chapter 3 103 trypsin. Therefore, the effect of collagenase on a test cell monolayer was assessed.
C2C12 cells were seeded as monolayers onto wells of a 24 well plate at 1 × 104
cells well-1. After the cells had attached to the surface, a collagen gel was
overlaid. After 24 hrs of incubation to allow the pH to equilibrate, the gels were digested with 2.5 mg ml-1 collagenase for 0, 30, 60, or 90 min, which was quenched
with EDTA at the incubation end point. The supernatant containing digested gel was removed from each well, transferred to a fresh well, and stained with a viability kit to determine whether any C2C12 cells were captured during gel digestion, whilst the remaining monolayer was also stained, to ascertain the extent of damage, and whether it remained intact.
The FITC fluorescence area (µm2)was measured for each image and converted into
a percentage of the total image area (measured as 551623.228 µm2 for each image
using ImageJ) as a proxy of monolayer integrity. The percentages were analysed using one-way ANOVA to compare the integrity of the monolayer during each collagenase incubation to that of the control (0 min). No significant differences were observed for any time point (p > 0.05). A qualitative analysis of the images is summarised in Table 3.4.
Figure 3.9 Percentage of FITC signal from images taken of monolayers below gels treated with collagenase (0 min, n = 2; 30, 60, and 90 min, n = 4).
0
30
60
90
0
20
40
60
80
100
120
Incubation time/min
P
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Collagenase test using C2C12 monolayers
Incubation time Monolayer Media/gel digest fraction
0 min Monolayer largely intact,
with some peeling from the surface visible. High
viability.
Some cell growth from (presumably) transferred cells in
suspension. High viability.
30 min Monolayer largely intact,
with some peeling from the surface visible. High
viability.
Cell clumps visible, presumably from cells interacting with the collagen. Some cell growth on
tissue culture surface. Low viability.
60 min Monolayer largely intact
with a few gaps visible. High viability.
Cells all in clumps, with no growth on the tissue culture
surface. Very low viability.
90 min Monolayer largely intact
with a few gaps visible. High viability.
Cells all in clumps, with no growth on the tissue culture
surface. Very low viability.
Table 3.4 Qualitative analysis of collagenase digestion.
Analysis used images taken of C2C12 monolayers (beneath collagen gels) following incubation with collagenase, and images of culture media/gel digest supernates containing cells and gel following digestion.