Particle Preparation

A common problem experienced was pockets of trapped air inside the pores causing them to float, to overcome this problem, particles were left in a vacuum oven to allow any that were floating to sink, this was anywhere between a few hours to overnight. They were then sterilised using a strict procedure in a biological hood. Initially washed in 100% ethanol, then centrifuged for several minutes a gradual solvent exchange process was carried out with 70% ethanol and PBS, followed by centrifuging again, and this process was repeated for 50%, 25% ethanol-PBS, before a 100% PBS was added. Following this by two washes in media each with a centrifuging step in- between. The particles were stored in cell medium in the fridge to be used when required.

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4.6. Actuator Setup

To stimulate the magnetic particles a linear actuator (L16 Linear Actuator 140mm, 35:1, 12V with Potentiometer Feedback) was modified so an arm of neodymium magnets with (0.8 kg of pull) could be attached perpendicular to it (See Figure 35). The cable was connected to a linear actuator control board (Firgelli Automations), and a 12 V power supply. Thus, by using the provided software the speed and frequency could be controlled to a high degree. The construction was rudimentary; the flasks were held by a bar clamp, and taped to a box – used to elevate the flasks above the movement of the actuator. The actuator itself was held down using several cable ties onto a test-tube rack, and a Blu Tack counterbalance to level the magnetic arm. To test the setup, microcarriers in water were moved using the arm. It was found that depending on the distance between the magnets and particles, they could move the greater distances of the flasks when close and little when further away. 3 mm was the maximum distance that demonstrated any displacement of the particles (Figure 36).

Figure 35: Image showing the modified linear actuator setup, and three T-25 flasks coupled using a G-clamp. The illustration on the right is for clarity is an isometric drawing of the critical components of the setup.

Figure 36: Timelapse of moving particles at a maximum speed of 3.2 cm/s, what can be seen is the movement of the magnetic particles to the left of the T-25 flask as the magnets pass beneath the surface.

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4.7. Cell Culture

4.7.1. Fibroblasts

DMEM Medium containing: L-Glutamine, 200 mM; Fungizone, Penicillin-Streptomycin and Fetal Calf Serum was mixed. A T-75 containing fibroblasts (patient: 298) was cultured in 15 ml of medium until they were 70 – 80% confluent, at passage 2.

4.7.2. HESMPs

4.7.2.1. Media Preparation

Expansion medium: Alpha-MEM (α-MEM), Foetal Bovine Serum (FBS), Penicillin (10,000 units)-Streptomycin, (10 mg/mL), L-Glutamine (200mM).

Passage Medium: Expansion medium, Fibroblastic Growth Factor Basic Recombinant Human Protein (hFGF) (10 μg/mL)

Osteogenic Medium: Expansion medium, 0.5 M beta-glycerolphosphate solution (βGP) 5 mg/mL Ascorbic Acid 2-Phosphate solution (AA2P), 10 μM Dexamethasone solution (Dex).

4.7.2.2. Cell culture

A T-75 flask was coated with 2.5 mL of gelatin solution (0.1 w/v% gelatine solution) and left for 30 minutes. The cells were defrosted by holding the frozen vial until it thawed; it was then transferred with 12 ml of warm expansion medium. The vial was rinsed into the same medium to ensure most of the cells were transferred. After centrifuging at 1,000 rpm for 5 minutes, the supernatant was carefully removed. The cells were carefully aspirated in 12 ml of fresh expansion medium which was supplemented with hFGF, and transferred into the gelatine coated T-75 flask, and left to incubate. The cells were passaged using the supplemented medium and were used inoculated when they were 70 – 80% confluent.

4.7.3. Cell Counting

The medium was removed from the flask, and 5-6 ml of Trypsin-EDTA was added to the T-75 flask in order to detach the cells, they were then incubated for 5 minutes. The cells were checked under a microscope to see if they had detached; if not, they were encouraged to do so by knocking the side of the flask against a firm surface. The now cell suspension was swiftly diluted with approximately 10 ml of medium to neutralise the trypsin and transferred into a universal tube. It was centrifuged, for approximately

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~ 5 mins at 10,000 rpm. The medium was decanted, while ensuring the pellet of cells remained stationary in the universal tube. A concentrated cell suspension was created by gently aspirating and purging 1 ml of medium until the pellet had re-dispersed. A Neubauer haemocytometer was used to estimate the number of cells in the suspension; this along with a glass coverslip was prepared by cleaning with 70% IMS solution and then drying. Moisture was applied to the coverslip, to help it fix to the surface of the haemocytometer (Flip upside down to check it has stuck down properly). 10 µl of the concentrated suspension was pipetted between the coverslip and haemocytometer. The grid was focused using a microscope (10x objective) so that the cells within the squares can be counted. Cells were calculated by 𝑇𝑜𝑡𝑎𝑙 𝑁𝑜. 𝑐𝑒𝑙𝑙𝑠 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑥 104 _{to get cells per millilitre.}

4.8. Cell Seeding

The particles were firstly prepared in a biological hood at all times to ensure they remained sterile, by stirring and pipetting 1-2 ml into each T-25 flask used. The medium in each T-25 was replaced by an expansion medium and left in an incubator for up to an hour. This period was adequate to prepare and count the cells, ready for culture.

Cells were counted, and 250,000 cells were seeded randomly into each area of the flask, before being put back into the incubator. The medium was changed three days later, by removing 3 ml and adding 3 ml of fresh medium back in, this was done for a week to allow the cells to proliferate and form clumps. After 7 days the cells were carefully transferred into, new flasks using Pasteur pipettes, the medium was also replaced with osteogenic medium (See above). The experiment was started a day after transfer.

4.9. Cell Stimulation

Two different methods of stimulation were employed, the first to demonstrate the idea, and the second utilised the magnetic response.

4.9.1. Orbital Shaker

EHA-IBOA particles containing no iron were used to demonstrate the initial concept of shear stress-induced differentiation. Cells were seeded in six T-25 flasks; 3 static controls and 3 dynamic culture flasks. They were left for 3 days allowing the

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aggregation of particles, before the first media change. They were shaken 30 mins a day at 50 rpm, for 7 days. Fresh media was replenished every 3 days by firstly removing 3 ml, and then adding 3 ml, keeping the total volume at 5 ml.

4.9.2. Modified Linear Actuator

The culturing process remained the same as before. Iron embedded microcarriers were stimulated at 100, 50 and 10 % speed for 30 mins daily for 6 days, and the experiment was stopped on day 7. Refer to Table 10 for settings.

Table 10: Actuator settings programmed into the Firgelli actuator software, include settings for all experiments done.

Speed (cm/s) Accuracy (%) Retract Limit (%) Extend Limit (%)

Cycle/ Minute 3.2 (100%) 96 10 80 13

1.6 (50%) 96 10 80 10

0.3 (10%) 96 10 80 4