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Influence of Adsorbed Proteins on Cell Culture

Chapter 3۞Results & Discussion

To exclude any form of bias, preliminary experiments were conducted to ensure satisfactory calibrations, functioning and performance of each stage of the investigation. All findings are interpreted, categorised and discussed in each respective section. A total of 60 BCC samples were prepared for the complete range of experiments needed for the project, with triplicates and duplicates of each configuration at each stage of the experimental design, where possible.

In relation to the hypothesis of the influence of adsorbed proteins on cell culture quite intriguing and visible developments were found. The adsorption nature of transferrin has shown particle preference in-turn affecting cellular interactions with a distinctive surface-related influence.

3.1 SEM Visualisation

SEM evaluation was performed with a FE-SEM ZEISS SUPRA 40 VP. Imaging took place at two stages of the experimentation process including after the fabrication of the BCCs to determine the success of fabrication, and finally after three days of fibroblast culture to observe cell morphologies. Firstly, appearance of the BCCs show a large area coverage of crystals with the larger particles accommodating easily in the centre while the smaller particles showed tendencies of accumulating around the peripheral regions (Figure 3.1).

Figure 3.1: The four different combinations of BCC combinations under a magnification of 5kX for A and B; to 10kX for C and D. A – D: Si5/PMMA, Si5/PSC, Si2/PMMA and Si2/PSC respectively.

The majority of the BCCs exhibited regular crystal morphologies with high surface coverages with the largest crystals located at the centre. Each crystal consisted of a monolayer of large silica particles with a multilayer of surrounding small PMMA or PS particles. However, several distortions in structure were noticed, but mostly towards the periphery of the crystal. This was expected but the observed trend was unique. This could possibly be due to the progression of the evaporation process from the centre to the edges. This did not present any hurdles as most of the evaluation was performed on the widespread regular crystal zone.

The heat treatment approach in order to stabilise the crystals for cell culture seemed ideal as it inflicted minimal melting or fusion of the smaller particles and ensured fixation and clear distinction of the BCCs on the glass surface. This is in relative to the cell culturing results observed and will be discussed further in the later part.

A revelation into the topographical features was the difference between glass, TCPS and BCC substrates. Glass and BCC share a common trait of consistency of micro features whereas TCPS discloses a landscape scattered with micro-grooves and furrows (Appendix C). This may be why it currently takes the place of being the most prominent substrate of choice for tissue culture systems.

Figure 3.2 SEM images of fibroblast cells attached on the surface of TF adsorbed BCCs. BCCs under a magnification of 1kX for A, C and D; 5kX for B.

A – D: Si5/PMMA, Si5/PSC, Si2/PMMA and Si2/PSC respectively.

Two sets of BCCs were subjected to fibroblast cell culture. One set were deposited on glass with a 5% PS coating and the other set deposited on glass without a PS coating. Contrary to previous observations made by Wang et al. (2016), the BCC samples fabricated on the PS coating formed a film with the BCCs fixed to the surface and were easily washed away during the fluorescent staining procedure. BCCs manufactured directly on the glass substrates were more stable. Signs of media related damage was seen on a few samples of combinations Si2/PMMA and Si2/PSC. The mode and duration of heat treatment may hold the key to this difference findings. The thickness of the PS is a possible avenue for further investigation as well. The illustration (Figure 3.2) above is not ideal but confirms the attachment of fibroblast cells on the BCC substrates. The cells in the images above are a by-product of the cells being subjected to chemical fixation, staining of the cells in preparation for fluorescence microscopy. It still retains its integrity in displaying the attachment of cells on the surface of the BCCs. The differences in attachment numbers and morphology is shown in the fluorescence microscopy results shown below.

3.2 MALDI Analysis

A relatively contemporary strategy of employing the standard MALDI onto a diverse surface analysis approach, surprisingly yielded positive results. The m/z (M+H+) values for the detected proteins may not be equal to that seen in the standard MALDI (Appendix D) but nevertheless it gives the signature of a biomolecule. Working with a non-conductive surface (BCC), the protein signal around 80,000Da (TF) generated becomes a product of the ionisation and desorption of the biomolecule purely from irradiation by the laser. The best signal (Figure 3.3) was achieved on BCC combination 4 with combination 3 featuring a similar signal at a laser power setting of 150 with peak detection limit extended to 100,000Da. BCC combinations 1 and 2 did not yield impressive results but the presence of proteins on the surface has been confirmed through XPS assessment discussion below. This is believed to be caused by strong binding of TF to the large particle comprising the BCC surfaces or TF getting trapped in between the interstitial spaces between the small and large particles (Griesser et al. 2004). Further tuning involved settings such as pulsed extraction parameter (80,000Da) and a linear detection operation mode.

Figure 3.3: Transferrin peak identified on BCCs at approximately 80,000Da by Surface MALDI technique.

Instrument configuration and setup was the key to obtaining quality results. Standard protocols use the conductive sample plate for identification. This provides a high potential enabling rapid ejection of biomolecules upon irradiation. These results prove that the same principle can be applied to diverse surfaces for detection purposes with appropriate calibrations and setup.

Taking advantage of this protein identification program, standard MALDI has found wide applications in bacterial identification and antibody resistance testing. Bacterial identities are a reflection of characteristic ribosomal protein patterns present in an organism hinged on growth attributes and eccentricity. Microorganisms are verified by comparing attained standard MALDI results against a spectral database using variable algorithms (Wieser & Schubert 2016).

If required, further examination of cellular adhesion communication could be possible by removing the cells and analysing the secreted ECM on the BCC surfaces. This would reveal intimate details regarding how cells perceive and distinguish between surfaces including adhesion preferences (Rabe, Verdes & Seeger 2011).

3.3 XPS Analysis

Exemplary quantitative results derived from XPS show in detail the composition of all the substrates including the controls. Sifting through the peaks detected in the XPS survey spectra allow identification of each element present on the fabricated surfaces. Peaks at 101eV, 285eV and 530eV correspond to the elements silicon (Si 2p), carbon (C 1s) and oxygen (O 1s), respectively, which is the chemical composition of the different substrates. Traces of sulphur (S 2p) (168eV), nitrogen (399eV) (N 1s) and zinc (1020eV) (Zn 2p) were also detected on the TCPS control surface, which is believed to be a product of the surface treatment during the manufacturing stage. The control glass surface showed signals corresponding to magnesium (89eV). It is well known for glass to contain trace elements, but it was an interesting result.

Figure 3.4: Substrate composition comparison.

The chemical composition of the BCCs (Figure 3.4) depicts a transitional connection between the laboratory tissue culture gold standard TCPS, and the commonly used glass slides. These values are shown in detail in the following table.

Comparison of these results with the XPS of Polymers Database (Graham Beamson and David Briggs) showed congruence with minimal drift from the expected binding energies. A comprehensive examination of the carbon spectra for each BCC combination set highlighted the nature of bonding on the surface of the colloidal particles.

Preliminary studies conducted to measure the difference in binding characteristics for a duration of 1 hour and 3 hours led to the justification for a 1-hour adsorption period with monotonous results.

Table 3.1: Substrate elemental distribution.

Oxygen 11.66 56.92 31.61
Carbon 85.62 16.05 59.18
Silicon 1.22 23.02 8.08
Other 0.84 4.02 1.15

The intricate types of bonds not visible in the survey spectra are uncovered by high resolutions analysis (Figures 3.5A, 3.5B) of the carbon spectra. Although they may seem similar at first glance, a closer look depicts a myriad of connections. The most prominent of these is the ‘C-C’ bond with a registered binding energy of 285.0 eV.

Figure 3.5 A: High resolution carbon 1s spectra for Silica and PMMA BCCs.

Glancing at the monomers for PMMA and PS, we can clearly see the diverse carbon connections present within each molecule.

PMMA: – [CH2C(CH3)(CO2CH3)]n –  PS: – [CH2CH(C6H5)]n –

Figure 3.5 B: High resolution carbon bonding between Silica and PSC BCCs.

Carbon double bonds are regularly resolved at 284.7 eV, oxygen being electronegative in comparison to carbon, pulls the atoms closer resulting in higher binding levels of 286.0 eV (C-O) and 289.0 eV (O-C=O). Exclusive to PS is the pi-electron shake up (π- π* at 291.0 eV) originating from the resonance in the benzene ring structure.

Figure 3.5C: Pi-electron shake up at 291.0 eV in PS.

Even though peaks tagging adsorbed TF have been detected through MALDI, the first two combinations of BCCs are missing this signal. The confirmation of TF adsorption is confirmed by the presence of an amine bond (C-NH2) peak in the XPS spectra observed in all the samples at 400eV. Comparison of these results and the MALDI confirm that there must be strong binding on the surface preventing matrix encapsulation of protein molecules during MALDI sample preparation, which is needed for detection during laser irradiation.

Figure 3.6: Protein confirmation through XPS.

Traces of nitrogen from the control samples were investigated further with confirmation of protein adsorption (Figure 3.6) when contrasting the respective intensities (Appendix E). Throughout the acquisition of the XPS results, multiple high resolution spectra charge accumulation on the surface of samples was recognised. This causes narrow peaks to be distorted and seem wider. Possible causes for this were voted to be generated from the nature of the substrate (insulating/semiconducting) or from the presence of adventitious carbon. Adventitious carbon is a thin layer of carbon material that is often found on samples exposed to air (Mangolini et al. 2014).

3.4 SPR Analysis

Binding characteristics of biomolecules is easily detected with sensitive instrumentation like SPR. Iterating internal algorithms also provided valuable layering quantification additionally. Experimental adoption of the SPR sensor slide with coatings of the particle used to fabricate BCC substrates furnished dynamic results.

Knowing the refractive index of TF being ~1.6 (McMeekin, Groves & Hipp 1964), refractive index increment property (dn/dc) of 0.190ml/g (Zhao, Brown & Schuck 2011) and the TF isoelectric point of ~5.5 (Stibler & Jaeken 1990), equating the binding characteristic is can be achieved.

Figure 3.7 A: Changes in TIR exhibited by preference binding of TF on different substrates.

Between the two wavelengths (670 and 785nm) employed by SPR, optimum results were achieved at 670nm (consistency) and all readings with TF binding are with regard to this wavelength. It is obvious that when offered a choice of surfaces to bind to, proteins take the most advantageous platform but when forced to into interaction with surfaces, will eventually bind but with very little effort. By nature of all biomolecules having preferences, the biased binding of TF becomes apparent (figure 3.7A).

Corresponding to the change in degree of total reflective angles for each substrate surface, the quantity of TF deposited is seen below (Figure 3.7B).

Figure 3.7 B: Thickness of TF layers on different polymers.

The comparative thickness of TF on silica and PS particle materials falls within a small range except for PMMA. The large deposition of TF exhibited on PMMA is believed to be caused from the expansion of the molecules allowing excess uptake of TF to form a thicker layer.

3.5 Epifluorescence Microscopy of Fibroblast Attachment

Fluorescence microscopy of fibroblast interactions with the BCC surfaces and controls was performed and proved to be an add-on bonus during the experimentation timeline that heralded promising results, however at the current stage, it is best to present them in a qualitative manner. With a an Inverted Nikon Microscope, fluorescent markers such as DAPI (nucleus) and Phalloidin-TRITC (actin-cytoskeleton) were detected after being irradiated with a high-intensity mercury arc lamp at two distinct wavelengths of 405nm and 532nm respectively (Kubitscheck 2013).

Figure 3.8A: Fluorescence images after 3-day culture of fibroblast cells on controls (A and B) and on transferrin adsorbed BCC combinations (C and D). A) TCPS control, B) Glass control, C) Si5/PMMA and D) Si5/PSC.

Differences in exposure times for the fluorescence dyes resulted in different intensities (Figure 3.8). Despite this, it is quite apparent that there is a high influence of TF on the activity of the fibroblasts and morphology of the cells. On the controls, the cells seem to be content with independent growth with a uniform appearance. A clear distinction can be made of each individual cell which has adhered to the surface. It is visibly evident from Figure 3.8A that TF has a multiplying effect on fibroblasts as there is a logarithmic increase in cell numbers (Appendix F). What may not be so apparent is the fact there is a 3-dimensional network formed.

Figure 3.8B: 3-dimensional fibroblast network resulting from the influence of TF adsorbed to BCCs.

From independent spreading to a more collective growth pattern is recorded. Strong belief is that this phenomena is resulting from TF acting as an insulin-like growth factor-binding protein-3 (IGFBP-3) binding protein triggering this response. It is plausible to conclude that with the DNA catalytic and growth potentiating attributes, the cells are being stimulated to grow allowing development of a complex 3-dimensional network (Weinzimer et al. 2001). Comparing these results to the original aim of this research of defining the aspect of proteins involved in the culture medium of iPSC generation protocols, we can conclude that proteins portray a myriad of characters allowing the replacement of the feeder cells stage in iPSC methods. A deeper understanding of protein capabilities is called for future research developments.

Chapter 4۞Conclusion & Forecast

Culminating from all the results discussed previously, EICAA generated nanostructured substrates are viable cell culturing systems. They provide a favourable nano-topographical landscape appreciated by cells, are cheap and easy to fabricate. Further investigation into the prevalence for the requirement of a PS film to stabilise the substrate in cell culture media is required. BCC substrates or dual nature substrates require extensive research before commercial availability.

A combination of complimentary techniques including Surface MALDI, XPS and SPR helped understand the behavioural patterns of essential proteins with fluorescence and electron microscopic techniques providing exceptional visual records of cell attachment behaviour on the surfaces. This research document represents a pathway for the adoption of sophisticated instrumentation and investigating the role of transferrin in fibroblast behaviour.

The progress of this research has exceeded its initial conception and design with the activities of fibroblast cells being visually tracked in relation to TF protein adsorbed on BCC substrate surfaces. Contemplating on the outcome of this argument, we can refine our approach at understanding and developing ideal biocompatible surfaces for prostheses in medical applications and in tissue engineering and regenerative medicine applications (e.g. iPSC generation from fibroblasts) improving the overall quality of disease therapy using stem cells.

Future developments with this gained knowledge are needed in terms of more controlled cell growth studies given that there were clear differences in the response of fibroblasts with or without adsorbed TF and on BCCs. Modulation of cell functioning through cytokine based methods will pave a road to achieving higher control and prevention of anomalies or deformities in the human genome or with life-style associated disease states.

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Ͽ Index Ͼ

Amphotropic Pathogen (bacteria or virus) capable of infecting a wide range of hosts or species of cell lines.
Annexed Extension; Attached to something larger.
Cenozoic Era Age of Mammals (65.5 million years ago till present) – UCMP
Chimera/Chimaera A single life form produced from multiple genetic parents.
Ecotropic Pathogen (bacteria or virus) capable of infecting a narrow range of hosts or species of cell lines.
Haemochromatosis Iron overload caused by an inherited recessive condition causing joint pain and general malaise.
Hemopexin Evolutionary plasma based proteins.
Hypotransferrinemia Transferrin deficiency in RBCs caused by accumulation in the heart and liver.
In vitro Latin for ‘Within the glass’. Experimentation/study conducted within a controlled environment outside of a living organism.
In vivo Latin for ‘Within the living’. 

Experimentation/study conducted using/ within a whole, living organism.

Mesozoic Era Age of Reptiles (251 – 65.5 million years ago) – UCMP
Morphic In a specific shape or form.
Multipotent Animal cells capable of developing into one or more cell types but are further limited than pluripotent cells.
Palaeozoic Era Geologic era or Time of Ancient life (542  to 251 million years ago) – UCMP
pH Scale of determining the acidic or basic nature of an aqueous solution. Acidic (0 – 7), Neutral (7), Basic (7 – 14).
Plasmon A quantum of plasma oscillations of the electron density in a conducting medium.
Pluripotent Animal cells capable of differentiating into all cell types except that forming a placenta or an embryo.
Proliferation Growth and multiplication of cells with rapid spreading.
Quantum The minimum amount of any physical entity involved in an interaction.
Senescence The process of deterioration associated with age. Generally, cells enter a state of growth suspension without cell death.
Somatic Cells of the body excluding germ cell lines; Diploid in nature (two copies of each chromosome).
Totipotent Animal cells capable of differentiating into all the cell types, tissue, organs, etc capable of forming a complete new individual.

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