PRIMEROS AUXILIOS, TRATAMIENTOS Y PROGRAMAS DE SALUD

Assays were carried out at day 1 and 7 using Live Dead kits for mammalian cells (Life Technologies). After washing all samples in warm PBS, a mixed solution of ethidium homodimer-1 and calcein was prepared in a ratio of 4:1 and 300μl of this solution and incubated for 15min. After this period, samples were washed in PBS three more times, before being left in PBS for microscope imaging.

94

4.2.14 Immunostaining

All cell encapsulated hydrogel samples were washed with PBS before fixation in 4% paraformaldehyde (PFA) (Sigma Aldrich) at room temperature for 30min. Samples were stored overnight at 4°C in PBS at this stage if required. Samples were then incubated in 0.1% triton x-100 (Sigma Aldrich) for 30min at room temperature. Blocking was carried out in 1% bovine serum albumin (BSA) (Sigma Aldrich) in PBS for 1hr at room temperature. For monocyte samples, the primary antibody for CD206 (R&D Systems) was diluted at 1:100 in PBS with 1% BSA and samples were incubated at 4°C in a humidified chamber overnight. The Alexa 488 or Alexa 594 conjugated secondary antibodies (Molecular Probes) were added at a dilution at 1:200 in PBS with 1% BSA and incubated for 1hr at room temperature in a dark humidified chamber. The primary and secondary antibody addition steps were repeated for 27E10 (R&D Systems) before adding 4, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc.) at 1:1000 dilution for 10min at room temperature whereby samples were ready for observation under the microscope. Between each stage 3x5min washes in PBS were carried out. For astrocyte samples, DAPI was added at 1:2500 dilution as well as Cellmask 647 membrane stain (Sigma Aldrich) at 1:1000 dilution in a PBS 10% goat serum (Gibco BRL) solution for 10 minutes at room temperature whereby samples were ready for observation under the microscope. Between each stage 3x5min washes in PBS were carried out.

4.2.15 ELISA Assays

The methods used here were identical to those described in Section 3.2.7.

4.2.16 Statistical Analysis

All data is expressed as mean ± standard deviation. T-tests or 2 way anova tests were performed where appropriate with p<0.05 being viewed as significant.

95

Results

4.3.1 Fabrication of Microchannels

GelMA is a biocompatible photocrosslinkable hydrogel that has been shown to support cell growth when cells are in contact with its surface or encapsulated within its structure159_{. It is also an interesting material for drug delivery applications as its porous}

structure can be fine-tuned to control the rate of drug release into the surrounding environment160,161_{. By optimising the photocrosslinking time, mechanical properties of}

the gel can also be exploited to easily fabricate and retain microchannels within the structure. The concept and process for fabricating this system is illustrated in Figure 4.1.

Figure 4.1. Construction of dual-layered microfluidic system and its concept. A) Schematic diagram illustrating the concept of the transformation from dry insertion state to drug delivering implantation state. B) Preparation procedure of microchannel in GelMA hydrogel construct surrounded by microporous PDMS membrane.

96

Before the hydrogel system design could begin, the sacrificial fibre fabrication method needed to be selected. Initially, 250µm and 500µm agarose fibres were printed using a glass capillary system. A 4% concentration of agarose in PBS was chosen as higher concentrations would not be drawn into the glass capillaries easily and lower concentrations resulted in more breakable fibres. This technique had some success when printing simple straight channels on non-dried systems (Fig. 4.2A). However, once loop systems were utilised and gels had been dried and reswollen, fibres were repeatedly breaking inside the gel instead of being removed as a result of the added force required. The addition of Triton-X to the solution in various concentrations had no effect on the removal success. The fibres were also shrinking and deforming during the drying stage resulting in a smaller microchannel (Fig. 4.2B).

Figure 4.2. Agarose microchannel optimisation. A) 250µm microchannel formed by removal of 4% agarose fibre and successfully filled with dye; B) Cross-section of microchannel post drying and reswelling stage showing shape formation and shrinkage.

97

As the objective is to bioprint these fibres in exact configurations in future, an alginate bioprinting system was also explored. A biaxial needle system allowed Calcium Chloride to be printed alongside the alginate itself causing it to crosslink in situ. There were several problems with this. In order to allow the fibre to print where it was supposed to, the rate needed to be slowed to a point where the fibre was unevenly printing (Fig. 4.3A). As well as this, printing directly onto the hydrogel was not possible as there was not sufficient traction to anchor the fibre resulting in it sliding along the surface as the printing path was followed. Finally, hair was investigated as an option as it is strong and flexible and so can be easily manipulated into any shape. It retained a constant thickness of 80±16μm channel that was unaffected by the drying andreswelling stage (Fig. 4.3B). It tended to float to the top of the hydrogel so crosslinking of the hydrogel had to be completed in two halves to ensure the anchorage of the fibre in the middle of the gel. The addition of 100μm microconnectors to the mould ensured exact placement of fibre and allowed easy precise filling of the microchannels.

Figure 4.3. Alternate sacrificial fibre formation strategies.A) Bioprinted alginate fibre exhibiting uneven nature; B) Hair showing uniform diameter.

98

4.3.2 Hydrogel Optimisation

In addition to optimising the properties of the sacrificial fibre, the properties of the hydrogel also affected the microchannel structure integrity over an extended period of time. Concentrations of below 5% GelMA resulted in channels leaking and not being able to retain their shape after a number of days of infusion (Fig. 4.4A). It was also important to ensure that a loop system could be easily created and the channel filled successfully (Fig. 4.4B) as highly crosslinked gels (10% GelMA, 40s UV) broke when more complex shaped fibres were removed.

Figure 4.4. Microchannel integrity in GelMA. A) Demonstration of microchannel failure in weak GelMA gel; B) successfully filled looped microchannel.

During the developmental process it was also noticed that the drying and reswelling stages were having an adverse effect on the integrity of the hydrogel, particularly if they were left for more than 3 days in the dry state. After these preliminary studies had been carried out, three different hydrogel formulae were investigated: 5% GelMA, 10% GelMA and 10% GelMA/2%PEG. The addition of the PEG as a long chain crosslinking monomer between GelMA chains was to provide extra crosslinking and resistance to cracking upon drying. Firstly, their mechanical properties with varying degrees of

99

photocrosslinking post drying and reswelling were analysed. The compressive moduli of 5% GelMA, 10% GelMA and 10% GelMA/2% PEG were 2.83±0.40kPa, 3.26±0.45kPa and 6.41±0.67kPa, respectively (Fig. 4.5A). The reswelling profiles of the three formulations were also assessed in order to ascertain if they would exert undue pressure on the brain and if their reswell time had the potential to be problematic in surgery as sometimes implants have to be removed and reinserted several times during this time162_{. All three}

formulations had a similar reswelling profile with total reswell within 4hr in PBS (Fig. 4.5B).

Figure 4.5. Hydrogel optimisation. A) Compressive modulus of three different concentrations of GelMA/PEG hydrogel composites (n=6). B) Reswelling profiles of three different concentrations of GelMA/PEG hydrogel composites (n=5).

The thicknesses of the gels in their dry state were 210±40µm, 430±20µm and 350±20µm respectively. After 72hr drying in air, 10% GelMA/2%PEG was the only formulation which remained entirely crack free in all samples. SEM images taken of this hydrogel (Fig. 4.6)

100

show the porous structure of the gel with pores of 90±37µm in diameter, as measured across 3 different cross-sectional images.

Figure 4.6. SEM image showingGelMA/PEG porous structure.

4.3.3 PDMS Membrane Construction

Particularly at lower concentrations, GelMA can degrade quickly over time and diffusion through the gel can be quick163_{. For these reasons, as well as preventing excessive}

reswelling post-insertion and allowing even greater control over the drug release, a porous PDMS membrane layer was added to the microchannel containing GelMA gel. PDMS is also non-toxic and non biodegradable164,165 _{and the reduced surface area for}

diffusion through the hydrogel based microfluidic system further slowed the release of active molecules. The spin coating process over the silicon wafer with a pillar system created a porous membrane, the spacings and sizes of which could be manipulated. One recurring problem was ensuring that all pores were fully open as often excess PDMS resulted in pits being formed instead of pores (Fig. 4.7). An extra stage of removing any excess PDMS post spincoating using a glass slide was added to the protocol at this point.

101 Figure 4.7. PDMS Membrane formation. Microscope image showing open pores (dark) and closed pits (light).

The technique to combine the hydrogel layer with the PDMS membrane also had to be carefully thought through. Plasma bonding was a suitable method by which to do this as it is relatively simple, results in a strong bond and negates the need for glue or extra chemicals122_{. GelMA cannot be activated by oxygen even in its dry state so a thin mould}

of PDMS was left surrounding the dry GelMA which could be activated and bonded with the membrane. However, the membrane itself was too thin and fragile to be utilised in the plasma bonder alone, so a method of anchoring the membrane in some way became necessary. A method of partially curing PDMS membrane frames which could then be pressed onto the PDMS loaded wafers and added to the oven to completely cure was devised. This ensured the membrane could not only be safely placed in and sufficiently activated in plasma bonder but also be easily removed from the silicon wafer mould without tweezers or the risk of ripping in the first place (Fig 4.8). This thicker frame could be removed at a later stage to minimise the thickness of the whole system.

102

Figure 4.8. Construction of PDMS membrane and frame.Schematic demonstrating addition of partially cured PDMS frame to membrane pre-curing.

4.3.4 Chip Development

Once the individual properties of the dual layered microfluidic system had been designed it was time to devise something we could use for our cell experiments. The chip itself was designed as a square piece with one single cylindrical microchannel of 80±16μm through the hydrogel in order to simplify the calculations for diffusion (Fig. 4.9A). The appearance of the chip itself as well as the microchannel and porous membrane under the microscope are shown in Fig. 9B-E with the hydrogel stained green.