Repercusiones del cuidado en la vida del cuidador

This section demonstrates the methods of the experiment and sample preparation.

6.2.1 Methods outline

The off-axis digital holographic microscopy (DHM) system, introduced in chapter 5, was used as the imaging tool offering two and a half dimensional

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(2.5D) images/videos of unstained RBCs. In this study, we investigated the infected RBCs with multiple measurements. First, the blood smear samples, both healthy and infected RBCs, were imaged by DHM. The obtained volumetric images, which stand for the static morphologies, were analyzed quantitatively. Secondly, the dynamic deformability measurement of the RBCs was conducted in a simple microfluidic channel with RBCs adhered on the bottom of the channel (adhesion protocol can be referred in chapter 6.2.2). The changing shear stress controlled by a pressure pump (model) was used to deform the RBCs. Three types of live RBCs were investigated totally, the uninfected RBCs, the artificial inelastic RBCs, and infected RBCs. Table 6.1 demonstrates the experimental setting.

Sample Morphology (static) Deformability (dynamic) Uninfected RBCs Blood smears

RBCs adhered assay in microchannel Inelastic RBCs

Infected RBCs Blood smears

Table 6. 1 Experimental setting.

6.2.2 Double container imaging chamber for infected RBCs in microfluidics For live infected RBCs, the malaria parasites are living inside the RBCs (small portion of them are in the solution outside the RBCs that needs to be handled in a sterile environment. In order to handle those samples in a non-sterile environment (non-Physical Containment 2 Facilities, (non-PC2)), we proposed the use of a double container that stores sample for biosecurity control (in accordance to Biological Safety Policy of Australian National University and Australian Government Department of Health). We especially designed a secured transparent double container to hold samples with microfluidic capabilities. Figure 6.2.a) shows the DHM system and b) presents the photo of the container. Figure 6.2.c) demonstrates a cross section of side view of the design and equipped pressure pump system (AF1 dual-microfluidic and vacuum pump, Elveflow). Two filter discs (0.2 µm, Sterile, Thermo Fisher Scientific) were used at the entrance to prevent infected RBCs and parasites from escaping from the container. A microfluidic reservoir (50 mL Falcon Tube 1/1 port, Elveflow) was used for the air passage and to prevent any liquid from going to the pressure pump when there

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is negative pressure applied. To fabricate the container, acrylic sheets were used as the material for good optical transparency and isolation. The sheets were laser cut into designed pieces and assembled by an adhesive (cyanoacrylates). The bottom part, where the microfluidic chip and sample reservoirs sit, can be removed prior to the experiment for convenient installation.

Figure 6.2 DHM Imaging setup with double container imaging chamber for live infected RBCs experiments. (a) Photo of DHM system (b) Photo of double container on microscopy stage. (c) Diagram of design of double container and pressure pump system.

6.2.3 Sample preparation

In this section, sample preparation procedures are listed out, which include fixed RCBs sample (blood smears), artificially inelastic RBCs, and live infected RBCs as well as live healthy RBCs. Fresh O+ blood were donated from the Red Blood Cross, Canberra (ACT). And the laboratory strain (CS2) is original from reference 30.6.2.3.1 Blood smears

Glass slides (1.2mm, Livingstone) were cleaned prior to blood smears. Slides were dipped in acetone, rinsed with deionised water before being dipped in methanol and rinsed again with deionised water. Slides were dried with nitrogen gas before preparing blood smears. The smears were fixed with methanol (100% or absolute) and dried completely before imaging.

93 6.2.3.2 Artificially inelastic RBCs

Uninfected red blood cells were artificially stiffened to varying degrees by different concentration of glutaraldehyde. Fresh 25% glutaraldehyde (Electron Microscopy Sciences) was diluted through serial dilution down to a concentration of 0.01, 0.02% (v/v) in Phosphate-buffered saline (PBS). Uninfected red blood cells in supplemented RPMI at 4% haematocrit (HCT) were centrifuged at 500 xg for 5 minutes at room temperature to pellet the cells. Later an equivalent amount of diluted glutaraldehyde was added. The sample was incubated for 10 minutes at room temperature. The glutaraldehyde solution was then removed from the cells by centrifugation at 500 xg for five minutes at room temperature and rinsed in PBS thrice before resuspending in PBS to a 4% HCT.

6.2.3.3 Malaria infected RBCs

Continuous culture of parasites in vitro:

Plasmodium falciparum (described as parasites in the following text) were incubated and grown in RPMI 1640 medium (Gibco) which was supplemented with GlutaMAXTM II 0.38% (w/v) AlbuMAXTM II (Invitrogen), 2.5% pooled heat inactivated human serum, 20 µg/mL Gentamicin (Gibco), 480µM hypoxanthine, 10mM D-glucose and 4% haematocrit of O+ blood. Cultures were maintained in 10ml or 30ml culture dishes kept in a hypoxic gaseous environment of 1% O2, 5%

CO2, 94% N2 at 37℃. A continuous culture of parasites was maintained by

diluting the parasitemia to 0.2% and feeding with fresh blood and media every 48 hours. In the following text, the parasite culture refers to the mix of parasite and fresh blood and RPMI 1640 media.

Magnet purification of parasites:

Although in the same parasite culture, the stage of infection among all the RBCs population is not the same (mix of the trophozoites stage, schizont stage, ring stage and uninfected state). The purification of the parasites needs to be conducted to provide relatively uniform parasite culture. Parasite in trophozoites and later stage were purified from the ring stage and uninfected RBCs through an iron wool column placed in a magnetic field as described in the paper 31. Purified parasites culture was recovered in supplemented RPMI 1640 media for an hour at 37℃.

94 Synchronisation of parasites via sorbitol lysis:

Ring stage parasite culture was obtained via sorbitol lysis 32 18 hours post infection, which selectively lyses mature stage parasites. Cell pellet was obtained from the mixed culture later and was incubated with 5% D-sorbitol for 10 minutes and subsequently recovered in supplemented RPMI.

6.2.3.4 Diluted RBCs PBS solution for microfluidic chip infusion

The RBCs samples were diluted in PBS solution at a ratio of 1:10 to reach the lower concentration of the RBCs in solution. The infusion was taken immediately after the dilution. The procedure is aimed to avoid dense RBCs population over the channels, and to make sure there is no overlap RBCs in the microscopic FOV.

6.2.3.5 Microfluidic chip fabrication and surface modification

The PDMS microfluidic devices were fabricated by soft lithography process described earlier in chapter 4. The molded PDMS chunk was bonded on the coverslip to serve as the microfluidic chip, which allows short working distance objective lens to be used to image the channel inside. The dimension of the microchannel is at 100 µm width and 25 µm high. The channel inside was treated before the experiment, in order to provide the adhesive surface for RBCs stick to. Firstly, pure ethanol was flowed into the channel for 10 mins. 4% APES (3-Aminopropyltriethoxysilane) in ethanol serves as the adhesion promoter 33 was perfused for 15 min. after that, the 50-100 µg/ml Con-A solution (Concanavalin A, Sigma ) was filled up with the channel for 2 hours room temperature (or overnight 2°C). The microfluidic channel was rinsed with PBS slowly (7.5

µl/min) before experiments.