Variables, medidas y manipulaciones

In the standard IVG model, I showed that adding collagen to the cells enhances their survival.

I wanted to examine the role of collagen in the EVG model to determine whether the findings in the agar model were consistent in the alginate microsphere system. Therefore, I made PBMCs suspensions in alginate either without or containing collagen and stimulated with Mtb. Each suspension was them sequentially bioelectrosprayed to generate microspheres containing PBMCs either with or without collagen. Supernatant was harvested from the spheroids and cell survival was assessed by LDH assay (figure 49). PBMCs that were mixed with collagen survived better than PBMCs with alginate only.

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Figure 48: Collagen addition to the microspheres resulted in less cytokines detected in the EVG model.

Mtb-infected PBMCs encapsulated using the bioelectrosprayer showed less Th1 cytokines (IL-1β and IL-12), Th2 cytokines (IL-5 and IL-6) and chemokine (IL-8). However, MCP1 showed minimal decrease in concentrations after adding collagen to the cells. Mean concentration was analysed from 3 experimental replicates per donor, and data shown are representative of results from 2 different donors. Error bars = standard deviation.

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Figure 49: Mtb-infected cells survived better when grown in the presence of collagen in the EVG model.

Mtb-infected PBMCs were mixed with type I collagen and encapsulated in microspheres and incubated. Supernatant of these spheres were harvested and the LDH assay was performed to assess cell survival. PBMCs in contact with collagen showed less LDH toxicity than cells without collagen. Data are representative of 3 experimental replicates per donor, and data shown are representative of results from 2 different donors. Error bars = standard deviation.

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Discussion

I have adapted a 3D cell culture model to further develop the classical monolayer IVG model of TB. I have impregnated freshly isolated PBMCs in collagen gel matrix and observed the progression recruitment of the cells in the TB-infected wells that formed a 3D structure. The cellular aggregates formed in response to Mtb infection and were absent in the uninfected wells. The kinetic pattern of cellular recruitment to the developing lesion in this model was similar to what was observed in the previous standard IVG model. By day 3, Mtb-infected cells start to form small aggregates which then developed more obviously by day 7.

The granuloma-like structure was confirmed by staining cells with CellTracker green that diffuse into live cells and stain cytoplasm in green. Combining images acquired by z-stack technique revealed that the formed aggregates in the Mtb-infected wells were detected at different levels demonstrating their 3D property. I harvested supernatants from wells of both collagen and agar 3D matrix gels and measured the concentration of LDH to assess cell viability. PBMCs grown within a 3D collagen gel matrix survived better than those impregnated in a 3D gel matrix composed of agar. When profiling MMPs in the supernatants of the 3D IVG model, I found TB infection leads to upregulation of MMP-1, 7, -8 and -9.

However, I was not able to replicate similar results when repeating the experiment multiple times.

In this chapter, I tried taking my cell culture studies further to resemble the environment in humans more closely. The previous standard culture system showed insight into the early events in TB granuloma formation. However, enhancing the granuloma model by impregnated cells within a matrix gel would allow for more understanding of the cell-matrix interaction and minimise the difference between cell culture and the normal physiological conditions of the lung tissue (Pampaloni et al., 2007). Furthermore, the formation of a 3D gel

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matrix would provide a suitable environment for the MMPs to function locally on different matrices.

Other researchers have also developed 3D IVG model and found results consistent with the results I observed. For example, Kapoor et al. reported an IVG of TB showing the development of Mtb dormancy in the granuloma which can be then resuscitated by immune suppression (Kapoor et al., 2013). They showed that human leukocytes formed granuloma-like aggregates at day 8 after infection with Mtb, though granulomas were smaller than what I observed in my model. These granulomas found to be secreting cytokines like IFN-γ, TNF-α and IP-10 reproducing the in vivo scenario.

Although the 3D cell culture model is favourable, it is associated with some technical and biological challenges. Dissolving the 3D matrix to isolate the cells or to study the local environment was one of the issues I was trying to address. Collagenase was used by other investigators to dissolve the collagen matrix but in my situation was not appropriate. This is because adding collagenase would lead to false results regarding concentrations of MMPs that I wanted to measure in this model. One of the possibilities of being unable to develop a reproducible 3D IVG model was that MMPs adhere to the matrix components and not diffuse into the supernatant efficiently. Therefore, I designed a matrix trapping experiment to identify any effect of matrix component that trap MMPs within it. MMPs appeared not to be trapped within the 3D gel except for MMP-9 that thought to be held within the collagen matrix.

The results of this 3D IVG model of TB confirm the findings I showed in the standard IVG model. The kinetics of the leukocytes recruitment and progressive accumulation into a granuloma-like structure was similar in both models. Furthermore, the 3D model also confirms the survival signal provided by the cell-matrix interaction and that collagen is important in enhancing cell survival. However, the MMP secretion profile in the 3D IVG model could not be relied on as it was not reproducible when repeating the experiment.

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Therefore, I was chose not to try to further develop this model to further study the cytokine profile and the role of MMPs in this model and moved to the bioelectrosprayer model instead.

Then, I moved to a novel engineered TB model of IVG where cells are encapsulated and sprayed to generate microspheres. Different type of cells can be studied and different matrices can also be added to the cells using a bioelectrosprayer, though there was the inherent risk of working with, an as yet, unproven model. Here, I have infected PBMCs with Mtb and the formed microspheres were monitored for 14 days and supernatants were collagen as cell survival enhancer as PBMCs mixed with collagen survived better than those without collagen.

The MMPs secretion profile showed that Mtb infection upregulated MMP-1, -7, -8 and -9.

This is also consistent to the secretion profile of MMPs in the standard IVG model where I found upregulation of MMP-1, -7 and -9. When profiling the cytokines in this EVG model, Th1 cytokines, IL-1β and IL-12 were upregulated in the Mtb-infected PBMCs which is consistent with the standard IVG results. Th2 cytokines, IL-4, IL-5 and IL-10 were found to be low in concentrations though increased in TB infected microspheres more the uninfected.

Chemokines and growth factors were also upregulated in TB. These findings are consistent with my previous in vitro models of TB and also with others published work (Kapoor et al., 2013). Therefore, the EVG model of TB is able to recapitulate the in vivo findings in relation to cytokine and chemokine secretion by immune cells in response to Mtb infection.

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In summary, I have taken the standard IVG model further and replicated a 3D model that showed that cell-matrix interaction is important in cell survival. However, due to some obstacles the role of MMPs in this model could not be revealed. The EVG model is a novel approach and may help provide valuable insight into TB pathogenesis. I have demonstrated the granuloma-like structure within microspheres and that adding collagen to the cells improves their survival.

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